Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes.

As suspensions of cells freeze, the electrolytes and other solutes in the external solution concentrate progressively, and the cells undergo osmotic dehydration if cooling is slow. The progressive concentration of solute comes about as increasing amounts of pure ice precipitate out of solution and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. The consensus has been that slow freezing injury is related to the composition of the solution in these channels and not to the amount of residual liquid. The purpose of the research reported here was to test this assumption on human erythrocytes. Ordinarily, solute concentration and the amount of liquid in the unfrozen channels are inversely coupled. To vary them independently, one must vary the initial solute concentration. Two solutes were used here: NaCl and the permeating protective additive glycerol. To vary the total initial solute concentration while holding the mass ratio of glycerol to NaCl constant, we had to allow the NaCl tonicity to depart from isotonic. Specifically, human red cells were suspended in solutions with weight ratios of glycerol to NaCl of either 5.42 or 11.26, where the concentrations of NaCl were 0.6, 0.75, 1.0, 2.0, 3.0, or 4.0 times isotonic. Samples were then frozen to various subzero temperatures, which were chosen to produce various molalities of NaCl (0.24-3.30) while holding the fraction of unfrozen water constant, or conversely to produce various unfrozen fractions (0.03-0.5) while holding the molality of salt constant. (Not all combinations of these values were possible). The following general findings emerged: (a) few cells survived the freezing of greater than 90% of the extracellular water regardless of the salt concentration in the residual unfrozen portion. (b) When the fraction of frozen water was less than 75% the majority of the cells survived even when the salt concentration in the unfrozen portion exceeded 2 molal. (c) Salt concentration affected survival significantly only when the frozen fraction lay between 75 and 90%. To find a major effect on survival of the fraction of water that remains unfrozen was unexpected. It may require major modifications in how cryobiologists view solution-effect injury and its prevention.     

Permeability of the 17-day fetal rat pancreas to glycerol and dimethylsulfoxide

Experimentally induced diabetes in rats can be reversed by the transplantation of several fresh or frozen-thawed fetal pancreases. An important question to both the mechanistic and practical aspects of cryobiology is the role played by the permeation of protective additives during freezing, thawing, and subsequent dilution. Answers require knowledge of the kinetics of permeation of the specific additive into the cell or tissue. In this paper, we report isotopic measurements of the rate of permeation of 2 M glycerol and 1 and 2 M dimethylsulfoxide (Me₂SO) into 17-day fetal pancreases at 0 and 22 °C. In Me₂SO, equilibrium was achieved in about 10–15 min at 0 °C and in less than 10 min at 22 °C. In glycerol, equilibrium was attained in about 60 min at 22 °C; but at 0 °C permeation was only 65% complete after 180 min. In general, Me₂SO permeated 10–30 times more rapidly than glycerol at 0 °C, and glycerol permeated about 10 times more rapidly at 22 than at 0 °C.The kinetics of permeation were more characteristic of a two-compartment than a single-compartment system. In all probability, the two compartments are the intercellular space and the intracellular space. The permeability data suggest that each compartment occupies about half the total volume.     

Adenosylhomocysteinase:Adenosine complex

Adenosylhomocysteinase from yellow lupin seeds forms a specific complex with adenosine. The complex can be isolated either by nonequilibrium or equilibrium gel filtration. It is also adsorbed on nitrocellulose disks. Dissociation constant of the complex determined by nitrocellulose filter assay is 5 × 10^?8M.     

Temperature dependence of the survival of human erythrocytes frozen slowly in various concentrations of glycerol.

One widely accepted explanation of injury from slow freezing is that damage results when the concentration of electrolyte reaches a critical level in partly frozen solutions during freezing. We have conducted experiments on human red cells to further test this hypothesis. Cells were suspended in phosphate-buffered saline containing 0-3 M glycerol, held for 30 min at 20 degrees C to permit solute permeation, and frozen at 0.5 or 1.7 degrees C/min to various temperatures between -2 and -100 degrees C. Upon reaching the desired minimum temperature, the samples were warmed at rates ranging from 1 to 550 degrees C/min and the percent hemolysis was determined. The results for a cooling rate of 1.7 degrees C/min indicate the following: (a) Between 0.5 and 1.85 M glycerol, the temperature yielding 50% hemolysis (LT50) drops slowly from -18 to -35 degrees C. (b) The LT50's over this range of concentrations are relatively independent of warming rate. (c) With glycerol concentrations of 1.95 and 2.0 M, the LT50 drops abruptly to -60 degrees C and to below -100 degrees C, respectively, and becomes dependent on warming rate. The LT50 is lower with slow warming at 1 degree C/min than with rapid. With still higher concentrations (2.5 and 3.0 M), there is no LT50, i.e., more than 50% of the cells survive freezing to-100 degrees C. Results for cooling at 0.5 degrees C/min in 2 M glycerol were similar except that the LT50s were some 10-20 degrees C higher. A companion paper (Rall et al., Biophys. J. 23:101-120, 1978) examines the relation between survival and the concentrations of salts produced during freezing.     

An Haemophilus influenzae mutant which inhibits the growth of HP1c1 phage

Summary A strain of Haemophilus influenzae, called hpm ^- inhibits the growth of phage HP1c1 but not S2. This inhibition is overcome by HP1c1ph mutants. Phage HP1c1 adsorbs normally to hpm ^- cells but only a small fraction of infected cells produce phage with a normal burst size or become lysogenic. When hpm ^- strains lysogenic for HP1c1 are induced, 100% of the cells yield phage. There is no degradation of phage DNA after infection of hpm ^- cells and HP1c1 can normally grow when its DNA is introduced into hpm ^- by transfection. The most probable explanation is that in hpm ^- cells the penetration of phage DNA is blocked. The hpm ^- property behaves as as unstable mutation.     

Effects of AET and MEA treatment of mice on the first day of pregnancy

Porton female mice were injected intraperitoneally with 2-aminoethylisothiouronium bromide hydrobromide (AET) or cysteamine hydrochloride (MEA) in a dose of 40 mg/kg of body weight on the first day of pregnancy. On the last, nineteenth, day of gestation, taking into consideration females in whose uterus live fetuses were observed, the increase in their body weight throughout pregnancy, the number of fetuses in the uterus, the body weight of fetuses, and placental weight were found smaller in mice treated with AET or MEA, than in control ones. Among the injected compounds, AET appeared to be less toxic than MEA.