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A Perspective Suggested by Peer-Reviewed Literature a of How Glycosaminoglycans May Mediate Cellular Activities Including Proliferation and Transformation D. Grant, Ph.D., Turriff AB53 Scotland UK A topic which is believed to continue to be of public interest was the subject of a presentation made at Marischal College Aberdeen in 1982 by Nancy E. Woodhead (a member of the W.F. Long, F.B. Williamson Polysaccharide Research Group). This document contains an edited transcript of my shorthand notes made at the time to which I now append an update of selected peer-reviewed literature in this field and also an edited version of a hypothesis which is thought to be of related interest (“Ascorbate and Nitric Oxide in Redox Control of Heparan Sulphate…” which was originally posted on the internet [dg4,5,8] in 2000 on a now-discontinued server. ================================================= ==================== “Glycosaminoglycans and Cellular Transformation” Prior to 1982 literature reports were classified by Nancy Woodhead as follows: 1. Experiments directly relating glycosaminoglycans (GAGs) to cell growth control and transformation.

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A Perspective Suggested by Peer-Reviewed Literature a

of How Glycosaminoglycans May Mediate Cellular Activities Including Proliferation and Transformation

D. Grant, Ph.D., Turriff AB53 Scotland UK

A topic which is believed to continue to be of public interest was the subject of a presentation made at Marischal College Aberdeen in 1982 by Nancy E. Woodhead (a member of the W.F. Long, F.B. Williamson Polysaccharide Research Group). This document contains an edited transcript of my shorthand notes made at the time to which I now append an update of selected peer-reviewed literature in this field and also an edited version of a hypothesis which is thought to be of related interest (“Ascorbate and Nitric Oxide in Redox Control of Heparan Sulphate…” which was originally posted on the internet [dg4,5,8] in 2000 on a now-discontinued server. =====================================================================

“Glycosaminoglycans and Cellular Transformation”Prior to 1982 literature reports were classified by Nancy Woodhead as follows:

1. Experiments directly relating glycosaminoglycans (GAGs) to cell growth control and transformation.

2. Alterations in GAG contents of cultured cells after transformation.

3. Alterations of GAG contents of mammalian tumors compared to normal tissue; [this provides more material than is available from cells in culture].

4. Changes in complex composition of certain cell surface GAGs; [especially of HS)]

5. Searches for a specific changes in function of cell surface GAGs; [possible functional change due to regional alterations in GAG structure].

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1. Includes reports of experiments relating GAGs to cell growth control and cellular transformation; starting from 1932:

Year Author

1932 Zakrezewski Reported that heparin suppresses the growth of normal embryonic tissue and Jensen sarcoma tissue.

1957 Sister M. Lippman Reported experimental evidence that heparin can act as a mitotic inhibitor. [This was an in vivo study of the effect of how administration of subcutaneous heparin affected the measured size of Ehrlich Ascites tumors in rats. It was shown that heparin, under the conditions studied, produced a 40- 50% regression of these tumors]. 1960 Ozzello et al. Umbilical cord extracted hyaluronate (HA) or chondroitin sulfate (CS) promoted the growth of human mammary carcinoma cells in culture. Any of these substance taken singly promoted tumor growth.

1964 Costachel et al. Heparin prevented mitosis. Evidence for intracellular action of heparin. Heparin also caused formation of microvili on cell surfaces.

(Is this an abnormal effect of heparin or is at an apparent normal intracellular function of heparin-like molecules?) It was apparent that the phenomenon was probably not a unique property of EDTA (a high affinity Ca2+

chelator) which had been previously known to produce microvilli formation. This outcome may simply be caused by the removal of Ca2+ from the cell surface. (Heparin-like molecules may have this function in vivo).

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1966 Takeuchi CS enhanced of tumor growth. Hydrocortisone inhibited the growth of tumors, but this inhibition was prevented by the presence of CS.

1974 Takeuchi et al. Heparin CS and HA : maintained cells in culture after they degenerated in normal culture medium. Some effect of CS and HA on cell surfaces was suggested to increase their potential for affecting cell growth.

1975 Olin in et al. ? Salt effects ? (and related sources of salt e.g. heparin etc.) can apparently [link to this document lost] can exert a concentration-dependent control effect on tumor cell growth. Large doses – inhibited epithelial cell growth but low cell surface doses accelerated growth. E.g. 50-100g/ml level (at) cell surface inhibited cell growth while 0.5g/ml promoted growth.

Summary of the pre-1975 findings

1. Polyanions can enhance tumor growth by protecting cell surface antigenic sites.

2. The production of new connective tissue giving GAGs is favorable to cancer cell growth More GAGs (if present)… they can be chosen for cancers to (allow cells to stick?)

3. Effect of heparin on nucleoproteins. Evidence shows that heparin interacts with proteins in the nucleus (and thereby affects DNA) and so (affects) protein synthesis.

4. Heparin removes Ca from cell surfaces. Cellular proliferation might be affected by this mechanism.

Studies which report an

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Alteration in the GAG content of cultured cells after transformation

1966 Ishimoto et al. Avian sarcoma virus in chicken embryo fibroblasts led to (i) 5x increase in HA synthesized, (ii) extracellular HA increases.

1973 Satoh et al. Herpes type II or SV40 virus transforming virus in hamster embryo fibroblasts led to

(i) increased HA(ii) ” HS proportion of total GAGs

1977 Hopwood and Dorfman SV40 in human skin fibroblasts led to

(i) increased HA; increased HS, decreased dermatan sulfate (DS).(ii) HA acid synthesis is inversely proportional to

cell density in normal but not in transformed cells.

1978 Dietrich et al. Primary and permanent cell lines have an(i) (Associated) increased CS and increased HS

in permanent cell lines(ii) (Associated) increased DS and increased HS

in primary cell lines. These results were similar for transformed (cell lines) (these are equivalent to permanent?)

1979 Mikuni-Takagali and Toole Rous sarcoma virus (RSV) transformed chondrocytes, showed: (i) less cell surface GAGs;

(ii) HS (was) shed into the cell culture medium. 1980 Ninomaya et al. Chemical transformation of liver parenchymal cell clones led to: Increased CS;

production of (usually less) HS. Suggested reason/(consequences) for these changes:

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(1) CS acts as stimulant for cell division, with HS and DS having different roles such as recognition and adhesiveness.

(2) HS may act as a negative control element growth.(3) Loss of activity of GAG degrading enzymes or increase in GAG synthesizing enzymes:

not a direct effect of transformation.

Assessed Mammalian Tumors 1978 Chiarugi et al. 100 cases of human cancer studied: normal and neoplastic GAG content compared:

(i) All neoplastic tissue showed changed

(ii)GAG contents.(iii) Malignant tissue showed larger

changes than non invasive tumors(iv) Most common effects - increase in CS or increase in HA or decrease in HA or CS.

Often an increase (occurs in) total GAG.

(1) Increase in HA and/or CS is a characteristic abnormality of GAGs in cancerous tissue.

(2) Increase in total GAG in tumor tissue gives overall change in negative charge associated with the tissue.

--- --- --- Studies Reporting Changes in Chemical Structure of Cell Surface GAG Following Cellular Transformation

1975 Underhill and Keller 3TS cell and SV40 transformed cells

(i) HA and CS not changed.(ii) HS from transformed cells elutes at lower

ionic strength from anion exchange column.

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1978 Nakamura et al. AH130 Ascites hepatoma Cell-associated GAG 93% HS (extracellular) fluid ” 58% HS, 26% HA, 16% CS Cell associated

(i) HS less sulfated.(ii) HS (is) highly heterogeneous (as indicated by electrophoresis).

1978 Winterbourne and Mora 3TS/SV3TS cells(i) HS elutes from anion exchanger at lower

anionic strength.

(ii) 35S/3H ratio lower.

(But) no change in overall turnover rate of HS.

1979 Chandreschan and Davidson Normal human breast cell line (compared with) human breast carcinoma cell lines. Cancer cell lines - (i) Mainly CS and HS.

(i) Heterogeneous HS.(ii) Size Charge (and) NAc/N-SO3 altered.

1980 Keller et al. 3TS/SV3T3 (Following transformation) (i) HS charge density is lower. (ii) 8% decrease (occurs) in O- Sulfate.

1981 Winterbourne and Mora 3TS/SV3TS

(i) HS charge-density is lower.(ii) O-sulfate containing disaccharides of HNO2

–degraded HS; the same.

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(iii) O-sulfate-containing oligosaccharides of HNO2 degradedHS show lower 6-O sulfation.

(iv) Overall sulfation – a decrease occurs in 6 (-O-sulfate).

CONCLUSION(S TO BE) DRAWN FROM THE ABOVE EXPERIMENTS 1. Change in sulfate incorporation into GAGs may reflect change in

substrate PAPS pool sizes.2. T antigen* (SV40 early gene product) may be responsible for change

in HS structure.3. Ca2+ binding of the cell surface may be altered by lower HS charge.4. Changes in HS structure may lead to altered binding to cell surface

protein e.g. fibronectin (this is of interest since fibronectin is not found on many transformed cells).

* Binds heparin therefore could possibly change the structure of HS on the cell surface

HS was found only on cells expressing T antigen. -- -- -- -- -- --

Specific Change in Properties of Cell Surface GAGs

1982 Fransson et al. (comparing) 3T3/8V3TS and PyY 3TS cells(i) HS (is) heterogeneous(ii) HS has a lower change density (in transformed cells)(iii) HS iduronate/glucuronate bearing N-sulfate segments

– (are less common in transformed cells). Increased heterogeneity is (also) associated with reduction in self-aggregation properties.

1982 Culp and Dorfman Highly N-sulfated HS sequences (bind) most efficiently to fibronectin affinity columns.

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1983 Stamatoglou and Keller 3TS/8V3TS HS elution from fibronectin (or) collagen. No difference between normal and transformed cells (when using physiol.) NaCl Only heparin will displace HS from collagen (but) heparin and DS can displace HS from fibronectin.

1982 Castellot et al. Smooth muscle cell growth is inhibited by heparin like substances.

Endothelial cells exposed to heparinoids are released from growth inhibition.

Heparitinase released from cells or platelets may cause stimulation of mitosis by degrading heparin like compounds.

If you get damage to an endothelial surface to a blood vessel then the platelets will adhere to this surface (but only if they) release heparinase and so stimulate cell growth. The release of thrombin also will occur under these conditions.

It should be noted that:Cancer – (is the) uncontrolled growth of cells. [Loss of cell social system control].Transformation - (is the) event leading to cancerous growth of cells.Metastasis – (is the critical-for-cancer-mortality) loss of cells from tumor surface to form subsidiary tumors elsewhere.N.b. Inflammatory conditions - (are those conditions where) CS and HS also tend to increaseN.b. also: GAGs seem always to become structurally altered at cellular transformation.

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Fibronectin ???? Could such evidence point in the wrong direction?

The important events in cellular transformation may be associated (most specifically) with the GAGs.

{Note added later. The current paradigm is that (usually multiple) mutation(s) of DNA produce cellular transformation and neoplasia. Whilst this mechanism is undoubtedly the primary cause of cancer the follow-on effect on the glycosylation system is putatively also an additional critical part of the etiologies of these diseases; especially the transformation-associated alteration of extracellular proteoglycan glycosylation regulatory functions afforded by HS may actually cause the greatest damage to the organism by allowing uncontrolled cellular growth angiogeneiss and metastasis to occur}. N.b. The HS information encoded “processing system” is now thought to be a major epigenetic driver.Chiarugi et al. had noted in 1974 (Biochim Biophys Acta. 345 283-293) that “ The effect of N-sulfated polyanions on tissue growth in vivo and in vitro has been recognized since 1932” (this was intimated by Zakrezewki loc. cit }

But (at what stage) in the transformation process are the HS or other GAGs related to the change in events involved ?

The initial important change must be there - the insertion of (some) new genetic interaction.

Perhaps a clue can be gained from the circumstances under which Ca is chelated out ( cf . thrombin, antithrombin and the heparin effect)

Mammalian GAGs - can be anti-inflammatory -- can be anti-cancer.

(Cf.) A consideration which might be of relevance is how cross-linked carrageenan beads can (behave) like proteoglycan complexes; (they are set in cells but they are not actually in the cells).

a The above information was dapted from notes made during a lecture/discussion literature survey of how GAGs (especially heparan sulfate (HS)) participated in the etiology of cellular transformation and neoplasia presented to the Marishcal College (Aberdeen) Polysaccharide Research Group* on 18 November 1982 by Nancy E. Woodhead (then a graduate student member of the W.F. Long and F.B. Williamson Polysaccharide Research Group) Cf also NE Woodhead et al., IRCS Medical Science : Biochemistry ; Cancer; Cell and Molecular Biology; Connective Tissue, Skin and Bone ; Pathology [IRCS Med. Sci., 14 427-428 (1986) (Heparan sulfates from fibroblasts exhibiting a temperature-dependent transformed growth trait). (This was a comparative study of normal vs. chemically-transformed cells which exhibited a transformed growth trait at 37oC. The transformed cells reverted to a normal growth pattern at a lower temperature. The observed changes in heparan sulfates may have mediated this phenotype change.These results remain of topical interest.

*[The hypothesis that cellular proliferation is, at least in part, determined by the binding of Ca2+ ions to cell surface glycosaminogycans was, for about thirty years, a principal research

focus for a polysaccharide research group headed by W.F. Long and F.B. Williamson at the University of Aberdeen.

Barré L. et al. reported in 2006 (FASEB J. 20 E963-E975) that a Ca2+ dependent pathway (involving the completion of the glycosaminogycan protein linkage tetrasaccharide formation)

controls chondroitin and heparan sulfate proteoglycan biosynthesis].

An Addendum (vide infra) lists a fairly random selection of more recent similar topic peer-reviewed literature

References

Citations given in the 1982 lecture by N.E. Woodhead are listed below in the order in

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which they appear in my notes (which were taken at the time written down in a University of Aberdeen Lab. Notebook 16/3/82-26/11/82 [Page heading “Nancy’s Talk”] retained by D Grant AB53 UK.); these are also listed in the “The Heparan Sulphates of Control, Virally-Transformed and Chemically-Transformed Fibroblasts” Nancy Elizabeth Woodhead Ph.D. Thesis University of Aberdeen, , 1985. It should be noted that this thesis also reported for the first time on how a chemical carcinogen (N-methyl-N′-nitro-N-nitrosoguandine)transformation of cells gave rise to similar diminution of HS sulphation to that observed with viral transformation or as is commonly found in HS extracted from cancer tissue. A previous Aberdeen University Ph.D. Thesis by H.H.K Watson (1980) had also dealt with related researches. Additional references to those discussed in the 1982 lecture and which further support the hypothesis that the etiology of

neoplasia could depend on alteration of GAG composition following cellular transformation which were Chapter 1.8

[“Glycosaminoglycans in Cancer”] cited of the above Thesis are:

Kuroda et al. (1974) [Rat tumors] Cancer Res. 34 308-312;

Kupchella et al. (1981) [Rat tumors ] ibid., 41 419-424

Kojima at al. (1975) [Human hepatic tumor] ibid., 502-547

Horai et al. (1981) [Human lung tumor] Cancer. 48 2016-2021

Knudsen et al. (1984) [Murine tumors synthesized 20x the amounts of GAGs in vivo/in vitro] J Cell Biochem. 25 183-196

David and Van den Berg (1983) [Transformed mouse epithelial cells] J Biol Chem. 258 7338-7344; Eur J Biochem. 1989

178 609-617

Robinson et al. (1984) ibid., 253 668-793

Ohkuboka (1983) Cancer Res. 43 2712-7

Additional Related-Topic: Older References

Mitotic Gelation (Water Structure?)

Cf. Chiarugi V.P. et al. (1974) Biochim Biophys Acta. 345 283-293: “Heparin has been found to prevent ‘mitotic gelation’ “; (a periodic release of free heparin occurs in synchrony with the cell cycle);Augusti-Tocco G. and Chiarugi V.P. (1976) Cell Differentiation 5 (3)161-170 (Surface glycosaminoglycans as a differentiation cofactor in neuroblastoma cell culture) “ the switch from the round to the neuron-like cells can be obtained by a simple change of the culture conditions, which causes an increase of cell adhesion” this is accompanied by “ an increasing ability of cells to retain heparan sulfate”.

Control of Cellular Activities by Cell Surface GAG Selective Binding of Ca 2+

Cf. Long W.F. and Williamson W.F. loc. cit. andCf. Vannucchi S. et al. Biochem J. 1978 170 185-187.Table 4 of this article indicates that the relative Ca2+ binding capacity of commercial GAGs was found to beHA 1.00, heparin 2.76, HS 2.00, CS(A) 1.60, CS(B) 1.67 and CS(C) 1.80.

Role of Ascorbate as a Heparan Sulfation Control Agent

Watson and Edward (1980) Biochem Soc Trans. 8 134-136

(Cf. Edward and Oliver (1983) [Ascorbate boosts HS sulfation] ibid., 11 383; ibid. 12 304

{this was confirmed by Kao et al. (1990) Exp Mol Pathol. 1990 53 1-10}and may be part of the mechanism by which ascorbate

demonstrates anti-cancer activity as indicated, e.g. by Cameron and Pauling (1985) PNAS 75 4538-4542 cf. ibid., 79 3685-

3689})

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Esko J.D. Rostand K.S. Weinke J.L.(1988) (Tumor formation dependent on proteoglycan biosynthesis) Science. 241 1092-

1096; cf. Barré L. et al., (2006) 20 E963-E975 (Evidence of calcium-dependent pathways in the regulation of human 1,3-

glucuronosyltransferase-1 (GlcAT-I) gene expression : a key enzyme in proteoglycan synthesis) FASEB J. [“These GAG chains

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are important regulators in a wide range of biological events, such as matrix deposition, intracellular signaling, morphogenesis,

cell migration normal and tumor cell growth”]

Fedarko N.S. Ishihara M and Conrad H.E. (1989) (Control of cell division in hepatoma cells by exogenous heparan sulfate

proteoglycan) J Cell Physiol. 139 287-294 PMID 2715188

(Additional related references accessed by Marion Ross a later member of the Aberdeen University polysaccharide group))

Zimina N.P. et al. (1987) Biokhimia. 52 (5) 856-861 [All types of sulfated GAGs in actively proliferating tissues (except

regenerating tissues) have a reduced degree of sulfation)

Kosir M.A. and Culp L.A. (1988) Surg Forum Med. 39 424-426

Matuoka K et al. (1984) Cell Structure and Function 9 357-367 [“1. HS plays a particular function in contact regulation of cell

proliferation.

Transformation-related changes in the structure of HS molecules do not much affect the function of HS. 3. The cellular

transformation, however, is accompanied by alteration in the growth regulating system sensitive to extracellular HS”.

Heparin inhibited growth in both normal and transformed cells]

Cf. also Long WF and Williamson FB (1979) (Glycosaminoglycans, calcium ions and control of cell proliferation)IRCS Journal Med Sci. 7 429-434; Cf. also Med Hypoth. 11 285-308 and ibid., 13 385-394

Most of the relevant Aberdeen University Polysaccharide Research Group publications including those of N.E. Woodhead (up to 2003) which had been undertaken in part to advance the above hypothesis were listed by Professor W.F. Long atweb. abdn.ac.uk/~bch118/publications2003march.doc

NE Woodhead et al. also conducted in vivo and in vitro studies relating to the Long-Williamson GAG divalent metal ion animal cellular control hypothesis (cf., Biochem J. 1986 237 281-284)

HS, the ubiquitous component cell surfaces and extracellular matrices of animals, may affect transformation of cells and also be a controller of their proliferation, at least in part, via HS-mediated control of the activities of (Ca2+ and Zn2+) metal ion signaling activities.

Zakrezewki Z. (1933) Z Krebsforsch. 36 513-521

Lippman M. (1957) Cancer Res. 17, 11-14

Ozzello L. et al. (1960) ibid., 20, 600-605

Costachel O et al. (1964) Exptl Cell Res. 34 542-547

Takeuchi J. (1966) ibid., 26, 797-802

Takeuchi J. et al. (1976) ibid., 36, 2133-2139

Olin (source not found)

Obrink B. et al. (1975) Conn Tiss Res. 3 187-193

Ishimoto N. et al. (1966) J Biol Chem. 241 2052-2057

Satoh C. et al. (1973) Proc Natl Acad Sci USA. 70 54-63

Hopwood J.J. and Dorfman A. (1977) J Biol Chem. 252 4771-4785

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Dietrich C.P. and Armelin H.A. (1978) Biochem Biophys Res Commun. 84 794-801; Cf. Dietrich C.P. and DeOca H .M. ibid., 80 805-812;

Mikuni-Takagaki Y. and Toole B.P. (1979) J Biol Chem. 254 8409-8415

Chiarugi V.P. et al. (1978) Cancer Res. 38 4717-4721

Underhill C.B. and Keller J.M. (1975) Biochem Biophys Res Commun. 63 448-454; cf., J Cell Physiol. 89 53-64Nakamura N. et al. (1978) Biochim Biophys Acta. 538 445-457

Winterbourne D.J. and Mora P.T. (1978) J Biol Chem. 253 5109-5120 (1981) ibid. 256 4310-4320

Keller L. et al. (1980) Biochemistry. 19 2529-2536

Fransson L.-Å. and Havsmark B. (1982) Carbohydr Res. 110 135-144

Stamatoglou S.C. and Keller J.M. (1982) J Cell Biol. 96 1820-1823

Castellot J.J. et al. (1982) J Biol Chem. 257 11256-11260

ADDENDUM

Selected Later References which further extend knowledge of the (heparin/HS involvement in animal cell proliferation and transformation) research topic

Zhou H. et al. (M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression) PloS ONE. 2011 6 (6) e21106

Chao B.H. et al.(Clinical use of the low-molecular-weight heparins in cancer patients: focus on the improved patient outcomes)Thrombosis. 2011 2011:530183; PMID 22084664 Borsing L. et al.(Sulfated hexasaccharides attenuate metastasis by inhibition of P-selectin and heparanase)Neoplasia. 2011 13 (5) 445-452

Casu B. et al.(Heparin-derived HS mimics that modulate inflammation and cancer)Matrix Biol. 2010 29 (16) 442-452

Raman K and Kuberan B (Chemical tumor biology of HS proteoglycan)Curr Chem Biol. 2010 4 (10) 20-31[“Heparan sulfate is a profound target for developing novel cancer therapeutics because modifying HS chains would affect Hpa, Hsulf-1, Hsulf-2, H-sulf-2, and 3-OST {these are key enzymes involved in HS biosynthesis and catabolism} activity in tumor cells, which in turn would affect angiogenesis, growth factor signal over amplification, and tumor growth, invasion and metastasis”]

Barash U. et al.(Proteoglycans in health and disease: new concepts for heparanase function in tumor progression and metastasis)FEBS J. 2010 277 (19) 3890-3903

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Zacharski L.R. and Lee A.Y.(Heparin as an anticancer therapeutic)Expert Opin Investgated Drugs. 2008 17 (7) 1029-1037

Pumphrey CY et al.(Neoglycans, carbodiimide-modified glycosaminoglycans: a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis)Cancer Res. 2002 62 (13) 3722-3728

Engelberg H.Cancer. 1999 85 (23) 257-272[Heparin is a potential anti-cancer drug]Cf. e.g. Goldberg I.D. Ann N Y Acad Sci 1986 463 289-291 Coombe D.R. and Kett W.C.(Heparan sulfate-protein interactions: therapeutic potential through structure-function insights)Cell Mol Life Sci. 2005 62 410-424[Cf. also the later review by Casu et al. loc. cit.) is of especially relevance by outlining why “GAGs and particularly heparin/heparin -HS-like structures are now attracting considerable interest as a source of new therapeutics for the treatment of infectious diseases, inflammation and allergic diseases and cancers”; attempts to provide a critical review of the historical development emphasises the complexity of the biochemistry of HS (which is confirmed to be a major master system which inter alia controls embryo development wound healing, hemostasis and the immune system) and points out that in vitro studies may not reproduce the in vivo conditions especially as regards the present of metal cation cofactors and pH both of which can have profound effects on the microstructure of HS chains on which outcome of the interactions with the target proteins depend”].

ANGIOGENESIS

Ritchie J.P. et al., (SST0001, a chemically modified heparin, inhibits myeloma growth and angiogeneis via disruption of the heparanase/syndecan-1 axis)Clin Cancer Res. 2011 17 (6) 1382-1393 Logie J.J. et al. (Glucocorticoid-mediated inhibition of angiogenic changes in human endothelial cells is not caused by reductions in cell proliferation or migration)PloS ONE. 2010 5 (12) e14476;[Anti-angiogenic actions of glucocorticoids may be in part mediated by induction of thrombospondin-1 (TSP-1); this in turn implicates a key role of HS in this process as cf. Feitsma K. et al. who had indicated in J Biol Chem. 2000 275 (13) 9396-9402 (Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. The microstructure of the HS cell surface binding sites for thrombospondin-1(TSP-1) (which are responsible for TST-1 endocytosis) contains a trisulfated 2-O-sulfated iduronic acid-N-sulfated 6-O-sulfated glucosamine disaccharide unit which is distinct from the HS structure which is required for e.g. basic fibroblast growth factor binding].

Lee Y.D. et al.(Antiangiogenic activity of orally absorbable heparin derivatives in different types of cancer cells)Pharm Res. 2009 26 (12) 2667-76; PMID 19830530;

Jakobsson L. et al.,(Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis)Developmental Cell. 2006 10 625-634[Cf. Medical News Today (www.medicalnewstoday.com/articles/43115.php(“New discovery about role of sugar in cell communication…” “A research team from Uppsala Univeristy has uncovered an entirely new mechanism for how communication between cells is regulated. By functioning like glue, a certain type of sugar in the body can make cell communication more effective and stimulate the generation of new blood vessels. The discovery paves the way for the development of drugs for cancer and rheumatism, for example”…)]

Linhardt R.J.

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(Heparin-induced cancer cell death)Chem Biol. 2004 11 (4) 420-422

Blackhall F.H. et al.(Binding of endostatin to endothelial HS shows a differential requirement for specific sulfates)Biochem J. 2003 375 (1) 131-139[Endostatin is believed to inhibit angiogenesis (and hence tumorigeneis) by a mechanism which appears to involve the binding of endostatin to HS and for which 6-O sulfates played a dominant role in site selectivity];

Folkman J. (Angiogeneiss inhibition and tumor regression caused by heparin or a heparin fragment in the presence of cortisol)Science. 1983 221 719-725;Cf. Crum R. et al., (A new class of steroids inhibits angiogenesis in the presence of heparin or HS fragments)Ibid., 1985, 230, 1375-1378Cf. Schachtschabel D.O. and Sluke G. (Effect of cortisol on glycosaminoglycan synthesis and growth of diploid, human fibroblasts (WI-38) in relation to in vitro aging)Z Gerontol. 1984 17 (3) 141-149 PMID 6475191[Contrary to the medium the pattern of the cell surface GAGs was changed by 1.4x10-7M cortisol with an increase in HA synthesis and a decrease in that of sulfated GAGs; this effect of cortisol is equivalent to a “counter aging” influence]

HEPARIN AFFIN. REGULATORY PEPTIDE (HARP) [Pleiotrophin]Vacherot F. et al.,(Involvement of heparin affin regulatory peptide in human prostate cancer)Prostate. 1999 38 126-136Cf. Heroult M et al. (HARP binds to VEGF and inhibits VEGF-induced angiogenesis)Oncogene. 2004 23 1745-1753Lee T-Y. Folkman J. and Javaherian K.(HSPG (heparan sulfate proteoglycan)-binding peptide corresponding to the exon 6a-encoded domain of VEGF (vascular endothelial growth factor [VEGF]) inhibits tumor growth by blocking angiogenesis in murine model)PloS ONE. 2010 5 (4) e9945;Cf. Chen J.-L et al.(Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor (VEGF), angiogenesis and metastasis of arthotopic implantation of human gastric cancer}World J Gastroenterol. 2007 13 (3) 457-461[N-desulfated heparin inhibits tumor metastasis and angiogeneis via an inhibition of the expression of VEGF

(Possible Therapeutic Potential of (Non-Animal-Sourced) HEPARINOIDS (HEPARIN-LIKE SULFATED POLYSACCHARIDE) Zaslau S et al., Amer J Surg. 2006 192 (5) 640-643(Pentosan polysulfate (Elmiron): In vitro effects on prostate cancer cells regarding cell growth and vascular endothelial growth factor production);[This heparinoid is a sulfated beech wood xylan also known as SP54]

Cf. Noda H et al.(Antitumor activity of polysaccharides and lipids from marine algae)Nippon Suisan Gakkaishi 1989 55 (7) 1265-1271

EXT (Heparan sulfate related) putative TUMOR SUPPRESSOR

Kitagawa H. et al.(The tumor suppressor EXT-like gene EXTL2 encodes… a key enzyme for the chain initiation of heparan sulfate)

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J Biol Chem. 1999 274 (20) 13933-13937;Lind T. et al.(The putative tumor suppressors EXT1 and EXT2 are glycosyltrqansferases required for the biosynthesis of heparan sulfate)J Biol Chem 1998 273 (41) 26265-26268

Rahmoune H. et al.Biochem Soc Trans. 1996 24 (3) 355S[While the usual effect of cellular transformation is a lower production of heparan sulfate of decreased degree of sulfation, cellular transformation can also be accompanied by an augmentation of heparan sulfate biosynthesis (with increasing cell surface as well as culture medium released heparan sulfate) relative to normal cells)]Cf. Biochem J. 1998 273 33 21111-21114[A MCF-7 tumor cell HS microstructure which binds laminin-1 (implicated in tumor-host adhesion) was identified (IdoA(2-O-SO3

-)-GlcNSO3-(6-O-SO3

-)]5[IdoA(2-O-SO3-)-AManR(6S-O-SO3

-was generated using a hydrazinolysis/deaminative procedure which cleaves deacetylated N-acetylglucosaminic bonds).

Liuzzo J.P. and Moscatelli D.(Human leukemia cell lines bind basic fibroblast growth factor (FGF) on FGF receptors and HS: Down modulation of FGF receptors by phorbol ester)Blood. 1996 87 (1) 245-255

HEPARAN SULFATE AT CELL NUCLEUS

Buczek-Thomas J.A. et al.(Inhibition of histone acetyltransferase by glycosaminoglycans)J Cell Biochem. 2008 105 (1) 108-120

Hsia E. et al.(Nuclear localization of basic fibroblast growth factor is mediated by HS proteoglycans through protein kinase C signaling)J Cell Biochem. 2003 88 (6) 1214-1225

Richardson T.P. et al.(Regulation of HS protoglycan nulcear localization by fibronectin)J Cell Sci. 2001 114 (9) 1613-1623

HEPARANASE (Effect at Nucleus)

Purushothaman A et al., (Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive phenotypeJ Biol Chem. 2011 286 (35) 30377-30383

Yang Y. et al.(Heparanase enhances local and systemic osteolysis in multiple myeloma by upregulating the expression and secretion of RANKL)Cancer Res. 2010 70 (21) 8329-8338

Chen L. and Sanderson R.D.(Heparanase regulates levels of syndecan-1{HS proteoglycan} in the nucleus)PloS ONE. 2009 4 (3) e4947[Although HS function within the nucleus is not well understood there is emerging evidence that it may act to repress transcriptional activity]

Mani K et al.(Tumor attenuation by 2(hydroxynapthyl)--D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products)

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Glycobiology. 2004 14 (5) 387-397[N.b. the HS oligomers which were found to signal to the nucleus in these studies showed anhydroMan end groups. These are formed during (nitric oxide ascorbate Cu/Zn facilitated) nitrosative cleavage i.e. the (partly) non-enzymic cleavage of HS (to give putative hormone-like HS fragments) from un-substituted GlcNH2 groups in HS pre-primed for nitrosative cleavage. (While such structures are known to occur in HS although the mechanism of their insertion e.g. during primarly biosynthesis details are still unknown but putatively may aberrantly be augmented as a result of non-enzymic de-N sulfonation during acidosis or redox metal ion dyshomeosasis of precursor GlcN-SO3

- groups. Or perhaps by other mechanisms which putatively contribute to the aetiologies of neoplasia and other degenerative diseases}]

HEPARANASE - GROWTH FATOR PHOSPHORYLATIIONCohen-Kaplan V. et al.,(Heparanase augments epidermal growth factor receptor phosphorylation: correlation with head and neck tumor progression)Cancer Res. 2008 68 (24) 10077-85

HEPARANASE -METASTASIS

Cohen E. et al. (Heparanase is overexpressed in lung cancer and correlates inversely with patient survival)Cancer. 2008 113 (5) 1004-1011;Faye C. et al.(Molecular interplay between endostatin, integrins and heparan sulfate)J Biol Chem. 2009 284 (33) 22029-22040Cf. McKenzie EA. Br J Pharmacol 2007 151 (1) 1-14

Liu D. et al.(Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis)PNAS USA 2002 99 (2) 568-573[Specific different HS microstructure mixtures generated in vivo by exogenous heparinase apparently signal for opposite growth effects on tumor cells was suggested by the observation of the effect of difference between heparinases I and III when injected into mice with B16BL6 melanoma; while the HS oligomer mixture arising from heparinase III digestion caused tumour cell inhibition that produced by heparinase I digestion caused tumor cell growth to be enhanced]

Vlodavsky I and Friedman Y. (Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis)J Clin Invest. 2001 108 (3) 341-347

CELLULAR IMMUNE SYSTEM ANTI-TUMOR RESPONSE

Dziarski R.(Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin)J Immunol. 1989 143 (1) 356-365

INORGANIC CALCIUM ION : COFACTOR IN HEPARAN SULFATE ACTION

Ca 2+ etc. chelation, heparan sulfate/chondroitin sulfate ratio METASTASIS Cf. Tímár J et al.(Modulation of heparan-sulfate/chondroitin-sulfate ratio by glycosaminoglycan biosynthesis inhibitors affects liver metastatic potential of tumor cells)Int J Cancer. 1995 62 755-761[Ethane-1-hydroxyl-1-1-diphosphonate [EHDP] a well tolerated pharmaceutical (and a Ca2+ binding ligand) and other (different mechanism) GAG biosynthesis inhibitors, were apparently able to diminish tumor metastasis (by putatively Ca2+ dependent) inhibition of heparan sulfate biosynthesis. It should be noted that EHDP and related bisphosphonates osteoporosis therapeutic use also has been indicated to produce an extended life expectance by an unknown mechanism; it should also be noted that bisphosphonates per se have been reported to demonstrate anti-metastatic effects in e.g. in prostate cancer cf. e.g. Montaque R et al., Eur Urol. 2004 46 (3) 389-401]

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Kan M. et al.J Biol Chem. 1996 271 26143-26148[Divalent metal ions are essential cofactors for basic fibroblast growth factor assembly] for correct interaction between heparan sulfate and L-selectin as well as 2 and 3 integrins and for annexinV assembly on cell surfaces];

Valencia-Sánchez A. et al. Mol Androl. 1995 7 (1,2) 57[Heparan sulfate Ca2+ flux control during capacitation and modulation of acrosome reaction by heparin]

Takeuchi Y. et al. J Biol Chem. 1990 265 (23) 13661-13668[Extracellular Ca2+ concentration regulates the distribution and transport of heparan sulfate proteglycans and heparan fragments in a rat parathyroid cell line]Vandewalle B. et al.J Cancer Res Clin Oncol. 1994 120 (7) 389Ca2+ enhanced HS proteoglycan activity which modulate tumor cell growth

Hayashi M. and Yamada K.M.J Biol Chem. 1982 257 5263-5267[Divalent cations are required for heparin binding to fibronectin]

Boehm T. et al.(Zinc binding of endostatin is essential for its anti-angiogenic activity)Biochem Biophys Res Commun. 1990 252 (1) 190-194;

The roles of Ca2+ and other metal ions in the inorganic biochemistry of heparan sulfateallows for a system of Ca2+ Zn2+, Cun+ etc. activity regulation (and perturbation of this toxic Mn+ metal ions) could be relevant inter alia to a fuller understanding of how GAGs contribute to animal tissue homeostasis and non-specific immune protection and wound healing.

The following text was obtained from internet files (dg2/dg4/dg5 and dg8/ )which are no longer accessible.

dg2

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Note by D Grant PhD - April 1999 Modified June 2000 Originally prepared for seminar (arranged by Prof KEM McColl University of Glasgow)

Linked Heparan Sulphate & Nitric Oxide Biochemistry in Autoimmune & Degenerative Diseases

----- ----- ----- ----- ----- ----- -----

A review article by Stichtenoth & Frolich (1998) British J Rheumatology 37 246-257 emphasises the important role of nitric oxide (and by inference, nitrite and nitrous acid) in the aetiology of rheumatic diseases; development of more effective inducible NO synthases might, it seems, lead to new therapies for degenerative diseases (including cancer?).

Cellular immune responses may give rise to overproduction of NO. The severity of the arthritic disease was correlated with the amount of excess nitrate secreted (ref 1), a significant part of which will derive from immunologically secreted NO by way of intermediate formation of nitrous acid.

Understanding the circumstances contributing to inappropriate reaction of such nitrous acid with heparan sulphate (discussed by Vilar et al (1997) (ref 2)) seems, in addition to the required search for more effective NO synthase inhibitors, to be fundamental to the design of successful intervention strategies against such diseases.

The rapid, highly specific, reaction of heparan sulphate with nitrous acid has been known for many decades. It was formerly somewhat puzzling, prior to the discovery of the ubiquitous importance of NO to mammalian biochemistry (dubbed by Science "molecule of the year 1992") that this reaction was so specific for such a key multicellular animal polysaccharide; an additional, previously unsuspected, direct degradation of Glc2NAc in glycosaminglycans by NO at usual physiological pH reported by Vilar et al (ref 2) was perhaps influenced by traces of redox metal ions present as impurities in their samples.

That thiocyanate is an efficient catalyst of nitrosylation by NO (ref 9) suggests that traces of iron normally present in such reaction conditions may be present in the transition state complex of the rate determining reaction.

Iron overload and iron biochemistry malfunction are generally implicated in arthritic and other degenerative diseases (Andrews et al (1989) ref 8). Dietary factors are believed to contribute to the phenomenon of iron overload which has shown strong epidemiolgical correlation with cardiovascular diseases (Salonen (1992) ref 8).

Although heparan sulphate as normally present in N-SO3 or NAc forms, which are relatively resistant to nitrous acid damage (in absence of specific enzymes or other catalysts) the NH2 forms, however (cf ref 2c) react rapidly with nitrous acid. N-SO3 but not NAc is hydrolysed by H+. Metal ion complexation to heparin is accompanied by release of protons which may become trapped locally thereby acting as catalysts for desulphonation of adjacent (N)-SO3- groups. An additional desulphonation impetus over and above the amount of protons released is, however, believed to accompany the binding of certain redox metals to heparin/heparan sulphate. The

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degree of proton release increases with increased strength of complexing of the metal ions to heparin/heparan sulphate.

It may therefore be hypothesized that catalysis of the de-N sulphonation of heparin by redox metal ions previously believed to promote disease exclusively by way of generation of reactive oxygen free radicals should be included as a possible tissue damage factor of such metal ions via catalysis of NO and NO metabolite degradation of heparan sulphate (Grant, 1996, unpublished). Such redox active metal ions are likely to be Fe, Cu, Ti and V but related effects might be perhaps occur with other ions also detected by mass spectroscopy in pharmaceutical heparin (such as Cr, Co, Ga, Zr, Mo, Sn, Mo, La, Nd, Pb and Ce, reported in brief by Grant et al 1987 ref 3d)) as well as Al3+ which has a very high affinity for heparin and might likewise perturb heparan sulphate biochemistry (Grant et al 1992, unpublished) as might also noble metal complexes (such as anti-rheumatic Au complexes and anti-cancer Pt complexes) currently employed therapeutically (cf Grant et al, 1996 ref 3aii).

Cu(II)-hydrogen peroxide as been reported to alter heparin specifically but a similar reaction with Fe(II) was found to be less specific (Perlin et al ref 3a); selective degradation of heparin by Fe2+ -EDTA-ascorbate has also been reported by Lahiri et al (ref 3a).

Abnormal biosynthesis of heparan sulphate was reported by Kaji et al (ref 3a) to occur under the influence of Pb and by Cardenas et al (ref 3a) in the presence of Cd. Another ion which may adversely affect glycosaminoglycan biosynthesis is F-(e.g. Susheela & Jha work).

On the other hand ascorbate was found to greatly stimulated the normal biosyntheis of heparan sulphate (Kao et al ref 8b).

It would be of interest to determine if, in similar cell culture experiments, ascorbate deficiency also caused biosynthesis of abnormally microstructured heparan sulphate and to compare the reactivity towards NO and its metabolites of such normally and abnormally produced heparan sulphate.

Heparan sulphate depolymerization in vivo must be a physiologically normal process such depolymerization augmenting heparitinase depolymerization; oligosaccharides produced will contribute to the inhibition of calcification of blood vessel walls (and in the urinary system (refs 3)) and provide anti-viral, anti-prion anti-oxidant and anti-excess-nitrite (?) protection. Controlled splitting of heparan sulphate by NO metabolites may also serve other poorly understood cellular functions including those involved in neurological activity and perhaps be involved in the aetiology of such diseases as Alzheimers disease (Fukuchi K et al 1998 ref 7a) and prion diseases (cf Diringer H & Ehlers B (1991) ref 7a) such as bovine spongiform encephalopathy in which heparan sulphate biochemistry is apparently intimately involved (heparin appears to control prion protein toxicity by normalising defective prion conformation; this may be a normal function, prion disease may then be in part attributable to a defect in this normalization control).

(The effectiveness of heparin and heparin analogues such as pentosan polysulphate as anti-pathogen agents may derive from participation of oligosaccharides in various immune responses (cf prolongation of vascular graft survival by low molecular

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weight heparin (i.e. heparin fragments) apparently via a mechanism involving NO (ref 1b)) as well as from inhibition of pathogen cell attachment and inhibition of viral reverse transcriptase etc.; the heparin/heparan sulphate mimic, pentosan polysulphate can also prevent scrapie in mice (ref 7a)).

Since the heparin/heparan sulphate polysaccharide family seems to have widespread function in mammalian biochemistry, abnormal processing by NO and its metabolites might be of critical importance to understanding various diseases. A corollary to this is that it is highly likely that tissue NO will be held under strict physiological control and malfunction of such control(s) may promote degenerative diseases.

Anti-nitrite control protective systems may then be at least as, but perhaps more extensively required for general health as are antioxidative control mechanisms.

Cytokines, Heparan Sulphate and NO Metabolites ---------------------------------------------- Expression of the inducible NO synthase is promoted by certain cytokines (TNFalpha, IFNgamma, IL-1 and IL-2) and suppressed notably by cytokines TGFbeta, IL4, IL8, IL10 and IL13 (ref 1).

These two sets of cytokines may act as part of a servo control mechanism to monitor and control excess "active N". Heparan sulphate binding of IL2 (ref 6a) and IL8 (ref 6) involves known specific heparan sulphate microstructural participation) which is involved in the action of most or all of these cytokines indicating possible feedback mechanisms involving oligosaccharide signalling akin to those apparent from the work of Yanagashita (cf ref 6c).

Extensive research has also recently elucidated the microstructural requirements of FGFs binding to heparan sulphate (e.g. Brinkman et al 1998 ref 6d). Inappropriate damage to heparan sulphate will perturb such growth factor signalling and potentially impair wound healing.

Helicobacter, IL10, Nitrite and Stomach Cancer ---------------------------------------------- Helicobacter pylori has been established to be involved in stomach cancer. Growth factor signalling perturbation by H pylori linked heparan sulphate was suggested (ref 5) but perturbation may also occur by way of NO metabolite alteration of the heparan sulphate microstructure thus further depleting stomach mucosal replendishment. This organism in common with many other disease organisms is believed to be attached to the host tissue, at least initialy, via a heparan sulphate link (ref 7); such attachment may adversely alter heparan sulphate participation in cytokine activity; pathogen killing by immuno secreted NO may be more potentially damaging to adjacent heparan sulphate molecules if additional catalysts such as Fe and Pb ions are are present.

Genetically IL-10 deficient mice were proposed to be a good animal model (with Helicobacter felis) of the H pylori induction of stomach cancer in man (ref 4); the wild-type mice with normal IL10 capabilities were reported not to develop the disease in marked contrast to the IL10 deficient mice, suggesting an involvment of induced synthesis of NO and nitrous acid induction by H pylori in this disease.

Summary ------- Peroxy nitrite, formed from NO and superoxide anions, seems now to have become accepted (ref 1) as likely promoting disease via DNA damage. Heparan

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sulphate, also an information holding specific sequence polymer, requires correct sugar sequences (cf Rosenberg et al 1998, ref 10) for proper functioning and will likewise potentially promote disease when damaged.

Nitrous acid yields S-nitroso compounds, N-nitroso carcinogens and causes inappropriate depolymerization of de-N sulphonated heparan sulphate; this may be exacerbrated under local acidosis situations in chronic inflammation and be promoted by by the activity of Fe and other redox active and toxic metal ions.

Addendum I

Ascorbate Dietary Supplement to Reduce Excess Nitrite? ------------------------------------------------------ The oxidation of NO to HNO2 is reversed by ascorbate which may be the key anti-nitrous acid tissue protective agent in vivo and be the basis of epidemiological linked dietarty ascorbate morbidity data in man Cameron & Pauling, 1976, 1978; NIH Conference Proc 1991 ref 8c. There may also be specialized roles for isoforms of ascorbate and ascorbate sulphate and other simple sugar cycle related molecules (pyruvate?) in such oxidation reduction protection.

Ascorbate apparently also greatly increases heparan sulphate biosynthesis in fibroblast cell culture (Kao et al 1990 (ref 8b)); ascorbate then may have a primary function as a heparan sulphate protection agent.

Is Nitrite Control Involved in Therapeutic Effects of Glucosamine and Other Drugs Effective for Control of Rheumatic Diseases? -------------------------------------------------- An apparent succesful therapeutic use of glucosamine sulphate and related substances for rheumatic joint dieseases may derive, at least in part, from an anti-NO/nitrous acid effect. A previous hypothesis had suggested a heparan sulphate boosting role for oral glucosamine (ref 8a). Again the importance of protecting heparan sulphate is indicated to be a key factor for successful control of such diseases.

Salicylate and other non-steroidal anti-inflammatory drugs also exhibit anti-NO activity (ref 1). Auranofin inhibits inducible NO synthase production (ref 1a). Anti-active N activity may be an important modus operandi of these anti-inflammatory agents.

Is Tyrosine-Cysteine Activity Involved in Anti-Nitrite Protection? ------------------------------------------------------------------ That tyrosine nitrate is an indicator of the presence of peroxynitrite (ref 1) may point to an involvement of tyrosine in anti-nitrite control. Since thiol groups are likely nitrite -> NO reductants, cysteine tyrosine clusters in proteins discussed by Grant et al (1989, ref 3a) may synergise tyrosine and sulphur oxidation-reduction activity for this function; (in vitro experiments to test for such anti-nitrite activity might be worthwhile).

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References ----------

(1) Stichtenoth DO Frolich JC (1998) British J Rheumatology 37 246-257 (1a)Yamashita M et al (1998) Portland Press Proc 15 (Biology of Nitric Oxide part 6) 242 CA 198 144647w (1b) Teranishi K Poston RS Reitz BA Robbins RC (1999) Transplant Proc 131(1/2) 103-105 Elesvier Science Inc CA 131 27696s (2) Vilar RE Ghael D Li M Bhagat DD Arrigo LM Cowman MK Dweck HS Rosenfeld L (1997) Biochem J 324 473-479 Ghael D Mileva M Dweck HS Rosenfeld L (1997) Biochem Med Biol Int 43(1) 183 CA 128 59743j (2a)Van den Born J et al (1992) J Biol Chem 270 (50) 31303 (3) Laihev JP (1996) Biometab. 9(1)10 CA 124 109969c (3a)Perlin et al (1994) Carbohydr Res 255 183 CA 120 299165a Lahiri B Lau PS Pousada M Stanton D Danishevsky I (1992) Arch Biochem Biophys 293 54-65 Kaji T Yamamoto C Saskamoto M (1991) Toxicology 68(3) 249 Cardenas A Bernard A Lauwerys R (1992) ibid 76 219-231 (3ai)Grant D Long WF Williamson FB (1989) Med Hypotheses 28, 245-253 (3aii)Grant D Long WF Williamson FB (1996) Biochem Soc Trans 24(2)204s CA 125 48565q (3b) Heparan sulphate is a potent inhibitor of calcium oxalate crystallization in vivo Yamaguchi S et al (1993) Urol Res 21 (3) 187 CA 119 243946t cf heparan sulphate of normal subjects more active than that of stone formers CA 120 188831v cf Grant D Long WF Williamson FB (1989) Biochem J 259 41-45 and Grant D Long WF Williamson FB (1992) Med Hypotheses 38 49-55 (3d) Infrared spectra of heparin-cation complexes Grant D Long WF Williamson FB (1987) Biochem J 244 143-149 (4) Berq DJ et al (1998) Am J Pathol 152(5)1377 CA 129 107408q (5) Ascencio F Hansson HA Larm O Wadstroem T (1995) FEMS Immunol Med Microbiol 12 (3-4) 265 (6) IL8 Spillman DS (with Lindahl U) et al (1998) J Biol Chem 273 (25) 15487-15493 (6a) IL2 Najjam S et al (1998) Glycobiology 8(5) 509 (6a1)IFNgamma Sader R et al (1998) J Biol Chem 273 (18) 10919 (6b) Yanagashita M (1998) Trends Glycosci Glycotechnol 10 (52) 57 (6c) Tanaka Y Aso M (1998) ibid 10(52)153 (6d) Brinkman YG et al (with JT Gallagher) (1998) Glycobiology 8(5) 463 CA 129 63209s (7) Chmiela M Paziak-Domavska B Wadstroem T (1995) AMPIS 103(6) 469-74 CA 124 53455n

(7a) Prion/scrapie factor Chemoprophylaxis of scrapie in mice Diringer H Ehlers B (1991) J Gen Virol 72 451-468

(Alzheimers disease Fukuchi K et al (1998) Front Biosci 3, D327-337 CA 129 3327e)

Some Pathogens Reported to Bind to Heparan Sulphate

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--------------------------------------------------- HIV e.g. De Clercq E (1993) Med Res Dev 13 (3) 229 CA 119 40051z Choay J et al (1993) J Med Chem 361 217 CA 120 8887m

Bovine foot and mouth virus Jackson T et al (1996) J Virol 70 (8) 5282 CA 125 139432k Bovine herpes I, pseudorabies virus GIII Liang et al (1993) Virology 194(1)233 CA 118 252458 Li Y (1996) J Virol 70(3) 2032 Pseudorabies Hanssens FP et al (1993) J Virol 671s 1 4492 CA 119 200885e Bovine herpes virus 4 Vanaeiplasch A et al (1993) Virology 196(1) 232 CA 119 200907p Herpes simplex CA 120 103695f Laquere S et al (1998) J Virol 72(7) 6119 CA 129 147550v h cytomeglavirus Compton T et al (1993) Virology 193 (2) 834 CA 118 231423m Price et al (1995) Immunol Cell Biol 73 (4) 305

Leishmania Donovani Butcher BA et al (1992) J Immunol 148 (9) 2879

Streptococcus pyrogenes M6 CA 120 129161d

Staphylococci CA 120 200396s

Bordetella pertussis CA 120 214133r

Hepatitis C Hems MW et al (1993) J Virol Methods 42 (1) 127 CA 119 1911k (8) Salonen JT et al (1992) Circulation 86(3) 803-811 cf Iron chelation cancer therapy Taetle R Honeysett M Bergeron R (1989) J Natl Cancer Inst 81 1229 cf Reizenstein P (1990) Med Oncol & tumor pharmacother 7 (2/3) 67-68

Andrews FJ Blake DR Morris CJ (1989) p145-175 in Iron in immunity, cancer and inflammation Ed M de Sousa & JH Brock John Wiley & Sons, Chichester etc

(8a) McCarty MF (1997) Med Hypotheses 48 (3) 245 CA 127 12883u (8b) Kao J Huey G Kao R Stern R (1990) Exp Mol Pathol 53 1-10 (8c) Ascorbate and cancer Proc Conf at NIH Bethseda MD USA Sept 10-12 1990 Am J Clin Nutr 54 (Suppl) 1113s-1329s Cameron E Pauling L (1976) PNAS USA 75 4538-4542; ibid 79 3685-3689 Cameron E & Pauling L (1979) Cancer and vitamin C Linus Pauling Institute of Science and Medicine, Palo Alto L Pauling (1985) Chem in Brit 21(1) 27

Ascorbate and the common cold Pauling L (1968) Science 160 265 Pauling L (1985) Vitamin C and the common cold, revised edition, Foundation for Nutritional Advancement, New York;

(9) Fan T-Y Tannenbaum SR (1973) J Agric Food Chem 21 (2) 237-240 (10) Control of biochemistry via specific sequence heparin/ heparan sulphate microstructure; haemostasis/lipid processing and growth factor signalling (cardiovasculature) Shworah NW et al (Rosenberg RD) (1998) Trends Glycosci Glycotechnol 10 (52) 175, cf Vascular smc Mishrc-Gories et al (1998) ibid 10 (52) 193 different microstructures for log growth bFG activation and aFGF activiation at confluence (neuroepithelial cells); Brinkman YG ey al (Gallagher JT) (1998) Glycobiology 8 (5) 463 Mammary epithelial cells distinct heparan sulphate microstructures forsets of growth factors Rahmoune H et al (Gallagher JT) (1998) Biochemistry 37 (17) 6003

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Addendum II Maintenance of heparan sulphate throughout evolution Ferrerro TMD et al (with Dietrich CP) (1993) Int. J Biochem 25 (6) 1219 CA 120 50475a; cf original heparan sulphate cellular recognition hypothesis: Dietrich CP Nader HB Straus AH (1983) Biochem Biophys Res Commun 111 (3) 865-871

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CHEMICAL ABSTRACTS (Heparin/Heparan Sulfate) LITERATURE SURVEY

(from documents which had been posted on ukonline websites during 2000)

_______________________________________________________________________________________________________________________________

dg4 (Document originally posted on the Internet in 2000 together with associated documents dg5 amd dg8)---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN"><!-- saved from url=(0121)http://209.85.135.104/search?q=cache:7kSouJC8zHUJ:web.ukonline.co.uk/dgrant/dg4/+/dgrant/dg4/&hl=en&ct=clnk&cd=1&ie=UTF-8 –

Ascorbate and Nitric Oxide In Redox Control Of Heparan Sulphate Structure of Relevance to Cancer

http://www.google.com/search?q=cache:7kSouJC8zHUJ:web.ukonline.co.uk/dgrant/dg4/+/dgrant/dg4/&amp;hl=en&amp;ct=clnk&amp;cd=1&amp;ie=UTF-"Heparan Redox Hypothesis." from home-based continuation of a literature survey following laboratory studies of heparin/heparan sulphate <BR>(with WF Long and FB Williamson Department of Molecular and Cell Biology University of Aberdeen)

CONTENTS 1. Summary 2. Introduction 3. ACTIVE NITROGEN

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Derivatives in Chemistry and Biology 3.1 Physiological utilisation of nitric oxide 3a Active nitrogen derivatives as promoters of degenerative diseases 3.2 Various potentially biologically active nitrogen derivatives 3.3 Nitrogen compounds as aqueous system denaturants 3.4 Nitrogen-centred free-radicals 3.5 Metal-catalysed effects in active nitrogen formation? 3.5.1 Iron and copper 3.5.2 Vanadium and titanium 3.5.3 Nickel and cobalt 3.5.4 Molybdenum 3.6 Peroxynitrite 3.7 Cysteine-tyrosine active nitrogen traps? 4. ASCORBATE Redox metal effects 5. Summary of HEPARIN/HEPARAN SULPHATE BIOCHEMISTRY 5a Activation of heparin/heparan sulphate from nitrous acid cleavage by removal of protective groups from glucosamine nitrogens

5b Evidence for the relevance of heparin/heparan sulphate biochemistry to cancer. Cancer-related metal ion binding by heparan sulphates

5c Effects of oxygen, glucose, toxins (including toxic metals) & ascorbate on heparan sulphate biosynthesis 6. Conclusions 7. References 8. Appendix a. Transition metal ion catalysis of reactive nitrogen damage b. Heparin/heparan sulphate brain development and function Neurodegenerative diseases including Alzheimer's disease (AD) Introduction Heparin/heparan sulphate in AD Zinc in AD Cognitive function in AD effect of Ateroid Neurite outgrowth Lack of correlation between extracted brain heparan suphate and AD Transferrin receptor function and heparan sulphate Fenton reaction free-radical damage in AD etc c. Supramolecular structure of heparin/heparan sulphate and binding

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potential

d. Silicates and heparin/heparan sulphate 9. Heparin/heparan sulphate involvement in prion diseases 10. Water binding to heparin/heparan sulphate <BR>Hofmeister effects on water structure Modulation of crystallisation by heparin/heparan sulphate 11. Established laboratory use of nitrous acid for heparin/heparan sulphate structure evaluation 12. Binding of pathogens to heparan sulphate 13. Acknowledgements

1. SUMMARY The prevention of deaminative cleavage of heparan sulphate by Vitamin C and its derivatives is postulated to be critically important to health by blocking dysfunction which may occur in heparan sulphate biochemistry under iron overload, pathogen induced acidosis and thiocyanate intoxication. Critically required heparan sulphate biosynthesis is suggested to be sensitive to cellular redox status involving ascorbate and nitric oxide.

2. INTRODUCTION Linus Pauling promoted the use of Vitamin C as an anticancer, antiviral and pro-cognition agent but lacked a convincing mechanism by which to account for such wide benefits to health (additional to antioxidant activity and promotion of collagen hydroxylation-dependent increased extracellular matrix resistance to tumour metastasis). It is becoming apparent that many tissue protective roles depend on heparin/heparan sulphate biochemistry, including highly microstructural-dependent growth factor signalling (cf Perrimon & Bernfield 2000) which might be abrogated by nitrous acid derived from nitric oxide and this could underpin the epidemiological and other evidence (cf Numerous authors, 1991) for an anti-cancer role of ascorbate (which is a highly efficient anti-nitrous acid agent). Although nitrous acid deaminative cleavage has been employed for decades and as a diagnostic test for heparan sulphates (and pathological nitric oxide related dysfunction of heparan sulphate biochemistry and possible metal ion catalysis of this has been evident for some time (Grant 1992) more recent reports (Vilar et al 1997; Ghael et al 1997) confirm that deaminative cleavage of heparan sulphate is of likely importance to pathology .

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Ascorbate anti-nitrite activity is most likely due to the rapid chemical reduction of nitrous acid to nitric oxide in agreement with the results of Tsao (1991) who showed that both D- and L- ascorbate had similar anti-tumour effectiveness in an animal model, indicating a pure-chemical (e.g. redox control) not an 'essentially biochemical' mechanism; ascorbate nevertheless is indicated to have further cancer-related biochemical effects. Dehydroascorbate, diketogulonic acid and copper ascorbate apparently display individually different anti-tumour activities; dehydroascorbate may directly inhibit cellular mitosis (Edgar 1970) perhaps involving control of cellular redox potential which also influence the biosynthesis of heparan sulphate of altered sulphation and chain length.

Copper ascorbate perhaps sensitises some tumour cells for subsequent destruction.

Ascorbate has been reported to directly increase the biosynthesis of heparan sulphate chains of higher degrees of sulphation (Kao et al 1990, cf Malemud et al 1978) this may be related to cellular redox control effects; apoliprotein E is also reported (Paka et al 1999) to boost the biosynthesis of more highly sulphated complex microstructured anticoagulant heparan sulphate chains in endothelial cells.

Lipolysis, recently reported (Anderson et al 1999) to be modulated by nitric oxide in human adipose tissue is known to be influenced by the heparin/heparan system, further potentially linking ascorbate, heparan sulphate, lipid and nitric oxide biochemistry and redox chemistry.

3. ACTIVE NITROGEN DERIVATIVES IN CHEMISTRY AND BIOLOGY

3.1 Physiological Utilisation of Nitric Oxide

Nitric oxide homeostasis in blood is believed to be controlled by binding to iron in erythrocyte haemoglobin. Ascorbate is, however well understood to provide protection against the formation of carcinogenic nitrosamines produced from dietary nitrite and is also likely to be involved more widely in the physiological control of the nitric oxide air oxidation product nitrous acid.

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Arginine-derived nitric oxide, produced by nitric oxide synthases upon stimulation by agents like calcium ions, calmodulin, cytokines and tumour necrosis factor, is utilised physiologically as a chemical messenger (for guanylate cyclase) potentiating endothelial relaxation (which is reported by Kouretas et al 1998 to be influenced in immune responses by non-anticoagulant heparin via inhibitioin of nitric oxide synthase by a guanine nucleotide regulatory protein); nitric oxide is also involved in mitochondrial activity, gene expression, apoptosis and haemostasis, and is a neurotransmitter with putative involvement in learning (Hawkins et al 1998) and lipid metabolism (Anderson et al 1999).

3a Nitrogen-Containing Carcinogens

The majority of compounds known to be carcinogenic seem to be N containing, and include aromatic amines, alkyl nitrosoamides, aryl dialkyl triazines, carbamates, azo and acridine dyes, nitrosamines and pyrrolizidine alkaloids. Some types of nitrogen compounds demonstrate both pro- and anti-cancer effects (cf Ferguson 1975).

Although nitrate ester and hydrazine derivatives have long been known to generate (therapeutically useful) cardiovascular effects, now known to be due to nitric oxide formation (Servent et al 1989), nitrogen oxide chemistry was traditionally thought to mainly impinge on biology in terms of noxious byproducts of hydrocarbon and tobacco combustion.

Active nitrogen intermediates are implicated in the aetiology of rheumatoid diseases where urinary secreted nitrate exceeds ingested nitrate in amounts correlated with the severity of the disease and inhibitors of inducible nitric oxide synthase are show promising therapeutic activity against such diseases (Cochran 1994, Stichentoch & Frollich 1998).

That active nitrogen intermediates derived from nitric oxide produced by autoimmune effects are likely implicated in the degradation of cartilage components in these diseases concurs with the

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putative use of glycosaminoglycan-selective colorimetric assay for improved diagnosis of rheumatoid diseases (Kery et al 1992) and with the apparent therapeutic success of dietary fish oil (an active component, docosahexaneoic acid, inhibits macrophage production of nitric oxide/nitrous acid (Jeyarajah et al 1999). Glucosamine or glucosamine sulphate dietary supplements are in common use to combat osteoarthritis (cf Theodosakis et al 1997) by apparently enhancing the biosynthesis of heparan sulphate (McCarty 1995).

Nitrous acid produced from nitrites present as food preservatives is a known inducer of stomach cancer (cf Cameron & Pauling 1979) and the above discussion of the role of nitrous acid in the aetiology arthritic diseases can logically be extended to carcinogenesis, in keeping with the notion that biochemistry seems highly centred on the unique properties of organic nitrogen compounds of which nitric oxide /nitrous acid is a part, capable of reacting with various organic nitrogens in proteins, nucleic acid bases as well as in amino sugars such as those in glycosaminoglycans.

A complex nitrogen oxidation-reduction chemistry exists with nine stable oxidation states including molybdenum-dependent microbiochemistry influenced by a wide range of redox metal (iron, copper, titanium, vanadium, chromium, perhaps nickel) and acid/base(zinc) catalysis.

In the deaminative cleavage of glucosamines, unstable intermediate diazonium derivatives are believed to be formed (as is general from primary amines) and may be accompanied by other unstable intermediates such as intrinsically unstable nitrite esters (cf Barton et al 1977). Nitrous acid itself is believed to equilibrate with more reactive N2O3 but other species may also be involved in similar equilibria Nitric oxide is readily oxidized to nitrous acid which, however, can be efficiently reduced back to nitric oxide by ascorbate. Nitrous acid reacts with amines, e.g. with polysaccharide glucosamines to cause deaminative cleavage of the polymer chains. Aromatic and aliphatic primary amines are affected differently by nitrous acid, the former produces diazonium, the latter nitrogen gas. Secondary amines produce (often carcinogenic) nitrosamines. Nitrite is commonly assessed by the Greiss reaction.

3.2 Various potentially biologically active nitrogen derivatives

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In addition to nitric oxide and its metabolites, other potentially damaging active nitrogen species include nitrogen-centred free-radicals, various nitrogen oxides, peroxides (especially peroxynitrite, Beckman 1990), oxyacids, hydrides, halides, pseudohalides and such species as cyanides, cyanates, isocyanates, thiocyanates, isothiocyanates and nitrenes. Amine derivatives should also be included (cf hydroxylamines have similar DNA mutagen effect to nitrous acid cf Mahler &amp; Cordes 1966 and their formation was considered to explain the carcinogenicity of aromatic amines) and hydrazine, azides and small ring heterocycles (e.g. azirines, aziridines and oxaziridines); redox metal complexes of the above nitrogen species as well as other long-known entities - activated molecular nitrogen and polymeric adducts of hydrogen atoms and nitric oxide, sulphur-containing Fremy's salt (cf Emeleus & Anderson 1946) and thionitrite.

Polyamines should also be included, as their formation may be linked with the promotion of stomach cancer by Helicobacter pylori (cf Xu et al 1998; Linsalata et al 1998).

An indication of potential biological reactivity of suspected active nitrogen chemical species might be revealed by their nitrogen nmr chemical shifts; damaging species conceivably having the most downfield shifted resonances (cf Mason 1983).

3.3 Nitrogen Compounds as Aqueous System Denaturants

Numerous nitrogen compounds can be considered as aqueous solution denaturants, relevant to biological damage.

Urea and guanidinium have this property shared with amines, such metal complexes as cuprammonium, ammonium salts, nitrosylated derivatives, cyclic amino oxides (Johnson et al 1966) and thiocyanate.

3.4 Nitrogen-Centred Free Radicals

Chloramines (formed by immune response myeloperoxidase hydrogen peroxide and chloride generated hypochlorite) are reported (Hawkins & Davies 1998) to yield potentially damaging nitrogen-centred free-radicals (especially from lysine) which are repairable by reduction by ascorbate.

Such repair could be a component of ascorbate tissue protection under autoimmune disease promotion conditions.

Nitric oxide (NO) is a stable free-radical. The chemistry of NO , however, includes redox shifts to NO+ (nitrosium) and NO- (nitroxyl).

3.4.1 R-S-H Adducts of NO

Cellular Redox Status, NO and Ascorbate Controlling Effect

NO homostasis may involve linked oxidation-reduction of S-H groups including those in redox metal complexes (cf Stamler et al 1992); involvement of glutathione perhaps in concert with ascorbate might be anticipated. Synergy between various such species may be relevant to the tissue damage mechanisms. The effect of exogenous metals on the stability of such NO adducts may be relevant to the deleterious effects of iron overload (cf Singh et al 1996).

Redox input into altered heparan sulphate microstructure at both the

biosynthetic stage and post synthetic stage is believed to be of high importance for normal and disease and therapeutic-related control of cellular activity.

3.4.2 NO as an Antioxidant

It has long been known that nitric oxide is an efficient antioxidant, e.g., radical chain reactions were detectable by stopping them by its injection(Hinshelwood 1957)and similar broad antioxidant function is also believed to be relevant to biology (Kanner et al 1991).

3.5 Metal Catalysis (Redox and Acid/Base Effects) in Active Nitrogen

Chemistry

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Significance of NO deaminative cleavage of heparan sulphate at physiological pH

3.5.1 Iron and Copper

Vilar et al (1997) reported the deaminative cleavage at physiological pH of aceylated glucosamines by nitric oxide gas in the presence of phosphate buffer but not in the presence of imidazole buffer. The former situation is often affected by trace transition metal catalysts, especially by iron and copper, absent from the latter situation. Further experiments are required to test this possibility. Previously it had been though that acid pH <5 was required for deaminative cleavage.

The details of the nitric oxide derivatives (perhaps involving effects of thiol (and metal) adducts) which are responsible for endothelial relaxation(Stamler et al 1992)) may also require further experimental work.

Factors which influence the reactivity of nitric oxide metabolites include thiocyanate (Fan & Tannenbaum 1973) may also be a function of trace redox metal catalysts (Chalis et al 1978; Park & Aust 1998).

Redox metal complexes formed with the active nitrogen intermediates also serve as antibiotics by binding and deactivating pathogen redox centres but nitric oxide is scavenged by binding to iron in haemoglobin without deactivating it; immonium oxide formation has been proposed to account for the catalase-like activity of haem nitroxides and related compounds (Mehlhorn &amp; Swanin 1992). 3.5.2 Vanadiuim, Titanium (& Chromium)

Redox metal activity analogous to Fenton-iron hydroxyl radical production which is observed for V(IV), Cr(III), Ti(III) analogously to the well-known Fe(II) reaction with hydrogen peroxide as discussed by Bodini & Sawer (1976) may also generate highly reactive nitrogen-centred free-radicals (such as from Cr(II) perchlorate studied by Wells & Salam (1968); Cr(III), however, which is highly reducing, would normally be expected to be rapidly oxidised by dissolved oxygen , e.g. , in the stomach.

The occurrence of redox processes involving protons such as

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TiO2+ 2H+ = e = Ti3+ + H2O indicate that Ti(III) and probably V(III) to be potentially more strongly reducing under neutral than pH 1 conditions where they may more readily stimulate active nitrogen formation.

3.5.2.a Possibly relevant V biochemistry

Although V(III) is usually stable in aqueous solution only at pH <3, it may, however, be stabilised at high pH values by ligands such as, e.g. may occur in tunicate blood. The ligand contained in fungal chlorooxidase shows V bound by three non-solvent oxygens, a histidine and a N3 azide group (Messerschmidt & Wever 1996) and the V(IV) in amavadin has NO and CO2 - binding groups (S,S)-2,2-(hydroxyimino)dipropionic acid (Garner et al 1999).

Bromo/iodoperoxidases containing V(V) in their active centres occur in brown and red seaweed and lichen.

Horse muscle contains vanadyl ATP which mimics insulin effects.

Some isopolyvanadates demonstrate heparin-like inhibition of inositol (1,4,5)triphosphate receptors but with low specificity (Potter & Nahorskji 1992).

A vanadium-containing ascorbate complex was indicated to be the oubain-like factor in human urine (Kramer et al 1998).

3.5.3 Nickel (& Cobalt)

Is the redox chemistry of Ni altered by biological ligands a possible risk factor for heparan sulphate deaminative cleavage?

A reported Ni(II) induced alteration in EGF and beta TGF mediated growth control during malignant transformation of human kidney epithelial cells(Mollerup et al; 1996) could conceivably have a deaminatively cleaved heparan sulphate link e.g. via active nitrogen generated from redox processes facilitated by biological ligand stabilized Ni(III).

3.6 Peroxynitrite

Peroxynitrite formed from nitrous acid and superoxide anion is believed to be an important reactive nitrogen species responsible for the carcinogenic DNA damage apparently previously ascribed to the effects of hydroxyl radicals (Beckman et al 1990) which may explain various carcinogenic effects of particulates including that of iron-rich varieties of asbestos (Park & Aust 1997).

Peroxynitrite is also implicated in the aetiology of various

neurodegenerative diseases (Beasl 1997; Cookson & Shaw 1999; Love 1999)including (familial) motor neuron disease (Rosen et al 1993; Calder 1995)this providing an explanation for the pivotal involvement of genetically defective superoxide dismutase-I.

Key polysaccharide heparan sulphates implicated in antioxidant defense (cf Adachi et al refs)are also likely to be subject to peroxynitrite damage as well as to deaminative cleavage by other active nitrogen species.

3.7 Are Clusters of Aromatic- and Sulphur-Containing Amino Acids in Proteins

Active Nitrogen Traps?

A postulated antioxidant function of conserved clusters of tyrosines, cysteines and methionines in proteins (Grant et al 1989a) might be logically extended to include anti-active nitrogen function (in addition to the previously known nitrene reactivity) this according both with these groupings being potential indirect scavengers of peroxynitrite by deactivating hydroxyl radical precursors as well as likely acting as direct scavengers since nitro tyrosine is observed on reaction with peroxynitrite as are analogous derivatives from chloramines (Hawkins & Davies 1998).

The formation of hydroxyl radicals by ascorbate, oxygen and Cu(II) led to free radical activated adducts producing randomisation around aromatic-cysteine S-S bond systems including insulin and oxytoxin.

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<P>Cysteine-tyrosine systems have been postuated to be involved in cysteine directed signal transduction (crosstalk) of growth factors in gene expression(Nakashima 1996) inappropriate perturbation of which by active nitrogens might be relevant to the aetiology of cancer but normal involvement in nitric oxide in such signalling might be anticipated.

4. Ascorbate

As with the biochemical role of heparan sulphates it is commonly stated that the biological function of ascorbic acid is not yet established. A role in redox control seems likely directly and indirectly inputting into heparan sulphate biochemistry.

Ascorbate is believed to be a key aqueous phase extracellular antioxidant; this function is extended now to it performing key anti-active nitrogen protective functions. The antioxidant function is supposed synergistic with uric acid and with lipid-soluble tocopherol and glutathione; ascorbate is likely to provide aqueous phase extracellular antioxidant and anti-active nitrogen protection augmented by related intracellular activities of ascorbate sulphates and perhaps other ascorbate derivatives which may be later identified.

An established role of ascorbate is in the in vivo utilisation of ascorbate is in

dopamine → norepinephrine conversion.

Adrenal glands and eye lens have a high ascorbate concentration.

In studies directed to probe the possibility of damaging effects of high ascorbate dietary supplementation; the consensus of opinion seems to be that damage may easily be demonstrated in vitro but less easily in vivo.

Minetti et al (1992) found no evidence for hydroxyl radical formation in esr studies of ascorbate free-radicals in moderately iron overloaded plasma although this had been previously observed.

Phosphate and other ligands for iron may catalyse the iron redox damage in presence of oxygen, cf, ascorbate, Cu(II) and oxygen promoted hydroxyl radical formation in vitro, scrambling disulphide bonds in insulin and oxytocin (Inoue & Hirobe 1987).

Podore et al (1998) discussed cellular DNA damage initiated by ascorbate which might, after all, have been of a protective nature (cf discussion by Levine, Salonen et al 1998).

Older work identified pro-cancer effects of ascorbate (Fukushima et al 1983,1987).

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Other possibly detrimental effects of excess ascorbate dietary supplementation lie in the increased risk of calculi formation from oxalate crystals (ascorbate metabolite) and increased ascorbate promoted collagen crosslinking which might (in some instances adversely) affect arterial walls (Dwyor 2000) and perhaps also reduce the permeability of Bruch's membrane causing visual damage.

Vitamin C dietary supplementation releases iron from insolubles such as oxalates and carbohydrate complexes (cf Sharma & Marthur 1995).

Ascorbate deficiency likely impairs the correct biosynthesis of the extracellular matrix components collagen and glycosaminoglycans including heparan sulphate (cf Kao et al 1990); syndecan-1 mediated differential collagen interaction may be affected (cf Sanderson et al 1994). Ascorbic acid has standard oxidation reduction potential of 0.058V pH7 and has pKa = 4.2 (C3-OH); a di-anion is also formed by dissociation of C2-OH.

An X-ray crystallographic structure (Hughes 1973) of the Tl salt showed de-localisation of electrons between lactone ring carbons 1-3, the anionic charge being shared between O(1) and O(3) atoms.

NMR studies show apparently various co-existing forms of ascorbate including the possibility of the formation of oligomers and polymers (Matusch 1977). Further simple to perform NMR studies are warranted.

Ascorbate is irreversibly transformed at higheer pH values into ketogulonic acid and by browing reactions.

Ascorbate sulphate esters, e.g., 2-O-sulphate , may have sulphotransfaerase properties and their oxidation reduction properties are likely to be relevant to tissue protection. <P>Evidence of anti-active nitrogen protection by ascorbate is afforded by the improvement of endothelial function by reversal of the oxidation of nitric oxide by free radicals in patients with chronic heart failure (Hornig et al 1998) and the likely role in anti-nitrite/nitrosamine carcinogenesis (e.g. Kirchner and Hopkins 1991).

The activity of ascorbate derivates such as ascorbalaminic acid present in plants (Couchman et al 1972) may also be relevant to protection by dietary 'ascorbate' against excess nitrous acid in the stomach.

The oxidation of ascorbate by dissolved oxygen is iron and copper catalysed(cf e.g. Minetti et al 1992) proceeding via the formation of ascorbyl and hydroxyl free radicals. Redox recycling by ferrioxidase activity in caeruloplasmin may contribute to iron overload dependent rapid oxidation of ascorbate in plasma. Copper-centred enzymic activity is also responsible for the ascorbyl oxidase activity of plants.

Hydroxyl radicals generated from ascorbate peroxide and Cu(II) rearrange substituents around aromatic containing groups attached to disulphide bonds in peptides such as insulin (Inoue & Hirobe 1987) and cause microstructural alteration (perhaps to N-sulphonate groups) in heparin detectable by abrupt reduction in the anti-Factor Xa activity (a similar reaction with iron results in less selective alteration (Liu & Perlin 1994).

Ascorbate may protect against damaging oxidative activity by higher oxidation states of chromium (Connett & Wetterhahn 1983) and perhaps vanadium.

Older work on the degradative activity of ascorbate is likely to have been influenced by the ability of trace redox metals (usually present unless special measures are taken to eliminate them) to generate hydroxyl radicals (as in the degradation of gastric and salivary mucins and hyaluronic acid etc in cartilage, vitreous humour and pneumonococci capsular polysaccharides reported by Roberson(1941) by ascorbate + hydrogen peroxide in phosphate buffer).

The formation of ascorbyl radicals/ascorbate degradation was found to be catalysed in a dose-dependent manner by Cu+, Cu2+, Fe2+, Mn2+, Fe3+ as well as by Zn2+ but not by Na+, K+ ,Ca2+ or Mg2+. Transition metals are normally strongly protein-bound in serum with the possible exception of Cu2+. Metal ions may regulate (Satoh &amp; Sakagaimi 1997) and be regulated by the biological activity of ascorbate (e.g. the iron homeostasis mechanism in lens epithelial cells which includes ascorbate-boosted de novo synthesis and likely ascorbate-assisted iron binding to ferritin (Goralska et al 1998)).

Vanadyl ascorbate complexes may be involved in ATPase regulation (possibly relevant to human health) being detected in human urine (Kramer et al 1998).

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Maillard reactions, including threose ascorbate decomposition produts of abnormal ascorbate metabolism, may have a role in lens caractognesis (galactose exposed lenses showed enhanced crystallin-bound Schiff base-linked ascorbate degradation products, (Saxena et al 1996)).

Cellular ascorbate activity (e.g. in erythrocytes) may mimic or augment that of NADH and glutathione having a role in the maintenance of oxidation-reduction potential.

High dose ascorbate therapy is believed to counter infection by augmenting such NAD(P)H high energy electron provision, e.g., for providing the respiratory burst of phagocytes (Cathcart 1991).

Oxalate

Ascorbate eventually metabolises to oxalate which may crystallise under defective anti-mineralization inhibitor conditions (cf Singh 1993).

5. SUMMARY OF HEPARIN/HEPARAN SULPHATE BIOCHEMISTRY

The most highly evolved glycosaminoglycans seem to be the heparan sulphates(mainly from the surfaces of most animal cells and extracellular matrix) and heparin (from mast cells and leukocytes but stored in endothelial cells (Hiebert & McDuffie 1989). Heparan sulphates, probably obligatory for multicellular animal organisms, are broadly similarly constituted to heparin but have characteristic differences (Gallagher et al 1985).

The assembly of heparan sulphates has recently been indicated to be more complicated than previously thought, requiring multiple

N-deacetylase/N-sulphotransferase (NDST) isoenzymes (Aikawa et al 1999). Precise sequence-dependent heparan sulphate microstructure is likely a fundamental component of much growth factor signalling (Krufka et al 1996) and heparan sulphate proteoglycans (HSPGs) are fundamental to the distribution and processing of extracellular signals that pattern fields of cells in developmental biology. The HSPGs syndecans and glypicans are associated with cell surfaces and perlecan (and agrin) with basement membranes(Perrimon & Bernfield 2000).

Perlecan core protein synthesis and sulphation of heparan sulphate side chains were reduced by Pb2+

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suggesting that this toxin inhibits endothelial cell proliferation by lowered response to bFGF (Fujiwara &amp; Kaji 1999).

Nitrous acid perturbation of heparan sulphate dependent growth factor signalling in mucosal replendishment has been suggested (Ascensio et al 1995) to promote stomach cancer.

Although many biological processes (e.g. as listed by Belford et al 1992 who gave prior references)are known to have potential inputs from heparin/heparan sulphate biochemistry, it is still usually stated that the main biochemical role of these substances has yet to be established. Apart from growth factors small ions are bound to heparin/heparan sulphate which may function in part as a conveyor belt for their distribution and this function may be influenced by a form of hydration dependent phase change behaviour discussed below.

The original function of glycosaminoglycans may have been for provision of a correct water and ionic biological environment similar to the believed function of polysaccharides secreted by bacteria, the extracellular polysaccharides of algae and even the polyanionic humic acid which confer biofriendly hydration and mutinutrient reservoir properties on soils.

Evolutionary pressure in development of multicellular organisms would require more complex functions such as the provision of anchoring sites (a 'workbench' or processor function) for a variety of effectors required to orchestrate such complex processes as haematopoiesis (cf Gupta et al 1998) and anticoagulant /coagulant activities including antithrombin (III) binding and processing (cf Rosenberg & De Agnostini 1992; Bourin & Lindahl 1993); their distant ancestral precursors

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evidently resembled bacterial capsular polysaccharides

(Cf [GlcUA→GlcNAc]n which can be structurally manipulated in vitro to yield heparin analogues (Lormeau et al 1992).

An unusually evolved flexibility of iduoronate rings may facilitate adoption of multiple environmentally directed conformations by heparin, heparan sulphate and dermatan sulphate (e.g. Sanderson et al 1995; Grant et al 1991). A similar evolution of increased functional complexity is also evident in the extracellular polysaccharides of plants (McNeil et al 1984).

It is this structural complexity of the highly evolved heparan sulphate structures, dependent on highly specific polysaccharide microstructures (cf Perrimon & Bernfield 2000) requiring correctly arranged substituted glucosamine sugars, which renders them especially susceptible to damage by nitrous acid generated by the cellular immune response to pathogens which bound to cell surface heparans sulphate chains.

Formation of complexes between heparin/heparan sulphate and proteins is often critical for stabilising protein conformation and aiding correct functionality, such complexes are formed by polysaccharide binding to groups of basic amino acids which include highly consderved consensus sequences such as the Trp-Ser-Pro-Trp sequence bounded by basic amino acids (Guo et al 1992). Consensus sequences for glycosaminoglycan recognition were determined as X-BB-X-B-X and X-BBB-XX-B-X where B is a basic residue and X is a hydrophobic residue (Cardin & Weintraub 1989).

Faham et al reported the first crystal structure of a protein-GAG complex(for bFGF-heparin); computer simulation of its receptor docking

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(Bitomsky &Wade 1999) can lead to insight into such complex formation, perhaps improved by including solvation effects. It is evident that nitrous acid or other active nitrant attack on vulnerable amides may affect both parts of the binding sites.

Heparin/heparan sulphate proteoglycans are believed to be involved in

haemostasis, fibrinolysis, capillary permeability, matrix assembly, potentiation of mitogenic, chemotactic, neurotrophoc and angiogenic activities, inter and intracellular communication, cellular recognition, immunological signalling, adhesion, differentiation, proliferation, wound healing, apoptosis, gene expression, capacitation, morphogenesis, embryogenesis, nerve and brain development and function (neurite outgrowth, synaptic function, myelination) the provision of links between extra and intracellular space, phosphatidyl inositide anchoring and cytoskeleton function, lipid processing, glomerular filtration and antioxidant protection, involving both the intact polymers and, at least in some instances, fragments.

(Oligosaccharides formed during normal heparin/heparan sulphate turnover have, however, likely specific intracellular functions following endocytosis as well as protecting the blood and urinary systems against damage by crystal formation and inhibiting viral, bacterial and prion infection).

It may be no coincidence that nitric oxide is also implicated in similar functions and therefore active nitrogen processing of heparin/heparan sulphate may be a normal part of such biological activity. The presence of tightly bound Cu2+ in heparin and heparan sulphate but not in other GAGs (Rej et al 1990) may

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conceivably influence such 'active nitrant' processing.

Nitric oxide synthase activity is reported (Kouretas et al 1998) to be modulated by non-anticoagulant heparin (of possible therapeutic value) via a guanine nucleotide regulatory protein mechanism .

Nitric oxide production by bovine endothelial cells has been correlated with shear stress (Korenaga et al 1992); heparan sulphate is also thought to be involved in flow sensing (e.g. by polymer coil conformation alteration caused change in Ca2+ and Na+ balance, studied by Siegel et al 1998). The biosynthesis of endothelial heparan sulphate proteoglycans was reported to be greatly influenced by flow, although no feedback effect of polymer fragments could be detected (Grimm et al 1988).

5a.

ACTIVATION OF HEPARAN SULPHATE FOR NITROUS ACID CLEAVAGE BY REMOVAL OF PROTECTIVE GROUPS FROM GLUCOSAMINE NITROGENS

Initial proton-catalysed removal of the sulphonate groups, or reaction of acyl groups with hydrazine, yields glucosamines which are much more highly reactive towards nitrous acid than are the acetyl or sulphonate substituted glucosamines of native heparan sulphate.

The stability of the N-sulphonate groups (studied with heparin) towards(nitrous) acid catalysed hydrolysis is affected by counterion binding by a mechanism which is believed to involve hydration changes and phase boundary effects. Alternative explanations (due to Manning) in terms of a

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critical charge density, above and below which outer or inner ion-binding occurs, was not substantiated by detailed analysis; additional catalysis of such effects may occur by iron, copper and perhaps vanadyl ions.

A tightening or loosening of the hydration sheath is engendered by cation-dependent binding with alteration in the accessibility of the N-sulphonate groups and susceptibility towards nitrous acid cleavage. The standard procedure uses replacement of counterions by pyridinium ion to render the molecule more readily de-N-sulphonated (an example of synergy between nitrogen-based reagents in the degradation of heparin). This counterion alteration effect evidently induces the formation of a less hydrated form of the heparin molecule. These situations may mimic pro-disease scenarios related to heparan sulphate damage.

Unsubsituted glucosamines in heparin/heparan sulphate (which will undergo immediate nitrous acid scission) were originally believed to be absent from tissue heparan sulphates or to be extraction artifacts, since the currently accepted mechanism of the biosynthesis of heparin/heparan sulphate requires that all glucosamine amino groups must be either N-acetylated or N-sulphonated (Kjellen et al 1992); such a rigid requirement seems, however, to be in disagreement with the recent reports of the presence of unsubstituted glucosamines, immediately deaminatively cleavable by nitrous acid at pH 4.6, in heparan sulphate present in endothelial cells although absent from heparan sulphate similarly obtained from CHO cells (Norgard-Sumnichi & Varki 1995, cf Van den Born et al 1995).

The presence of unsubstituted glucosamines in vivo may be evidence for the occurrence of pathological post-synthetic modification. The

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free amino groups in pharmaceutical heparin detectable from nitrophenylation, included both amino acid end groups and glucosamines (Arai et al 1993). Older work (Linker &amp; Hovingh 1973) reported unexpectedly larger amounts of unsubstituted glucosamines in heparan sulphate from amyloid liver but not from healthy tissues which may indicate that catalysis of the de-N-sulphonation by e.g. quaternary nitrogen counterions or trace transition metals occurred in the acidic proteolysis extracts or even a disease-related elevation of free glucosamine residues in heparan sulphate biosynthesis. Further studies of heparan sulphate alteration by active nitrogen (including possible iron and copper catalysis) in conjunction with effects of other metal ions, notably zinc, aluminium as well as silicates are warranted in this context.

Heparan sulphates with sulphate-rich segments, which inhibit inappropriate arterial well cellular proliferation, were diminished in arteriosclerosis patients. Oligosaccharides produced by nitrous acid deaminative cleavage of heparan sulphate at pH 3.9 showed marked antiproliferative activity against smooth muscle cells (of relevance to the prevention of arteriosclerosis) but this property was absent from oligosaccharides produced by cleavage of heparan sulphate conducted at pH 1.5 (Schmidt et al 1992).

Extended sequences of 7-8 N-sulphonated disaccharides, in which a proportion of the iduronic residues are sulphated at C-2, appeared to be required for cell adhesion and migration (Walker et al 1996).

The following cancer-related situations (cf Ferguson 1975) may also conceivably involve heparan sulphate-related signalling processes: conversion of carcinogenic aromatic amines to N-

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sulphonated derivates (possibly mimicking heparin/heparan sulphate structures) was apparently required to induce cancer and various non-alkylating antileukaemic drugs possess a potency apparently related to the possession of O-N-O triangulation sites reminiscent of N-sulphonate centred binding in heparin/heparan sulphate.

5b.EVIDENCE FOR THE RELEVANCE OF HEPARIN/HEPARAN SULPHATE BIOCHEMISTRY TO

THE AETIOLOGY OF CANCER

The effect of heparin and related compounds on cell and tissue growth has been recognised since 1932. Release of heparin/heparan sulphate from cells was found to be in synchrony with the cell cycle (Chiarugi et al 1974).

Heparin and heparinoids have demonstrated substantial antitumour effects in animal models (Zacharski 1988) and are likely to have similar effects in humans with potential therapeutic benefit (Engleberg 1999). Plant xylan derived heparinoids have demonstrated anti-tumour activities (Wessel et al 1998).

Some anti-cancer drugs while supposed to act by deactively crosslinking of DNA may (or in addition) block proliferation-related cell surface sites as appears to be the case for the inhibition of cell proliferation by Suramin(Nakajima et al 1991) a non-polysaccharide heparinoid (Grunicke 1991).

At least two members of the EXT family of 'tumour suppressors' were found to encode D-glucosyl and N-acetylglucuronyl transferases required for the chain elongation of heparan sulphate (Lin & Lindahl et al 1998).

The HSPG syndecan-1 is reduced in cervical cancer where excessive expression supressed the growth of the cancer cells (Nakanishi et al 1999).

Heparin (but much less so de-N-sulphonated heparin) was indicated to have multiple potential immuno-enhancing effects, including stimulation of IL-1 mediated activities inducing increased cytotoxic responses against histocompatible tumours in mice (Dziarski 1989, 1992).

Heparin/heparan sulphate binding growth factors appear to be involved in cellular physiology of relevance to cancer. Heparin affinity regulated peptide (HARP) was associated with epithelial cells in prostate cancer but not with those of normal or benign hyperplasia and may be involved in the regulation of prostate tumour cell proliferation (Vacherit et al 1999).

Heparin selectively inhibits cell cycle phases in endothelial cell proliferation (Kimura et al 1992) and regulates protein tyrosine

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phosphorylation in vascular smooth muscle cells(Mishra & Castellot 1999).

Heparan sulphate-dependent internalisation and catabolism likely controls the in vivo availability of bFGF (Colin et al 1999) and bFGF alters the intracellular distribution of the GAG degradation products (Tumova et al 1999).

Many pathogens are known to initially interact with the host via a heparan sulphate linkage (David 1998); this includes organisms which are known to promote cancer (especially noteworthy is Helicobacter pylori which may perturb b-FGF heparan sulphate signalling putatively linked to stomach cancer by Ascensio et al (1995) in agreement with more virulent strains demonstrating increased heparan sulphate binding ability (Chmiela et al 1995)).

Ascorbyl radicals were increased with increasing H pylori acitivity in these tissues (Drake et al 1996) in accord with for an anti-oxidant/nitrant anti-cancer role of ascorbate in countering immunologically produced anti-bacterial responses which if inadequete would lead to inappropriate degradation of heparan sulphates.

Cancer cells often show a large difference from normal cells in their surface heparan sulphates both in amount and in microstructure, most characteristically by a decrease in amount and decrease in charge density of the surface bound heparan sulphates. This is likely to be a consequence of altered biosynthesis as well as augmented degradation both by enzymic and non-enzymic (including oxidant/nitrant) routes. In the cell culture model of cancer, an alteration in heparan sulphate biosynthesis is found in both cellular transformations produced by tumour viruses and by nitrosoguanidine derivatives (cf Woodhead et al 1987). More recent studies identify types of HSPG putatively involved (e.g., syndecan-1 studied by Nakanishi in 1999). Oncogenic transformation may also be accompanied by biosynthesis of heparan sulphates of increased charge density (Rahmoune et al 1996).

High grade glioma derived cells expressed markedly increased amounts of hyaluronic acid and heparan sulphate (Steck et al 1989).

The cancer inducer phorbol ester triggers HSPG-dependent tumour cell adhesion (Timar et al 1996). Heparan sulphate moieties, and not the core protein, of a phosphatidyl inositol anchored proteoglycan, initiated the mouse melanoma cell adhesion to fibronectin (Drake et al 1992).

The relative virulence and metastatic potential of cancer cell lines often correlates with their secreted heparanase acitivites (Nakajima et al 1986-1992). Katz et al(1996) showed that inhibition by heparin of lung colonization by in vitro virally transformed 3T3 cells rendered highly tumorigenic by in vitro passage, was achieved by the inhibition of the transformed cell heparanse activity. The degradation of basement membrane by prostate tumour heparanse is believed to be a key factor in the malignancy and potentially may be countered by inhibition of the GAG-ase (Kosir et al 1999). Basement membrane heparan sulphates studies of implanted Lewis lung carcinomas showed that more highly metastatic cancer cells possessed greater basement membrane organization, this being perhaps asttributable to the effects of accompanying heparan sulphates with greater overall sulphation (Nakanisha et al 1992).

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Heparin/heparan sulphates provide binding sites for the major extracellular superoxide dismutase (cf Adachi et al) and were indicated to possess intrinsic antioxidant effects (Grant et al 1987b) demonstrating some intrinsic superoxide dismutase activity (Grant et al 1988) and also binding trace transition metal (e.g. iron) ions thus countering potential free-radical and peroxynitrite DNA damage (Ross et al 1992).

5c CANCER-RELATED METAL ION BINDING BY HEPARAN SULPHATES

Heparan sulphate biochemistry seems to be implicated in various cation transportations of relevance to cancer.

Transferrin glycosylation is believed to include heparan sulphate side chains (Fransson et al 1984)and tumour cells may have more heparan sulphate directed iron entry. Anti-tumour chelation therapy may include interruption of such iron transport.

Trace bound redox metals may contribute to the superoxide dismutase activity of heparin although activity was found to remain after attempting their removal and sequestration (Grant et al 1988).

Altered heparan sulphate in tumour cells allows radioctive tracer tumour imaging (gallium was believed to mimic iron binding; (cf transferrin binding sites are believed to have heparan sulphate side chains). Gallium ions stick selectively to tumour and inflammatory lesions via binding to heparan sulphates((Kojima et al 1983; Table I of this article reports that heparan sulphate exhibited much higher binding than heparin) cf also Hama et al 1983 and 1984) endocytosis and degradation of gallium-heparan sulphate was indicated to occur in lysosomes.

Therapeutic utilisation of gallium nitrate for the normalization of hypocalcemia of malignancy is reported (Warrell et al 1991) superior to other treatments such as ethane 1-hydroxyl,1,1-diphosphonate (a blocker of CaCO3 crystal nucleation of similar activity to pyrophosphate or heparan sulphate Grant et al 1989b). A complex calcium-sensitive heparan sulphate cell engulfment has been demonstrated in parathyroid cells (Takeuchi, Yanagishita et al 1990) and similar mechanisms may also apply to other cell types.

Formation of crystals capable of surface enrichment of bound redox-metals capable of initiating free radical cascades as has been hypothesised for asbsetos-linked cancerigenesis may also apply to other pathological crystals. Heparin/heparan sulphate has a role in the prevention of such crystallisation but N-sulphonate depeleted heparin was found to be much less effective (Grant et al 1989b). Calcium-rich crystals may also generate mitogenic signals directly, pathological calcification may therefore be both a cause and an effect of malignancy (cf discussion of relevant literature by Grant et al 1992b).

In summary, metal ion homeostsis is likely subect to alteration by disease processes which affect and are affected by heparan sulphate microstructures.

5e. EFFECTS OF OXYGEN, GLUCOSE, TOXINS & ASCORBATE ON HEPARAN SULPHATE MICROSTRUCTURE

Cell culture experiments have indicated that heparan sulphate biosynthesis may be sensitive to cellular oxidation-reduction status but the details vary between cell types. Heparan sulphates from bovine arterial endothelial cells showed dependence of heparan sulphate biosynthetic patterns on oxygen pressure, hypoxia inducing reduction in chain length and decrease in sulphation (Karlinsky et al 1992). Chlorate is used routinely in vitro cell cultures to decrease sulphate incorporation into heparan sulphate, monensin having similar effects (Sampaio et al 1992). Ascorbate, however, specifically boosted the biosynthesis of heparan sulphate in fibroblast cell culture and increased sulphate incorporation into the polymer (Kao et al 1992); a mechanism involving redox status seems indicated.

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Ascorbate thus may both potentially promote biosynthesis as well as provide protection against inappropriate post-secretion damage to heparan sulphate by active oxygen/nitrogen. (Priior work had demonstrated that ascorbate increased collagen and glycosaminglycan synthesis).

5ei Effect of glucose on heparan sulphate biosynthesis

In a model of diabetic nephropathy glucose augmentation in mouse glimerular epithelial culture differentially reduced the biosynthesis of various heparan sulphate proteoglycans (Morano et al 1999)(this effect likely impairs glomerular filtration).

5eii Effect of toxins on heparan sulphate biosynthesis

Toxins (e.g. lead (Kaji et al 1991) cadmium (Cardenas et al 1992) and endotoxin (Dietrich et al 1996) reduced heparan sulphate biosynthesis. There may be an additional, normal, age-related diminution of heparan sulphate as a proportion of the total glycosaminoglycans at arterial walls where a linear decrease with age has been reported (Murata &amp; Yokoyama 1989).

The effects age, excess glucose, toxins and ascorbate deficiency(through dietary insufficiency or in smokers) may impair heparan sulphate function.

6. Research into intervention of carcinogenesis and other degenerative diseases using the above model might reasonably consider heparan suphate replacement therapy via administration of preformed heparins, semisynthetic heparinoids or dietarty supplementa of ascorbate, glucosamine or glucosamine sulphate or provision of additional protection by other ascorbate derivatives, improved inducible nitric oxide synthase inhibitors used in conjunction with redox metal chelation therapy.

(Perhaps inhibitors of inducible nitric oxide synthases studied for rheumatic disease therapy e.g. dietary components, warrant investigation as anti-cancer agents).

The hypothesis of a central role of redox status for cellular activity control in which heparan sulphate microstructure is involved offers a new framework for research.

(originally posted on the internet on 2000 at http://web.ukonline.co.uk/dgrant/dg4)45454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545454545

APPENDIX

8.a.

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TRANSITION METAL CATALYSIS OF REACTIVE NITROGEN DAMAGE (further notes) 8a-1. Iron & Copper Catalysis of Nitrosylation & Deaminative Cleavage. 8a-2. Nitrous Acid Reduction to Nitric Oxide by Ascorbate. Effect of Redox Metals. 8a-3. Anti-Cancer Effects of Nitrite. 8a-4. Deaminative Cleavage of Heparin/Heparan Sulphate at Physiological pH. 8a-5. Thiocyanate Effects. 8a-6. Phosphate Effects. 8a-7. Multi-Ion Binding Properties of Heparin/Heparan Sulphate. 8.a-7-1. Pathological Species May Seek Nutrients from Heparin/Heparan Sulphate. 8.a-7-2. Spark Source Mass Spectrometry of Heparin. 8.a-7-2-1. Ppm Elements Before and After Cation Exchange. Possible Pathologically-Relevant Redox Metals. 8.a-7-2-2 Titanium. 8.a-7-2-3. Chromium. 8.a-7-2-4. Vanadium. 8.a-7-2-5. Manganese. 8.a-7-2-6. Molybdenum. 8.a-7-2-7. Silver. 8.a-7-2-8. Lanthanum, Neodymium, Zirconium & Yttrium. 8.a-7-2-9. Cerium & Antimony. 8.a-7-2-10. Mercury & Cadmium. 8.a-7-2-11. Aluminium. 8.a-7-3. Nickel Effects. 8.a-7-4. Anions in Heparin. 8.a-7-4-1. Phosphorus 8.a-7-4-2. Silicon. 8.a-7-4-3 Boron. 8.a-7-5. The Binding of Aluminium & Fluoride to Heparin 8.a-7-6. Importance of Nucleation Factors & Lipids in Induction of Polysaccharide Supramolecular Structures. 8.a-7-6-1. Hydrophobic Polyanion Binding Conditions. 8b. Heparan Sulphate, Brain Development, Function & Neurodegenerative Diseases Including Alzheimer's Disease (AD). 8b-1. Introduction. 8b-2. Heparin/Heparan Sulphate in AD. 8.b-2-1. Learning & Heparin/Heparan Sulphate.

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8.b-2-2. Zinc in AD. 8.b-2-3. Cognitive Function in AD. Therapeutic Use of Heparin/Heparan Sulphate. 8.b-2-4. Neurite Outgrowth Stimulation by Heparin/Heparan Sulphate. 8.b-2-4-1 Possible Heparin/Heparan Sulphate-Dependent Growth Factor Effects in AD. 8.b-2-5. Extracted Brain Heparan Sulphate - Lack of Correlation with AD. 8.b-2-6. Possible Transferrin Receptor Function. Aluminium & Heparin/Heparan Sulphate in AD. 8.b-2-7. Fenton Reaction/Free-Radical Damage in Neurodegenerative Diseases. 8c. Supramolecular Structure of Heparin/Heparan Sulphate PGs and their Binding Potential. 8c-1 Silicates in GAGs. 9. Involvement of Heparin/Heparan Sulphate in Modulation of Prion Protein Activity. 10. Water Binding to Heparin/Heparan Sulphate. 10a. Hofmeister Effects on Water Structure. 11. Modulation of Crystallization by Heparin/Heparan Sulphate. 12. Established Laboratory Use of Nitrous Acid for Heparin/Heparan Sulphate Analysis. 13. Binding of Pathogens to Heparan Sulphate. 14. References. 15. Acknowledgements. 8a-1. Iron & Copper Catalysis of Nitrosylation & Deaminative Cleavage. Dietary nitrite is traditionally thought to lead to carcinogenic nitrosamine formation; iron-nitroso complexes (imparting a characteristic red colouring to cured meat) potentially enhance nitrosamine formation under iron overload conditions e.g. exacerbate by chronic (autoimmune) nitric oxide production which may contribute to the aetiology of various degenerative diseases including cancer, arthritis and Alzheimer's disease in which inappropriate nitrous acid deaminative cleavage of heparin/heparan sulphate polysaccharides, (important mediators of immunological signalling, haemostasis, cardiovascular function and wound healing) may be involved. Iron may catalyse such deaminative cleavage both by pre-priming heparan sulphate via de-N-sulphonation (unpublished observations of the author) and by directly catalysing the nitrous acid depolymerizaton process.

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Available iron, indicated by serum ferritin, may increase naturally with age in the absence of specific observable disease (Jarrett et al 1989) according with a redox-metal centred reactive oxidant (or nitrant) hypothesis of ageing. The depletion of heparan sulphate in atherosclerotic arteries increased linearly with age (plot of data in Table 4 of Murata & Yokoyama (1989)) this effect may be related to an iron dependent heparan sulphate degradation in atherosclerosis. In vitro experimental evidence for iron and copper salt catalysis of nitrosoamine formation, although not widely researched, was provided by the studies of Challis et al (1978). Evidence for possible transition metal-related reactive nitrogen damage to health is :- an increased incidence in men than pre-menstrual but not post-menstrual women of upper stomach cancer supposed to be promoted by nitrosamines (McColl 1999); an iron-catalysed nitric oxide/nitrous acid DNA damage implicated (Park & Aust 1998) in the mutagenicity of iron-rich asbestos and an iron-overload correlated mortality of Eastern Finnish men (from serum ferritin epidemiological studies reported by Salonen et al 1992). A possibly related ability of heparin to inhibit free-radical reactions initiated by Fe(II) was found (Ross et al 1992) to be dependent on the integrity of heparin, damage produced to heparin by nitrous acid eliminating this protective effect. 8a-2. Nitrous Acid Reduction to Nitric Oxide by Ascorbate. Effect of Redox Metals. Ascorbic acid influences the availability of nitrous acid by efficiently reducing it (back) to nitric oxide. Iron and copper ions impair this anti-nitrite protection by catalysing the reaction of ascorbic acid/ascorbate with molecular oxygen (to give dehydroascorbic acid). 8a-3 Anti-Cancer Effects of Nitrite. Nitrite, supplemented by that produced by (molybdenum enzyme-dependent) reduction of dietary nitrate, can also have an anti-cancer effect (Benjamin 1997) probably by killing cancer-inducing bacteria. The balance of beneficial and damaging influences of nitrite/nitrous acid is suggested to be a function of the activation of spurious redox metals capable of enhancing nitrosylation and deaminative cleavage of heparin/heparan sulphate, balanced by the availablilty of reductants, principally ascorbate for blocking these reactions and the

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effectiveness and availability of redox metals in the oxidative depletion of ascorbate. 8a-4. Deaminative Cleavage of Heparin/Heparan Sulphate at Physiological pH. Nitric oxide, previously not thought capable of producing cleavage of N-sulphonated or N-acetylated heparin/heparan sulphate at physiological pH, is now established from the work of Vilar et al (1997) to undergo cleavage by nitric oxide gas in vitro, under conditions of phosphate, but not imidazole buffering. The effect of trace iron and copper impurities normally present in phosphate buffers (Nader & Krishnamoorthy 1983, cf Singh et al 1996) is surely relevant to this situation. Further investigation is required. 8a-5. Thiocyanate Effects Thiocyanate, augmented in the saliva and serum of smokers (Boyland & Walker 1974; Zhang et al 1997) is a potent catalyst of nitrosylation (McColl et al 1997) facilitating the formation of nitrosium cations, and possibly additionally complexing with and enhancing the deaminative catalytic characteristics of thiocyanate metal complexes. The effect of thiocyante on the deaminative cleavage of heparin/heparan sulphate requires investigation. Thiocyanate-promoted denaturation of biological hydrogen-bonds may allow deep penetration of associated redox metal complexes such as those of copper, chromium, vanadium and iron (the bright red iron (III) thiocyanate complex is well-known sensitive laboratory test for ferric ions) should be considered as potential catalysts of heparan sulphate cleavage by active nitrants. 8a-6. Phosphate Effects. Phosphate containing ligands may increase damaging activity of redox metals both by oxidative and nitrant routes. Phosphate, polyphosphates and phosphate ester-liganded redox metals are likely to both enhance nitrosylation reactions and also to increase the depletion of ascorbate by oxidation processes. The form and availability of more active forms of liganded phosphate may be critical to such catalytic processes. Experimental studies are needed to assess catalysis of such reactions as well as the relevance of perhaps related stochastic scrambling, long known to be a characteristic of abiological phosphate/polyphosphate and related systems (Van Wazer 1959); applied to DNA this results in mutagenic effects, as yet is unevaluated by biochemists).

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8a-7. MULTI-ION BINDING PROPERTIES OF HEPARIN/HEPARAN SULPHATE. The more highly charged segments of heparan sulphate (sometimes as heparin-like segments) may provide multi-trace-metal binding sites which provide antioxidant protection as well as nutrient ion reservoirs. 8a-7-1. Pathological Species May Seek Nutrients from Heparin/Heparan Sulphate. Inappropriate active nitrogen depolymerization may provide pathological organisms with the otherwise growth-limiting nutrients. The large number of pathological organisms which become attached to heparan sulphate may do so in part to gain such nutrient advantage. 8a-7-2. Spark Source Mass Spectrometry of Heparin. Numerous transition metals assayed by spark source mass spectroscopy in ('sodium') heparin from mixed mammalian sources with a very high trace metal loading (the trace metals are presumably present in the original animal polysaccharide as well as being picked up during processing e.g. from tap water (a major drug company provided for academic study a presumably non-commercial high-ash heparin (far outwith the allowable specification believed to be a common preparation 'problem'; EDTA is apparently used to diminish the unacceptably high toxic ash contents of some preparations and residual EDTA poses an contaminant problem in pharmaceutical heparin)) in addition to the presence of major cations reported by Grant et al (1987a) which were (ppm before/after cation exchange resin percolation):- nickel(170/5), molybdenum(7/0?), chromium (30/1); the sample also contained vanadium; manganese, however, was below the detection limit of 1ppm (in ppm before/after Tl+ replacement on a cation exchange column). 8.a-7-2-1. Ppm Elements Before (x) and After (y) Cation Exchange. (listed thus (x/y*)) Cations Iron & Copper Caesium & Rubidium iron(1100/10*), Cs 9/0*, copper(730/,5*) Rb 3/0.2* Lead, Arsenic & Tin Pb 16/4*, As 15/1*, Sn 5/0.3* Possible Pathologically-Relevant Redox Metals.

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8.a-7-2-2. Titatnium Titanium showed up relatively abundantly in heparin at (>390/4*). If present in a polymerized oxy-bridged Ti oxy/hydroxy species it would be less damaging than if, e.g. ‘liganded’ by halide. 8.a-7-2-3. Chromium Cr in heparin was (30/1*). Part of the anti-cancer role of ascorbate might be to counter pro-oxidant effects as in the chemical reduction of Cr(VI) which is likely to be responsible for ascorbate protection against chromate induced cancer (Connett & Wetterhahn 1983). 8.a-7-2-4. Vanadium Vanadium, found in lesser amounts than the above elements in mammalian heparin (3/?*), could partly originate from fungal sources where it is abundant, forms similar medium stability complexes with thiocyanate to thiocyanate-iron(III) (but iron(II) interacts more weakly)(Bahta et al 1997) but may sometime pose a 'vanadium overload' threat. 8.a-7-2-5. Manganese 8.a-7-2-6. Molybdenum Molybdenum dependent enzymic action is involved in the oxidation-reduction biology of nitrogen compounds. Mo in heparin was (7/?*). 8.a-7-2-7. Silver Silver occurs in mammalian heparin. Ag (4/0.5*), and may be similarly involved in thiocyanate-related pathology. 8.a-7-2-8. Lanthanum, Neodymium, Zirconium & Yttrium. La 7/1*,W 5/0*, Nd 5/0*, Zr 5/0.3*,Y 3/0* were present in significant amounts in the studied heparin. 8.a-7-2-9. Cerium and Antimony Cation exchange replacement of cations by Tl+ appeared to scarcely remove Ce (7/3.5*) and Sb (2/1.7*); it must be assumed therefore that these elements occur in anionic or form unusually strongly bound adducts with heparin or are artifacts of the analytical procedure. Inappropriate release of such stored metals may stimulate inappropriate, pro-disease, redox activity. 8.a-7-2-10.

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Mercury and Cadmium Mass spectral analysis shows that although mercury is below the detection limit in the above mammalian animal heparin, both mercury and cadmium have been reported (Muzzarelli 1983) to be present in chitosan evidently via marine pollution; intoxication from this source and dental fillings have been listed in Internet discussions of mercury toxicity to be a major causative factor in AD. Further work is required to fully epidemiologically quantify such trace metal loading of heparin/heparan sulphate and other mammalian GAGs and to determine possible pathological links. 8.a-7-2-11. Aluminium The sample was not assayed for Al because of the use of an aluminium powder sampling procedure. Further work is required to assess Al contents of high ash and normal pharmaceutical heparin. 8a-7-3. Nickel Effects. Nickel metal provokes hypersensitivity in some persons. Nickel ions normally do not demonstrate well-defined aqueous solution redox behaviour, but heterocyclic N-containing ligands have been observed to stabilise otherwise difficult-to-produce Ni(III) (Lindol, 1975) which may redox cycle with Ni(II) leading to the formation of active-oxygen and active-nitrogen species. As Ni is employed in mammalian enzyme centres, relevant Ni-dependent heparan sulphate interaction might be anticipated. Further studies are required to test this possibility. Ni in heparin was relatively abundant (170/?*). 8a-7-4. Anions in Heparin. Major, likely anionic 'contaminants' detected by mass spectrometry in the above heparin sample were also surprisingly largely removable by cation exchange resin treatment, including 2000ppm chloride, 130 ppm bromide, 10 ppm iodide and surprisingly 890 ppm fluoride (removable to 4ppm upon Tl+ exchange, therefore likely to be attached to heparin-bound cations such as Al3+). 19-F NMR studies might be of value in establishing the mode of binding of such fluorine to heparin. Strong binding of heparin to iodide, chloride and sulphate has previously been described in a Patent claim (Fo-We, 1962). In a 1980 review Jaques stated that heparin exhibits "strong binding of counterions of sodium, potassium, ammonium, quaternary ammonium radicals, and co-ions such as sulfate, phosphate and acetate". Sulphate anions in heparin were found not to be removed

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by dialysis (Simon 1982) as would be expected if present as a consequence e.g. of de-N-sulphonation; unpublished observations of the author confirmed that a proportion of the 35S labelled sulphate in heparin may behave anomalously on gel filtration separation in keeping with the formation of a bound sulphate anion-heparin complex. 8.a-7-4-1. Phosphorus The above analysis also showed the presence of 440ppm phosphorus (which was reduced to 30ppm on cation exchange resin treatment), likely present as phosphate ((1348ppm as PO43- or other P(V) oxy-species derivatives) in heparin. Additional interest of the high phosphorus content of heparin is related to a likely influence of such phosphate and related species in enhancing damaging metal redox behaviour. Condensed phosphates if bound to heparin/heparan sulphate would be expected to provide strong metal ion binding sites. Calcium in the studied heparin was 30000ppm which was reduced to 30 ppm upon passage through the cation exchange resin column in agreement with the chemical environment of heparin-bound phosphorus being possibly present as a calcium P(V)oxy-species-heparin complex. Ir spectra of a heparin solution peptized hydroxyapatite (obtained in studies of the inhibition of crystallization of this substance by heparin) confirmed the presence oxy-phosphate attached to the heparin chains in such a way as to significantly alter the heparin ir asymmetric S-O vibration absorbance bands (Grant et al 1992, unpublished). 8.a-7-4-2. Silicon The mass spectral results of heparin discussed above also showed 5900ppm Si, 100ppm being retained bound to the heparin after passage through a cation exchange resin. Clearly a large part of the silicon is likely to be present as silicate and be bridged to heparin by cations such as Ca2+ and Al3+ . Schwarz (1973) found that polyuronides, including GAGs, contained strongly linked (molybdate-inactive) silicate, (e.g. as a crosslinking silicic acid derivative, 29-Si NMR would be usefully employed to determine the environment of Si in such complexes); this author reported that heparan sulphate had 427ppm Si which could not be removed with 8M urea. Grant et al (1992f) presented experimental evidence for the importance of multi-phase phenomena in various cation (including

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calcium)-heparin interaction as an altenative explanation to the Manning hypothesis for the existence of critical binding behaviour. Such a phase change mechanism may explain why inorganic anions are often irreversibly bound to the highly anionic polysaccharides (of additional likely relevance to understanding the mechanism of DNA and RNA cation binding where a similar situation pertains) and may further be of relevance to the possible gating of ion, including proton, conduction by such polymers as well, perhaps being relevant to understanding of the apparently unrelated phenomena such as the physical chemistry of the formation of pathological amyloid (heparan sulphate containing) deposits in which metal ion loading may have some causative participation (cf, Ca2+ but not other M2+ except non-physiological Cd2+ mediated the association of human serum amyloid P component with heparan and dermatan sulphate (Hamazaki 1987)). A mechanism (Long & Williamson 1979) of control of cellular proliferation by heparin/heparan sulphate interactions with Ca2+ might be extended to include the co-binding of phosphate and silicate to Ca2+ (perturbable by such cations as Al3+?) to heparin/heparan sulphate and be of further possible relevance to the involvement of GAGs in calcification. 8.a.-7-4-3. Boron. The mass spectrometric results showed that the starting heparin contained <25ppm B, likely as borates or related anions, reducible to <1ppm after cation exchange. 8.a-7-5. The Binding of Aluminium & Fluoride to Heparin. A previous indication by Kojima et al (1983) that Al3+ binds strongly to heparin/heparan sulphate was confirmed by Grant et al (unpublished results obtained using potentiometric and osmotic methods). Both heparin/heparan sulphate biochemistry and (more controversially) the effect of aluminium intoxication, have been implicated in neurodegenerative diseases such as AD (e.g. Lehmann 1992). Fluoride intoxication at high levels is reported to alter the GAG content of bones (Susheela & Jahr; Priince & Navia) which might include the biological effects of Al3+-polysaccharide binding (perhaps causing perturbation of Ca2+-heparan sulphate binding?). Biologically-relevant effects of Al3+ plus F- include adenylate cyclase activation by F- (involving trace Al3+ or Be2+ co-catalysis cf, e.g, Martin, 1988). 8.a-7-6.

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Importance of Nucleation Factors & Lipids in Induction of Polysaccharide Supramolecular Structures. Further ideas of relevance of binding of anions to heparin/heparan sulphate might be an involvement of these polysaccharides in various currently poorly-understood physiological nucleation phenomena and disease-related alteration in such activity due to erroneous supramolecular structural distorting effects. Since Ca/Mg pyrophosphate granules are believed to nucleate calcification (e.g. Taylor et al 1990) such pyrophosphate granules attached to glycosaminoglycans may have similar properties. Nucleation initiating catalysts may control specific glycosaminoglycan phase change; iron impurities are believed to be initiators of carrageenan supramolecular structure formation (Williamson FB, Aberdeen, personal communication). 8.a-7-6-1. Hydrophobic Polyanion Binding Conditions. Physiological phase changes in the pericellular environment clearly involve the physical chemistry of lipids. Whereas under normal physiological saline conditions, metal ions are often only relatively weakly bound by mono and polysaccharides, in non-aqueous media including pericellular lipid-rich environments, large conformational alterations are found upon cation sugar interaction, suggesting that this is unusually strong in character (cf Tajmir-Riahi 1989), The nmr and infrared spectra of various heparin metal ion complexes provide some insight into details of polyanion-cation-water interaction. 13-C nmr spectra are influenced by a time averaging of fast cation exchange processes exemplified by a dominant effect of Na in mixed Na/Ca heparin and by a surprisingly masked effect of paramagnetic ions although an apparent specific binding of paramagnetic Cu(II) shows up more clearly in 1-H nmr spectra (Rej et al 1990). 205-Tl nmr shows a relatively uninformative distorted single broad absorption indicating a poorly resolved distribution of cation environments. Solid samples studied by ir provide evidence of cation-specific interaction with clear dependence of the importance of cation-dependent water binding under these conditions. Binding of heparin to basic polypeptides in hydrophobic media show the importance of glue-like activities of water molecules associated with the cation/polyanion complex (Grant et al 1992). 8b HEPARAN SULPHATE, BRAIN DEVELOPMENT, FUNCTION & NEURODEGENERATIVE DISEASES INCLUDING ALZHEIMER'S DISEASE (AD).

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8b.-1 Introduction. Heparan sulphate proteoglycans, nitric oxide (Salvemini et al 1998), and perhaps redox systems which will include Zn2+ effects (perturbed by Al3+ ?) ions are implicated in the pathogenesis of AD and related diseases as well as in normal memory function. A chronic inflammatory state of the brain may occur in AD and might explain why patients on anti-inflammatory drug therapy for rheumatoid arthritis appear less susceptible to AD (McGeer et al 1990) agreeing with a likely involvement of immunologically produced nitric oxide/nitrous acid /redox metal heparan sulphate damage in AD. Inappropriate modulation of heparan sulphate by nitric oxide metabolites influenced by trace metal ions (perhaps including inappropriate initiation of polyanion phase change as discussed in the previous section) might instigate neurological malfunction and contribute e.g. to the formation of amyloid deposits in various amyloidoses which have the common non-covalently linked basement membrane heparan sulphate PG component (Narindrasorasak et al 1991). Neutralization of inappropriately formed nitric oxide generated nitrous acid by ascorbate reduction or by sacrificial deaminative cleavage of glucosamines may provide essential protection against such effects. Iron (iron ions are especially potent potential redox and phase change damaging agents) is reported to be selectively accumulated with aluminium in neurofibrillar tangles (NFI) of AD (Perl et al 1992). Membrane spanning heparan sulphate proteoglycans are thought likely to be important regulators of activity-dependent modulation of neuronal connectivity (Laun et al 1999) perhaps linking cytoskeletal elements with the pericellular environment. Nitrous acid also can potentially disrupt neurotransmitter monoamine oxidase activity directly deaminately cleaving dopamine and its metabolites and cause DNA cross-linking (Kirchner & Hopkins 1991). Liver amyloid was reported to show a very high level of de-N-sulphonated heparan sulphate chains absent from other heparan sulphates (Linker & Hovingh 1977) allowing uncontrolled nitrous acid cleavage. 8b-2. Heparin/Heparan Sulphate in AD. Fukuchi et al 1998 recently reviewed the involvement of heparin/heparan sulphate (in beta-amyloid protein, neurite plaque, NFT tau protein fibril formation and ApoE4 lipid processing) in AD

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but did not deal with the effects of nitric oxide and its metabolites in this disease which is now suggested (as a main theme of these notes) to damage heparan sulphate molecules under conditions pertaining to such diseases. 8.b-2-1. Learning and Heparin/Heparan Sulphates. Nitric oxide neurotransmitter activity may be involved in learning (Hawkins et al 1998, cf also Breen 1992, Fukuchi et al 1998). N-CAM glycoprotein function in synaptic connectivity was found to be heparin-dependent (Bullock & Rose 1992) (Cole et al 1986) with a possible memory function release of heparan sulphate (Sugara & Der 1994); injection of heparin into the adult rat hippocampus induced seizures (Mudher et al 1998); the heparan sulphate proteoglycan syndecan-3 was implicated in neuron extracellular matrix interaction in the cellular mechanism responsible for synaptic plasticity, associated with cortactin src type and fyn tyrosine kinases, playing a crucial role in long term potentiation (LTP) (enzymic cleavage of heparan sulphate as well as the addition of heparin prevented development of LTP in rat hippocampal cells studied by Laun et al, 1999); heparan sulphate may also provide resevoirs for growth factors, zinc and neurotransmitters. 8.b-2-2. Zinc in AD. Zinc is a potential modulator of heparin/heparan sulphate activity. Zinc is an intracellular messenger causing protein kinase C (PK) to translocate to polymerised actin components in the cytoskeleton (Zalewski et al 1990). A neurological function in sustained cellular responses involves PKC is long-term potentiation of synaptic transmission in the hippocampus (Ackers et al 1986); this activity is also heparan sulphate dependent (Laun et al 1999). Phosphorylation of cytoskeletal elements modulatable by heparin/heparan suphate may be implicated in short-term memory. Involvement of zinc APP-heparan sulphate interactions (Multhaup 1994) in AD therapy (Masters et al 1993) suggest possible modulation of APP activity by heparin/zinc. Zinc supplementation could apparently impair congnitive function in AD patients, restorable on cessation of the zinc supplementation (cf Smith et al 1994). The reported anti-heparinase activity of Zn2+ (cf Patent Spec RO96508) needs to be investigated further, but indicates a possible role of Zn2+ in the modulation of heparin/heparan sulphate signalling via alteration of its degradation rate.

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Are there linkages between Zn2+ - heparin/heparan, Zn2+- ascorbate, Zn2+- finger linked biochemistry Zn in SOD (Cu,Zn SOD), and effects of Zn2+ on NO biochemistry? Zinc disturbance in AD may include a function of specialized Zn2+-GAG binding (Grant et al 1992f). Zn2+ may counter a neurological protection afforded by heparin (considered to be against proteolytic cleavage of APP, the integral trans-membrane protein that is released from cells in culture following proteolytic cleavage) but nitric oxide related mechanisms might also be relevant. Selective Zn2+ binding to heparin (e.g. Woodhead et al 1986), an unusual proton binding effect produced uniquely by Zn2+ with heparin (Grant et al 1992f) (and perhaps a role in proton conduction) may have relevance for zinc-dependent heparan sulphate processing. Zn2+ catalysis of de-N-sulphonation of heparin by co-binding of metals and the role of Zn fingers in proteins in modulation of gene expression may be part of overall Zn2+ controlled mechanisms which include, at some stage, heparan sulphate-linked redox ascorbate and nitric oxide biochemistry and nitric oxide modulation of metal-protein binding (cf Draper et al 1996). Zn2+ at 50 nM (the effect saturated at 70 micro M) promoted heparin binding to amyloid precursor protein APP and abolished a protective effect of heparin against its proteolytic cleavage. Zinc dietary supplementation was reported to impair cognitive function in AD patients (Masters et al (1993). Heparan sulphate was separated from other GAGs by its greater readiness to form Zn2+ cross- linked phospholipid precipitates (Wu & Cohen 1984). Parrish & Fair (1981) reported that Zn2+ was bound to heparin but less readily by chondroitin 4 and 6 sulphates, dermatan sulphate or hydaluronic acid. NMR studies by Whitfield & Sarkar (1991) suggested that Zn2+-carboxyl interactions controlled the conformation of the iduronate rings of heparin monosaccharides and Li+ ions appear similarly to affect iduronate conformations in heparin preparations (Grant et al 1991; perhaps these observations are relevant to heparan sulphate mechanisms in neurological physiology. It might further be hypothesized that similar effects might include nucleation of phase change related to cycloskeletal elements involved neurological function and in memory function, further modulatable by nitric oxide. 8.b-2-3 Cognitive Function in AD.

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Therapeutic Use of Heparin/Heparan Sulphate A heparan sulphate containing GAG preparation ('Ateroid') has been reported by Ferrero et al (1989) to improve cognitive function in AD patients. A mechanism which might be relevant to such effects by blocking by exogenous heparin of heparan sulphate beta amyloid peptide interaction was suggested by Leveugle et al (1994). Vascular malfunction involving heparin/heparan sulphate and nitric oxide is also possibly involved the aetiology of neurodegenerative diseases. Heparin induced endothelial cell cytoskeletal reorganization was suggested to be a possible mechanism for vascular relaxation (Mandil et al 1995) and nexin-2 an isoform of the AD amyloid beta protein precursor secreted in large quantities by platelets upon vascular injury may be potentiated by heparin/heparan sulphate. Glia-derived nexin-1 binds to heparin (Rovelle et al 1992). The nmr spectrum of heparin was perturbed by Gd3+ binding in a less selective manner than by Cu2+ (Rej et al 1990). Cu in heparin may contribute both to its SOD and angiogenic activity (Raju et al 1982). 8.b-2-4. Neurite Outgrowth Stimulation by Heparin/Heparan Sulphate. Neurite outgrowth is stimulated by heparin (e.g. Sedden et al 1994) and heparan sulphate (Dow et al 1992). A heparin-binding domain in APP is also thought to be involved in regulation of neurite outgrowth (Smith et al 1994) in which there is a likely autocrine role of heparan sulphate proteoglycans (Dow & Riolelle 1992) perhaps via a thrombospondin route (O'Rouke et al 1992). 8b-2-4-1. Possible Heparin/Heparan Sulphate-Dependent Growth Factor Effects in AD. Since the brain has long been recognized as a plentiful source of mitogenic acitivity and is a rich source of FGFs (Gimenea-Gallego et al 1985) subject to modulation by heparan sulphate (or by exogenous heparin), heparan sulphate proteoglycans are likely also to control bFGF activity in developing brain (Hondermark et al 1992) and be of relevance to neurodegenerative disease as is implied by the usefulness of a bFGF binding assay (Peiry et al 1992) to study the incidence of heparan sulphate in plaques in AD and other neurodegenerative diseases. Regulation of nerve growth factor (NGF) (is likely to be heparan sulphate-dependent (Damon et al 1992). Cellular therapy to achieve NGF replacement has been proposed for AD therapy (Hawkins,

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press report 2000). NGF was reported to change the invasive properties of neuro-ectoderm melanoma cells, possibly dependent on heparanase activity (Maschetti et al 1999); such activity, as discussed above, may also be Zn2+-dependent. 8.b-2-5. Extracted Brain Heparan Sulphate - Lack of Correlation with AD. Although brain heparan sulphates differed from those of other organs (Lindahl et al 1995), little difference was detected between the sugar composition and partial sequences of heparan sulphates from AD and control brains, indicating that the amount and primary microstructure of heparan sulphates may not contribute directly to AD; however the complexity of heparan sulphate biochemistry, involving the possibility of multiple cation and anion binding as well as multi-phase behaviour, does not rule out some future establishment of a direct heparan sulphate (supramolecular) structural link with neurodegnerative disease pocesses. It is suggested that, in addition to the polysaccharide microstructure, the inorganic content of the normal and AD heparan sulphates needs to be examined by e.g. spark source mass spectroscopy. Of particular interest is aluminium and silicon contents since there are putative links of these with the disease. (Cf silicic acid has been suggested to exert a protective role in aluminium neurotoxicity cf Birchall & Chappell 1989). 8.b-2-6. Possible Transferrin Receptor Function, Aluminium & Heparin/Heparan Sulphate in AD. Transferrin receptors which contain heparan sulphate side chains (Fransson et al, cf Gallagher et al 1986 and Hu & Reogoeczi 1992) are 67-Ga3+ markers for brain aluminium transport and coincide with the areas of the brain vulnerable to AD (although unaltered in abundance in AD) (Edwardson et al 1990). 67-Ga3+ binding which was much more strongly bound to heparan sulphate than to other GAGs (Kojima et al 1983) likely mimicking effects of Al3+. Al3+ is also implicated in dialysis dementia and Guam disease. Al3+ may inhibit calcium-mediated proteolysis of cytoskeletal proteins and induces NF to form complexes thought to be via phosphate ester binding (Nixon et al, 1990). Cis-aconitate is thought be a likely pathological carrier of Al in AD and Al-citrate backs up transferrin and ferritin binding (Lehmann 1992). 8.b-2-7.

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Fenton Reaction /Free-Radical Damage in Neurodegenerative Diseases. Complex interactions involving heparin/heparan sulphate are seen to be involved directly and indirectly in control of both redox balance, antioxidant and other enzymic activity. Redox balance of which glutathione, ascorbate, nitric oxide and multiple antioxidant activity is part, is suggested as a theme of these notes to be a dominant factor in heparin/heparan sulphate biochemistry, pro-disease, abnormal heparan sulphate arising from its perturbation which include abnormal iron, aluminium and silicon loading which could adversely affect the pro-antioxidant activity and proteolytic modulatory actions of heparin/heparan sulphate. Al3+ directly deactivates SOD (Shainkin-Kestenbaum et al 1989) and brain proteases (Nixon et al 1990) and perhaps other relevant Ca2+, Cu(II) and Zn2+ dependent mechanisms. In AD, Down's syndrome, amylotrophic lateral sclerosis, Parkinson's and Guam dementias (Leveugle et al 1994), affected neurons apparently over-synthesise lactoferrin (involved in iron and aluminium transport). Antioxidant SOD genetic insufficiency has been identified in the familial form of amylotropic lateral sclerosis (Rosen et al 1993, cf Calder et al 1995), in agreement with an augmented superoxide initiated damage involving reactive nitrogen species including peroxynitrite (Cookson & Shaw 1999, cf Love 1999 who reviewed oxidative stress in brain ischemia). Iron, phosphate (and aluminium) intoxication effects, however, may also be implicated in the above disease processes. As discussed in section 3.5 above, reactive oxygen free-radicals generated by iron-Fenton reactions are likely to damage GAGs. Further evidence of this is provided by Nagasawa et al 1992 who observed such damage to hyaluronic acid and heparin/heparan sulphate in less sulphated regions. Another example is the Cu(I) hydroxyl radical formation damage to hyaluronic acid in metallosis of the eye (Sterk et al 1985). 8c. Supramoleular Structure of Heparin/Heparan Sulphate PGs and their Binding Potential. Ca2+ (Liang et al 1982, Dais et al 1987) and Zn2+ binding the heparin may be critically dependent on the cooperative effects of supramolecular structure and arrayed N-sulphonate groups (Grant et al 1992c,f) to which crosslinking by small amounts of e.g. Al3+ could lead to disruption of the requirements for protein folding and binding.

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Nitric oxide damage to heparan sulphates in neurodegenerative disease may enhance such inappropriate binding activities. 8c-1. Silicates in GAGs. Silicates have been found to be associated with aluminium in AD plaques (Edwardson et al 1990). Although aluminium and silicate involvement in AD may be related to Zn2+ or Ca2+ homeostasis, silicon is an essential nutrient although the details of its biochemistry are still obscure and (inorganic) silicates are believed to occur in normal association with glycoaminoglycans (Iler,1979). It is conceivable that silicate acquisition may be a normal function of GAGs. The effect, if any, of Al3+ on this is worthy of study. Differences in the amounts of bound silicates and phosphates by glycosaminoglycans from different species organs and diseases and work-up procedures might have contributed to difficulties in comparing metal ion binding and other activities of such GAG preparations. 9. Involvement of Heparin/Heparan Sulphate in Modulation of Prion Protein Activity. Prion proteins have a heparin/heparan sulphate perspective (by differentially binding to heparin-like molecules, the synthesis and metabolic fate of prion proteins in scrapie infected cells is inhibited by reversing their phenotype back to normal (Gabizone et al 1993)). Part of the normal cellular function of prion proteins may be conceivably of relevance to memory function as they are evidently implicated in neurodegenerative diseases, principally scrapie but also Kuru, Creutzfeld-Jacob and Gerstmann-Straussler-Scheinkler diseases (Prusnier 1995). The anti-scrapie effect of heparin and related molecules was established by workers (Diringer & Ehlers 1991) who do not, however, agree with the prion hypothesis (Diringer 1985). A further factor which does not seem to have been considered is the effect of non-physiological metal intoxication of heparin/heparan sulphate from the perspective of prion protein biochemistry. 10. WATER BINDING TO HEPARIN/HEPARAN SULPHATE. The number of water molecules, strongly bound to sulphate half-ester groups of heparin was found to the highly dependent on the counterions present, averaging six for sodium, three for potassium and one for calcium. A quasi phase change mechanism of counter ion binding to heparin/heparan sulphate may have relevance to switchable ion and proton pumping. Commercial sulphonated

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polymers find use as proton conductors, such activity being dependent on cation-dependent water binding to the arrays of sulphonate groups (Colomban 1992) which is analogous to a similar effect of counterions on the stoichiometry of water molecule binding to sulphate half-ester groups in highly sulphated heparin-like molecules. Hydration changes may also be relevant to the coil conformation changes thought to be responsible for signalling by sodium/calcium - heparan sulphate chains in the proposed mechanism of sensing of blood flow in vivo (Siegel et al 1998) and perhaps other servo-control mechanisms, evidently employing potentially involving heparan sulphate conformation -dependent effects, such as control of calcium/heparin binding sites in parathyroid cells (Takeuchi et al 1990). 10a HOFMEISTER EFFECTS ON WATER STRUCTURE The Hofmeister series ranks the relative protein denaturation abilities of aqueous salt solutions. Water aggregate structures present in various metal salt solutions, deducible from near infrared spectroscopy, also varied with the Hofmeister series (Luck 1969) indicating that the ionic content of aqueous solutions affects the aggregation state of hydrogen bonded water molecules thought to be composed, at physiological temperature, of several hundred H2O units but salt ions 'melt' these aggregates according to their Hofmeister activities. Flickering clusters of such labile hydrogen-bonded arrays may also behave in a determinisitic chaotic manner, e.g. being influenced by phase boundary conditions which although poorly understood, may be relevant to biology (which usually restricts such considerations to hydrophobicity indices) and even to the mechanism of memory storage and retrieval. Heparinized surfaces may create bio-friendly non-denaturing surfaces by providing correct hydrophilicity/hydrophobicity in addition to providing anticoagulant protection as well as binding sites for antioxidants and growth factors. Heparin/heparan sulphate, as well as other biological polyanions, can be considered to provide Hofmeister-series activity modulation through Hofmeister-effect-empowered-hydration. This is thought to be related to the established morphological role of heparin/heparan sulphate and related polysaccharides in calcification and to a hypothetical role in e.g. cytoskeletal controls. The entropic driven properties of quasi phase change behaviour (such consideration are also perhaps relevant to the reversible binding and diffusion of bFGF on HSPGs of basement membranes

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(Dowd et al 1999) and evently an unconventional discussion of water structure as an information carrier in organisms (Schwabl 1994) are possibly related the the unusual thermodynamic properties of water-rich gels perhaps underlying the evolution of sulphated polysaccharides for flexible ligand binding, the basis of developmental biology (Comper 1994). Another example of such gel effect is the intestinal mucous coat with an unstirred water layer (Smithson et al 1981). 11. MODULATION OF CRYSTALLIZATION BY HEPARIN/HEPARAN SULPHATE. A biological function of glycosaminoglycans and related polysaccharides in calcification has been well established. A similar morphological role is possible with other biological mineral and protein structures. This includes cytoskeletal element dependent fundamental cell activity such as of the mitotic spindle (Roussel et al 1990) and perhaps also involves the nucleus (Bhavanandan & Davidson 1975). Seeded calcite crystallisation studies demonstrated that lower molecular weight or de-N-sulphonated heparins (such as might be produced as a result of nitric oxide/nitrous acid reaction) were much less effective calcification inhibitors than intact highly sulphated polymers (Grant et al 1989b). A pro-disease effect of pathological in vitro crystallisation and its prevention by heparin-like molecules has been discussed by Grant et al (1992b). The ability of structurally sound heparan sulphates, but not pathologically altered heparan sulphates, both to inhibit crystallisation and to act as seeds for the induction of correct protein folding could be related phenomena. Rate determining processes during crystallization may be dependent on relative hydration energies in transition states and related cation and hydration-dependent polysaccharides may provide enthalpic driven proximity and flexibility for ligand binding requirements of multicellular organisms, which is at the basis of developmental biology (Comper 1994) and presently hypothesized to the an important fundement of the pathology of degenerative diseases and autoimmune processes. Foreign particles (such as asbestos fibres) or abnormal solids (e.g. amyloid fibres) may provoke immunological reaction including nitric oxide/nitrite formation unless adequately deactivated by suitable biopolymers including heparan sulphate which may restore an acceptably inert structure.

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12. ESTABLISHED LABORATORY USE OF NITROUS ACID FOR HEPARIN/HEPARAN SULPHATE ANALYSIS Nitrous acid has long been known to be a useful organic chemical reagent, employed by synthetic organic chemists to allow accurately defined molecular transformations to be achieved, e.g. yielding alcohols from aliphatic primary amines, diazo compounds from aromatic primary amines and nitrosoamines from secondary amines. Prior to the current awareness of nitric oxide and its metabolites as being highly pertinent to biochemistry and medicine, the high specificity of the nitrous acid deaminative cleavage of heparan sulphates encouraged chemists to employ this reaction to confirm the presence or absence of heparin/heparan sulphates in biological samples and to further characterise the fragments obtained by gel filtration etc. The reaction is normally employed to allow scission of the N-sulphonated glucosamines leaving the N-acetyl glucosamines intact. Hydrazine treatment, however, selectively de-N-acetylates which when followed by nitrous acid treatment at pH 4, provides further heparan sulphate fragments for analysis. 13 BINDING OF PATHOGENS TO HEPARAN SULPHATE Such binding is common for many pathogens allowing therapeutic intervention to be achieved by heparin and heparinoids by blocking the heparin binding sites and by other mechanisms including inhibition of reverse transcriptases. Helicobacter pylori (among the many heparan sulphate binding organisms) has a marked pro-cancer effect, enhanced in cyclotoxin associated antigen CagA strains. Exposure of gastric epithelial cells to H pylori induced activation of the transcription factor protein 1 and activation of the proto-oncogenes c fos and c jun. This might be a crucial step for the induction of neoplasia (Meyer-Ter-Veh et al 2000). Heparin/heparan sulphate biochemistry might also be implicated in H pylori induction of cancer e.g. through perturbation of growth factors e.g. by the displacement of FGFs from heparan sulphate PG sites by bacterial heparan sulphate binding peptides (Ascencio et al 1995). Anti-proliferative effects of heparin on c fos and c jun in arterial walls appeared to be indirect perhaps via alteration of other cell cycle events (CA 120 124557c).

14.

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REFERENCES. Vide infra).

15. ACKNOWLEDEMENTS Grateful thanks are due to Professor KEL McColl and Ms J Grant (Glasgow), Drs Roger Worthington, Frank Williamson and Bill Long (Aberdeen) as well as to the Carnegie Foundation for the Universities of Scotland, for assistance. Spark source mass spectrometric results were kindly provided by Dr Bacon (Macaulay Inst Aberdeen) originally at the instigation of Colin Moffat (Aberdeen). * Mass spectroscopic results are given thus : (parts per million by weight (ppm) in untreated heparin/ppm in heparin after cation exchange on a Tl+ loaded Amberlite IR 120 column).

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References

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cancer in the US has decreased by 75% since 1930..(due to) increased use of domestic refrigerators and decreased reliance on nitrates to preserved bacon and other meat foods. Vitamin C prevents nitrosoamine formation and it is reasonable to assume that the trend towards an increasing consumption of fruits and vegetables rich in vitamin C is also responsible for the downturn in the incidence of stomach cancer. The mortality from stomach cancer in Japan is the highest in the world being eight times that in the US (partly related to nitrite effect?) cf also Anon Chem in Brit 1987 (Dec) 1158 Microwaved bacon has less nitrosamines than fried bacon since the nitrite formation of nitrosoamines is optimum at 185C) Campbell JH Rennick RE Kalevitch SG Campbell GR (1992) Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype Exp Cell Res 299 156-167 Candy JM Oakley AE McArthur FK Taylor GA Mountfort SH Edwardson JA (1994) Microanalytical and molecular approaches to the study of aluminium in relationship to Alzheimer's disease Life Chem Rep 11(1) 55-69 CA 121 285822s Cardenas A Bernard A Lauwerys R (1992) Incorporation of [35-S]sulfate into glomerular membranes of rats chronically exposed to cadmium and its relation with urinary glycosaminoglycans and proteinuria Toxicology 76 (3) 219 CA 118 75023y Heparan sulphate - Cd2+ interaction Cardin AD Weintraub HJR (1989) Molecular modeling of protein-glycosaminoglycan interactions Arteriosclerosis 9(1) 21-32 Consensus sequence for GAG recognition X-BB-X-B-X and X-BBB-X-X-B-X where B is a basic and X is a hydrophobic residue Castillo GM Templeton DM (1992) Structure and metabolism of multiple heparan sulphate proteoglycans synthesized by the isolated rat glomerulus Biochim Biophys Acta 1136 119-128 Cathcart RF (1991) A unique function for ascorbate Medical Hypotheses 35 32-37 Massive ascorbate doses reverse the usual direction of the ascorbate glutathione redox couple so that GSSG is reduced to GSH Massive ascorbate doses can substitute for NADPH Cerutti (1985) Science 277 375 Cu ascorbate DNA damage Challis BC Edwards A Hunma RR Kyrtopoulos SA Outram JR (1978) IARC Sci. Publ. 19 (Environ. Aspects N-Nitroso Compd.) 127-142 CA 89 196583x Fe and Cu etc catalysis of nitrosamine formation Chiarugi VP Vannuchi S Urbano P (1974) Biochimica et Biophysica Acta 345 283-293 Exposure of trypsin-removable sulphated poyanions on the surface of normal and virally transformed BHK(21/C13) cells

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Carcinogenesis 17(3)559 Ascorbyl radicals detected for first time in human gastric mucosa are increased in H pylori infection Drake S Klea DJ Michelson DJ Oegema TR Furcht LT McCarthy JB (1992) J Cell Biol 117(6)1331 Heparan S and not its core protein of PI anchored HSPG initiates mouse melanoma cell adhesion to fibronectin Draper et al (1996) BioEssays 18(1) 549 Modulation of nitric oxide by metal protein regulatory activities Drzeniek Z Stocker G Siebertz B Just U Schroeder T Ostertag W Haubeck H-D (1999) Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells Blood 93(9) 2884-2897 Duthie GG (1992) Food Sci Technol 298) 21 CA 116 171158e Dziarski R (1989) Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin J Immunol 143 365 Dziarski R (1992) Synergistic enhancement of T-cell responses and IL1 receptor expression by IL1 and heparin and dextran sulphate Cell Immunol 145(1) 100 CA 118 57833b Edelberg JM Pizzo SV (1990) Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator mediated plasminogen activation Biochemistry 29 2509-5911 Edgar JA (1970) Nature (London) 227 24 Dehydroascorbic acid and cell division cf Schapiro HM (1972) J Surg Res 13 138 (redox balance in the body: an approach to quantitation) Edwardson JA Oakley AE Pullen RGL McArthur FK Morris CM Taylor GA Candy JM (1990) Aluminium in Food and the Environment p20-36 Ed R Massey D Taylor (Royal Society of Chemistry Special Publication No 73 Proc symposium (Environment and Food Chemistry Groups of the Industrial Division of the Royal Society of Chemistry, London, 17 May 1988)). Other contributions to this symposium are relevant to discussion of possible health risks of aluminium. Elenius et al (1992) J Biol Chem 267 (9) 6435 CA 116 208559k Emeleus HJ Anderson JS (1946) Modern Aspects of Inorganic Chemistry George Routledge & Sons London cf p78, p430 Engelberg H (1999) Actions of heparin that may affect the malignant process Cancer (NY) 85(2) 257-272 243 ref review. Anti-cancer effects include mestatasis/angiogenesis inhibition of vascular endothelial gf tissue and platelet activating factors anti-thrombin (effect on NK cell activity tumours/fibrin formation) tumour cell adhesion to vascular endothelium selectin chemokine activation oncogenic viruses matrix metalloproteinases, serine proteinases heparinases blocks oncogenic activation by ornithine decarboxylase

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polypeptides of protoheparan sulfate from human skin fibroblasts and the transferrin receptor may be identical or closely similar Fransson L-Å Coster L Carlstedt I Malmstrom A (1985) Domain structure of proteoheparan sulphate from confluent cultures of human embryonic skin fibroblasts Biochem J 231 683-687 Fransson L-Å Havsmark B (1982) CA 98 16981e Carbohydr Res 110(1) 135 Modification of heparan sulphate by N-desulphonation and N-acetylation abolished chain-chain interaction Correct secondary structure is required for self-association of heparan sulphate Fransson L-Å (1989) (Heparin/heparan sulphate may act as biological cation 'conveyor belt') personal communication Fredj-Reygrobellit D et al (1994) Opthalmic Res 26(6) 325 CA 122 230721c Heparan sulphate analog sulphonate for wound healing with bFGF in eye Frege J et al (1990) (cf Susheela HK et al refs) Effect of fluoride on bone GAG Frenette GP Ruddon RW Krzesicki RF Naser JA Peters BP (1989) J Biol Chem 264 3078-3088 (Role of ascorbate in hydroxylation of proline residues and in secretion of type IV collagen) Fritze LMS Reilly CF Rosenberg RD (1985) An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells J Cell Biol 199 1041-1049 Fritz TA et al (1994) J Biol Chem 269(46)28809 CA 121 249190e Mn required for one GlcNAc transferase for heparan sulphate PG synthesis Fujiwara Y Kaji J (1999) Toxicology 133(2,3) 159 CA 131 154569 Pb alters HSPG biosynthesis Fukushi Ki Hart M Li L (1998) Alzheimers disease and heparan sulfate proteoglycan Frontiers in Bioscience (on line computer file) 3 d327-337 CA 129 3327e Available URL:http://www.bioscience.org/1998/v3/d/fuchuchi/d327-337.htm Fukushima S Imaida K Sakata T Okamura T Shibata M Ito N (1983) Cancer Res 43 4454 Sodium ascorbate is a promotor in rat bladder carcinogenesis initiated by N-Bu-N-(4-OH-Bu)nitrosamine; Fukushima S Shibata M-A Shirai T Kurata Y Tamano S Imaida K (1987) ibid 47 4821-24 Ascorbic acid + K carbonate promotes cancer; Fukushima S Ogiso T Kurata Y Shibata M-A Kakizoe T (1987) Cancer Lett 35 17-25 Calcium ascorbate or fatty acid ascorbate esters do not promote cancer from Hayatsu H et al (1988) Mutation Res 202 429

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Gabizone R et al (1993) Heparin like molecules bind differentially to prion proteins and change their intracellular metabolic fate J Cell Physiol 157(2) 317 CA 120 28472a Gallagher JT Walker A (1985) Molecular distinctions between heparan sulphate and heparin Biochem J 230 665-674 Polysaccharide structures analysed by complete nitrous acid degradation Gallagher JT Lyon M Steward WP (1986) Structure and function of heparan sulphate proteoglycans Biochem J 236 313-325 Gallagher JT Turnbull JE Lyon M (1992) Patterns of sulphation of heparan sulphate Int J Biochem 24(4) 553 CA 117 42780u Gallagher JT Turnbull JE (1992) Glycobiology 2(6)523 CA 118 161146x Review (58 ref) plasma membrane HSPGs and their role in regulating activity of bFGFs Garner D 1999 RSC Annual Conf report by D Bradley in The Alchemist (Chemweb News RoundUp Fungal trap 10 sept 1999) Ghael S Mileva M Dweck HS Rosenfeld L (1997) Biochem Mol Biol Int 183 CA 128 39743j Nitric oxide -> nitrous acid degradation heparan sulphate at pH 5 Gilat D Hershkoviz R Goldkorn I Cahalon L Korner G Vlodavsky I Lider O (1995) Molecular behaviour adaps to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T cell adhesion molecule, depending on the local pH J Exp Med 181(5) 1929 CA 122 288881w Heparanase acts either as a pro adhesive mol e.g. for CD4+ h lymphocytes or degrades ECM Gimenez-Gallego G Rodkey J Bennett C Rois-Candelore M DiSalvo J Thomas K (1985) Brain-derived acidic fibrobalst growth factor: complete amino acid sequence and homologies Science 230 1385-1388 Gitay-Goren H Soker S Vlodavski J Neufeld G (1992) CHECK Vascular EGF - heparin interaction local pH dependent (cf also effect of Ni(II) in EGF effect in malignant transformation (Molerup et al 1996) perhaps GAG-related?) Gleizes P-E Noaillac-Depeyre J Amalric F Gas N (1995) Basic fibroblast growth factor FGF-2 internalisation through the heparan sulfate proteoglycans-mediated pathway: An ultrastructural approach Eur J Cell Biol 66(1) 47-59 CA122 205582a bFGF internalisation via heparan sulphate PG Goedert M Jakes R Spillantini MG Hasegawa M Smith MJ Crowther RA (1996) Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans Nature 383 550-553

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Gomez-Pinnilla F et al (1995) Regulation of astrocyte proliferation by FGF2 and heparan sulphate in vivo J Neurosci 15 3(Pt1) 2021 CA 122 205604j Goralska M Harned J Fleisher LN McGahan MC (1998) The effect of ascorbic acid and ferric ammonium citrate on iron uptake and storage in lens epithelial cells Exp Eye Res 66(6) 687-97 Grant D Long WF Williamson FB (1985) Role of GAGs in cellular adhesion of relevance to the cancer state Biochem Soc Trans 13 cf also ibid 16 1029 Grant D Long WF Williamson FB (1987a) Infrared spectroscopy of heparin-cation complexes Biochem J 244 143-149 Grant D Long WF Williamson FB (1987b) Pericellular heparans may contribute to the protection of cells from free radicals Med Hypotheses 23(1) 67-71 Grant D Long WF Williamson FB (1988) Effect of heparin on dismutation of superoxide anion Biochem Soc Trans 16 1030-1031 Grant D Long WF Williamson FB (1989a) A comparison of the antioxidant requirements of proteins with those of synthetic polymers suggests an antioxidant function for clusters of aromatic and bivalent sulphur-containing amino acid residues Medical Hypotheses 28 245-253 Grant D Long WF Williamson FB (1989b) Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization Biochem J 259 41-45 CA 110 171240x Grant D Long WF Williamson FB (1990) The dependence on counter-cation of the degree of hydration of heparin Biochem Soc Trans 18(6) 1283-1284 Grant D Long WF Moffat CF Williamson FB (1991) Infrared spectroscopy of heparins suggests that the region 750-950cm-1 is sensitive to changes in iduronate residue ring conformation Biochem J 275 193-197 Grant D Long WF Williamson FB (1991) Heparin-polypeptide interaction Biochem J 277 569-571 Grant D Long WF Williamson FB (1992a) Near and fundamental region IR spectroscopy of heparan sulphates isolated from the surface of normal and transformed fibroblasts Making Light Work Adv Near Infrared Spectrosc Int Conf Near Infrared Spectrosc 1991 (pub 1992) 633 Ed I Murray IA Cowe VCA Weinheim Germany CA 118 164498z Grant D Long WF Williamson FB (1992b) Degenerative and inflammatory diseases may result from defects in antimineralization mechansims afforded by glycosaminoglycans Medical Hypotheses 38 49-55

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Grant D Long WF Moffat CF Wiliamson FB (1992c) A study of Ca2+-heparin complex-formation by polarimetry Biochem J 282 601-604 Grant D Long WF Moffat CF Williamson FB (1992d) Cu(2+)-heparin interaction studied by polarimetry Biochem J 283 243-346 Grant D Long WF Williamson FB (1992e) A potentiometric titration study of the interaction of heparin with metal cations Biochem J 285 477-480 Grant D Long WF Williamson FB (1992f) Zn2+-heparin interaction studied by potentiometric titration Biochem J 287(3) 849-53 CA 117 226049u Phase change mechanism; reversal of the initial pH change Grant D (1993) Disfunction of sulphated polysaccharides in degenerative diseases (Report to the Carnegie Trust for the Universities of Scotland, 24/8/93) Grant D (1996) (unpublished ir studies of heparin de-N sulphonation cataysed by iron and copper ions; this produces heparin with GlcNH2 primed for rapid nitrous acid degradation) Grant D Long WF Williamson FB (unpublished) Relative pH decrease; heparin + cation: Mg2+ 1, Mn2+ 1.14, Fe2+1.34, Ca2+ 1.75, Cu2+ 3.0, Fe3+ 3.24, Al3+ 4.54 Grant D Long WF Moffat CF Williamson FB (unpublished) Anion content of heparin (Glaxo 008) from spark source mass spectrometry (with thanks to J Bacon, Macaulay Inst) results ppm, (and after pasage through cation exhange resin column in parenthesis) Silicate etc as Si 5900(100), Cl 5600(1000), F 890(4), phosphate etc as P 440(30) Br 130(0.7), arsenate etc as As 15(1), I 10(0) cf Grant D (2000) Chemweb preprint server biochemistry paper submitted (Multi-ion content of heparin SUMMARY Spark source mass spectrometry (SSMS) of a (sodium) heparin reveals the presence of 38 additional counterions; comparison of their amounts before and after cation exchange treatment allows residual binding strengths to be classified as: small amounts of relatively strongly held K, phosphorus (likely as phosphate), Ni, Co, Zn, Cr, and Ag as well as Pb and Sn; somewhat less strongly held were more abundant Mg, Fe and Cu. Non-physiological elements included difficult-to-remove Sr, La and Ce. Such sequestration of small amounts of large numbers of ions to heparin/heparan sulphate suggests possible physiological and pathological significance for cellular nutrition, ionic transport and detoxification). Grimm J Keller R de Groot PG (1988) Lamellar flow induces cell polarity and leads to rearrangement of proteoglycan metabolism in endothelial cells Thrombosis and Haemostatsis 60(3) 437-441

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culture Toxicology 68 249-257 Pb decreases heparan sulphate biosynthesis cf Fujiwara & Kaji (1999) Karlsson K Marklund SL (1988) Extracellular superoxide dismutase association with cell surface bound sulphated glycosaminoglycans Basic Life Sci 49 (Oxygen Radicals in Biol Med) 647-50 CA 111 131597b cf Biochem J 242 55 Karlinsky JB Rounds S Farber HW (1992) Circ Res 71(4) 782 CA 118 78363v Effects of hypoxia on heparan sulphate in bovine aortic and pulmonary artery endothelial cells (Hypoxia [3% oxygen] induced decreased sulphation of heparan sulphate but increased amount of AT(III) sites; higher molecular weight anticoagualnt active heparan sulphates were synthesized under hypoxic conditions) Kery V Orlovska M Stancikova M Risko M Zlnay D (1992) Urinary glycosaminoglycan excertion in rheumatic diseases Clin Chem 38(6) 841-846 Urinary GAGs det by dimethyl methylene blue but not alcian blue highly diagnositc for rheumatic diseases RA>OA> ankylosing spondylosus> normal patients Kimura I Nagaura T Naitoh T Kobayashi S Kimura M (1992) Jpn J Pharmacol 60(4) 369-75 CA 118 73444n Heparin inhibits the progressive phase of sub-culture endothelial cell proliferation in rat aorta Kinnuren T Lindahl U et al (1996) J Biol Chem 271(43) 2243 Kirchner JJ Hopkins PB (1991) Nitrous acid cross-links duplex DNA fragments through deoxyguanosine residues at the sequence 5'-CG Kjellén L Lindahl U (1991) Annu Rev Biochem 60 443-465 Kjellén L Lindahl U (1991) proteoglycans: structures and interactions Annu Rev Biochem 60 443-475 Lists proposed functions of proteoglycans including negative charge, mechanical support, regulation of cell migration and aggregation, role in development and stabilisation of synaptic structure, endothelial regeneration, stabilisation of basement membranes, modulation of collagem fibrillogenesis including transparency of cornea, regulation of cell growth, urinary trypsin inhibition, provision of filtration barrier, role in morphogenesis, link to cytoskelaton and ECM, mediation of adhesion and morphogenesis, assembly of matrix phosphatidyl-inositol linkage, resevior for bFGF, regulation of blood coagulation, mediation of transferrin, uptake role in antigen presentation Kjellén L Pettersson I Unger E Lindahl U (1992) Two enzymes in one: N-deacetylation and N-sulfation in heparin biosynthesis are catalysed by the same protein Adv Exp Med Biol 313 (Heparin Relat Polysaccharides) 107-11 CA 118 75739m

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Van den Born J et al (1995) Diabetologia 38(2) 161 CA 236801a Glomerular HSPG proteinurea Van den Born J et al (1996) J Biol Chem 271(37) 22802 CA 125 272673j NAc domains Ab HSPGs disease cf E coli capsular polysaccharide [GlcUA->GlcNAc]n Van der Voorst R et al (1999) Heparan sulphate modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase cMet J Biol Chem 274(10) 6499 CA 130 336182h Van Wazer JR (1958) Phosphorus and its Compounds Vol I Interscience New York Van Wazer (1962) Structural re-organization through ligand exchange American Scientist 50 450-472 Venktaraman G et al (1996) PNAS USA CHECK bFGF dimerization - heparan sulphate Vilar RE Ghael D Li M Bhagat DD Arrigo LM Cowman MK Dweck HS Rosenfeld L (1997) Nitric oxide degradation of heparin and heparan sulphate (both nitrous acid and nitric oxide degrade heparin; buffer dependence of nitric oxide degradation at physiol pH suggests unidentified catalyst or inhibitor) Biochem J 324 473-497 Vlodavsky T (CHECK) et al (1995) Invasion Metastasis 14 (1-6) 290 cf Kaatz BZ (1995) ibid 276 Vlodavsky I Michaeli IR Mohzen M Bar-Shavit R Catane R Elere HPT (1992) Adv Exp Med Biol 315 (heparin and related polysaccharides) 317 CA 117 225598k Walker A Gallagher J (1996) Biochem J 317(3) 871 CA 125 188757d Structural domains of heparan sulphate for specific recognition of the C terminal heparin binding domain of human plasma fibronectin (HEP II) Warrell RP Jr Murphy WK Schulman P O'Dwer PJC Helller G (1991) J Clin Oncol 9(8) 1467-75 Randomized-double blind gallium nitrate compared with etridronate for acute control of cancer related hypercalcaemia Warren C Manley (1984) Measurement of heparan sulphate in normal human serum by laser nephelometry Clin Chim Acta 137(3) 355 CA 100 188260v Heparan sulphate in blood serum of healthy humans was 1.6mg/L Wanake Shinker A (1994) CHECK Heparin binding growth factors in neural development and injury Kenkyu No Shinpo 38(6) 896 CA 122 130206v Pleiptrophin and midkine synthesized in neurons, mainly localized to the cellular suface, may function as ECM molecules in an autocrine and/or paracrine fashion, upregulated in injury

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Weksler BB (1990) Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostacyclin H synthase and prostacyclin synthase J Cell Physiol 142 514-522 Wells CF Salam MA (1968) The kinetics of the reaction of chromium(II) with hydrazine, hydroxylamine and hydrazoid acid in perchlorate media: the formation of halegeno- and sulphato- complexes of chromium(II) J Chem Soc (A) 1568-1575 Cr(II) causes formation of N-centred free radicals and Fenton like chemistry Wei W et al (1999) CA 130 309799h Overexpression of nitric oxide synthase I related to lymph node metastasis in non small cell lung carcinoma Wessel HP et al (CHECK) (1998) Carbohydr Mimics 417-431 Ed Y Chapleux Wiley VCH Verlag GmbH Weinheim Germany CA 130 182657c From GAGs to heparinoid mimetics with antiproliferative activity (Review with 37 refs on the preparation of heparinoid mimetics as anti-tumour and smooth muscle cell inhibitors) Willenbourg DO Parish CR (1988) Inhibition of allergic encephalomyelitis in rats by treatment with sulfated polysaccharides J Immunol 140 3401-3405 Whitfield DM Sarkar B (1992) Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation Biopolymers 32 597-619 Whitfield DM Sarkar B (1992) Metal binding to heparin monosaccharides: D-glucoasamine-6-sulphate, D-glucuronic acid, and L-iduronic acid J Inorg Biochemistry 41 157-170 Williamson FB (1989) personal communication Williamson MF Long WF Williamson FB (1992) The effect of heparin on the UV absorption properties of Fe(II) and Fe(III) Biochem Soc Trans 20(4) 360s Winterbourne DJ Mora PT (1981) Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulphate with reduced degree of sulfation J Biol Chem 256(9) 4310-4320 Wong SF Halliwell B Richmond R Skowroneck WR (1981) The role of superoxide and hydroxyl radicals in the degradaton of hyaluronic acid induced by metal ions and by ascorbic acid J Inorg Biochem 14(2)127-34 Ascorbic-induced depolymerization is inhibited by desferrioxamine, catalase and scavengers of hydroxyl radical Woodhead NE Long WF Williamson FB (1986) Heparan sulphates from fibroblasts exhibiting a temperature-dependent transformed growth trait IRCS Med Sci 14 427-428 cf Biochem Soc Trans 12(2)

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300 CA 100 189661p (chemical transformation affected by N-methyl-N'-nitro-N-nitroguanidine) Woodhead NE Long WF Williamson FB (1986) Binding of zinc ions to heparin Biochem J 237 281-284 Wrenchall LE Cerra FB Singh RK Platt JL (1995) Heparan sulfate initiates signals in murine macrophages leading to divergent biological outcomes J Immunol 154(2) 871-80 CA 122 79080v Heparan sulphate modulates cellular immunological macrophages with cytokine signalling PGE2, IL6 activation of tyrosine kinase, nuclear factor kappa B, protein kinase C Wright TC Jr Castellot JJ Petitou M Lormeau J-C Choay J Karnovsky MJ (1989) Structural determinants of heparin's growth inhibitory activity Interdependence of oligosaccharide size and charge J Biol Chem 264(3) 1534-1542 2-O sulphate not required for antiproliferative activity Wu Van-Yu Cohen MP (1984) A competitive binding assay for measurement of heparan sulphate in tissue digests Anal Biochem 139 218-223 WuDunn D Spear PG (1989) Initial interaction of Herpes Simplex virus with cells is binding to heparan sulphate J Virol 63(1) 52-58 Wunde A et al (1995) Thromb Res 78(2)139 CA 122 255935p Anti-atheroma effect of heparin Xu G Yuan M-B Zhong X-J Fu S-Ji Tang S-L Zhao L Sun Y-P Jian P (1998) Huaren Xiaohua Zashi 6(2) 125-127 CA 130 294939c Polyamines increase with Helicobactor pylori infection in stomach (Perhaps some nitrous acid degradation of relevance to cancer?) Yanagishita M (1992) Metabolism of plasma membrane associated heparan sulphate proteoglycans Adv Exp Med Biol 313 (Heparin related polysaccharides) 113 Yanagishita M (1992) Glycosylphosphatidylinositiol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotoic and intracellular degradative pathways J Biol Chem 267(14) 9505-9511 Yanagishita M Hascall VC (1992) Cell surface heparan sulfate proteoglycans J Biol Chem 267(14) 9451-9454 Yang L Yang Y-C (1995) Heparin inhibits the expression of IL11 and granulocyte-macrophage colony-stimulating factor in primate bone marrow stromal fibroblasts through mRNA destabilization Blood 86(6) 2526 CA 123 246454z Mechanism for regulating cytokines via heparin induced mRNA degradation Yoshizumi M Kourembanas S Temizer DH Cambria RP Quertermous T Lee M-E (1992) Tumor necrosis factor increases transcription of the heparin-binding epidermal growth factor-like

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growth factor gene in vascular endothelial cells J Biol Chem 267(14) 9467-9469 Zalewski PD et al (1990) Synergy between zinc and phorbol ester in translocation of protein kinase C-V to cytoskeleton FEBS Lett 273(1-2) 131-4 4 Zhang G Li B Fan J Feng S (1997) Determination of trace thiocyanate in body fluids by a kinetic fluorimetric method Talanta 44 1141-1147 Zhang L Keuchi Y Lu J Rosenberg RD (1999) Anticoagulant heparan sulphate precursor structure in F9 embryonal carcinoma cells J Biol Chem 274(9) 5681 CA 120 323265n Demonstrates for the first time the processing assembly mechanism of specific sequences in heparan sulphate Structure differences around potential 3-O sulphate acceptors in the active and inactive anticoagulant versions of heparan sulphate Zou S Magura CE Hurley WL (1992) Heparin-binding properties of lactoferrin and lysozyme Comp Biochem Physiol 103B(4) 889-895 Binding of biotinylated heparin to lactoferrin and lysozyme in presence of Na, Ca, Cu, Zn, Fe(II) and Fe(III) showed complex buffer, pH and especially cation dependent behaviour. Heparin binding to lactoferrin unaffected by less than 10 microM Zn or Cu but was enhanced at Cu of 100 microM and inhibited above 500 microM and was inhibited at all Zn > 10 microM; Fe(III) was inhibitory at all concentrations but Fe(II) enhanced binding slightly at 10 microM. May be of significance for effect of e.g. iron overload on antimicrobial propeof studied proteins or modulation of heparin/ heparan -------------

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