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Mitochondrial calcium uptake regulates cold preservation-induced Bax translocation and early reperfusion apoptosis.
Anderson CD, Belous A, Pierce J, Nicoud IB, Knox C, Wakata A, Pinson CW, Chari RS
(2004) Am J Transplant 4: 352-62
MeSH Terms: Apoptosis, Calcium, Caspase 3, Caspases, Cryopreservation, Cytochromes c, Hepatoblastoma, Humans, Indicators and Reagents, Mitochondria, Protein Transport, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-bcl-2, Reperfusion Injury, Ruthenium Red, Tumor Cells, Cultured, bcl-2-Associated X Protein
Show Abstract · Added January 10, 2014
Mitochondrial calcium (mCa + 2) overload occurs during cold preservation and is an integral part of mitochondrial-dependent apoptotic pathways. We investigated the role of mCa + 2 overload in cell death following hypothermic storage using HepG2 cells stored in normoxic-hypothermic (4 degrees C) or hypoxic (< 0.1% O2)-hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 microM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 degrees C. Cytoplasmic cytochrome c levels were studied by Western analysis and by fluorescent microscopy after transfection of cytochrome c-GFP expression plasmid. Immunofluorescence determined the intracellular, spatio-temporal distribution of Bax, and TUNEL staining was used to evaluate cell death after 180 min of rewarming. Caspase activation was evaluated using Western analysis and a caspase 3 activity assay. Bax translocation, cytochrome c release, and early rewarming cell death occurred following hypothermic storage and were exacerbated by hypoxia. Caspase 3 activation did not occur following hypothermic storage. Blockade of mCa + 2 uptake prevented Bax translocation, cytochrome c release, and early rewarming cell death. These studies demonstrate that mCa + 2 uptake during hypothermic storage, both hypoxic and normoxic, contributes to early rewarming apoptosis by triggering Bax translocation to mitochondria and cytochrome c release.
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17 MeSH Terms
Reversed activity of mitochondrial adenine nucleotide translocator in ischemia-reperfusion.
Belous A, Knox C, Nicoud IB, Pierce J, Anderson C, Pinson CW, Chari RS
(2003) Transplantation 75: 1717-23
MeSH Terms: Adenosine Triphosphate, Animals, Blotting, Western, Bongkrekic Acid, Calcium, Cryopreservation, Intracellular Membranes, Liver, Male, Mitochondria, Mitochondrial ADP, ATP Translocases, Osmolar Concentration, Rats, Rats, Sprague-Dawley, Reperfusion Injury
Show Abstract · Added January 10, 2014
BACKGROUND - Graft dysfunction as a result of preservation injury remains a major clinical problem in liver transplantation. This is related in part to accumulation of mitochondrial calcium. In an attempt to sustain cell and mitochondrial integrity during ischemia, intramitochondrial F(0)F(1) adenosine triphosphate (ATP) synthase reverses its activity and hydrolyzes ATP to maintain the mitochondrial transmembrane potential (mdeltapsi). It is not known how cytoplasmic ATP becomes available for hydrolysis by this enzyme. The authors hypothesized that mitochondrial adenine nucleotide translocator (ANT) reverses its activity during ischemia, making cytoplasmic ATP available for hydrolysis by F(0)F(1) ATP synthase.
METHODS - Rat livers were perfused with cold University of Wisconsin solution at 4 degrees C (39.2 degrees F)through the portal vein and processed immediately or after 24 hr of cold storage. Mitochondria were separated by differential centrifugation. ATP-dependent mitochondrial calcium-45 (45Ca)2+ uptake was determined after incubation with ATP (5 mM) or adenosine diphosphate (ADP) (5 mM) with or without 15 microM of bongkrekic acid (BA), an ANT blocker; the nonhydrolyzable analog of ATP (adenosine 5'-beta,gamma-imidotriphosphate [AMP-PNP]) served as the negative control. All measurements were performed in triplicate. Student t test, P<0.05 was taken as significant.
RESULTS - Inhibition of ANT by BA prevents mitochondrial Ca2+ accumulation in the presence of ATP and high 45Ca2+ concentrations, and increased extramitochondrial 45Ca2+ stimulated mitochondrial 45Ca2+ uptake in the presence of ATP but not ADP, AMP-PNP, or BA.
CONCLUSIONS - These data demonstrate that ANT plays an important role in mitochondrial Ca2+ uptake under ischemic conditions by reversing its activity and allowing transport of extramitochondrial ATP into the matrix for hydrolysis by reversed F(0)F(1) ATP synthase.
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15 MeSH Terms
A simple method for freezing and storing viable tissue fragments.
Hayward SW
(1998) In Vitro Cell Dev Biol Anim 34: 28-9
MeSH Terms: Cryopreservation, Freezing, Humans, Male, Prostate
Added December 10, 2013
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5 MeSH Terms
Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease.
Wagner JE, Rosenthal J, Sweetman R, Shu XO, Davies SM, Ramsay NK, McGlave PB, Sender L, Cairo MS
(1996) Blood 88: 795-802
MeSH Terms: Actuarial Analysis, Acute Disease, Adolescent, Adult, Anemia, Aplastic, Blood Donors, Blood Preservation, Child, Child, Preschool, Cryopreservation, Female, Fetal Blood, Genetic Diseases, Inborn, Graft Survival, Graft vs Host Disease, HLA Antigens, Hematopoietic Stem Cell Transplantation, Histocompatibility, Humans, Infant, Infant, Newborn, Leukemia, Male, Severity of Illness Index, Treatment Outcome
Show Abstract · Added December 10, 2013
To reduce the morbidity and mortality associated with unrelated donor bone marrow (BM) transplantation and potentially extend the pool of suitable donors, cryopreserved unrelated donor umbilical cord blood was considered as an alternate source of hematopoietic stem cells for transplantation. Patients with leukemia, BM failure syndrome, or inborn error of metabolism were eligible for a phase I clinical trial designed to estimate the risk of graft failure and severe acute graft-versus-host disease after transplantation of umbilical cord blood from unrelated donors. As of December 21, 1995, unrelated donor umbilical cord blood was used to reconstitute hematopoiesis in eighteen patients aged 0.1 to 21.3 years weighing 3.3 to 78.8 kg with acquired or congenital lympho-hematopoietic disorders or metabolic disease. Patients received either HLA-matched (n = 7) or HLA-1 to 3 antigen disparate (n = 11) grafts collected and evaluated by the New York Blood Center (New York, NY). The probability of engraftment after unrelated donor umbilical cord blood transplantation was 100% with no patient having late graft failure to date. The probability of grade III-IV acute graft-versus-host disease at 100 days was 11%. With a median follow-up of 6 months (range, 1.6 to 17 months); the probability of survival at 6 months is 65% in this high risk patient population. We conclude that cryopreserved umbilical cord blood from HLA-matched and mismatched unrelated donors is a sufficient source of transplantable hematopoietic stem cells with high probability of donor derived engraftment and low risk of refractory severe acute graft-versus-host disease. Limitations with regard to recipient size and degree of donor HLA disparity remain to be determined.
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25 MeSH Terms
Development of a bioartificial liver: properties and function of a hollow-fiber module inoculated with liver cells.
Rozga J, Williams F, Ro MS, Neuzil DF, Giorgio TD, Backfisch G, Moscioni AD, Hakim R, Demetriou AA
(1993) Hepatology 17: 258-65
MeSH Terms: Animals, Artificial Organs, Cell Separation, Cell Survival, Cryopreservation, Dogs, Equipment Design, Female, Forecasting, Liver, Male, Models, Theoretical, Rats, Swine
Show Abstract · Added March 20, 2014
We have developed a bioartificial liver support system utilizing hollow-fiber bioreactor, plasmapheresis and microcarrier cell culture technologies. Liver cells were obtained through portal vein perfusion with ethylenediaminetetraacetate or ethylenediaminetetraacetate/collagenase. A mathematical model of mass transport in a hollow-fiber module, at various plasma flow velocities and system configurations, was developed. The bioartificial liver's ability to carry out specific differentiated metabolic liver functions was tested in vitro and in vivo. A reproducible large-animal model of acute ischemic liver failure was developed. Most major first-generation cyclosporine and 19-norterstosterone metabolites were isolated after substrate addition to the bioartificial liver in vitro. After bioartificial liver treatment for 6 hr (with dog or pig liver cells), dogs with acute liver failure had significantly lower serum ammonia and lactate levels and significantly higher serum glucose levels than did control animals treated with a bioartificial liver system inoculated with microcarriers alone. In addition, bioartificial liver-treated animals had significantly higher mean systolic blood pressures than did controls. Liver cell viability at the end of the 6-hr in vivo experiment was greater than 90%.
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14 MeSH Terms
Cryopreservation of human brain tissue.
Robbins RJ, Torres-Aleman I, Lebranth C, Bradberry CW, Deutch AY, Welsh S, Roth RH, Spencer D, Redmond DE, Naftolin F
(1990) Exp Neurol 107: 208-13
MeSH Terms: Brain, Cell Survival, Cryopreservation, Dopamine, Humans, Mesencephalon, Neurons, Substantia Nigra, Tegmentum Mesencephali, Tyrosine 3-Monooxygenase
Show Abstract · Added May 27, 2014
Tissues from products of conception were examined to determine the feasibility of obtaining viable neural tissue after suction abortion at 9-12 weeks of gestation. The ventral mesencephalon, a prototype region whose maturation can be monitored and which is a potential tissue for transplantation, was identified in 32 of 120 cases. The tissue was then screened for the presence of infectious agents, while being held at -196 degrees C in cryopreservative solutions. Three of 32 specimens were found to be contaminated by normal vaginal bacteria; all other viral, fungal, and mycoplasma testing was negative. Thawed brain fragments retained high viability after storage in liquid nitrogen and when grown in vitro exhibited neuronal morphology, tyrosine hydroxylase immunoreactivity, and dopamine production. We have demonstrated that human fetal brain tissue can be cryopreserved in a manner which not only retains viability but allows normal phenotypic differentiation after thawing.
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10 MeSH Terms