Effect of polyethylene glycol on lipid peroxidation in cold-stored rat hepatocytes

A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause peroxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondialdehyde, MDA). Hepatocytes were stored in the UW solution +/- glutathione (GSH) or +/- polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37 degrees C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 +/- 0.731 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 +/- 0.564 nmol/mg protein. When freshly isolated hepatocytes were incubated (37 degrees C) in the presence of iron (160 microM) MDA formation was maximally stimulated (3.314 +/- 0.941 nmol/mg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmol/mg protein at 24 h and 0.752 nmol/mg protein at 48 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement and activity of cytosolic deoxyribonucleoside-activated nucleotidase in various cell types from rat liver and spleen

The enzyme activity was measured in hepatocytes, Kupffer cells, endothelial cells and spleen cells. Hepatocytes showed proportionality between enzyme activity and cytosol concentration, but with Kupffer cells, endothelial cells and spleen cells the specific activity decreased with decreasing cytosol concentration when the amount of cytosol protein in 250 microliters incubation mixture was below 80, 60 and 20 micrograms, respectively. The specific activities in hepatocytes, Kupffer cells, endothelial cells and spleen cells were 2, 16, 18 and 115 nmol/min per mg of cytosol protein, respectively.     

Hypothermic preservation of hepatocytes : I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation (r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermic incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.     

曼地亚红豆杉‘Hicksii’花粉活力检测条件优化和适宜诸藏温度分析

采用单因素实验设计对适宜于曼地亚红豆杉‘Hicksii’(Taxus media‘Hicksii')花粉活力检测的TTC染色法和离体培养法的实验条件进行了选择,并采用优化条件研究了在25℃～-196℃条件下储藏13周花粉活力的变化.结果表明:采用TTC染色法测定的花粉活力均高于离体培养法.在TTC染色法的3个影响因素(染液pH、TTC浓度和染色温度)中,染液pH对检测结果有极显著影响,而染色温度和TTC浓度则无显著影响,但温度对染色速率有影响.在离体培养法的3个影响因素(培养基中蔗糖添加量、H3BO3和Ca(NO3)2浓度及培养温度)中,蔗糖添加量对检测结果有极显著影响,在含质量体积分数15％蔗糖的培养基上花粉活力最高,而在含质量体积分数20％和25％蔗糖的培养基上花粉均不能萌发;在含100和200 mg ·L-1H3BO3的培养基中添加200 mg·L-1Ca(NO3)2均能显著提高花粉活力;培养温度对花粉萌发速率有影响但对花粉活力没有明显影响.TTC染色法的最优检测条件为:用5.0g·L-1TTC染液(pH 7.0)在35℃下染色2.0h;离体培养法的最优检测条件为:用含质量体积分数15％蔗糖、100 mg·L-1H3BO3和200 mg·L-1Ca(NO3)2的培养基暗培养4d.在25℃、4℃、0℃、-20℃、-80℃和-196℃条件下储藏13周,‘Hicksii'花粉活力和保持时间有明显差异,其中,于-80℃和-196℃储藏3d 花粉就丧失活力;于25℃和-20℃储藏7周、4 ℃储藏10周,花粉仍有一定的活力;而在0℃条件下花粉活力最高,且储藏11周花粉仍有活力.推测曼地亚红豆杉‘Hicksii’花粉对低温的抗性较差,0℃为其适宜的储藏温度.     

Dose-related involvement of CCK in bombesin-induced pancreatic growth.

We examined the role of CCK in bombesin-induced pancreatic growth in rats using the CCK receptor antagonist L-364,718. Rats (155 +/- 1 g, 8-10 per group) received subcutaneous injections every 8 h for 5 days with bombesin (0.6, 1.7 and 5 nmol/kg) or bombesin in combination with L-364,718 (1 mg/kg). After 5 days the pancreas was removed and pancreatic weight, protein content, DNA, amylase and chymotrypsin contents were determined. Bombesin produced a significant increase (48-475%) of pancreatic weight, tissue contents of protein, DNA, amylase and chymotrypsinogen (F = 82, P less than 0.001). When a large dose of bombesin (5 nmol/kg) was combined with L-364,718 a significant inhibition (up to 70%) of all tissue parameters was observed (P less than 0.001). L-364,718 did not affect the growth response to a small dose of bombesin (0.6 nmol/kg). Plasma CCK levels 15 min after a single injection of bombesin (0.6, 1.7 and 5 nmol/kg) were significantly increased in response to the 5 nmol/kg dose (2.0 +/- 0.7 to 3.4 +/- 0.8 pM, F = 6.9, P less than 0.01). No increases of CCK plasma levels were found in response to the 0.6 and 1.7 nmol/kg doses of bombesin, corresponding to the lack of effects of L-364,718 on growth parameters at these doses. Measuring the time-course of CCK plasma levels after a single injection of 5 nmol/kg bombesin revealed an increase from basal values of 1.4 +/- 0.3 pM to maximal levels of 3.5 +/- 0.5 pM after 15 min (F = 7.1, P less than 0.001). Values returned to basal after 60 min. These results suggest that low doses of bombesin act directly at the acinar cell or through release of non-CCK growth factors whereas high doses of bombesin act in part through CCK release.     

External Na-independent Ca extrusion in cultured ventricular cells. Magnitude and functional significance

The relative magnitudes and functional significance of Ca extrusion by Na-Ca exchange and by an Nao-independent mechanism were investigated in monolayer cultures of chick embryo ventricular cells. Abrupt exposure of cells in 0-Nao, nominally 0-Cao solution to 20 mM caffeine produced a large contracture (3.94 +/- 0.90 micron of cell shortening) that relaxed with a t1/2 of 8.60 +/- 1.22 s. An abrupt exposure to caffeine plus 140 mM Na resulted in a contracture that was smaller in amplitude (1.53 +/- 0.50 micron) and relaxed much more rapidly (t1/2 = 0.77 +/- 0.09 s). An abrupt exposure to caffeine in 0-Nao solutions produced an increase in 45Ca efflux that persisted for 20 s, and a net loss of Ca content, determined by atomic absorption spectroscopy (AAS), of approximately 4 nmol/mg protein, within 35 s. A comparable net loss of Ca was demonstrated in the presence of 100 microM [Ca]o. The abrupt exposure of cultured cells to 0 Nao in 1.8 mM Ca produced a Ca uptake, estimated with 45Ca, of 3.2 nmol/mg protein X 15 s, but produced no increase in cell Ca content (AAS). In cells in which a 30% increase in Nai was produced by 5 min exposure to 10(-6) M ouabain, the abrupt exposure to 0 Nao produced a Ca uptake of 6 nmol/mg protein X 15 s and an increase in Ca content (AAS) of 4 nmol/mg protein. We conclude that there is an Nao-independent mechanism for Ca extrusion in these cells, presumably a Ca-ATPase Ca pump, with a limited Ca transport capacity of no more than 2 nmol/mg protein X 15 s. This is five times smaller than the demonstrated maximum capacity of the Na-Ca exchanger in these cells. The relaxation of twitch tension in these cells seems to be dependent primarily on sarcoplasmic reticulum uptake of Ca, with a secondary role provided by the Na-Ca exchanger. The Ca pump appears to contribute little to beat-to-beat relaxation.     

Critical evaluation of methods used for the isolation of rat liver hepatocytes for metabolic studies.

Hepatocytes were isolated from normal fed, fasted and alloxan diabetic animals. The best cell preparations were obtained by using low concentrations of collagenase (10–20 mg) and exposing the liver for a very short period of time (10–15 min). Addition of hyaluronidase significantly decreased the glycogen content of the isolated hepatocytes. Glucagon (10⁻¹²M) stimulated glycogenesis in hepatocytes containing high glycogen whereas, in cells containing low glycogen much higher concentration of glucagon was needed (10⁻⁹M). Addition of insulin (100 μunits) stimulated both glycogen and protein synthesis in isolated hepatocytes containing high glycogen. Under these conditions glycogen synthase activity was stimulated by 40%. Incorporation of ¹⁴C phenylalanine into protein was linear for only 3–4 hr in cells containing low glycogen whereas, in cells containing high glycogen incorporating was linear for 8–10 hr. These studies suggest that intracellular glycogen plays an important role in the hormonal regulation of metabolism in hepatocytes.     

Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes.

Normal adult rat liver cells have been successfully cultured as monolayers without subjecting donor animals to a partial hepatectomy before cell isolation. Coating plastic tissue culture dishes with acid soluble calf skin collagen increases the efficiency of cell attachment. Hepatocytes form a monolayer in 24 hr in serum-free L-15 medium although 10% fetal calf serum for 24 hr increases efficiency of attachment. In serum-free medium the mono-layer remains viable for at least one week without added insulin. Cultured cells maintain tyrosine transaminase and four of the five urea cycle enzymes at levels above or equal to freshly isolated cells for 72 hr. Adenylyl cyclase activities are maintained for at least 72 hr, and are stimulated by epinephrine, glucagon and fluoride. Tyrosine transaminase activity is increased in cultured cells by glucagon and dexamethasone, but urea cycle enzymes are not.     

Degradation of 2,4,6-Trinitrophenol (TNP) by Arthrobacter sp. HPC1223 Isolated from Effluent Treatment Plant

Arthrobacter sp. HPC1223 (Genebank Accession No. AY948280) isolated from activated biomass of effluent treatment plant was capable of utilizing 2,4,6 trinitrophenol (TNP) under aerobic condition at 30 °C and pH 7 as nitrogen source. It was observed that the isolated bacteria utilized TNP up to 70 % (1 mM) in R2A media with nitrite release. The culture growth media changed into orange-red color hydride-meisenheimer complex at 24 h as detected by HPLC. Oxygen uptake of Arthrobacter HPC1223 towards various nitro/amino substituted phenols such as dinitrophenol (1.2 nmol/min/mg cells), paranitrophenol (0.9 nmol/min/mg cells), 2-aminophenol (0.75 nmol/min/mg cells), p-aminophenol (0.4 nmol/min/mg cells), phenol (0.56 nmol/min/mg cells) and TNP (2.42 nmol/min/mg cell) was analysed, which showed its additional characteristic of broad substrate catabolic capacity. The present study thus report a novel indigenous bacteria isolated from activated sludge utilized TNP and has broad catabolic potential towards substituted phenols.     

Inhibition of colloid cell swelling in rabbit kidney cortex by disodium glycerophosphate.

Solution based on disodium glycerophosphate have been assessed for ability to inhibit cellular swelling during hypothermic storage of slices of rabbit renal cortex. It was found that slices stored in glycerophosphate media for 3 days gained about 50% of their control weight, whereas control slices stored in chloride based media gained 50% of their control weight after only 2 hr of storage and gained about 128% of their initial weight after 3 days. Electrolyte content of slices stored at 4 °C reflected the change in hydration. When slices were warmed after various storage times, it was found that viability was much better preserved in glycerophosphate saline than in chloride based saline, although the slices stored in glycerophosphate media contained more sodium prior to warming than slices stored in conventional media. Glycerophosphate solutions appear to be suitable for routine use in experiments with renal cortical slices in the vicinity of 0 °C when cell volumes are to be held constant for prolonged periods.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

Effect of cisplatin on the plasma membrane phosphatase activities in ascites sarcoma-180 cells: a cytochemical study

To study the effects of cisplatin [cis-dichlorodiammine-platinum (II)] on tumor cells in the presence or absence of the immune system, animals with ascites sarcoma-180 tumor burden were treated with therapeutic dose levels (9 mg/kg). Similarly, ascites sarcoma-180 cells were maintained in tissue culture media containing the same levels of the drug. Cell samples were taken from the animals at 12-hr intervals for 3 days, whereas samples were drawn from the tissue cultures at 15-, 30-, 45-, and 60-min and at 2-, 3-, 4-, and 5-hr intervals. Treated and untreated cells from in vitro and in vivo experiments, when checked for alkaline phosphatase, 5'-nucleotidase, Ca2+-ATPase, and Na+-K+-ATPase, show a gradual decrease in activity on the plasma membrane. It takes about 60 min for inactivation of any enzyme in vitro, whereas it takes 2 days in in vivo experiments. Quantitative analysis show alkaline phosphatase activity drops from 9.7 to 4.9 nmol in just 15 min, and drops further to 0.79 nmol after 2 hr. Inactivation of various plasma membrane enzymes, resulting in permeability changes, is probably responsible for cell death.     

Chloroform mineralization by toluene-oxidizing bacteria.

Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.     

Artificial gravity and functional plasticity of nerve system. L-[14C]-glutamate uptake by nerve terminals from rat cerebellum and cerebral hemispheres under hypergravity stress

We have investigated the effects of altered gravity on the kinetic parameters of glutamate transport activity. We observed no differences in Km values for cerebellum and cerebral hemisphere nerve terminals (synaptosomes) between control rats- 18,2 +/- 7,6 micromoles (cerebellum), 10,7 +/- 2,5 micromoles (cerebral hemispheres) and animals exposed to hypergravity- 23,3 +/- 6,9 micromoles (cerebellum), 6,7 +/- 1,5 micromoles (cerebral hemispheres). The similarity of this parameter for the two studied groups of animals showed that affinity of glutamate transporter to substrate in cerebellum and cerebral hemispheres was not sensitive to hypergravity stress. The maximal velocity of L-[14C]-glutamate uptake (Vmax) reduced for cerebellum synaptosomes from 9,6 +/- 3,9 nmol/min/mg of protein in control group to 7,4 +/- 2,0 nmol/min/mg of protein in animals, exposed to hypergravity stress. For cerebral hemisphere synaptosomes the maximal velocity significantly decreased from 12,5 +/- 3,2 nmol/min/mg of protein to 5,6 +/- 0,9 nmol/min/mg of protein, respectively.     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Metabolic Fate of 14C-Labeled Glutamate in Astrocytes in Primary Cultures

The metabolic fate of L-[U-14C]- and L-[1-14C]glutamate was studied in primary cultures of mouse astrocytes. Conversion of the uniformly labeled compound to glutamine and aspartate was followed by determination of specific activities after dansylation with [3H]dansyl chloride and subsequent thin layer chromatography of the dansylated amino acids. Metabolic fluxes were calculated from the alterations of specific activities and the pool sizes, which were likewise measured by a dansylation method. Formation of 14CO2 from [1-14C]glutamate was determined by the trapping of CO2 in hyamine hydroxide in a gas-tight chamber, which is, in the known absence of glutamate decarboxylase activity in the cultured astrocytes, an unequivocal expression of the metabolic flux via alpha-ketoglutarate to CO2 and succinyl-CoA. The metabolic fluxes determined by these procedures amounted to 2.4 nmol/min/mg protein for glutamine synthesis, 1.1 nmol/min/mg protein for aspartate production, and 4.1 nmol/min/mg protein for formation and subsequent decarboxylation of alpha-ketoglutarate. The latter process was unaffected by virtually complete inhibition of glutamate-oxaloacetic transaminase with aminooxyacetic acid, indicating that the formation of alpha-ketoglutarate occurs as an oxidative deamination rather than as a transamination. This suggests that the formation of alpha-ketoglutarate from glutamate represents a net degradation, not an isotopic exchange.     

ULTRACYTOCHEMICAL AND BIOCHEMICAL STUDIES OF PLASMA MEMBRANE ATPASE IN THE INDIVIDUAL PARTS OF THE MATURE,DORMANT SEED OF PISUM SATIVUM

The location and activity of a K⁺-ATPase in mature, dormant peas were investigated using two ultracytochemical techniques, as well as biochemical assays of plasma membrane fractions from separate seed parts. Both the Wachstein and Meisel (1957) and the Ernst (1972) cytochemical methods showed plasma membrane-associated reaction product located primarily on the exterior surfaces of the entire pea embryo, except for the stem apex and tip-most cells of the radicle. No plasma membrane-assocated reaction product was found in the seed coat, which typically consists of cells with degenerating protoplasts. Biochemical results showed the highest specific K⁺-ATPase activity in the radicles (775 nmol Pi/mg protein/hr), followed by epicotyls (168 nmol Pi/mg protein/hr) and cotyledons (147 nmol Pi/mg protein/hr). It is proposed that the entire pea embryo may function in the active absorption of nutrients during the initial phases of germination. Additional functions of the enzyme may include cell wall loosening prior to cell elongation, regulation of cytoplasmic pH, and the generation of turgor.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).