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Biomedical subjects

B J Fuller

Publications and source records attributed to B J Fuller.

At least 91 records · Page 5Linked to original sources
PubMed1993

Reoxygenation following hypoxia stimulates lipid peroxidation and phosphatidylinositol breakdown in kidney cortical slices.

Reoxygenation of hypoxic (120 min at 37 degrees) rabbit kidney cortical slices in vitro resulted in a rapid increase in lipid peroxidation and phosphatidylinositol hydrolysis. No changes in phosphatidylinositol breakdown occurred during hypoxia or upon reoxygenation in the absence of calcium. Incubation of renal slices with carbon tetrachloride resulted in increased lipid peroxidation but had no effect on phosphatidylinositol breakdown. It is concluded that altered intracellular calcium homeostasis during reoxygenation is involved in mediating increased phosphatidylinositol hydrolysis through activation of a specific phospholipase C, but that oxidative stress per se does not have a significant effect on the inositol phosphate secondary messenger response in this model system.

Animals↗
PubMed1993

Metabolic activity of freshly prepared and cryopreserved hepatocytes in monolayer culture.

The successful use of isolated hepatocytes for transplantation will, no doubt, require cryopreservation of the cells. However, cryopreservation results in the loss of viability of isolated hepatocytes. In this study a method is described that allows recovery of viable hepatocytes after cryopreservation. Freshly prepared and cryopreserved hepatocytes (1.8 M Me2SO in Euro-Collins solution) were suspended in Dulbecco's culture medium and allowed to form monolayers in Primaria T-25 culture flasks. Viable hepatocytes produced monolayers after 3 h of incubation and viable cultures could be maintained for up to 7 days as judged by trypan blue exclusion. Metabolic viability was measured by incorporation of radiolabeled isoleucine into proteins. Maximal protein synthetic capabilities in freshly prepared hepatocytes was obtained 24 h after culturing. Cryopreserved hepatocytes showed a decrease in protein synthetic capabilities after 24 h culturing. However, longer times of culture resulted in regenerating the capacity of these cryopreserved cells to carry out protein synthesis to levels similar to those of freshly prepared and cultured cells. Thus, incubation of monolayer cultures of cryopreserved hepatocytes for at least 48 h provides a means for reversing the injury caused by freeze-thaw stress and the regeneration of initial viability. This technique may provide a method that is suited for the use of cryopreserved hepatocytes for clinical transplantation.

Animals↗
PubMed1993

Control of intracellular pH in mammalian liver at hypothermia: evidence for a relationship with energy metabolism.

The metabolic status of rat livers during hypothermia has been studied using 31P magnetic resonance spectroscopy. Perfusion with oxygenated buffer at 6-8 degrees C allowed maintenance of ATP, while pH increased to values in the range 7.7-7.9. In organs depleted of ATP by a short (2 h) period of cold ischemia, pH fell to 6.92 +/- 0.10. If these livers were reperfused with hypoxic buffer at hypothermia, two distinct responses were noted. In one group (responders), there was evidence of ATP resynthesis and in these organs pH returned to 7.90 +/- 0.28. In the second group (non-responders), there was no recovery of ATP synthesis and pH remained depressed at 6.97 +/- 0.07. In another group, adenine nucleotides were severely depleted by 24 h of cold ischemia, and in these livers there was again no significant recovery of ATP synthesis during hypoxic reperfusion and pH remained at 7.03 +/- 0.25. These results suggest that (a) there is an apparent relationship between energy metabolism and control of intracellular pH in the hypothermic mammalian liver, and (b) that intracellular pH may shift in liver at hypothermia to values predicted by the alpha-stat hypothesis.

Adenosine Triphosphate↗
PubMed1992

Vitrification of mouse oocytes using short cryoprotectant exposure: effects of varying exposure times on survival.

The effects on oocyte viability of varying the duration of exposure to cryoprotectants before rapid cooling to -196 degrees C were examined, using the vitrification protocol of Nakagata. A very short exposure (15 sec) was found to be optimal, resulting in an overall rate of development from vitrified oocytes to hatching blastocysts of 31.8%. Very high rates of survival (77-89%) of oocytes exposed to the cryoprotectant media, but without the vitrification, together with extreme variability in results between straws in the vitrified groups, suggest that losses in viability during vitrification may result from ice damage during devitrification of the medium.

Animals↗
PubMed1992

Measurements of the membrane water permeability (Lp) and its temperature dependence (activation energy) in human fresh and failed-to-fertilize oocytes and mouse oocyte.

The volumetric response of oocytes during rapid alterations of the extracellular osmotic environment were recorded using video microscopy. From these observations, the kinetics of water loss for human and mouse oocytes were determined over the temperature range 37 to 10 degrees C, including 37, 30, 20, and 10 degrees C. The changes in diameter of oocytes were measured over a 5-min period and a computer model was used to derive values for membrane water permeability (Lp) and inactive volume (Vb) and to compare the experimental data to the predicted values. The results for the mouse oocyte Lp were comparable to values determined by other methods. However the human data, for both failed-to-fertilize and fresh oocytes, have a wide range of values with large standard deviations. The Lp values at the various temperatures were used to calculate the Arrhenius activation energy (Ea). An Ea value of 9.48 kcal/mol was found for the fresh mouse oocyte, whereas the activation energy for human oocytes was extremely low, 3.73 kcal/mol for fresh oocytes and 1.93 kcal/mol for failed-to-fertilize oocytes.

Animals↗
PubMed1992

Deterioration of cold-stored tissue specimens due to lipid peroxidation: modulation by antioxidants at high subzero temperatures.

It is often necessary to store tissue specimens in subzero conditions for assay in batches. During storage at -20 degrees C we found that sufficient lipid peroxidation occurred in rat liver homogenates in phosphate-buffered saline to affect subsequent malondialdehyde assays. This peroxidation did not occur at -196 degrees C. The ratio of oxidized to reduced glutathione increased with storage at -20 degrees C and the level of conjugated dienes increased progressively. The addition of a specific free radical scavenger, superoxide dismutase (200 u/ml) reduced the level of malondialdehyde (P < 0.001) during -20 degrees C storage for periods of 28 days but failed to prevent the changes in the glutathione ratio or dienes. Storage in a less specific free radical scavenger, 0.25 molar sucrose/EDTA, instead of phosphate-buffered saline totally prevented the malondialdehyde production over similar storage periods.

Animals↗
PubMed1992

Fertilization and embryonic development of human oocytes after cooling.

Injury to living cells resulting from rapid cooling to temperatures at or near 0 degrees C has long been recognized, and the phenomenon, which is termed 'cold shock', has been known to occur in some mammalian gametes. Although human embryos have been successfully stored at low temperatures, cryopreservation of the human oocyte is proving to be more difficult. Whether or not this lack of success is a direct result of cellular injury brought about by 'cold shock' is the purpose of the current investigation. Human oocytes were cooled, in the absence of cryoprotectants, at two different cooling rates (-3 degrees C/min and -1000+ degrees C/min) to a temperature of 0 degrees C and rewarmed prior to insemination. In both cases fertilization after cooling was similar to the rates achieved in a routine in-vitro fertilization and embryo transfer procedure. After cooling at -3 degrees C/min, the rate of fertilization was 19/22 (86%) and after cooling at -1000+ degrees C/min, 9/9 (100%), with non-cooled control rates of 62/87 (71%) and 35/50 (70%) respectively. Fertilized oocytes from both groups were successfully cultured for a further 24 h before termination of the experiment.

Cryopreservation↗
PubMed1991

Vitrification of mouse oocytes: improved rates of survival, fertilization, and development to blastocysts.

Rall and Fahy's (1985) vitrification procedure for the cryopreservation of 8-cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1% of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8% developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4% of vitrified oocytes and 72.4% of nonvitrified VS1-exposed oocytes developing to blastocysts; 85.7% of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes to 5-8 degrees C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure.

Animals↗
PubMed1991

The effects of cryopreservation on protein synthesis and membrane transport in isolated rat liver mitochondria.

Protein synthesizing activity and membrane transport were examined in fresh and cryopreserved isolated rat liver mitochondria. In the presence of 0.6, 1.2, and 1.8 M final concentrations of dimethyl sulfoxide (Me2SO), both metabolic parameters were considerably inhibited in the fresh samples and even more inhibited in the cryopreserved specimens. However, simple exposure to this penetrating cryoprotectant, followed by its subsequent removal by washing, did not seem to affect significantly the examined functions. When different freeze-thaw regimes were investigated, it was observed that optimal recovery of protein synthesis and membrane transport functions were obtained when fast freezing took place in the absence of Me2SO.

Adenosine Triphosphate↗
PubMed1990

The effects of cryopreservation on membrane integrity, membrane transport, and protein synthesis in rat hepatocytes.

The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.8 M of Me2SO during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me2SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na+,K+)ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of L-[U-14C]isoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of alpha-amino[1-14C]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180 min, subsequent addition of labeled alpha-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at significantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined.

Amino Acids↗