Optimized recirculation survival of mouse carrier erythrocytes.

Carrier mouse erythrocytes prepared by a hypotonic dialysis technique and reinjected into mice have a 24 hour survival of approximately 50%. Twenty-four hour survival can be improved substantially to 74% by removing the more fragile erythrocytes by a hypotonic wash treatment. The mean cell volume of the carriers prepared by this modification is significantly (p less than 0.01) different from cells prepared by the standard method with a isotonic wash treatment. Carriers prepared by the hypotonic treatment wash modification exhibit a different 50% hemolytic value (15% difference) from isotonically prepared carriers, and normal erythrocytes. Carrier-erythrocytes removed from mice 24 hour post-injection exhibit an osmotic profile that is independent of the treatment. Carriers were also prepared by another modification of the encapsulation procedure and held in a permeable state overnight before resealing and annealing. Carriers prepared in this manner showed a much lower 24 hour survival (13%).     

Intracellular trehalose improves the survival of cryopreserved mammalian cells

We report that the introduction of low concentrations of intracellular trehalose can greatly improve the survival of mammalian cells during cryopreservation. Using a genetically engineered mutant of Staphylococcus aureus alpha-hemolysin to create pores in the cellular membrane, we were able to load trehalose into cells. Low concentrations (0.2 M) of trehalose permitted long-term post-thaw survival of more than 80% of 3T3 fibroblasts and 70% of human keratinocytes. These results indicate that simplified and widely applicable freezing protocols may be possible using sugars as intracellular cryoprotective additives.     

Reaction of ozone with sulfhydryls of human erythrocytes

Oxidation of GSH by ozone yielded 60% GSSG. Exposure of human erythrocytes to ozone caused oxidation of intracellular GSH. Between 4 and 6% of the administered ozone caused GSH oxidation. No more than 30% of the GSH oxidized by ozone could be accounted for by GSSG in the erythrocyte. The GSSG formed in the erythrocyte was rapidly reduced and the pentose phosphate pathway was stimulated. When GSH and unsealed erythrocyte ghosts were simultaneously exposed to ozone, 6–11% of the oxidized GSH could be accounted for as mixed disulfide of protein and GSH. When GSH and cytoplasmic proteins from the erythrocyte were simultaneously exposed to ozone, 5–7% of the oxidized GSH could be accounted for as mixed disulfide. Ozone generated membrane protein disulfide crosslinks when erythrocyte ghosts, but not intact erythrocytes, were exposed. Ozone had no effect on glucose uptake and did not change oxyhemoglobin content of the erythrocytes.     

Factors affecting the survival of mouse embryos cryopreserved by vitrification

Preimplantation stage mouse embryos have been used to examine the response of a simple multicellular system to cryopreservation by the complete vitrification of the suspension. Successful vitrification requires the use of a solution of cryoprotectants that is sufficiently concentrated to supercool and solidify into a glass at practicable cooling rates. Factors that influence the survival of embryos include the concentration and composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution. High rates of survival are obtained when embryos are dehydrated prior to vitrification in solutions composed of saline plus multimolar concentrations of either mixtures of permeating cryoprotectants (e.g. dimethyl sulphoxide-acetamide-propylene glycol) or single permeating cryoprotectants (propylene glycol or glycerol). Full permeation of cryoprotectants into the cells is not necessary and may lead to chemical toxicity and osmotic injury. Partial permeation and osmotic shrinkage concentrates the endogenous cytoplasmic macromolecules and greatly increases the likelihood of intracellular vitrification. Vitrification is a practical approach for embryo cryopreservation and offers new opportunities to examine fundamental aspects of cryoprotection and cryoinjury in the absence of freezing.     

The possibility of increasing the precision of biochemical diagnostics of Gaucher disease

Estimation of chitotriosidase activity is proposed for final diagnostics of Gaucher disease. Using this method the diagnosis has not been confirmed in one patient of 25 ones with this preliminary diagnosis. The problems of complex diagnostics of Gaucher disease are discussed.     

Pinacidil enhances survival of cryopreserved human embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.     

Pinacidil enhances survival of cryopreserved human embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.     

近地层臭氧浓度升高对杂交稻颖花形成的影响

依托全球唯一的稻田开放式空气中臭氧浓度增高系统平台,以汕优63和两优培九为供试材料,设置大气背景臭氧浓度和高臭氧浓度（比大气背景臭氧浓度高50%）两个浓度水平,研究FACE条件下高O₃浓度对杂交稻颖花形成的影响.结果表明：高O₃浓度使汕优63和两优培九每穗颖花数分别减少28朵和34朵,下降幅度分别为15%和13%.从稻穗构成看,高O₃浓度胁迫下杂交稻每穗颖花数减少主要与每穗2次枝梗颖花数明显减少有关,对每穗1次枝梗颖花数的影响较小,因此高O₃浓度胁迫下水稻每穗1次枝梗颖花数占全穗的比率增加,每穗2次枝梗颖花数占全穗的比率降低.从颖花形成看,高O₃浓度胁迫下杂交稻每穗颖花数下降主要是颖花（特别是2次颖花）的分化受到抑制所致,而颖花的退化数不增反降.上述结果表明,采取相应措施削弱高O₃浓度胁迫对颖花分化的抑制作用可能是近地层高O₃浓度条件下减少杂交稻产量损失的关键.     

Biochemical indices of erythrocytes cryopreserved with protection by 1,2-propanediol and glycerin

Based on the data concerning the content of ATP, 2,3-DFG, K+, and Na+ in depreserved red cells stored at 4 degrees C in resuspending media TsNIIGPK 8b and 8v it has been established that blood preserved under protection of 1,2-propanediol is comparable with that preserved under glycerin protection as regards its quality and is even better from the standpoint of some other indicators. Medium TsNIIGPK 8v may be used as resuspending medium for blood exposed to low temperature preservation under protection of 1,2-propanediol.     

Studies on the possibility of increasing resistance to cocoa swollen-shoot virus by breeding

In tests using seed, the resistance of cocoa progenies to primary infection with cocoa swollen-shoot virus by mealybugs was stable to inoculum pressure and effective against several virus strains. The results suggested that the resistance would be effective throughout the cocoa growing areas of Ghana. There were indications that resistance factors from different cocoa populations could be accumulated to give progenies of higher resistance than presently available.     

Natural selection through survival alone,and the possibility of Gaia

Here I advance two related evolutionary propositions. (1) Natural selection is most often considered to require competition between reproducing “individuals”, sometimes quite broadly conceived, as in cases of clonal, species or multispecies-community selection. But differential survival of non-competing and non-reproducing individuals will also result in increasing frequencies of survival-promoting “adaptations” among survivors, and thus is also a kind of natural selection. (2) Darwinists have challenged the view that the Earth’s biosphere is an evolved global homeostatic system. Since there is only one biosphere, reproductive competition cannot have been involved in selection for such survival-promoting adaptations, they claim. But natural selection through survival could reconcile Gaia with evolutionary theory.     

On the mechanism of injury to slowly frozen erythrocytes.

When cells are frozen slowly in aqueous suspensions, the solutes in the suspending solution concentrate as the amount of ice increases; the cells undergo osmotic dehydration and are sequestered in ever-narrowing liquid-filled channels. Cryoprotective solutes, such as glycerol, reduce the amount of ice that forms at any specified subzero temperature, thereby controlling the buildup in concentration of those other solutes present, as well as increasing the volume of the channels that remain to accommodate the cells. It has generally been thought that freezing injury is mediated by the increase in electrolyte concentration in the milieu surrounding the cells, rather than reduction of temperature or any direct action of ice. In this study we have frozen human erythrocytes in isotonic solutions of sodium chloride and glycerol and have demonstrated a correlation between the extent of damage at specific subzero temperatures, and that caused by the action at 0 degrees C of solutions having the same composition as those produced by freezing. The cell lysis observed increased directly with glycerol concentration, both in the freezing experiments and when the cells were exposed to corresponding solutions at 0 degrees C, showing that the concentration of sodium chloride alone is not sufficient to account quantitatively for the damage observed. We then studied the effect of freezing in anisotonic solutions to break the fixed relationship between solute concentration and the volume of the unfrozen fraction, as described by Mazur, P., W. F. Rall, and N. Rigopoulos (1981. Biophys. J. 653-675). We confirmed their experimental findings, but we explain them differently. We ascribe the apparently dominant effect of the unfrozen fraction to the fact that the cells were frozen in, and returned to, anisotonic solutions in which their volume was either less than, or greater than, their physiological volume. When similar cell suspensions were subjected to a similar cycle of increase and then decrease in solution strength, but in the absence of ice (at 20 degrees C), a similar pattern of hemolysis was observed. We conclude that freezing injury to human erythrocytes is due solely to changes that occur in the composition of their surrounding milieu, and is most probably mediated by a temporary leak in the plasma membrane that occurs during the thawing (reexpansion) phase.     

Effects of ozone exposure on the lungs and the erythrocytes of rats and monkeys: relative biochemical changes.

Ozone exposure (0.5 p.p.m., 8 hours daily for 7 days) resulted in a 20-26% (P less than 0.05) increase in the level of reduced glutathione (GSH), and the activities of the GSH peroxidase system in rat lungs. The increases were of smaller magnitude (10-15%) in the lungs of ozone-exposed monkeys. No significant changes were observed in these parameters in the erythrocytes of ozone-exposed and control animals of the two species. The results suggest that rats may be more sensitive to ozone than monkeys in terms of biochemical lesions in the lung, and that ozone effects are manifested primarily in the lung.     

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.