Effect of a vitrification protocol on the lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities and the hatching rates of Zebrafish (Danio rerio) and Turbot (Scophthalmus maximus) embryos

Vitrification, is the most promising option for the cryopreservation of fish embryos but requires high concentrations of potentially toxic cryoprotectants. In this study, embryos from Turbot and Zebrafish, each in two developmental stages, were submitted to a four stepwise cryoprotectant incorporation protocol. After incubation in the vitrificant solution (5M dimethyl sulfoxide, 2M methanol, 1M ethylen-glycol and 10% sucrose) embryos were loaded in straws and plunged into liquid nitrogen. The activity of two cytoplasmic enzymes, LDH and G6PDH, and the hatching rates were analyzed in control embryos, those subjected to the cryoprotectant solutions and in frozen/thawed embryos. Results showed that the cryoprotectants incorporation protocol did not have important effects on the analyzed enzymatic activities, which remained at similar levels to that in control embryos but significantly reduced the hatching rates. Turbot was less sensitive than Zebrafish to the toxic effect of the cryoprotectants, achieving hatching rates of 74.8% in comparison with fresh control embryos at G stage, whereas in Zebrafish only 17.7% of hatching was reported with five somites-treated embryos. In Turbot, G stage was more resistant to the cryoprotectants and thus more convenient for further vitrification studies. After vitrification no survival was recorded and enzymatic activities dropped significantly, particularly in Zebrafish, indicating cell damage and loss of cytoplasmic enzymes. Nevertheless, total cell lysis was not produced, and once again Turbot was more resistant to the effect of vitrification, particularly at the later stage. In that stage, Turbot embryos showed around 50% of G6PDH activity after vitrification, in comparison with the control, indicating the preservation of some cellular activity after freezing-thawing, despite the loss of developmental ability.     

黑鲷精子的超低温冻存及DNA损伤的SCGE检测

以0.5 mL的麦细管为冻存管和DMSO为抗冻剂进行超低温冷冻黑鲷精子,对冻精核DNA的损伤情况进行单细胞凝胶电泳（SCGE）检测,其结果表明,以Cortland溶液为稀释液,5%、10%、15%及20%DMSO为抗冻剂的超低温冻存的黑鲷精子活力、受精率与鲜精无显著差异。其中以10%DMSO为抗冻剂的冻存效果最佳,冻精的激活率、运动时间、寿命及受精率分别达（92.91±1.25）%、（39.90±2.70）min、（53.82±2.84）min及（89.35±1.99）%;而以25%及30%DMSO为抗冻剂时,冻精活力及受精率显著下降。SCGE检测结果显示,DMSO浓度为5%、10%、15%及20%时,黑鲷冻精与鲜精的彗星率及损伤系数差异不显著;DMSO浓度为25%及30%时,冻精与鲜精的彗星率及损伤系数差异显著;冻精的彗星率与抗冻剂DMSO浓度成正相关。黑鲷鲜精及冻精核的DNA损伤主要为轻度和中度损伤,重度损伤比例较低,完全损伤仅存在于25%及30%DMSO为抗冻剂的冻精中,且比例低。分析认为,较高浓度的DMSO是引起冻精核DNA损伤的主要原因。     

Effects of cooling and equilibration in DMSO,and cryopreservation of mouse oocytes,on the rates of in vitro fertilization,development, and chromosomal abnormalities

In a previous study, we have shown that the cryopreservation of mouse oocytes caused increases in the rates of degeneration and of digynic polyploid embryos, while the fertility of frozen-thawed oocytes was decreased. In this study, we have attempted to determine the different stages in the complete freezing-thawing process which are deleterious for the oocytes and the subsequent zygotes. IVF assays showed that DMSO decreased the fertility of oocytes, whereas cooling to 0°C had no effect. DMSO, used at 0°C, was less deleterious for oocytes. Thus, the prefreezing manipulations seem to be important for the quality and fertility of oocytes. However, neither DMSO nor cooling increased the incidence of chromosomal abnormalities in embryos obtained from inseminated exposed oocytes. Therefore, the increased frequency of polyploidy observed in embryos after the cryopreservation of mouse oocytes must correspond to disruption occurring during the freezing-thawing process.     

Comparative study on the percentage of recovery, survival and mode of sequestration of fresh and frozen preserved (-196 degrees C) human platelets

Platelet concentrates amounting to 142 X 10(9) +/- 11 X 10(9) cells are prepared by cytopheresis from one liter of blood. Ten of the concentrates are labelled with 51Cr and reinfused autologously. The other ten concentrates are frozen with a controlled rate freezer in a medium containing 10% DMSO and 5% glucose for 7 days at -196 degrees C. The fresh platelets have a 51Cr 24 hrs. recovery of 68 +/- 7% and a survival rate (T 1/2) of 10 +/- 1 days. The frozen preserved platelets have a 51Cr 24 hrs. recovery of 54 +/- 6.1%, a T 1/2 of 8.1 days, and significantly increased liver sequestration. There is a correlation between the reversal reaction in vitro after hypotonic shock and the 51Cr 24 hrs. in vivo recovery of fresh platelets and the 51Cr 1st hr. recovery of frozen platelets.     

The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test

The most commonly used toxicity test worldwide is the acute Daphnia magna test. The relevance of acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity in D. magna exposed to chromium, cadmium, and diazinon was evaluated in connection with this standard test. We found no link between enzyme activities and immobility. Concentrations of Cr(6+) up to 280 microg/L had no effect on AChE and GST activities, while 20% immobility was observed. At concentrations of 20-25 microg/L of Cd(2+) AChE activity was increased by about 50%. The effect of diazinon on both enzymes was insignificant up to concentrations that caused 27% immobility. Consequently, while the use of AChE and GST activities is recommended when the mode of action of chemicals is studied, the value of these biomarkers in routine acute toxicity tests is limited because the relationship between enzyme activities and immobility of D. magna exposed to different chemicals is unclear.     

Divergent Regulation of Acetylcholinesterase and Butyrylcholinesterase in Tissues of the Rat

Abstract: Investigating the possibility that acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are regulated in a coordinated manner, we have examined the natural variation in activity of these two enzymes in several tissues of adult male Sprague-Dawley, Fischer-344, and Wistar-Furth rats. Both enzymes varied greatly in mean activity among brain, diaphragm, atria, serum, superior cervical ganglia, and liver. In Sprague-Dawley rats there were also large individual variations with up to a fivefold range of AChE activities and up to a 100-fold range of BuChE activities in a given tissue. Individual variations in cholinesterase activities appeared to be smaller in the inbred Fischer-344 or Wistar-Furth rats. Experiments with internal standards of partially purified AChE and BuChE indicated that the individual variations probably reflected differences in the intrinsic content or specific activity of the tissue enzymes. Comparison of the AChE activities in different tissues of a given group of rats failed to reveal statistically significant correlations in any strain (i.e., the relative activity of any one tissue was no guide to the relative activity of any other tissue in the same rat). This result indicates that the regulation of AChE is tissue-specific. By contrast, BuChE activity showed highly significant correlations among the majority of the tissues examined in the Sprague-Dawley rats, implying that widely dispersed factors can affect the regulation of this enzyme. Body-wide regulation is not necessarily the rule, however, since only a single tissue pair in the inbred Fischer rats and none of the pairs in the Wistar-Furth rats showed significant correlations of BuChE activity. In general, AChE and BuChE activities were not correlated with each other to a statistically significant degree. We conclude that the control of these enzymes normally involves different mechanisms and is strongly affected by the genetic background of the sample population.     

Studies on cryopreservation of Cryptosporidium parvum

Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.     

Effect of cryopreservant combinations on the motility and morphology of curimba (Prochilodus lineatus) sperm

This study investigated the application of intra- and extra-cellular cryoprotectant combinations on the quality of curimba Prochilodus lineatus semen subjected to cryopreservation. Semen treatments were tested with 8% DMSO or methanol as intracellular cryoprotectant, 5% egg yolk or lactose as extracellular cryoprotectant and 5% BTS. These cryoprotectant combinations are suitable for curimba but have not been tested at the lesser concentrations proposed or in combination with BTS. Semen samples collected from 19 curimbas were diluted into one of four cryoprotectant combinations: DMSO+yolk; DMSO+lactose; methanol+yolk; and methanol+lactose. After dilution, semen samples were cryopreserved in 0.5 mL straws for 10 days in a liquid nitrogen tank. Semen was thawed in a water bath at 60°C for 8s. We evaluated the quality of fresh, diluted (pre-freezing) and post-freezing semen according to sperm motility rate (%) and duration (s). Sperm morphology was also analyzed in thawed semen. Sperm motility rate decreased progressively after dilution and thawing. The motility rate in post-freezing semen was higher in the treatments using DMSO+lactose and methanol+yolk. Sperm motility duration in post-freezing sperm was greater in the treatments using methanol rather than DMSO as intracellular cryoprotectant, irrespective of the extracellular cryoprotectant used. Abnormality frequency in thawed sperm was less in semen treated with egg yolk than with lactose. Thus the use of methanol intracellular cryoprotectant is recommended along with yolk extracellular cryoprotectant in the cryopreservation process for curimba semen.     

Comparative electron cytochemical and biochemical study of ATPase and beta-glycerophosphatase activity in thymocytes, leukocytes and erythrocytes]

A study was made of the effect of procedures (freezing-thawing prior to incubation, prefixation with formaldehyde and glutaraldehyde, incubation with DMSO) on the activity of ATPase and beta-glycerophosphatase in leucocytes and erythrocytes of man, and of the effect of these procedures and of homogenization on ATPase activity in the cells of the rat thymus. The homogenization of rat thymocytes decreases ATPase activity by 15%. A repeated freezing-thawing results in a 15% decrease of ATPase activity in the cells of the rat thymus. The homogenization of rat thymocytes decreases ATPase activity in rat thymocytes, in a 2% decrease in human leucocytes, and in a 21% increase in human erythrocytes. Beta-glycerophosphatase activity in leucocytes and in erythrocytes increases thereby by 89 and 38%. Incorporation of 5% DMSO into the medium increases ATPase activity in human leucocytes and erythrocytes by 17 and 16%, while thymocytes this activity drops by 27%. Beta-glycerophosphatase activity increases thereby in leucocytes by 26 and in erythrocytes by 11.5%, resp.     

Effect of cooling and freezing,the two first steps of a freezing protocol,on the fertilizing ability of the rabbit sperm

The effect of different phases of a freezing protocol on the fertilising ability of rabbit spermatozoa was evaluated. An extender containing 1.75 M DMSO and 0.05 M sucrose (final concentration) was used to freeze rabbit sperm. In the first experiment, the results obtained with fresh and cooled (5 degrees C for 45 min) sperm were compared; no differences were observed between fresh and cooled semen for any of the parameters studied: fertility rate (78% vs. 91% for fresh and cooled sperm, respectively), and normal embryos two days after insemination (6.8 vs. 8.5 normal embryos for fresh and cooled sperm). In the second experiment, the results obtained with fresh semen and sperm which had passed the first two steps of a freezing protocol (5 degrees C for 45 min and -30 degrees C for 30 min, and thawed at 50 degrees C for 15 s) were compared; the differences between them were obtained for fertility rate (94% vs. 61% for fresh and frozen sperm, respectively) and normal embryos two days after insemination (7.8 vs. 3.8 fresh and frozen sperm). These observations indicated that the differences in the results obtained with fresh and cryopreserved sperm were produced during the second step of the freezing protocol, and that apparently no toxic effect of DMSO was produced.     

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.     

Immobilization of microorganisms with PVA hardened by iterative freezing and thawing

In order to utilize polyvinyl alcohol (PVA) as a gel matrix for the immobilization of microorganisms, PVA was subjected to iterative freezing-thawing. The effects of the procedure on the mechanical characteristics of the PVA hydrogel and the stability of the immobilized microorganisms were investigated. PVA showed rubber-like elasticity after iterative freezing-thawing. Gel strength increased with the iteration number of freezing-thawing until seven iterations. Although the activities of both the free and immobilized cells decreased during the iteration of freezing-thawing, addition of cryoprotectants such as glycerol and skim milk was effective for preventing the decrease in activity.     

Freezability of Iberian ibex (Capra pyrenaica) spermatozoa according to the glycerolization temperature and plasma testosterone concentration

Ibex spermatozoa can be successfully frozen using glycerolated media. However, no information is available regarding the most effective method of glycerol addition in this species. The aim of the present work was to evaluate the effect of the glycerolization temperature on the response to freezing-thawing of ibex spermatozoa collected by electroejaculation. The effect of the interaction glycerolization temperature x plasma testosterone concentration was also evaluated. The spermatozoa used in this work came from six adult ibexes maintained in captivity. Each ejaculate was divided into two aliquots in a Tris-egg yolk-based medium. One fraction was subjected to single step dilution with 5% glycerol at room temperature (23 °C). The other fraction was diluted in two steps, first by dilution at room temperature with an extender identical to that described above but without glycerol, followed by the addition of glycerol after cooling to 5 °C. The glycerolization temperature did not affect any sperm variable after thawing. Heterospecific artificial insemination involving domestic goats, revealed no differences in the fertilization rate for frozen-thawed spermatozoa diluted by the one or two step procedures (18.2% vs. 20.0%). The interaction glycerolization temperature x plasma testosterone concentration had no affect on the freezing-thawing of the sperm cells. The results revealed, however, that high plasma testosterone levels during the pre-rutting season may interfere with the freezing-thawing process, having a negative influence on sperm cryosurvival.     

Effects of curcumin on the social behavior,blood composition,reproductive hormones in plasma and brain acetylcholinesterase in cadmium intoxicated mice

Cadmium (Cd) exposure can induce acute lethal health-related threats in humans since it has an exceptional ability to accumulate in living organism tissues and cause toxicological effects. Curcumin (Cur) on the other hand has a wide variety of biological activities and several studies have suggested its potential therapeutic or protective effects against several ailments and infections.To study the effect of Cur on the toxicity of Cd, Swiss–Webster strain male and female mice (sixty each) were divided into 6 groups of ten each at random. Group-1 served as the naïve control and received no treatment. Group-2, 3 and 4 were the experimental controls and were administered once a day with a single oral dose of 50% dimethyl sulfoxide (DMSO), Cur (300 mg/kg) or Cd (100 mg/kg) respectively, for 2 weeks. Group-5 and 6 received Cur and Cd in combination once a day orally for 2 weeks except that Cur in a dose of 150 and 300 mg/kg to group 5 and 6 respectively, was administered one hour before Cd administration to both groups.After treatment period, the male animals were subjected to social standard opponent test and females were subjected to the tube restraint tests and thereafter, their blood was collected to measure the blood composition indices and level of reproductive hormones. The animals were sacrificed to collect their brain for the estimation of acetylcholinesterase (AChE).Results indicated that Cd significantly increased nonsocial activities in males and latency to first bite in females, whereas the social activities in males and the number of bites in females were significantly decreased. All measured indices of blood composition and levels of progesterone (female) and testosterone (male) in blood and AChE in their brain tissues were significantly decreased due to Cd treatment.However, administration of Cur along with Cd had an ameliorating effect on all the behavioral and biochemical parameters studied herein and reduced the toxicity of Cd significantly and dose-dependently. Thus, Cur may be beneficial for general health and for protection from Cd intoxication.     

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AIF4-and different types of heparin

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca²⁺ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60°C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45°C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.     

Freeze preservation of platelets using hydroxyethyl starch (HES); a preliminary report.

A comparative study has been made of platelets stored by freeze preservation following treatment with dimethyl sulfoxide (DMSO) or hydroxyethyl starch (HES) with fresh platelets and platelets stored at 4 °C for 48 hr. The indices studied were platelet recovery, pH, light microscope morphology, platelet Factor 3 (PF₃) availability and the hypotonic stress response. The DMSO preserved platelets gave a better response to hypotonic stress and incurred lesser degrees of membrane damage as demonstrated by PF₃ availability. There was however a significantly higher recovery of platelets treated with HES; with DMSO the osmotic damage inflicted during removal caused considerable lysis. Platelets frozen by DMSO or HES gave consistently better in vitro results than platelets stored at 4 °C for 48 hr. A preliminary clinical trial of HES preserved platelets has confirmed haemostatic effectiveness in vivo. HES being relatively nontoxic, platelets can be infused immediately after thawing and with minimal post thaw manipulation, thus maintaining a relatively closed system. It is concluded that cryopreservation with HES is a practical and effective means for long term platelet storage.     

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Platelet cryopreservation. 1. In vitro aggregation studies

Platelets were harvested by a Hemonetics Model-30 discontinuous cell separator from 20 normal volunteers and were cryopreserved in the presence of 5% DMSO at a controlled rate of freezing of -1 degrees C/min and stored in liquid nitrogen for up to 3 months. A significant loss of platelets occurred at the platelet concentration step through adhesion of platelets to the bag walls. A small reduction in aggregation associated with this was also seen and may reflect some damage to the platelets during the pheresis procedure. A small, but significant loss of platelet aggregation was seen with all agents following cryopreservation. Mean percentage aggregation post-thaw for all the agents was 75.4% (range 74-78%) and platelet recovery was approximately 90%. No significant changes in aggregation or recovery were seen over the 3 months' storage period. The cryoprotectant DMSO was shown to have no deleterious effect on platelet function in vitro.     

Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.