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Life Science JourTUll, the Acta Zhengzhou University Oversea Version, is an international journal with the purpose to enhance our natural

and scientific knowledge dissemination in the world under the free publication principle. The journal is calling for papers from all who are associatedwith Zhengzhou University~ home and abroad. Any valuable papers or reports that are related to life science-in their broadest sense-are wel-

come. Other academic articles that are less relevant but are of high quality will also be considered and published. Papers submitted could be re-

views, objective descriptions, research reports, opinions/debates, news, letters, and other types of writings. Let's work together to disseminateour research results and our opinions.

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&litor-in-Chief: Shen, Changyu

Associate Editors-in-Chief: Ma, Hongbao ;Xin, Shijun; Li, Qingshan

Editors (in alphabetical order): An, Xiuli; Cai, Zeling;Chen, George; Cherng, Shen;Duan, Guangcai;Edmondson, Jingjing Z. ; Li, Xinhua;

Li, Yuhua; Lindley, Mark; Liu,Hongmin; Liu,Zhanju; Qi, Yuanrning; Sun, Yingpu; Tang, Wenxue; Wang, Lidong; Wen,Jiangoo;Zhang, Jianying; Zhang, Kehao; Zhang, Shengjun; Zhang, Xueguo; Zhang, Xuewen; Zhang, Zhan; Zhang, Zhao; Zhu, Huaijie ;Xue, Changgui

Website and Cover Design: Shi, Lifeng; Ma, Hongbao

Printer: Zhengzhou Yuxing Printing Limited Company

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li~~1'!IIIntroductions to Authors

1. General Infonnation

0) Goals: As an international journal published both in print and oninternet, Life Science JourTUll is dedicated to the dissemination of

fundamental knowledge in all areas of life science. The main purpose

of Life Science JourTUll is to enhance our knowledge spreading in theworld under the free publication principle. It publishes full-length pa-

pers (original contributions), reviews, rapid communications,. andany debates and opinions in all the fields of life science.

(2)Wbat to Do: The Life Science Journal provides a place for discus-

sion of scientific news, research, theory, philosophy, profes.,ion andtechnology that will drive scientific progress. Research reports and

regular manuscripts that contain new and significant information ofgeneral interest are welcome.

(3)Wbo: All people are welcome to submit manuscripts in life sciencefields. Papers of other fields are also considered.

( 4) Publication Costs: US$30 per printed page of an article to defray

costs of the publication will be paid by the authors when it is accepted.

Extra expense for color reproduction of figures will be paid by authors( estimate of cost will be provided by the publisher for the author's ap-

proval). For the starting, publication fee will be waived for the cur-

rent issues and they will be supported by Zhengzhou University.(5)Journal Copies to Authors: One hard copy of the journal will beprovided free of charge for each author.

(6)Additional Copies Bought by Authors: Additional bard copies and

offprints could be purchased with the price of US$4lissue (mailing andhandling cost included).

(7) Distributions : Web version of the journal is freely opened to the

world without payment or registration. The journal will be distributed

to the selected libraries and institutions for free. US$5/issue hard copyis charged for the subscription of other readers.

(8) Advertisements: The price will be calculated as US$400/page,i.e. US$200/a half page, US $ 1O0/a quarter page, etc. Any size of theadvertisement is welcome.

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tials, title, journal, year, volume, issue, and pages etc.Referem:e Exmnples :

Journal Article: Hacker J, Hentschel U, Dobrindt U. Prokary-otic chromosomes and disease. Science

2003;301(34) :790~3.

Book: Berkowitz BA, Katzung EG. Basic and clinical evaluation

of new drugs. In: Katzung EG, ed. Basic and Clinical Pharma-cology. Appleton & Lance Publisher. Norwalk, Connecticut,USA. 1995 :60 - 9.

00) Submission Address: editor@sciencepub. net, Marsland Press,2316 Gunther Avenue, Bronx, NY 10469, The United States, 718-513-0385.

01 ) Reviewers: Authors are encouraged to suggest 2 - 8 competent re-viewers with their name and email.

3. Manuscript Preparation

Each manuscript is suggested to include the following components butauthors can do their own ways:(1) Title page: including the complete article title; each author's full

name;institution(s) with which each author is affiliated, with city,state/province, zip code, and country; and the name, complete mail-ing address, telephone number, facsimile number (if available), and

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(3) Keywords. (4) Introduction. (5) Materials and Methods. (6)

Results. (7) Discussions. (8) References. (9) Acknowledgments.

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LI Life ScienceJournal, Volume 2, Number 1,2005 ISSN: 1097 - 8135

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CONTENTS~

Pages

1.Preface

Changyu Shen

2. Life and Immortality: A Comparison of Scientific, Christian, and Hindu Concepts

]ingjing Z. Edmondson

3. Nature of Life

Hongbao Ma, Shen Cherng

4. MINlREVIEW: A Puzzle of the Effect of Magnetic Field on Biological Cells

Hsien Chiao Teng

5. Expression and Purification of a Human Tumor-Associated Protein Annexin V

Kaijuan Wang, Liping Dai, Yan ]ia, ]ianying Zhang'~

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6. Clinical Investigation in Improved Quality of Life with Medroxyprogestrone Acetate in

Patients with Advanced Lung Cancer

Liping Wang

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7. Expression of the Antigenic Gene TspEI of Trichinella spiralis in Skinand Muscle of BALB/c Mice

ling Cui, Zhongquan Wang, Hongwei Zhang, Guoqiang Zhao, Haiyan Wei, Huamin Han..

8. The Endocrine Disorder by Smoking Inhalation

Sulin Wang, Xiufang Chen, Shen Cherng

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9. Study of Physiologically Required Selectivity Coefficients of Potentiometric

Sensors in Clinical Assays

Wei Zhang

~ 10. Comparison of Patients' Psychological Status between Controlled Seizures

and Uncontrolled Seizures by Symptom Checklist 90

Meizhen Sun, Wei Wang, Yuxi Liu, Kerang Zhang, Xiaofeng Ren

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2-6

7-15

16 - 21

22- 26

27 - 29

30-36

37 - 39

40-45 L

46-48

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Life Science Journal, Volume 2, Number 1,2005

49 - 54

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ISSN: 1097 - 8135

11. Changes of Left Ventricular Dysfunction and Cardiomyocyte Apoptosis inLosartan-treated Heart Failure Rats

Chunjing Fu, Hua Tian, Longhui Guo, Youtian Huang, Qi Shen

12. The Radiosensitive Therapy for Colorectal CanFer

Zifa Wang, Tracy Cook, David Blumberg

B.B-type NatriureticPeptide in Predicting Short-term Mortality in Patients with

Acute Coronary Syndromes: A Preliminary Report

Tongwen Sun, Lexin Wang, Shuxiang Zhang, Yanzhou Zhang

14. Transposition of Pedicled Adrenal for the Treatment of Cushing's Disease

Baoping Qiao, Gaoxian Zhao, Weixing Zhang, Haiyang Jiang

15. Study of Remanent Activated Sludge Used as Biosorbent (I) Effect of pH on Zeta Potential

of Dissociative Bacteria and Its Application in the Adsorption of Activated Sludge

Zhanhang He, Shujuan Ye, Yueyang Fan, Guangfeng Hong, Zhengshan Tian

16. Simvastatin Improved Matrix Metalloproteinase Mediating Ventricular- Remodelingin Rats after Myocardial Infarction

Jinying Zhang, Xiang Cheng, Yuhua Liao, Baojun Lu

17. EGF Receptor Tyrosine Kinase Inhibitor Tyrphostin AG1487 Induce Human Tongue

Cancer Cells Tca8113 Cell Cycle at Gl Phase and Apoptosis

Xinguang Han, Bogui Wen

18. Phylogenetic Relationship of Tetraogallus Inferred from Sequences of

Cytochrome b Gene

Yifeng Gong, Jinfu Wang, Hongyan Li, Li Wang, Runlin Ma

19. Anti-tumor of ILIT Combined with Cisplatin in a Rat Glioma Model

Bin Liu, Bilian Jin, Ling Li

Call for Papers

Author Index, Subject Index

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Life Science Journal, 2 (1 ) ,2005, Shen , Preface

Preface

Changyu Shen

President, Zhengzhou University, Zhengzhou, Henan 450001 , China

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Through the joint efforts of Zhengzhou University and the North American Alumni Associa-tion of the University, the Acta Zhengzhou University Overseas Version Life Science Journal isbeing made available to all readers. This journal not only offers an interface between all scientistsin the life sciences fields, but also opens an academic window to the world for Zhengzhou Univer-

sity. Furthermore, the journal will become a linkage between alumni of Zhengzhou Universityabroad and their alma mater.

In reviewing social history it becomes evident that human beings have always been involvedin exploring the origins both of material substance and of life. These explorations have been in theforefront of scientific progress, and technological innovations that bring about revolutionarychanges in human society stem from principal breakthroughs in the basic sciences. While in the20th century developments in the physical sciences took leading and dominant roles in scientific de-velopment, in the 2Pt century the core of scientific progress may gradually shift to the life sci-ences. The exploration of life includes the discovery of genetic material and the birth of molecularbiology, which advances provide humans with opportunities for understanding. the origin andmystery of life. Such work also gives rise to promising developments in biological engineeringtechnology. The atlas of the human genome is absolutely an important milestone in the history ofthe life sciences. Within the context of these developments, the establishment of Life Science

Journal will provide an additional arena for life science research.Life Science Journal is an interdisciplinary and international journal, and will publish aca-

demic theses, research reports, academic critiques and reviews. The journal will report on thenewest tendencies, academic trends, and research achievements in the life sciences both in Chinaand abroad, and will serve as a medium of communication between Zhengzhou University re-searchers and those in other countries of the world.

There are no limitations and no borders for science. Moreover, as the Chinese saying has it,

"The mountain, only by not rejecting any dirt becomes so high, and the ocean, only by not refus-ing any water, becomes so deep." In comparable fashion, we strongly believe that through ourhard work, the Life Science Journal, by promoting the progress of the life sciences, can play itsrole in revealing the mysteries of life, and in contributing to the health of human beings and thewelfare of society.

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Life Science Journal, 2 (1 ), 2005, Edmondson, Life and Immortality

Life and Immortality: A Comparison of Scientific,Christian, and Hindu Concepts

pations of future cosmic evolution. On the onehand, astronomers anticipate that if the averagedensity of matter in the universe should prove to beless than a certain critical value, the expansion ofthe physical universe that began some fifteen billionyears ago with the explosion of a point of infiniteheat and density (the Big Bang) will continue toinfinity (" open universe") (Barrow, 1991; Grib-bin, 2000; Morris, 1993). Science anticipates thatwithin an "open universe" the stars (including theearth's sun), of which receding galaxies are com-posed, will eventually burn themselves out, the u-niverse will lapse into a cold darkness, and life onthe planet earth will cease. In such case, the atomsof former human bodies could presumably continueas components of the dead earth, and human physi-cal immortality might thus in a sense be sustained.However, the expectation that our dead earth willcontinue to be involved in an infinite cosmic expan-sion, thereby preserving that immortality, can forliving persons never be more than an unverifiableanticipation, for the one reason that no personswould remain alive during the later (dead earth)phases of that expansion in order to verify it, aswell as for the more general reason that any infiniteexpansion iPso facto denies the possibility of itsown confirmation (that is, since infinity can neverexceed itself, by the same token it can never be ex-ceeded by a confirming observer "beyond" itself).

Scientific anticipation holds, as another possi-bility, that if the density of matter in the universeshould prove to be greater than the critical value,gravitational forces will eventually cause the expan-sion of the universe to reverse itself and come to an

end in an awesome implosion (Big Crunch)( Greene, 2003). If the Crunch occurs it would

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Jingjing Z. Edmondson

Institute of Communication Studies,Zhejiang University,Hangzhou,Zhejiang 310028, China;

jjze@zju. edu. cn

Abstract: In this paper conceptions of immortality provided by two of the world's leading religions, Christianity andHinduism, are critically compared with each other, as well as with the possibilities of atomic immortality implied bymodem physical science, with the aim of ascertaining how these religious and scientific views mayor may not becompatible or mutually supportive. [Life Science Journal. 2005;2( 1):2 - 6J (ISSN: 1097 - 8135).

Keywords: atomic science; Christianity; Hinduism; immortality; life

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Everything in earthly existence, including hu-man life in all of its facets, is involved in a processof ongoing change. Hence, permanence seemsunattainable, and thereby especially desirable. Thewish for immortality thus becomes one of the mostimportant original reasons for the appearance of re-ligions, and the motives of many scientific researchfields can also be traced to this motive. Since veryancient times humans have wondered if after their

deaths in this world they might continue to existforever in some next and unchanging condition. Mypurpose in this paper is to contrast a modern scien-tific conception of the possibility of human immor-tality with some more traditional religious views ofthe same.

Scientific discoveries made during the last twocenturies have determined that the fundamental

building blocks of the physical universe as we nowfind it consist of submicroscopic" atoms", each ofwhich in turn has a complex inner structure of ele-mentary "particles" and" forces" (Brennan,1992). A further solidly established scientific de-termination is that since the establishment of atoms

during one stage of cosmic evolution they have beeninvolved in a universal process of rearrangement.Thus individual humans in their physical aspect areaggregates of atoms that formerly and variouslyconstituted elements of other creatures or objects,and upon the death and decomposition of humans'bodies, their atoms, rather than simply dissipatinginto nothingness, become parts of subsequent crea-tures or objects, and so forth indefinitely. In thissense deceased humans achieve an "atomic immor-

tality" .However, atomic immortality is theoretically

problematic in view of alternative scientific antici-

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mean that the universe is finite (" closed uni-

ver~"). In the case of the Crunch the atoms in-volved in the expansion of the universe would pre-sumably be transformed and compressed back into apre-atomic point of infinite density of the sort thatled tothe Big Bang. The radical transmutation ofmatter-energy involved in the Crunch would pre-sumably disallow retention of any identifiable rem-nants of given human bodies; hence it would be ex-travagant in this case to maintain that the atomsthat had provided persons with an extension oftheir existence beyond their deaths would providetrue immortality. What would transpire after theCrunch is unknown, although some astronomershave conceived the possibility of a series of BigBangs and Big Crunches, and therewith the evolu-tion and devolution of a series of universes, or of

one "oscillating universe" (Jastrow, 1992). But inthe case of transformations of universes back into

points of infinite heat and density, it is difficult toimagine that any atoms that had once been parts ofhuman bodies within a given universe could survivethe changes from one universe to the next; thus inthe scenario of an oscillating universe human im-mortality would be denied.

Nonetheless, anticipations of atomic immortal-ity are presumably meaningful to some people. Yetit seems probable that such concepts are insuffi-ciently satisfying to most people, who need to an-ticipate an immortality involving not only, in oneor another fashion, their post-mortem physical sur-vival, but also the survival in some sense of theconsciousness of self which they experience whilealive. Such survival is absent from atomic immor-

tality, which may involve some of the atoms of adeceased person's body being recycled as compo-nents of other living persons' bodies, yet withoutinvolving any synchronous transmigration of thefirst person's consciousness. Understandably,then, humans throughout their history have devel-oped religious beliefs that accommodate the antici-pation of a continuing post-mortem consciousness.To exemplify the multitude of religious beliefs, pastand present, the present discussion will focus onthe doctrines of two of the world's major living re-ligions, Christianity and Hinduism.

Christianity holds that one eternal God (J eho-vah) created the universe, including all of its formsof life. Although regarded as a spiritual being, thisGod is nonetheless conceived of as anthropomor-

phic, typically as Caucasian, and as having malegender (as the Christian scripture, the Bible, putsit: "God created man in his own image.") (TheNew English Bible, 1971). Christian doctrine isstrikingly different in a number of ways from scien-

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tific conceptions of being. Scientific knowledge,however imposing it may be, remains teleologicallyneutral, affording no insight into any ultimate pur-pose, or lack thereof, of the universe. But Chris-tian doctrine holds that God had a purpose in creat-ing the universe, whereby it is concluded that theuniverse itself, including the lives of humans, alsoserve an ultimate purpose. With respect to God'sown purpose, Christians maintain that he freelycreated the universe, not from any necessity of self-completion, but out of love for the humans who areparts of that creation, while the fundamental pur-pose of human lives is to love God in return, out ofgratitude for their existence.

Christianity maintains that God, as part of hiscontinuing creative activity, adds to each new hu-man body an immaterial" soul", inclusive of"mind" ,which latter is the seat of a person's con-sciousness. After the first two humans were creat-

ed, the subsequent chain of human sexual repro-duction has been less a matter of direct causation byGod than simply a result of humans acting in accor-dance with the biological "natural law" establishedby God; however, the addition of souls to new per-sons (at their moments of conception in their moth-ers' wombs) remains a direct and purely divine ac-tion. Whereas a strictly materialist conception ofexistence does not deny individual human con-sciousness as an attribute of a' living person's"mind" , it holds that mind is no more than an ex-ceptionally complex manifestation of a person'sphysiology; "mind" reduces, as it were, to physi-ology, and physiology reduces to atoms, and sothere is no mind or consciousness as a reality in ad-dition to body. One might suppose that if con-sciousness is thus merely an effect of a person'sbodily atoms, then the particular atoms of a givenperson's body that subsequently become compo-nents of other persons' bodies would transfer atleast some part of the first person's consciousnessto those other persons. However, the conscious-ness-qualities of such transferred atoms becomenewly and differently operative when incorporatedinto a new and different whole person, else the col-lective effect of any given person's accumulatedatoms could only be that of an incoherent melange.That is, on strictly materialist terms any given per-son's consciousness is the result only of a particu-lar, coherent, and unique assemblage of atoms thatare melded into a nontransferable consciousness of"self". This situation is reminiscent of the fact that

atoms of themselves, in separation from living or-ganisms, have no "life" .Whether or not life consti-tutes (in accordance with the biological philosophyof "vitalism") a stratum of being in excess of the

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and to paraphrase a famous statement made (in an-other context) by Albert Einstein, "God (by whichEinstein meant simply a supreme order in nature)does not play dice with the universe". However, itseems the anthropomorphic Christian God does in-deed play dice in the case of humans with their freewill, as he has endowed them with even odds formorally ruining themselves with devastating effect.In a related consideration, human earthly experi-ence often involves suffering, and if asked whyGod, who is regarded by Christians as both all-powerful and loving, allows this to happen, Chris-tians contend that without the challenge of retain-ing, through tests of suffering, their reciprocal lovefor God, that love would be merely trivial and vac-uous.

Referring again to atomic immortality, sciencemay someday achieve the great success of satisfyinghuman curiosity concerning the components andmechanisms of the physical universe, and therewithbetter clarify the possibilities of atomic immortali-ty. But, again, there is no reason to expect thatscience would thereby afford any clue as to a finalpurpose of the universe - or that it would yield anyrationalizations wherewith to justify the sufferingsof living humans - or that it would provide peoplewith the dramatic stimulus of being engaged in amoral struggle to achieve a full-body and conscious-ness-retentive immortality - whereas Christian be-lief provides all of these benefits.

It is instructive to consider a contrasting reli-gious perspective provided by Hinduism. The latterincludes an exceptionally rich abundance of diverseand often conflicting beliefs, and its discussion heremust be restricted principally to the doctrines of oneof its many schools, that of Advaita Vedanta(Isayeva, 1993). The fundamental concept of thisschool is that of brahman as the uncreated and

eternal ground of all being. Brahman, like theChristian God, is the spiritual and generativesource of the universe, including all of its forms oflife. But unlike the anthropomorphic, Caucasian,and male Christian God, brahman is regarded asamorphous, racially indistinct, and gender neutral.While undetectable as such to human sensory per-ception, brahman nonetheless thoroughly pervadesall things; the individual objects and creatures ofthe world as humans empirically perceive these areactually only transient manifestations of the intan-gible and unifying brahman itself. Moreover,brahman encompasses not only the things andcreatures of this world, but also an extensive rosterof gods. Yet the Advaita viewpoint (" Advaita"means "non-duality") is conceived of as monistic,as the gods are regarded merely as personalized re-

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sums of physical atoms compnsmg bodies, it re-mains that atoms manifest life only as constituentsof such bodies. As for Christian doctrine, even if itmay grant that persons' bodies of themselves arecomposed of temporary combinations of universallysimilar atoms, it maintains that each person's soulis an extra-atomic and unique element, which pro-vides each person with a self-consciousness-reten-tive immortality (Attwater, 1961).

Christians anticipate that humans will experi-ence that immortality either in a paradisiacalrealm-a place or state of being-for those personshaving led lives acceptable to God, or in a realm ofsuffering for those who have not. Leading lives ac-ceptable to God requires beliefs and moral behaviorin line with certain injunctions, which, it is main-tained, God has revealed to humans and which arerecorded in the Bible. However, it is further as-serted that God endows humans with free will,whereby they can choose to obey or disobey thoseinjunctions. According to the Christian historicalscenario, in the past many humans indeed dis-obeyed the injunctions, whereupon God sent his in-carnated son Jesus to earth to undergo a sacrificialdeath by torture in atonement for humans' sins ofdisobedience. It is genuine belief in the reality andpurpose of this sacrifice, plus true repentance forone's sins, that qualifies a person to receive God'ssaving" grace" (mercy) and thereby gain access tothe preferable kind of immortality (Chamberlin &Feldman, 1961).

According to Christian doctrine it will be atthe time of a "Last Judgment" that God, in the as-pect of his resurrected son Jesus, will effect theconsignment of individuals either to heaven or hell.In conjunction with the Judgment God will reani-mate the material bodies of deceased persons asthose bodies were at their point of physical primewhile on earth, to be joined with their souls beforeentry into one of the supernatural realms. At theclose of the Judgment God will bring to an end thephysical universe of human earthly experience. Andjust as God existed eternally before his creation ofthe realm of human earthly experience, so he willcontinue to exist eternally after its annihilation,while the conjoined and individually identifiablebodies and souls of humans will also endure forever

in one or the other of the supernatural realms(Smart & Hecht, 1982).

If God in his omnipotence intentionally createdhumans with the capacity freely to obey or disobeyhis injunctions, one can only assume he realizedthat some humans would disobey them, and thushave their reconstituted bodies consigned to puni-tive torture in hell. Relevant to this consideration,

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flections of various aspects and activities of the onesupreme and unifying reality of Brahman (Menon& Allen, 1960).

The concept of brahman as all pervasive in-volves the corollary that brahman is the fundamen-tal reality of each individual person's" soul", or"self" , erson, the atman, like the Christian soul,never dies. However, unlike the Christian soul,the atman becomes joined with a succession oftemporary bodies in the course of an indefinitelylong series of bodily births and deaths. Thus, an-other feature of Advaita (which it shares with oth-er branches of Hinduism) is the belief in reincarna-tion, the cycles of which are known as samsara. Itmight appear that the Hindu concept of reincarna-tion is congruent with scientifically conceived atom-ism, insofar as the latter envisages the cosmic recy-cling of atoms, some of which pass from livingbody to living body. However, not only is the rein-carnation of the Hindu atman a metaphysical pro-cess, but unlike the fortuitous destinations of recy-cled material atoms, anyone transmigration of agiven atman proceeds only from one specific personto another.

Moreover, totally unlike mere physical atomicrecycling, Hindu reincarnation is intimately boundup with morality. As the atman is joined time andtime again with a new body, the qualitative experi-ence of anyone of its incarnations is determined bykarma, which is the cumulative record of the

thoughts and actions, morally considered, of theindividual atman ' s series of lives. Hinduism em-

braces a conception of the right way of living(dharma), and the ways in which a person's be-havior in his or her past incarnations adhered to ordeviated from the precepts of dharma generatedgood or bad karma. The moral quality of a livingindividual's own actions supplement his or her re-ceived karma, and the new total is embodied in theperson's next incarnation and determines the initialquality of that next life. At the end of anyone ofan atman ' s incarnations, the body of that incarna-tion reverts to a nonspecific, unmanifested aspect ofbrahma (Deutsch, 1968).

If, over a (usually extensive) mnnber of reincar-nations a person's accumulation of good karma is suffi-cient, the cycles of samsara can be brought to an end.Desires motivate human actions, which in turn producekarma, but karma-producing desires result at bottomfrom a person's sense that he or she is ultimately a sep-arate entity left alone to pursue egocentric cravings. Butif a person gradually and profoundly comes to grasp theidentity of atman with brahman, then the person'skarma - producing desires are stilled, whereupon theperson's atman is released from samsara, an achieve-

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ment that constitutes liberation, or rnoksha. Followingthe attainment of rnoksha ,and the subsequent death

of a perEOn' s last physical body, the perEOn' s atmancontinues to exist forever as a supramundane componentof brahman. By contrast with human experiencewith-in samsara , rrwksha provides a new state of continuousserenity, within which individuals enjoy immortality.In Hinduism there is no state or condition of hell as a

counterpart to rnoksha. In the Hindu view enlightenedpeople achieve rnoksha, while unenlightened people,whose bad actions produce bad karma, simply continueto undergo repeated, and EOmetimes qualitatively worsereincarnations, until such time as they may reverse theprocess and eventually become themselves enlightened(Klostennaier, 1989).

The Hindu concept of immortality is differentin a number of other ways from the Christian con-cept. Moksha is a purely spiritual liberation thatentails no equivalent of Christian reconstituted bod-ies. In addition, while Hindu immortality involves,as in the Christian case, the retention of conscious-ness, in the Hindu conception that consciousnessexpands from a personal to a universalized con-sciousness. The self-consciousness of the atman at

the level of bodily involvement is elevated to identi-ty with the universal consciousness of brahman.The Christian belief is that persons who achieve im-mortality retain their personal identities while com-ing directly to see and know God., Yet such personseven in their new supernaturally enlightened appre-hension of God do not become identical with God,

nor do they ever comprehend exhaustively the ulti-mate mystery of God. In a sense, then, theachievement of moksha may be the more profoundand comprehensive immortality, since persons whoattain moksha are thoroughly at one with brah-man, with ultimate reality beyond any distinctionor disjunction between divine and human.

Within the compass of the foregoing discussion,what can be said, in sum, about anticipations of humanimmortality? All perEOnscan expect as inevitable the at-tainment of "atomic immortality," which, however,cannot be expected to provide people with an immortal-ity that is consciousne.'iS-retentive. The promise of thelatter is characteristically an a<.;pectof religious belief.But this recognition immediately poses the major prob-lem of there being no objective basis for asserting thevalidity of anyone religious anticipation of immortalityrather than any other. IRvotees of given religions-Christianity, Hinduism, andrnafiy others-typicallyregard their mutual confinnation of the beliefs they holdin common as evidence that those beliefs are true. But

this is not the universal interperEOnal validity of manyobjectively established scientific findings, and theworld's religious groups remain splintered among them-

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be a poor substitute for some firm and elaboratelyconceived religious-doctrinal conviction, but suchhope does not necessarily imply its own futility.

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selves and parochial in their doctrines. From which cir-cumstance it follows that the only objective fact revealedby the long human historical record of conflicting reli-gious beliefs is that of the psychological occurrence ofthose beliefs.

To focus these issues from a somewhat differ-

ent angle, today there are scientifically educatedChristians, Hindus, and devotees of other reli-

gions, who recognize the validity of atoms as fun-damental constituents of the physical world (whocan deny, in the face, for example, of atomic pow-er plants and atomic bombs, the validity of atomicscience?). But while the reality of atoms-andtherewith atomic immortality, such as it may be-appears to be a necessary part of any explanation ofexistence, the question remains whether it is a suf-ficient explanation of reality. For persons who be-lieve in the validity of samsara and karma, or inthe reality of Christian indestructible souls, or inany of a further extensive assortment of religiousconcepts, atomism of itself is obviously not a suffi-cient explanation of the full fabric of being (actuallymany prominent physicists have believed, on a per-sonal mystical basis, that existence in its broadestterms is determined by forces that are transcenden-tal or spiritual in nature) (Wilber, 1985). Ofcourse, comprehensive but mutually contradictoryversions of the fundamental attributes of universal

existence - including the eschatological scenarios ofvarious religions, as well as the stance of a rigorousmaterialism that excludes such scenarios-cannot a-

like be true, although if anyone comprehensiveversion if indeed true, it is ipso facto true for allpersons, as we all share one and the same universe.

I realize that numerous persons' endure the fre-quent and sometimes tragic frustrations of their lifeexperience with courage and poise as a consequenceof their religious faith of whatever variety. Surelysuch faith typically-and understandably-receivesits greatest impetus from anticipations of immortali-ty, since these provide a sense of reassurance thatthe adversities which people experience in theirearthly lives amount to more than a random absur-dity, as these adversities will be justified and com-pensated for in a future and better existence. Hav-ing these realizations in mind, I submit merely thaton the hasis alone of objective, nonsectarian com-prehension, perhaps the best any person can do isto hope that humans are destined to experience a(preferably benign) immortality, although the ex-act cosmic status and specific characteristics of thatimmortality remain, for persons still in this life,totally unsearchable. Simple hope may emotionally


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Correspondence to;Jingjing Z. EdmondsonInstitute of Communication Studies

Zhejiang UniversityHangzhou, Zhej iang 310028, ChinaTelephone : 011-86-571-8796-9884Email: [email protected]

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References

1. Attwater D, ed., A Catholic Dictionary (The CatholicEncyclopaedic Dictionary). Macmillan Company, NewYork, USA. 1961:1- 531.

2. Barrow J. Theories of Everything: The Quest for Ulti-mate Explanation. Fawcett Columbine, New York,USA. 1991: 1 - 285.

3. Brennan R. Dictionary of Scientific Literacy. John Wiley& Sons, New York, USA. 1992:19-20,22-3,287.

4. Chamberlin R, Feldman H, eds. , The Dartmouth Bible:An Abridgment of the King James Version, with Aids toIts Understanding as History and Literature, and as aSource of Religious Experience. Houghton Mifflin Com-pany, Boston, USA. 2nd ed. 1961: xxix-xliv.

5. Deutsch E. Advaita Vedanta: A Philosophical Recon-struction. East-West Center Press, Honolulu, USA.1968:3 -110.

6. Greene B. The Elegant Universe: Superstrings, HiddenDimensions, and the Quest of the. Ultimate Theory.Vintage Press, New York, USA. 2003:3 - 387, 413-414.

7. Gribbin J. The Search for Superstrings, Symmetry, andthe Theory of Everything. Little, Brown and Company,New York, USA. 2000:3 -199.

8.lsayeva N. Shankara and Indian Philosophy. State Uni-versityof New York Press, Albany, New York, USA.1993:1-255.

9. Jastrow R. God and the Astronomers. W. W. Norton &Company, New York, USA. 2nded. 1992:7-107.

10. Klostermaier K. A Survey of Hindusim. State Universityof New York Press, Albany, New York, USA. 1989:373 - 381.

11. Menon Y, Allen R. The Pure Principle: An Introductionto the Philosophy of Shankara. Michigan State Universi-ty Press, [East Lansing, Michigan], USA. 1960: 1 -127.

12. Morris R. Cosmic Questions: Galactic Halos, Cold DarkMatter, and the End of Time. John Wiley & Sons, NewYork, USA. 1993:1-194.

13. Smart N, Hecht R. Sacred Texts of the World: A Uni-versal Anthology. Crossroad Publishing Company, NewYork, USA. 1982:91-124,179-230.

14. The New English Bible with the Apocrypha. Oxford U-niversity Press, New York, USA. 1971: 1 - 6.

15. Wilbur K, ed. , Quantum Questions: Mystical Writingsof the World's Great Physicists. Shambhala Publica-tions, Boston, USA. 1985:3-208.

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Nature of Life

HongbaoMal, Shen Cherni

1. Department of Medicine, Michigan State University, East Lansing,

MI 48823, USA; hongbao@msu. edu

2. Department of Electrical Engineering, Chengshiu University, Niaosong,

Kaohsiung, Taiwan 833, ROC;cherngs@csu. edu. tw

Abstract: Life is a physical and chemical process. From ontology aspect, the world is timeless and the life exists for-ever as any other lxxJy in the nature. The nature of life is that life is a process of negative entropy, evolution, au-topoiesis (auto-organizing), adaptation, emergence and living hierarchy. Up to now, there is no scientific evidenceto show that life lxxJy and non-life lxxJy obey the same natural laws. But, all the researches are made by the meth-ods of biology, biochemistry and molecular biology, etc. It is very pO&'iiblethat the life and non-life are essential dif-ferent in the biophysics, i. e. the quantum level. In the future, it is possible to make artificial life by either biologi-cal method or electronic technique. [Life Science Journal. 2005: 2( 1) :7 - 15] (ISSN: 1097- 8135) .

Keywords: entropy: evolution: existence; life; nature

Contents

1. Introduction2. Definition of Life3. Essential Conceptions of Life4. Life in the Timeless Wodd5. Life as Negative Entropy6. Life as Autopoiesis (Auto-organizing)7. Evolution and Creation8. Adaptation9. Emergence10. Living Hierarchy11. Continuum or Dichotomy12. Apoptosis13. Artificial Life14. Matter and Form of Life15. Life and Mind16. Life and Quantum17. Origin of Life18. Discussions

1 Introduction

Life is unique in the known universe, which isin a diversity of forms ranging from bacteria to hu-man. The life organisms exist in everywhere of theearth. The first forms of life on earth spontaneous-

ly arose out of a preexisting prebiotic chemicalsoup. Individual living organisms maintain theirself-identity and their self-organization while con-tinually exchanging materials and energy and infor-mation with their environment. It is really differ-ent between the life and non-life bodies, but no-

body knows what the exact difference it is, even

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this is one of the most important issues that attract-ed people in the whole human history. There aremillions of people working in life science research-es, many with Ph. D. degree. More money hasbeen spent in the life science studies than that spentin any other fields. Nature, Science, and other bigjournals published more papers in life science thanthe papers in any other topic. But, there are veryfew people thinking about the nature of life. Thistopic has attracted thinkers since the beginning ofhuman history, but ignored by the modern society.Most philosophers ignore the issue today, perhapsbecause it seems too scientific. At the same time,

most scientists also ignore the issue, perhaps be-cause it seems too philosophical. The nature of lifeis not clear for the current intelligence. It is a topicof philosophy, and also of biology (Bedau, 2005).However, it is very difficult to get financial supportfor the study of nature of life.

2 Definition of Life

It is difficult to give an exact definition for thelife, as the nature of life is not clear. As the refer-ences, here I give the definition from some dictio-nanes:

( 1) Spiritual existence transcending physicaldeath; the period from birth to death; the qualitythat makes living animals and plants different fromdead organisms and inorganic matter. Its functionsinclude the ability to take in food, adapt to the en-

vironment, grow, and reproduce (Encarta@ WoddEnglish Dictionary, 2005).

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metabolism (Crick, 1981). Kuppers pointed lifeas: metabolism, self-reproduction and mutability(Kuppers, 1985). Maynard Smith gave life twoproperties: metabolism and parts with functions(Maynard, 1986), and Ray cited two aspects:self-reproduction and the capacity for open-endedevolution (Ray, 1992).

Mayr thought that the process of living couldbe defined by a list of the kinds of characteristics bywhich living organisms differ from inanimate mat-ter: (1) All levels of living systems have an enor-mously complex and adaptive organization. ( 2 )Living organisms are composed of a chemically u-nique set of macromolecules. (3) The importantphenomena in living systems are predominantlyqualitative, not quantitative. (4) All levels of liv-ing systems consist of highly variable groups of u-nique individuals. (5) All organisms possess histor-ically evolved genetic programs which enable themto engage in teleonomic processes and activities.(6) Classes of living organisms are defined by his-torical connections of common descent. (7) Organ-isms are the product of natural selection. (8) Bio-logical processes are especially unpredictable(Mayr, 1982). '(9) Life is continuum. (10) Alllife organisms are programmed to death naturally,which is called apoptosis (Ma, 2005b).

Schrodinger persisted that the second law ofthermodynamics plays key role in' the process ofmetabolization. The following sentences give hisopinions: What is the characteristic feature of life?When is a piece of matter said to be alive? When itgoes on doing something, moving, exchanging ma-terial with its environment, and so forth, and that

for a much longer period than we would expect aninanimate piece of matter to keep going under simi-1ar circumstances. How does the living organismavoid decay? The obvious answer is: By eating,drinking, breathing and assimilating. Linguistical-ly, the scientific term of life is metabolism. The es-sential thing in metabolism is that the organismsucceeds in freeing itself from all the entropy(Schrodinger, 1969).

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(2) The condition that distinguishes animalsand plants from inorganic matter, including the ca-pacity for growth and functional activity (CompactOxford English Dictionary, 2005).

(3) The property or quality that distinguishesliving organisms from dead organisms and inani-mate matter, manifested in functions such asmetabolism, growth, reproduction, and response tostimuli or adaptation to the environment originatingfrom within the organism (Dictionary. com,2005).

3 Essential Conceptions of Life

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The biological world is viewed as a hierarch oflevels. These levels include chemicals, organelles,cells, organs, organisms, and ecologies. There arethree conceptions for life: as a loose cluster of prop-erties, a specific set of properties, and metaboliza-tion. There are many other opinions of life, such asthat life is something of autopoiesis and self-replica-tion, etc. Several hundred years ago, peoplethought that there was a vitalism inside life bodiesthat keep the body to be a life. The scientific re~suits absolutely denied the existence of vitalism.The demise of vitalism told us that no super physi-cal substance or force or spirit to distinguish any lifefrom non-life. For all we know, all life phenomenaobey to all the natural laws (physical and chemical)that adapted to the non-life world. There is no anyextra natural law for the life world only. Life is nomore unified than a collection of overlapping prop-erties from overlapping disciplines, such as, bio-physics, biochemistry, molecular biology, genet-ics, evolution, ecology, cytology, microbiology,physiology, anatomy and heredity ,etc. However,the biophysics is poor result.

Farmer and Belin listed eight characteristics ofthe life: process, self-reproduction,' informationstorage of self-representation, metabolism, func-tional interactions with the environment, interde-pendence of parts, stability under perturbations,and the ability to evolve. According to Farmer andBelin, life is a pattern of spacetime, rather than thespecific identities of the atoms ( Farmer, 1992).

Taylor described the properties of life: "Eachproperty by itself, even when considered with oth-ers, is unable to clearly delineate the living fromthe non-living, but together they do help to charac-terize what makes living things unique." (Taylor,1992) .

Monod listed three characteristics of life:

teleonomic or purposeful behavior, autonomousmorphogenesis and reproductive invariance (Mon-od, 1971). Crick focused on the points related to:self-reproduction, genetics, evolution and


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4 Life in the Timeless World

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As it was described in another paper "The na-ture of time and space": From the ontology (ornaturalism) angle, time and space are absolute (ex-isted) and the universe is a timeless world, whichmeans that all the past, the present and the futureexist eternally. Everything in the universe will nev-er change. Time and motion are nothing more thanillusions. In the universe, every moment of everyindividual's life-birth, death, and anything in be-

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tween-exists forever. Everyone is eternal. Thatmeans each and everyone of us is immortal. The u-niverse has neither past nor future. All the thingsin the past, present, and future exist forever. Theconcepts of past, present and future are dependedon the human brain (Ma, 2003). Life is something( substance) existing in the timeless world. Sothat, all the life processes are the simple existenceof something in the universe, like a movie in atape, exist already and forever. This is the essentialnature of life, in the ontology point. Under thetimeless principle, there is only existence in the u-niverse, not something complexity and other thingsimplicity. The life is not more complex than non-life from the ontological concept. However, in thetimeless world, there are natural connections a-

mong the all the existence. All the scientific stud-ies, philosophical ratiocinations and religious believeare the trial to reveal the natural rules.l

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5 Life as Negative Entropy

The second law of thermodynamics was for-mulated in the middle of the last century by Clau-sius and Thomson, which could be formulated infour different ways: (1) Heat cannot flow from acolder body to a hotter one without energy input;(2) Entropy must increase in a closed system; (3)No cyclic process can convert heat entirely to work;( 4) In any cyclic process the heat Q transferred tothe system from its surroundings at the temperatureT must obey an inequality: p dQ/T < 0 (Ma,2003). Above the four points, the principle con-cept of the second law of thermodynamics is to saythat in the closed system all the natural processesincrease entropy (decrease order). So, the secondlaw of thermodynamics can be called the entropylaw or law of entropy. However, life violates sec-ond law of thermodynamics. In natural world, lifeprocess is negative entropy one. In the life process,the entropy decreases, which means that the orderincreases. More importantly, there is no evidenceto say that the entropy decrease of life costs by theentropy increase of environment. The conclusion isthat the life process does not obey the second law ofthermodynamics. For all we know that all life phe-nomena obey to all the natural laws that adapted tothe non-life world. How can we say that life vio-lates the second law of thermodynamics? Is thereany conflict? The answer is that there is no conflicthere. As it was described in the article "The nature

of time and space", "the second law of thermody-namics is a statistical result, , the basic statis-tical principles and the second law of thermodynam-ics are useful tools in human practice, but they are

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not the true natural existence" (Ma, 2003). Thefact is that the life process does not obey the secondlaw of thermodynamics, but it obeys all the naturallaws. The second law of thermodynamics is not anatural law, but a technical tool.

6 Life as Autopoiesis (Auto-organizing)

Autopoiesis is the process whereby an organi-zation produces itself. An autopoietic organizationis an autonomous and self-maintaining unity whichcontains component-producing processes. The com-ponents, through their interaction, generate recur-sively the same network of processes which pro-duced them. An autopoietic system is operationallyclosed and structurally state determined with no ap-parent inputs and outputs. A cell and an organismis an autopoietic system. Autonomy is the conditionof subordinating all changes to the maintenance Ofthe organization. Self-asserting capacity of livingsystems maintain their identity through the activecompensation of deformations. Allopoiesis is theprocess whereby an organization produces some-thing other than the organization itself. An assem-bly line is an example of an allopoietic system( Varela, 2005). Life is an emergent property ofautopoietic, dissipative systems. Life is an au-topoiesis (auto-organizing) complex, which can or-ganize itself without energy input, even without in-formation input. Active life process costs energyand uses information. However, the cost of energyis not the requirement of energy by the second lawof thermodynamics. It cannot stay long periodwithout energy and information input. Aftet awhile without exchange energy and informationwith outside world, the active life will die.

7 Evolution and Creation

Evolution theory is one of the most importanttheories in science. Evolution of life shows a re-

markable growth in complexity. Simple prokaryoticone-celled life leads to more complex eukaryotic sin-gle-celled life, which then leads to multicellularlife, then to large-bodied vertebrate creatures withsophisticated sensory processing capacities, and ul-timately to highly intelligent creatures that use lan-guage and develop sophisticated technology as hu-

man. Creation theory says that life)s not evolutionbut created by God, and all the species do notchange forever. The interest thing is that many sci-entists are strongly believe creation in their non-work time, which means that the scientists believeBible when they are in their churches in their reli-gious time (normally in the weekend) or when theyspend time in their Bible studies. However, these


ing systems.The important feature for all life is the evolu-

tionary process of adaptation. For the evolution, itis sometimes the blind operation of natural selec-tion, sometimes the general process of evolution,and sometimes the adaptation produced by the evo-lution. Normally the life should have the ability toadapt appropriately to unpredictable changes in theenvironment. It is the force of adaptation and selec-tion that makes the evolution happens. The adapta-tion is supple.

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scientists never do anything following creation the-ory in their work time, which means that they nev-er do any experimental or publish any thing in theacademic journals 0f teach students to support cre-ation opinions. In the work time they need to dosomething that positive for their life as their incomecomes from the work, and non-work time they cando anything what they want.

Gene transfer is to transfer a gene from oneDNA molecule to another DNA molecule, which

can change the genetic background of an organismin anyway we want (Ma, 2005a). The evolutionhappens naturally, and also can happen artificiallyby gene transfer technique. Cloning creates a ge-netically identical copy of an organism, which canbe done in all the kinds of living things, includinghuman being. Transgenic animal and clone for thestudy of gene regulation and expression has becomecommonplace in the modern biological science now(Pinkert, 1999). The sheep Dolly was the world'smost famous clone animal, but it was not the first

one. Many animals-including frogs, mice, sheepand cows had been cloned before Dolly. Plants havebeen often cloned since ancient people. Human i-dentical twins are also clones. Dolly was the firstmammal to be cloned from an adult cell, rather

than an embryo. This was a major scientificachievement of Dolly, but also raised scientific andethical concerns. Since Dolly was born in 1996many other animals have been cloned from adultcells, such as mice, pigs, goats and cattle. Cloningby interspecies nuclear transfer offers the possibilityof keeping the genetic stock of those species onhand without maintaining populations in captivity( Lanza, 2002) and change the species, but alsopossibly creates the risk of biological calamity (Ma,2004) .

8 Adaptation

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Adaptive evolutionary explanations are familiarto all of us from elementary school biology. A clas-sic application of adaptationism is to explain the gi-raffe's long neck as an adaptation for browsing a-mong the tops of trees, on the grounds that naturalselection favored longer-necked giraffes over theirshorter-necked cousins. There are alternatives to

adaptive explanations, such as explanations appeal-ing to allometry, genetic drift, developmental con-straints, genetic linkage, epistasis, and pleiotropy.The presupposition that a trait is an adaptation andso deserves an adaptive explanation is usually treat-ed as unfalsifiable. The adaptationist perspective onevolution emphasizes natural selection's role in cre-ating the complex adaptive structures found in liv-


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Both living systems and artificial life modelsare commonly said to exhibit emergent phenomena.Emergent phenomena share two characterizations:they are constituted by and generated from under-lying phenomena, and they are autonomous fromthose underlying phenomena. There are three mainpoints for emergent properties. The first key pointof emergence is simply the idea of a property thatapplies to wholes or totalities but does not apply tothe component parts considered in isolation. Thesecond key point of emergence is to insist that e-mergent properties are supervenient properties withcausal powers that are irreducible to the causalpowers of micro-level constituents. The third keypoint of emergence is poised midway between theother two. '

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Living phenomena fall into a complex hi.erar-chy of levels, what can be called the vital hierar-chy. Even broad brush strokes can distinguish atleast eight levels in the vi tal hierarchy: (1) ecosys-tems, (2) communities, (3) populations, (4) or-ganisms, (5) organ systems (immune system, car-diovascular system), (6) organs (heart, kidney,spleen), (7) tissues, and (8) cells. Under the lifehierarchy, there are molecules, atoms and quantathat are substance but not life constituents. Items

at one level in the hierarchy constitute items athigher levels. Individual organisms are born, livefor a while, and then die. The vital hierarchy raisestwo basic kinds of questions about the nature oflife. First, we may ask whether there is some in-herent tendency for living systems to form hierar-chies. Why are hierarchies so prevalent in the phe-nomena of life? The second question concerns therelationships among the kinds of life exhibitedthroughout the vital hierarchy. Are there differentforms of life at different levels, and if so then howare these related? How are they similar and differ-ent? Which are prior and which posterior? What is

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the primary form of life?The theory supple adaptation reveals a two-tier

structure with connected but different forms of life.

The first tier is the primary form of life-the sup-plely adapting systems. At the second tier, entitiesthat are suitably generated and sustained by such asupplely adapting system branch off as different butconnected secondary forms of life. These secondaryforms of life include organisms, organs, and cells.

11 Continuum or Dichotomy

Can things be more or less alive? Serious re-flection about life quickly raises the questionwhether life is a boolean property (zero or one)-whether it is a continuum property . We can saythat a rat is alive and a rock is not alive. But it is

difficult to say some condition of living body is aliveor not, such as a virus which is unable to replicatewithout a host and spores or a frozen cell which re-main dormant and unchanging indefinitely but thencome back to life when conditions become suitable.

Furthermore, we all agree that the original lifeforms somehow emerged from a pre-biotic chemicalsoup, and this suggests that there is very little, ifany, principled distinction betweeR life and non-life. In fact, life is continuum and it can be more orless alive. There is no absolute line between life and

non-life. If life is considered as supple adaptationthe most important life/non-life distinction involvesa continuum because the activity of supple adapt-ability comes in degrees.

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12 Apoptosis

For all the things existed, including the lifecells in the earth and universe itself, there is a timeto live and a time to die. There are two ways inwhich cells die: (1) Cells are killed by injury ordisease. (2) Cells suicide. Programmed cell deathis also called apoptosis, which is cell suicide. Apop-tosis is a mechanism by which cells undergo deathto control cell proliferation or in response to DNAdamage. Some types of cancers, such as B-cellchronic lymphocytic leukemia, follicular lymphoma(Tsujimoto, 1985) and tumors infected by humanT-cell leukemiallymphoma virus-1 (Hengartner,2000) are characterized by defects in apoptosis

leading to immortal clones of cells. Other malig-nancies have defects in the apoptotic regulatory

pathways such as p53 (Kaufmann, 2001).Apoptosis can be triggered by the following in-

ternal signals: (1) In a healthy cell, the outermembranes of its mitochondria express the proteinBcl-2 on their surface. (2) Bcl-2 is bound to amolecule of the protein Apaf -1. (3) Internal dam-

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age to the cell (e. g. , from reactive oxygen species)causes: Bcl-2 to release Apaf-1; a related protein,Bax, to penetrate mitochondrial membranes, caus-ing; cytochrome c to leak out. (4) The releasedcytochrome c and Apaf-1 bind to molecules of cas-pase-9. (5) The resulting complex of cytochromec, Apaf-1, caspase-9 and ATP is called the apopto-some. (6) These aggregate in the cytosol. (7)Caspase-9 is one of a family of over a dozen caspas-es. They are all proteases. They get their name be-cause they cleave proteins-mostly each other-ataspartic acid (Asp) residues. (8) Caspase-9 cleavesand activates other caspases. (9) The sequential ac-tivation of one caspase by another creates an ex-panding cascade of proteolytic activity, which leadsto digestion of structural proteins in the cytoplasm,degradation of chromosomal DNA, and phagocyto-sis of the cell.

Apoptosis can be triggered by external signalsalso: (1) Fas and the TNF receptor are integral

membrane proteins with their receptor domains ex-posed at the surface of the cell. (2) Binding of thecomplementary death activator (FasL and TNF re-spectively) transmits a signal to the cytoplasm thatleads to activation of caspase 8. (3) When cytotox-ic T cells recognize their target, they produce moreFasL at their surface. This binds with Fas on sur-

face of the target cell leading to its death by apopto-SIS.

Apoptosis is a universal event in the universe,that happens in all the life bodies and azoic thingsin the universe, including the universe itself. Tounderstand apoptosis clearly will be importan.t tothe understand of the basic nature laws (Ma,

2005b). Apoptosis is the nature of life, and apop-tosis is also the nature of nature!

13 Artificial Life

Could robot do all the things what human do?Could artificial electronic life play all the functionswhat the organic life play? Up to now, nobody cananswer these questions.

In 1966, John von Neumann made the firstartificial life model with his famous creation of a

self-reproducing, computation-universal entity us-ing cellular automata. John von Neumann was pur-suing many problems that are important in the arti-ficiallife today, such as understanding the sponta-neous generation and evolution of complex adaptivestructures. Originally, cybernetics applied twotools to the living system studies: the use of infor-mation theory and a deep study of the self-regulato-ry processes. Information theory typifies the ab-stractness and material-independence of artificial

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dimensional manifold), and also in Bohr's andHeisenberg's quantum world. Now it may be con-sidered that matter and space are unified.

Advances in the new sciences suggest a furthermodification of this assumption about the nature ofreality. In light of what scientists are beginning toglimpse regarding the nature of the quantum vacu-um, the energy sea that underlies all of spacetime,it is no longer warranted to view matter as primaryand space as secondary. In the modern conceptthere is no absolute matter, but only a matter gen-erating energy field.

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life, and self-regulation is one of the hallmarks ofliving systems studied in artificial life.

Biology studies have provided rich knowledgeabout actual living systems. Physics and mathemat-ics have had a strong influence on artificial life, es-pecially in the study of complex systems. Statisticalmechanics and dynamical systems theory have im-proved artificial life' s methodology.

The real artificial life should be organic life,same as the natural life. Right now, people can syn-thesize simple organic molecules such as sugar andamino acids from the inorganic carbon, hydrogenand oxygen. Just after the technique developing,people will have the ability to make the real cells,tissues, organs and animals even a real human. Thiswill be the real artificial life-everything is same asthe natural life.

14 Matter and Fonn of Life

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The advent of the field of artificial life has fo-

cused attention on a set of questions about the roleof matter and form in life. On the one hand, cer-tain distinctive carbon-based macromolecules playacrucial role in the vital processes of all known livingentities; on the other hand, life seems more like akind of a process than a kind of substance. Fur-thermore, much of the practice of artificial life re-search seems to presuppose that life can be realizedin a suitably programmed computer. This raises anumber of related questions: Can a computer playall the functions of the organic life play? Is the nat-ural life just substance properties what the sub-stance has or life has independent proper that per-forms by the substance? Functionalism captures thetruth about life. Furthermore, there is no evidentreason why the functional structure specified thetheory could not be realized in a suitably structuredcomputational medium. If so, then a computerized"life" could in principle create a real, literally livingentity. In fact, a computer can play many func-tions of the organic life play, but could not play allthe functions of the organic life play, because thematter is essential different. The natural life is de-

pendent on the substance of the life bodies.According to the classic science, there are two

independent existences in the world: matter andspace. Matter occupies space and moves about in itand it is the primary reality. Space is a backdrop orcontainer. Without furnished by material bodies, itdoes not enjoy reality in itself. This common senseconcept goes back to the Greek materialists and itwas the mainstay also of Newton's physics. It hasbeen radically revised in Einstein's relativistic uni-verse (where spacetime became an integrated four-


15 Life and Mind

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It is an essential philosophical questionwhether there is any intrinsic connection betweenlife and mind. Viruses, plants, bacteria, worms,animals and human have various kinds of sensitivityto the environment, various ways in which this en-vironmental sensitivity affects their behavior, andvarious forms of inter-organism communication.Various kinds of what one could call mental capaci-ties are present throughout the biosphere. Further-more, the relative sophistication of these mental ca-pacities seems to correspond to and explain the rela-tive sophistication of those forms of life. It is rea-sonable to ask whether life and mind have some

natural connection. The process of evolution estab-lishes a genealogical connection 'between life andmind, but life and mind might be much moredeeply unified. Since all forms of life must cope inone way or another with a complex, dynamic, andunpredictable world, perhaps this adaptive flexibili-ty inseparably connects life and mind. In fact, themind comes from brain that composes by the organ-ic molecules and the organic molecules compose byinorganic matter. But, there is no evidence to saythat the inorganic matter in the living organism isdifferent from the inorganic matter out the livingorgamsm.

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.I16 Life and Quantum

Up to now, no scientific evidence to show thatlife body and non-life body obey the different natu-ral laws. By the classic physics and chemistry,there is no essential difference discovered in life and

non-life. There is no lifeline defined by modern sci-ence, this means that we neither qualitate norquantitate life by any current scientific method.However, all the researches are made by the meth-ods of biology, biochemistry and molecular biology,etc., which means that all current biological andneurobiological descriptions of the life and brain arebased on Newton's physics, even if it is well

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known that Newton's physics has its limitations.Biophysics has started for several decades and it didnot get many achievements. Up to now, nobodytried to reveal the nature of life under the quantumlevel. It is reasonable to think about that the lifeand non-life are essential different in the bio-physics, i. e. the quantum level. The life phe-nomenon, especially consciousness, is unlikely toarise from classical properties of matter. Quantumtheory allows for a new concept of matter altogeth-er, which may well leave cracks for life and con-sciousness, for something that is not purely materi-al or purely extra-material. Interactions with thequantum vacuum may not be limited to micro-parti-cles: they may also involve macroscale entities,such as living systems. The recognition of opennessis returning to the natural sciences. Traffic be-tween our consciousness and the rest of the worldmay be constant and flowing in both directions.Everything that goes on in our mind could leave itswave traces in the quantum vacuum, and every-thing could be received by those who know how totune in to the subtle patterns that propagate there.

All the life organisms compose by organicmolecules plus their inner environment such as in-organic water and ions (and specific fields) insideand outside the cells. The whole life world finallycomposes by an organic world, and even though allorganic molecules compose by inorganic substance.But, nobody knows if the water in alive cells andaround cells is same or different from the water faraway from the cells (under the living meaning). Itis possible that the inorganic environment of livingcell is different from non-living environment in thequantum level. This is the principle task for bio-physics doing to reveal the nature of life.

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17 Origin of Life

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When the earth formed about 4. 6 billion yearsago, it was a lifeless place. A billion years later itwas teeming with organisms such as blue-green al-gae. How did life begin? The discovery of self-replicating RNA was a critical milestone on theroad to life. Before the mid-17th century, mostpeople believed that God had created humankindand other organisms by mud. For the next twocenturies, those ideas were subjected to increasinglysevere criticism.

In 1903, Svante Arrhenius proposed that lifeon the Earth was seeded by spores originating fromanother planet. In 1905, the astronomer SimonNewcomb proposed that because the Earth was arepresentative planet orbiting a representative starSun, life could be abundant throughout the uni-

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verse (Zubay, 2000). But up to now, there is nodiscovery of the life existing in another planet.

All living things consist of similar organiccompounds. Proteins in all organisms are consistedby one set of 20 amino acids. These proteins in-clude enzymes that are essential to live, develop andreproduce, and the protein that essential to the or-ganism structure. Organisms carry their genetic in-formation in nucleic acids RNA and DNA, and usethem as the same genetic code. This code specifiesthe amino acid sequences of all the proteins andpeptides in each organism. The nucleotides consistof a sugar (deoxyribose in DNA and ribose inRNA), a phosphate group and one of four differentbases. In DNA, the bases are adenine (A), gua-nine (G), cytosine (C) and thymine (T). InRNA, uracil (U) substitutes for T. The bases con-stitute the alphabet, and triplets of bases form thewords as the genetic codes. As an example, thetriplet CUU in RNA instructs a cell to add theamino acid leucine to a growing strand of proteinwhen the protein is synthesized. Organisms storegenetic information in nucleic acids that specifiedthe composition of all sythesized proteins. It relieson proteins to play the biological metabolism pro-cesses.

There is a paradox. Nowadays nucleic acidsare synthesized only with the catalyzing of pro-teins, and proteins are synthesized only with thecoding of nucleic acids. It is impossible that pro-teins and nucleic acids arose spontaneously in thesame place at the same time. It is also impossible tohave one without the other. And so, at f.irstglance, one might have to conclude that life couldnever have originated by chemical means. In thefact, RNA came first and established what is nowcalled the RNA world-a world in which RNA cat-alyzed all the reactions necessary for a precursor oflife's last common ancestor to survive and repli-cate. RNA has developed the ability to code aminoacids to synthesize proteins. The modem RNAviruses are still use RNA as their genetic codes.The ribonucleotides in RNA are more re<\dilysyn-thesized than are the deoxyribonucleotides in DNA.Moreover, DNA could evolve from RNA and thentake over RNA' s role as the heredity. In fact,RNA came before proteins. In 1983 Thomas Cechat University of Colorado and Sidney Altman atYale University discovered the first known ri-bozymes, enzymes made of RNA. The first ri-bozymes identified could do little more than cut andjoin preexisting RNA. Nevertheless. --

As the experiments to reveal the original originof life in the Earth, in the early 1950s StanleyMiller, working in the laboratory of Harold C.

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Urey at the University of Chicago, did the first ex-periment to clarify the chemical reactions that oc-curred on the primitive earth. In the flask at the

bottom, he heated water and forced water vapor tocirculate through the apparatus. The flask at thetop contained an atmosphere consisting of methane( C~), ammonia (NH3) , hydrogen (Hz) and the

circulating water vapor. Next he exposed the gasesto a continuous electrical discharge, causing thegases to interact. Water soluble products of thosereactions then passed through a condenser and dis-solved in the mock ocean. The experiment yieldedamino acids and enabled Miller to explain how theyhad formed. For instance, glycine appeared afterreactions in the atmosphere produced simple com-pounds formaldehyde and hydrogen cyanide thatparticipated in the set of reactions that took place.For the above experiments, one heavy critics is thatthe so called amino acid products coming from bac-teria contamination. Bacteria exist everywhere inthe Earth and it is very possible to get the bacterialcontamination in the experiments.

Stem cell is the origin of an orgnism' slife.Stem cells have the remarkable potential to developinto many different cell types in life bodies, thatare exciting to scientists because of their potentialto develop into many different cells, tissues and or-

gans. Stem cell is totipotent and it is a single cellthat can give rise to progeny that differentiate intoany of the specialized cells of embryonic or adulttissue. The ultimate stem cells (fertilized egg) di-vides to branches of cells that form various differen-

tiated tissues or organs. During these early deci-sions, each daughter cell retains totipotency.Through divisions and differentiations the embry-onic stem cells lose totipotency and gain differenti-ated function. During normal tissue renewal in

adult organs, tissue stem cells give rise to progenythat differentiate into mature functioning cells ofthat tissue. Stem cells losing totipotentiality areprogenitor cells. Except for germinal cells, whichretain totipotency, most stem cells in adult tissueshave reduced potential to produce cells of differenttypes (Ma, 200Sc).

18 Discussions

.There are plenty of puzzles about the concept

of life. The concrete objects ready to hand are usu-ally easily classified as living or non-living. Fishand ants are alive while candles, crystals and cloudsare not. Yet many things are genuinely puzzling toclassify as living or not. Viruses are one borderlinecase, biochemical soups of evolving RNA strings inmolecular genetics laboratories are another. Ex-


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traterrestrial life forms, if any exist, might wellnot depend on DNA-encoded information or, in-

deed, any familiar carbon chemistry processes.How would we recognize extraterrestrial life if wefound it? We have no reason to suppose it will haveany of the accidental characteristics found in famil-iar forms of life. What, then, are the essentialproperties possessed by all possible forms of life?The search for extraterrestrial life needs some an-

swer to this question, for we can search for life onlyif we have a prior conception of what life is.

The phenomena of life raise a variety of subtleand controversial questions. Early life forms some-how originated from pre-biotic chemical soup. Doesthis imply that there is an ineliminable continuum

of things being more or less alive, as many sup-pose? Another subtle question concerns the differ-ent levels of living phenomena, such as cells, or-gans, organisms, ecosystems and asks in whatsenses the concept of life applies at these variouslevels. Does the essence of life concern matter orform? On the one hand, certain distinctive carbon-based macromolecules playa crucial role in the vitalprocesses of all known living entities; on the otherhand, life seems to be more in the nature of a pro-cess than a kind of substance. The relationship be-tween life and mind raises another question. Whenwe consider plants, bacteria, insects, and mam-mals, for example, we apparently find differentkinds of mental activity, and it seems that differentdegrees of behavioral sophistication correspond todifferent levels of intelligence. Might the variousforms of life and mind be somehow connected.? Toanswer questions like these above and make sense ofthe puzzling phenomena of life, we need a soundand compelling grasp of the nature of life. Can anyproperty embrace and unify not only life's existingdiversity but also all its possible forms? What is thephilosophically and scientifically most plausible wayto account for the characteristic life-like features of

this striking diversity of phenomena? How can weresolve the controversies about life? The concept oflife as supple adaptation, explained below, is myattempt to address these issues.

Notice that our ordinary, everyday concept oflife does not settle what the true nature of life is.Thus, we are not concerned here with careful de-

lineation of the paradigms and stereotypes that wecommonly associate with life. We want to knowwhat life is, not what people think life is. Glassdoes not fall under the everyday concept of a liquid,even though chemists tell us that glass really is aliquid. Likewise, we should not object if the truenature of life happens to have some initially coun-terintuitive consequences.

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Four questions are important to answer: (1)How are different forms of life at different levels ofthe vital hierarchy related? (2) Is there a continu-um between life and non-life? (3) Does life essen-tially concern a living entity's material compositionor its form? (4) Are life and mind intrinsically con-nected?

For now, many people, including biologistsand other scientists are still believing that God cre-ated the life, even they never publish any academicarticles to describe that. The ridiculous things arethat many biologists always write articles and teachstudents evolution in their work time but believecreation theory (deny evolution) in their weekendchurch time. Depending on the academic articles,they make their career and life, but depending onthe Bible, they come back non-experiment believe.The fighting between science and religion is still aheavy topic in the modern time.

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Correspondence to:Hongbao Ma138 Service RoadB410 Clinical CenterMichigan State UniversityEast Lansing, Michigan 48824The United StatesTelephone: 517-432-0623Email: [email protected]

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References1. &tgley R, Fanner JD. ~tana:Jll5 Emergence of a

MetaWism,in Langtonet al.1992:93-140.2. Bedau MA. hup: I Iwww.reed.edu/~mab/papersllife.

OXFORD. html. 2005.

3. Encarta'" World English Dictionary.hup: Ilencarta. msn.rom!enmet/featuresldictionaryIDictionaryResults. aspx?refid= 1861696586.2005.

4. Compact Oxford English Dictionary. hup: Ilwww.askoxford.com/concise-oedllife? view= uk.2005.

5. Crick F. Life Itself: Its Origin and Nature. New York,USA. 1981.

6. Dictionary.com.http://dictionary.reference.com/search? r

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= 66&q= life. 2005.7. Farmer D, Belin A. Artificial Life: The Coming Evolu-

tim, In Artificial Life II. C. G. Langton, et aI.,(eds.). Addison-Wesley: Redwood City, CA, USA.1992:815 -40.

8. Hengartner MO. The biochemistry of apoptosis. Nature2000;407: 770 - 6.

9. Kaufmann SH, Hengartner MO. f'rq!;rammed cell death: aliveand well in the new millennium. Trends Cell Bioi 2001; 11:526- 34.

10 . Lanza RP, Dresser BL, Damiani P. Cloning Noah's Ark, inUnderstanding Cloning. &ientific American, Inc. and ByronPress Visual Publications, Inc. 2002: 24 - 35.

11. KUppers BO. Molecular Theory of Evolution: Outline of aPhysico-Chemical Theory of the Origin of Life. Berlin,German. 1985.

12.MaH. The natureof timeand space.Nature and Science2003;I( 1) :1- 11.

13. Ma H. Technique of Animal Clone. Nature and Science2004;2(1) :29 - 35.

14. Ma H,Chen G. Gene transfer technique. Nature and Sci-ence 2005a;3(1) :25 - 31.

15.Ma H,Chen G.Apoptosis. Nature and Science 2005b;3(2):1 - 4.

16. Ma H, Chen G. Stem cell. The Journal of American Sci-ence 2005c; 1(2) :90 - 2.

17. Maynard Smith J. The Problems of Biology New York.USA. 1986.

18. Mayr E. The Growth of Biological Thought. Cambridge,Massachusetts USA. 1982: 52 - 4.

19. Monad]. Chance and Necessity. New York, USA. 1971.20. Pinkert CA, Murray JD. Transgenic farm animals, in Trans-

genic Animal in Agriculture, Murray JD, Anderson GB,Oberbauer AM, McGlaughlin MM (eds). CABI Publishing.New York, NY, USA. 1999:1-18.

21. Ray T. An Approach to the Synthesis of Life, Chapter 3, inLangton et al.1992:371-408.

22. S:hrOOinger E. What is Life? Cambridge, MassachusettsUSA. 1969:74-6.

23. Taylor C. "Fleshing Out" Artificial Life II,in Langton,et al.1992:25 - 38.

24. Tsujimoto Y,cmsman J ,Jaffe E,Croce CM. Involvement ofthe bcl-2 gene in human follicular lymphoma. &ience 1985;228:1440-3.

25. Varela F. http://pespmc1.vub.ac. bel ~I ALWffiIESIS.html.2005.

26. Zubay G. Origins of Life on the Earth and in the Cosmos.Academic Press, New York, USA. 2005: xix.

. 15 . editor@sciencepub. net ,II

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tic human in tenns of average and maximum elec-tric field intensities. The calculation revealed the

induced dosimetry amount for various major organsupon exposing to extremely low-frequency electro-magnetic fields. The whole body induced maximumcurrent density is about 21 micro amp per metersquare from a 50 Hz to a 60 Hz source frequencyand from a 1 amp per meter to a' O. 1 Gauss sourcestrength. Comparatively, the cell culture dosimetryfor low frequency magnetic fields study by Hart(Hart, 1996) has shown the induced current densi-

ty averagely about O. 9 micro amp per meter squarefrom a 60 Hz source of frequency and a 1 Gausssource of field strength. Accordingly, the inducedeffect between the human body and the cells inculture clarified the interaction mechanisms in-volved could be different. The mechanisms where-

by ELF electromagnetic field stimulates changes inbiological functions of cells in culture may notguarantee that would cause the same biological ef-fect in vivo. Many researchers therefore switched

their interest to the biological effect of very highfrequency band of wireless communication protocoljust because of the support funding being hard tocontinuous for ELF biological effect study.

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Abstract: The mechanism for interactions of magnetic field, particularly in extremely low frequency with biologicalcells is still puzzle to the scientific researchers. Our investigations have guided to the speculation of roles of the mag-netic field for the cell-cell communication in physiological responses. To explain the complexity, the experimentalevidences and observations and their associated theories have been included in this review. The result of a disruptionof the homeostatic regulation of the cells responding to a specific strength and frequency of magnetic field can be asthe signal that triggers signal transduction to modulate the cell-cell communication. This review leads into thestochastic systems in cell, for instance, ion channels on cell membrane, providing a basis for signal amplification todisrupt the cell homeostatic regulation. [Life ScienceJournal. 2005; 2(1) :16 - 21] (ISSN: 1097- 8135).

Keywords: biological effect; cell; magnetic field

1 Introduction

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Biological cells have been shown to respondlow frequencies electromagnetic fields as well as inchemical and biochemical reactions ( Dobson,1996). Puzzle of the energy of the fields is too lowto the noise in cellular level to produce observableeffects (Adair, 1991; 1992; 1994). Therefore, thephysical mechanism of primary interaction betweenthe magnetic fields and the biological target sites,such as electrical charge in motion, molecules struc-ture with magnetic moments and the application ofFaraday law, generating local electrical current by avarying magnetic field has mainly concluded onlyamplification mechanisms can solve the puzzle. Aswe know, the interaction should be very weak atfield strength less than 10 Gauss and the frequencybelow 100 Hz. If considering the stochastic reso-nance model, the amplification of weak electromag-netic interaction signals can be modulated by exter-nal fields (J ung, 1993). By considering ferromag-netic transduction model, the minimum appliedmagnetic fields would produce a torque on a bio-genic magnetite particle coupling via the cytoskele-ton to the ion gates. The deformation of the cellmembrane and the closing of the ion gates wouldoccur quickly enough to compensate for the forcedopening of the gate as long as the frequency of theforcing field was below 100 Hz, regardless of thestrength of the applied field. Dawson et al. (Daw-son, 1996) used scalar potential finite differencecode for low frequency electromagnetic computa-tions and to model induction in anatomically realis-


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frequency (ELF) electromagnetic fields may pre-sent a health hazard to workers and the public. Thecontroversial and contradictory finding in the scien-tific research, especially from epidemiological stud-ies is puzzle (EPRI, 1994). Research of ELF elec-

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tromagnetic fields interaction caused biological ef-feet in biological system includes experimental in-vestigations on both in vivo and in vitro. In pre-sent, the researchers have been interested in bothto the electric fields and magnetic fields as well as i-dentifying a possible mechanism for ELF acting as acancer initiator, promoter or co-promoter. On theother hand, epidemiological studies have shownvery weak connection between ELF exposure andleukemia, brain cancers, breast cancer and lungcancer. Almost all related research studies were in

flaws, for instance, numbers of cases were too lowto look at cancer subtypes, lack of specific expo-sure, lack of reliability of data, lack of statisticalpower and lack of control for repeaters. There is noreliable supporting data for an association betweenELF exposure and cancer risk in the public. Thereis no conclusive evidence so far from the epidemio-logical evidence that electric or magnetic fieldscause a risk of cancer. The researcher only holds apossibility of the risk of cancer of ELF electromag-netic fields occupational exposures. Oppositely, ex-perimental investigations with cellular systems haveshown that electromagnetic fields can interact withbiological systems. Several researchers have con-firmed cellular effects involving the movement ofcalcium ions through cellular membranes underELF interaction (Fewtrell, 1994). The signifi-cance of this effect as it relates to possible adversehealth outcomes is still not understood. Direct ef-

fects on significant cellular molecules, such asDNA, have not been observed. No direct muta-genic or carcinogenic effects on animals have beenobserved. Current research has shifted to focusingon the role of ELF (particularly magnetic fields) asa tumor promoter or co-promoter. However, it isstill no effects were observed on mice exposed with-out the chemical promoter yet. The overall viewobtained from the research literature indicates that

while some biological effects of exposure to ELFelectric and magnetic fields occur, there are no re-sulting adverse health effects from these exposures.In the United States, a large number of researchpapers and overview reports have been produced a-long with numerous conferences over the past 15years. Unfortunately, the findings remain contro-versial and contradictory. There is insufficient datato determine if a cause and effect relationship ex-ists. The National Council on Radiation Protection

and Measurements (NCRP) in Bethesda, Mary-land, set up a committee chaired by Dr. W. RossAdey to review the possible health effects of ELF.The National Academy of Sciences committee,chaired by Dr. Charles Stevens, Salk Institute,California released a report in 1996 concluding no

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clear, convincing evidence exists to show that resi-dential exposures to electric and magnetic fields(EMFs) are a threat to human health. The UnitedKingdom's National Radiological Protection Board(NRPB) established an Advisory Group on Non-Ionizing Radiation in 1990 to review the scientificevidence and determine the extent to which this ev-idence suggests possible health risks. The Interna-tional Non-Ionizing Radiation Committee (INIRC)of the International Radiation Protection Associa-tion (IRPA) in cooperation with the EnvironmentalHealth Division of the World Health Organization(WHO) developed recommendations for 50/60 Hzelectric and magnetic field exposure limits. At the8th Congress of the IRPA in May 1992, the IRPAestablished a new independent scientific organiza-tion, the International Commission on Non-Ioniz-ing Radiation Protection (ICNIRP) as a continua-tion of the former IRPA/INIRC. In April 1998,ICNIRP published guidelines for limiting electro-magnetic field exposures for frequencies up to 300GHz, including 50/60 Hz.

3 Energy Transduction in Cell

The mitochondria in cell contain the series of

catalysts known as the respiratory chain collecttransport reducing equivalents to react with oxygenfor forming water. The reducing equivalents,-H orelectrons, are made from oxidation of carbohy-drate, fatty acids and amino acids. The cell systemcouples respiration to generate the high energy in-termediate A TP, termed oxidative phosphorylationinvolved NADH, Succinate, Ubiquinol, Ferrocy-tochrome and ATP synthase five protein lipid en-zyme complexes. Three different mechanisms in-cluding chemical coupling hypothesis, the confor-mational coupling hypothesis and the chemiosmotichypothesis have been proposed to explain the ener-gy transfer between electron transport and ATPsynthesis (Menendez, 1996). Accordingly, elec-tron transport along the respiratory chain can be thesource of electromagnetic field to exert forces on theproton. Through these forces, electromagnetic cou-pling hypothesis is proposed to describing the pro-tons are moved from the mitochondrial matrix to

the exterior. In the mean time, the protons movedby the field toward the inner membrane passthrough its protein components plays the role ofproton channel. Just as if in a motor, ELF triggersthe electrical energy, transfer of electrons along therespiratory chain, to produce proton translocation,which is mechanical energy, be used in cell system.


channel and be transported to plasma membrane ofthe cell. Before pairing process, hemi channels areclosed to avoid leakage of cellular contents and en-try of extra-cellular materials. During the pairing ofconnexons and aggregation into plaques at the plas-ma membrane, connexin 43 is phosphorylated atleast twice and connexons are attracted to those lo-cated on the adjacent cells. Two connexons join inan end-to-end manner to form a complete channel.The channels aggregate into large gap junctionplaques open to connect two cells for cell-to-cellcommunication and is called gap junctional intracel-lular communication (GJIC), which can be modu-lated by environmental factors, such as drugs, X-ray, electromagnetic fields etc. Since the functionof the GJIC, cultured cells coupled in vitro exceptthe stem cells and cancer cells (Trosko, 1991;2001) . Furthermore, in basic signal transduction,we want to know what the gap junction growthregulatory signal within cells is. The cAMP is avery important gap junction signal molecule. Thissignal molecule can pass between cells through gapjunction channels and affects cell growth. The lev-els of the cAMP can oscillate within the cell andgenerates oscillation in growth control. The ampli-tude of the oscillations would be dampened byGJIc. However, cAMP is not the only signalmolecule that may affect GJIc. Increasing drugpenetration and dispersal in tumors would increaseGJIc. Gap junction channels in cell membrane cancreate a stochastic two-state system, opening orclosing of the channels, to amplify weak signalcaused by ELF fields.

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4 Stochastic Resonance

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Several resonance theories, for instance, ioncryotron resonance (Liboff, 1991), parametric res-onance were proposed to describe the biological ef-fect caused by ELF fields. For the problem of syn-chronization of the relaxation time and the noise, itis hard to find the evidence of resonance in cell sys-tem. Nevertheless, because of ionic channels of cellmembranes are elemental molecular switches forchannel states, open or close, in spite of the smallsize of the channel, high resolution ion current de-tection allows us to watch electrical activity of asingle ionic channel isolated within a micrometerpatch of a cell membrane. The state-transition be-havior between the open and close states of an ionchannel characterizes many biological processes.The experimental results suggested that the poten-tial oscillation could be driven by the oscillation inthe intracellular concentrations of cyclic AMP andcalcium in two-state model system (channel closingand opening). The signal-to-noise ratio increaseswhen the state-transition rate of membrane channelinfluenced by the frequency response of the intra-cellular sensing system. An applied ELF (electricor magnetic field) field is an important factor toperturb the rate of state-transition of membranechannel in opening or closing. Jung (1993) has de-veloped stochastic resonance driven process for mul-tichannel systems and shown the optimal choice ofparameters can lead to signal amplification. Fromthe stochastic resonance theory, time average of thetotal current through a set of N channels can becalculated. To access the magnitude of amplifica-tion of the responding signal caused by externalELF field in cell, the membrane with N voltagegated channels and biochemical oscillator (calciumoscillation) must be considered. Because of thismodulation, the external ELF field signal is trans-formed into a periodic component in the ion currentacross the membrane. As we have focused in thestudy on the primary mechanisms that a biologicalcell can use to amplify weak external influences, asystem of identical ion channels embedded in amembrane and synchronously modulated can signif-icantly amplify the original signal. The amplifica-tion of the signal to noise ratio is proportional to thesquare root of the number of channels modulatedand inversely proportional to the square root of thesum of the average channel relaxation times.

5 Role of Gap Junctions

In cell, six connexin 43 subunits oligomerze inthe Golgi apparatus into a connexon, called hemi


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The diffusive current equation for connexin 43channels can be written as J

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j

i

< I> = f,kP(k)k=!

where probability P(k) indicates total m channelsis taken into account for opening k channels fromall cell-to-cell communications on the surface of thecell mono layer. Therefore,,

P( k ) = m : (PO)k ( pe ) m - k (2 )k!(m-k)!

dpo = repe - rOpO (3)dt

dpe = rOpo- repe (4)dt

where re is the rate of changing from c-state to 0-

state and rO is the rate of changing from o-state toc-state of the connexin 43 channels activating total-

lyon the cells mono layer surface. Generally, rO

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does not have to be same with rCsince the life timesof the o-state and c-state may vary. To clarify thephysical meaning, we further assume the currentthrough an open channel as i. The diffusive currentcaused by GnC channels can be rewritten as

< I> = miP~ (5)where P~ is the modulated probability for o-state byexternal ELF field signal. According to theory ofJung (1993), the power spectral component origi-nated from the signal is given by

(mi)2 00 I ISk = ~ {;1 Cq 8(w - qWk)

Cq is the Fourier expansion coefficients of P~.In comparison with equations (3) and (6),

the signal-to-noise ratio (SNR) of the characteristicfrequency of the cell system can be depicted (Gal-vanovskis, 1997).

SNR =I

signal amplitude1

2

background amplitude

=A2I Jm ~ rOrc 1

2 (7)t:..w(rO + rC)

where m is the number of channels, A is the ampli-tude and t:..wis the bandwidth of the external ELFfield signal.

7 Specific Inhibit and Promote of GJIC

Many researchers have developed new tech-niques to inhibit or promote the expression of a tar-get gene in culture cells and animals. ELF magneticfield treatment is one of the main factors that scien-tists are interested. We proposed a new way to sep-arate the background and signal and revealed theevidence of existing stochastic resonance systemburied in biological cells by GnC essay under ELFmagnetic fields. Specific inhibit of GnC withinmouse osteoblast cells in culture under the exposureof ELF magnetic field (Teng, 2002) depicted sev-eral possibilities, blockage of connexin gene expres-sion, connexin gene knockout and transfection ofdefective connexin genes. Additionally, specific en-hancement of GnC within mouse osteoblast cellsunder the exposure of different doses of ELF mag-netic field (Hart, 1996) can be transfected of func-tional connexin genes.

8 SNR Spectrum

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ltll

By using of the probe of Gauss-meter, cells-in-duced magnetic fluctuation can be shown as

IB;I= IBl ,Bl"" ,Bloool (8)Equation (8) contains the cellular response signal ofthe reaction to the external ELF magnetic field(Teng, 2003). The sampling time was 0.0005

http://www.sciencepub .org

(6)

second. The probe was located at the distance 10- 4

m perpendicularly to the center of single layer ofthe cell surface in culture dish. The Gauss-meterwas manufactured by F. W. Bell Company (seriesof 9550) in Florida of USA. Oscilloscope was man-ufactured by Agilent Company (54621A) and canbe used to convert IB;I to voltage sequenceIv;1as

I V;I = I VI, V1,"', v1oool (9)Matlab and Fortran programming were used forpower density spectrum analysis of these voltage se-quences. IBi I for the first control was taken 2000

times per second at the distance 10- 4 m perpendic-

ularly to the culture dish with only medium in it.Ceo-field control IBi I was taken with the same

sample rate at the distance 10- 4 m perpendicular tothe empty culture dish recording local geomagneticfield fluctuation. The corresponding IVi I andIVi I can be obtained by the same way as IVi Ipreviously. Furthermore, trial signals,

ni ( n) = Ai x sin( Win) ,lHz:(;wi:(;60Hz,where signal amplitude Ai = F X V rnax' F is theadjustablefraction factor and Vmax is such, V rnax=max( IV;I), as to the maximum value of the se-quence IV; I.By taking into consideration of signalamplitudes at F values, for instance,F=O. 7,0.4,and O. 03 respectively, the corresponding ampli-tudes would be

AO.7=0. 7X V max'AO.4=0.4x Vrnax'AO.O3=0.03x Vmax

for a given trial signal at ELF Wi(1 Hz:(; Wi:(;60Hz). We computed autocorrelation function ofIV;:t ni ( n ) I and its Fourier transforms to obtaintheir corresponding signal-to-ratio ratio. The SNRspectrum, then, for IV;:t ni ( n ) Iat frequencycould be simply a second order equation as

aX(Swi(F»2+bxSwi(F)+c=0 (10)Accordingly, substituting the SNR at different

F (different amplitudes at frequency) into the e-quation, we can solve unknowns a, band c. If c-value is much bigger than zero, then, the SNR ofthe intrinsic signal peaked at Wi is detected (Teng,2005). The value of c is about O.07.

In the paper by (Galvanovskis, 1997), theSNR could be written as

SWi(F) =(FxVmax)2XmXQX (2: )Wi r ° r C

when the life time of c-state and o-state equal to

10 - 6 second. Under optimal condition, the quality

factor Q = ::approximately equals to 100 at 60

~~


jLife Science Journal, 2 (1 ) ,2005, Teng, A Puzzle of the Effect of Magnetic Field on Biological C€lls

vealed the possibility of signal transduction path-ways perturbed by ELF magnetic fields at differentcharacteristic frequencies and the stochastic reso-nance frequencies within' the cells. The result of amodulation of the GnC within the cells respondingto a specific strength and frequency of magneticfield can be as the signal that triggers signal trans-duction in cell.

JjJJ

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jJJ

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Hz with bandwidth ~w = 0.6 Hz and F = O. 6.The numbers of GnC channels are taken 1000 percell (Galvanovskisz, 1997). The SNR value is fitto the value calculated from SNR spectrum suggest-ed by Teng (Teng, 2005).

9 Discussion

We examined the role of the ELF magneticfield in the biological cells system. It is hard toimagine how the forces of evolution could have ledto biological mechanisms that respond to ELF mag-netic field in cells, because low frequency fieldshave not been present during virtually all of evolu-tion. So far, there is still no direct evidence thatELF magnetic field can affect the physiological end-points within cells in culture. However, enoughevidences have shown the biological effect that thecell responded to the interaction of externally ap-plied ELF magnetic field (Kaiser, 1996). SinceGnC affiliates with many physiological endpoints,changing of the characteristics of GnC causes thebiological effect within cells in culture. A confluentcell culture in a vertical magnetic field could receiveseveral different exposures. If the cells are nottightly jojnted to each other and to the walls, thenthe system is homogeneous. Different cell typescould e~perience very different induced current andelectric field distributions under the same magneticfield exposure in similar dishes. Gap junctions jointthe cells and are open to allow current paths con-nect the cell interiors. They produce the currentpaths throughout the interiors of the cells in the cellculture. Most of the time, two dimensional model

is oversimplify the current density distribution thatis actually experienced by cells in a culture dish.Value for the induced current density, in effect, isthe top surface perpendicular to the applied mag-netic field. When the magnetic field is applied a-long the surface of the cells, perpendicular to thenonnal direction of the surface, the homogeneoussystem would be inapplicable around the centralplanes, but it could be used away from the centralregion.

10 Conclusion

~

If a cell culture is exposed to a vertical mag-netic field for a relatively long period, the exposurereceived by the cells may change during the courseof the experiment. Trusko et al. (Trosko, 2001;Upham, 1998) originated the perfusion of the dyeproduced by the function of the GnC in cell cultureto study the cell physiology. T eng et al. first pro-posed the signal-to-noise SNR spectrum calculationof the near magnetic field in 2002. They have re-


Correspondenceto:Hsien Chiao TengDepartment of Electrical EngineeringChinese Military AcademyFengshan, Taiwan 830, ROCTelephone: 011886-7747-9510 ext 134Email: [email protected]

References1. Adair RK. Constraints on biologicaleffects of weak ex-

tremely low frequencyelectromagneticfield. Physical RevA 1991;43:1039-48.

2. Adair RK. Criticism of Lednev' s mechanism for the in-fluence of weak magnetic fields on biological systems.Bioelctromagneics 1992; 13 :231 - 5 .

3. Adair RK. Biological responses to weak 60 Hz electricaland magnetic fields must vary as the square of the fieldstrength. Proc Natl Acad Sci 1994;91 :9422 - 5.

4. Dobson JP, Grassi P. Magnetic properties of human hip-pocampal tissue: evidence for biogenic magnetite in thehumanbrain. BrainResBull1996;39:255- 9.

5. Dawson T, Stuchly MA. Analytic ~alidation of a threedimensional scalar potential finite difference code for lowfrequency magnetic induction. Appl Comput ElrctonmagnSoc] 1996; 11 (3) : 63 -71.

6. EPRI. Biology and electric and magnetic fields: biologicalmechanisms of interaction. Printed by Gradint Corpora-tion, Cambridg, Massachusetts. 1994.

7. Fewtrell C. Calcium oscillation in non-excitable cells, inAnn Rev of Physiology, Vol. 55, Editor Hoffman JF,Annual Review, Inc. Palo Alto. 1994; 55: 427 - 54.

8 .Bruce N. Biomedical signal processing and signal model-ing. Wiley-Interscience Publication, John Wiley & Sons,Inc. New York, ISBN 0-471-34540-7. 2001.

9. Glaser R, Michalsky M, Schamek R. Is the ci + trans-

port of human erythrocytes influenced by ELF- and MF-electromagnetic fields? Bioelectrochemistry and Bioener-getics 1998 ;47: 311 - 8.

10. Hart F. Cell culture dosimetry for low frequency magnet-ic fields. Bioelectromagnetics1996; 17:48 - 57.

11. J ung P. Periodically driven stochastic systems. Phys Rep(Phys Lett) 1993 ;234: 175 - 95.

12. Kaiser F. External signals and internal oscillation dynam-ics: biophysical aspects and modeling approaches for in-teractions of weak electromagnetic fields at the cellularlevel. Bioelectrochemistry and Bioenergetics 1996; 41 : 3-18.

13. Liboff AR, Parkinson WC. Search for ion cyclotron reso-nance in an Na+ transport system. Bioelectromagnetics1991;12:77-83.

14. Menendez RG. An electromagnetic coupling hypothesis

JI

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J

1~

JI

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Life Science Journal, 2 ( 1 ) ,2005, Teng, A Puzzle of the Effect of Magnetic Field on Biological Cells

to explain the proton translocation mechanism in mito-chondria, bacteria, and chloroplasts. Medical Hypotheses1996 ;47 : 179 - 82.

15. Takb H, Shiga T, Kato M, Masada E. Biological andHealth Effects from E~pcsure to Power Line FrequencyElectromagnetic Fields-confinuation of Absence of AnyEffects at Environmental Field Strengths. IOS Press,Ohmsha, ISBN 4-274-90402-4C3047. 1999.

16. Teng HC, Cherng S, Trosko JE, Chang CC, Upham BL.Sensitivity of Osteoblast Cells to Inhibition of Gap Junc-tional Intercellular C.ommunication By ELF-EMF at 14Hz (The 24th Annual Meeting of the BioelectromagneticsSociety). 2002.

17. Teng HC, Cherng S. Mouse osteoblast cell sensitivity tothe AC magnetic field at 14 Hz. Nature and Science

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2003;l (1) :27 - 31.18. Te!1g HC. The molecular biological application of the

theory of stochaStic resonance: the cellular response to theELF AC magnetic field. Nature and Science 2005; 3 ( 1) :37 ~ 43.

19. Trosko JE,Chang CC,Madhukar BV. Modulation of In-tercellular Communication during Radiation and ChemicalCarcinogenesis. Radiation Research 1990; 123 :241 - 51.

20. Trosko JE, Chang CC. Role of stem cells and gap junc-tional intercellular communication in human carcinogen-sis. Radiation Research 20tH; 155: 175 - 80.

21. Upham BL,Deocampo ND, Wurl B, Trosko JE. Inhibitionof Gap Junctional Intracellular Communication by perfluo-rinated fatty acids is dependent on the chain length of thefluorinated taiL Int J Cancer 1998;78: 491 - 5.

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sponse to chemotherapy[ 4]. We have previously de-scribed the overexpression of annexin V in gastric

carcinoma tissues using proteome- based ap-proach[5]. In this study we have made an annexin

V expression construct, purified the protein andfurther determined the identity of the protein forthe purpose of studying its role in tumor cells[6].

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Life ScienceJournal, 2005, Wang, et ai, Expression and Purification of Annexin V

Expression and Purification of a HumanTumor-Associated Protein Annexin V

Kaijuan Wangl.2, Liping Dai1, 2, Yan Jial, Jianying Zhangl,2

1. Department of Epidemiology ,School of Public Health, Zhengzhou University,

Zhengzhou, Henan 450052, China

2. Proteomics Research Center, Zhengzhou University, Zhengzhou, Henan 450052, China

Abstract:Annexin V is a phospholipase A2. and protein kinase C inhibitory protein with calcium channel activity andan undefined role in cellular growth and differentiation. The proouct of annexin V gene is necessary for doing ex-periments to determine whether the tumor-associated annexin V protein can be used as a new molecular marker fordiagnosis and prognosis of gastric carcinoma. This study describes the procedures which were used for cloning andexpressing the human annexin Vasa maltose binding protein (MBP) fusion polypeptide in bacteria. The expres-sion plasmid for annexin V was constructed by ligation of the annexin V cDNA into the expression vector pMAL-c2x. The protein was expressed by E. coli strain TBl cells and purified by amylose affinity column chromato-graphy. The purity of the protein was assessed by SDS-PAGE and Western blot. The results showed that themolecular weight of the recombinant MBP-annexin V polypeptide was consistent with the calculated molecularweight, and that the purified protein appeared as an apparent single band of 77 kDa on the gel filtration column bySDS-PAGE. Our expression system allows the expression and purification of annexin V with MBP in high yieldwith no need of removal of the tag and gives pure protein in one purification step, and also makes it possible for thestructural and functional studies of these proteins. [Life Science Journal. 2005; 2 ( l) : 22 - 26] (ISSN: 1097-8135) .

Keywords: annexin V; expression; purification; pMAL-c2x

1 Introduction

-

Annexins are ea2+ and phospholipid-bindingproteins forming an evolutionary conserved multi-gene family. Each member of this protein familycontains a conserved protein core characterized byhigh alpha-helix content that includes the calciumand phospholipids binding sites, and a variable N-terminal domain that is specific in sequence andlength for each annexin. Although the structure ofthe protein core is highly conserved, different an-nexins exhibit a wide biochemical and functional di-versity, e. g., inflammatory response, membranefusion and exocytosis, ion channel regulation, andinhibition of blood coagulation[l]. Despite an abun-dance of experimental evidence suggesting that an-nexins are associated with a plethora of biologicalprocesses, the exact physiological function of an-nexins remains to be investigated. Annexin V is aphospholipase A2 and protein kinase C inhibitoryprotein with calcium channel activity and an unde-fined role in cellular signal transduction, inflamma-tion, growth and differentiation[2]. It has beenpreviously isolated as placental anticoagulant pro-tein[3]. Annexin V may be used to assess tumor re-

.http://www.sciencepub. org

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2 Materials and Methods

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Construction of pMAL-Annexin V: TotalRNA extracted from human placental by RnaseyMidi Kit (Qiagen, Valencia, CA, USA). The mR-NA was then reverse transcribed to cDNA usingAMV reverse transcriptase (Promega Co, USA)with 9-mers random primer. The cDNA encodingfor human annexin V (Accession # J03745)was

amplified by PCR using the upstream primer 5'-GCGAATTCA TGGCACAGGTTCTCAGAGGCA-3'designed with an upstream EcoR I restriction site( underlined), and the downstream primer 5'-GCGCTGCAGTTAGTCATCTTCTCCACA-3' de-signed with a down-stream Pst I restriction site(underlined). Both PCR products contain an 'A' atthe 3' end, the 965-bp DNA fragments from thePCRs were directly cloned into pMD T vector

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(TAKARA Bio Inc.) following standard proce-dures[7]. pMAL-c2x plasmid (New England Bio-labs) contains an Ampr gene, tac promoter, the lacIq gene, a polylinker, and the mal E gene, whichencodes for maltose binding protein (MBP). pMD-annexin V was digested with the restriction en-zymes EcoR I and Pst I (Promega Co. ,America),and pMAL was digested with EcoR I and Pst Iwith manufacturer's buffers at 37°C for 2 hours.The fragments from these restriction digests weregel-purified using the Qiaex II Gel Extraction Kit( Qiagen, Valencia, CA, USA) . The cut pMAL vec-tor was then ligated with the annexin V gene usingT4 DNA ligase (Promega Co. , USA). All ligationswere transformed into MAX efficiency E. colistrain TB1 cells. Bacterial cells were plated ontoLB-Amp plates and incubated overnight at 37°C. Acolony containing each of the constructs were inoc-ulated into LB-Amp culture medium overnight andthen midi-prepped using a Plasmid Midiprep Kit(Vitgene, China).

Verification of constructs: The recombinantplasmid was detected by restriction endonucleasesand specific PCR. The midi-prepped constructswere sequenced on an ABI 3700 Sequencer to verifycorrect splicing of the gene into th~ plasmid.Se-quence analysis was done with th~ program Se-quencher, software available from Gene Codes Cor-poration (Ann Arbor, MI).

Expression of fusion proteins: TB1 competentbacterial cells were transformed according to manu-facturer' s instructions with the sequenced MBP-Annexin V[8]. Cells were plated onto LB-Ampplates and grown overnight at 3TC. The overnightculture was added to 900 ul 50 % glycerol to makeglycerol stocks. Overnight cultures were diluted 1:100 with pre-warmed LB-Amp media. The proteinexpression was induced by addition of lsopropyl-Be-ta-D-thiogalactoside (IPTG) to a final concentra-tion of O.2 mM when OD6ooreached O.4 - O.5.After a 6 h post-induction period, the cells wereharvested by centrifugation at 6,000 g at 4°C for20 min. One ml of both uninduced and induced cul-tures were spun down and resuspended in 100 ul 2X SDS-PAGE sample buffer and analyzed by 12%SDS-PAGE gel.

Purification of MBP fusion proteins: The fu-

sion proteins were purifiedbased on a protocol[9,10]as described (New England Biolabs, USA). Oneliter of cells transformed with the fusion constructwas induced to produce protein as described above.The cultures were spun down and resuspended in50 ml column buffer (20 mM Tris-Cl, 200 mMNaCl,l mM EDTA, 1 mM DTT). Resuspended

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cultures were frozen at - 20° C until purification.Frozen cultures were defrosted slowly in ice-coldwater. O.5 M DTT, 250 mM PMSF, and 100 X

protease inhibitor cocktail from Sigma were addedto the defrosted cultures to final concentrations of 5mM, O. 1 mM respectively. The mixtures weresonicated in short bursts of 10 seconds for 20 min-utes total[1l,12]. Sonicates were spun down, andthe supernatants were collected. 1. 5 ml amyloseresin beads (New England Biolabs, USA) wereadded to the supernatants and the protein was al-lowed to bind to the beads overnight on a rotaryshaker at 4°C. The beads were washed with col-umn buffer and protein was eluted with 100 mMmaltose in column buffer overnight at 4°C. Samplesfor SDS-PAGE analysis were taken from inducedculture, post-sonicate pellet, post-sonicate super-natant, supernatant after binding to amylase resinbeads, and eluted protein was analyzed using a12.5% SDS-PAGE.

Detection of recombinant proteins by westernblot: Electrophoretically separated proteins weretransferred to an Immobilon PVDF transfer mem-brane (pore size O. 45 pm, Millipore) by elec-trophoresis in transfer buffer (PBS, pH 7. 4, 200mM glycine, 20% methanol, 0.1 % SDS) using aTransblot cell (Biorad, USA) at 275 mA fo~ 30min. After transfer the non-specific binding siteson the membrane were pre-blocked by incubatingwith blocking buffer overnight at 4°C. The primaryrabbit anti-annexin V antibody (Abcam Ltd,USA) was diluted 1 : 300 in PBS and incubatedwith the pre-blocked membrane for 60 min at roomtemperature with constant shaking. Non-bound an-tibodies were removed by washing three times inwash buffer (0.05% Tween-20 in PBS) for a totalof 60 min. The antibody antigen complex on themembrane was detected by incubating the mem-brane with a secondary horseradish peroxidase-con-jugated goat anti-rabbit antibody (diluted 1: 5,000)for 1 hour at room temperature. The membrane wasthen washed as before and rinsed with PBS[13-15].Reactivity was visualized according to the manufact,. .urer s Instructlons.

3 Results

Verification of constructs of MBP-AnnexinV: As shown in Figure 1, the recombinant plasmidof pMAL-annexin V was digested into two frag-ments by EcoR I and Pst I which are 6. 7 kbpMAL vector fragment and O. 96 kb annexin Vgene fragment[15], and there was a single 7.0 kbfragment by the digestion of EcoR I alone. The0.96 kb annexin V gene fragment could be ampli-

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were readily expressed in TB1 cells. To obtain highAnnexin V expression, Figure 2 shows the TB1cellular content of Annexin V expressed in the dif-ferent conditions. After SDS-PAGE analysis, theMBP-Annexin V protein was detected at 77 kDa(42 kDa MBP + 35 kDa Annexin V = 77 kDa)by Coomassie blue staining. As shown in Figure 3,the level of expressed Annexin V was apparentlyaffected. Annexin V constituted approximately39. 9% of total cellular protein after IPTG induc-tion for 4 hours.

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fied by specific PCR. It was demonstrated that re-combinant plasmid of pMAL- annexin V was con-structed successfully. Sequencing analysis showedthat the construct, which was used for protein pu-rification contains the full-length 965 bp annexinV gene. The cDNA opening reading frame of an-nexin V gene contained 965 bp which coded for aprotein with 319 amino acids and a calculatedmolecular mass of 35,805 Da.

1 2 3

-10 kb-3 kb

-1 kb

-0.5 kb

Figure 1. The recombinant plasmid was verified by restriction en-

donucleases. Lane I, recombinant plasmid DNA was digested byEcoR I and Pst I; lane 2, recombinant plasmid DNA was digest-ed by EcoR I alone; lane 3, DNA molecular markers (10 kb).

1 2 53 4 6 7 8

-97.4Da

-66.2Da

-43Da

3IDa

-20Da

~

Figure 2. Expression of MBP-Annexin V fusion constructs. LaneI ,TBI (pMA) O. 2 mM IPTG induced 5 hours; lane 2, TBI(pMA) 0.2 mM IPTG induced 4 hours;lane3,TBI (pMA) 0.2mM IPTG induced 3 hours;lane 4, TBI (pMA) 0.2 mM IPTGinduced 2 hours; lane 5, 1TBI (pMA) 0.2 mM IPTG induced Ihours; lane 6, protein marker; lane 7, TBI induced 4 hours; lane 8,TBI (pMAL- c2x) un-induced 4 hours.

Expression of recombinant MBP-Annexin Vfusion protein: Annexin V was cloned into thepMAL-c2x expression vector, as described in mate-rials and methods. MBP-Annexin V constructs

http; / /www.sciencepub.org

~ [1] 39.877%

[2] 7.945%[3] 9.388%~

~:;

[4] 14.198%[5] 10.379%[6] 5.695%[7] 1.640%[8] 3.353%

[9] 7.525%

Figure 3. Contents analysis of the MBP- Annexin V fusion pro-

tein. Percentage of the each protein was showed in the figure.

Annexin V constituted approximately 39.9 % of total cellular pro-tein after IPTG induction for 4 hours.

Purification of recombinant MBP-Annexin Vfusion protein: The efficiency of the purificationwas dependent on appropriate amounts of equili-brated amylose resin. Due to the relatively bindingcapacity of the resin, we found that 1 ml crude ex-tract (approximately 10 mg protein) needs 1 mlofequilibrated gel slurry. Prolonged incubation timesof more than 15 min at 4t had to be used in orderto achieve optimal recovery of bound protein. Weobserved that the first washing steps containedlarge amounts of MBP-Annexin V complexes.Most of the MBP-Annexin V protein produced byIPTG induction was found in the post-sonicate pel-lets, and not in the post-sonicate supernatants. Al-most all of the protein contained in the supernatantswas able to bind to the amylose resin and could beeluted from the beads with a high efficiency. Afterpurification and SDS-PAGE analysis, the MBP-Annexin V protein was detected as a single band of77 kDa, arid it also immunoreacted with anti-An-nexin V polycolonal antibody, The result showedthat this protein was expressed and purified as asingle chain MBP-Annexin V fusion protein. Thefinal yield was 1. 9 mg of purified protein per liter

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culture cells and the purity of the preparation wasestimated to be over 95% as judged by SDS-PAGEanalysis. .

4 Discussion

This study describes the process of bacterialexpression and purification of a MBP-Annexin Vfusion protein. The results indicate that fusion pro-tein using the MBP purification system from NewEngland Biolabs can be readily produced in bacteriaand purified with high yield. The experimentswhich have been done in this study showed that thepost-sonicate supernatant contains cellular solublefractions from the rupture of cells. The appearanceof more protein in this component of the sonicateimplies that the number of cells that remained rup-tured after sonication is greater than the number ofcells that released their protein during sonication.The anti-Annexin V polyclonal antibody was usedto assess the specificity of annexin V protein byWestern blot analysis. The results demonstratedthat the purified Annexin V protein had strong re-activity with the polyclonal anti-Annexin V anti-body, suggesting that it could be used in the subse-quent experiments to characterize the gene annexinV in tenns of its function.

2 4 5 6.31

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Figure 4. Purification of recombinant MBP-Annexin V fusion pro-tein. Lanes 1 - 4, Purification of MBP-Annexin V protein fromtubes 1 - 4; lane 5, Molecular marker;lane 6, TB1 (pMAL-an-

nexin V) post-sonicate supernatants.

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Some other experiments are currently under-way to further investigate the function of AnnexinV. Its probable role in regulation of the expressionof the tumor-specific protein may make it as an in-

triguing subject of research[16]. This study only de-


scribes the procedure of expression and purificationof recombinant Annexin V, which will be useful forfurther investigations of Annexin V. The purifiedAnnexin V protein could be used as important ex-perimental material for the studies that will be per-formed to gain insight into the functioning of An-nexin V.

In summary, we reported a robust method toexpress, isolate and purify recombinant MBP-An-nexin V fusion protein in bacterial cells. Due to its

high soluble content, the ci+ -dependent phospho-lipid binding proteins have proven readily to expressand isolate in sufficient quantities for structural andfunctional studies. The protocol which was used inthis study allows the protein production of at least 2mg per liter of culture, and also makes it possiblefor the structural studies of these proteins.

Acknowledgments

This work was supported by Program for NewCentury Excellent Talents in University of HenanProvince of China, and partially by grants fromNational Natural Science Foundation of China ( #30471966) and Key Scientific Project of HenanProvince ( # 495033400) .

Correspondence to] ianying ZhangDepartment of EpidemiologySchool of Public HealthZhengzhou UniversityZhengzhou, Henan 450052, ChinaTelephone: 01186-371-6691-1354Email: [email protected]. cn or jianyingzhang@ hot-mail. com

References

1. Gerke V, Moss SE. annexins: from structure to func-tion. Physiology Review 2002; 82: 331 - 71.

2. Cuervo AM, Gomes AV, Barnes JA, Dice JF. Selectivedegradation of annexins by chaperonemediated au-tophagy. The Journal of Biological Chemistry 2000; 275:33329 - 35.

3. Kaplan R, Jaye M, Burgess WH,Schlaepfer DD, HaiglerHT. Cloning and expression of cDNA for human en-donexin II,a ci+ and phospholipid binding protein. TheJournal of BiologicalChemistry 1988;263: 8037 - 43.

4. Ogura Y, Krams SM, Martinez OM, Kopiwoda S. Radio-labeled Annexin V imaging; diagnosis of allograft rejectionin an experimental rodent model of liver transplantation.Radiology2000; 214 :795 - 800.

5. Wang KJ, Wang RT, Zhang JZ. Identification of tumormarkers using proteome based approach in gastric carcino-ma. World Journal of Gastroenterology 2004; 10: 2179-83.

6. D' Arceuil H, Rhine W,Crespigny A, Yenari M, Tait JF,Strauss WH, Engelhorn T, Kastrup A, Moseley M,

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Virological Methods 1999; 82: 129 - 36.12. Mohiti J ,Caswell AM ,Walker JH. The nuclear location of

annexin V in the human osteosarcoma cell line MG-63

depends on serum factors and tyrosine kinase signalingpathways. Experimental Cell Research 1997; 234: 98 -104.

13. BarwiseJL, Walker JH. Subcelluarlocalizationof annex-in V in human foreskin fibroblasts:nuclear localizationde-pends on growth state. FEES Letters 1996; 394: 213 -6.

14. Tzima E, Trotter PJ, Orchard MA, Walker JH. AnnexinV binds to the actin-based cytoskeleton at the plasmamembrane of activated platelets. Experimental Cell Re-search 1999;251 :185 - 93.

15. Krausz E, Graw J. A new cat reporter gene vector de-signed for rapid and efficient cloning of PCR products.Gene 1996; 177:99 -102.

16.Altier F, Maras B, Turano C. Nuclear matrix localiza-tion of Annexin V in chicken liver. BiolochemicalandBiophysicalResearch Communications 1996; 225: 448-54.

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Blankenberg FG. 99mTc Annexin V imaging of neonatalhypoxic brain injury. Strok 2000; 32: 2692 - 700.

7. Feher A, Boross P, Sperka T, Oroszlan S, T ozser ]. Ex-pression of the murine leukemia virus protease in fusionwith maltose-binding protein in Escherichia coli. ProteinExpressionandPurification2004;35:62- 8.

8. Furukawa T, Tanese N. Assembly of partial TFIIDcomplexes in mammalian cells reveals distinct activitiesassociated with individual TAT A box-binding protein-as-sociated factors. The Journal of Biological Chemistry2000;275(38) :29847 - 56.

9. Kenneth J, Turner, lain A, Robin. An interactive visualprotocol stimulator. Computer Standards & Interfaces2001 ;23:279 - 310.

10. Bruno H. Muller, Daniele Chevrier, Jean-Claude Boulain,Jean-Luc Guesdon. Recombinant single-chain Fv antibodyfragment-alkalinephosphatase conjugate for one-step im-munodetection in molecularhybridization. Journal of Im-munological Methods 1999; 227 : 177 - 85.

11. Llames L,Goyache J ,Domenech A,Avila A, Suarez EG,Lucia G. Rapid detection of specific polyclonal and mono-clonal antibodies against bovine leukemia virus. Journal of

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Life ScienceJournal, 2 ( 1 ), 2005, Wang, Investigation in Improved Quality of Life with MedroxyprogestroneAcetate

Clinical Investigation in Improved Quality ofLife with Medroxyprogestrone Acetate in Patients

with Advanced Lung Cancer

Liping Wangl.2

1. Department of Pulmonary Medicine, Xiehe Hospital, Tongji Medical College, Huazhong University

of Science and Technology, Wuhan, Hubei 430022, China

2. Department of Oncology, First Affiliated Hospital, Zhengzhou University,


Abstract: Objective. This study was to investigate the effect of medroxyprogesterone acetate (MFA) in 50 caseswith advanced lung cancer. Methods. 98 cases with advanced lung cancer were randomly divided into two groups,50 cases of the therapeutic group undergoing routine chemotherapy plus taking MFA, and 48 cases of the controlledgroup undertaking routine chemotherapy alone. Results. In the therapeutic group, 78.-0% cases showed improve-ment of appetite, 60.5% cases gained weight, and the score of Kamofsky rose 59.1 % (P<O. 01). Conclusion.MFA has beneficial effects to improve quality of life in patients with advanced lung cancer while its side effect is notobvious. [Life Science Journal. 2005;2(1):27-29J OSSN: 1097-8135).

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Keywords : MFA; chemotherapy; lung neoplasm; quality of life

1 Introduction

Dystrophia and increasing weight loss are com-mon complications of advanced cancer, which areaggregated by Nausea,' vomiting and Anorexia in-duced by chemotherapy, usually they constitute oneof the main causes that prevent patients from con-

tinuing chemotherapy. To reduce the side effects ofchemotherapy, improving quality of life in patientsof advanced cancer during chemotherapy is one im-portant aspect in present clinical investigation ofcancer. From late 1980s, except intestinal and ex-traintestinal high nutrition and other supportivetherapy can improve weight of patients with can-cer, the synthesized drug of progesterone-Medroxyprogesterone Acetate (MPA) can remedyAnorexia and weight loss, so indirectly improvedthe effects of anti-tumor therapy. Now reports asfollows from the results of clinically randomized in-

vestigation of our department in recent years.


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2. 1 Clinical data98 patients with advanced lung cancer, from

October 1998 to May 2002, were all diagnosed byhistodiagnosis. There were 53 males and 45 fe-males, the median age was 60. 5 years old (From17 to 78 years old), in which there were 32 pa-


tients with small cell lung cancer and 66 with nonsmall cell lung cancer; 21 patientsin stage ill and77 in stage N. The average Karnofsky scores were62. 5 in the therapeutic group and' 64.2 in the con-trolled group. The standards by which patients canbe chosen were: CD not accepting hormonetherapy;(Z) without history of hypertension diabetes and

thrombosis; CD not accepting enteral or parenteralhigh nutrition (including transfusion of blood andsome albuminoid biologic preparation); @ withnormal function of liver, kidney and blood routine.2.2 Methods

Both chemotherapy schemes used cisplatin plusetoposide for two cycles at least. Patients in thetherapeutic group began to have MPA of 500 mgtwice a day, for 14 days continually when routinechemotherapy started. Patients in the controlledgroup were treated by routine chemotherapy plususual supportive therapy. Improvement of ap-petite, changes of weight and changes of Karnofskyscores after treatment were investigated separately.2. 3 Statistic analysis

Statistic analysis adopted chi-square test (x2-test). '"

3 Results

3.1 The amount of food that patients consumedand weight

The standard to evaluate the appetite of pa-


The result shows that appetite of patients began toimprove after using MFA for 2 to 3 days. Inwhich, the patients in the therapeutic group were78.0% (39/50), and the patients in the controlledgroup were 10.4 (5/48). There was significance ofdifference by comparing the two groups (Table 1).

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tients is: it can be divided into three groups on thebasis of changes of the amount of food that patientsconsumed everyday after treatment. Improvement:the amount improved no less than O. 1 kg. Nochange: the amount change less than O. 1 kg. Re-duction: the amount reduces no less than O. 1 kg.

3. 2 Changes of Kamofsky scoreChanges of Karnofsky scores can be classified

into: improvement, the Karnofsky scores after ther-apy increased no less than 10; no change, thescores changed less than 10; reduction, the scoresreduced on less than 10. The scores of Karnofskyin the therapeutic group increased in 29 patients,which is 58. 0 % (29/50). However the scores ofKarnofsky increased in 11 patients in the controlledgroup, which is 22.92 % (11148). There was sig-nificance of difference by comparing both groups

(P<O.Ol) (Table 2).3.3 Bad effect

Among 50 patients that took MPA, four pa-tients (8 percent) got slightly depressed edema andrecovered after taking uretica and three patients'(6 percent) blood glucose increased. Among 23 fe-male patients, 2 patients have vagina herrmorrhageafter stopping taking MFA. Vascular obstructivedisease, liver and renal function damage couldn'tbe found.

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and promote albumin assimilation, but also palliatecancer pain, reduce toxicity and side effects thatchemotherapeutic agents generate on marrow andgastrointestinal tracts and consequently developquality of life during chemotherapy and tolerance toit completely. It is indicated that from 30 percentto 100 percent advanced cancer patients get nega-tive nitrogen balance while MPA can improve ap-petite, raise absorption to protelh'~'heat and sodi-um, keep positive nitrogen balance. In the thera-peutic group, 39 (78; 0 pe~~ellt) patients showedimprovement of appetite, 32 (60. 5 percent) pa-tients gained weight and 29 '( 58. 0 percent) pa-tients' score of Kamosfsky rose. Living quality of

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Anorexia, weight loss, strength loss, hypoal-buminemia and so on are main factors that affect

quality of life of advanced cancer patients. As a ma-jor therapy to conquer advanced cancer, chemother-apy is hindered because that myelosuppression andgastrointestinal tract toxicity caused by chemother-apy can reduce furtherly patient's quality of life.MFA is a kind of synthetic progestogen that canpromote albumin assimilation. It is indicated inclinical study in recent 10 years that large dose ofMFA can not only improve appetite, raise weight


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Table 1. Changes of appetite and weight in both groups

Changes of appetite Changes of weight

Group Cases Improvement ReductionNo Rate of

Improvement ReductionNo Rate of

change effectiveness change effective( cases) ( cases)

(cases) (%)( cases) (cases)

(cases) (%)

The

therapeutic 50 39 2 9 78.0 32 3 15 64.0group

Thecontrolled 48 5 31 12 10.4 6 3 39 12.5

group

Group CasesScores Scores no Increasing Increasing Rate of

reducing change 10 20 effectiveness( % )-

The therapeutic 50 4 17 18 11 58.0group

The controlled48 12 25 9 2 22.92

group

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therapeutic group improved more than controlgroup significantly (p < 0.01) .

To sum up, cachectic syndrome like anorexiarelated with cancer and toxicity caused bychemotherapy are important factors that affects liv-ing quality of advanced cancer patients. Comparedwith singly using enternal feeding, parenteral hy-peralimentation or other heteropathies that can im-prove advanced cancer patients' nutritional stateand toxicity caused by chemotherapy, MPA can notonly stimulate appetite, raise weight, promote al-bumin assimilation, raise strength and spiritualstate but also be characterized by convenient use,no pain and long term use. So that, MFA can notonly treat enzyme sensitive tumor but also improvequality of life of cancer patients during chemothera-py completely if it was accurately and reasonablytaken by intermediate and advanced cancer pa-tients. So it is an effective medicine worthy spread-

ing to improve quality of life advanced lung cancerpatients.

Correspondence to:Liping WangDepartment of OncologyFirst Affiliated Hospital

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Zhengzhou UniversityZhengzhou, Henan 450052, ChinaTelephone: 01186-0371-6516-5757Email:[email protected]

References1. Splinter TA. Cachexia and cancer, a clinician's view. Ann On-

coI1992;3:525-527.

2. Lu SL,Sheng MY. The effect of MFA on quality of life of can-

cer patients. Journal of Oncology 2001 ;7( 4) :239 - 40.

3.Jiang YR, Lan Xl, Yang GH, et al. Investigation of Medroxy

progesterone acetate used in advanced cancer patients. Journalof Treatment and Prevention of Tumour 2001 ;8(5) :556 -7.

4. Li J ,Li XQ, Li JB. et al. The effect of Fu Zheng Bao Zhen Soup

in quality of life of cancer patients undergoing chemotherapy orradiotherapy. Journal of Zhejiang Traditional Chinese MedicalCollege 2000;24(3) :23 - 5.

5. He XX, Xia YQ, Xiao JC, et al. Effective investigation of

Medroxyprogesterone acetate four-combined therapies in cancer

patients anorexia. Journal of Chinese Oncology Clinical and Re-covery 2000;7(5) :83 - 4.

6. Lelli G, Angelilli B, Giambiasi ME,et al. The anabolic effect of

high dose Medroxyprogesterone acetate in oncology. PhannacolRes Commun 1983; 15(6) :561 - 8.

7. Liu XY,Fang J, Li JT. Improved quality of life with Medrox-

yprogestrone acetate in lung cancer patients undergoingchemotherapy. Journal of Chinese Newdrug 1994;3(2) :25-7.

8.Eeller E, Tattersall M, Lunmley T, et al. Improved quality of

life with megestrol acetate in patients with endocrine in sensi-tive advanced cancer. J Ann Oncol 1997; 8 (3) : 277 - 83.


main source of Trichinella infection for humans inChina (Wang et al., 1998). The transmission ofT. sPiralis via household garbage is a main featureof the epidemiology of swine trichinellosis in China(Wang and Cui, 2001b) . Vaccination of swine a-gainst T. sPiralis could provide an alternative toprevent the risk of human infection. The develop-ment of vaccines capable of preventing swine frombecoming infected would thus make a substantialcontribution towards the ultimate goal of disease e-limination.

In order to control trichinellosis, vaccines havebeen developed based upon irradiated- or ultravio-let-attenuated infective first stage larvae (Agyei-Frempong & Catty, 1983; Nakayama et al.,1998), autoclaved larvae (Eissa et al., 2003),antigens from different life-cycle stages (Darwish etal. , 1996; Aucouturier et al. , 2001), and a syn-thetic peptide (Robinson et al. , 1995; McGuire etal. , 2002). In each case, high levels of specific an-tibodies against Trichinella were induced in exper-imental animals, but failed to protect these animalsfully against infection. In addition, the attenuatedvaccine is impractical for field use.

DNA vaccination has been used to deliver a va-riety of parasite antigens to both small and large an-imal species, and the protective efficacy of antibod-ies generated in response to DNA vaccines has beenshown in several challenge models (Donnelly et al: ,1997; Rothel et al., 1997; Zhang et al., 2001;

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Life ScienceJournal, 2 (1 ), 2005, Cui, et aI, Expression of the Antigenic Gene TspEl

Expression of the Antigenic Gene TspEl of Trichinellaspira Iis in Skin and Muscle of BALB/c Mice

Jing Cuil, Zhongquan Wangl, Hongwei Zhangl, Guoqiang Zhao2, Haiyan Weil, Huamin Hanl

1. Department of Parasitology, Zhengzhou University, Zhengzhou, Henan 450052, China

2. Departni'~nt,of Mciobiology and"Immunology ,Basic Medical College,. .Zhengzliou University, Zhengzhou, Henan 450052, China

Abstract: Trichinellosis is a serious zoonosis with a worldwid~ distribution and pork is still the most common sourceof human trichinellosis. Vaccines are needed to control this disea;e in §wine. The recombinant eukaryotic expressionplasmid, pcDNA3- T spE1, containing a gene encoding a 31 kDa an.\JgenQfrom T. spiralis was constructed. BALBIc mice were immunized with plasmid DNA vaccine by intramuscular injection and via gene-gun delivery. The tran-scriptional activity of the pcDNA3- TspE1 in skin and muscle at the site of inoculation was detected by RT-PCR us-ing gene specific primers. Protein expression from the TspE1 gene in ski!) and muscles at the site of inoculation wasdetected by immunohistochemistry and indirect fluorescencent antibody test (IFAT), respectively. The results indi-cated that the recombinant plasmid pcDNA3- TspE1 was successfully transcribed and expressed in skin and muscle atthe site of inoculation of mice. Thus, the plasmid encoding 31 kDa antigen may be of value for further developmentof a DNA vaccineagainst swine trichinellosis. [Life ScienceJournal. 2005;2(1) :30 - 36J (ISSN: 1097- 8135).

Keywords: Trichinella sPiralis; DNA vaccine; gene-gun delivery; expression;mice

1 Introduction

.

Trichinellosis, one of the most serioushelminthic zoonosis, is still considered to be endem-ic in many parts of the world. Human acquire thedisease by ingesting raw or insufficiently cookedmeat containing Trichinella larvae. Outbreaks oftrichinellosis have occurred in many areas aroundthe world over the past 20 years, even though theveterinary public health efforts have focused on thecontrol and eradication of the disease for more thana century. The global prevalence of trichinellosis isdifficult to evaluate, but it is estimated that asmany as 11 million people may be infected(Dupouy-Camet, 2000) . More than 10000 cases ofhuman trichinellosis were reported by the Interna-tional Commission on Trichinellosis from 1995 toJune 1997 and about 10000 porcine infections werereported by the Office International des Epizootiesin 1998 (Dupouy-Camet, 2000). Thus, trichinel-losis has been regarded as emerging or re-emergingdisease in both developed and developing regions(Murrell and Pozio, 2000). The disease is particu-larly problematic in some parts of China. Since thefirst patient with trichinellosis was recorded in Ti-bet in 1965 (Huang, 1965), there have been 548outbreaks reported from 12 provinces, autonomousregions or municipalities (P/AIM)of China during1966 -1999 (Wang and Cui, 200la). Pork is the

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Dumonteil et al. , 2003). DNA vaccination has theattraction of enabling antigen expression to remainin a eukaryotic cell, increasing the probability thatexpress~d antigen will be correctly glycosylated andgenerate conformation-specific antibodies.Trichinella antigens used as recombinant-proteinvaccines have been identified, and demonstrated ca-pable of eliciting host protective humoral immuneresponses (Arasu , et al., 1994 ; Sun, et al.,1994). In our previous study, the structural gene(TspEl) encoding 31 kDa antigen of T. spiral iswas cloned and expressed in the. prokaryotic ex-pressing vector, pGEMEX-l. High specific anti-body against the recombinant 31 kDa protein was i-dentified by Western blot, and Trichinella-specificantibody was induced in mice immunized with thefusion protein (Cui et al. , 2002). Production andanalysis of a DNA vaccine encoding the 31 kDaantigen of Trichinella spiralis in skin and muscleof BALB/c mice is described in this paper.

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'" 2.1 MiceMale BALBI c mice aged 4 - 6 weeks were ob-

tained from the Experimental Animal Center ofTongji Medical College, Huazhong University ofScience and Technology (Wuhan, China) and wereraised in plastic micro-isolator cages before and afterimmunization. Anti- Trichinella antibodies werenot detected by ELISA using secretory-excretory(ES) antigens of T. sPiral is muscle larvae in allmice before immunization.2.2 Parasite

T. spiral is used in this study was obtainedfrom a swine source in the Henan Province of Chinaand was maintained by serial passage in Sprague-Dawley (SD) rats every 6 - 8 months. Each ratwas orally infected with 500 T. spiralis larvae.2.3 Generation of anti-31 kDa fusion proteinpolyclonal sera

Anti-31 kDa fusion protein polyclonal mousesera, used as positive control sera in IFAT, wereobtained from BALB/c female mice injected in-traperitoneally on two occasions 2 weeks apart with60 fig (20 fig each injection) of 31 kDa fusion pro-tein in complete Freund's adjuvant. Sera were col-lected 28 days after the second immunization andstored at - 80t until use.2.4 Plasmid construction

The recombinant eukaryotic expression plas-mid pcDNA3-TspEl was constructed as previouslydescribed (Cui et al. , 2004). In brief, the larvaeof T. spiralis in skeletal muscles were collected byacid-pepsin digestion. The target gene encoding the

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31 kDa protein was prepared by RT -PCR from T.spiralis muscle larval RNA and cloned into thepUC18 vector. The positive clones were examinedfor the presence of a correct size insert by double di-gestion with BamH I and Hind III and single di-gestion with Eco RI, and also by PCR using genespecific primers. The target gene was sub-cloned in-to the eukaryotic expression vector pcDNA3 (Invit-rogen, CA, USA). The pcDNA3 vector, whichcontains the human cytomegalovirus (CMV) pro-moter and an ampicillin-resistance gene, was usedfor the DNA vaccination studies. After transform-ing Escherichia coli strain JM1O9, the recombinantclones were selected on LB plates containing 100fig/ml ampicillin overnight at 37t and plasmidDNA was extracted by the alkaline lysis method(Maniatis et al., 1982). The colonies containinginserts of the appropriate size in the right orienta-tion were identified by electrophoresis of PCR prod-ucts using gene specific primers. An 876 bp frag-ment was obtained after the recombinant plasmidpcDNA3-TspEl was digested by BamH I andHind III and as expected. Nucleotide sequencingwas carried out to confirm the authenticity of theinsert.

The recombinant plasmids were maintainedand propagated in E. coli JM109 cells. The endo-toxin-free plasmid DNA was purified from bacterialcells using a plasmid purification kit (Qiagen ChinaRepresentative Office, Shanghai, China). ThepcDNA3 plasmid without insert was also purifiedfor use as a control. The purified plasmids werestored at - 20t until use. .2. 5 Production of DNA coated gold beads for

immunizationUsing protocols developed for the'Hilos Gene

Gun System (Bio-Rad, CA, USA), pcDNA3-TspEl DNA and the control, pcDNA3 DNA, wereprecipitated onto gold beads (1.6 fim average diam-eter) and used to coat the inner surface of plastictubing. The tubing was cut into a half inch lengthand stored dry at 48t until required. The quantityof gold and DNA comprising each immunizing"shot" was adjusted to produce the 1 mg DNA/O. 5mg gold "shots" for use in the immunization.2.6 Immunization of mice

Male BALBI c mice, aged 4 - 6 weeks (10 perexperimental group), were anesthetized and immu-nized. For intramuscular vaccination, 50 fig of pureplasmid DNA (in 50 fil of PBS) were injected intothe left and right quadriceps of each leg, using a 1ml tuberculin syringe fitted with a 28 G needle;The control animals received only PBS or pcDNA3.The mice were sacrificed 2 weeks after the primaryintramuscular inoculation of DNA vaccine or pcD-

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ed with 10% nonnal goat serum at room tempera-ture for 5 min to block non-specific binding. Thesections were then incubated with sera from mice

infected with T. sPiralis (diluted 1: 50 in PBS) ornonnal mouse serum at 4'C overnight. Sectionwere washed in O. 01 M PBS three times and ex-

posed to biotinylated goat anti-mouse IgG, followedby treatment with the SABC and stained with di-

aminobenzidine (DAB) with 0.15% hydrogen per-oxide. Counterstaining was perfonned with haema-toxylin.

2.9 Indirect fluorescencent antibody test (IF AT)The expression of the TspEl antigens in mus-

cles at the site of inoculation in mice was detected

by IFAT. The mice were sacrificed 2 weeks afterthe initial intramuscular inoculation of DNA vaccine

and the muscles at the site of inoculation were sepa"rated and stored at - SO°C. Then, 4 pm thick offrozen sections were cut, collected on gelatinizedslides, and fixed with cold acetone for 10 min to

prevent detachment of the sections during the fol-lowing procedures. IF AT with frozen sections ofmuscles was carried out as previously described(Cui et al., 1999). Briefly, the frozen sectionswere incubated with 1 : 10 dilution of sera from

mice immunized with recombinant fusion protein orinfected with T. spiral is at 37'C for 30 min. Afterwashing with PBS, the sections were incubatedwith 1 : 16 dilution of goat anti-mouse IgG conju-gated to fluorescein isothiocyanate (FITC) at 3TCfor 30 min. Sera from nonnal mice were used as

negative control. Slides were examined under fluo-rescent microscope (Olympus, Tokyo, Japan).When a yellow green fluorescence staining appearedon the section, the reaction was defined as IF ATpositive.

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NA3 alone. For vaccination by gene-gun delivery,mice were anesthetized, their abdomens shaved,wiped with damp gauze and" shot" with the GeneGun using 300 psi of helium gas. DNA vaccine dos-es of 3 fJ-g (three shots) per mouse were used forepidennal inoculation. Controls mice were immu-nized similarly with pcDNA3 alone. The mice weresacrificed 4 h, S h,24 h,3 d,5 d and 7 d after the

initial inoculation of DNA vaccine or pcDNA3 aloneby gene-gun delivery.2. 7 Detection of transcriptional activity of the

pcDNA3- TspEl in skin and musclesTo detennine the duration and site of antigen

presentation to the host, RNA was extracted fromthe skin and muscles of immunized mice and sub-

jected to RT-PCR to detect evidence of transcrip-tion of the parasite DNA in transfected skin andmuscle cells. Skin and muscles at the site of inocu-lation were excised at various intervals after DNA

immunization, frozen on dry ice and stored at- SO'C. The tissuewas pulverizedwith mortar andpestle into powder and total messenger RNA wasextracted using a commercial kit (Invitrogen, CA,USA). The extracted mRNA was treated withDNase I (RNase-free, Promega) to prevent PCRpriming from plasmid a genomic DNA for 30 min at3TC and subjected to AMV reverse transcriptionreaction (Promega, WA, USA) for 60 min at42°C. The reverse transcribed material was PCR

amplified with primers specific to the encoding re-gion of 31 kDa protein (using the thennal cycleprotocol: 2 min 94°C; 35 cycles of 94 °c for 30 sec,55°C for 30 sec and 72°C for 40 sec, and tenninat-ing with a 5 min extension step of 72°C). The re-sultant RT-PCR products were examined in 1.5 %agarose electrophoresis gels.2. 8 Immunohistochemical staining

The expression of TspEl gene in skin at thesite of inoculation was assayed by immunohistoche-mical staining. The abdominal skin of vaccinatedmice was excised and embedded in paraffin and 5fJ-mthickness of sections were prepared. After de.paraffinization, the sections were processed to un-mask the antigens by conventional microwave ovenheating in 10 mg/ml citric acid buffer (pH 6. 0)and subsequent detergent treatment using poly-oxyethylene sorbitan monolaurate in PBS for 30min. In order to observe the expression of TspElantigen in epidermis, immunohistochemical stain-ing of 5 fJ-m paraffin sections was perfonned withthe Strept-Avidin- Biotin-Peroxidase complex( SABC) method (DGBio Co, Bej ing , China).Briefly, 5 fJ-m paraffin sections were treated with3 % hydrogen peroxide at room temperature for 10min to remove endogenous peroxidase and incubat-


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3.1 Detection of mRNA for TspEl in skin andmuscle of DNA immunized mice

RT-PCR products were obtained only from theskin samples of the pcDNA3- TspEl immunizedmice at S h post-immunization (Figure 1), but notobtained at 4 h, 24 hand 72 h post-immunization.As expected, there was no TspEl transcriptionalactivity in the skin samples from the control miceimmunized with pcDNA3. These results demon-strate that detectable transcriptional activity ofDNA transfected skin cells is very transient andconfined to the site of immunization. mRNA was

extracted from muscle of pcDNA3- TspEl immu-nized mice 2 weeks following injection and was ana-lyzed by RT-PCR using 31 kDa antigen-specificprimers. The transcriptional activity of TspEl gene

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was clearly visible in muscle tissue from mice inject-ed with pcDNA3-TspE1 but not in those mice in-jected with control plasmid (Figure 2).

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Figure 1. RT- PCR analysis of mRNA in skin of BALBI c mice im-munized with pcDNA3-TspEl and pcDNA3 plasmid. Mice were

immunized with 3 fig of indicated plasmid DNA by gene-gun de-livery. Skin at the site of inoculation was excised 8 h after the ini-tial inoculation of DNA vaccine or pcDNA3 alone. The mRNAwas isolated from the muscles and used for RT-PCR.

Lane 1: pcDNA3- TspEl ; Lane 2: pcDNA3; Lane M: high molecu-

lar weight biomarker.

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Figure 2. RT-PCR assay on RNA extracted from muscle twoweeks after immunization with pcDNA3-TspEl and pcDNA3plasmid (1. 5 % agarose gel electrophoresis). Lane 1: DNAmarker; Lane 2: RNA extracted from quadriceps muscle after im-munization with pcDNA3-TspEl; Lane 3: RNA extracted fromquadriceps muscle after immunization with pcDNA3.


3.2 Detection of TspEl protein in skin at the siteof inoculation

The results of immunohistochemical stainingshowed that specific brown round particles wereseen among cells and in the cytoplasm of epidermialcells in all of the mice immunized with pcDNA3-TspE1 at 8 h post-immunization, but not in themice immunized with pcDNA3 (Figure 3). Sec-tions of T. sPiralis muscle larvae as positive controlwere reacted with sera from the infected mice. Nodeep brown particles were observed when sectionsfrom the immunized and the infected mice were re-acted with PBS or normal mouse sera (not showedin the figure).3 .3 Expression protein in muscle at inoculation

sites of miceThe results of IFAT showed that the frozen

section of inoculation site of mouse muscle 2 weeksafter the initial intramuscular inoculation of pcD-NA3-TspE1 reacted with sera from mice immu-nized with recombinant fusion protein or infectedwith T. sPiralis. The fluorescence did not appearon the frozen section of inoculation site of mouseskin with only empty plasmid pcDNA3 (Figure 4).

4 Discussion

Both conventional protein vaccines and DNAvaccines (normally constituted of a naked DNAplasmid) elicit antibody responses, however, DNAvaccines have the additional advantage of stimulat-ing cytotoxic T cells because the host is producingthe antigenic protein intracellularly, thereby fa"cili-tating presentation of the antigen in the context ofMHC class I molecules. Development of cellular im-munity is important in fighting intracellularpathogens, giving DNA vaccines a clear advantageover protein vaccines. Another advantage of a DNAvaccine is its ease of administration and production.DNA is easily produced in large quantities withgreat purity, minimizing the risk of vaccine con-tamination with potential pathogens (McDonnelland Askari, 199Q). Moreover, DNA can be readilyintroduced into tissues by DNA-coated microprojec-tiles through particle-mediated epidermal delivery(PMED), which facilitates easy epidermal admin-istration and avoids the use of needles (Williams etaI, 1991). Additionally, DNA vaccines may resultin expressed antigens that resemble native antigensmore closely than do antigens in conventional vac-cines because manufacturing techniques can alterepitopes and reduce antigenicity. DNA vaccinesmay also be safer than some live attenuated vac-cines, particularly in immunocompromised hosts.


Life ScienceJournal, 2 ( 1 ), 2005, Cui, et al ,Expression of the Antigenic Gene TspEl

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c. Negative control of pcDNA3 expressed in skin tissue of mouse at 8 h after gene-gun injection (400 x ) .

d. Positive control of immunohistoqhemicaI staining of skeletal muscle of mouse infected with T. spiralis (400 x ), arrow showing thelarva stained as brown.

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Figure 4. IFAT showing the antigenic protein expression of TspEl gene in muscles 2 weeks after the initial intramuscular inoculation of

recombinant plasmid pcDNA3- T spEl.

a. Section of muscle carrying pcDNA3- TspEl reacted with sera from mice immunized with the recombinant fusion protein (200 x ) .b. Section of muscle carrying pcDNA3-TspEl reacted with sera from mice infected with T. sPiralis (200 x).

c. Section of muscle carrying empty plasmid pcDNA3 reacted with sera from mice immunized with the recombinant fusion protein (100x)

d. Section of muscle carrying empty plasmid pcDNA3 reacted with sera from mice infected T. sPiralis (200 x ) .

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Furthennore, several antigen genes could be in-cluded on one plasmid, reducing the total numberof vaccinations that must be administered. Initialstudies suggest that DNA vaccines can be adminis-tered safely and without overt toxicity (Tacket etal. ,1999; Roy et al. ,2001).

DNA immunization represents a novel methodfor generating the in situ expression of vaccine anti-gens and is now a widely reported means of gener-ating immune responses. The administration ofplasmid DNA via intramuscular, intradennal orgene gun delivery routes has been shown to stimu-late T helper cells, cytotoxic T cells (CTL) andantibodies specific to the plasmid encoded antigen(Donnelly et al., 1997). The protective role forantigen-specific antibodies induced by genetic im-munization has been manifestly demonstrated in avariety of challenge models against parasitic infec-tion (Zhang et al, 2001; Dumonteil et al. , 2003).However, up to date, DNA vaccines againstTrichinella infection have not been reported in theliterature.

We selected DNA vaccine as a strategy to pre-vent swine trichinellosis. The recombinant eukary-otic expression plasmid pcDNA3-TspE1 containedthe gene encoding a 31 kDa antigen of T. spiraliswas constructed. BALB/c mice were immunizedwith plasmid DNA vaccine by intramuscular injec-tion and via gene-gun delivery. The transcriptionalactivity of the pcDNA3-TspE1 in skin and musclesat the site of inoculation was detected by RT-PCRusing gene specific primers. The expression ofTspE1 gene in skin and muscles at the site of inocu-lation was confinned by immunohistochemistry andiFAT, respectively. These results showed that therecombinant plasmid pcDNA3-TspE1 was success-fully transcribed and expressed in skin and musclesat the site of inoculation of mice. Thus, the plas-mid encoding 31 kDa antigen may be of value forfurther development of DNA vaccine against chal-lenge infection with T. spiralis.

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Acknowledgments

This study was supported by a grant(20023100014) from Department of Education,and grant (0524410037) from Department of Sci-ence and Technology, Henan Province, China.

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Correspondence to:Jing CuiDepartment of ParasitologyMedical College, Zhengzhou UniversityZhengzhou, Henan 450052, ChinaFax: 01186-371-6699-7182

http://www.sCiencepub.org

Email:[email protected]

References

1.Agyei-Frempong M, Catty D. The measurement of anti-gens released by radiation-attenuated Trichinella spiralislarvae. Parasite Immunol1983 ;5(3) :289 - 303.

2. Arasu P, Ellis LA, Iglesias R, Ubeira FM, Appleton JA.Molecular analysis of antigens targeted by protective anti-l:x:xIiesin rapid expulsion of Trichinella spiralis. MolBiochem Parasitol1994: 65(2) :201 -11.

3 . Aucouturier J, Deville S, Perret C , Vallee I, Boireau P.Assessment of efficacy and safety of various adjuvant for-mulations with a total soluble extract of Trichinella spi-

ralis. Parasite 2001: 8 (2 Supp!) :S126 - S132.4.Cui], Wang ZQ, Han HM, Wei HY, Zhang HW, Li

YL. Expression of DNA vaccine against Trichinella spi-ralis in mammalian cells. Chin J Parasitol Parasit Dis2004; 22(5): 266 -70.

5. Cui J, Wang ZQ, Wang HZ, Zhao 00, Zhang HW.Cloning and expression of the antigen structural geneTspEl of Trichinella spiralis pre-encysted larvae. Chin JParasitol Parasit Dis 2002:20(5) :278 - 80 (in Chinese).

6.Cui], Wang ZQ, Zhu W, Zhang RG. Seroepidemio-logicl study of Trichinella sPiralis infection in centralChina. Helminthologia 1999:36(4) :235 - 9.

7. Darwish RA, Sanad MM, Youssef SM. Immunization a-gainst Trichinella spiralis using antigens from differentlife-cycle stages experimental study in mice. J Egypt SacParasitol1996 ;26( 1): 19 - 26.

8. Donnelly]], Ulmer JB, Shiver JW, Liu MA. DNA vac-cines. Annu Rev Immunol 1997: 15 : 617 - 48.

9. Dumonteil E, Maria Jesus RS, Javier EO, Maria delRosario G. M. DNA vaccines induce partial protection a-gainst Leishmania mexicana . Vaccine 2003; 21 ( 17 -18) :2161 - 8.

10. Dupouy-Camet J. Trichinellosis: a worldwide zoonosis. VetParasitoI2000;93(3-4):191-200. .

11. Eissa MM, el-Azzouni MZ, Baddour NM, Boulos LM.

Vaccination trial against experimental trichrinellosis usingautoclaved Trichinella spiralis larvae vaccine (A TSL V) .J EgyptSacParasitol2003;33(1) :219- 28.

12. Huang FC. Report of one case of human trichinellosis.Chin J Intern Med 1965: 13(4) :392.

13. McDonnellWM, Askari FK. DNA vaccines. N Engl JMed 1996;334(5) :42 - 5.

14. McGuire C, Chan WC, Wakelin D. Nasal immunization

with homogenate and peptide antigens induces protectiveimmunity against Trichinella spiralis. Infect Immun2002;70(12):7149-52.

15. Murrell KD, Pozio E. Trichinellosis: the zoonosis that

won't go quietly. Int J Parasitol 2000: 30 (12 - 13):1339 - 49.

16. Nakayama H, Inaba T, Nargis M, Chisty M, Ito M,Kamiya H. Immunization of laboratory animals with ul-traviolet-attenuated larvae against homologous challengeinfection with Trichinella britovi. Southeast Asian J.

Trop Med Public Health 1998: 29(3): 563 - 6.17. Robinson K, Bellaby T, Chan WC, Wakelin D. High

levels of protection induced by a 40-mer synthetic peptidevaccine against the intestinal nematode parasiteTrichinella spiralis. Immunology 1995:86(4) :495 - 8.

18. Rothel JS, Boyle DB, Both GW, Pye AD, Waterkeyn

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JLife Science Journal, 2 (1), 2005, Cui, et al ,Expression of the Antigenic Gene TspEl

livered by a gene delivery device. Vaccine 1999; 17(22) :2826- 9.

23 . Wang ZQ, Cui J. Epidemiology of human trichinellosis inChina during 1964 - 1999. Parasite 2001a; 8 (2 Supp!):S63 - S66.

24 . Wang ZQ, Cui J. Epidemiology of swine trichinellosis inChina. Parasite200lb;8 (2 Suppl):S67-S70. .

25. Wang ZQ, Cui J, Li HS, Jin XX, Wu F, Xue CG, MaoFR. Some observations on trichinosis in China.

Helminthologia1998;35(1):27 - 9.26. Williams RS, Johnston SA, Riedy M, DeVit MJ, McEl-

ligott SG, Sanford JC. Introduction of foreign genes intotissues of living mice by DNA-coated microprojectiles.ProcNatlAcadSciUSA1991;88(7) :2726- 30.

27. Zhang Y, Taylor MG, Johansen MV, Bickle QD. Vac-cination of mice with a cocktail DNA vaccine induces a

Thl- type immune response and partial protection againstSchistosoma japonicum infection. Vaccine 2001 ;20(5 -6) : 724 - 30.

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JG, Wood PR, Lightowlers MW. Sequential nucleic acidand recombinant adenovirus vaccination induces host-pro-tective immune responses against Taenia ovis infection insheep. Parasite ImmunoI1997;19(5) :221-7.

19. Roy MJ, Wu MS, Barr L]. Induction of antigen-specificCDS + T cells, T helper cells, and protective levels ofantibody in humans by particle-mediated administration ofa hepatitis B virus DNA vaccine. Vaccine 2000; 19(7-8):764-78.

20. Sambrook J, Russell DW. Molecular Cloning: A Labora-tory Manual, 3rd ed. NY: Cold Spring Harbour Labora-tory. 2001: 1. 31 - 1. 42.

21. Sun S, Xu W, He N, Sugane K. An antigenic recombi-nant fusion protein from Trichinella spiralis induces aprotective response in BALB/c mice. J Helmintholl994;68(1):89-91.

22. Tacket CO, Roy MJ, Widera G, Swain WF, Broome S,Edelman R. Phase I safety and immune response studiesof a DNA vaccine encoding hepatitis B surface antigen de-

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Life Science Journal, 2 (1), 2005, Wang, et al, Endocrine Disorder by Smoking Inhalation

The Endocrine Disorder by Smoking Inhalation

SulinWangl, XiufangChen2, Shen Cherni

1. Basic Medical Science Research Center, Zhengzhou University, Zhengzhou, Henan 450052, China

2. Department of Medicine, Henan Xiandai Medical Research Institute, Zhengzhou, Henan 450052, China;

cherng@msu. edu

Abstract: The smoking inhalation reduced the secretion levels of blood serum, testosterone (T), luteinizing hor-mone (LH), and follicle stimulating hormone (FSH) of the rats. Prolactin level was reduced under the long-term

regular smoking inhalation for female rats. The histology analysis of the testis of the male rats was performed to ex-am the degree of lesion by smoking. Conclusively results revealed that smoking inhalation causes the endocrine dis-order. It took long time to recover from endocrine disorder if stopped smoking. [Life Science Journal. 2005 ;2( 1):37-39J (ISSN: 1097-8135).

Keywords: endocrine disorder; smoking; smoking inhalation

1 Introduction

Smoking is one of the important factors to af-fect the human health. The smoking may cause the

organ system acting abnormally. Chemicals insmoking inhalation causing the endocrine disorderof the human body have been discussed for twodecades. The main purpose of this report is to con-firm the levels of endocrine disorder by smoking.

2 Material and Method

36-male and 36-female Wistar rats were divid-ed into three types of group as smoking inhalation,control and natural recovery group respectively.Each group has 12 rats and the weight of them wasbetween 180 g to 220 g. All the animals were pro-vided by Henan Experimental Animal Center(Zhengzhou, China). Hongxi cigarette, made inHenan Ruzhou Cigarette Manufacture Company,and the home-made inhalation box, sized as 1740mmX 1100 mm X 1500 mm with two 2 mm X 3mm air holes on the two vertex of topside diagonalline were used to provide smoking inhalation. Thenicotine amount of Hongxi cigarette is 1. 1 mg.The amount of tar is 17 mg per 84-mm-Iongcigarette as well. The experimental test kits oftestosterone (T), luteinizing hormone (LH), folli-cle stimulating hormone (FSH) and prolactin wereprovided by Tianjin Depu Biomedical TechniqueCompany. The SM-696 )'-measure machine wasmade in Shanghai Nuclear Research Institute, Ri-huan Equipment Manufactory Company. Six ratswere kept in one cage. Two cages designed to fitinto the smoking inhalation box. Four oieces of

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cigarette were tightened as a bundle. A piece ofbundle cigarettes were hanged and burned forsmoking in the cage. Changing the bundle-cigaretteevery 15 minutes. It was scheduled to burn up 32pieces of cigarette in 50 minutes, twice a day forfirst 38 days. And then once a day for another 38days for 76 days, we sacrificed the animals bycoeliac-injection of ketamine for checking the levelsof T, LH, FSH and prolactin for female and thevisceral coefficients of the testis arid epididymis formale. The data were analyzed by SPSS 10.0 pro-gram with u test (Ridit analysis), student t testand expressed the results as x + s (standard er-ror). Visceral coefficient is defined as the ratio ofthe weight of organ to the body. The histologystudy was followed up by the standard procedures.

3 Results

Table 1 and Table 2 have shown the level ofT,LH,FSH and prolactin in control, natural recov-eryand smoking inhalation group. The natural re-cover group did not show any significant differencein comparison with the control (P > O. 05) . Othershave shown the significant difference. The data ofvisceral coefficients of testis and epididymis formale rats (x:t s ) presented in Table 3. Figure 1depicted the normal histology structure of testis forrats control group. Spermatogonium cells, Stertolicells, primary spermatocytes and secondary sper-matocyte all could be seen in seminiferous tubles.Sperms were in tubli recti and Laydig cells were al-so seen in rete testis. Minor lesions were found inFigure 2. Histoplasmosis and less sperms were de-picted for 50% rats in smoking inhalation group.In Figure 3, seminiferous tubles presented to be

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Table2. In comparisonto the level of FSHand Prolactin forfemale rats ex :t s)

Type of the group. n FSH (lUlL) Prolactin (p. gIL)Control 12 1. 45 :t O.34 2.76 :t 1. 08

Smoking inhalation 12 1. 02 :t 0.31 * 3. 13 :t 1. 16

Natural recovering 12 1.06 :t O.33 * 3. 08 :t 1.21

Note: in comparison with control statistically, * P<0.05

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thinner than nonnal, much less primary spennato-cytes and secondary spennatocytes were found.Spenn and spenn cells disappeared. However,Spennatogonium cells still could be seen for 25 %rats in smoking inhalation group. Necrosis of sper-matocyte was seen clearly. In Figure 4, all cellsnecrosis was seen clearly in serious lesions for 25 %rats in smoking inhalation group. For natural re-covery group, in comparison with smoking inhala-tion group, we were not able to find significant dif-ference. All the lesions were very similar(p >0.05).Table 1. In comparisonto the level of T and LH for femalerats (x:t s)Type of the group n T (n molIL) levelLH (lUlL) levelControl 12 4.86:t 2. 29 1. 74:t O.66Smokinginhalation12 2.1:t 1.12 * * 1.06:t O.41*

Natural recovering 12 2.3 :t 0.97 * * 1. 18 :t 0.36'Note: in comparison with control statistically, * P<0.05,

* *P<O.O1

Figure 1. The nonrnl testis histolqsy microsropic picture (HE x 400)

Figure 3. The serond degree lesion of the testis of the male rats (HE x200)

4 Discussion

. The effect of the smoking inhalation to human re-productive endocrine, special for the male becomes moreand more concern to the most of smokers. The experi-mental data revealed that the quality and quantity of the

sperms being influenced by smoking[2.3]. This report


Table 3. The visceral coefficients of testis and epididymis formale rats (x:t s)

Type of the group n Testis (g/kg) Epididymis (g/kg)

Control 129.78:t1.29 4.45:t0.33

Smokinginhalation12 8. 59:t O.98* 4.05 :tO.26* *

Natural recovering 12 8.66 :t 0.79* 4. 10 :t 0.27 *

Note :in comparison with control statistically, * P < O.05 ,* * P<O.O1

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Figure 2. Minor lesions of the testis of the male rat caused by smokinginhalation (HE x 400)

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Figure 4. The serious lesion of testis of the male rat under smoking in-halation (HE x 200)

also revealed that the smoking could cause the differentdegrees of lesion on testis of the male rats, which isconsistent to the research results all around the

world[4l. The cigarette smoking includes about 400different chemicals that may affect the human or-

gan[5,6]. The mechanism of the lesion occurred in testis

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tissue may be originated by the long tenn increasing of

anti-oxidize~P] and the level of plasma endothelin-l [8],which may cause the vas:xx:>nstrictionto insufficientlyprovide the blood to the organ. llis may be the reasonthat smoking can cause the cell shrinkage of the testis.By C£erre, plasma endothelin-l is the chemical to causemast vas:xx:>nstriction.Plasma endothelin-l causes vaso-

mnstriction in organs including testis of the male rat.Therefore, the supply of the blood may be insufficientfor the function of testis.

5 Conclusion

llis re{X)rtpresents the result of decreasing I:xxlylevel T, which is mnsistence with the research result

that Wei did in the year of 2000[9,10]. The synthesis oftestosterone is in the endoplasmic reticultnn (ER) of in-terstitial cell and mitochondria. In the mean time, thesynthesis of testosterone is alSJ involved hY{X)thalamic-

pituitary unit. Zhou[11] suggested Hffi may inhibit the

secretion of testosterone and was mnfinned by our ex-

periments[ 12]. The smoking may alSJ affect the cruu-ac-teristics of Leydig cells, which indirectly affect the se-cretion of testosterone and caused the decrease of sennn

mncentration. The analysis of the data from natural re-mvery group revealed the existence of the mechanismfor remvery if smoking is stopped in the long run.However, the detail mechanism is still not clear and theremvery effect was slow in mmpariSJn with the damageof the organs. The further research will be focused onthe mechanism of the remvering process of the stoppedsmoking.

CoITespondence to:Shen CherngIRpartment of Medicine


Henan Xiandai MedicalResearch InstituteZhengzhou, Henan 450052, ChinaTelephone: 517-349-2362 (Michigan, USA)Email:cherng@msu. eclu .

References1. Rajpurkar A, Li HK, Dhabuwala CB. MorplTInetric analy-

sis of rat testis following chronic expcsure to cigarette smoke.

Journal of Environmental Patholcgy, Toxicolcgy and Oncolo-

gy(JEP1D) 2000;19(4):363-8.2. :k;!;'i: , ~.lL ~. P1HmXf}JH11::m: fllij] a9~ nlol. 9=t00~

~'*~;!; 2005;5(2):242-3.3. 5!E~~,~1k~ ,5Ui~,~. iQ,tffllXf~f£frH1U!J::l:a9~

nloJbHtl:ktt1iJf~. 9=t$~f4,,¥:,2002;8(1) :35 ~7.4. Guven CM, Can B, Ergun A, et aI. Ultrastructural effects of

cigarette smoke on rat testis. European Urolcgy 1999; 36:645-9.

5. QUa SE, Xu B, Ong CN, et aI. Effect of cadmium andcigarette smoking on human semen quality. Int J Ferti!Menopausal Stud 1994; 39 :292 - 9.

6. Vine MF,Magolin BH,Morriffin HI,et aI. Cigarette smokingand sperm density: a metaanaloysis. Fertil Sterill994; 61: 35-~. .

7. Rajpurkar A, Dhabuwala CB, Yang J, et aI. Chronic cigarettesmoking induces an oxidant-antioxidant imbalance in thetestis. Journal of Envirorunental Patholcgy, Toxicolcgy andOncolcgy(JEP1D) 2000; 19(4) :369- 73.

8. Goerre S,Staehli C,Shaw S,et aI. Effect of cigarette smokingand nicotine on plasma endothelin-lleVe!s. J Cardiovase Phar-macolI995;26:S236-S238.

9.~W*'I, .fflj1::~, Ifftf,~. P&fflIxt~'l1frfrft~~,frfTJj]~~]j'd~~IPJ~nloJa91iJf~.9=t00~,f4,*~;!; 2000;14(4):237- 9.

10. Tarnimi R, Mucci LA, Spana; E, et aI. Testa>teroneandoestradiol in relation to tobacco smoking, body I)1aSS index,

energy consumption and medicine intake among adult men.

European Journal of Cancer Precention 2001; 10:275 - 80.11. .fflj~ , ~WT JL, ~ Jj 1'- , ~. I*J &: ~xt* M. fiij J9jJlIJ }Jf1HUliiJ

1::Ji\ta9~nloJ.9=t00~,f4,*~~ ,2001; 15(3): 160- 2.12. I~~. *~;fIJ .lIJkffllxtME'I1/J\M.1::m:I*J7H1HU1Ea9~

nlol.~ffl*~,*m(I2'f,*JI!i) 2002;37(2):164-7.

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Life Science Journal, 2 (1), 2005, Zhang, Physwwgically Required Selectivity Gxfficients of Potentiometric Sensors

Study of Physiologically Required Selectivity Coefficients

of Potentiometric Sensors in Clinical Assays

26246 FieldstoneDrive, Novi, MI 48108, USA;[email protected]

Abstract: Required selectivity mefficients of ion-selective electrodes (ISEs) in physiological analysis are studied based on the

ronventional Nirolski- Eisenman equation and a newly proposed ronsistent approach with the worst allowable error (1 % ) .The physiologically required selectivity mefficients from both methods are rompared in this work. The revised ronsistent

approach gives out a more reasonable description of selectivity even in case of the unequally charged primary and interfering

ions. An Mi+ -selective electrode using ionophore E1H 5504 is tested to measure the selectivity of free Mi+ against the

interfering ions in physiological levels. Obviously the required selectivity mefficients derived from the ronsistent approach

shows the more accurate description of selectivity than the ronventional one. For evaluating the applicability of ISEs in

physiological solution, the ronsistent required selectivity mefficients provide the re<L'iOnablecriteria especially regarding the

primaryand interferingionsof differentcharges. [Life ScienceJournal. 2005;2(0 :40 - 45J (ISSN: 1097- 8135).

Keywords: selectivity mefficients; ion selective electrodes; magnesium; physiological assay; ETH 5504

1 Introduction

~I

Potentiometric ion-selective electrodes (ISEs)based on neutral inonphore are widely used in clinical

assays and abo integrated in diagnostic analyzers[1-3].The selectivity pattern of any ion-selective electrode isclearly the most important character that often reflects

whether a sensor may be reliably employed in analysingthe target electrolyte over other coexisting interferingions. One important application of ISE is detectingelectrolyte ion activities in whole blood or serum in

which a small mncentration range of electrolyte ex-tremely mandates the measuring accuracy. The ISEshould achieve a sufficient selectivity pattern of the pri-mary ion over coexisting interfering ions (semndaryions) to meet the physiological requirement of worst al-

lowable error of 1 % [4]. Conventionally, the requiredselectivity coefficients for physiological analysis are de-rived from the well-known semi-empirical Nimlsky-Eisenman (N-E) equation[5,6]:

E - E o RT I ( K jX>tz.lz.)- i + F n ai + ij aj' }z.z

where E is the measured electromotive force (EMF) ofthe electrode in the sanrple, all mnstant potential mn-

tributions are included in E? ; K?j\s the selectivity coef-ficient; Zi and Zj are the charges of primary ion I and

interfering ion ] ; ai and aj are the sanrple activities of Iand]; and R, T and F have their usual meanings. Itis well known that the selectivity coefficient is, in theo-ry, a mnstant parameter for a particular electrode, irre-

spective of the charges of primary and interfering ionssince K?jt is a function of EO values respectively for two


(1)

ions of interest.

Although the N-E equation is, with some excep-

tions[7,8], accurate for ions of the sanre charge, the se-lectivity coefficient of ISEs derived from this equationmay lead to an inmnsistent description for unequallycharged primary ion and interfering ion. For instance,taking primary ion as interfering ion and vice versa does

not give the sanre predicted electrcrle potential[9]. Infact, when the monovalent and divalent ions mnsidered

as primary and interfering ions, this discrepancy canamount to 8 mV or more than 30 % activity variation.Such discrepancy of selectivity is a serious limitation of

N-E equation that may lead to wrong practical predic-tions. A lot of discussions on this problem have beenmnducted and many new modification approaches of N-

E equation have also been proposed[8,1O,1l]. In 1998, a

mnsistent fonnalism was proposed by Zhang et al[9]with modifying the mnventional N-E equation with in-

troduction of mnsistent selectivity coefficient KijfJS

E = E? + W'ln( aillz; + KiJrcaYz)) (2)

where KijfJSis defined as mnsistent selectivity coefficientand

KeafJS = (Kl!'ts ) lIz;ZJ ZJ

With this modification, the selectivity coefficient KijfJSis mmpletely independent of the charge number of theions involved.

In measuring ion activity in intercellular fluid sanr-pIes, ion-seleCtive electrode should specifically respondto the activity of target ion with a sufficient discrimina-tion over the CD-existinginterfering ions. The minimum

selectivity requirement of ISEs determines the applica-

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Life Science Journal, 2 ( 1 ), 2005, Zhang, Physiologimlly Required Selectivity Gefficients of Potentiometric Sensors

~ bility of certain ISEs and the accuracy of assays. Thephysiologicallyrequiredselectivitymefficientsare calcu-lated assuming an allowableerror of 1% (0. 01) in theworst caseof a maximumquantity of interferingion anda minimum quantity of primary ion within physiologicalrange.

Traditionally, the required selectivity mefficientsare calculatedbasedon the a:mventionalNimlsky-Eisen-man equation, assuming an allowable error of 1%(0. 01) in the worst caseof a maximum quantity of in-terfering ion and a minimum quantity of primary ionwithin the physiologicalrange by the following equa-tion[12]:

~lL

...

(..

...

C'"l10.

r..

l (Krt)r equiml~ 0.01 . ai,/min

. )z. za.' }

} ,rnax10. or

..II,.

log(Kr)requim!~ log(O.01 . a~,~)a" }} ,rnax

~ When electrode is calibrated by physiological back-ground S)lutions, the variationsof interfering ion activi-ty around its medium value need to be mnsidered. Thedifference between the activity of the calibrator aj ,cal

d th unkn 1.

(z./z. z./z

)an e own S) utIon aj' aj:d. - aj' j , mn-tributes to the actual interference. Then, Eq. (5) canbe then changed to:

..

IL

l,'-LIl a. ..,nun

log(K\~?)requirOO~ log I 0.01. z/z z/z I (6)aj:d. - aj' }

Obviously, the required selectivity mefficients in Eq.(5) and (6) are inaccurate in case of unequal charge

numbers of the mncemed ions (Zi oF Zj ), which is o-riginated from the discrepancy of the mnventional N-Eequation.

In order to get the mnsistent selectivity criteria forphysiological assay with lSEs, the mnsisterit required

selectivity mefficients are derived from the newly modi-fied N- E equation for a given allowed relative error,

1 % (0. 01), in the measurement of physiological S)lu-tion[2] :

[

lIz.

]

a. ,',nun

log(Kij)requiml ~ log 0.01 . 1kaj,r.rlJx

..

\.I..,

lI.

L

lI.......

~L...

... With the physiological background calibration, the mn-sistent required selectivity mefficient is described as:

liz,

jlog(Kij)requim!~logl 0.01' lIZai'mh,lIz (8)

a } -a. }} ,rnax }

It is shown that Eq. (7) and (8) are obviouslydifferentfrom Eq. (5) and Eq. (6). For primary and

interfering ions of different charges (Zi oF Zj ), the mn-

sistent required selectivity mefficients log ( K\jt) requirOOgives out the reliable cri~eria to determine the applicabil-

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ity of lSEs, which gets rid of the discrepancy from the

mnventional N-E equation. In this work, an Mi+ -se-

lective electrode with iononphore EIH 5504[13] wasused to check the selectivity mefficient in physiologicalmeasurements.

(4)

2 Experiments

2.1 ReagentsAll chemicalswere of analytical reagent grade or

higher purity. The followingchemicalswere purchasedfrom Fluka Chemie AG, Buchs, Switzerland: MgClz,eao2, KO, NaO, KTpOPB (potassium tetrakis(4-chlorophenyl)bonite), PVC (poly( vinyl chloride),high molecular weight) and tetrahydrofuran (1HF,distilled prior to use). The Mi+ -selective ionophoreEIH 5504 (1, 3, 5-tris [ 10-(methyl-7, 9-dioxo-6, 10-diazadecyl)benzene]) and the plasticizer EIH 8045(12-( 4-ethylphenyl)dodecyl-2-nitrophenyl-ether) weresynthesizedas describedin['Z,14].Ibubly distilled waterwas used throughout. .

2.2 Membranepreparationand EMF measurementThe PVC-basedMi + -ISE membranewas pre-

pared as describedelsewhere[13,15]using 1 wt% Mi+ -selective ionophore; 155 mol% (relative to theionophore) lipophilicanionic sites KTpOPB; 65 wt%plasticizer, EIH 8045; and 33 wt % PVc. Theworking electrode was assembledby cutting disks of 7mm diameter out of the membrane (thickness is about150 pm). The disk was mounted into a Phillips elec-trode body (ISE - 561, GlasblasereiMoller, ZUrich).A S)lutionof O.1M MgC12was used as the internal fill-

i~ S)!utionfor the Mi + -selectiveelectrode.After mnditioning the electrode in O. 1M MgC12

for 24 hours, EMF measurementswere conductedwithfollowing cell: Hg, I-I&O2 I KO (satd.) MMKO(3.0 M) MM sample S)lution II membrane II internalS)lution (MgClz, 0.1 M) IAgO, Ag.

The calomel reference electrode corresponded tothe free-flowing double-junctiontype[16].The cell po-tential was measuredusing an Apple nGS (Apple Com-puter, Cupertino, CA, USA) equipped with a 7150Digital Multimeter (~lartron Instrumentation, UK).The single-ion molal molar activities in physiologicalelectrolyte situation were calculated using the Pitzerprogram[17].The selectivitymefficientswere measuredbased on the IUPAC recommendedprocedureof sepa-rate S)lutionmethod.All EMF valueswere correctedfor

changes in the liquid-junctionpotential, Ej' using theHendefS)n formalism[18].All potentiometric measure-mentS were performed at ambient temperature(~25°C) .2.3 The main cationsin physiologicalsolution

In physiological fluids, the main cations are Na + ,

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K+ , ci + and Me?+ which play important roles in.bi-ological functions. In human blood serum, the intercel-lular concentrations of these cations vary in certainrang~" that are listed in Table 1. In calculating the re-quired selectivity coefficients of ISEs for physiologicalassays, an allowable measuring error of 1. 0% is con-sidered with minimum level of a physiological molar ac-tivity. The required selectivity coefficients for detectingmain inorganic cations in physiological solution are cal-culated assuming an allowable error of 1. 0 % at theminimum level of a physiological molar activity of thedetecting ion. The sensors are measured with- andwithout the calibration of physiological ion background(Dc, assuming variation of the background ion molalitylevel around the mean physiological points, Table 1).

The calculation without background calibration(Eq. (5) and Eq. (7» is based on the maximum level

max. level

6.0X 10-4

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4.8X 10-3

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of interfering ion aj,rrnxand the minimum level of pri-mary ion ai ,min' For obtaining the required selectivitycoefficients with the background calibration (Eq. (6)and Eq. (8», the minimum level of the primary ion

ai,min and the medium level of the interfering ion aj,calis considered for the background calibration.

3 Results and Discussion

3. 1 G)Jnparison of methods for calculating the re-quired selectivity coefficients

The required selectivity coefficients of ISE for dif-ferent primary ions are calculated based on the conven-tional N-E equation and the modified consistent N-E e-quation. The obtained required selectivity coefficientswith and without the physiological background calibra-tion (Eq. (6) and Eq. (8» are compared in Table 2.

Thble1. The main cation levels in physiological 'solution

min. level Medium level

'Fdble 2. Comparison of the required selectivity coefficients of ISEs for measuring the main inorganic cations in physiological solutions,assuming an allowable error of 1 . 0 % at the lower level of the physiological range of the detecting ion. .

Conventional( 1 KiD!) . ' Consistent ( 1 KiD') .

~ ~,~ ~ ~,~

Secondary no with Secondary no withion calibration calibration ion calibration calibration

Cation

Mi+

Ca2+

K+

Na+

PrimaryIon

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Concentration c (M) 3.0X 10-4 4.5X 10-4

molar activity a (M) 1.1XlO-4 1.6xlO-4

Concentration c (M) LOX 10-3 1.15 X 10-3

molar activity a (M) 3.4X 10-4 3.9xlO-4

Concentration c (M) 3.3xlO-3 4.05 X 10-3

molar activity a (M) 2.4X 10-3 2.9xlO-3

Concentration c (M) 0.134 0.138

molar activity a (M) 0.099 0.103

Ca2+ -2.4 -1.5 Ca2+ -2.2 -1.0

K+ -0.9 -0.3 K+ -1.4 -0.6

Na+ -3.8 -2.6 Na+ -2.9 -1.4

Mi+ -1.8 -1.1 Mi+ -1.9 -0.9

K+ -0.6 -0.0 K+ -1. 3 -0.5

Na+ -3.5 -2.3 Na+ -2.8 -1.2

Mi+ -2.8 -1.8 Mi+ -2.8 -1.8

Ca2+ -2.9 -1. 7 Ca2+ -2.9 -1. 7

Na+ -3.6 -2.1 Na+ -3.6 -2.1

Mi+ -1.2 -0.2 Mi+ -1.2 -0.2

Ca2+ -1. 3 -0.1 c".l+ -1. 3 -0.1

K+ -0.5 0.3 K+ -0.5 0.3

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Because the modified consistent N- E equation re-moves the influence from the ion charge-dependence,the consistent required selectivity coefficient

(Kijn.')requiredreasonably illustrates the preference of thesensor to the primary ion over interfering ions, especial-ly for measuring the unequally charged primary and in-terfering ions. It can be seen from Table 2 and Table 3that when monovalent ion (K + or Na+) is consideredas the primary ion, two methods give out the equal re-

quired selectivity coefficients (Kijn.s )rffjuired

(K~Y) rffjuired'However, the consistent coefficient val-

ues of log (Kijn.s)requiredfor divalent primary ions

(Mi+ or Ca2+) are significantly different from the

conventional ones of log (Kijt)rffjuired' The logarithm of

the required selectivity coefficient for Mi+ over Ca2+changesfrom - 2. 4 to - 2. 2 and the one for Mi +

over K+ from - O.9 to - 1. 4. As well, the log

(Kijt) '1"'1uiredfor Mi + over Na + decrea.'3esfrom - 3. 8to - 2. 9. Such differences lead to the nece&sityof ap-

plying the consistent formulism of the required selectivi-ty coefficient for divalent primary ions.

3.2 Mi + -selective electrode and the required selec-tivity coefficients in physiological analysis

In recent years, studies of developing selective

Mi+ potentiometric sensors have been intensively con-ducted in many groups and the adequate discrimination

between Mi+ and Ca2+ is still the main target[19-21].Suzuki et al[22] synthesised a selective Mi+ ionophoreK22B5 which logarithm of selectivity coefficient of

Mi + over Ca2+ was reported as - 2. 5 measuredwiththe IUP AC recommended separate solution method

(SSM)[23]. The inadequate selectivity against monova-

lent cations (Na + and K+) of this kind of Mi +ionophoresmade the correspondingMi + membraneelectrodes hard to be applied in physiological assays.

Spichiger et al developedan Mi + selective electrodebased on ionophore ETH 5504 which has high selectivi-

ty over ci+ , K+ and Na + while its high lipophilicityprevents ETH 5504 leaching from plasticized PVCmembrane.So far, many Mi + selective microelec-

trodes basedon variousMi + ionophores have also been

reported in measuring the physiological systems[24-28].In serum samples, the intercellular Mi+ molar

activity range (0. 46 ~ 0.66 M) is much lower thatCa2+ level range (1. 01~ 1. 26 M) and the discrimina-tion of Mi+ over Ca2+ is very critic for accurate mea-

surement of Mi+ in physiological assays. The selectiv-

ity coefficients of Mi+ -selective electrode based onionophore ETH 5504 was determined according to theIUPAC recommended separate solution method

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(SSM)[23] as well as the newly modified consistentSSM[9].

In order to ensure the selectivity coefficients of

Mi+ -ISEs to reflect the "worst case" in physiologicalsituation, the minimlllll level of Mi + and the maxi-mlllll levels of interfering ions were used in calculatingthe selectivity coefficients. EMF values of the minimlllll

physiological level Mi+ were compared with that ofmaximlllll physiological levels of other interfering ions,

Ca2+ ,Na+ and K+ separately (Figure 1). Two sets

selectivitycoefficientsfor Mi+ - ISE, logK~.J and

logK~:J , were calculated based on both the conven-tional N-E method and the modified consistent N- E

method. As shown in Figure 2, the selectivity coeffi-

cients (SSM) of Mi + -selective electrode based on bothmethods were obtained by using electrolyte solutions ofstandard concentration (0. 1 M) and of physiologicalmedilllll levels of primary and interfering ions (Table1).

It can be seen from the results in Figure 2 that the

selectivity pattern of Mi+ -selective electrode withETH 5504 were measured based on both approaches ofthe conventional and the modified consistent approach-

es. The selectivityof Mi + against ci + reaches - 1. 0~ - 1. 8 with conventionalSSM approach and - 1.0~ -1. 2 with the consistent method. llith values sat-

isfied the physiologically required selectivity coefficientscalculated from Eq. (6) (conventional approach,

loge Kijt) rffjuired= - 1. 5) and Eq. (8) (consistent ap-

proach, log( Kijn.s)rffjuired= - 1. 0) with physiologicalbackground calibration. However, for the direct mea-surements without background calibration, the selectiv-ity still has space to improve. Regarding the interferingmonovalent cations of K+ and Na+, this Mi +- ISEshowed the adequate selectivity over K+ even for non-calibrationmeasurementof Mi + either with conven-

tional method (log( Kijt) rffjuired= - O.9) and consistentmethod (log ( Kijn.s)required= ~ 1. 4). However, thediscriminationagainst Na+ of this Mi + -ISE also re-quires the physiological background calibration so as to

satisfy the physiological requirement (with background

calibration, log (Kijt) required= - 2. 6 andloge K'jjn.s)required= - 1. 4). According to this compari-son, it is obviousthat the physiologicalbackgroundcali-bration is really needed for using the Mi+ -ISE withionophore ETH 5504 to measure free intercellularMi + level.This requirement can predict the necessaryenhancing space of selectivity pattern for developing fu-ture Mi + -ISEs in direct measurements in physiologicalassays.

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Life S::ienceJournal, 2 (1), 2005, Zhang, Physiologically Required Selrrtivity Qyefjicients of Potentimnetnc Sensors J)

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~

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0 M

~~ -2..s ca~K ~

,/Na "" /~"'---_..--

-1

-3

-4

-5 (a)conventional N-E (h)consistent N-E ,IJ

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~

.....'""-E.~;:c"....'Scr"....

~

~"0c:

;:c".~6-"....

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~K~~Ca ~~//Na

Figure 2. The comparison of the Mi + -selectivity coefficients with required selectivity coefficients.

(a) Conventional N-E method; (b).. Consistent N-E method.

4 Q)nclusion

-In conclusion, the physiologically required selec-

tivity coefficients of ISEs for physiological analysis werestudied by comparing the conventional N- E method andthe modified consistent N-E method. The modified

consistent method eliminates the discrepancies originat-ed from the conventional N-E equation for unequallycharged primary and interfering ions. Indeed, the con-sistent required selectivity coefficients for the divalent

primary ion (Mi+ or ea2+) gives out a more accurate

calculation that the conventional one and provides a rea-S]nable criteria for evaluating the applicability of ISEs in

.http; / /www.sciencepub. org

physiological assays. However for the monovalent pri-

mary ion (K+ or Na + ), the required selectivity coeffi-cients calculated with two methods are identical. For

comparing the perfonnances of ISE in the physiologicalS]lution with the required selectivity pattern, the elec-trode was checked in the" worst case" of minimum level

of primary ion and maximum level of interfering ion inphysiological S]lution. Indeed, the modified consistentrequired selectivity coefficients are more accurate thanthe ones derived from the conventional N-E equation,which provides the more reliable criteria for evaluatingthe applicability of ion-selective electrodes in physiologi-cal assays.

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Life Science Journal, 2 (1), 2005, ZIulng, Physiologically Required Selectivity Gxfficients of PotentiO/11£tricSensors

Correspondence to:Wei Zhang26246 Fieldstone Drive

Novi, Michigan 48108, USAllelephone:734-355-9682Email: [email protected]

References1. Bakker E, Prestch E. Anal Chern 2002:74;420A.

2. Spichiger UE, Chemical Se=rs and Bi==rs for Medicaland Biological Applications. Wiley VCH, Weinheim, Ger-many 1998. .

3. Bakker E, Diamond D, Lewenstam A, Pretsch E. AnalChim Acta 1999: 393 ; 11.

4.Spichiger UE, Eugster R, Haase E, Rumpf G, Gehrig P,Schmid A, Rusterholz B, Simon W. Fresenius J Anal Chern1991:341;727.

5. Nicolsky BP. Zh Fiz Khim 1937; 10;495.6. Eisenman G, Rudin m, Casdy Ju. Science 1957: 126;

831.7. Umezawa G, Umezawa K, S3.toH. Pure Appl Chern 1995;

67;507.8. Bakker- E, Meruva RK, Pretsch E, Meyerhoff M. Anal

Chern 1994:66;3021.9. Zhang W, Fakler A, Demuth C, Spichiger UE. Anal Chim

Acta 1998:375;211.10. Cattrall RW, Drew DM. Anal Chim Acta 1975: 77 ;9.11. Bagg], Vinen R. Anal Chern 1972: 44 ; 1773.12. Spichiger UE. ETH Thesis No. 8803, 1989.

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ll.J..

J.~

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13. Zhang W, Jermy L, Spichiger UE. Analytical Sciences2000;16;11.

14. Eugster R, Rosatzin T, Rusterholz B, Aebersold B, Pedraz-za U, Ruegg D, Schmid A, Spichiger UE, Simon. AnalChim Acta 1993:289; 1.

15. Oesch U, Br=ka Z, Simon W. Anal Chern 1986; 58;2289.

16. DolmerRE, WegmannD, Mon WE, SimonW. kal Chern1986:58;2585.

17. Pitzer KS. Geochim Cosmochim Acta 1984:48;723.18. Henderson P. Zeitschrift f(ir Physikalische Chemie 1907: 59;

118.

19. Maj-Zurawska M. Chemia Analityczna 1997:42; 187.20. Toth K, Lindner E, Horvath M, Jeney J, Pungor E, Bitter

I, Agai B, Toke L. Electroana1ysis 1993:5;781.21. Spichiger UE. Electroana11993:5;739.22. Suzuki K, .Watanabe K, Matsumoto Y, Kobayashi M, S3.to

S, Siswanta D, Hisamoto H. Anal Chern 1995; 67 ;324.23. Guilbault GG, Durst RA, Frant MS, Freiser H, Hansen

EH, Light TS, Pungor E, Rechniz G, Rice NM, RohmTJ, Simon W, Thomas DR. Pure Appl Chern 1976: 48;129.

24. Zhang W, Trutmarm AC, Luethi D, McGuiganJAS. Mag-nesium Bulletin 1995:17;125.

25.Gunzel D, Schlue WR. Biomatals 2002;15;237.26. Zhang X, Fakler A, Spichiger UE. Electroana11998: 10;

1174.

27. Gunzel D, Muller A, Durry S, Schlue WR. ElectrochimicaActa 1999;44;3785.

28. Spichiger UE, Fakler A. Electrochimica Acta 1997; 42;3137.

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Life £ience Journal, 2 (1), 2005, Sun ,et al, Camjx1risonof Patients' Ps}cholofiical Status

Comparison of Patients' Psychological Statusbetween Controlled Seizures and Uncontrolled Seizures

by Symptom Checklist 90

Meizhen Sunl, Wei Wangl, Yuxi Lit?, Kerang Zhani, XiaofengRen4

1. Department of Neurology and Psychiatry, Tongji Affiliated Hospital of Tongji Medical College,

Huazhong University of Science and Technology, Wuhan, Hubei 430030, China

wwang@tjh. tjmu. edu. en or meizhensun23@yahoo. com

2. Department of Neurology, First Affiliated Hospital of Shanxi Medical University, Taiyuan ,Shanxi, China

3. Department of Psychiatry, First Affiliated Hospital of Shanxi Medical University, Taiyuan ,Shanxi, China

4. Department of Veterinary, Northeast Agricultural University, Harbin, Heilongjiang, China

[email protected]

Abstract: Purpose. Psychological treatment is one of the most important treatment options available to patients withepilepsy. It is vital to determine whether long-term psychological treatment is necessary. The purpose of this studyis to compare the psychological status of patients' in which seizures are controlled with that of patients who experi-ence uncontrolled seizures by Symptom Checklist 90 (SCL 90). Method. Patients with epilepsy were divided intotwo groups: Controlled seizure group (n = 64 ,seizure free 1 - 5 years), and uncontrolled seizure group (n = 61).The two groups were matched in gender, age, duration of disorder, education and personality .(Eysenck personalityquestionnaire, EPQ). SCL 90 was used to test psychological status and a social support questionnaire used to assesssocial support status. Result. Compared with the average in the Chinese population, the factor scores of somatiza-tion (mean 1. 65 :t O. 57 SD) , obsessive-compulsive (mean 2. 08 :t O. 79 SD) , depression (mean 1. 90 :t O. 85 SD) ,anxiety (mean 1.80:t O.74 SD), hostility (mean 1.84:t O. 97 SD), photic anxiety (mean 1.58:t O.59 SD), andpsychotic tendencies (mean 1. 60 :t O. 59 SD), were significantly higher in the uncontrolled seizures group(P < O.05) ; the factor scores of obsessive-compulsive (mean 2. 01 :t O. 65 SD), hostility (mean 1. 68 :t O. 61SD) ,photic anxiety (mean 1.41 :t 0.42 SD) , and psychotic tendencies (mean 1. 53 :t O. 55SD) were significantly higherin the controlled seizure group (p< 0.05). However, only the factor score of somatization was significantly higherin the uncontrolled seizure group than that of the controlled seizures group (mean 1. 33 :t O. 25 SD, P < O.05). At"the same time, there was no significant difference in social support between the two groups (P >0. 05). The scoreof each group was below 35 (mean'25.67:t4. 97 SD for controlled seizures and mean 24. 38 :t4. 53 SD for uncon-trolled seizures). This result indicates that both groups were short of good social support. Conclusion. Patients withepilepsy need long-term psychological treatment and good social support. [Life Science Journal. 2005; 2( 1) : 46-48J (ISSN: 1097-8135).

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the psychological status of patients' in whichseizures were controlled with that of patients whoexperienced uncontrolled seizures by the SymptomChecklist 90 (SCL 90) and social support question-nalre.

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Keywords: epi lepsy; seizure; psychology; Symptom Checklist 90; social support

1 Introduction

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Epilepsy is a chronic neurological disease. Asin other chronic conditions, more attention should

be paid to the quality of life (QOL) of patientswi th epilepsy. Psychological functioning is one ofthe most important factors in the health-related

quality of life model[l]. Lots of studies show thatthere are psychological disorders in the patientswith epilepsy. But there are few articles on thestudy of the psychology of patients with controlledepilepsy. Symptom Checklist 90 (SCL 90) is a ma-

jor measure of mental health symptoms[2] .The present study was designed to compare


2 Patients and Methods

2. 1 The criteria of patients and groupsPatients were considered suitable for the study

if they met the following criteria: Age> 12 years,education> 6 years, and had a normal intelligencelevel. If patients had history of psychiatric illnessor were on psychiatric medication, they would beexcluded.

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Life S::ienceJournal, 2 ( 1 ), 2005, Sun, et aL, UrmjX1risonof Patients' Psy;fwwgicaL Status

Patients with epilepsy were chosen. in twogroups. Group one (controlled seizure group) con-sisted of 64 patients known to be seizure free for 1- 5 years. Group two comprised 61 subjects whocontinued to experience seizures. The two groupswere matched in gender, age, duration of disorder,education and personality.2.2 The explanation of questionnaire

Eysenck personality questionnaire (EPQ)(Table 1): SCL 90 was used to test psychologicalstatus and social support status was assessed with asocial support questionnaire. The patients recruitedfrom community. The SCL 90 and EPQ are trans-lated versions. They are validated and self-reportinventory. .

Table 1. The conditions matched in two groups

Controlled seizure group UnccntrolledseizuregroupFemale:n =24; Female: n =24;

male:n=40 male:n=37

Mean: 24 . 6 years Mean: 25 . 8 years

Gender

... AgeDuration of

disorder

Education >6 years: n = 64 >6 years: n = 61

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Mean: 6.4 years Mean: 10. 1 years

Pers:rnlity(rmm:t SJ)

P:5.44:t 3. 08 P:6. 39:t 3. 91

E: 10. 65:t 4.48 E: 11. 25:t 5. 31

N:1O.06:t4.93 N:ll.23:t5.61

L: 13. 12 :t 3. 62 L: 12. 65 :t 4. 31

P> O. 05: Personality (P, E, N, L) of two groups comparedwith each other. P is the abbreviation of psychoticism; E is theabbreviation of Extroversion; N is the abbreviation of neuroti-cism; L is the abbreviation of lie.

2.3 Statistical methodsThe factor scores of EPQ and SCL 90 in two

groups compared with the averages within the Chi-nese population respectively, and compared eachother used paired t- test.

3 Results

Compared with the averages within the Chi-

nese population[2], the factor scores of somatiza-tion, obsessive-compulsive, depression, anxiety,hostility, photic anxiety, and psychoticism weresignificantly higher in the uncontrolled seizuregroup (p < O. 05); the factor scores of obsessive-compulsive, hostility, photic anxiety, and psy-choticism were significantly higher in the controlledseizure group (p < O. 05). However, only the fac-tor score of somatization was significantly higher inthe uncontrolled seizure group than that of the con-trolled seizure group (Table 2). At the same time, .there was no significant difference in social supportbetween two groups (P >0. 05). The scores from


both groups were below 35 (Table 3). This sug-gests that both groups were short of good socialsupport.

Table 2. Factor scores of the SCL 90 in the two groups

Factor lhmtrolled ~ group Cilltrdled~ group

Mean :t SD P Mean:t SD P

1.65:t0.57 <0.05 1.33:t0.25 P<0.05Somatization

Obls:,sive-com- 2.08:t o. 79 <0.05 2.01:t O.65 <0.05

pu Slve

Depression 1. 90 :t O.85 < 0 . 05

Anxiety 1.80:t0.74 <0.05

Hostility 1. 84:t O.97 <0.05 1. 68:t O.61 <0.05

Photic anxiety 1.58:t0.59 <0.05 1.41:t0.42 <0.05

Psychoticism 1. 60:t O.59 <0.05 1. 53 :to. 55 <0.05

P<O. 05:Compared with norm of Chinese; * P<O. 05: som-atization in uncontrolled seizure group compared with that in con-trolled seizure group.

Table 3. Social support scores of the two groups

Uncontrolled seizures Controlled seizure Pgroup (mean:t SD) group (mean:t SD)

Socialsupport 25.67 :t 4.97 24. 38 :t 4. 53 >0.05

P > O. 05: Social support scores of two groups compared eachother.

4 Discussion

There are specific measurement parameters forthe efficacy of treatment of epilepsy. Currentseizure frequency is one of the most important pre-dictors. Psychological factors, as well as the Quali-ty-of-life (QOL) of the patient, are also important

to achieve an accurate prognosis[3]. Research. hasconfirmed that patients with epilepsy inevitablyhave psychological disorders such as depression andanxiety, as such symptoms accompany the diagno-sis of epilepsy. It is important to understand the re-lationship between the frequency of epilepsy andthe psychological symptoms presented. For exam-ple, in caseswhere seizures are controlled, do psy-chological symptoms disappear or decrease drastical-ly? It is necessary to determine whether a long-term psychological treatment is required? We com-pared the psychology status of patients in whomseizures were uncontrolled with that of patients inwhom seizures were controlled ( seizure-free 1 - 5

years), by Symptom Checklist 90 (SCL 90). Wefound the factor score of somatization .0 be signifi-cantly higher in the uncontrolled seizure group thanthat of controlled seizure group. It is suggested thatthe patients still present many psychological symp-toms in the controlled seizure group and need long-term psychological treatment. Somatization may bethe factor that can easily change in patients with 1- 5 seizure-free years.

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Tongji Medical CollegeHuazhong University of Science and TechnologyWuhan, Hubei 430030, ChinaEmail:[email protected]. cn or

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Psychological status is related to the frequencyof epilepsy in patients. The more frequently thatseizures occur, the poorer the psychological status

of epileptic patients is expected to be[4,5]. Howev-er, the patient's perception of seizure severity has astronger relation to psychosocial adjustment than

actual seizure frequency[ 6- 11]. Our results indicatethat epileptic patients continue to present psycho-logical symptoms even though their epilepsy hasbeen seizure-free for 1 - 5 years. There are somereasons for these results: fear of repeat seizures;lack of confidence in the future; lack of social sup-port; and additional factors.

Depression and anxiety are the major factors

causing psychological distress in epilepsy[12-I4].These factors were shown to be significantly higherin the cohort that experienced uncontrolled seizurescompared with the average in the Chinese popula-tion. However, the scores for the group in whomseizures are controlled were not significantly differ-ent from the population average. This indicatesthat depression and anxiety are to some extent re-lated to the frequency of epilepsy. Gramer hasstudied the influence of depression on seizure severi-

ty[15]. He found that patients with depression re-ported higher levels of perceived severity and dis-tress from seizures than those patients without de-pression experiencing similar types of seizures.

However, Attarian, studying the relationshipbetween depression and intractability of seizures,found that patients with epilepsy have a higherprevalence of depression than the general popula-tion, the intractability of the seizure disorder doesnot seem to be an independent risk factor for the

occurrence of depression[16]. There is no relation-ship perceived between the severity of depressionand monthly seizure rate. Therefore attentionshould be paid to both seizure severity and depres-SIon.

Social support plays an important role in health

and good mood[17,18]. Hence we investigated thelevel of social support experienced by the twogroups. We found that in both the uncontrolledseizure group and the controlled seizure group thelevel of social support was low. Thus it is suggestedthat patients with epilepsy need long-term psycho-logical treatment and good social support.

~ Correspondenceto:

Wei Wang


References1. Baker GA. Assessment of quality of life in people with epilep-

sy: some practical implications. Epilepsia 2001; 42 (Suppl.3):66-9.

2. Brooks R. The reliability and validity of the Health of the Na-tion Outcome Scales: validation in relation to patient derivedmeasures. Aust N Z J Psychiatry 2000:34(3) :504 -11.

3. Tang Q, Cheng Z, Yuan A, et al. The application and analy-sis of SCL-90 in China. Chinese Journal of Clinical Psychology1999 :7(1): 16 - 20.

4. Herodes M, Oun A, Haldre S, et al. Epilepsy in Estonia: aquality of life study. Epilepsia 2001:42(8) :1061-1073.

5. Elliott I. Psychosocial functioning in adolescents with complexpartial seizures. Axone 1992; 13 :72 - 6.

6. Baker GA, Jacoby A, Buck 0, et al. Quality of life of peoplewith epilepsy: a European study. Epilepsia 1997;38(3):353-62.

7. Collings JA. Epilepsy and well-being. Soc Sci Med 1990a:31:165 -70.

8. Collings JA. Psychoscial well-being and epilepsy: an empiricalstudy. Epilepsia 1990b:31:418-26.

9. Collings JA. Correlates of well-beings in a New Zealandepilepsy sample. N Z Med J 1990c: 103 :301 - 3.

10. Hennann BP, Whitmann S. Psychosocial predictors of inter-ictal depression. J Epilepsy 1989;2:231-7.

11. Hermann BP, Whitmann S, Wyler AR, et al. Psychosocialpredictors of psychopathology in epilepsy. Br J Psychiatry1990; 156 :98 -105.

12. Smith OF, Baker GA, Dewey M, et al. Seizure frequency,patient perceived seizure severity and the psychosocial conse-quences of intractable epilepsy. Epilepsy Res 1991: 9: 231 -41.

13. Piazzini A, Canger R. Depression and anxiety in patients withepilepsy. Epilepsia 2001 :42 (Suppl1) :29 - 31.

14. Ettinger AB, Weisbrot OM, Nolan EE, et al. Symptoms ofdepression and anxiety in pediatric epilepsy patients. Epilepsia1998; 39( 6) :595 - 9.

15. Hermann BP, Trenerry MR, Colligan RC, et al. Learnedhelplessness, attributional style, and depression in epilepsy.Epilepsia 1996:37(7) :680 - 6.

16. Gramer JA, Blum 0, Reed M, et al. The influence of comor-bid depression on seizure severity. Epilepsia 2003: 44 ( 12) :1578 - 84.

17. Attarian H, VaWe V, Carter J, et al. Relationship betweendepression and intractability of seizures. Epilepsy Behav2003 :4(3) :298 - 301.

18. House JS, Landis KR, Umberson O. Social relations andhealth. Science 1988:241:640-5.

19. Goyne JC, Downey G. Stress, social support and the copingprocess. Ann Rev Psychology 1991:42:401-26.

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Changes of Left Ventricular Dysfunction and Cardiomyocyte

Apoptosis in Losartan- treated Heart Failure Rats

Chunjing Fu1, Hua Tian2, Longhui Gu03, Youtian Huang1, Qi Shen4

1. ~partment of Pathophysiology,Medical Collegeof ZhengzhouUniversity,Zhengzhou,Henan 450052, China

2. Department of Pathophysiology, Medical College of Weifang, Weifang, Shandong 261042, China

3. Department of Cardiovascular Surgery, Second Affiliated Hospital, Zhengzhou University,


4. Department of Computer, Secondary Hygienic School, Zhengzhou University,


Abstract: Objective.This study is to determine whether angiotensin II (Ang II) I type receptor (ATIR) retarderinhibits cardiomyocyteapoptosisand attenuates left ventricular ( LV) dysfunction in the chronic heart failure rats.Methods.40 rats of health Sprague-Dawley (SD) were divided into 4 groups randomly. Group I was sham-operat-ed group (n =8). Group II was heart failure group (n = 12). Group ill was losartan treated group 1 (n = 10) .Group N was losartan treated group 2 ( n = 10). Besides group I , other 3 group models of heart failure were estab-

lished by part constriction of abdominal aorta of rats. After 6 weeks, group ill and group N were treated by gavageof losartan 10 mg. kg- 1 .d - 1and 30 mg. kg- 1. d - 1 respectively. Group I and group II were gavaged by normalsaline (NS). After 14 weeks, the parameters of hemodynamic and LV remodeling were detected. Ang II of plasmaand cardiomyocyte were measured by radioimmuneoassay. Cardiomyocyte apoptosis were stained in situ by usingTUNEL. The expression of BcI-2, Bax and procaspase-3 protein of cardiomyocyte was determined by Westernblot. Results. The Left ventricular end-diastolic pressure(L VEDP) of group II was 7.2 mmHg higher than that of

group I (P<O. 01)in If! weeks. Left ventricular weight( LVW)/ Body weight (BW) of group II was O.42 mg/ghigher than that of group I (P < O.01). Ang II of plasmaand cardiomyocyteof group II were 135.31 pg/ml and94.4 pg/g higher than that of group I respectively (P < 0.01). The exponent of cardiomyocyte apoptosis of groupII was 12.11 ')I()()higher than that of group I (P<O. 01). When group II comparedwith group I , the expressionof cardiomyocyte Bax of group II was increased significantly while expression of BcI-2 and procaspase-3 decreased

significantly(P< 0.01). When group ill and groupN compared with groupII , the indexes above mention havestatistical significance(P < 0.01 or P < O.05), and the index changes are related to losartan by treated dose. Con- .elusion. The results indicate that heart failure is associatedwith LV dysfunction and cardiomyocyteapoptosis in-volvingactivation of procaspase-3, and increased BaxlBcl-2 ratio in the rat heart. ATIR retarder attenuates LVdysfunction in the chronic heart failure rats through decreasingcardiomyocyteapoptosisand changing expressionofapoptosis-relatedproteins. [LifeScience]ournal.2005;2(1) :49- 54] (ISSN: 1097- 8135).

Keywords: apoptosis; heart failure; losartan; rat

1mal experiments are disagreement. A number ofgenes have been identified that regulate the apop-totic process. The Bcl-2 proto-oncogene family iscritical for the regulation of apoptosis[3]. &1-2family members come in 2 functional categories,that are inhibiting apoptosis 0. e. , Bcl-2) and in-duce apoptosis (i. e., Bax). The relative abun-dance of proapoptotic and antiapoptotic proteins de-termines the susceptibility to cell death. Relation-ship between these proteins and Ang II is ambigu-ous. Losartan is AT1R retarder which mostlytreats hypertension in clinic. Mechanism of it inpressure overload by constriction of abdominal aortaof rats partly is indistinct in inducing cardiomyocyteapoptosis.

I'--

Introduction

An important character heart failure is neuro-hormonal activation. Neurohormonal activation isclosely related with cardiac apoptosis. Enhance-ment of circulation and local Ang II are importantfactors[iJ. The receptor of Ang II also is in-creased[2]. Although the available evidences sug-gest that apoptosis can be induced in cardiomyocyteby a variety of stimulation including pressure over-load, most of models are used to acute heart failuremodels. The relationship between Ang II andapoptosis are ambiguous. Many studies use culturedcells. However, results of clinical studies and ani-

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boiled for 5 minutes in 10 volume of acetic acid(1 mollL)/HCl (20 mmol/L), and homogenizedat high speed (PT 1200, Polytron ). The ho-mogenate was then ultracentrifuged at 27 kg at 4°C, and the supernatant was stored at - 20°Cuntilradioimmunoassay.

Detection of apoptosis: In five animals fromeach group, myocardial apoptosis was assessed us-ing a commercially available method, which relieson terminal deoxynucleotidyl transferase (T dT )-mediated digoxigenintagged dUTP-biotin end-label-ing of 3' -OH DNA ends generated by DNA frag-mentation in situ. In brief, 6 pm tissue sectionswere deparaffinized in xylene, and rehydratedthrough graded ethanol and distilled water. Thesections were then incubated for 1 min at 22°C in e-quilibration buffer. The equilibration buffer wasthen replaced by TdT solution and incubated for1 h at 3T C in a humidified chamber. The slideswere then washed in phosphate-buffered saline(PBS, pH 7.4) and incubated with anti-digoxi-genin antibody conjugated to horseradish peroxi-dase, washed with PBS and further incubated withdiaminobenzidine and hydrogen peroxide. For neg-ative controls, TdT enzyme was replaced with dis-tilled water in the labeling reaction. Myocardial nu-clei were considered apoptotic only if they displayedboth TUNEL stain positive and appropriate nuclearmorphology (condensation and/or ,fragmentation).Five sections from each specimen were examined.At high magnification ( X 400), five random fieldsper section from noninfarcted, remodeled regionswere examined to calculate the number of apoptoticnuclei per 1000 total nucented. Specimens wereread in a blinded fashion.

Western blot analysis: For Western blots,frozen LV myocardium was pulverized in liquid ni-trogen and homogenized in a lysis buffer containing100mMNaCl, 50mMTris(pH7.4), 0.5mMTriton X -100, 1 mM dithiotreitol, 50 mM NaF,0.5 mM NaVO3, and an EOTA-free protease in-hibitor cocktail (Roche). Protein homogenateswere centrifuged at 14,000 rpm for 10 min at 4°C,and the supernatants were used for Western blot-ting. After determining protein concentrations bythe Bradford Method (Bio-Rad), 30 fJ-gof protein/sample were denatured by boiling for 5 min in aloading buffer containing 0.25 M Tris (pH 6.8),20 % glycerol, 4% SOS, and 0.05 % bromophenolblue, and subjected to 10% SOS-PAGE. Followingelectrophoresis, the separated proteins were trans-blotted on to PVDF membranes (Immobilon P,Millipore). The membranes were blocked with10% normal goat serum (preimmune s~rum,OAKO) for 1 h at 22°C. The membrane was then

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In the present study, we delineated the losar-tan role and changed in Bax, Bcl-2 and procaspase-3, in pressure overload by part constriction of ratsabdominal aorta, critical steps involved in deathsignaling pathways.


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Animal and design:The healthy male SO rats(BW 100 - 110 g) were provided by HenanProvince Experiment Animal Center. 40 rats ofhealth SO were divided into 4 groups randomly.Group I was sham-operated group (n = 8).Group II was heart failure group (n = 12). Groupill was losartan treated group 1 (n = 10). GroupN was losartan treated group 2 (n = 10) . Besidesgroup I, other 3 group models of heart failurewere established by part constriction of abdominalaorta of rats. Rats were anaesthetized by 2 % bu-taylone 40 mg/kg abdominal injection. After ab-dominal operation, upside of renal artery, an injec-tor pinhead (internal diameter O. 80 mm) wasplaced around the abdominal aorta. After ligatingpinhead and abdominal aorta, pinhead was drawnout. Rats of sham-operated group only separate ab-dominal aorta but didn't constrict abdominal aorta.After 6 weeks, group ill and group N were treated

by gavage of losartan 10 mg' kg-1'd-1 and 30 mg'kg - 1. d -1 respectively. Group I and group II were

gavaged by NS.Determination of cardiac function: After 14

weeks, all animals were killed. The animals wereanaesthetized by 2 % butaylone abdominal injectionof 40 mg/kg before killed, and arterial blood pres-sure was measured directly via the left carotidartery. LV hemodynamics was measured by insert-ing a PE 50-catheter through the free LV wall intothe left ventricle. Pressures were registered with atransducer and an amplifier. LV contractility wasobtained from the ventricular pressure curves.Hemodynamic measurements were performed, thehearts were removed, and ventricles were dissectedfree, weighed and kept in liquid nitrogen until fur-ther analysis.

Determination of LV remodeling: BW andheart weight (HW) and LVW were measured after14 weeks. Heart quality index is HW/BW. LVquality index is LVW/BW.

Determination of Ang II contents of plasmaand myocardium:Ang II was measured by radioim-munoassay. Plasma samples were extracted inphenyl-encapped cartridges, washed with 1 ml dis-tilled water, eluted with 0.5 ml methanol,lyophilized, and reconstituted. For extraction oftissue Ang II, samples were pulverized frozen,


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incubated with primary antibody and then with sec-ondary antibody for 2 h each. Signals were revealedwith chemiluminescence using the ECL-detectionsystem (Amersham). Quantification of the signalswas performed using NIH image. Rabbit polyclonalIgG antibodies against human Bcl-2 (cross reactivewith rat) were used at a concentration of 5 fig/ml.

Statistical analysis: All results are presented asmeans:t SD. Differences among the 4 groups ofrats were tested by a one-way ANOVA. Compari-son between groups was performed with the multi-ple comparison Student-Newman-Keuls test. Pvalues < 0.05 were considered statistically signifi-cant. The detection of specific protein binding waspreformed with enhanced chemiluminescence West-ern blot detection system.

3 Results

Hemodynamicand weight: Because rats sufferfrom acute heart failure, dead rats were 4 in group IIinthe experiment course. Becauserats suffer from chronicheart failure, there were 2 dead rats in group ill and

group N respectively. There was no dead rat in group!.After 14 weeks, LVEDP and:t dp/dtrnax were signifi-cantly lower in group II,compared with group 1, con-finning the presenceof LV dysfunction. LVEDP and:tdp/dtrnax have statistical significantly group ill, com-pared with groupII~Along with increaseddoses,aoove-mentioned indexeswere more significantly(Table 1).

BW of group II rats was lower than that ofgroup I rats. Heart quality indexesand LV quali-ty indexes in group II rats were markedly higherthan that in group I rats. Heart quality index andLV quality indexes in group ill rats were lowerthan that in group II rats. Along with increaseddoses, above-mentioned indexes were more signifi-cantly (Table 2).

Ang II contents of plasma and myocardium:Ang II of plasma and myocardium in the group IIrats was higher than that in the group I rats (p <0.01). Above-mentioned index was obviously de-creased in the group ill rats than that in the groupII rats (P<O.Ol). Along with increased doses,above-mentioned index was significantly different(Table 3).

Table 1. Effects of losartan on hemodynamic indexes

Group LVSP (mmHg) LVEDP (mmHg) + dp/dtmax(mmHg/s) - dp/dtmax(mmHg/s)

I 123.6:1::15.2 5.7:1::1.8 4853.7:1::411.7 3817.3:1::262.8II 105.5:1::12.4c 12.9:1::1.81 3862.1:1::436.61 3283.8:1::247.81

III 119.7:1::12.3 9.5:1::2.1 4402.7:1::352.6 3544.1:1::193.7

N 120.9:1:: 13.5d 7.64:1::1.5d 4829. 3:1::309.7d 3788. 8:1::220. 9d

Mean:t SD, 'P<O. 05 vs group I and group IIIand groupN /P<O. 01 vs group I and group III and group N;d P<O. 05 vs groupIII I :sham-operated group (n = 8) ; II :heart failure group( n =8) ; III: losartan treatment group 1( n = 8) ; N : losartan treatment group2 ( n = 8). L VEDP: left ventricular end-diastolic pressure; LVSP: left ventricular systolic pressure; dp/ d1max:the maximal rate of rise of

left ventricular pressure; -dp/dtmax: the maximal rate of drop of left ventricular pressure

Table2. Effects of losartan on BW, and ratio of heart weight and BW, and ratio of LV weight and BW

Group BW(g) HWIBW(mg/g) LVWIBW(mg/g)I 386.6:t38.5 3.17:t0.29 2.34:t0.25II 346. 1:t 26. l' 3.86 :t 0.361 2.76 :t 0.361III 359.5:t39.4d 3.49:t0.25d 2.46:t0.32d

N 372.8:t34.8 3.19:t0.38 2.35:t0.24

Mean:t SD, cP < 0 .05 vs group I and group III and group N ;dP < 0 .05 vs group II and group N ;1P < 0 . 01 vs group I and group IIIand group N .

I : sham-operated group (n = 8) ; II : heart failure group (n = 8) ; III: losartan treatment group 1 ( n = 8); N: losartan treatment group

2 ( n =8) . BW: body weight; HW : heart weight; LVW : left ventricular weight.

Table 3. Effects of losartan on contents of Ang II and index of apoptosis

Plasma AngII Myocardium AngII Index of apoptosis(pg/ml) (pg/g) (%0)

I 105. 17 :t 24.79 214.2 :t 75 4.02 :t 0.78

II 240.48:t 31. 24' 308.6:t 59' 16.13:t 1.17'

III 175.47 :t 38. 50 263. 6 :t 65 13. 57 :t 1. 14

N 128.14:t 42. 71£ 229.7:t 601 11.32:t 1. 34£

Mean:t SD, 'P<O. 01 vs group I and group N ;lp<O. 05 vs group III.I :sham-operated group (n =8) ; II :heart failure group ( n = 8) ; III: losartan treatment group 1( n = 8) ; N : losartan treatment group 2(n =8).

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Apoptosis of cardiomyocyte: The apoptotic in-dex decreased (p < O. 01) after treatment withlosartan. Along with increased losartan doses, theapoptotic index was significant (Table 3). Theapoptotic index decreased (P < O.05) in group N ,compared with group ill.

Expression of BcI-2, Bax and precaspase-3:Bax expression diminished after treatment withlosartan (P < O.01) (Figure 1). On the contrary,Bcl-2 expression increased after treatment withlosartan (p < O. 01) (Figure 1). As a conse-quence, the ratio Bax/Bcl-2 decreased after treat-ment with losartan. Procaspase-3 was decreased inheart failure group rats, compared with sham-oper-ated group and losartan treatment group rats (P <0.01) (Figure 1).

Lane 2 4

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Figure 1. Effects of losartan on expression of apoptosis relativeprotein. Losartan up-regulate Bcl-2 expression and down-regulat-ed Bax expression significantly. The size of molecular weightmarker is shown on the right, and the names of proteins are

shown on the left respectively. Lane 1 : group I ; lane 2: group II ;

lane 3 : group ill: land 4 : group N. Bcl-2 26kD, Bax 21kD, proca-pase-3 32kD.

4 Discussion

.

This experiment's results show that losartandecrease cardiomyocyte apoptosis, as evidence bydecrease in Bax to Bcl-2 ratio, caspase-3 activationprofile, accumulation of TUNEL-positive nuclei.Losartan attenuates LV dysfunction in the heartfailure rats through decreasing cardiomyocyte apop-tosis and changing expression of apoptosis-relatedproteins. LV hypertrophy is an adaptive cardiac re-sponse to the imposition of pressure overload to the

heart[4]. The initial benefits of cardiac hypertro-phy, such as normalization of wall stress andpreservation of systolic force generation, may beoffset during the late stages of chronic hemodynam-ic overload due to progressive cell loss, which maylead to deterioration of cardiac function[5]. The re-

sults show that left ventricle has taken place hyper-trophy and cardiomyocyte of left ventricle has apop-tosis at the same time. Left ventricle hypertrophy


and cardiomyocyte apoptosis remarkable decreaseafter losartan treatment. Along with increase ofdoses, above-mentioned indexes are significantly.Theses results show that cardiomyocyte apoptosisand left ventricle remodeling have a strong correla-tion. These results support important role of apop-tosis in heart failure. Contractive function of LVobvious decline because left ventricle have a mass of

cardiomyocyte apoptosis. These results illuminatethat the relation of LV dysfunction and cardiomy-ocyte apoptosis is close.

Currently, the most popular model, rapid ven-tricular pacing, is simple to produce and represent acondition that is not frequently found in humanheart failure. Also the physiological changes do notpersist beyond removal of the pacing stimulus andventricular dilatation occurs without an initial hy-

pertrophic response[6]. Other methods include vol-ume overload and coronary ligation. These studiesdo not calculate heart failure as a chronic process.Therefore, we have developed a reproducible modelof chronic heart failure that results in marked sys-tolic dysfunction in this study. This model is simi-1ar with clinical syndrome and is successful fromhemodynamic assessment. This is perfect model toreplicate heart failure.

The mechanism of Ang II evocable apoptosis isdisputed. Several observations suggest that A TIstimulation mediates apoptosis through extracellularsignal-regulated kinase (ERK) inhibition[7], ce-ramide accumulation, activation of mitogen-activat-ed protein kinase phosphatase 1 (MKP1) with sub-sequent inhibition of mitogen-activated protein.ki-

nase, and Bcl-2 dephosphorylation[S]. Apoptosiscan be blocked by PD-123319 and PD-123177[9],

whic? are specific ATI blockers. In contrast, someauthors[lO] have shown that AT1 blockade with

losartan can equally protect from apoptosis. AT1stimulation can lead to an increase in Fas, togetherwith a fall in constitutive NO synthase and Blc-2levels. Similar observations have been made in the

hearts of spontaneously hypertensive rats[ll]. TheACE inhibitor captopril reduced apoptosis in spon-taneously hypertensive rats with CHF[12]. Our re-sults show that Ang II of plasma and myocardiumare increased in heart failure rats. But these resultsare decreased after losartan treatment. At the same

time, Bcl-2 and Bax have taken place huge changesafter losartan treatment. Because losartan is selec-

tive AT1R blockade, we support AT1 stimulationcan lead to an increase in Bax, together with a fallin Blc-2 levels. These results show that Ang II isan important factor in inducing cardiomyocyteapoptosis. Because losartan is a selective AT1 re-

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ceptor inhibitor, we think that induced apoptosis ofAng II is through AT1 receptor in heart failure bypart constriction of abdominal aorta of rats.

Cardiac apoptosis can proceed via either death-

receptor or mitochondrial-dependent pathways[13],either of which activates specific caspases, ulti-mately resulting in cell death. The death-receptorpathway proceeds when extracellular death signalligands, such as TNF-a, FasL, bind to their spe-cific cell membrane receptors. Mitochondrial-de-pendent pathways, initiated by the release of cy-tochrome c from mitochondria, are closely regulat-ed by the Bcl-2 family of proteins, which has bothanti-apoptotic (e. g. Bcl-2 and Bcl-XL) and pro-apoptotic (e. g. Bax and Bcl-XS) members. Thecellular balance between anti-apoptotic and pro-apoptotic Bcl-2 family proteins is an important de-terminant of cell survival or death[14,15]. In this

study, our results show that anti-apoptotic Bcl-2and pro-apoptotic Bax have taken placed hugechanges. Above-mentioned index presents oppositechanges after losartan treatment. Several lines ofevidence indicate that myocardial apoptosis con-tributes significantly to remodeling of failing my-ocardium. Caspases, which are activated in variousstages of HF, are the key effectors molecules forapoptosis. Cellular caspases exist as inactive precur-sors and need proteolytic cleavage for activation.Caspase activation co-localizes to apoptotic areas andprecedes DNA degradation and the development of

apoptotic morphology[16]. After a death signal,Bax can associate with the mitochondria to induce

the release of cytochrome c and formation of theapoptosome complex in association with caspase-9activation. Furthermore, caspase can also influencethe contractile machinery of myocytes through

cleavage of troponin[!7]. This can result in contrac-tile dysfunction. Indeed, overexpression of caspase-3 induces contractile dysfunction in mice. Further-more, caspase-3 transgenic mice showed increasedinfarct size and a pronounced susceptibility to

die[18]. Therefore, activated caspase-3 lead to con-tractile dysfunction in rats. Contractile dysfunctionof heart obvious ameliorates after losartan. These

results show that losartan improve heart functionthrough decreasing cardiomyocyte apoptosis.

Pathophysiologic mediators of remodeling,such as catecholamines , Ang 11[15], myocardial

stretch[15,19], inflammatory cytokines[14], and ox-

idative stress[20] can all induce cardiomyocyte apop-tosis. In animal models of HF[16,20,2J] and human

end-stage cardiomyopathy[22] , remodeled my-ocardium displays apoptotic cardiomyocyte loss,variably associated with enhanced expression of

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ulation of Bax[14.15]. However, apoptosis in HF isnot limited to cardiomyocytes. Indeed, a recent

study of canine-pacing tachycardia HF[20] demon-strated progressive apoptosis of myocytes, endothe-lial cells, and fibroblasts indicating that bothparenchymal and interstitial cell loss occur duringmyocardial remodeling. Although earlier studies re-ported apoptotic (TUNEL-positive) nuclei in fail-ing hearts as high as 35 %, most have reportedrates less than 1 % [20-22]. Consistent with these

prior studies, we consider that these are a strongcorrelation betvyeen LV remodeling and apoptoticrate. Our data indicate that increased apoptosis infailing myocardium occurs concomitantly with in-creased activity of mitochondrial pathways. Thechanges in apoptosis-related proteins suggest thatcaspase-3 activation and Bcl-2 down regulation act-ed upstream of Bax and caspase-9. After a deathsignal, Bax can associate with the mitochondria toinduce the release of cytochrome c and formation ofthe apoptosome complex in association with cas-

pase-9 activation[23].In summary, this study also provides direct

evidence that anti-apoptotic Bcl-2 is downregulatedin failing myocardium, indicating a shift in the reg-ulatory balance of the Bcl-2 family proteins favoringapoptosis, and suggesting attendant activation ofmitochondrial-dependent pathways.

Our data indicate that Bcl-2 expression ismarkedly decreased in failure hearts. Losartanameliorates contractile function of LV dysfunctionin the heart failure rats. The mechanism may bedecrease cardiomyocyte apoptosis through balanceof the Bcl-2 family proteins. I

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Correspondence to:Chunjing Fu,Department of PathophysiologyMedical College of Zhengzhou UniversityZhengzhou, Henan 450052, ChinaTelephone: 01186-371-6691-3922Email:[email protected]. en

References1. Hasegawa K, Iwai-Kanai E, Sasayama S. Neurohormonal

regulation of myocardial cell apoptosis during the developmentof heart failure. J Cell Physiol 2001; 186( 1) : II - 8.

2. van Kats JP, Duncker DJ, Haitsma DB, et al. Angiotensin-

converting enzyme inhibition and angiotensin II type 1 recep-tor blockade prevent cardiac remodeling in pigs after myocar-dial infarction: Role of tissue angiotensin II. Circulation2000; 102(26): 1556 - 63.

3. Martin DA, Elkon KB. Mechanisms of apoptosis. Rheum DisClin North Am 2004;30(3) :441- 54.


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Life Science Journal, 2 (1), 2005, Fu, et aI, Changes of Left Ventricular Dysfunction and Cardiomyxyte Apoptosis

4. Katz AM. Maladaptive growth in the failing heart: the car-diomyopathyof overload. Cardiovasc Drugs Ther 2002; 16(3) :245 - 9.

5. Sawa Kostin, Lieven Pool, Albrecht Elsasser, et al. My-ocytes die by multiple mechanisms in failing human hearts.Circ Res 2003 ;92: 715 - 24.

6. Heinke MY, YaoM, ChangD, etal. Apoptosisofventricu-lar and atrial myocytes from pacing-induced canine heart fail-ure. Cardiovasc Res 2001 ;49(1): 127 -134.

7. Lehtonen JY, Daviet L, Nahmias C, et al. Analysis of func-tional domains of angiotensin II type 2 receptor involved inapoptosis. Mol Endocrinol1999; 13: 1051 - 60.

8. Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 re-ceptor mediates programmed cell death. Proc Natl Acad SciUSA 1996;93:156-60.

9. Li D, Yang B, Philips MI, et al. Proapoptotic effects ofANG II in human coronary artery endothelial cells: role ofATI receptor and PKC activation. Am J PhysiolI999;276:H786 - H792 .

10. Li D, Tomson K, Yang B, et al. Modulation of constitutivenitric oxide synthase, Bcl-2 and Fas expression in cultured hu-man coronary endothelial cells exposed to anoxia-reoxygena-tion and angiotensin II: role of ATI receptor activation. Car-diovasc Res 1999 ;41: 109 -15.

11. Fortuno MA, Ravassa S, Etayo JC, et al. Overexpression ofBax protein and enhanced apoptosis in the left ventricle ofspontaneously hypertensive rats: effects of ATI blockade withlosartan. Hypertension 1998;32:280 - 6.

12. Li Z, Bing OH, Long X, et al. Increased cardiomyocyteapoptosis during the transition to heart failure in the sponta-neously hypertensive rat. Am J PhysioI1997;272:H2313-H2319.

13. Haunstetter A, Izumo S. Apoptosis. Basic mechanisms andimplications for cardiovascular disease. Circ Res 1998; 82:1111-29.

14. Parissis ST, Adamopoulos S, Antoniades C, et al. Effects oflevosimendan on circulating pro-inflammatory cytokines and

.I


soluble apoptosis mediators in patients with decompensatedadvanced heart failure. Am J Cardiol 2004; 93 (10) : 1309 -

12.

15.Filippatos G, Tilak M, Pinillos H, Uhal BD. Regulation of

apoptosis by angiotensin II in the heart and lungs. Int J MolMed 2001 ;7(3) :273 - 80.

16. Black SC, Huang J Q, Rezaiefar P, et al. Co-Iocalization of

the cysteine protease Caspase-3 with apoptotic myocytes afterin vivo myocardial ischemia and reperfusion in the rat. J MolCell Cardio11998;30 :733 - 42.

17. Communal C, Sumandea M, de Tombe P, et al. Functional

consequences of caspase activation in cardiac myocytes. ProcNatl Acad Sci USA 2002; 99: 6252 - 6.

18. Condorelli G, Roncarati R, Ross J Jr, et al. Heart-targeted

overexpression of caspase-3 in mice increases infarct size anddepresses cardiac function. Proc Natl Acad Sci USA 2001;14;98(17) :9977 - 82.

19. Annarosa Leri, Yu Liu, Baosheng Li, Fabio Fiordaliso, et al.

Up-regulation of ATI and ATI receptors in postinfarcted hy-pertrophied myocytes and stretch-mediated apoptotic celldeath. Am J Pathol 2000; 156: 1663 -72.

20. Cesselli D, Jakoniuk I, Barlucchi L, Beltrami AP, Hintze

TH, Nadal- Ginard B, et al. Oxidative stress-mediated car-

diac cell death is amajor determinant of ventricular dysfunctionand failure in dog dilated cardiomyopathy. Circ Res 2001 ; 89 :279 - 86.

21. Leri A, Liu y, Malhotra A, Li Q, Stiegler P, Claudio PI',

et al. Pacing-induced heart failure in dogs enhances the ex-pression of p53 and p53-dependent genes in ventricular my-

ocytes. Circulation 1998; 97: 194 - 203.22. Yung CK, Halperin VL, Tomaselli DF, Winslow RL. Gene

expression profiles in end-stage human idiopathic dilated car-

diomyopathy: altered expression of apoptotic and cytoskeletalgenes. Genomics 2004;83(2) :281 - 97.

23. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG,

Youle RJ. Movement of Bax from the cytosol to mitochondriaduring apoptosis. J Cell Bioi 1997; 139: 1281 - 92.

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Life Science Journal, 2 (1), 2005, Wang, et al, Radiosensitive Therapy for Colorectal Cancer

The Radiosensitive Therapy for Colorectal Cancer

Zifa Wangl,2, Tracy Cook1, David Blumbergl

1. Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh,

PA 15232, the United States

2. Orient Organ Transplantation Center, Tianjin First Central Hospital, Tianjin 300192, China

Abstract: The colorectal cancer is one of the most common cancers in the United States. Surgical resection is appro-

priate as the first component of treatment. However ,only less than half of the patients are cured by primary ;esec-tion. Neoadjuvant radiation can decrease tumor size before surgery, and can enable patients previously deemed unre-sectable, to undergo curative surgical resection. Unfortunately since many tumors have hypoxic areas or genetic mu-tations that enable them to be radioresistant, only 22 - 44 % of rectal cancers will respond to neoadjuvant radiation.To overcome the radioresistance of tumors, multiple drugs have been tested as potential radiosensitizers. 5-Fluo-rouracil is one of the most widely used radiosensitizers, which enhances radiosensitization of colorectal cancer due toalteration of cell kinetics. Nitric Oxidize is a promising potential radiosensitizer. AdiNOS treatment of HCf-116 tu-mors significantly delayed tumor doubling time and growth when combined with single or multifractionated radia-tion. Other radiosensitizers, such as, Protein Kinase C-specific Inhibitor PKC412 , Survivn, Caffeine, Bromod-eoxyuridine and Iododeoxyuridine are also briefly discussed in this review. [Life Science Journal. 2005; 2 ( 1) : 55 -60] (ISSN: 1097 - 8135) .

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Keywords: colorectal cancer; neoadjuvant radiation; radioresistance; radiosensi tizers

1 Introduction

The colorectal cancer is the third leading causeof cancer in the United States with approximately150,000 new casesannually. The primarymodalityfor treatment for colorectal cancer is surgical resec-tion. For every 100 patients initially evaluated, 45

are cured by primary resection[l]. Even though re-markable progress has been made in the treatmentof the colorectal cancer during the past twodecades, approximately 44 % of patients with colon

cancer will present with stage III or IV disease[2] .Neoadjuvant radiation can decrease tumor size be-fore surgery, enabling a greater chance of obtaininga tumor-free surgical margin and can enable pa-tients previously deemed unresectable, to undergo

curative surgical resection[3]. Several studies haveexamined preoperative irradiation alone as a neoad-juvant regimen for the treatment of rectal cancer.The European Organization for Research andTreatment of Cancer studied randomized patientswith T2 - T4 tumors to preoperative radiation or

surgery alone. Although the radiation dosage of3,450 cGy was lower than the standard dosage giv-en today, there was a 50 % reduction in the localrecurrence rates in patients treated with radia-

tion[4]. The first prospective, randomized con-trolled study documented that preoperative therapyreduces local recurrence and improves survival[5].

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44 % of rectal cancers will respond to neoadjuvant

therapy[6]. To overcome the radioresistance of tu-mors, multiple drugs such as, £luorodeoxyuridine,caffeine, and nitric oxidize have been tested as po-tential radiosensi tizers[ 7- 9]. In this article, we re-

view some potential radiosensitive agents in colorec-tal cancer.

2 Molecular Mechanisms of Radiosensitization

Radiation is considered to have ionizing poten-tial. The radiation used in radiation therapy (RT)produces several hundred thousand ionization eventsper cell per gray. The absorbed energy causes ejec-tion of primary electrons that go on to ionize othermolecules leading to a complex chain reaction.DNA is the most important target for RT. The evi-dence from experiments shown that irradiation ofthe nucleus, but not the cytoplasm, results in celldeath. The extent of initial DNA damage and thepersistence of this damage are presumably criticalfactors in determining the cellular response to radia-tion. In the process, free radicals, which are neu-tral atoms or molecules that have an unpaired elec-tron, are generated. Because of their unpaired elec-trons, free radicals are very reactive and can reduceor oxidize biological molecules and break theirchemical bonds. Since the most abundant moleculein cells is water, the most common free radicals

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cers, medulloblastomas, melanoma, breast can-

cers, prostate cancers, renal cell cancers, gradesIII and IV brain tumors, ovarian, head and neck

cancers). Radiosensitivities could roughly be divid-ed into two groups: the more radiosensitive groupand the more radioresistant group. The intrinsic ra-diosensitivity of human tumor cells exists amongdifferent histological classes of neoplasm. If, how-ever, tumors contained on average 20 percent hy-poxic cells, the dose needed for equivalent cellkilling increasedby about a factorof 2. 6 - 2. 8. Al-so, there was no correlation between the rankingsof relative radiosensitivities of the various classes oftumor cells at high doses (as in radiosurgery) to thesensitivity at low doses (as in conventional fraction-ated radiotherapy) [18].

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that are generated in a cell after exposure to ioniz-ing irradiation are reactive oxygen species (ROS).Biological agents, such as growth factors, cy-tokines, monoclonal antibodies to cell surface recep-tors, can alter molecular pathways within a cell.Chemotherapeutic and physical agents, such as hy-perthennia, hypoxia and radiation itself, can acti-vate pathways that can also affect the outcome.

An important consequence of the involvementof free radicals in radiation damage is that oxygenplays a major role as a modifier of radiation respons-es. Oxygen influences the nature of the free radi-cals and the lesions that are fonned. The peroxidesand hydroperoxides, in particular, inhibit repair.Under hypoxic conditions the cells are typically 2.5- 3 times more resistant to irradiation than in the

presence of oxygen[10]. A number of studies havedocumented the importance of hypoxia to the out-come of RT. At the molecular level, hypoxia in-duces expression of a number of genes, in particulargenetic programs that are under the control of hy-poxia inducible factor (HIF-l). Some of these(such as erythropoietin, vascular endothelialgrowth factor and tumor necrosis factor alpha(TNF-a)) are clearly aimed at increasing angiogen-esis and increasing oxygen delivery. Hypoxia cantherefore play an important role in driving angio-genesis and tumor expansion. Other hypoxia-in-duced genes (such as p53) are part of a stress re-sponse that encourages cells to undergo cell deathbyapoptosis. Acting in this way, hypoxia serves asa selective force to favor expansion of cells mutated

in p53[1l].Expression or knockout of proto-oncogenes,

tumor-suppressor genes, cytokines, cytokine recep-tors, cell adhesion molecules, redox-active genesand many other genes that are important in deter-mining cell behavior, can influence the outcome of

irradiation[1l-14]. For example, transfer of genesfor growth factors or growth factor receptors thatcause cell proliferation can often achieve radiationresistance. A dominant negative approach anti-sen~, or antibody directed against, for exampleEGFR, can result in cellular radiosensitization and

the level of expression of EGFR by a tumor can de-

tennine radiocurability[15 -17]. Radiosensitizationoften results from transfer of cytokine genes or re--ceptors that slow cell cycle progression or encourageapoptosis.

Radiosensitivity is also related to histologicalclasses. Leith studied the in vitro X-ray radiationsurvival characteristics of 181 cell lines from 12 dif-

ferent classes of exponentially growing human tu-mor cells (sarcomas, lung cancers, colorectal can-


3 Radiosensitive Agents in Colorectal Cancer

3. 1 Fluorodeoxyuridine5-Fluorouracil (5-FU) is one of the most

widely used chemotherapeutic agents, and is knownto be a radiosensitizer. The combination of fluo-

ropyrimidines and radiation has resulted in in-creased control of colorectal cancer in the clinic. In

1980 the North Central Cancer Treatment Groupand the Mayo Clinic compared adjuvant combina-tion chemotherapy and irradiation to radiationalone. Patients undergoing combiration chemother-apy and irradiation had an improvement in both dis-ease-free and overall survival rates compared withthose undergoing irradiation alone[19]. Crane com-

pared the outcome from preoperative chemo~adia-tion and from radiation therapy in the treatment ofrectal cancer in two large, single-institutional expe-riences. Multivariate analysis of the patients inthese groups showed that the use of concurrent5-FU with preoperative radiation therapy for T3and T4 rectal cancer independently increases tumorresponse and may contribute to increased sphincterpreservation in patients with low rectal cancer[20].More recently, 5- Flurouracil has been shown to be aradiosensitizer and acts in part to increase the sus-ceptibilityof tumor cells to the damaging effects ofradiation[21]. Combined radiation with 5-FU-based

chemotherapy is more efficacious than radiationalone in patients with squamous cell cancer of theanus. Similarly, combined chemoradiation has beenshown by Minsky et al. to be more effective thanradiation therapy as a neoadjuvant regimen in pa-

tients with rectal cancer[22]. Minsky and colleaguescompared two groups of unresectable patients whowere nonrandomly treated with combined chemora-diation or radiation alone. In patients undergoingchemoradiation (n = 20), there was a higher com-

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plete pathologic response compared with the 11 pa-tients who underwent radiation alone (20 % vs.

0% ).The radiosensitization by 5-fluorodeoxyuridine

is in part due to alteration of cell kinetics and redis-tribution of cells throughout the cycle. In laborato-ry preliminary work showed that 2 h exposures ofHT 29 human colon carcinoma cells to relativelylow levels of 5-fluorodeoxyuridine resulted in ex-tended thymidylate synthase inhibition after thedrug was removed (up to 30 h after treatment withO. 5 microM 5- fluorodeoxyuridine). The low cyto-toxicity associated with this treatment simplified ef-forts to test the effects of extended thymidylate

synthase inhibition on radiosensitivity of HT 29cells. Although thymidylate synthase was com-pletely inhibited at the end of the 2 h exposure, anincrease in the radiosensitivity of the cells was notevident until 16 h after the removal of drug. Flow

cytometric analysis showed that cells accumulatedin early S phase over time, and the increase in radi-ation sensitivity of the entire population followedthe increase of the proportion of cells in early Sphase, a relatively radiosensitive phase of the cellcycle. This treatment schedule was compared with24 h continuous exposure, and was found that thesame maximum increase in radiosensitivity was

achieved by both treatment strategies. However,more cytotoxicity was associated with continuous

exposure[23] .Adenoviral transduction of the Escherichia coli

uracil phosphoribosyltransferase (UPRT) gene in-duced marked sensitivity in human colon cancercells to 5-FU. Kayama investigated the efficacy ofvirally directed UPRT and 5-FU to enhance the ra-diosensitivity of HT 29 human colon cancer cells.In vivo chemoradio-gene therapy using the UPRT /5- FU /radiation system showed tumor regressive ef-fects even against large HT 29-established subcuta-neous tumors in nude mice[24] .

3.2 Protein kinase C-specific inhibitor PKC412The cellular response to ionizing radiation is

governed by the DNA-damage recognition processbut is also modulated by cytoplasmic signal trans-duction cascades that are part of the cellular stressresponse. Growth-promoting protein kinase C ac-tivity antagonizes irradiation-induced cell death,and, therefore, protein kinase C inhibitors mightbe potent radiosensitizers. The antiproliferative andradiosensitizingeffect of the novel N -benwylatedstaurosporine analogue PKC412 was tested in vitroagainst genetically defined p53-wild type ( + / + )and p53-deficient (- / -) murine fibrosarcomacells and in vivo against radioresistant p53 - /-

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murine fibrosarcoma and human colon adenocarci-

noma tumor xenograft (SW480, p53-mutated).PKC412 sensitizedboth p53 + / + and p53 - /-tumor cells in vitro and in vivo for treatment with

ionizing radiation but with a different mechanism ofradiosensitization depending on the p53 status. Inp53 + / + , cells combined treatment with PKC412and ionizing radiation drastically induced apoptoticcell death, whereas no apoptosis induction could beobserved in p53-deficient cells in vitro and in histo-logical tumor sections. Combined treatment result-ed in an increased ~ cell cycle distribution in p53- / - cells at PKC412 concentrationsthat did notalter cell cycle distribution when applied alone. Invivo, a minimal treatment regimen during 4 con-secutive days of PKC412 (4 X 100 mglkg) in com-bination with ionizing radiation (4 X 3 Gy) exerteda substantial tumor growth delay for both p53-dis-functional tumor xenografts and showed that theclinically relevant protein kinase C inhibitorPKC412 is a promising new radiosensitizer with a

potentially broad therapeutic window[7].3.3 Survivin

Spontaneous apoptosis has been shown to pre-dict tumor response to radiochemotherapy in rectalcancer in vivo. Recently, a novel member of theinhibitor of apoptosis protein family, designatedsurvivin, was identified. The inverse correlation of

survivin-expression with spontaneous and radiation-induced apoptosis suggests that survivin is an im-portant inhibitor of apoptosis in colorectal cancercell lines. Analysis.of survivin mRNA or proteinexpression may therefore provide predictive irifor-mation on radio- and chemoresistance of individual

colorectal tumors[2S]. Rodel investigated the impact

of survivin expression on tumor cell apoptosis inthree colorectal cell lines of different intrinsic ra-

diosensitivities. In vitro analysis revealed higher

spontaneous and higher radiation-induced apoptosisrates in the radiosensitive line (SW 48), as com-

pared with the more resistant line (SW 480).SW 480 was characterized by a higher spontaneous

expression and a pronounced induction of survivin48 h after irradiation, whereas survivin expressionwas low when untreated and not increased after ir-radiation in the most radiosensitive line SW 48.3.4 Caffeine

Boonkitticharoen investigated the effect of caf-feine, the methylated xanthine, in sensitizing thelethal action of ionizing radiation in vitro in humancancer cells. Plateau phase cultures of colon adeno-carcinoma, after absorbing doses of 2 Gy, survived

at a rate of 56. 30 per cent for colon cancer[26] .3.5 Bromodeoxyuridine and iododeoxyuridine

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man colorectal cancer corroborate this inverse rela-

tionship between iNOS expression and tumor pro-gression[35]. Lack of iNOS in knock-out mice pro-motes intestinal tumors further substantiating therole of iNOS in host defense against colorectal can-cer[36]. Given these studies, the use of iNOS genetransfer would be a rationale means to improve theimpairment in tumor defense mechanisms that uti-lize NO.

Although NO is a promising radiosensitizer,and NO itself induce apoptosis, the use of NOdonors to augment the effects of radiation in vivohas significant limitations, since in vivo adminis-tration of these agents results in systemic hypoten-sion and may increase tumor perfusion and oxy-genation, potentially promoting tumor growth[37].Overexpression of iNOS in tumors by localized di-rect intratumoral injection of the iNOS gene has thepotential of minimizing the systemic side effects ofNO while maintaining the salutary tumoricidal ef-fects of high output paracrine NO release. We havepreviously examined the effects of direct intratu-moral gene delivery of iNOS combined with bothsingle and multifractionated irradiation on growthof HCT-116 colorectal tumors in nude mice. Adi-NOS treatment of HCT-116 tumors significantly(P :s;;;O.005) delayed tumor doubling time andgrowth when combined with single or multifrac-tionated radiation in nude mice. We have previous-ly demonstrated that adenoviral delivery of the iN-OS gene enhances radiation-induced apoptosis incolorectal cancer cells[38]. We have also demon-strated that overexpression of the human induciblenitric oxide synthase gene by adenoviral gene deliv-ery radiosensitizes both human colorectal cancercells and tumors associated with increased apoptosisin nude mice[39]. The mechanism of NO radiosensi-tization may be that NO increases angiogenesis andthen increases oxygen delivery.

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Bromodeoxyuridine (BrdU) and iQdo-deoxyuridin (IdU) have similar the radiosensitizingeffects on colorectal cancer. Miller conducted con-currently to characterize its effects on the shape ofthe radiation survival curves of cells of two humancolon cancer cell lines, HT 29 and HCT 116. Theefficiency of radiosensitization by BrdU, expressedas a function of percentage thymidine replacement,was lower when compared to IdU in both cell lines.The major radiosensitizing effect of BrdU was man-ifest as an increase in the initial slope (alpha), justas observed for IdU. However, with BrdU, incontrast to IdU, an increase in curvature (re-

. pairable damage) was also evident. Cells of themore radiosensitive line, HCT 116, showed lesssensitization by either BrdU or IdU than cells ofthe more radioresistant line, HT 29. These resultswere consistent with the proposed mechanism of ra-diosensitization being an increase in the single-hitcharacter of low-LET radiation. The radiosensi-tizing effects of both analogs were largest in thelow-dose region of the survival curve[27].3.6 Nitric oxidize

Nitric oxidize (NO). is another potentialpromising radiosensitizer. Multiple studies usingNO donors have examined its effects in radiosensi-tizing tumor cells. Initial studies examining NOdonors indicated that NO enhanced the radiosensi-

tivity of hypoxic mammalian cells in vitro[28].NO's radiosensitizing property was first demon-strated in 1957 by Howard Flanders in Nature[29].Flanders interest in NO was based on the fact thatlike oxygen, NO had a reactive electron making it afree radical. He hypothesized and demonstratedthat NO because of this property could effectivelysubstitute for oxygen as an electrophile and sensitizebacteria to radiation under anaerobic conditions.Subsequently others have shown that NO could alsoradiosensitize normal human cells[28,3o,31J.NO wasfound to be as effective as oxygen in radiosensitizinghypoxic mammalian cells[3O]. One group demon-strated that cytokines could induce endogenous NOproduction and radiosensitize hypoxic breast cancercells[32].

NO itself also has effect on colorectal cancer.In one large study of colorectal cancers, iNOS ac-tivity and protein expression correlated inverselywith advanced stage of disease[33]. Pre-malignantcolorectal adenomas may have the highest iNOS ac-tivity. iNOS overexpression in these polyps is asso-ciated with a specific point mutation in the p53gene, suggesting that NO may function to initiatedevelopment of colorectal cancer rather than stimu-lating cancer progression[34]. Other studies in hu-


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Correspondenceto:Zifa WangOrient Organ Transplantation CenterTianjin First Central HospitalTianjin 300192, ChinaTelephone: 01186-22-2362-6560Fax: 01186-22-2368-2662Email:wangz£[email protected]

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jan NA, Read TE, Myerson RJ. The addition of continuousinfusion 5-FU to preoperative radiation therapy increases tu-mor response, leading to increased sphincter preservation in

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21. Rotman M,Aziz H. Concomitant continuous infusion chemotherapyand radiation. Cancer 1990; 65 :823 - 35 .

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23. Miller EM, Kinsella Tl. Radia;ensitization by fluorodeoxyuridine:

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24. Koyama F, Fujii H, Mukogawa T, Ueno M, Hamada H,Ishikawa H, Doi S, Nakao T, Matsumoto H, Shimatani H,

Takeuchi T, Nakajima Y. Chemo-radio-gene therapy for col-orectal cancer cells using Escherichia coli uracil phosphori-

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Rodel F. Spontaneous and radiation-induced apoptosis in col-orectal carcinoma cells with different intrinsic radiosensitivi-

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of radiosensitization by halogenated pyrimidines. II. Ra-diosensitization of human colon cancer cells by bromodeoxyuri-dine. Radiat Res 1992;131:90-7.

28. Mitchell lB, Wink DA, DeGraff W, Gamson 1, Keefer LK,

Krishna Me. Hypoxic mammalian cell radiosensitizati,m bynitric oxide. Cancer Re.'i 1993 ;53 :5845 - 8.

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tizing of hypoxic tumor cells in vitro by nitric oxide. Int 1Radiat Oncol Biol Phys 1996: 36: 377 - 83.

31. Dewey DL. Effect of oxygen and nitric oxide on the radiosen-

sitivityof human cells in tissue culture. Nature 1960; 186: 780-2.

32.1anssens MY, Van den Berge DL, Verovski VN, Monsaert

C, Storme GA. Activation of inducible nitric oxide synthaseresults in nitric oxide- mediated radiosensitization of hypoxic

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SM, Harrington AM, Shields PG, Felley-Bosco E, HussainSP, Harris CC. Relationship between p53 mutations and in-

ducible nitric oxide synthase expression in human colorectalcancer. 1 Natl Cancer Inst 1999; 91: 86 - 8.

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sion: lessons from experimental tumors. Cancer and Metasta-sis Reviews 1998;17:91-106.

36. Scott DJ, Hull MA, Cartwright EJ, Lam WK, Tisbury A,

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inducible nitric oxide synthase gene enhances radiation-induced

apoptosis in colorectal cancer cells via a caspase-dependentmechanism. Nitric Oxide 2003; 8: 119 - 26.

39. Wang Z, Cook T, Alber S, Liu K, Kovesdi I, Watkins SK,

V odovots Y, Billiar TR, Blumberg D. Adenoviral gene trans-

fer of the human inducible nitric oxide synthase gene (iNOS)enhances the radiation response of human colorectal cancer as-

sociated with alterations in tumor vascaularity. Cancer Re-search 2004; 64( 4) : 1386 - 95.

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Poulsom R, Markham AF, Bonifer C, Coletta PL. Lack ofinducible nitric oxide synthase promotes intestinal tumorigene-sis in the APC(Min/ + ) mouse. Gastroenterology 2001; 121:889 - 99.

37. Van de Casteele M, Hosli M, Sagesser H, Reichen J. Intra-portal administration of glyceryl trinitrate or nitroprusside ex-erts more systemic than intrahepatic effects in anaesthetisedcirrhotic rats. J Hepatol1999;31:300-5.

38. Chung P, Cook T, Liu K, Vodovotz Y, Zamora R, Finkel-stein S, Billiar T, Blumberg D. Overexpression of the human

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Life Science Journal, 2 (1 ), 2005, Sun, et al, Short-term Prognostic Value of B-type Natriuretic Peptide

B-type Natriuretic Peptide in Predicting Short-term

Mortality in Patients with Acute Coronary Syndromes:A Preliminary Report

Tongwen Sunl, Lexin Wangl,2, Shuxiang Zhan~, Yanzhou Zhang!

1. Department of Cardiology!Department of Emergency, First Affiliated Hospital,

Zhengzhou University, Zhengzhou, Henan 450052, China

2. Charles Sturt University, New South Wales 2650, Australia

3. Department of Imernational Exchange and Cooperation, Zhengzhou University, Zhengzhou,Henan 450001, China

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Abstract: Objective. This study was designed to investigate the usefulness of B-type natriuretic peptide (BNP) inpredicting the short-tenn mortality in patients with acute coronary syndromes (ACS). Methods. A total of 106 pa-tients with ACS, whose blood BNP concentration were measured with Triage BNP test, within 1 - 3 days after on-set of ischemic symptoms, were divided into two groups: the survival and the non-survival group, according to theresults of 4-week follow-up. Results. The blood BNP concentration was significantly higher in the non-survival thanin the survival (P<O.OOO1) group; univariate analysis showed that BNP (~172 ng/L, median) and Killip class(II - N) were prognostic factors of the short-tenn cardiac death in patients with ACS (P < 0.0005 and P = o.001); stepwise logistic regression analysis indicated that smoking and BNP (~ 596 ng/L, 75 % percentile) wereindependent predictors of short-tenn cardiac death in patients with ACS (OR= 5. 5,P = O. 028; OR= 21.19, P<0.0005). Conclusion. BNP might predict the 4-week mortality in patients with ACS. [Life Science Journal.2005; 2( 1) :61 - 64] (ISSN: 1097- 8135).

Keywords:B-typelbrain natriuretic peptide; acute coronary syndrome; prognosis

1 Introduction

Acute coronary syndromes (ACS) encompass a

continuum of cardiac ischemic events, ranging from

unstable angina pectoris with no biochemical evi-

dence of myocardial necrosis to ST-elevation acute

myocardial infarction (AMI) [1]. Acute predictionof the mortality in patients with ACS is critically

important to facilitate the application of preventa-

tive measures. B-typelbrain natriuretic peptide

(BNP), which is mainly secreted by ventricular

myocytes, is increased in patients with systolic ordiastolic heart failure[2,13]. Numerous clinical trials

have demonstrated that BNP could be used to diag-

nose cardiac dysfunction[4,S]. It has also beenshown that an increase in BNP values is associated

with a higher mortality and morbidity rate in pa-tients with ventricular failure or ACS[6 -10]. In this

study, we investigated the usefulness of blood BNP

in predicting 4-week cardiac death in patients withACS.

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Patient selection

Patients were included if they presented within72 hours after onset of ischemic discomfort and met

one or more of the following criteria: electrocardio-graphic changes (ST-segment depression or eleva-tion of at least 0.5 mm, T-wave inversion of atleast 3 mm in at least three leads, or left bundle-

branch block), elevated levels of cardiac markers,or a history of coronary disease.

One hundred and six patients admitted to thecoronary care unit (CCU) at the First AffiliatedHospital of Zhengzhou University, were enrolled inthe study between September 2003 and May 2004.Thirty-three patients had ST elevation myocardialinfarction (STEMI), 7 had non-ST elevation my-ocardial infarction (NSTEMI) and 66 had unstable

angina pectoris (UAP). Seventy-one patients weremale and 35 were female. Mean age was 62 years(37 - 85). They were divided into 2 groups ac-

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cording to the 4-week follow-up results: the sur-vival ( n = 93, 88 %) and the non-survival (n =13, 12 % ) .2.2 Measurement of blood BNP level

A point-of-care test of fluorescence immunoas-say for the quantification of BNP was used (BiositeDiagnostics Inc, USA), 2 ml of intravenous bloodwas collected at the early morning after 1 - 3 dayon admission and BNP was determined within 20

min. The range of measurement was 5 - 5000 ng/L.2.3 Statistical analysis

Data of BNP were presented with categoricalvariables and with continuous variables. The differ-

ence of circulating BNP between the survival andthe non-survival were compared by Wilcoxon signedrank test. Categorical variables were compared byx2-test. Both univariate and stepwise multivariateLogistic forward regression analysis were used to e-valuate the prognostic value of the parameters. Thecriterion for inclusion in the regression equation wasP < 0.05. The criterion for exclusion from the re-

gression equation was P > O. 1. A value of P <0.05 was considered statistically significant. Alldata analysis was performed using the StatisticalPackage for Social Sciences (SPSS 11. 0) .

3 Results

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The mean of BNP for all patients was 511.05:t 799. 57 ng/L (5 - 5000 ng/L), median was 172ngiL, and quartile range was 37. 5 - 596 ng/L.There were 12 patients whosecirculating BNP wasabove 172 ng/L (median) and 10 patients whosecirculating BNP was above 596 ng/L (75 per-centile) in the non-suverial ( n = 13). Patients withcirculating BNP above 172 ng/L and those withcirculating BNP above 596 ng/L had a mortality of23.1 % (12/52) and 40.0% (10/25) at 4 weeks,respectively. Rank correlation analysis demonstrat-ed that the circulating BNP levels and Killip classwere positively correlated with cardiac death (r =0.429, P<0.0005;r=0.316, P=O.OO1).

The circulating BNP level in the non-survivalgroup was significantly higher than the survivalgroup (median 1169 vs 126 ng/L, U = 148, P <0.0001, Figure 1). Univariate analysis (Table 1)showed that BNP (~172 ng/L, median) and Kil-lip class ( II- N) were prognostic factors of short-term cardiac death in patients with ACS(P<O.OOl and P=O.OO1).

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Figure 1. Comparison of circulating B-type natriuretic peptide be-

tween the survival and non-survival groups.

Table 1. Comparison between the survival and non-survival

groups

Variables

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Stepwise logistic forward regression analysisdemonstrated that smoking and circulating BNP(above 596 ng/L,75 percentile) were independentpredictors of cardiac death at 4 weeks (OR = 5. 5,95% confidence interval 1. 20 - 25. 30, P =0.028; OR = 21. 19, 95% confidence interval4. 53- 99. 06, P < O.0005); covariateswere age,sex, smoking, drinking, history of heart failure,history of myocardial infarction, hypertension, dia-betes mellitus, Killip class and circulating BNP(above 596 ng/L). The risk of death in patientswith smoking and circulating BNP level above 596ng/L were 5. 5 times and 21 times higher than non-smokers and those with circulating BNP level under596 ng/L, respectively.

2000

1500

1000 ...500

Survivals Non-survivals

Sex(men/women)

Age( <60060)

Smoking

Drinking

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... 4 Discussions

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BNP is a 32-amino-acid polypeptide secretedby the cardiac ventricles in response to increasedstretch or wall tension[3,4]. Its diverse actions in-

clude natriuresis; vasodilation, inhibition of therennin-angiotensin-aldosterone system, and inhibi-tion of sympathetic nerve activity[8]. Previousstudies have shown that BNP could be used to diag-nose and to evaluate the prognosis of congestiveheart failure[6]. This study showed that BNP is anindependent predictor of short-term prognosis inpatients with ACS. The risk of death in patientswith circulating BNP level above 596 ng/L was 21times higher than those with circulating BNP levelunder 596 ng/L.

A recent study found that the base-line level ofBNP was correlated with the risk of death, heartfailure, and myocardial infarction 10 months afterthe ischemic event[7] . In patients with non-ST ele-

vation ACS, the mortality at 7 day and 6 month al-so increased significantly in patients with BNPabove 80 ng/L[8]. Our preliminary study on 4-week mortality yielded similar results, indicatingthat measuring the circulating BNP levels within 3days of ischemic event can give clinical physicianscritical information in evaluating the severity of thedisorder. For patients with very high circulatingBNP levels (above 596 ng/L), regardless theseverity of ischemic symptoms, physicians shouldpay great attention to these patients and treat themaggressively, in order to decrease mortality, andimprove the short-term prognosis.

It is unclear why BNP is elevated in our pa-tients with ACS who did not have noticeable heart

failure. The release of BNP from myocardial cells isprovoked by a variety of stimuli, including hyposi-a, ischemia, increased wall stress, and dilation ofventricles[IO]. In rats, rapid induction of ventricu-lar BNP gene expression and BNP production wasfound in the infarct region and the ischemic periin-farct region. BNP expression was also identified inthe nonischemic surrounding myocardium 4 hoursafter ischemia or infarction[!l]. Transient increase

in BNP secretion can be found in patients undergo-ing percutaneous transluminal coronary angioplasty(PTCA)[I2]. Above studies indicate that myocar-dial ischemia can induce synthesis and secretion ofBNP, even without myocyte necrosis and overt left

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ventricular dysfunction.The pathophysiologic mechanisms and clinical

significance that inducing BNP synthesis and secre-tion by myocardial ischemia are follows[IO]. First,elevation of BNP may result from acute ventriculardysfunction caused by myocyte necrosis, and therisk associated with elevated levels could therefore

be related to the effect of ventricular impairment onmortality or to a new or progressive heart failure.Second, elevation of BNP levels may result from a-cute myocardial stretch caused by ischemia withoutmyocyte necrosis, and the risk associated with ele-vated levels could therefore be related to the extent

of ischemia and the consequent risks of future in-farction and arrhythmia.

This study also demonstrated that smokingwas a significant predictor of cardiac death in pa-tients with ACS. Smoking and hyperlipidemia werethe main clinical risk factors for coronary spasm a-mong Chinese[13]. Brummett's study indicated thatdepressive symptom, smoking, and sedentary be-havior were independent predictors of mortality inpatients with coronary artery disease[14].This studyaccords with above two studies.

The limitations of this study are that the sam-ples were relatively small and the time of follow-upwas very short. Larger and double-blinded trialsmay be required to validate the values of BNP inthe risk stratification for patients with ACS.

5 Conclusions

Measuring blood BNP within 1- 3 days of ad-mission could provide important clinical informationfor predicting the prognosis of ACS. The risk ofdeath in patients with circulating BNP level above596 ng/L is 21 times higher than those with circu-lating BNP level under 596 ng/L. Therefore, ag-gressive treatment should be applied to patientswith very high circulating BNP levels.

Correspondenceto:Tongwen SunDepartment of CardiologyFirst Affiliated HospitalZhengzhou UniversityZhengzhou, Henan 450052, ChinaTelephone: 01186-13838516916Fax: 01186-371-697-0906

Email: [email protected]

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(14):1014-21.

8. Morrow DA, de Lemos JA, SabatineMS, et al. Evalua-

tion of B-type natriuretic peptide for risk assessmentin

unstable angina/non-ST-elevation myocardial infarction.J AmColiCardiol2003;41(8): 1264~ 72.

9. Sabatine MS, Morrow DA, de Lemos JA, et al. Multi-

marker approach to risk stratification in non-ST-elevation

acute coronary syndromes: simultaneous assessment of

troponin I, C-reactive protein, and B- type natriuretic

peptide. Circulation 2002: 105(11): 1760 -7.10. White HD, French JK. Use of natriuretic peptide levels

for risk assessment in non-ST-elevation acute coronary

syndrome. J Am Coli Cardiol 2003 ;42( 11): 1917 - 20.11. Hama N, Itoh H, Shirakami G, et al. Rapid ventricular

induction of brain natriuretic peptide gene expression in

experimental acute myocardial infarction. Circulation1995: 92(10) : 1558 - 64.

12. Tateishi J, Masutani M, Ohyanagi M, et aI. Transient

increase in plasma brain (B-type) natriuretic peptide after

percu taneous transluminal coronary angioplasty. ClinCardio 2000 ;23(7): 776 - 80.

13. Xiang DC, Kleber FX. Smoking and hyperlipidemia are

important risk factors for coronary artery spasm. ChinMed J 2003; 116(4 ):510 - 3.

14. Brummett BH, Babyak MA, Siegler IC, et al. Effect of

smoking and sedentary behavior on the association be-

tween depressivesymptoms and mortality from coronaryheart disease. Am J Cardio 2003:92(9) :529 - 32.

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References

1. Ross R. Atherosclerosis: an inflammatory disease. N En-

gl J Moo1999;340(1) :115- 26.2. Yasue H, Yoshimura M, Sumida H, et al. Localization

and machanism of secretion of B-type natriuretic peptidein comparison with those of A-type natriuretic peptide innormal subjects and patients with heart failure. Circula-

tion 1994; 90(1):195-203.

3. Yoshimura M, Yasue H, Okumura K, et al. Different

secretion patterns of atrial natriuretic peptide and brain

natriuretic peptide in patients with congestive heart fail-ure. Circulation 1993; 87(2):464-9.

4. Wei TM, Zeng CL, Chen LP, et al. Role of B-type na-triuretic peptide in the diagnosis of left ventricular dias-

tolic dysfunction in patients with hypertension. Eur JHeart Fail 2005;7 (1) :75 - 9.

5. Multinational Study Investigators. Rapid measurement of

B-type natriuretic peptide in the emergency diagnosis of

heart failure. N Engl J Med 2002:347(3): 161-7.6. Anand IS, Fisher LD, Chiang YT, et al. Changes in

brain natriuretic peptide and norepinephrine over time

and mortality and morbidity in the Valsartan Heart Fail-

ure Trial (Val-HeFT). Circulation 2003: 107 (9) : 1278-83.

7. de Lemm JA, Marrow DA, Bentley JH, et al. The

prognostic value of B-type natriuretic peptide in patients

with acute coronary syndromes. N Engl J Med 2001 ;345

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Life Science Journal, 2 (1), 2005, Qiao, et al, Transposition of Pedicled Adrenal for Treatment of Cushing's Disease

Transposition of Pedicled Adrenal for the Treatment

of Cushing's Disease

Baoping Qiao, Gaoxian Zhao, Weixing Zhang, Haiyang Jiang

Department of Urology, First Affiliated Hospital, Zhengzhou University,

Zhengzhou, Henan 450052, China;

Abstract: Objective. To explore a rational surgical treatment for the Cushing's disease. Methods. 68 patients withCushing's disease were treated in our department with a new operation designed by authors between October 1990and October 2004. All the patients pr(5ented typical Cushing's syndrome. The level of 24 hour urinary excretion of17- hydroxycorticosteroids (17 -OHCS) fluctuated from 40 - 125 flmol/24 h (62. 5 i: 27. 8). The diurnal rhythm ofthe plasma cortisol level was lost in all cases. In the operation, left adrenal gland was freed from surrounding tissuewith the tissues between upper pole of adrenal and diaphragm was saved. Thus, a pedicle about 1. 5 = in widthand4 - 6 cm in length could be formed and the gland could be transposed into dorsal subcutaneous tissue through11th intercostal space. Then right total adrenalectomy was performed. Postoperative histological diagnoses were bi-lateral adrenocortical hyperplasia. Adrenocortical substitution therapy was administered after operation and stoppedcompletely 10 to 14 postoperative days in all patients. Results. All of patients recovered well after operation. 56 pa-tients have been followed for six months to nine years. 52 patients were well. In these patients, plasma and urinarycortisol values had returned to normal levels and all clinical signs of Cushing's disease were absent. 42 patients hadbeen followed up more than 3 years. The level of 24 hour urinary excretion of 17-0HCS decreased to 22.6 i: 9.2

flmol/24 h (P<O.OOl) in 3 postoperative years. Hypoadrenocorticism developed in 4 patients and skin hyperpig-mentation was observed. The patients required adrenocortical replacement therapy. No recurrent Cushing's sYn-drome was observed in all patients. Conclusions. It's simple to make a vascular pedicle on upper pole of the adrenaland transpose the pedicled adrenal through 11th intercostal space into the dorsal subcutis. Second operation is easyto perform if relapse of syndrome occurs. Nowadays, this procedure may be a rational technique for the treatment ofCushing's disease. [Life Science Journal. 2005:65 - 67J (ISSN: 1097 - 8135).

Keywords: adrenal: Cushing's disease: surgical treatment

1 Introduction

It' s well recognized that Cushing's disease iscaused by overproduction of cortisol. The majorityof cases (85 %) are due to bilateral adrenocortical

hyperplasia stimulated by overproduction of pitu-itary adrenocorticotropic hormone (ACTH) .Though many treatments for Cushing's disease are

presented, there is no ideal one up to now[l]. Inthe clinical practice, we have found that adrenalvascular and the surrounding tissue are very abun-dant and the superior adrenal artery and vein withtheir surrounding tissues can be dissected andformed a vascular pedicle which is about 4 - 6 cm inlength. Therefore, an operation with transpositionof pedicled adrenal beneath dorsal subcutaneous wasdesigned. Form October 1990 to October 2004, 68cases of Cushing's disease were treated with thistechnique and satisfactory results have beenachieved.

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2 Operative Technique

Under extradural anesthesia, 11th intercostal

incision is employed. The left adrenal is operatedon firstly. The dorsal and upper parts of Gerota' sfascia are incised and Gerota' s fascia is dissected.

The adrenal gland is exposed and explored todemonstrate the hyperplastic adrenal gland. Thegland is freed laterally, inferiorly, anteriorly, pos-teriorly and medially. The tissues between upperpole of adrenal and diaphragm are saved as much aspossible for protection of superior adrenal artery andvein that come from inferior phrenic vessels. Thefreed gland is pulled upwards and laterad. If themedial parts of tissues are felt tense, some of themcould be divided. Then, a satisfactory pedicle isformed and sometimes several little vessels that passinto gland or its surrounding tissues can be seen.The pedicleis about 1.5 cm in width and 4 - 6 cmin length, so the gland can be transposed into dor-sal subcutaneous tissue though 11 th intercostalspace easily. The bases attach to diaphragm as afan. The way of the transposition ought to be as

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and utilize them. Our experience reveal that it ispossible to moderately free superior adrenal artery

and vein 4 - 6 cm long. According to literature[2],the superior adrenal artery comes from inferiorphrenic artery that is the main blood supply for a-drenal and the branches of it form a vascular circle

around adrenals with other artery. The venousdrainage of adrenal gland is almost exclusively via

the inferior adrenal vein. The sup~rior, medial andinferior adrenal arteries all paired by their relativeveins. Generally superior, medial veins don't takemain role in adrenal vein drainage, but when inferi-or adrenal vein is obstructed, they can exert

drainage function[2,3]. Therefore the blood circula-tion in upper pole of adrenal may be maintained andthe necrosis can be avoided providing superiorartery and vein are preserved. Between upper poleof adrenal and diaphragm, there are a lot of connec-

tive tissues. Superior adrenal vessel passes throughthem. In order to form a satisfactory pedicle, thesetissue should be preserved as much as possible. Inthis group of patients, the length of pedicle wasabout 4 cm in children and 5 - 6 cm in adults. Insome pedicles there were several vessels to be seenand oozing of blood on severed gland edges was visi-ble in some cases, but these was not obvious in oth-

er cases. In the following up the patients, wefound that the adrenal function in these patientswas similar. Therefore, we realiz~d that it's easyto form a vascular pedicle on the upper pole of a-drenal and it's not important for survival and func-tion of transposed adrenal if oozing of blood on sev-ered edges is visible or not, providing the pedicleare thick enough.

It's important for this procedure to form a sat-isfactory vascular pedicle. An appropriate operativeapproach is a key. Anterior transabdominal ap-proach was employed on first patient. It was diffi-cult to expose the upper pole of adrenal gland andmake a 5 cm long pedicle. When we transposed thispedicled adrenal into dorsal subcutis, the pediclefelt tension as the way is longer. From then on,11 th intercostal incisions were employed routinely.Through 11th intercostal space and external lateraledge of sacrospinal muscle, the transposition way isstraight and short for fixing gland into the dorsalsubcutis with flaccid pedicles. Therefore, we rec-ommend 11th intercostal incision for this proce-dure.

About the problems of ,the amounts of re-mained adrenal, traditionally 10 - 20 % of adrenalare remained in subtotal adrenalectomy and maymeet the daily needs of human bodies. In this

group, more glands were remained[4]. 90% of each

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short as possible to reduce the pedicle tense. If di-aphragm is in the way of transposition, parts ofcrus of diaphragm should be divided. This proce-dure makes the transposition way shorter andstraighter remarkably. The pedicle is fixed with su-tures with nearby intercostal muscle. 10 % of a-drenal is removed for histological examination. Theremained adrenal is fixed beneath the dorsal sub-

cutis. The gland is marked with silver clips for X-ray location. Then right total adrenalectomy is per-formed. If the transposition on left gland is unsatis-factory or unsuccessful, a transposition on right a-drenal could be performed as procedures above, butit's more difficult than on the left due to the prox-imity of the liver and vena cave. Adrenocorticalsubstitution therapy was administered after opera-tion and stopped completely 10 to 14 postoperativedays in all patients.

3 Clinical Material

.I

We reviewed the records of 68 patients withCushing's disease treated in our department be-tween October 1990 and October 2004. Cases of

pituitary adenomas were excluded. Fifty-three ofpatients were female and fifteen were male, andtheir ages rangedfrom 11- 50 years (mean 36. 5) .All the patients presented typical Cushing's syn-drome. The level of 24 hour urinary excretion of17-OHCS fluctuated from 40 - 125 (62. 5 :t 27. 8)!1mo1l24 h. The diurnal rhythm of the plasma cor-tisol level was lost in all cases. Skullroentgenography and tomography of the sella turci-ca were performed to exclude the presence of a pi-tuitary tumor. However, no pituitary abnormalitywas demonstrated. Studies to localize adrenal hy-perplasia with tomography were also performed.Postoperative histological diagnoses all were bilater-al adrenal adrenal cortical hyperplasia. 56 patientshave been followed for six months to nine years. 52patients were well. In these patients, plasma andurinary cortisol values had returned to normal levelsand all clinical signs of Cushing's disease were ab-sent. 42 patients have been followed up more than3 years. The level of 24 hour urinary excretion of17-OHCS decreased to 22. 6 :t 9. 2 !1mo1l24 h (P <O. 001) in 3 postoperative years. Hypoadreno-corticism developed in 4 patients and skin hyperpig-mentation was observed. The patients required a-drenocortical replacement therapy. No recurrentCushing's syndrome was observed in all patients.

4 Discussion

It's well known that the vessels of adrenal are

so multiple and variable that it is difficult to dissect


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adrenal gland was remained. The reason for this isthat we remained 30 % of gland in the operation infirst several patients and hypoadrenocorticism de-veloped in 2 patients. Thereafter, we try our bestto remain more glands. Up to now, we don't findany cases of hyperadrenocorticism. We think thereasonable explanations are that: (1) Media and in-ferior adrenal vessels are divided completely and su-perior adrenal vessel is divided partially in the oper-ation. The blood supply to the adrenal is not good,only parts of upper pole gland can survive postoper-atively no matter how many glands are preserved.(2) The best place for the development of adrenalis its anatomy location. Dorsal subcutis is not suit-able for its development.

In brief, we conclude that it's simple to makea vascular pedicle on upper pole of the adrenal andtranspose the pedicled adrenal through 11th inter-costal space into the dorsal subcutis successfully.Second operation is easy to perform if relapse ofsyndrome occurs. Nowadays, this procedure maybe a rational technique for the treatment of Cush-t

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Correspondenceto:Baoping QiaoDepartment of UrologyFirst Affiliated HospitalZhengzhou UniversityZhengzhou, Henan 450052, ChinaTelephone: 01186-371-6516-9151Email: [email protected] . en

References1. Bloom LS, Libertino. Surgical management of Cushing's

Syndrome. Urol Clin North Am 1989; 16: 547 - 65.2. Jiang HY, Zhao GX, Qiao BP, et al. T ransposi tion of

pedicled adrenal gland for the treatment of Cushing's dis-ease-A new surgical technique (Report of 6 cases). ChinJ UroI1991;12:423-5.

3. Guz BV, StraHon RA. Operative approaches to the adrenalgland. Urol Clin North Am 1989;16: 527 - 34.

4. Zhang W, Zhao G, Meng Q, et al. Transposition of bilat-eral pedicled adrenal gland for the treatment of Cushing'sdisease. Chin J Surg 2000; 38 : 192 - 3.

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Life Science] oumal , 2 ( 1 ) , 2005, He, et al , Remanent Activated Sludge Used as Biosorbent (I) Effect of pH

Study of Remanent Activated Sludge Used as Biosorbent (I)

Effect of pH on Zeta Potential of Dissociative Bacteria

and Its Application in the Adsorption of Activated Sludge

on [4]. Moreover, the significance of the problemstudied consists in providing theoretic groundworkand thinking direction for recycle investigation ofremnant activated sludge.1.2 Structure and chemistry component 6f bacte-

rial cell wall

Bacteria have special structure of cell wallwhich other organisms have not. Substance consti-tuting the framework of bacteria is peptidoglycancomposed of peptide chain and amino sugar chain.And add other components such as amylose, mu-ramic acid, protein, mucopolysaccharide, and thenbacterial cell wall with different kinds of function

has been made up[S] .

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Department of Chemistry,Zhengzhou University, Zhengzhou, Henan 450052,China;

hezhanhang@zzu. edu. en

Abstract: The thesis researched the effect of pH value on the adsorbability of activated sludge and on zeta potentialof di&'SOciativebacteria in it. We found that with the change of pH, the changing trend of the two was consistent.Considering the cell waJl as functionary target, the author discussed the chemistry reaction between acid or alkaliand peptidoglycan or teichoic-acid of the cell wall. From this, the author explained the changing trend of bacterialzeta potential, and expounded the relation between it and the adsorbability of activated sludge. [Life Science Jour-nal. 2005 ;2( 1) :68 -71 ] (ISSN: 1097 - 8135).

Keywords: biosorbent; activated sludge; zeta potential; dissociative bacteria

1 Theory

.

1. 1 The surface zeta potential of bacteria in acti-vated sludge

There are many sorts of microorganism, but

most are bacteria[l]. The chemical components ofbacterial cell wall and polymer on the surface of thecell decide that the surfaces of bacteria are elec-

tronegative. So bacteria are the main reason whyactivated sludge has adsorbability.

Activated sludge possesses adsorbability,which is related with the zeta potential of bacteria

in it[2] . And the value of zeta potential is tied upwith the value of charge on bacterial surface. Thevalue of zeta potential is determined by dint of elec-trophoresis phenomenon of electriferous particulatewith the function of electric field. From the Helmo-

hohz-smolachowski formula[3], it has direct ratio

with electrophoresis velocity of electriferous partic-ulate. So it can be calculated by determining thevalue of the velocity.

In this article, we studied the effect of pH val-ue on the zeta potential of bacteria in activatedsludge. And our purpose, for one thing, it was toresearch how to change the charge value of bacteriaand improve the adsorbability. For another, it wasimportant for the research of microbe ecology to in-vestigate the optimum pH condition for microbe todevelop. The reason was that pH value directly af-fected the microbe's variety, quantity and life ac-tivity, mode of metabolization and also type of pro-duction metabolized, surface speciality and so



2. 1 MaterialReagent: NaOH (AR), HCL (AR) , Active

carbon (The No.3 Chemistry Reagent Factory ofTianjin City); Fresh activated sludge; Reactivebrilliant red X-3B (C. I. Reactive Red 2) ;Acid lakeblue A (C. I. Acid Blue 7) (The No.3 ReagentFactory of Shanghai) .

Apparatus: TL - 5. 0 desk style centrifugalmachine (Shanghai Centrifugal Machine GraduateSchool, Shanghai, China); 79 - 1 magnetic forceheating blender (Jintan Medical Treatment Appara-tus Factory, Jintan, China); BDL-B surface poten-tial particle diameter apparatus (Shanghai ShangliSurvey Apparatus Factory, Shanghai, China); pH-pXFL:G1 acidity meter (Fanlong Instrument Cor-poration, Shanghai, China); 721 spectral pho-

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tometer (The No.3 Analysis Apparatus Factory ofShanghai, Shanghai, China).2. 2 Method

(1) Pretreatment of sludge: Remove some waterof the fresh activated sludge by centrifugal(2000 rpm, 3 min), and wash it four timeswith distilled water. Then get the abluentwet sludge (containing water 93 % ) .

(2) Effect of pH value on zeta potential of disso-ciative bacteria in activated sludge: Get 20 g

wet sludge every share, place in 100 cm3 dis-tilled water, and adjust the pH to needed val-ue. Then separate it by centrifugal and mea-sure pH value of the clear liquid above, anddetermine respectively the surface zeta poten-tial of six kinds of dissociative bacteria, that

is staphylococcus, micrococcus, diplococcus,

spirillum, bacillus and capsul-micrococcus[6] .(3) Effect of pH on adsorbability of activated

sludge: Separately place 2 g wet sludge in 50

cm3, 100 mg/L reactive brilliant red solutionand acid lake blue solution. And adjust the

pH value using O. 5 mol/L HCl. When thesystem reaches adsorption equilibration, de-termine pH and absorbency of the solution atthe most adsorption wavelength of the corre-sponding dyestuff with 721 spectral photome-ter. Then calculate the adsorption value ofactivated sludge and elimination rate of reac-tive brilliant red and acid lake blue at differ-

ent pH.( 4) Compare of adsorbability of activated sludge

and active carbon: Dry some active carbon( - 200 mesh) at 105°C for 4 hours in oven.Take 0.15 g dried active carbon every share,

at pH = 6 and 3, separately place in 50 cm3reactive brilliant red solution (250 mg/L)and acid lake blue solution (300 mg/L).When it comes to adsorption equilibration,determine its absorbency. Simultaneously, atthe same condition determine the adsorptionof 1. 5 g dried activated sludge at pH = 3.

Calculate respectively the equilibrium concen-tration and adsorption value.

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With alteration of pH, the changing circs ofsurface zeta potential of the six kinds of bacteria areshown in Figure 1.

We can see from Figure 1 that when pH re-duces, zeta potential (minus means the surface ofbacteria take negative charge) of the six kinds of

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bacteria are all decreasing. At low pH, the chang-ing trend is similar to that of surface potential of ac-

tivated sludge granule reported[3]. And the differ-ences between them just prove that zoogloea is notentirely equal to dissociative bacteria in character.

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Figure 1. Effect of pH on zeta potential of dissociative bacteria inactivated sludge

The changing trend of zeta potential of thedissociative bacteria in Figure 1 can be explained by

the chemical reaction betweeb H+ and peptidogly-can. When add acid to the dissociative system ofbacteria, on the one hand, amido and imido groupin peptidoglycan are protonized and then take posi-tive charge. On the other hand, ionization equili-bration of carboxyl moves to the reverse directionand thus the amount of carboxyl negative ion is re-duced. Therefore, while pH of the system is min-shing, the negative charge of cellular surface is de-creasing, and bacterial velocity of electrophoresis isalso decreasing with the function of electric field. Itresults in reduction of bacterial surface zeta poten-tial.3.2 Effect of pH on adsorbability of activated

sludgeAlteration of pH value makes change of the

adsorbability of activated sludge. Figure 2 showsadsorption value of reactive brilliant red and acidlake blue by activated sludge at different pH value,and the change of elimination rate of the twodyestuffs in the experimental condition.

It can be seen from Figure 2 that the adsorp-tion value is increased when pH value minishes.And the elimination rate takes on the same chang-ing trend. Take reactive brilliant red for example,when pH reduces to about 4. 7, the adsorption ofactivated sludge increases promptly. And when pHreduces to about 3.0, the adsorption goes on to in-crease but the change is not obvious. At pH = 3. 0 ,


negative ion dyestuff. In water solution, elec-

tronegative sulfonic group ( - SO3-) they ionizedlinks with electropositive amido ( - NH3+) in pep-

tidoglycan by ionic bond[7]. When pH decrease, thezeta potential decrease, that is the negative chargeof bacterial surface reduces. And it is advantageousfor it to contact closely with the molecule ofdyestuff, and for adsorption to happen. So the ad-sorption value is increased.

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the adsorption adds to 29.47 mg/g, and the elimi-nation rate adds to above 95 %. The changing sta-tus of acid lake blue is similar. Therefore pH =3 isan appropriate critical point of adsorption.

Comparing Figure 2 and Figure 3, we canknow that with the change of pH, the changingtrend of adsorption of activated sludge is in accordwith that of dissociative bacterial surface zeta po-tential. Reactive brilliant red and acid lake blue are

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Table 1. Compare of adsorbability of activated sludge and active carbon

Active carbon Activated sludge

3.3 Compare of adsorbability of activated sludgeand active carbon

As show in Table 1, the aQsorption of activecarbon is better at rather low pH condition. How-ever, the adsorption of activated sludge is muchhigher than that of active carbon when pH = 3.This suggests that the adsorption of acidic activatedsludge on dyestuffs is more rapid in pace, better incapability and effect.

~4 Conclusion

To sum up from above, the change of pH con-dition makes surface zeta potential of bacteriachange, and so does the adsorbability of activated


sludge. Thereby, we can let activated sludge havebetter adsorbability by adjusting the pH.

Remanent activated ~ludge is the object fordisposing in City Sewage Disposal Factory. If it canbe used as sorbent after simple treatment, then notonly the cost is very cheap but the effect is rathergood. And this nicely accords with the research anddevelopment direction of sorbent for dyestuffs-sorbent with high effect and low cost.

Correspondence to:Zhanhang HeDepartment of ChemistryZhengzhou UniversityZhengzhou, Henan 450052,

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Concentrations Concentration Concentration C.oncentration Concentration Concentraticm

mgll mgll mgll mgll mgll

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(250 mgll)

Acid Lake Blue79.1 31. 8 89.4 17.5 108.7

(300 mgll)

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Telephone: 01186-371-6776-6027Email: [email protected]. en

References

1. Shi J, XU Y, Zhang S. Environment Microbiology.Shanghai: Publishing Company of East China NonnalSchool, 1993:243 - 4.

2. Sadowski Z. Technical note effect of biosorption of Pb(II), Cu(II) and Cd(II) on the zeta potential and floc-culation of nocardia SP. Minerals Engineering 2001: 14(5): 547 - 52.

3. Lu G, Zhong J. Effect of inorganic electrolyte and organ-ic high molecular flocculant on the surface zeta-potentialof sludge. Liaoning Chemical Industry 1999; 28 (1): 38-40.

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4. Liu W, He B, Zhang X. Effect of biology pretreatmenton zeta-potential of colloid in water coming from organicpollution source. China Feedwater and Drainage 1996;12 (4): 27 - 9.

5. Ju X. Experimental method of microbe chemistry classifi-cation (Japanese, translated by FANG Shuang).Guiyang: Guizhou Renmin Publishing Company. 1989:8-18.

6. Wuxi Light Industry College, Huanan Engineering Col-lege, Tianjing Light Industry College etc. Microbiology.Beijing: Light Industry Publishing Company. 1980:22-3.

7. Compiling Office of Dystuff Appliance Manual in Shang-hai Spinning Industry Bureau, Dystuff Appliance Manu-al. Beijing: China Spin Publishing Company. 1994,: 337-8 (first volume), 175 (second volume).

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Life Science Journal, 2 (1 ), 2005, Zhang, et al, Simvastatin Improved Matrix Metalloproteinase

Simvastatin Improved Matrix Metalloproteinase Mediating

Ventricular Remodeling in Rats afterMyocardial Infarction

Jinying Zhang1, Xiang Cheni, Yuhua Lia02, Baojun Lu1

1. Department of Cardiology, First Affiliated Hospital, Zhengzhou University,

Zhengzhou,Henan 450052, China;jyzhang@zzu. edu. cn

2. Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University

of Science and Technology ,Wuhan, Hubei 430022, China

Abstract: Objectives. It was observed that effect of simvastatin on both matrix metalloproteinase (MMP)-2,9 and

type I collagen mediating ventricular remodeling in rats after acute myocardial infarction (AMI). Methods. The

AMI model of rat was made by ligature of left anterior descending coronary artery, and the animals were divided in-

to three groups: simvastatin treatment group (MI-S), myocardial infarction control group (MI-C) and sham group

(Sham). The animals were feed four weeks. The mRNA expression of MMP-2 and MMP-9 in noninfarcted zone of

left ventricle (LV) were determined by reverse transcription-polymerase chain reaction (RT-PCR), and cardiac

type I collagen in noninfarcted zone were measured by immunohistochemistry, cardiac function was determined by

echocardiographyand hemodynamics analysis. Results. After four weeks, mRNA expression of MMP-2 and -9 and

type I collagen in LV post-myocardial infarction (M!) groups were more than Sham group (P<O. 05) ,and the in-

dices in MI-S group were significantly lowered than those in MI -C group (P < O. 05), while higher than Sham

group (P<0.05). Compared with Sham group, hemodynamics analysis showed that left ventricular end-diastolic

pressure (L VEDP) significantly increased (P< 0.01), while systolic blood pressure (SBP), diastolic blood pres-

sure (DBP), left ventricul~r systolic pressure (LVSP) and LV pressure:t dp/dtmax significantly decreased

(P<O. 05), and there was no change for heart rate (HR) in MI-C group. Compared with MI-C group, the

LVEDP significantly lowered (P < O. 05), and LV pressure:t dp/dtmax obviously increased (P < 0.05), while

HR, SBP, DBP and LVSP did not alter in MI-S group. Compared with Sham group, echocardiography showed.

that left ventricular end-diastolic diameter (L VEDd) significantly increased, and both fractional shortening (FS)

and ejection fraction (EF) significantly reduced (P < 0.05, respectively) in MI-C group. Compared with MI-C

group, simvastatin significantly decreased LV dilatation and improved LV function (P< 0.05). Conclusion. Sim-

vastatin could attenuate mRNA expression of MMP-2 and MMP-9 in LV 4 weeks after MI-rats, reduced collagen

synthesis, and improved cardiac function. [Life Science]ournal. 2005 ;2(1) :72 -76J (ISSN: 1097 - 8135).

activity of MMPs is increased in both experimental

MI and clinical dilated cardiomyopathy[6J. As ex-tracellular matrix degradation may play an impor-tant role in LV remodeling, MMP inhibiting has e-merged as a potential therapeutic strategy for pa-tients at risk for development of congestive heartfailure. Preliminary data proposed that administra-tion of an MMP inhibitor may decrease left ventric-ular enlargement in pacing-induced models of con-gestive heart failure and in spontaneous heart failurein rats[7]. The effects of MMP inhibition in the

post-MI period are incompletely understood. Thepresent study evaluated the effects of administration

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Keywords: simvastatin; acute myocardial infarction; matrix metalloproteinase- 2, 9 ; RT -PCR; immunohistochemistry;hemodynamics; echocardiography; rat

1 Introduction

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The extent of primary ischemic necrosis aswell as the later effects of distending forces and thecardiac tissue healing process influenced ventricular

dilatation after myocardial infarction (MI) [1- 3J.The activity and dynamic expression of matrix met-alloproteinases (MMPs) may affect lots of the mor-phological changes that happen after MI at both in-

fracted and peri-infarcted wnes[4,5J. MMPs aremembers of a family of enzymes that degrade spe-cific extracellular matrix (ECM) components; the


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of oral simvastatin in early left ventricular remodel-ing as assessed by transthoracic echocardiographyand hemodynamics after experimental MI in rats.


2.1 Infarction model and experimental groupsWistar rats weighing 200 - 240 g were anes-

thetized by intraperitoneally injection of sodiumpentobarbital (30 mg/kg) intubated, and ventilat-ed with a small-animal respirator. The left anteriordescending coronary artery was ligated proximatelywith a 7 - 0 silk suture after a left anterior thoraco-tomy. Sham-operated rats underwent the identicalprocedure without ligation of coronary artery. Thefollowing experimental groups were studied: CD

Sham-operated (Sham) (n = 10); (2)MI control(MI-C) (n = 12); (J)MI simvastatin (MI-S) (n= 12). Simvastatin (40 mg/kg body weight) wasgiven by gastric gavage 24 hours after the anesthe-sia, then continued for 4 weeks (40 mg/kg perday). An equal amount of normal saline was givento the other two groups every day.2.2 Measurement of MMP-2, 9 mRNA expres-

sion by RT-PCRThe infarcted heart was sectioned into nonin-

farcted zone by visual inspections. Total RNA wasreversely transcribed into first-strand cDNA afterisolation by using TRlzol reagent. MMP-2, 9 andGAPDH gene expression were analyzed in nonin-farcted area. The sense primer (S) and the anti-sense primer (A) for MMP-2,9 were as follows:MMP-2 S,S'-A~Gf -3' ,A,S'-CbAGCAMAGC:'AI'CAfCD\C-3' (348bp-production).MMP-9 S, S'-MCITTGlAGGGTCD:;fTCfG-3' ,A,5'-cccrGTGAGTGGGTfGGAIT -3' (469bp-produc-tion) .GAPDH S, 5'-11ITGXIGACATCAAGMGGTGG-3',A S'-CAcrAarrGTTGCTGIA-3' (213bp-produc-tion) .

PCR amplification was performed by addingeach cDNA sample 2 fll to 20. S fll of reaction mix-ture. Each cycle consisted of denaturation at 94. Cfor 40 seconds, annealing for 40 seconds (MMP-2,9 at 60.C, GAPDH at S6.C) , extension at n.cfor 1 min, and final extension at 72.C for S min.Each PCR product was separated by electrophoresison a 1. S% agarose gel and tested by a digital imageanalysis system (GSDSOOO, UVP, England) .Each amplified cDNA fragment was counted forsemiquantitative evaluation by normalization withthe GAPDH band.2.3 Measurement of type I collagen by immuno-

histochemistryParaffin-embedded myocardium specimens in


noninfarcted zone were serial sectioned into a thick-ness of 3 flID. The section was incubated with pri-mary antibodies [anti-rat type I collagen (Mono-son) (1 : 100)] which were stored at 4'(;overnight. Incubation with biotinylated second an-tibody was performed at room temperature for 30min. Immunoreactivity was evaluated under the mi-croscope using the HPIAS-2000 software.2.4 Echocardiography and hemodynamics analy-

sisTwo-dimensional echocardiography was per-

formed on each rat before surgery and 4 weeks aftersurgery with a 10 MHz (short focus) transducer.Long-axis, short-axis, and apical four-chamber im-ages were measured. The LV end-diastolic andend-systolic volumes (EDV and ESV, respectively)were aJlculatedby the rmdified SimImn's method[8].Cardiac output was calculated as (EDV -ESV ) /1000 X heart rate; the LV ejection fraction (LVEF)was determined as (EDV-ESV) /EDV X 100 % .The fractional shortening (FS) was measured atthe short-axis image. Hemodynamic studies wereperformed after the animals were anesthetized withan intraperitoneal injection of 1. 0 g/kg urethan.Through the right common carotid artery, acatheter filled with heparin solution was insertedinto LV and hemodynamic data recorded.2. 5 Statistic analyses

The data are given as mean:t SD. The multi-variate ANOVA was used to determine the overall

difference between the three independent groups.The statistical significance between groups was de-termined using a post hoc BonferronilDunn test. Avalue of P < O.OSwas considered significant.

3 Results

3.1 RT-PCR analysis of MMP-2,9 geneCompared with Sham operation group, the

mRNA expression of MMP-2 and MMP-9 signifi-cantly increased in noninfarcted zones of MI-Cgroup (P<O.OS). MMP-2 and MMP-9 expressionwere obviously decreased in MI-S group(P < O.OS), but still higher than those in Shamoperation group (p < O.OS) (Figure 1).3. 2 Immunohistochemistry analysis of type I

collagenCompared with Sham operation group, type I

collagen was markedly increased in noninfarctedzone of MI-C group (p < 0.01). Compared withMI-C group, type I collagen was significantly low-ered in MI-S group (P<O. OS) (Figure 2).3.3 Hemodynamics analysis

Table 1 displays hemodynamic data at 4 weeksafter operation. In the MI group, LV end-diastolic

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compared with the Sham operation group. LVEDPdecreased(P < 0.05) and :t dp / dt max significantlyincreased (P < O.05) in the MI-S group comparedwith the MI-C group. Other variables were not dif-ferent between both MI groups.

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pressure (L VEDP) significantly increased (p <O.01), systolic blood pressure (SBP), diastolicblood pressure (DBP) , LV systolic pressure(L VSP) and LV pressure maximal rate of rise andfall (:t dp/dtmax) noticeably decreased(p < 0.05) , while heart rate (HR) did not change

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of MI-C group and MI-S group 4 weeks post-MI. Quantitative analyses mRNA of MMP-2,9, * P<O.Ol vs. Sham group,# P<O. 05 vs. MI-C group.

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ocardiurn of MI-C group: (B) and MI-S group: (C) 4 weeks pcst-MI(rnagnificationx 200): (D) Quantitative image analy-

ses of type I mllagen production, * P< 0.01 vs. Sham group, # P< 0.05 vs. MI-C group.

Table 1. The effects of simvastatin treatment on hemodynamics

HR SBP DBP LVSP

Groups n (bpm) (mmHg) (mmHg) (mmHg)

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MI - S 12 377 :1:19 11O. 1:!::9. 6 * 90. 3 :1:7. 1 * 112. 8 :1:11. 1*

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3.4 Echocardiographic studiesFigure 3 shows representative short -axis e-

chocardiographic images from a Sham-operated ani-mal, an untreated MI rat and a simvastatin-treatedMI rat. Compared with Sham operation group,there were significantly increased LVEDd (P <O.01), markedly decreased FS (P < 0.01) and EF(P<O.O1) in the MI-C group. Although these pa-rameters also differed from the Sham operationgroup in the MI-S group, simvastatin significantlyattenuated LV dilatation and improved LV functioncompared with the MI-C group (P<O. 05) (Table2) .

Table 2. The effects of simvastatin treatment on echocardio-

graphic measurements

Groups n LVEDd (mm) EF (%) FS (%)Sham 10 4.3 ::1::0.2 87.2::1::3. 5 53.4::1::3. 3

MI - C 12 7.8::1::0.4 * 42. 1::1::3.9 * 21. 2 ::I:: 2. 1*

MI-S 12 5.6::1::0.3# 49.7::1::4.1# 27.5::1::2.2#

Compared to Sham group, * P < O. 05; Compared to MI-Cgroup, # P< 0.05

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MMPs are a family of zinc-depending endopro-teinases that specifically degrade ECM components.They are important enzymes which degrade matrixin the cardiac remodeling process after MI. The ac-tivity increase of MMPs leads to reduction of ECM,destruction of cardiac supporting structure and ven-tricular dilatation[9]. MMPs modulate synthesis ofcollagen, the rise of MMPs activity enhances fibro-sis, and fall of MMPs activity decreases fibro-sis[lO]. Clinical research confirmed[l1] that MMPswere obviously associated with stability of

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atherosclerotic plaque, the plasm MMPs markedlyrose in patients with coronary heart disease, and in-hibition of MMPs activity may increase plaque sta-bility. The experimental study proposed thatMMPs participated in the remodeling process afterMI, and were activated 1 day after MI. Activityand gene expression of MMP-2, 9 markedly in-

creased during remodeling process after MI[12,13],and application of MMPs inhibitors could decreaseventricular dilatation in rats after .MI and improve

cardiac performance[ 14-17]. It was further showedthat the rat with MMP-9 deficiency existed residualnecrosis expansion, the healing process retard afterMI[17,18] .

Statins are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, which arecapable of lowering the serum cholesterol level andare successfully used to treat hypercholesterolemiaand atherosclerosis. Moreover, the ability of statinsto lower the mortality and morbidity of cardiovas-cular diseases has been ascribed not only to theircholesterol-lowering activities but also to a numberof additional effects, including improving endothe-lial cell function, enhancing fibrinolysis, and an-tithrombotic activity. In addition, a number of im-portant anti-inflammatory effects of statins havebeen reported.

This study confirmed that mRNA expressionof MMP-2, 9 was significantly higher in MI rats,and simvastatin could obviously decrease mRNA

expre&'Sion of MMP-2, 9. It was assumed that sim-vastatin could not only stabilize atheroscleroticplaque, lower the incidence of unstable angina andMI, but also alleviate cardiac fibrosis, restrict de-

velopment of ventricular remodeling and heart fail-

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metalloproteinase inhibition with congestive heart failureimproves left ventricular geometry and pump function.Circulation 1997:96(Suppl I) :I - 520.

8. Schiller N B, Shah P N, Crawford M. Recommadationsfor quantitation of the left ventricle by two-dimensional e-chocardiography. ] Am Sac Echocardiogr 1989; 2 ( 5 ) :358 - 67.

9. Spinale FG, Coker ML, Bond BR, et al. Myocardialmatrix degradation and metalloproteinase activation in thefailing heart: a potential therapeutic target. CardiovascRes 2000:46(2) :225 - 38.

1O.Li YY, Feng YQ, Kadokami T, et al. Modulation ofmatrix metalloproteinase activities remodels myocardialextracellular matrix in TNF-a transgenic mice. Circulation1999:100(Suppl):1752.

11. Brown DL, Desai KK, Vakili BA, et al. Clinical andbiochemical results of the metalloproteinase inhibitionwith subantimicrobial doses of doxycycline to prevent a-cute coronary syndromes ( MIDAS) pilot trial. Arte-rioscler Thromb Vasc Bioi2004; 24(4) :733 - 8.

12. RomanicAM, Bums-Kurtis CL, Gout B, et al. Matrixmetalloproteinase expression in cardiac myocytes followingmyocardial infarction in the rabbit. Life Sci 2001 ;68(7):799 - 814.

13. Hojo Y, Ikeda V, Veno S, et al. Expression of matrixmetalloproteinase in patients with acute myocardial in-farction. ]pn Circ] 2001 :65(2): 71 - 5.

14. Rohde LE, Ducharme A, Arroyo LH, et al. Matrixmetalloproteinase inhibition attenuates early left ventricu-lar enlargement after experimental myocardial infarctionin mice. Circulation 1999:99(23) :3063 -70.

15. Villarreal F], Griffin M, Omens]. Early short-termtreatment with doxycycline modulates postinfarction leftventricular remolding. Circulation 2003: 108 (12) : 1487-92.

16. Podesser BK, Siwik DA, Eberli FR, et ai, ET(A)-re-ceptor blockade prevents matrix metalloproteinase activa-tion late postmyocardial infarction in the rat. Am] Phys-iol Heart Circ PhysioI2001:280(3) :H984 - H991.

17. Heymans S, Luttun A, Nuyens D, et al. Inhibition ofplasminogen activators or matrix metalloproteinase pre-vents cardiac rupture but impaires therapeutic angiogene-sis and causes cardiac failure. Nat Med 1999; 5 ( 10 ) :1135 - 42.

18. Ducharme A, Frantz S, Aikawa M, et al. Targeteddeletion'of matrix metalloproteinase-9 attenuates left ven-tricular enlargement and collagen accumulation after ex-perimental myocardial infarction. ] Clin Invest 2000; 106(1):55-62.

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ure and improve prognosis patient with MI by in-hibiting expression of MMPs. However, it is un-known whether statins directly or indirectly inhib-ited expression of MMPs.

Acknowledgments

National Natural Science Foundation (No.30370574); Henan Province's Creation TalentProjection of Medical Science & Technology, Chi-na (No. 2002116); Zhengzhou University's Scien-tific Research Development Fund, China (No.2004021) .

CoITespondenceto:Jinying ZhangDepartment of CardiologyFirst Affiliated Hospital,Zhengzhou UniversityZhengzhou,Henan 450052, ChinaTelephone :01186-371-6516-9392

01186-135-0383-0283Email:[email protected]. en

References

1. Pfeffer ]M, Pfeffer MA, Fletcher P], et al. Progressiveventricular remodeling in rat with myocardial infarction.Am] Physiol1991 :260(5 pt 2): H1406 -14.

2. Pfeffer MA, Braunwald E. Ventricular remodeling aftermyocardialinfarction: experimentalobservationsand clin-ical implications. Circulation 1990:81(4): 1161-72.

3. Rumberger ]A. Ventricular dilatation and remodelingaf-ter myocardialinfarction. Mayo Clin Proc 1994: 69(7) :664 -74.

4. Tyagi SC, Ratajska A, Weber KT. Myocardial matrixmetalloproteinases: localizationand activation. Mol CellBiochem 1993: 126 ( 1) :49 - 59.

5.Cleutjens]PM, Kandala]C, GuardaE, etal. Regulationof collagen degradation in the rat myocardium after in-farction. ] Mol Cell CardioI1995:27(6): 1281- 92.

6. Thomas CV, Coker ML, Zellner ]L, et al. Increasedmatrix metalloproteinase activity and selective upregula-tion in LV myocardium from patients with end-stage di-lated cardiomyopathy. Circulation 1998: 97 ( 17) : 1708 -

15.7. SpinaleFG, KrombachRS, CokerML, et al. Matrix

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EGF Receptor Tyrosine Kinase Inhibitor Tyrphostin AG1487Induce Human Tongue Cancer Cells Tca8113 Cell Cycle

at G1 Phase and Apoptosis

..Xinguang Han1, Bogui Wen2

1. Department of Stomatology, First Affiliated Hospital, Zhengzhou University,

Zhengzhou, Henan 450052,China;xinguanghan@sina. com

2. Laboratory of Tumor Molecular Biology, Department of Pathology, Medical College,

Shantou University,Shantou, Guangdong 515031, China

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Abstract: Objective. This study was to investigate the effects of tyrphostin AG1487 on expression of activatedERK1I2, distribution of cell cycle and apoptosis in Tca8113 cells. Materials and Methods. Tca8113 cells were ex-posed to different concentrations of phosphotyrosine kinase inhibitor tyrphostin AG1487, and then immunocytoche-mistry , Western blot, flow cytometry, and electron transmission microscopy were employed to investigate the ef-fects of AG1487 on expression of activated ERK1/2, distribution of cell cycle and apoptosis of Tca8113 cells. Re-sults. The results of immunocytochemistry showed intensity of activated ERK1/2 reactivity in treated cells wasmarked decreased. These results were corresponded with the expression of activated ERK1/2 in Western blot: ex-pressions of activated ERK1I2 gradually reduced along with the increasing of concentration of tyrphostin AG1487.Cell cycle kinetic analysis demonstrated that AG1487 induced a delay in cell cycle progression and arrested at G1phase. Furthermore, AG1487 could induce a marked apoptosisof Tca8113 cells. All these effects were in a dose-dependent pattern. Conclusion. EGF receptor tyrosine kinase inhibitor tyrphostin AG1487 can inhibit the expres-sion of activated ERK1/2, induce cell cycle arrest at G1 phase and apoptosis. [Life Science Journal. 2005;2(1): 77- 84] (ISSN: 1097 - 8135) .

Keywords:Tca8113 cell;EGF receptor; ERK1I2; cell cycle;apoptosis

Abbreviations:EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MAPK, mitogen-activat-ed kinase; ERK, extracellular signal- regulated kinase

1 Introduction

The EGF receptor belongs to the erbB familyof four closely related cell member receptors: EGFreceptor (erbBl), erbB2, erbB3 and erbB4. Allthese receptors are transmembrane glycoproteinsthat consist of an extracellular ligand-binding do-main, a transmembrane domain, and an intracellu-lar domain with tyrosine kinase activity for signaltransduction [1- 3] . After ligand binding, the tyro-sine kinase of the receptor is activated, and initiatesa cascade of intracellular events[4]. These events

lead to recruitment and phosphorylation of severalintracellular substrates, such as extracellular signal-

regulated kinase (ERK), which lead to the mito-

genic signaling and other cellular activities[1,4].Many studies have shown EGF receptor is highlyexpressed in malignent tumors, and is associatedwith progression of malignant tumors and poorprognostic features. EGF receptor and its down-stream signaling pathways are, therefore, becom-

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ing the promising antitumor targets for cancer ther-apy.

A variety of different approaches are currentlybeing used to target the EGF receptor, the mostpromising strategies include to prevent ligand bind-ing and to inhibit autophosphorylation of EGF re-ceptor. Therapeutic mAbs targeting the extracellu-lar domains of EGF receptor have similar affinity

binding to EGF receptor as EGF and TGF-a, com-pete with these ligands for receptor binding, andblock activation of receptor tyrosine kinase induced

by EGF and TGF-a[5,6]. Small molecule inhibitorsare generally reversible competitors with respect toATP for binding to the intracellular catalytic do-

main of the tyrosine kinase[5]. Some studies hadshown that small molecule inhibitors not only com-

pete with ATP in the classical mode of action butalso induce the formation of inactive, unphosphory-lated EGFR dimmers[5,7].

Both in vitro and in vivo studies have demon-

strated that EGF receptor targeted agents could in-

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with goat anit-mouse IgG-HRP or goat anti-rabbitIgG-HRP antibodies (Boster, Wuhan, China) for1 h at room temperature. Coverslips Were visualizedusing 3-3' -diaminobenzidine as a chromogen for 5min. Then the specimens were dehydrated and cov-erslips were mounted on the glass slides usingmounting media. Positive and negative controlswere included in each staining run.2.3 Western blot

Western blot was perfonned as reported previ-ously[13]. Cells were seeded in parallel and underthe same conditions. When cells grew to monolay-ers, PRMI medium was refreshed and phosphoty-rosine kinase inhibitor tyrphostin AG1487 wasadded at the indicated concentrations (0 nM, 50nM, 100 nM, 200 nM) and incubated for 2 h.Then the cells were scraped into 0.5 ml of cold ly-sis buffer [50 mM Tris-HCI (pH8. 0), 150 mMNaCI, 0.1% SDS, 100 !Ig/ml PMSF, 1 !Ig/mlAprotinin, 1% NP - 40, O. 5% sodium deoxy-cholate], and protein was extracted. The concen-tration of protein was detennined by Bradfordmethod. Western blots were perfonned to detectthe expression of total ERK1/2 (Sigma 1: 10 ,000 )and activated ERKl/2 (Sigma 1: 10,000) .2.4 Flow cytometry

Tca8113 cells were cultured at same conditionand then exposed to indicated concentrations of tyr-phostin AG 1487 (0 nM, 50 nM, 100 nM, 200nM) and treated for 24 h, 48 hand 72 h respec-tively. Cells were washed with PBS, trypsinizedand harvested, and then fixed with pre-cold ( -20'C) ethanol for 2 h. The cells were centrdugedfor 5 min at 1500 nnp and quickly removed the su-pernate, washed with PES and added RNase 50 !II(20 !Ig/ml) for 10 min at room temperature, thenstained with propidium iodide (PI, sigma) 200 !II(5 ng/ml) for 20 min, washed with PBS and re-suspended. After filtering cells through 100 !Impore size mesh, cell cycle distribution was analyzedby a flow cytometer (Becton Dickinson FAC Sort).2.5 Electron transmission microscopy

T ca8113 cells were cultured as above and ex-posed to different concentrations of tyrphostinAG1487 (0 nM, 50 nM, 100 nM, 200 nM) for 48h, then washed with PBS, trypsinized and harvest-ed. Cells were fixed with 2 % glutaraldehyde solu-tion. Ultrathin Epon plastic sections were preparedand stained with uranyl acetate followed by leadcitrate and stabilized for transmission electron mi-croscopy (HITACHI. H-300). Changes ofTca8113 cells after exposing to tyrphostin AG 1487were clearly identified by its characteristics.

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hibit the processes involved in tumor growth andprogression, including proliferation, apoptosis,

metastasis and angiogenesis[4,5,8]. All these effectsare correlated with the blockade of EGF receptorsignaling. ERKs, as one of the important signalingmolecule that lies downstream of EGF receptor,usually be highly expressed in malignant tumor

cells[9]. Some studies demonstrated EGF receptortargeted agents could inhibit the activation of EGF

receptor and ERKs in malignant tumor cells[ 10-12] .For further study the EGF receptor agents ef-

fects on malignant tumor cells, we adopted smallmolecule inhibitor of EGF receptor tyrphostinAG1487 to treat human tongue cancer cells(Tca8113), and then to investigate the effects ofAG1487 on expression of activated ERK1/2, dis-tribution of cell cycle and apoptosis in Tca8113cells.

2 Material and Methods

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2. 1 Tumor cell lines and regentsThe cell line of human squamous cell carcino-

ma of tongue, Tca8113 cells, were obtained fromShanghai Institute of Cell Biology, Chinese Acade-my of Sciences and cultured in PRMI medium 1640(GmCO) containing 10% heat~inactivated FES,100 units/ml penicillin G and 100 unites/ml strep-tomycin at 37'C in humidified air containing 5%COz. Polyclonal anti-EGF receptor antibodies ob-tained from Boster Biological Technology Co.(Boster, Wuhan, China), anti-total ERKl/2 anti-bodies and anti-activated ERK1/2 antibody ob-tained from Sigma Chern. Co. (St. Louis, MO,USA), Tyrphostin AG 1487 obtained from Cal-biochem (San Diego, CA, USA).2.2 Immunocytochemistry

Cells were seeded on coverslips coated withpoly-L-Iysine placed in six culture plates under thesame conditions. On the next day, PRMI medium1640 was refreshed and tyrphostin AG 1487 wasadded at indicated concentration (100 nM) to thecultures for 2 h. Then the slides were' removedfrom the culture plates, and cells on coverslips werefixed in 2 % parafonnaldehyde solution with O. 1%Triton X-1O0 for 30 min at room temperature.Nonspecific protein reactivity was blocked with10% goat serum (SABC) in PES for 10 min. Andendogenous peroxidase reactivity was blocked in3 % hydrogen peroxide solution for 10 min. Incu-bation with primary antibodies were made at roomtemperature for 1 h at following dilutions: EGF re-ceptor (1: 100), activated ERKl/2 (1: 1000) andtotal ERKl/2 (1: 1000). After being washed withPES three times, the coverslips were incubated


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3 Results

3.1 Expression of EGFRThe results of immunocytochemical staining

demonstrated EGF receptor protein of Tca8113cells was mainly in the cytoplasm and on the plasmamembrane. There was no significant differenceabout expression of EGF receptor in before and af-ter treated cells (Figure la, b). Unfortunately, wedid not detect the expression of activated EGF re-ceptor. But in a few of studies shown AGl478 andother EGFR inhibitors could inhibit EGFR phos-phorylation without reduced expression of EGFRprotein[2,14].3. 2 Expression of activated ERKl/2 and total

ERKTo study the effects of tyrosine kinase in-

hibitor tyrphostin AGl487 on EGF receptor signal-ing of Tca8113 cells, we adopted immunocyotche-mistry and Western blot methods to detect the ex-pression of total ERK1I2 and activated ERK1I2.The results of immunocytochemistry showed thatthere was no significant difference in intensity oftotal ERKI/2 reactivity in Tca8113 cells before andafter treated with AGl487 (100 nM) (Figure lc,d). As for activated ERK1I2, it was marked de-creased of Intensity of reactivity in nucleus andmade nucleus to be a shadow (Figure Ie, f). Thesere~ults were corresponded with the expression oftotal ERKI/2 and activated ERKI/2 in Westernblot (Figure 2): Along with the increasing of con-centration of tyrphostin AG-1487, expression of ac-tivated ERKI/2 was reduced or abolished in West-ern blot. But there was no significant difference in

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expression of total ERKI /2.3. 3 Distribution of cell cycle after treatment

with AG1487In order to investigate the effects of AGl487

on cell cycle in Tca8113 cells, we employed differ-ent concentrations of AGl487 to treat the cells fordifferent time (0 h, 24 h, 48 h, 72 h). The re-sults suggested that the percentage of cells in GIphase was correlated with the concentration of tyr-phostin AGl487 and treating time. Along with theincreasing of concentration of tyrphostin AGl487and treating time, the percentage of cells in GIphase also increased (Figure 3). When exposingTca8113 cells to 100 nM AGl487 for 72 h, thepercentage of cells in GI phase reached to maxi-mum (77.57%, Table O.3.4 Effect of AG1487 on apoptosis of Tca8113

cellsSome studies demonstrated EGF receptor tar-

geted agents could induce tumor cells apoptosis invivo or in vitro, and this effect was correlatedwith inhibition of EGF receptor signaling pathway.In the present study, effect of tyrphostin AGl487on apoptosis of Tca8113 cells was also studied. Theresults showed EGF receptor tyrosine kinaseAGl487 could induce apoptosis of Tca8113 cells,and the effect was correlated to the concentration oftyrphostin AG1487. Along with the increase ofconcentrations of AG1487, the number of apoptoticcells gradually increased (Figure 4a, b). Compar-ing with untreated cells, the number of apoptoticcells was nearly 7 fold in Tca8113 cells treated withhigh concentration of AGl487 (200 nM) (Figure5) .

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TimeCell cycle (%)

Gl s G2

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(hour) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM) (nM)

24 32.35 34.65 38.81 47.70 54.89 50.78 49.17 45.64 12.76 14.57 12.02 6.67

48 32.15 45.50 60.67 74.04 55.20 48.34 33.48 21.07 11. 90 6.16 5.85 4.89

72 32.35 75.64 77.57 54.04 54.85 18.06 17.19 38.05 10.90 6.30 5.23 7.91

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( x 400). a. EGF receptor immunochemical staining in untreated cells. b. EGF receptor immunochemical staining in treated cells.c. Total ERK1I2 immunochemical staining in untreated cells. d. Total ERK1I2 immunochemical staining in treated cells. e. Activated

ERK1I2 immunochemical staining in untreated cells. f. Activated ERK1I2 immunochemical staining in treated cells.

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tFigure 2. Expression of total ERKll2 and activated ERK1I2 in Tca8113 cells treated or untreated with AG1487. Tca8113 cells pretreat-ed with 0, 50, 100 and 200 nM AG1487 in an equal amount of DMSO (final concentration 0.5 %) for 2 h at 37'(;. Cell lysates wereprepared and Western blot analyses were perfo=ed with antibodies to total ERK1I2 and activated ERKll2.

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ing to different concentrations (0 nM, 50 nM, 100 nM, 200 nM) of AG1487 and for different time (24 h, 48 h, 72 h), and cells werethen harvested and stained with propidium iodide as described under Materials and Methods. Their cell cycle distribution was analyzed byflow cytometer. a. Cell cycle distribution in Tca8113 cells untreated with AG1487. b. Cell cycle distribution in Tca8113 cells treated

with AG1487 in concentration 50 nM for 24 h. c. Cell cycle distribution in Tca8113 cells treated with AG1487 in concentration 1O0nM

for 48 h. d. Cdl cycle distribution in Tca8113 cells treated with AG1487 in concentration 200 nM for 72 h.

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kinase induced a remarkable Gl cell cycle arrest,accompanied by a decrease in the percentage of cells

in the S phase in malignant tumor cells[13,14], andthis was associated with increased expression of p27(KIPl), which binds to and inactivates cyclin-de-pendet kinase-2 activity and produces cell cycle ar-

rest in Gl phase[14]. At present study, the percent-age of Tca8113 cells in Gl phase was studied aftertreated with tyrphostin AG1487 at different con-centrations and for different time, the resultsshowed the percentage of cells inGl phase was cor-related to the concentrations and treating time. A-long with the increasing of concentrations and pro-longing of treating time, the percentage of Gl cellsgradually increased. When exposing Tca8113 cellsto 100 nM AG1487 for 72 h, the .percentage ofcells in G 1 phase reached to m~xi)llum (77. 57 % ) .Further analysis suggested these /results were indose- and time-dependent patterns.

Apoptosis is a physiological mechanism of celldeath that occurs during normal development andunder certain pathological conditions[19,20]. Severaltypes of regulatory pathway can contribute to thecellular decision to enter apoptosis. These includeDNA damage, dominant death signals from" deathreceptors" such as Fas, tumor necrosis factor recep-tor, and block the signaling pathways from growthfactor receptor-activated kinase cascades[21]. Acti-

vation of the EGF receptor provides a potent sur-vival signal in many cell types. PI3K and ERKl/2are signaling molecules on two major downstream

pathways initiated by the activation of EGF recep-tor[22,23]. A few of studies demonstrated small

molecule inhibitor of the EGF receptor tyrosine ki-nase, for example ZD-1839 and AG1487, could in-'duce apoptosis of malignant tumor cells in vivo or

in vitro[16,24]. And this effect was also dose depen-dent. At present study, the percentage of apoptoticcells were calculated after exposing Tca8l13 cells todifferent concentrations of AG1487. The results

showed along with the increasing of concentrationsof AG1487, the percentage of cells proceedingapoptosis also increased. And the morphologicchanges of apoptotic cells, such as vesicle formationin the cytoplasm, nuclear condensation were also

influenced by the concentration of tyrphostinAG1487. All these results suggested tyrphostinAG1487 could induce apoptosis of Tca8113 cellsand the effect was in a dose dependent pattern.

In summary, EGF receptor signaling path-ways play an important role in regulating cellularprocesses in Tca8113 cells. Small molecule inhibitorof EGF receptor tyrosine kinase, AG1487, couldeffectively inhibit ERK1I2-MAPK pathway and re-

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4 Discussion

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Dimerization and phosphorylation of the epi-dermal growth factor (EGF) receptor are the initialand essential events of EGF or TGF-a-induced sig-nal transduction, so selective inhibition of tyrosinekinase activity is one of promising strategy for can-cer treatment. Tyrphostin AG1487, as one of thephospho tyrosine kinase inhibitors, has been used ina few of studies and showed it can induce inactive,unphosphorylated EGF receptor - erbB-2 het-erodimers, thereby sequester EGF receptor from

signaling interactions with other erbB corecep-tors[15]. These effects lead to the activation of

ERKs-MAPK (mitogen-activated kinase) be inhib-ited[12,16]. Biochemical and cell fractionation stud-ies have indicated that activation of ERKs and

translocation from the cytoplasm to the nucleus arerequired for growth factor-induced gene expression

and cell cycle entry[17]. At pre~ent study, wedemonstrated expression of activated ERKl/2 couldbe effectively inhibited by tyrphostin AG1487. Andthis inhibitory effect was in a does-dependant pat-tern. The result of immunochemical stainingshowed intensity of activated ERKl/2 reactivity innucleus marked decreased, it means translocation

process of activated ERKl/2 from cytoplasm to nu-cleus was inhibited by tyrphostin AG1487. Allthese results suggested small molecule inhibitor ofEGF receptor tyrosine kinase, AG1487, could ef-

fectively inhibit the ERK-MAP kinase pathway andtranslocation process of activated ERKl/2 inT ca8113 cells.

Besides EGF receptor regulating the growthand progression of human oral squamous cell carci-noma, the link between EGFR signaling and thecell cycle has also been identified[18]. A few ofstudies demonstrated both therapeutic mAbs andsmall molecule inhibitors of EGF receptor tyrosine


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duce the expression of activated ERKII2 in a does-dependent manner. Cell cycle kinetic analysesdemonstrate AG1487 induce a remarkable Gl cellcycle arrest, followed by a modest apoptotic re-sponse. All these results suggest the EGFR kinasespecific inhibitor is of potential to be developed intodrugs for squamous cell carcinoma treatment.

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Acknowledgments

We thank Vr. Guanwu Li (Laboratry of Tu-mor Molecular Biology, Medical College, ShantouUniversity) for excellent technical assistance; Wethank professor] unlong Lin (Medical College,Shantou University) for assistance with flow cy-tometry.

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effect and potentiation of cytotoxic drugs activity in hu-man cancer cells by W-1839 (Iressa), an epidennalgrowth factor receptor-selective tyrosine kinase inhibitor.Clin Cancer Res 2000; 6: 2053 - 63.

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Gl arrest in oral squamous carcinoma cell lines. Oncology2002;63 :92 - 8.

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Life ScienceJournal, 2 (1 ), 2005, Gong, et al, Phylogenetic Relationship of Tetraogallus Inferred from Sequences

Phylogenetic Relationship of Tetraogallus Inferred

from Sequences of Cytochrome b Gene

"Yifeng Gongl, ]infu Wangl, Hongyan Lil, Li Wantt, Runlin Ma3

\I'" 1. Ministry of Education Key Laboratory of Xinjiang Endemic and Ethnic Disease,

Shihezi University, Shihezi, Xinjiang 832003, China

2. Department of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, China

3. Institute of Genetics and Developmental Biology, Chinese Academy Sciences, Beijing 100101, China

Abstract:A phylogenetic tree of Neighbor-joining (N]) for the sequences of cytochrome b (Cyto b) gene was con-structed to study the phylogenetic relationship of the genus Tetraogallus. Numbers near the branches were boot-strap probability (BP) values coming from 1000 replications. Some bootstrap probability values were 92 % (Gi-turnix chinensis / Giturnix japonica ), 100 % (Tetraogallus altaicus / Tetraogallus himalayensis), and 100 %(Tetraogallus altaicus / Tetraogallus himalayensis / Tetraogallus tibetanus). The overall average distance was0.112, and average genetic distance among Tetralgallus was O.042. The genera Alectoris, Giturnix, Tetraogal-lus and Pucrasia formed a monophyletic group. The two genera, Alectoris and Giturnix, should have a commonancestor, which was the sister taxon of the ancestor of the genus Tetraogallus. The interspecific genetic distancesof the genus Tetraogallus were 0.012 (T. altaicus vs T. himalayensis), 0.067 (T. himalayensis vs T. ti-betanus) and 0.068 (T. altaicus vs T. tibetanus) respectively. By combining the geographical distribution pat-tern, morphological characteristics and the genetic distance among these species of the genus Tetraogallus. It canbe inferred preliminarily that Tibetan snow cocks were the primitive species among the three breeds; and that 1.7million years ago, one subspecies of Tibetan snow cocks, tibetanus with a deep body color, distributing in thesouthwest of Xinjiang and the midwest of Tibet, migrated towards the west and entered the Himalayan Mountain tobecome the present Himalayan snow cocks; and that later, another subspecies of Himalayan snow cocks, koslowi,

owning a light body color and often emerging in the Altai Mountain and the east of Kunlun Mountain, evolved intoAltai snow cocks. [Life ScienceJoumal. 2005 ;2(0 :85 - 89J (ISSN: 1097 - 8135).

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Keywords: snow cock; cytochrome b gene; phylogeny; mitochondrial DNA...~t."" 1 Introduction

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Snow cocks, the herbivore birds with high

medicinal value inhabiting in the highest sea level in

the world, are the children of the Tetraogallus un-

der Galliform, Phasianidae. Nowadays, five

species of snow cock have been found in Eurasia on-

ly. Except the foreign species, caspian snow cock

( T. caspius) and Caucasian snow cock, the other

three species including Altai snow cock (T. al-

taicus), Himalayan snow cock (T. himalayensis)and Tibetan snow cock (T. tibetanus), can all be

seen in Xinjiang Uygur autonomous region of China

( T. caucasicus) (Zheng, 2002). All of them hadbeen listed as the international first-grade endan-

gered and national second-protected wildlife.Presently, foreigners have little research about

theniexcept some researchers of Soviet Union whoonce had a shallow study on them at the beginning

of 20th century. In the 1990s, people began to re-

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search their domestication and reproduction in Xin-

jiang, Gansu and Qinghai. In China, researches

were promoted at the beginning of 1960s, and have

been limited by the domains of their ecological dis-

tribution and process. Now, we know that snow

cocks are an excellent group for biological studies.

However, the phylogenetic relationships among the

snow cock species, their phylogenetic position and

evolution process are still a secret that impedes ourfurther researches of snow cocks.

Mitochondrial DNA can be used as a molecular

marker since people have paid overwhelming atten-

tion to its traditional characteristics such as rigorous

maternal inheritance (Gyllensten, 1985), rapid

evolution (Vawter, 1986) and non-rearrangement

(Cann, 1987). In recent years, the polymorphic

analyses of mitochondrial DNA have become the ef-

fectual means for phylogeny reconstruction and tax-

onomy (Kirchman, 2000; Meyer, 1990) . For ex-

ample, cytochrome b (Cyto b) gene of mitochon-


solvedin TE (pH 8. 0) and stored at - 20"C for

later use (Ausubel, 1995).

2.3 PCR amplificationTotal genomic DNA was extracted from blood

using a modified protocol, and used as the templatein the polymerase chain reaction (PCR) amplifica-tions. The primers were designed using DNAMANsoftware according to the published Himalayansnow cock Cyto b sequence (Accession No.AY678108). The upstream primer (5' -CTATAC-TACggCTCCTACCTg-3') and downstream primer(5' -gTTTgggATTgAgCgTA ggATg-3') were usedin this study. Then, reactions were conducted involumes of 50 !iI, typically with a amplificationprofile as follows: 3 min at 95"C, 35 cycles of 45sec at 95"C, 45 sec at 52°C, and 3 min at n"C ,followed by a final extension at noc for 10 min us-ing the HiFi Ready-to-use PCR kit (Sangon, Cat.:#SK 2073, USA) on PTC-lOOTM Thermocycler.

2. 4 Purification, cloning, and sequencing ofPCR products

PCR products were purified from agarose gelsusing the EZ-I0 spin column PCR product purifica-tion kit (BBI, Cat. :#BS363, USA) and cloned di-rectly into pGEM- T-easy vector (Promega, Cat.:#A3600, USA). Sequencing was ,carried out withan ABI 3100 Genetic Analyzer, using the BigDyeTerminator Cycle Sequencing Kit according to themanufacturer's protocols.2.5 Sequence analysis and phylogenetic tree con-

struction

Mitochondrial Cyto b gene sequences from Al-tai and Himalayan snow cocks were generated inthis study. The other data was obtained from Gen-Bank or from the literatures (Table 1). All nu-

cleotide sequences were aligned with theCLUST AL X (version 1. 8) multiple alignmentprogram and refined manually. Phylogenetic analy-sis was performed with MEGA version 2. 1 (Ku-mar, 2001) and TREE PUZZLE version 5. O. Weapplied two different methods of phylogenetic anal-ysis, Maximum parsimony (MP) and Neighborjoining (N]), to ensure that our analyses were fitenough for the reality. Phylogenetic trees were in-ferred from the ML distances calculated with

Kimura 2-parameter with Crossoptilon auritum asoutgroup.

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Life ScienceJournal, 2 (1), 2005, Gong, et al, PhylogeneticRelationship of Tetraogallus Inferred from Sequences

drial DNA, is widely used for avian molecular phy-

logenetics at the levels of species and genus (Tu-

inen, 2000), which can be used to resolve the

problems bequeathed by morphological taxa

(Avise, 1994). In this study, snow cocks and

their close related genera were selected as the ex-

perimental materials in order to study the phyloge-

netic position of the genus Tetraogallus, under-

stand the phylogenetic relationships among the

available snow cock species (the foreign species of

T. caspius and T. caucasicus are very difficult to

find and sample, and the related sequence data can

not be found in other literatures), and infer the

evolution process of the Tetraogallus by combining

the molecular data, distribution patterns and mor-

phological traits.


2. 1 Materials and nucleotide sequencesAltai snow cocks and Himalayan snow cocks

were captured in the Altai Mountain and the areasof Tianshan, respectively. The blood samples werecollected. The other sequence data were retrievedfrom GenBank as shown in Table 1.

Tablet. The sequence data used in this study"

Code Latin name English name AccessionNo

AG Alectorisgraeca R<xkPartridge ZA8772

CAb Crossoptilonauriturn Blue Eared-Pheasant AF534552

CC Coturnix chinensis Blue-breastedQuail NCOO4575

CJ Coturnix japonica JapaneseQuail NCO03408

FF Framvlinusframvlinus Black Francolin AFO13762

PM Pucrasia rnacrolopha Koklass Pheasant AFO28800

IT1' Tetraogallus tibetanus Tibetan snow c<xk AY563128

TT2' Tetraogallus tibetanus Tibetan snow c<xk AY563130

TT3c Tetraogallus tibetanus Tibetan snow c<xk AY563133

Notes:a The information included the supplementary materials'

Latin names, English names and sequenceaccessionnumbers in the

GenBank, and, at the same time, the codes for these supplementarymaterials were formed by the two first letters of their Latin names in

this study. bThis specieswas selectedas outgroup in this study. ' Thedigitalsbehind the codes represent the individualswe selected in thisstudy,

~2.2 Extraction of total genomic DNA

The blood samples were digested with SDS/Protease K. Then the supernatant was extractedwith phenol/chloroform. Finally, ethanol precipi-tation would be preformed to condense the total ge-nomic DNA. The concentrated DNAs were dis-


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Life ScienceJournal, 2 ( 1 ), 2005, Gang, et ai, PhylogeneticRelationship of Tetraogallus Inferred fram Sequences10"

and all the other species was the biggest one of allthe genetic distances in the same lines. The inter-specific genetic distances of the genus Tetraogalluswere O. 012 (T. altaicus vs T. himalayensis),0.067 (T. himalayensisc vs T. tibetanus) andO.068 (T. altaicus vs T. tibetanus), respective-ly (Table 2). It is well known that phylogeneticreconstruction benefited from increased character

inclusion, which provided all characters sharing acommon evolutional history. In ;ec~nt avian geneticevolutionary researches, people propose that the ge-netic distance of Cyto b gene is bigger than O. 01( > O. 01) between species and smaller than O. 01«0.01) between subspecies (Krajewski, 1996).So, the result suggested that Altai snow cocks andHimalayan snow'cocks had an intimate relation-ship, and Altai snow cocks and Tibetan snow cockshad a distant relationship among the three species.The length of mtDNA Cyto b gene sequence in oursampled species was 535 base pairs, and the aver-age base composition of Tetraogallus was 25 %thymine (T), 35% cystine (C), 27.5% adenosine(A), and 13% guanine (G), and the T and A con-tents (52 %) was higher than that (47. 7 %) of Cand G. This composition was very similar to that ofother Snow cocks. At the same time, we foundthat no gaps were introduced into the Cyto b genealignment, and there were 227 sites that were vari-able ones, and conservative sequence composing of113 base pairs was discovered.

Table2.Geneticdistance(Lower-left)and standarderror (Upper-right)analyzedby MEGAversion2. 1 with Kimura2-pa-rameter"


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Snow cocks are an excellent kind of birds for

biological studies. There are five species of thisgenus living in Eurasia only, and T. altaicus, T.himalayensis and T. tibetanus can be seen in Chi-na except T. caspius and T. caucasicus. Tradi-tionally, Tetraogallus were classified into differentspecies according to their morphological characteris-tics and geographical distributions. Tibetan snowcocks, one of these species, can be divided into sixsubspecies whose distribution areas are composed ofHimalayan Mountain, Parmir Plateau, MishmiMountain and Tibetan Plateau. Nowadays, it issaid that there are O.2 million Tibetan snow cocks

in Tibet. Altai snow cocks are composed of twosubspecies, which can be discovered from AltaiMountain to the Northwest Territories of Mongoli-a. Himalayan snow cocks include four subspecies,which have a wide distribution includingAfghanistan, Turkey to Nepal, and the northwestterritories of China (Zheng, 2002). Nowadays,only shallow researches have been done. However,the evolutional relationship among the Snow cocksstill remain unclear, and even the evolution processof the whole genus is sometimes in doubt.

The overall average distance was O. 112, andaverage genetic distance among Tetralgallus was0.042. The genetic distances between C. auritum

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AGCC 0.141

CJ 0.139 0.121FF 0.136 0.150 0.161PM 0.129 0.149 0.151 0.112TA 0.135 0.143 0.138 0.127 0.133

illl 0.131 0.143 0.143 0.129 0.133 0.011TH2 0.135 0.145 0.145 0.132 0.136 0.013 0.006TH3 0.133 0.143 0.143 0.127 0.133 0.011 0.004 0.006ITI 0.126 0.139 0.138 0.134 0.129 0.068 0.067 0.070 0.068TT2 0.124 0.137 0.136 0.132 0.127 0.066 0.065 0.068 0.066 0.002Tr3 0.126 0.139 0.139 0.129 0.124 0.068 0.068 0.070 0.068 0.004 0.002CA 0.143 0.162 0.164 0.135 0.140 0.160 0.159 0.162 0.160 0.153 0.150 0.148

Notes:" The genetic distances (lower-left) and standard errors (upper-right) were analyzed using MEGA version 2. 1 with Kimura 2-parameter. The genetic distances between CA and all the other species were the biggest ones in the same line. The overall average dis-tance was O.112 and average genetic distance among TetralgaLlus was O.042. The interspecific genetic distances of the genus Tetraogal-lus were 0.012 (T. altaicus vs T. himalayensis), 0.068 (T. altaicus vs T. tibetanus) and 0.067 (T. himalayensis vs T. ti-betanus) respectively. b The species of Altai snow cock ( T. altaicus).' The speciesof Himalayan snow cock ( T. himalayensis) and Ti-betan snow cock ( T. tibetanus), and the digitals were the code of individuals used in this study.

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Life Science Journal, 2 (1), 2005, Gong, et al, Phylogenetic Relationship of Tetraogallus Inferred from Sequences

Figure 1. Phylogeny of Tetraogallus and its close related genera inferred from mitochondrial cytochrome b gene sequences in the analysis.

The tree topology was inferred from a Neighbor-joining (N]) distance matrix calculated with Kimura 2-parameter. Numbers near the

branches were bootstrap probability values coming from 1000 replications. Some bootstrap values were 92 % (Coturnix chinensis ICo-

turnix japonica), 100 % (Tetraogallus altaicus I Tetraogallus himalayensis), and 100 % [( Tetraogallus altaicus I Tetraogallus hi-

malayensis) I Tetraogallus tibetanusJ. Crossoptilon auritum was used as outgroup. The tree topology was also supported by Maximum-

Parsimony (MP) analyses.

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As the NJ tree shown that the genus Tetrao-

gallus fonned a sole clade, and which was well re-

solved with bootstrap percentage value 100 %. The

final results based on the molecular data supportedthat the genus Tetraogallus encompassed three

(except two species, T. caspius and T. caucasi-

cus) named groups given the rank of species: T.

altaicus, T. himalayensis and T. tibetanus.

And, our data suggested that the genera Alectoris ,

UJturnix, Tetraogallus and Pucrasia fonned a

monophyletic group. The bootstrap percentage val-

ue of the branch composed of the genera Tetrao-

gallus, Alectoris and UJturnix was 88 %. There-

fore, the two genera, Alectoris and UJturnix,

should have a common ancestor, which was the sis-

ter taxon of the ancestor of the genus Tetraogallus

(Figure 1). Both the plwlogenetic trees and the

genetic distances suggested that the Francolinus

francolinus was older than other studied species in

its evolutionary history.

The NJ tree, at the same time, suggested that

the genus Tetraogallus had evolved into two

branches with bootstrap percentage (100 % ): one

branch was composed of the Altai and Himalayan

snow cock with bootstrap percentage 000%), andthe other one was the Tibetan snow cock. This was

also supported by the data of MP analysis. On theother hand, there are also several lines of evidence

including distribution patterns and morphological

traits to support our point of view for the evolution-

ary pattern of the three snow cock species. i) Both

the Altai and Himalayan snow cocks have a similar

body fonn, about 60 centimeters in length, while


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the Tibetan snow cocks have a body fonn about 53

centimeters in length; ii) The two subspecies ofAltai snow cocks can be discovered from Altai

Mountain to the Northwest Territories of Mongoli-

a, and have an offwhite breast and belly. Hi-

malayensis, one subspecies owning a deepest body

colo~ of Himalayan snow cocks, can be detected inthe areas of Tianshan and northwest of Xinjiang

Uygur autonomous region, while another sub-

species, koslowi, owning a light body color, often

emerge in the Altai Mountain and the east of Kun-

lun Mountain. There are two subspecies holding alight body color, Przewalskii and henrici, can be

discovered in the east of their distribution area; and

one subspecies holding a deep body color, ti-

betanus, distributes in the southwest of Xinjiangand the midwest of Tibet.

Shields and Wilson pointed out that the diver-

gence rate of avian mitochondrial DNA Cyto b gene

is about O. 02 per million years (Shields, 1987).

Therefore, according to the conclusion, we can de-

duce the divergence time of snow cocks was 1. 7

million years ago when the terrain and environment

of Hengduan mountains region had a greatly

changes in the Pliocene. Considering its molecular

phylogeny and geographical distribution patterns,

and the evidences of the genetic distances between

species are all bigger than O. 01, and the results of

genetic distance (0. 12) between Altai snow cocks

and Himalayan snow cocks reveal that the two

species have a closer relationship. It is possible that

Tibetan snow cocks might be the primitive species

among the three breeds and originate in the Heng-

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., duan mountains region according to the witness of

geographical distribution pattern and morphologicalcharacter combining the genetic distances among

them. So we could propose that one subspecies ofTibetan snow cocks, tibetanus, holding a deep

body color, migrated towards the West and entered

the Himalayan Mountain to evolve into the present

Himalayan snow cocks. Later, another subspecies

of Himalayan snow cocks, koslowi, owning a light

body color, often emerge in the Altai Mountain andthe east of Kunlun Mountain to evolve into Altai

snow cocks.

Although the present study provided a good

start towards our understanding of the relationshipsof the snow cock breeds found in China, detailed

interrelationships and limits of the subgroups still

need further study. But in all our trees Tibetan

snow cocks occupy a relatively basal position. As

for Caspian snow cocks and Caucasian snow cocks,

we consider that our sample of Tetraogallus taxa is

insufficient to resolve the evolutional processes of

the foreign snow cock breeds, and that solid con-

clusions on the status of the total Tetraogallus

awaited inclusion of additional related taxa, and re-

quire further investigation. But, we can give a hy-

pothesis that, according to the geographical distri-

bution range and morphological character, Tibetan

snow cocks may be the most primitive breed among

the five snow cock species, and Caucasian snow

cock derived from Altai snow cocks. As to Caspian

snow cocks, they might derive from Himalayan

snow cocks. This hypothesis needs further investi-

gation and study.

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Endemic and Ethnic Disease

Shihezi UniversityShihezi, Xinjiang 832003, ChinaTelephone: 01186-993-205-8510Email:[email protected];[email protected]

References

1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seid-

man JG, Smith JA, Struhl K. Short Protocols in Molecu-

lar Biology (3rd edition). John Wiley & Sons, Inc.,

1995;30-5.

2. Avise JC. Molecular Markers, Natural History and Evolu-

tion Champman and Hall. New York, London, 1994;511.

3. Cann RL,Stoneking M, Wilson AC. Mitochondrial DNAand humanevolution.Nature1987;325(6099):31 - 6.

4. Gyllensten U, Wharton D, Wilson AC. Maternal inheri-

tance of mitochondrial DNA during backcrossing of two

species of mice. The Journal of Heredity 1985; 76 (5):321-4.

5. Kirchman J. Relationships among cave swallow papula-

tions determined by comparisons of microsatellite and cy-

tochrome b data. Mol Phylo Evol2000; 14(1): 107 - 21.6. Krajewski C, King DG. Molecular divergence and phy-

logeny: rates and patterns of cytochrome b evolution incranes. Mol Bioi Evol1996;13:21- 30.

7. Kumar S, Tamura K, Jakobsen lB, Nei M. MEGA:

molecular evolutionary genetics analysis software. Bioin-formatics 2001; 17: 1244 - 5.

8. Meyer A, Kocher TD, Basasibwaki P, Wilson AC.

Monophyletic origin of lake Victoria cichild fishes sug-

gested by mitochondrial DNA sequences. Nature 19.90;347( 6293) : 550 - 3.

9. Shields GF, Wilson AC. Calibration of mitochondrial

DNA evolution in geese. Journal of Molecular Evolution

1987 ;24(3) :212 -7.

10. Tuinen MY, Sibley CG, Hedges SB. The early history

of modern birds inferred from DNA sequences of nuclear

and mitochondrial ribosomal genes. Mol Bioi Evol 2000;17:451-7.

11. Vawter L, Brown WM. Nuclear and mitochondrial DNA

comparisons reveal extreme rate variation in the molecular

clock. Science 1986 ;234: 194 - 6.12. Zheng GM. A Check-list on the Classification and Distri-

bution of the Birds of the World. Beijing. Science Press,

2002; 1153 - 9.

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Anti-tumor of ILTT Combined with Cisplatin in aRat Glioma Model

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1. Department of Neurosurgery, Henan Provintial People's Hospital,

Zhengzhou, Henan 450003, China; brainI988cn@yahoo. com. en

2. Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University

of Science and Technology, Wuhan, Hubei 430081, China

Abstract: Objective. This study is to evaluate the therapeutic effect of interstitial laser thennotherapy (ILTT) com-bined with cisplatin chemotherapy on rat glioma model with deeply sited tumors. Methods. Cr,glioma cells were in-jected into the nucleus caudatus by stereotactic technique to induce transplanted gliomas in the rat brains. The ratsbearing glioma were treated with ILTT in combination with cisplatin. The survival time of rats was observed up to40 days after ILTT, and the tumor size of rats at 7th day after ILTT was measured. The data in different groupswere statistically analysed. Results. The mean survival period of rats was (26. I:!: 3.6) days in the control group,(27. 3 :!:3. 9) days in the cisplatin treated alone group, (30. 4:!: 5. 3) days in the ILTT alone group and (34. 4 :!:4.0) days in the ILTT combined with cisplatin chemotherapy group. At the 7th postoperative day, the maximum di-ameter of tumors measured in coronal sections of the brains were different between control group and experimentalgroups: (6. 2 :!:O.2) mm of the control group, (6. 2 :!:O. 1) mm of the cisplatin treated alone groups, (4. 8 :!:O. 2)mm of IL TT group and( 4.9 :!:O. 1) mm of the ILTT combined with cisplatin chemotherapy group. Conclusions.Based on the above described results, we would conclude that the combination of ILTT and cisplatin chemotherapymight provide a significantly greater antitumor effect in the treatment of glioma. [Life Science Journal. 2005; 2(1):90-93J (ISSN: 1097-8135).

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).Keywords: antitumor; cisplatin; interstitial laser thennotherapy; rat glioma

1 Introduction

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Interstitial laser thermotherapy is a new inva-sive method for treating cerebral tumors, especially

suitable for deep brain tumors[l]. Cerebral gliomais one of the most common tumors in central nerve

system. Glioma is difficult to be completely excisedby operation for its infiltrative growth, and thoselocated in deep or functional regions are more re-stricted with operations. The chemotherapy effectis unable to be ensured because most chemotherapydrugs are difficult to penetrate into blood-brain bar-rier which exists in central nerve system. It is nec-essary to explore novel treatments because the con-ventional therapy effect on cerebral glioma is unide-al. So we performed the experimental study ontreating rat cerebral glioma with ILTT combinedwith cisplatin. The experimental findings are re-ported as follows.


2. 1 Cerebral glioma modelC6 cell line of rat cerebral glioma (presented

kindly by Graduate School of Neurosurgery in


Shanghai) were cultured for passages with cultur-ing method of monolayer cells in complete culturesolution RP-1640 with 10 % bovine serum. Dl,lringphase of logarithmic growth, the cells which weredealt with digestive solution of O. 24 % pancretinand then washed with Hanks solution twice were

made into suspended solution for inoculation. 64healthy rats (offered by animal laboratory of TongjiMedicalUniversity) weighed300 - 350 g were fas-tened after anesthesia to head arc of ]iangwan type,stereotactic apparatus for rat brain. The indicationsof rat skull were exposed by operation and the inoc-ulated target was selected: at a point 1. 0 mm tothe front side of bregmatic midpoint, 3. 0 - 3. 5mm to the right side of sagital sutura, and to adepth of 5 mm under dura. Hole in skull was drilled

and 2. 0 X 106/15 [..llC6 cells (li vabili ty > 90 %) wereinoculated into each rat with micro-injector directedby stereotactic apparatus. The injection time wasover 10 minutes and the injector was slowly with-drawn after being kept for 5 minutes, then thescalp was stitched. The rats fed on food and waterfreely in cages. The 14-days rats after inoculationwere selected as cerebral glioma model in this ex-periment.

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2.2 Treatment method

At 14 days after inoculation of ~ cells, therats were randomly divided into 4 groups: 16 ratsin each group (10 of 16 were kept for observingsurvival time, 6 for measuring tumor diameter),Group A acted as control group (without treat-ment); Group B as cisplatin treatment group;Group Cas ILTT group; Group D as ILTT com-bined with cisplatin group. 1 fig/g cisplatin wasgiven to rats of group B by. intravenous injection ofrat tail, and afterwards each time equivalent dosageof cisplatin was given every 48 hours up to 3 times.1fig/g cisplatin was given to rats of group D by in-travenous injection of tail 30 minutes before ILTT ,and each time equivalent dosage of cisplatin wasgiven every 48 hours up to 3 times. Dexamethason(7 mglkg) was given by intramuscular injection at24 hours, 30 minutes before IL TT, and at 24hours after IL TT, respectively, to prevent cerebraledema. After IL TT, 40, 000 U of penicillin wasadministered by intramuscular injection each dayfor 3 days in sequence.2.3 Interstitial laser thermotherapy technique

At 14 days after inoculation of C6 cells, all ratswere fastened to head arc of ] iangwan type, stereo-tactic apparatus (products of Shanghai) for ratbrain after anesthesia with intra-abdominal injection

of 2 % pentobarbital sodium. Dura was excided a-long injector pathway after skull was exposed byoperation. One side of optic fiber with a core diam-eter of O. 5 mm was connected with laser and an-

other side (naked optic fiber) implanted into theselected target by stereotactic apparatus. Continu-ous Nd: YAG laser with wave-length of 1. 06 fim

and a power of 1. 5 W was given for irradiation for120 seconds, and equivalent quantity of laser wasgiven again after an interval of 120 seconds, thentreatment was finished.

2. 4 The measurement of largest transverse diam-eter of tumors

At 21 days after inoculation of tumor, ratswere perfused with alcoholic solution containingO. 5 % methylene blue through aorta of left ventri-cle, complete brain was taken out and fixed with10% formaldehyde, and then coronary section wasmade along the center of tumor (the center of opticfiber pathway). The largest transverse diameter ofeach tumor was measured on horizontal direction

(including pathological tissues such as necrosisetc. ).2. 5 Survival time

After treatment rats were recovered and raised

continuously. Rats in each group were observedeach day till rats died and the survival times (day)were written down. The survival time of rats with-

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out death was counted as 40 days (observation du-ration was 40 days). Two-sample test of nonpara-metric tests for multiple samples (Nemenyimethod) was applied to analyze the survival time ofeach group.

3 Results

3 .1 Survival time

Rats in all groups had no signs of nerve func-tion deficiency such as hemiplegia et al. and therewas no death in late period after treatment. Theaverage survival time is: Group A (26. 14 :t 3. 58)days, Group B (27. 26 :t 3. 90) days, Group C(30.40:t5.27) days, Group D (34.42:t 4. 05)days. By comparison, there is a significant differ-ence between group D and A (P<O. 01) and a dif-ference between group D and C( P < 0.05) . Thereis no significant difference among other groups.3 .2 The largest transverse diameter of tumors

Group A (6.2:t O. 2) mm, Group B (6.2:t0.1) mm, Group C (4. 8 :t O. 2) mm, GroupD(4. 9 :to. 1) mm.

4 Discussion

In recent several decades, the therapeutic ef-fect of malignant glioma is still unsatisfactory. It isdifficult to eradicate glioma for its infiltrativegrowth. Only biopsy can be usually performed andrelapse will occur shortly after operation if tumor issituated in deep position or functional region of

brain, the average survival time is 9 months[2]. Itis necessary to explore novel methods to enhancetherapy effect, so we carried out the experimentalstudy on cerebral glioma treated with IL TT com-bined with cisplatin. The biological behavior of C6cells of rat with cerebral glioma are similar to thatof human glioma, in recent years C6 cells are widely

applied to establish rat glioma model to expand ex-perimental research. C6 cell which was inoculatedinto caudatum could be acted as glioma model in

deep position of brain[3].Nd: YAG laser is a laser that' can transmit

through a £lectional optic fiber which can be im-planted into brain tumor tissue with brain tridimen-tional direction-finder. Its character of striking

penetration to tissue ensures to develop more potentthermal effect by contacting irradiation to tissueswith less scatter[4], so the laser can be a thermalresource for thermotherapy of brain tumors. It in-dicated in one research that thermal effect of Nd:Y AG laser with IL TT developed an globose coagu-lated necrotic region surrounded by a band of edema

zone[!]. It presented in our research that thermal

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effect in edema zone also could induce apoptosis ofbrain glioma (pending materials for publication).The effect on coagulating tumor with IL TT maykill out an absolute majority of glioma tissue as pos-sible as can, but a small quantity of tumor cells maysurvive in peripheral zone around effective focus forthe infiltrative growth of glioma. If further corre-sponded treatments such as irradiation orchemotherapy are not applied, the effect of IL TTwill be influenced because the residual tumor tissues

may grow quickly to develop a relapse of tumor. Itshows in this experiment that at 1 week after IL TTthe largest transverse diameter of tumors is lessthan 5 mm in treatment group, and more than 6mm in control group and cisplatin group; the aver-age survival time of rats loaded with tumor in singleIL TT group is longer than that of control group orsingle cisplatin group, but shorter than that ofgroup of ILTT combined with cisplatin.

Presently the chemotherapy of brain tumors isa hard nut to crack relatively, most chemotherapydrugs are restricted to penetrated into central nerve

system for blood-cerebral barrier[5,6], only thosedrugs with small molecular weight and with lipid-solubility can enter into tumor tissue across blood-

cerebral barrier. Although in vitro studies provedthat many chemotherapy drugs could influence theproliferation of glioma cells, they were not still usedfor therapy of brain glioma mainly because the

drugs can't overpass blood-cerebral barrier[7]. Re-searches have proved that thermotherapy couldopen blood-cerebral barrier and promote the trans-portation of chemotherapy drugs between blood and

tissues[8]. The experiment has showed the distri-bution of Evans Blue in thermal effect focus and its

surrounded tissues[9]. Depending on the results wededuced that permeability of blood-cerebral barrierin effective focus and its surrounded tissues en-

hanced, so cisplatin can enter effective focus and itssurrounded glioma tissues and accumulate to a defi-nite concentration on which cisplatin can induce cellapoptosis to kill tumor cells. So we applied cisplatinto kill residual tumor cells right after IL TT. Cis-platin is a kind of non-specific cell-cycle drugswhich can be activated in cells with light or heat

and its effect is not restricted by cell cycle[lO].At present the reports on therapy of brain

glioma with ILTT published increasingly, the ani-

mal experiment of EI - Ouahabi[l1] presented thatthe average survival time of rats with brain gliomatreated with single ILTT was a little longer thanthat of control group but there was no difference instatistics. ] ust as the analysis given by the author,the remained tumor tissue after ILTT and relapse


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of tumor may be the main cause for animal death.

Reports from each clinical research differed[12,13].Single ILTT was mostly applied for treatments inwhich temperature in therapeutic zones was con-trolled by computer system or was monitored by re-al-time imaging. Our experiment shows that al-though average survival time can be prolonged withsingle IL TT, there is no statistic difference bycomparison with average survival time of controlgroup. The average survival time of rats loaded withtumor can be more prolonged if cerebral glioma istreated with ILTT combined with cisplatin. Thereis a significant difference of average survival timebetween treatment group with ILTT combinedwith cisplatin and control group or single cisplatingroup(P<O.Ol and P<O. 05). So the therapeuticeffect of treatment group with ILTT combinedwith cisplatin is markedly superior to that of singleIL TT group.

The mechanism of tumor therapy with ILTTmay include several aspects as follows: (1) Coagu-lating tumor tissue: it mainly develops in therapeu-tic zones with higher temperature nearby opticfiber. Tumor cells in a state of nutrition deficiencyacquires energy mainly by anaerobic glycolysis, andlactic acid accumulates in tumor cells ; Vessels intumor tissue which is short of smooth muscle can't

dilate in accordance with the increase of tempera-ture; and the vessel structure is not intact and has alow tolerance of heat. All these characters enhance

the sensitivity of thermotherapy to tumor cells. (2)Inducing apoptosis of tumor tissue. (3) Activatingcytokines, inflammatory media and vascular actoivesubstances to influence blood supply for tumor and/or develop a direct killing effect. Even so, the ef-fect of single IL YY is still not ideal. Although av-erage survival time on treatment group with IL TTis longer than that of control group, there is nostatistic difference. It may be because that gliomagrows with infiltration state but ILTT creates aglobose effective focus, so residual tumor cells insome position lead to tumor relapse after the end oftreatment. The opening of blood-cerebral barrierenables chemotherapy drugs to enter tumor tissueto kill remained tumor cells.

IL TT is a palliative therapy for glioma. AfterIL TT the relapse of glioma is inevitable for residualtumor cells. If ILTT is combined with chemothera-

py and/or photodynamic therapy, it will helpful toraise the therapeutic effect of malignant cerebralglioma.

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\Henan Provincial People's Hospital7 Weiwu RoadZhengzhou, Henan 450003, ChinaTelephone : 01186-371-6884-8307Email: [email protected]

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... References1. Liu Bin, Ll Ling. Brain tumors of intE;rstitiallaser thennother-

apy. Chin J Laser Med Surg 2000; 9: 45 - 6.2. Walker MD, Alexander EJ, Hant WE, et al. Evaluation of

BCNU and/or radiotherapy in the treatment of an anaplasticgliomas a cooperative clinical trial. J Nurosurg 1987; 49: 333-6.

3. Peterson DL, Sheoidan PL, Brown WE. Animal models forbrain tumors: historical perspectives and future directions. JNeurosurg 1994; 80 :865 - 73 .

4.Daikuzono N, Suzuki S,Tajiri H,et al. Laserthennia: a newcomputer controlled contact Nd: YAG system for interstitiallocal hyperthennia. Lasers Surg Med 1988; 8: 254 - 8.

5. Takakura K, Abe H, Tanaka R, et al. Effects of ACNU andradiotherapy on malignant gliomas. J Neurosurg 1986; 64: 53-7.

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6. Watanabe M, Tanaka R, Hondo H, et al. Effects of antineo-plastic agents and hyperthennia on cytotoxicity toward chroni-cally hypoxic glioma cells. Int J Hyperthennia 1992;8: 131 -

8.7. Tanaka R, Kim CH, Yamasa N, et al. Radiofrequency hy-

perthennia for malignant brain tumors: preliminary results ofclinical trials. Neurosurgery 1987 ;21 :478 - 83.

8. Lin JC, Lin MF. Microwave hyperthennia-induced bloodbrain barrier alternations. Radiat Res 1982; 89: 77 - 87.

9.Sugiyarna K, Sakai T, Fujishima I,et al. Stereotactic inter-stitial laser hyperthennia using Nd- YAG laser. Stereotact

Funct Nurosurg 1990; 54 + 55; 501-505.10. Ha Xianwen. Laser surgery-mininally invasive surgical treat-

ment of tumors. Chinese J Laser Med Surg 1996;5: 119 - 20.

11. El-Ouahabi A, Ghttmann CRG, Hushek SG, et al . MRI

guided interstitial laser therapy in a rat malignant glioma mod-

el. Lasers Surg Med 1993; 13 ; 503 - 10.12. Menovsky T, Beek JF, van Gernert MJC, et al. Interstitial

laser thennotherapy in neurosurgery. A Review. ActaNurochir 1996; 138 : 1019 - 26.

13. Reimer P,Bremer C,Horch C. MR-monitored ILTT as a pal-

liative concept in patients with high grade gliomas: prelimi-

nary clinical experience. J Magn Reson Imaging 1998; 8: 240-4.

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Life Science Journal "J

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Acta Zhengzhou University Oversea Version,

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Life Science ]aurnal ,2 (1 ) ,2005 ISSN: 1097 - 8135

Authors

Blumberg, DavidChen,XiufangCheng, XiangCherng, ShenCook, TracyCui,Jing ,Dai, LipingEdmondson,Jingjing ZFan, YueyangFu,ChunjingGong, YifengGuo,LonghuiHan, HuaminHan, XinguangHe, ZhanhangHong, GuangfengHuang, YoutianJia, YanJiang, HaiyangJ in, Bilian

Pages55 - 6037 - 3972 - 76

7-15,37 - 3955 - 60

30 - 3622 - 26

2-668 - 7149 - 5485 - 8949 - 5430 - 3677-8468 - 7168 - 7149 - 5422 - 2665 - 67

90 - 93

Author IndexAuthors

Li, HongyanLi, LingLiao, YuhuaLiu, BinLiu, YuxiLu, BaojunMa, HongbaoMa, RunlinQiao, BaopingRen, XiaofengShen, ChangyuShen, QiSun, MeizhenSun, TongwenT eng, Hsien ChiaoTian,HuaTian, ZhengshanWang,JinfuWang, Kaijuan

Wang, Lexin

Pages85 - 8990 - 9372- 7690 - 9346 - 4872 - 76

7-1585 - 8965 - 6746 - 48

1-1

49 - 54

46 - 4861 - 6416 - 2149 - 5468 - 7185 - 8922 - 26

61 - 64

Authors

Wang,LiWang, LipingWang, SulinWang,WeiWang,ZhongquanWang, ZifaWei,HaiyanWen, BoguiYe,ShujuanZhang, HongweiZhang ,JianyingZhang, JinyingZhang, KerangZhang, ShuxiangZhang, WeiZhang, WeixingZhang, YanzhouZhao, GaoxianZhao, Guoqiang

Pages85 - 8927 - 2937 - 3946-4830 - 36

55 - 6030 - 3677-84

68 - 7130 - 3622 - 2672 - 7646-48

61 - 6440 - 45

65 - 6761 - 6465 - 6730 - 36

Keywords

activated sludgeacute coronary syndrome

acute myocardial infarctionadrenalannexin Vanti-tumor

apoptosisatomic science

biological effectbiosorbent

B-typelbrain natriuretic peptidecell

cell cycle

chemotherapyChristianity

cisplatincolorectal cancer

Cushing's disease

cytochrome b genedissociative bacteriaDNA vaccine

echocardiogiaphyEGF receptorendocrine disorder

entroIJY


Pages686172652290

49,772

1668611677272

9055658568307277377

Subject IndexKeywords

epilepsyERK1/2

ETH 5504evolutionexistence

expressIOn

gene-gun deliveryHinduismheart failure

hemodynamics

immortalityimmunohistochemistry

interstitial laser thermotherapyion selective electrodeslife

h'Xlrtan

lung neoplasm

magnesium

magnetic fieldmatrix metalloproteinase-2, 9mice

mitochondrial DNAMPAnature

neoadiuvant radiation

. 95

Pages46774077

22,30302

4972

2729040

2,74927401672308527

755

Keywords

phylogenyphy~ologjcal assaypMAL-c2X

prognOSIS

psychologypurification

quality of liferadioresistance

radiosensitizersrat

rat gliomaRT-PCRseizure

selectivity coefficientssimvastatin

smoking

smoking inhalationsnow cock

social support

surgjcal treatment

Symptom Checklist 90Tca8113 cell

Trichinella spiral is

zeta potential

Pages854022614622275555

49,729072464072373785466546773068

editor@sciencepub. net

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