Biomedical subjects

P A Wilce

Publications and source records attributed to P A Wilce.

At least 73 records · Page 4Linked to original sources

PubMed1988

Studies on regenerating liver and hepatoma plasma membranes--I. Lipid and protein composition.

1. Plasma membranes were isolated from normal liver, Morris hepatoma 7288C and regenerating liver, 6, 15, 24, and 48 hr after partial hepatectomy. 2. The cholesterol/phospholipid ratio was lower in regenerating liver 6 hr after partial hepatectomy (0.51) compared to the sham control (0.68), returning to normal after 15 hr. This was accompanied by a small increase in palmitic acid (16:0). There were no other changes in the lipid composition in regenerating hepatocytes in the first 48 hr after partial hepatectomy. 3. Analysis of lipid composition showed a higher cholesterol/phospholipid ratio in the hepatoma plasma membrane compared to normal liver accompanied by an increase in saturation of the fatty acyl groups of the phospholipids. There were also significant changes in the phospholipid classes. 4. There was no change in the two-dimensional electrophoretic profile of membrane proteins in the early stages of liver regeneration, however hepatoma membranes showed significant differences in protein profile. 5. These changes in the lipid composition of the hepatoma plasma membrane would have the effect of decreasing the average fluidity of the membrane and together with the changes in protein composition may be significant in the altered growth of the hepatoma. Changes in the lipid composition of the hepatocyte plasma membrane early in liver regeneration may reflect the onset of renewed cell division.

Animals↗

PubMed1988

Studies on regenerating liver and hepatoma plasma membranes--II. Membrane fluidity and enzyme activity.

1. Rat hepatocyte plasma membranes isolated from Morris hepatoma 7288C, normal and regenerating liver were labelled with the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. 2. Steady-state fluorescence polarisation measurements indicated an increased fluidity of the membranes in the early stages of regeneration, returning to normal levels after 48 hr. 3. There was a decrease in hepatoma plasma membrane fluidity compared to normal hepatocytes. Changes in fluorescence polarisation with temperature (Arrhenius studies) indicate an increase in the lower critical temperature for the membrane lipid thermotropic transition of hepatoma compared to normal liver plasma membranes. 4. These changes in membrane lipid fluidity alter the activation of some intrinsic and extrinsic membrane bound enzymes.

5'-Nucleotidase↗

PubMed1988

Amino acid oxidation in the tumor-bearing rat.

It has been proposed that the presence of a tumor leads to uncontrolled oxidation of the branched chain amino acids in the peripheral tissue of the host animal. This was investigated in Buffalo strain rats bearing the tumor, Morris hepatoma 5123tc. The rate of 14CO2 production from [1-14C] leucine was observed to be significantly increased in the tumor-bearing rats in vivo. This increase may be attributed to an observed increase in the activity of the rate-limiting enzyme of leucine oxidation (2-oxoisocaproate dehydrogenase) not only in host skeletal muscle but also in the tumor. In contrast, the oxidative capacities of host tissues (liver and muscle) towards tyrosine were, however, decreased by the presence of a tumor, an observation consistent with tumor induction of fetal-like changes in host tissue metabolism.

Animals↗

PubMed1987

Chronic consumption of ethanol increases glucose oxidation in isolated cerebral tissue of rat.

Tolerance to ethanol was induced in male Wistar rats by adding 15% ethanol to the drinking water for 3 and 9 months. The oxidation of [U-14C]glucose was measured in cerebral cortex prisms. 14CO2 production in prisms for ethanol-treated animals was markedly increased when compared to controls. Neither a single injection of a low dose of ethanol nor a comparable concentration of ethanol in vitro had this stimulatory effect.

Alcoholism↗

PubMed1987

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse uterine epithelial cells.

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase was studied in mouse uterine epithelium. The enzyme was rapidly inactivated during incubation with ATP/Mg2+ in vitro, and could be re-activated by incubation with partially purified rat liver phosphoprotein phosphatase. Enzyme activity was rapidly inhibited by mevalonate injection in vivo to approx. 30% of control. The percentage of total enzyme active in vivo was measured by inclusion of NaF in the isolation buffers. The percentage of enzyme active in vivo 18 h after stimulation by oestrogens remained at approx. 25% after inhibition of activity by mevalonate injection, cholesterol feeding or progesterone pretreatment. However, 9 h after oestrogen stimulation, cholesterol feeding inhibited enzyme activity to 57% of control, 94% of which was in the active form. We conclude that, although all components for a reversible phosphorylative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity are present in uterine epithelial cells, a role in the rapid changes in epithelial enzyme activity has not been demonstrated.

Animals↗

PubMed1987

Effect of ethanol on the GABA-benzodiazepine receptor in brain.

Chronic ethanol inhalation produced no change in the number or affinity of flunitrazepam binding sites on well-washed synaptic membranes prepared from male Quackenbush mice, but produced a significant decrease in the capacity of GABA to enhance flunitrazepam binding. This decrease was characterized by a higher EC50 (1.4 microM compared to 0.6 microM) and a lower maximal level of enhancement (162% compared to 172%) for tissue from the chronically treated animals compared to tissue from control animals. Acute ethanol treatment or ethanol incubated in vitro with the brain membranes did not produce changes in any of the flunitrazepam binding parameters. These results support other findings that chronic ethanol may affect the coupling of various sites on GABA-A receptor-ionophore complexes in brain.

Animals↗

PubMed1987

Hepatic cholesterol synthesis and hydroxymethylglutaryl CoA reductase activity after injection of methylazoxymethanol acetate.

Cholesterol synthesis and 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) in the liver of rats at various times (7, 22, 45 and 314 days) after injection with the carcinogen, methylazoxymethanol acetate (MAMA) is reported. Seven days after treatment, an increase in both cholesterol synthesis and HMG-CoA reductase activity was observed. Elevated HMG-CoA reductase activity and reduced dietary feedback was present 22 days after carcinogen. Cholesterol synthesis was normal at this time but dietary cholesterol failed to significantly reduce synthesis. Forty-five days after carcinogen both cholesterol synthesis and HMG-CoA reductase activity had returned to normal. Both parameters were normal 314 days after carcinogen. The enzyme gamma-glutamyl transferase was also elevated at 7, 22 and 314 days. These results indicate that HMG-CoA reductase activity and cholesterol synthesis exhibit different regulatory characteristics during the early stages of hepatocarcinogenesis initiated by MAMA injection.

Animals↗

PubMed1987

Protein synthesis in the tumour-bearing rat.

The rates of protein synthesis in liver, muscle and tumour of animals bearing either a rapidly growing (7288c) or slow-growing (5123c) hepatoma were determined. Liver protein synthesis was increased whilst, in contrast, protein synthesis in muscle decreased. Tumour protein synthesis comprised between 15% and 43% of total body protein synthesis. Since food intake of tumour-bearing animals was not significantly different from that of controls, these changes were not attributable to an anorexic component of the tumour-bearing state but may reflect a systemic tumour-mediated effect.

Animals↗

PubMed1986

Cholesterol synthesis and HMG CoA reductase activity during hepatocarcinogenesis in rats.

Two treatment regimes were used to produce preneoplastic foci (as determined by the presence of gamma-glutamyl transferase) in rat liver. Increased [14C]acetate incorporation into cholesterol and 3-hydroxy-3-methyl glutaryl CoA reductase activity were associated with high levels of gamma-glutamyl transpeptidase and foci formation. Dietary feedback inhibition of both [14C]acetate incorporation and 3-hydroxy-3-methyl glutaryl CoA reductase activity was reduced at a selected time when gamma-glutamyl transpeptidase activity was high. These changes could not be accounted for by a regenerative response in the liver.

2-Acetylaminofluorene↗

PubMed1986

The effect of ethanol on the lipoprotein metabolism of aortic smooth muscle cells.

The effect of ethanol exposure on the binding and metabolism of bovine low density lipoprotein by bovine arterial smooth muscle cells was studied. In cells exposed to ethanol (80 mM) for 48 hr or 14 days and incubated with low density lipoprotein for 24 hr there was a reduction in the amount of low density lipoprotein internalised at all concentrations of lipoprotein. There was no effect on the rate of degradation of the low density lipoprotein and no demonstrable changes in the amount of low density lipoprotein bound to the cell surface at high concentrations of low density lipoprotein. Similar results for internalisation and degradation were obtained in a time dependent study. Binding was shown to be reduced in the ethanol treated cells (48 hr) when low concentrations (5 micrograms/ml) of low density lipoprotein were incubated for short periods (less than 3 hr). Scatchard plot analysis indicated that this reduced binding may be due to a reduction of receptor numbers in these cells.

Animals↗

PubMed1985

A mechanism for the in vitro stimulation of adrenal cortisol biosynthesis by epidermal growth factor.

EGF stimulates adrenal steroidogenesis in ewes and in ovine adrenal slices. In vitro, The stimulation is blocked by the cholesterol synthesis inhibitors compactin and AY 9944. EGF stimulates the incorporation of [14C]acetate into cholesterol. EGF increases the activity of the rate limiting enzyme in cholesterol biosynthesis, HMG CoA reductase. EGF has no effect on the levels of any intermediates involved in the conversion of pregnenolone to cortisol, although ACTH produced changes consistent with 17 alpha-hydroxylase activation. We propose that EGF increases adrenal cortisol synthesis in vitro by a stimulation of cholesterol precursor biosynthesis mediated through activation of HMG CoA reductase.

Adrenal Cortex↗

PubMed1985

Effects of ethanol on the lipid composition of bovine vascular smooth muscle cells in culture.

Ethanol (50 mM) had no effect on the growth rate or viability of arterial smooth muscle cells over 3.5 days. The cholesterol:phospholipid ratio of the cells was unchanged after 7 days exposure. The major phospholipid components phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were unchanged by ethanol exposure. Sphingomyelin content fell significantly within 12 hr. There were major changes in the fatty acid composition of the phospholipids with a reduction in saturated fatty acids and an increase in unsaturated fatty acids.

Animals↗

PubMed1985

Effect of assay temperature on the kinetics of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in rat liver and Morris hepatoma 5123C.

A procedure is described for the assay of 3-hydroxy-3-methylglutaryl CoA-reductase (HMG-CoA reductase) in a large number of samples with minimal benchwork and within a 24-hr period. The Michaelis constants for HMG-CoA reductase were determined for microsomal enzyme from the liver of normal and cholesterol-fed rats and Morris hepatoma 5123C. The apparent Km D-HMG-CoA was ca. 3.5 microM and was not affected by assay temperature or cholesterol feeding. The apparent Km NADPH for microsomal HMG-CoA reductase was 10-15 microM and similarly was not affected by assay temperature. The Arrhenius plot parameters (activation energy and transition temperatures) were the same whether determined using the reaction velocity from fixed substrate concentrations or V from subtraction curves. This confirmed that values obtained using fixed saturating substrate concentrations are valid and not affected by a temperature-dependent alteration in the affinity of the enzyme for its substrates.

Animals↗

PubMed1984

Influence of ethanol on fatty acid composition of phospholipids in cultured neurons.

Animals chronically exposed to ethanol show changes in neural membrane lipids which may underlie the development of tolerance and physical dependence. The object of this study was to investigate changes in the fatty acid composition of neuronal phospholipids cultured in the presence of ethanol (55 or 110 mM) for periods up to 7 days. Decreases were observed in the percentage of individual and total saturated fatty acids, while the double bond index: total saturated fatty acid ratio, increased. These changes do not support the hypothesis that neural membrane lipid composition changes to counteract the fluidizing action of ethanol.

Animals↗

PubMed1984

Stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in mouse uterine epithelial cells by oestradiol-17 beta.

The characteristics of 3-hydroxy-3-methylglutaryl-CoA reductase from mouse uterine epithelial cells were studied. Preliminary experiments showed that enzyme activity was stimulated approx. 10-fold 18h after administration of 100ng of oestradiol-17 beta. This activity was associated with all particulate fractions of the uterine luminal cell. The Km for D-3-hydroxy-3-methylglutaryl-CoA was 5.54 +/- 1.12 microM. The detailed time-course of oestrogen stimulation showed two peaks of activity, 9 and 15h after hormone treatment. The DNA content of the epithelial cells doubled between 6 and 12h after hormone treatment, whereas the protein content increased linearly over the 18h period. The peak of enzyme activity at 9h is associated with early S phase of the epithelial cells; the peak at 15h may be associated with a second S phase or with mitosis. Pretreatment with progesterone for 3 days before injection of oestradiol-17 beta (a treatment which inhibits uterine epithelial DNA synthesis) reduced the oestrogenic stimulation of enzyme activity by 63%; progesterone treatment alone did not stimulate enzyme activity. These data suggest that uterine epithelial 3-hydroxy-3-methylglutaryl-CoA reductase may play an important role in the cell cycle in this tissue.

Animals↗

PubMed1983

Effect of ethanol on cholesterol and phospholipid composition of HeLa cells.

Chronic exposure of animals to ethanol leads to changes in membrane lipid composition which may be related to the development of tolerance and physical dependence. The object of the present study was to investigate this phenomenon at a cellular level. HeLa cells were grown in the presence of ethanol (86 mM) for periods of up to 9 days. Both the cholesterol and phospholipid concentration of these cells increased during this period but the cholesterol:phospholipid ratio remained unchanged. Among the phospholipid classes phosphatidic acid decreased while phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine increased rapidly, returning toward control values by 9 days. Significant decreases were observed in saturated (14:0, 16:0) and monoenoic (16:1, 18:1) fatty acids while the major polyenoic fatty acid (20:4) increased. It is concluded that cultured mammalian cells represent a useful model for investigation of the direct effects of ethanol on membrane lipid metabolism.

Cholesterol↗

PubMed1983

Reversible phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase in Morris hepatomas.

The reversible phosphorylation of microsomal 3-hydroxy-3-methylglutaryl CoA reductase in host liver and hepatoma 5123C has been investigated. The percentage of the total enzyme activity in vivo was similar in the normal liver, host liver and hepatoma 5123C. The inclusion of 30 mM EDTA and 10 mM mevalonic acid in assays of 3-hydroxy-3-methylglutaryl CoA reductase inactivation in vitro eliminated artifacts generated by the presence of mevalonate kinase. Inactivation of 3-hydroxy-3-methylglutaryl CoA reductase from normal liver, host liver and hepatoma occurred at a similar rate with similar half-times. We conclude that phosphorylation/dephosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase occurs in hepatomas and that the lack of dietary cholesterol feedback inhibition in the hepatomas is not a result of a defect in this particular aspect of the reversible phosphorylation system.

Adenosine Triphosphate↗

PubMed1983

Inhibition of sterol and DNA synthesis in peripheral blood lymphocytes by AY9944.

The effect of the inhibitor of cholesterol synthesis AY9944 on DNA synthesis was tested in phytohemagglutinin stimulated lymphocytes incubated in delipidated serum. AY9944 caused a decrease in cholesterol synthesis from [14C] acetate and an accummulation of [14C] 7-dehydrocholesterol. This inhibition of cholesterol synthesis resulted in an inhibition of DNA synthesis in a dose related manner. Inhibition could be partly reversed by added lipoprotein. Inhibition of DNA synthesis occurred if AY9944 was added at the early stages of blast transformation but not when the response was well established. These findings suggest that some endogenous cholesterol synthesis may be required for the complete phytohemagglutinin stimulation of lymphocytes.

Cholesterol↗