mitochondria paper

8/10/2019 Mitochondria Paper

1/23

The Structural and Functional

Characterization of

Mitochondria and Lysosomes

Through the Use of Dyes

Matt Granger

6819400

Dr. Erwin HuebnerBIOL 4560

Microtechnique


2/23

1

Mitochondria

The discovery of mitochondria in animals is credited to Richard Altmann, who in 1890

gave them the name bioblasts.1See figure 6 for Altmann drawing. It was not until 1898 that

they were given the name mitochondria, when Carl Benda used the Greek terms mitos

meaning thread, and chondros meaning granule, in reference to the visual description of

mitochondria.1Mitochondria in plants would later be discovered by Friedrich Meves, when he

was studying the European waterlily, Nymphaea alba.2

One of the first stains used on mitochondria was by Leonor Michaelis, not yet famous

for his work with Maud Menten on enzyme kinetics, who would use Janus Green B to vitally

stain mitochondria.3,4

In 1910, Claudius Regaud would develop a staining method for

mitochondria which utilized hematoxylin with an iron alum.5In 1924, Bailey and Davis

developed a method to progressively stain mitochondria utilizing Regauds fluid, washing,

dehydrating, embedding in paraffin, and staining with Mallorys phosphotungstic acid-

hematoxylin dye.6This stain results in purple mitochondria against a red background.6

Construction of a model of how mitochondria function took many years to develop. The

function of one mitochondrial protein, succinate dehydrogenase, was discovered by Torsten

Thurnberg, through the observation of the colour change during methylene blue oxidation.7,8

The function of another mitochondrial enzyme, cytochrome oxidase, was discovered by Arnold

Lazarow and Sherwin Cooperstein through the use of Janus Green B, which is a leuco dye and

would stain mitochondria blue when oxidized.9The first work to bring together what would

become known as the electron transport chain was accomplished by Keilin in 1925 in his work

showing that cytochrome b was the first electron acceptor.10,11

Keilin and Hartree would have


3/23

2

the order of the respiratory chain enzymes figured out by 1939 when they were able to show

that cytochrome awas actually two

separate proteins.10,12

In 1952, George Palade was the

first to describe the inner foldings of the

mitochondria, known as cristae via the use

of an electron microscope.13

In 1963, Nass and Nass would use

osmium tetroxide to discover that

mitochondria contain DNA.14

Nearly 20

years later, in 1981, Anderson et. al, would

sequence the Human mitochondrial genome, and discover that it was 16569 base pairs in

length, and contained heavy and light strands.15

37 genes would be discovered on the

mitochondrial genome that code for 22 tRNAs, 12S and 16S ribosomal RNA, cytochrome c

oxidase subunits, and many other proteins.15,16

While many scientists, some described above, have been able to develop staining

procedures for the mitochondria, there have been challenges to overcome to maintain the

morphological and functional properties of mitochondria with respect to fixation and cell

death.

17

Pocernich and Butterfield were able to show that the use of acrolein as a fixative

resulted in a loss of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activity.18

While researching post-mortem changes in guinea pigs, Anniko and Bagger-Sjbck found that

Figure 1. Electron micrograph of mitochondria from

guinea pig pancreas showing the inner and outer

membrane. Image made available by James D. Jamieson

and the Department of Cell Biology, Yale University

School of Medicine.


4/23

3

the first identifiable signs of autolysis in mitochondria in hair cells began to occur within 10 to

15 minutes after sacrifice.19

Many features and functions of the mitochondria have been able to be characterized

through the use of dyes, such as morphology, which can vary in different tissues from being

ovoid or spherical in hepatocytes of rats, to becoming tubular in fibroblasts in order to protect

themselves from autophagocytosis.20,21

Mitochondrial functions can also be analyzed through

the behaviour of dyes, such as leuco dyes that only exhibit colour when exposed to changes in

light, pH, concentration, temperature.22

Some of these dyes and how they have helped

researchers investigate the mitochondria will be

explored below.

Janus Green B

Janus Green B can be used as a vital stain, and

is made up of a diethylsafranine molecule connected

to a dimethylaniline molecule by way of an Azo

bond.23

Janus Green B is capable of undergoing multiple reduction reactions when kept in an

anaerobic environment, resulting in the production of violet, yellow, and red dye derivatives.23

Selective staining of mitochondria from pancreatic tissue requires an environment that is

partially anaerobic and which results in some the loss of some of the stain, as Lazarow and

Cooperstein observed in 1953 that only mitochondria near the periphery of the tissue had been

selectively stained blue.23

In 1969, through the use of spectrophotometric methods, Udvardy

and Holland determined that over 90% of Janus Green B was bound to coenzyme Q, a

lipoprotein that has the function of shuttling electrons between complex I and complex II in the

Figure 2. Janus Green B molecule. Courtesy

of Sigma Aldrich.

http://www.sigmaaldrich.com/catalog/pro

duct/aldrich/201677?lang=en&region=CA


5/23

4

electron transport chain.24, 25

Kensuke Baba developed a method to selectively injure

mitochondria by using a ruby laser to radiate HeLa cells stained with Janus Green B.26

Storb et.

al also used ruby lasers and Janus Green B to damage mitochondria from epithelial and baby

hamster kidney cells and determined that mitochondria were able to recover and reverse the

damage within 4 hours if the damage was not too severe.27

Gomori trichrome stain

The ingredients that the Gomori trichrome stain utilizes are chromotrope 2R,

phosphotungstic acid, and either fast green, light green or aniline blue, mixed in diluted glacial

acetic acid.28

Gomoris trichrome stain will result in nuclei being stained black, collagen being

stained either green or blue, and cytoplasm, keratin and muscle fibers being stained red.28

The

Gomori trichrome stain is a stain that has found use in histopathological analysis of

mitochondrial diseases.29

Gomori trichrome stain has also been useful in the visualizing of

dysfunctional astrocytes that are found in the hypothalamus and are associated with increased

lipid-binding proteins that may be associated with Alzheimersdisease.30When used to stain

muscle fibers, this trichrome stain shows what is known as red ragged fibers, where there is

high subsarcolemmal mitochondrial density due to abnormal cytochrome C oxidase function.31

Red ragged fibers tend to occur most often in type 1

muscle fiber.32

See Figure 7 for an image of the Gomori

trichrome stain showing this condition.

Methylene Blue

A stain that has actually been used as a form of

treatment for mitochondrial dysfunction due to

Figure 3. Structure of Methylene Blue.

Image courtesy of Sigma-Aldrich Co.

http://www.sigmaaldrich.com/catalog/pr

oduct/sial/m9140?lang=en&region=CA


6/23

5

Alzheimers disease is methylene blue (MB), as it is capable of crossing the blood-brain

barrier.33,34

A proposed mechanism that enables methylene blue to alleviate mitochondrial

dysfunction is thought to be from the ability of methylene blue to be reduced to MB by NADPH,

and subsequently be oxidized to MBH2by cyctochrome

coxidase.35

The result of this is a decrease in

superoxide radicals, and an increase both cellular

oxygen consumption and the number of cytochrome c

oxidase enzymes.35,36

Like JC-1, methylene blue

accumulation varies depending on membrane

potential, and is photochemically inactive when the

membrane is hyperpolarized.37

Rhodamine

Rhodamine-123 (R123) is another fluorescent dye that can be used to stain

mitochondria.38Many variations of rhodamine have been developed, such as Rho B, Rho 6G,

Rho 19, Rho 101, Rho 110, Rho 116.39

A derivative of Rho 19, C4R1, has been shown to

uncouple mitochondrial resulting in the stimulation of respiration and a decrease in

mitochondrial membrane potential.40

C4R1 also successfully prevented oxidative stress in the

brain after reperfusion.40

R123 has enabled changes in mitochondrial membrane potential to be

studied as R123 is positively charged and will accumulate in the mitochondria when the interior

is more negatively charged, or hyperpolarized.41

As mitochondria become depolarized,

fluorescence from R123 will be diminished.41

One Rhodamine derivative, dihydrorhodamine 6G

Figure 4. Structure of Rhodamine-123.

Courtesy of Thermo Fisher Scientific Inc.

http://www.lifetechnologies.com/order/c

atalog/product/R302


7/23

6

(DHR-6G), is capable of being oxidized to rhodamine 6G (R6G) and fluorescing when oxidized by

reactive oxygen species from cigarette smoke.42

JC-1

JC-1 is another dye that has enabled the study of mitochondrial membrane potential as

it is capable of changing colour with changes in pH, being orange when there is a high

membrane potential, and being green when there is a low membrane potential, this has made

it a useful stain for use in flow cytometry.43,44

However, there are issues with JC-1 as it is also

capable of changing from green to red as its concentration increases, so depending on how it is

used, concentration needs to be monitored closely.41

Diaminobenzidine

Diaminobenzidine is a stain that can be used specifically for cytochrome c oxidase, thus

enabling the determination of the location of the enzyme within mitochondria when analyzed

by an electron microscope.45

Seligman et. al utilized diaminobenzidine to determine that

cytochrome c oxidase was located on the outer leaflet

of the mitochondrial inner membrane facing the

mitochondrial matrix.46

Acridine Orange

10-N-nonyl acridine orange (NAO) is a cationic,

fluorescent dye that can be used to stain mitochondria

in live cells by binding to the anionic phosholipid, cardiolipin.47,48

Through the use of NAO,

researchers were able to determine the percent of cardiolipin in the of the outer leaflets of the

mitochondrial inner membrane to be approximately 57%.49

NAO has also been able to be

Figure 5. Structure of Acridine orange.

Image courtesy of Thermo Fisher

Scientific Inc.

http://www.lifetechnologies.com/order/c

atalog/product/A1301


8/23


9/23

8

Figure 7. Gomori trichrome stain. Normal muscle fibers on the

left. Red ragged muscle fibers on the right. Image courtesy of

Peter Takizawa, Director of Medical Studies, Department of Cell

Biology, Yale School of Medicine.


10/23

9

Lysosomes

In 1967, Christian de Duve had a paper published in Subcellular Particles, describing a

new organelle called the lysosome, which were thought to differ from mitochondria in that

they contained acid phosphatases, and were responsible for the acid hydrolysis of

materials.55,56

Through the use of injecting bacterial endotoxins fromA. aerogenes into rabbits,

Weissmann and Thomas were able to study the release of acid hydrolases from lysosomes.57

Lysosomes play a role in helping to create antigens that will be presented to the immune

system, thus enabling lymphocytes to target foreign bodies.58

Lysosomes also play a role in

autophagy where they fuse with phagosomes to degrade materials; this function may serve

play a part in neuroprotective mechanisms.59

Weissmann also noted that lysosome morphology

may change depending on whether there was the state of disease or not.60

Cancerous cells

have been observed to have altered levels of lysosomal enzymes such as cathepsins, which

have been visualized with GFP; and acid phosphatases, have been visualized with Nile blue

dye.61,62,63,64While these cancer-related lysosomal changes can result in dysfunctional

lysosomal enzymatic behavior, because of this there is also an opportunity to use these changes

to target malignant cells.65

Long-lived post-mitotic cells such as cardiomyocytes and neurons

are subject to lysosomal lipofuscin accumulation where processes such as autophagy of

mitochondria are disrupted resulting in the production of excessive reactive oxygen species,

which can result in senescence and cell death.66According to Staretz-Chacham et. al there are

over 50 different lysosomal storage disorders (LSDs), which have an combined incidence rate

approximately as high as 1 in 1500 live births, to as low as 1 in 7000.67

Most of these LSDs are a

result of dysfunctional lysosomal acid hydrolysis of molecules, and their subsequent


11/23

10

accumulation that can eventually result in organ failure.68,69

Many of these LSDs do not have

any forms of treatment available, or are difficult to treat because of their location, such as the

nervous system.68,70

Lysosomal autophagy dysfunction and subsequent autophagosome

accumulation has also been implicated in Alzheimers and Parkinsons disease,

respectively.71,72,73

For these reasons, the study of LSDs and lysosomes are of particular

importance.

Niemann-Pick Disease

Niemann-Pick type C is a lysosomal disorder due to a mutation in NPC1 or NPC2 genes

that results in cholesterol, gangliosides, and bis-monoacylgylcerol phosphate accumulating in

lysosomes, resulting in dysfunction.74

Techniques have been develop which make use of

recombinant antibodies, and immunofluorescent substrates, often of a proprietary blend,

lysosomal disease morphology can be imaged. The accumulated cholesterol in the lysosome has

been a target for characterizing Niemann-Pick through the use of filipin staining, as well as

utilizing a bacterial toxin that is capable of binding to cholesterol, perfringolysin O.75

Kwiatkowska et. al utilized a recombinant probe

consisting of perfringolysin O (PFO) and glutathione S

transferase (GST) that was successful in indicating the

cholesterol deposits that are common in Niemann-Pick

disease.

75

anti-GST IgY-peroxidase antibodies from

chickens were then bound to the recombinant probe and

visualized with SuperSignal West Pico Substrate.75

See

figure 9 for image of GST-PFO stain. A fluorescent

Figure 9. Lysosomal cholesterol labelled

with GST-PFO-antibody complex and

visualized with SuperSignal West Pico

Substrate.75


12/23

11

antibiotic, filipin, can also be used to stain lysosomal cholesterol from fibroblasts in Niemann-

Pick disease, to show the accumulation of cholesterol in neuron cell bodies and glia.76

Detection

of Niemann-Pick cholesterol deposits in lysosomes from blood smears have also been

successfully stained with filipin.77

However, this method has proven cumbersome because

fibroblasts must first be cultured, so Wakida et. al developed a technique that is much faster

through the utilization of bone marrow smears as a source for lysosomal cholesterol to perform

the filipin stain.78

See figure 10 below for image of filipin stain.

Rhodamine and Fluorescent Dextrans

A rhodamine based probe was developed by Lu et. al that responded to changes in pH,

and when exposed to an alkaline environment is colorless, but when the pH becomes acidic the

probe begins to fluoresce pink, thus enabling real-time observation of lysosomal pH changes.79

Use of an elastin-rhodamine complex was used to observe the activity of a lysosomal enzyme

known as elastase, from Human granulocytes.80

Another pH sensitive rhodamine probe that

was bound to morpholine was created by Shi et. al, that was able to show lysosomal when

induced by chloroquine.81

Other rhodamine-morpholine probes that become fluorescent after

cleavage by caspase-3 in order to observe apoptosis in cells have also been developed and were

useful both in vitro and in vivo.82

Kang-Kang et. al developed two rhodamine-based probes that

were showed no cytotoxicity, and were sensitive to changes in pH at 4.79, and 5.23,

respectively.

83

Fluorescent dextrans have succesfully used as a pH monitor in lysosomes, and

were able to further analyze that the pH of new phagosomes was alkaline, despite an

environmental pH of 6.5 and lysosomal fusion.84

Simultaneous delivery of sulforhodamine and

fluorescent dextrans together to an organelle were used to observe that cargo selection to


13/23

12

lysosomes is a selective process as the two compounds were delivered to lysosomes at

different rates, despite the same initial time of entry.85

Wang and Goren believed that the

temporal difference may have been due to the gelling of the dextrans, which may have

impeded lysosomal movement.85

Mycobactrium avium is capable of surviving inside lysosomes,

in order to take advantage of this for study, Oh and Straubinger labelled the surface of M.

aviumwith rhodamine and carboxyfluorescein to observe if M. aviumchanges the pH of

lysosomes in order to survive.86

Through the use of the properties of rhodamine, which is a pH

insensitive dye, while carboxyfluorescein is pH sensitive, and analysis of their fluorescent ratios,

Oh and Straubinger were able to determine that M. aviumwere exposed to a pH of

approximately 5.7.86

Ohkuma and Poole used fluorescent dextrans to observe typical

macrophage pH to be in the range of 4.7-4.8.87

Fluorescent dextrans have been successfully

used to observe M. tuberculosis interfere with lysosomal pH has in as well.88

Luzio et. al used

endosomes containing either rhodamine-dextran and Oregon-Green-dextran to observe

homotypic and heterotypic SNARE fusion via confocal microscopy.89Hasegawa et. al modified

rhodamine by linking it to cyclodextrin, making two hybrids that were sensitive to changes in

pH.90

Koshkaryev modified liposomes with octadecyl-rhodamine B, and used them to deliver a

fluorescent dextrin to lysosomes as a proof-of-concept in treating LSDs, and to demonstrate

that octadecyl-rhodamine B can improve delivery to dysfunctional lysosomes.91

From the same

lab as Koshkaryev, Thekkedeth et. al also successfully have used octadeceyl-rhodamine B to

improve delivery of a therapeutic dose of glucocerebroside to a dysfunctional Gaucher cell in

vitro which resultedin an increased amount of the therapeutic reaching the targeted

lysosomes.92

In 2011, a rhodamine derivative has also been fused to a lactam-ethylenediamine


14/23

13

to behave in a pH-sensitive manner that was also resistant to photo-bleaching by Wu et. al.93

Again in 2011, from the same lab, Li et. al developed a rhodamine-lactam hybrid that was

bound to mesoporous silica nanoparticles in order to observe lysosomal pH changes when

analyzed by flow cytometry.94

Glunde et. al developed a compound, 6'-O-lissamine-rhodamine

B-glucosamine, to fluorescently label lysosomes in order to understand the role they may play

in breast cancer metastasis.95

With this compound they were able to observe the co-localization

of 6'-O-lissamine-rhodamine B-glucosamine with lysosome associated proteins 1 and 2, as well

as CD63.95

Hama et. al developed a method utilizing a avidin-rhodamine X hybrid, where the

probe is activated only after endocytosis and subsequent lysosomal degradation.96

Using this

method, they were able to minimize background noise, and fluorescent pinpoint cancerous

microfoci within mouse peritoneal ovaries.96

Acridine orange

Through the use of acridine orange Urashima et. al were able to stain leukemic cells,

resulting in the cells staining orange or green which correlated to the metabolic activity of the

cells, with the orange cells containing more RNA, and having an improved prognosis of acute

lymphoblastic leukemia.97

Pittis et. al used acridine orange as a fluorescent marker to indicate

phagolysosomal fusion in patients with thalassemia major, and to show the efficacy of the

bacterial siderophore, deferoxamine, in restoring this loss-of-function in phagolysosomal fusion

in patients with thallasemia major.

98,99

Okamoto et. al were able to use pH-dependent

quenching of acridine orange to demonstrate that H+-ATPase isolated from rat liver can utilize

dATP as well as dGTP, and that Mn++ can result in 77% of the activity as seen when Mg++ is

used.100

Moorjani et. al measured the change in fluorescence of lysosomes labelled with


15/23


16/23

15

it is a suitable form of therapy.108

Kusuzaki et. al have also investigated the cytocidal induction

via visible light, or X-rays of musculoskeletal sarcomas treated with acridine orange due to non-

malignant cells being able to clear acridine orange due to not possessing an acidic environment

where cancerous cells have a growth advantage.109,110

They found that when acridine orange

accumulated in acidic organelles, like lysosomes, and when it was excited by by visible light or

radiation, that the excited acridine orange generated ozone from cytoplasmic oxygen.109,111

The

ozone will eventually rupture the lysosomes, resulting in the dumping of lysosomal enzymes,

and cell death.109

Research done by Zdolsek evaluated the effect that a variation in oxygen

levels had on the phototoxicity of acridine orange in lysosomes from mouse macrophages, as

well as the effect that sodium azide, a singlet oxygen quencher, to determine whether the

phototoxicity of acridine orange was due to the production of singlet oxygen or free radicals.112

Zdolsek found that elevated oxygen levels resulted in greater phototoxicity, and that cells

treated with sodium azide showed protection against irradiated acridine orange, indicating that

acridine orange produces singlet oxygen, as corroborated by Amagasa, Kobayashi and Ito, and

Suzuki et. al.112,113,114,115

The accumulation of acridine orange in musculoskeletal tumours was

studied with the intent of determining the mechanism of acridine orange accumulation in

musculoskeletal tumours.116

From observing the changes in extracellular and intracellular pH

via the fluorescence intensity of acridine orange, Matsubara et. al found that the accumulation

of acridine orange in cancerous cells was due to difference in intracellular pH and extracellular

or lysosomal pH.116

The antioxidative and lysosomotrophic properties of an acridine derivative,

9-amino-acridine-propanolol (9-AAP), were investigated in combination with acridine orange as

a fluorescent indicator.117

Dickens et. al found that when fluorescent cells were washed with 9-


17/23

16

AAP, that they became more dim, due to alkalization, and that 9-AAP may be useful in

protection against free radicals.117

The use of counting cells with antibodies has proven to be a

useful method, but it is also very time-consuming and expensive.118

Lovelace and Cahill

developed a faster, and less expensive method using acridine orange to as a highly specific

marker for counting microglia cells amongst astrocytes, due to the high lysosome content of

microglia and the selectivity of acridine orange for lysosomes.118

In 1992 Handrock et. al used

acridine derivatives have the ability to increase sulfated glycosaminoglycan storage in

lysosomes possibly due to their acridine ring.119

See figure 11 for image of Human fibroblasts

stained with

acridine orange.f120

Figure 11. Human fibroblasts stained with acridine orange to

show lysosomes.120

Figure 10. Lysosomal cholesterol from fibroblasts stained with filipin. Image

courtesy of Mayo Clinic. http://www.mayoclinic.org/medical-professionals/clinical-

updates/neurosciences/treating-rare-disease-niemann-pick-disease-type-c


18/23

17

References

1. Ernster, L.; Schatz, G. Mitochondria: a historical review. (1981) The Journal of cell biology vol. 91 (3 Pt 2) p.227s-255s

2. Meves, F. ber das Vorkommen von Mitochondrien bezw. Chondromiten in Pflanzenzellen. (1904). Ber. D.Deutsch. Bot. Ges. 22: 284286.

3. Bourne, G. Cytology and Cell Physiology. (2012) Elsevier Science p. 144. Michaelis, L.; Menten, M.L.; Johnson, K.A.; Goody, R.S. The original Michaelis constant: Translation of the 1913

Michaelis-Menten paper. (2011) Biochemistry vol. 50 (39) p. 8264-95. Gray, P. The microtomist's formulary and guide. (1954) Blakiston6. Bailey, P.; Davis, L.E. A Progressive Staining Method for Mitochondria.(1924) The Journal of medical research

vol. 44 (5) p. 535-538.1

7. Hederstedt, L.; Rutberg, L. Succinate dehydrogenase--a comparative review.(1981)Microbiological reviews vol.45 (4) p. 542-55

8. Thunberg, T. Studien ber die Beeinflussung des Gasaustausches des berlebenden Froschmuskels durchverschiedene Stoffe. (1909) Skandinavisches Archiv Fr Physiologie vol. 22 (2) p. 406-429

9. Lazarow, A.; Cooperstein, S.J. Studies on the enzymatic basic for the Janus green B staining reaction. (1953)Journal of Histochemistry & Cytochemistry vol. 1 (4) p. 234-241

10. Slater, E.C. Keilin, Cytochrome, and the Respiratory Chain. (2003) J. Biol. Chem. vol. 278 (19) p. 16455-1646111. Keilin D. (1925) Proc. R. Soc. Lond. B Biol. Sci. 98:312339.

12. Keilin D., Hartree E. F. (1939) Proc. R. Soc. Lond. B Biol. Sci. 127:167191.

13. Palade, G.E. The fine structure of mitochondria.Anat. Rec. 114, 427451 (1952)14. Nass, M.M; NASS, S. Intramitochondrial fibers with DNA characteristics. I. Fixation and electron staining

reactions.(1963) The Journal of cell biology vol. 19 p. 593-61115. Anderson, S.; Bankier, A.T.; Barrell, B.G.; de Bruijn, M.H.; Coulson, A.R.; Drouin, J.; Eperon, I.C; Nierlich, D.P.;

Roe, B.A.; Sanger, F.; Schreier, P.H.; Smith, A.J.; Staden, R.; Young, I.G. Sequence and organization of thehuman mitochondrial genome. (1981) Nature vol. 290 (5806) p. 457-65

16. Grossman L.I.; Shoubridge, E.A. Mitochondrial genetics and human disease. (1996) Bioessays 18: 983-91.17. Shen, Z.Y.; Shen, J.; Li, Q.S.; Chen, C.Y.; Chen, J.Y.; Yi, Z.Morphological and functional changes of mitochondria

in apoptotic esophageal carcinoma cells induced by arsenic trioxide.(2002) World journal ofgastroenterology: WJG vol. 8 (1) p. 31-5

18. Pocernich, C.B.; Butterfield, D.A. Acrolein inhibits NADH-linked mitochondrial enzyme activity: implicationsfor Alzheimer's disease. (2003) Neurotoxicity research vol. 5 (7) p. 515-20

19. Anniko, M.; Bagger-Sjbck, D. Early post-mortem change of the crista ampullaris. A light and electronmicroscopic study of the Guinea-pig. (1977) Virchows Archiv. B: Cell pathology vol. 25 (2) p. 137-49

20. Das, S.; Hajnoczky, N.; Antony, A.N.; Csordas G.; Gaspers, L.D.; Clemens, DL, Hoek, J.B.; Hajnoczky, G.

Mitochondrial morphology and dynamics in hepatocytes from normal and ethanol-fed rats.PflgersArch. 2012; 464:101109.

21. Rambold, A.S.; Kostelecky, B.; Elia, N.; Lippincott-Schwartz, J. Tubular network formation protectsmitochondria from autophagosomal degradation during nutrient starvation. (2011) Proceedings of theNational Academy of Sciences of the United States of America vol. 108 (25) p. 10190-5

22. Muthyala, R. Chemistry and Applications of Leuco Dyes. (1997) Springer Science & Business Media23. Lazarow, A.; Cooperstein, S.J. Studies on the mechanism of Janus Green B staining of mitochondria: I. Review

of the literature. (1953) Experimental Cell Research vol. 5 (1) p. 56-6924. Udvardy, A.; Holland, R.Disturbance of Energy Transfer as a Factor Promoting Changes in the BiologicBehavior of Tumor Cells. (1969) Cancer Res. vol. 29 (8) p. 1550-155

25. Turunen, M.; Olsson, J.; Dallner, G.Metabolism and function of coenzyme Q. (2004) Biochimica et biophysicaacta vol. 1660 (1-2) p. 171-99

26. Baba, K.SELECTIVE INJURY OF MITOCHONDRIA WITH JANUS GREEN B AND RUBY LASER LIGHT-ENZYMEMORPHOLOGICAL AND ULTRASTRUCTURAL STUDY. (1970) Pathology International vol. 20 (1) p. 59-78

27. Storb, R.; Amy, R.L.; Wertz, R.K.; Fauconnier, B.; Bessis, M. An electron microscopy study of vitally stainedsingle cells irradiated with a ruby laser microbeam. (1966) The Journal of cell biology vol. 31 (1) p. 11-29


19/23

18

28. Carson, F.L.; Hladik, C. Histotechnology: A Self-instructional Text.(2009) ASCP Press p. 165-16629. Olson, W.; Engel, W.K.; Walsh, G.O.; Einaugler, R. Oculocraniosomatic neuromuscular disease with "ragged-

red" fibers. (1972) Archives of neurology vol. 26 (3) p. 193-21130. Young, J.K.; Baker, J.H.; Muller, T. Immunoreactivity for brain-fatty acid binding protein in gomori-positive

astrocytes.(1996) Glia vol. 16 (3) p. 218-26

31. Reichmann, H.; Vogler, L.; Seibel, P. Ragged red or ragged blue fibers. (1996) European neurology vol. 36 (2) p.98-102

32. Attardi, G.Mitochondrial Biogenesis and Genetics, Part 2. (1996) Gulf Professional Publishing p. 51133. Mosconi, L.; de Leon, M.; Murray, J.; E, Lezi; Lu, J.; Javier E.; McHugh P.; Swerdlow, R.H.Reduced mitochondria

cytochrome oxidase activity in adult children of mothers with Alzheimer's disease. (2011) Journal ofAlzheimer's disease : JAD vol. 27 (3) p. 483-90

34 Peter C.; Hongwan D.; Kupfer A.; Lauterburg B.H. Pharmacokinetics and organ distribution of intravenous andoral methylene blue. (2000) Eur J Clin Pharmacol 56,247-250

35. Kelner, M.J.; Bagnell, R.; Hale, B.; Alexander, N.M.Methylene blue competes with paraquat for reduction byflavo-enzymes resulting in decreased superoxide production in the presence of heme proteins. (1988)Archives of biochemistry and biophysics vol. 262 (2) p. 422-6

36. Atamna, H.; Nguyen, A.; Schultz, C.; Boyle, K.; Newberry, J.; Kato H.; Ames B.N. Methylene blue delays cellularsenescence and enhances key mitochondrial biochemical pathways. (2008) FASEB journal : officialpublication of the Federation of American Societies for Experimental Biology vol. 22 (3) p. 703-12

37. Gabrielli, D.; Belisle, E.; Severino, D.; Kowaltowski, A.J.; Baptista, M.S.Binding, aggregation andphotochemical properties of methylene blue in mitochondrial suspensions. (2004) Photochemistry andphotobiology vol. 79 (3) p. 227-32

38. Baracca, A.; Sgarbi, G.; Solaini, G.; Lenaz, G. Rhodamine 123 as a probe of mitochondrial membrane potential:evaluation of proton flux through F(0) during ATP synthesis. (2003) Biochimica et biophysica acta vol.1606 (1-3) p. 137-46

39. Beija, M.; Afonso, C.A.M.; Martinho, J.M.G. Synthesis and applications of Rhodamine derivatives asfluorescent probes. (2009) Chemical Society Reviews vol. 38 (8) p. 2410-2433

40. Khailova, L.S.; Silachev, D.N.; Rokitskaya, T.I.; Avetisyan, A.V.; Lyamsaev, K.G.; Severina, I.I.; Il'yasova, T.M.;

Gulyaev, M.V.; Dedukhova, V.I. ; Trendeleva. T.A.; Plotnikov, E.Y. .; Zvyagilskaya, R.A.; Chernyak, B.V.;

Zorov, D.B.; Antonenko, Y.N.; Skulachev, V.P. A short-chain alkyl derivative of Rhodamine 19 acts as a

mild uncoupler of mitochondria and a neuroprotector. (2014) Biochimica et biophysica acta vol. 1837(10) p. 1739-47

41. Perry, S.W.; Norman, J.P.; Barbieri, J.; Brown, E.B.; Gelbard, H.A. Mitochondrial membrane potential probesand the proton gradient: a practical usage guide. (2011) BioTechniques vol. 50 (2) p. 98-115

42. Ou, B.; Huang, D.Fluorescent approach to quantitation of reactive oxygen species in mainstream cigarettesmoke. (2006) Analytical chemistry vol. 78 (9) p. 3097-103

43. Gravance, C.G.; Garner, D.L.; Baumber, J.; Ball, B.A. Assessment of equine sperm mitochondrial function usingJC-1. (2000) Theriogenology vol. 53 (9) p. 1691-703

44. Perelman, A.; Wachtel, C.; Cohen, M.; Haupt, S.; Shapiro, H.; Tzur A. JC-1: alternative excitation wavelengthsfacilitate mitochondrial membrane potential cytometry. (2012) Cell death & disease vol. 3 p. e430

45. Anderson, W.A.; Bara, G.; Seligman, A.M. The ultrastructural localization of cytochrome oxidase viacytochrome.(1975) The journal of histochemistry and cytochemistry : official journal of theHistochemistry Society vol. 23 (1) p. 13-20

46. Seligman, A.M.; Karnovsky, M.J.; Wasserkrug, H.L.; Hanker, J.S. Nondroplet ultrastructural demonstration ofcytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). (1968)The Journal of cell biology vol. 38 (1) p. 1-14

47. Garcia Fernandez, M.I.; Ceccarelli, D.; Muscatello, U.Use of the fluorescent dye 10-N-nonyl acridine orange inquantitative and location assays of cardiolipin: a study on different experimental models. (2004)Analytical biochemistry vol. 328 (2) p. 174-80

48. Erbrich, U.; Septinus, M.; Naujok, A.; Zimmermann, H.W. [Hydrophobic acridine dyes for fluorescence stainingof mitochondria in living cells. 2. Comparison of staining of living and fixed Hela-cells with NAO and


20/23

19

DPPAO].(1984) Histochemistry vol. 80 (4) p. 385-849. Petit, J.; Huet, O.; Gallet P.F.; Maftah, A.; Ratinaud, M.; Julien R.Direct analysis and significance of cardiolipin

transverse distribution in mitochondrial inner membranes. (1994) European Journal of Biochemistry vol.220 (3) p. 871-879

50. Zhang, G.; Kimura, S.; Murao, K.; Obata, K.; Matsuyoshi, H.; Takaki M. Inhibition of cytochrome c release by 10-

N-nonyl acridine orange, a cardiolipin-specific dye, during myocardial ischemia-reperfusion in the rat. (2010) American journal of physiology. Heart and circulatory physiology vol. 298 (2) p. H433-9

51. Gao, W.; Pu, Y.; Luo, K.Q.; Chang, D.C. Temporal relationship between cytochrome c release andmitochondrial swelling during UV-induced apoptosis in living HeLa cells. (2001) Journal of cell sciencevol. 114 (Pt 15) p. 2855-62

52. Rodriguez, M.E.; Azizuddin, K.; Zhang, P.; Chiu, S.; Lam, M.; Kenney M.E.; Burda C.; Oleinick N.L. Targeting ofmitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellularuptake and localization. (2008) Mitochondrion vol. 8 (3) p. 237-46

53. Jacobson, J.; Duchen, M.R.; Heales, S.J.R. Intracellular distribution of the fluorescent dye nonyl acridineorange responds to the mitochondrial membrane potential: implications for assays of cardiolipin andmitochondrial mass.(2002) Journal of Neurochemistry vol. 82 (2) p. 224-233

54. Erbrich, U.; Naujok, A.; Petschel, K.; Zimmermann, H W. [The fluorescent staining of mitochondria in livingHeLa- and LM-cells with new acridine dyes (author's transl)]. (1982) Histochemistry vol. 74 (1) p. 1-7

55. de Duve, C. Lysosomes: a new group of cytoplasmic particles, in Subcellular Particles, (T. Hayashi, editor), NewYork, Ronald Press Co., 1959, 128.

56. Hayashi, T. Subcellular particles. (1959) Ronald Press Co., New York57. Weissman, G.; Thomas, L.Studies on lysosomes. I. The effects of endotoxin, endotoxin tolerance, and

cortisone on the release of acid hydrolases from a granular fraction of rabbit liver. (1962) The Journal ofexperimental medicine vol. 116 p. 433-50

58. Lecker, S.H.; Goldberg, A.L.; Mitch, W.E. Protein Degradation by the Ubiquitin-Proteasome Pathway in Normaland Disease States. (2006) J. Am. Soc. Nephrol. vol. 17 (7) p. 1807-1819

59. Levine, B.; Kroemer, G.Autophagy in aging, disease and death: the true identity of a cell death impostor.(2009) Macmillan Publishers Limited vol. 16 (1) p. 1-2

60. Weissmann, G. The Role of Lysosomes in Inflammation and Disease. (2003) Annual Reviews 4139 El CaminoWay, P.O. Box 10139, Palo Alto, CA 94303-0139, USA

61. Mohamed, M.M.; Sloane, B.F. Cysteine cathepsins: multifunctional enzymes in cancer. (2006) Nature reviews.Cancer vol. 6 (10) p. 764-75

62. Linke, M.; Herzog, V.; Brix, K. Trafficking of lysosomal cathepsin B-green fluorescent protein to the surface ofthyroid epithelial cells involves the endosomal/lysosomal compartment. (2002) Journal of cell sciencevol. 115 (Pt 24) p. 4877-89

63. Dobrossy, L.; Pavelic, Z.P.; Vaughan, M.; Porter, N.; Bernacki, R.J.Elevation of lysosomal enzymes in primaryLewis lung tumor correlated with the initiation of metastasis. (1980) Cancer research vol. 40(9) p. 3281-5

64. Lin, C.W.; Shulok, J.R.; Kirley, S.D.; Cincotta, L.; Foley, J.W.Lysosomal localization and mechanism of uptake ofNile blue photosensitizers in tumor cells. (1991) Cancer research vol. 51 (10) p. 2710-9

65. Kallunki, T.; Olsen, O.D.; Jttel, M. Cancer-associated lysosomal changes: friends or foes? (2013) Oncogenevol. 32 (16) p. 1995-2004

66. Terman, A.; Kurz, T.; Navratil, M.; Arriaga, E.A.; Brunk, U.T.Mitochondrial turnover and aging of long-livedpostmitotic cells: the mitochondrial-lysosomal axis theory of aging. (2010) Antioxidants & redoxsignaling vol. 12 (4) p. 503-35

67. Staretz-Chacham, O.; Lang, T.C.; LaMarca, M.E.; Krasnewich, D.; Sidransky, E. Lysosomal storage disorders inthe newborn. (2009) Pediatrics vol. 123 (4) p. 1191-207

68. Vellodi, A.Lysosomal storage disorders. (2005) British journal of haematology vol. 128 (4) p. 413-3169. Greiner-Tollersrud, O.K.; Berg, T.Lysosomal Storage Disorders. (2000) Landes Bioscience70. Lavery, C. Role of patient support groups in lysosomal storage diseases. (2006) Oxford PharmaGenesis71. Nixon, R.A. The role of autophagy in neurodegenerative disease. (2013) Nature Medicine vol. 19 (8) p. 983-

997


21/23

20

72. Lee, J.H.; Yu, W.H.; Kumar, A.; Lee, S.; Mohan, P.S.; Peterhoff, C.M.; Wolfe, D.M.; Martinez-Vicente M.; Massey,

A.C.; Sovak, G.; Uchiyama, Y.; Westaway, D.; Cuervo, A.M.; Nixon, R.A. Lysosomal proteolysis andautophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. (2010) Cell vol.141 (7) p. 1146-58

73. Dehay, B.; Bov, J.; Rodrguez-Muela, N.; Perier, C.; Recasens, A.; Boya, P.; Vila M. Pathogenic lysosomal

depletion in Parkinson's disease. (2010) The Journal of neuroscience : the official journal of the Societyfor Neuroscience vol. 30 (37) p. 12535-44

74. Vance, J.E.Lipid imbalance in the neurological disorder, Niemann-Pick C disease. (2006) FEBS letters vol. 580(23) p. 5518-24

75. Kwiatkowska, K.; Marszalek-Sadowska, E.; Traczyk, G.; Koprowski, P.; Musielak, M.; ugowska, A.; Kulma, M.;

Grzelczyk, A.; Sobota, A. Visualization of cholesterol deposits in lysosomes of Niemann-Pick type Cfibroblasts using recombinant perfringolysin O. (2014) Orphanet Journal of Rare Diseases vol. 9 (1) p. 64

76. Vanier, M.Niemann-Pick disease type C. (2010) Orphanet Journal of Rare Diseases vol. 5 (1) p. 1677. Takamura, A.; Sakai, N.; Shinpoo, M.; Noguchi, A.; Takahashi, T.; Matsuda, S.; Yamamoto, M.; Narita, A.; Ohno,

K.; Ohashi, T.; Ida. H.; Eto, Y. The useful preliminary diagnosis of Niemann-Pick disease type C by filipintest in blood smear. (2013) Molecular genetics and metabolism vol. 110 (3) p. 401-4

78. Wakida, K.; Matsuyama, Z.; Suzuki, Y.; Sawada, M.; Tsurumi, H.; Kimura, A.; Hayashi, Y.; Hashizume, T.; Hozumi,

I.; Inuzuka T.[Diagnosis of adult type of Niemann-Pick disease (type C) in two brothers by filipin stainingof bone marrow smears]. (2004) N to shinkei = Brain and nerve vol. 56 (12) p. 1047-53

79. Lu, H.S.; Huang, S.Y.; Xu, Y.; Dai, X.; Miao, J.Y.; Zhao, B.X. A new fluorescent pH probe for imaging lysosomes inliving cells. (2014) Bioorganic & Medicinal Chemistry Letters vol. 24 (2) p. 535-538

80. Galdston, M.; Klein, N.; Rothstein, E. Human granulocyte lysosomal elastase activity using t-butyloxycarbonyl-L-alanine p-nitrophenyl ester and elastin-rhodamine as substrates. (1975) The American review ofrespiratory disease vol. 112 (5) p. 629-32

81. Shi, X.L.; Mao, G.J.; Zhang, X.B.; Liu, H.W.; Gong, Y.J.; Wu Y.X.; Zhou L.Y.; Zhang J.; Tan, W. Rhodamine-basedfluorescent probe for direct bio-imaging of lysosomal pH changes. (2014) Talanta vol. 130 p. 356-62

82. Wang, Z.Q.; Liao, J.; Diwu, Z.N-DEVD-N'-morpholinecarbonyl-rhodamine 110: novel caspase-3 fluorogenicsubstrates for cell-based apoptosis assay. (2005) Bioorganic & medicinal chemistry letters vol. 15 (9) p.2335-8

83. Kang-Kang, Y.; Kun L.; Ji-Ting H.; Hui-Huan Q.; Yong-Mei X.; Chen-Hui Q.; Xiao-Qi Y.Rhodamine-based

lysosome-targeted fluorescence probes: high pH sensitivity and their imaging application in living cells .(2014) RSC Advances vol. 4 (64) p. 33975-33980

84. Geisow, M.J.; D'Arcy Hart, P.; Young, M.R. Temporal changes of lysosome and phagosome pH duringphagolysosome formation in macrophages: studies by fluorescence spectroscopy. (1981) The Journal ofcell biology vol. 89 (3) p. 645-52

85. Wang, Y.L.; Goren, M.B. Differential and sequential delivery of fluorescent lysosomal probes intophagosomes in mouse peritoneal macrophages. (1987) The Journal of cell biology vol. 104 (6) p. 1749-54

86. Oh, Y.K.; Straubinger, R.M. Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometryto investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction. (1996) Infection and immunity vol. 64 (1) p. 319-25

87. Ohkuma, S.; Poole, B. Fluorescence probe measurement of the intralysosomal pH in living cells and theperturbation of pH by various agents. (1978) Proceedings of the National Academy of Sciences of theUnited States of America vol. 75 (7) p. 3327-31

88. Chicurel, M.; Garca, E; Goodsaid, F. Modulation of macrophage lysosomal pH by Mycobacteriumtuberculosis-derived proteins. (1988) Infection and immunity vol. 56 (2) p. 479-83

89. Luzio J.P.; Pryor, P.R.; Gray, S.R.; Gratian, M.J.; Piper, R.C.; Bright. N.A. Membrane traffic to and fromlysosomes. (2005) Biochemical Society symposium (72) p. 77-86

90. Hasegawa, T.; Kondo, Y.; Koizumi, Y.; Sugiyama, T.; Takeda, A.; Itoa, S.; Hamada, F. A highly sensitive probedetecting low pH area of HeLa cells based on rhodamine B modified beta-cyclodextrins. (2009)Bioorganic & medicinal chemistry vol. 17 (16) p. 6015-990.

91. Koshkaryev, A.; Thekkedath, R.; Pagano, C.; Meerovich, I.; Torchilin, V.P. Targeting of lysosomes by liposomes


22/23


23/23

22

110. Zheng, J. Energy metabolism of cancer: Glycolysis versus oxidative phosphorylation (Review). (2012)Oncology letters vol. 4 (6) p. 1151-1157

111. Kusuzaki, K.; Hosogi, S.; Ashihara, E.; Matsubara, T.; Satonaka, H.; Nakamura, T.; Matsumine, A.; Sudo, A.;

Uchida, A.; Murata, H.; Baldini, N.; Fais, S.; Marunaka, Y. Translational research of photodynamic therapywith acridine orange which targets cancer acidity. (2012) Current pharmaceutical design vol. 18 (10) p.

1414-20112. Zdolsek, J.M. Acridine orange-mediated photodamage to cultured cells. (1993) APMIS : acta pathologica,

microbiologica, et immunologica Scandinavica vol. 101 (2) p. 127-32

113. Amagasa, J. Mechanisms of photodynamic inactivation of acridine orange-sensitized transfer RNA:participation of singlet oxygen and base damage leading to inactivation. (1986) Journal of radiationresearch vol. 27 (4) p. 339-51

114. Kobayashi, K.; Ito, T. Wavelength department of singlet oxygen mechanism in acridine orange-sensitizedphotodynamic action in yeast cells: experiments with 470 nm. (1977) Photochemistry and photobiologyvol. 25 (4) p. 385-8

115. Kobayashi, K.; Ito, T. WAVELENGTH DEPENDENCE OF SINGLET OXYGEN MECHANISM IN ACRIDINE ORANGE-SENSITIZED PHOTODYNAMIC ACTION IN YEAST CELLS: EXPERIMENTS WITH 470 NM.(1977)Photochemistry and Photobiology vol. 25 (4) p. 385-388

116. Matsubara, T.; Kusuzaki, K.; Matsumine, A.; Shintani, K.; Satonaka, H.; Uchida, A. Acridine orange used forphotodynamic therapy accumulates in malignant musculoskeletal tumors depending on pH gradient.(2006) Anticancer research vol. 26 (1A) p. 187-93

117. Dickens, B.F.; Weglicki, W.B.; Boehme, P.A.; Mak, T.I.Antioxidant and lysosomotropic properties of acridine-propranolol: protection against oxidative endothelial cell injury. (2002) Journal of molecular and cellularcardiology vol. 34 (2) p. 129-37

118. Lovelace, M.D.; Cahill, D.M. A rapid cell counting method utilising acridine orange as a novel discriminatingmarker for both cultured astrocytes and microglia. (2007) Journal of Neuroscience Methods vol. 165 (2)p. 223-229

119. Handrock, K.; Laschke, A.; Lllmann-Rauch, R.; Vogt, R.D.; Ziegenhagen, M. Lysosomal storage of sulfatedglycosaminoglycans in cultured fibroblasts exposed to immunostimulatory acridine derivatives. (1992)Toxicology and applied pharmacology vol. 114 (2) p. 204-14

120. Dunlop, R.A.; Brunk, U.T.; Rodgers, K.J. Proteins containing oxidized amino acids induce apoptosis in human

monocytes. (2011) The Biochemical journal vol. 435 (1) p. 207-16

mitochondria paper

Documents