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Organochemistry of cryoinjury and cryophlyaxis 2. Noncolligative cryophylaxis by effective low molecular-weight cryoprotectants
ANNUAL
MEETING
action by glycerol and DMSO. (Supported. in part, by NSF Grant 15749.) of Cryoinjury and Cryo61. Organochemistry phlyaxis 2. Noncolligalive Cryophylaxis by Effective Low Molecular-Weight Cryoprotectants. W. N. FISHBEIN (Biochemistry Branch, Armed Forces Institute of Pathology, Washington. DC 20305).
The succinate cytochrome c reductase (SCcR) enzyme complex of mouse liver mitochondrial isolates provides a convenient assay for freezing damage. When suspended in 0.15 M KC1 and quenchthawed in droplet form, mitochondrial SCcR shows an optimum recovery (89%) in isopentane, and lower recovery (50%) at both faster (liquid propane) and slower (liquid Nz) cooling rates. Moreover, intramitochondrial ice was clearly demonstrable at supraoptimal cooling rates. and was absent at infraoptimal rates. Various concentrations of dimethylsulfoxide (DMSO) were employed to evaluate the nature of cryophylaxis. DMSO protected as well at supraoptimal as at infraoptimal cooling rates, and was most effective at 0.2-0.4 M. On both counts. colligative properties cannot account for the cryoprotection. The studies were extended to a series of diols to evaluate the contribution of specific molecular structures. Protection by 1,3-propanediol could be explained by colligative properties, since it appeared only at concentrations >l M, and increased gradually thereafter. At. these high levels, 1,2-propanediol (PG) and ethylene glycol (EG) were less effectirc; but at
ABSTRACTS (Cryobiology Research Institute, son, WI 53704).
RFD 5, Madi-
Certain species of yeast and bacteria have, on several previous occasions, been reported to tolerate, to limited extents, intracellular freezing and thawing. Sufficiently finely subdivided ice crystals appear, that is, not to damage the cells from within. Studies on six species of bacteria (Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus
aureus,
Escherichia
coli,
Pseudomonas
aeruginosa, Serratia marcescens) have been extended to permit corresponding conclusions with reference to freeze-drying. Neither the loss of intracellular ice during the freeze-drying (completed in each case at -4O”C), nor the subsequent desorption of water (completed in each case at 2O”C), nor the reentry of water during rehydration at room temperature appeared to be lethal. The 3 grampositive species survived freezing and freeze-drying from distilled wat,er suspension i50-70% survivals). The gram-negative bacteria required the presence of one or more solutes. Ficoll and dextran, free from low molecular-weight contaminants, were effective only in part. Combinations consisting of Ficoll or dextran and one or more compounds of lower molecular weight, sucrose or glycerol, for instance, permitted freeze-drying survivals as high as 100%. Traces of glycerol were more effective than larger quantities of sucrose. It appears, on the basis of the preliminary evidence that the glycerol conferred protection at the plasma membrane or at loci within the cells at sites inaccessible to sucrose. (Studies supported by NIH grants GM-15143 and RR-05740.) 63. Studies on the Tolerance of Isolated Nuclei to Drying by Sublimation of Ice In Vacua. R. L. RUBIN AND D. GREIFF (Department of Pathology,
The Medical College of Wisconsin, Milwaukee, WI 53233). The ability of isolated rat hepatic cell nuclei to incorporate YXabeled amino acids was used to measure the effects of cooling at lOO”C/min to a terminal temperature of -76°C prior to thawing at 37°C or freeze-drying. The order of active incorporation after freezing for samples prepared in sucrose and suspended in different reagents was as follows: dextran in sucrose > dextran in saline > glucose > phosphate buffer > sucrose > saline > PVP in glucose > PVP in sucrose = serum albumin. human; in samples prepared in DMSO plus glucose: sucrose > phosphate buffer > saline; and in samples prepared in PVP plus sucrose: dextran in sucrose > glucose > phosphate buffer > sucrose > saline > serum albumin, human. The order of active incorporation after freeze-
MEETING
action by glycerol and DMSO. (Supported. in part, by NSF Grant 15749.) of Cryoinjury and Cryo61. Organochemistry phlyaxis 2. Noncolligalive Cryophylaxis by Effective Low Molecular-Weight Cryoprotectants. W. N. FISHBEIN (Biochemistry Branch, Armed Forces Institute of Pathology, Washington. DC 20305).
The succinate cytochrome c reductase (SCcR) enzyme complex of mouse liver mitochondrial isolates provides a convenient assay for freezing damage. When suspended in 0.15 M KC1 and quenchthawed in droplet form, mitochondrial SCcR shows an optimum recovery (89%) in isopentane, and lower recovery (50%) at both faster (liquid propane) and slower (liquid Nz) cooling rates. Moreover, intramitochondrial ice was clearly demonstrable at supraoptimal cooling rates. and was absent at infraoptimal rates. Various concentrations of dimethylsulfoxide (DMSO) were employed to evaluate the nature of cryophylaxis. DMSO protected as well at supraoptimal as at infraoptimal cooling rates, and was most effective at 0.2-0.4 M. On both counts. colligative properties cannot account for the cryoprotection. The studies were extended to a series of diols to evaluate the contribution of specific molecular structures. Protection by 1,3-propanediol could be explained by colligative properties, since it appeared only at concentrations >l M, and increased gradually thereafter. At. these high levels, 1,2-propanediol (PG) and ethylene glycol (EG) were less effectirc; but at
ABSTRACTS (Cryobiology Research Institute, son, WI 53704).
RFD 5, Madi-
Certain species of yeast and bacteria have, on several previous occasions, been reported to tolerate, to limited extents, intracellular freezing and thawing. Sufficiently finely subdivided ice crystals appear, that is, not to damage the cells from within. Studies on six species of bacteria (Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus
aureus,
Escherichia
coli,
Pseudomonas
aeruginosa, Serratia marcescens) have been extended to permit corresponding conclusions with reference to freeze-drying. Neither the loss of intracellular ice during the freeze-drying (completed in each case at -4O”C), nor the subsequent desorption of water (completed in each case at 2O”C), nor the reentry of water during rehydration at room temperature appeared to be lethal. The 3 grampositive species survived freezing and freeze-drying from distilled wat,er suspension i50-70% survivals). The gram-negative bacteria required the presence of one or more solutes. Ficoll and dextran, free from low molecular-weight contaminants, were effective only in part. Combinations consisting of Ficoll or dextran and one or more compounds of lower molecular weight, sucrose or glycerol, for instance, permitted freeze-drying survivals as high as 100%. Traces of glycerol were more effective than larger quantities of sucrose. It appears, on the basis of the preliminary evidence that the glycerol conferred protection at the plasma membrane or at loci within the cells at sites inaccessible to sucrose. (Studies supported by NIH grants GM-15143 and RR-05740.) 63. Studies on the Tolerance of Isolated Nuclei to Drying by Sublimation of Ice In Vacua. R. L. RUBIN AND D. GREIFF (Department of Pathology,
The Medical College of Wisconsin, Milwaukee, WI 53233). The ability of isolated rat hepatic cell nuclei to incorporate YXabeled amino acids was used to measure the effects of cooling at lOO”C/min to a terminal temperature of -76°C prior to thawing at 37°C or freeze-drying. The order of active incorporation after freezing for samples prepared in sucrose and suspended in different reagents was as follows: dextran in sucrose > dextran in saline > glucose > phosphate buffer > sucrose > saline > PVP in glucose > PVP in sucrose = serum albumin. human; in samples prepared in DMSO plus glucose: sucrose > phosphate buffer > saline; and in samples prepared in PVP plus sucrose: dextran in sucrose > glucose > phosphate buffer > sucrose > saline > serum albumin, human. The order of active incorporation after freeze-