Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding

[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Toxicity oft-butylhydroperoxide in hepatocyte monolayers exposed to hypoxia and reoxygenation

Summary Inasmuch as it is known that the toxicity of anesthetic agents is potentiated by hypoxia and that the reductive metabolism of these agents results in the formation of lipid hydroperoxides, we investigated the toxicity of hydroperoxides under low-oxygen concentrations. We found that hypoxia exacerbates the toxicity oft-butyl hydroperoxide, shifting the dose-response curve oft-butyl hydroperoxide vs. lysis of hepatocytes approximately an order of magnitude to the left. Furthermore, although at the end of a 4-h exposure to 0.5% O₂ hepatocyte monolayers seemed normal by three indices (release of⁵¹Cr and serum glutamate transaminase or exclusion of trypan blue), they were completely lysed after an additional 20 h reoxygenation at 20%. O₂. In contrast, monolayers exposed to 2% O₂ for 4 h seemed normal after 20 h reoxygenation. However, cells exposed to both a subtoxic dose of hydroperoxide and 4 h of 2% O₂, although seeming healthy at the end of the hypoxic period, were completely lysed within 20 h after reoxygenation. The study was supported by grant OH 00978 from the National Institutes for Occupational Safety and Health, Atlanta, Georgia.     

Species variations amongst proteinases in liver lysosomes

Cathepsin B, H, L and D activities in liver lysosomes were compared between species. Although cathepsin B and D were detected in bovine, pig, chicken and rat liver, striking species differences were evident for cathepsin H and L. Cathepsin L activity was particularly high in chicken lysosomal extracts, but could not be detected in bovine and pig extracts. Whereas there was no significant cathepsin H activity in bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H activity.     

Cuticular lipids for species recognition of mole crickets (Orthoptera: Gryllotalpidae): II. Scapteriscus abbreviatus,S. acletus,S. vicinus,S. sp., and Neocurtilla hexadactyla

Qualitative analyses were made from whole-body cuticular extracts of Scapteriscus abbreviatus, Scapteriscus acletus, Scapteriscus vicinus, and Neocurtilla hexadactyla by isothermal and temperature-programmed gas chromatography. Adults of both sexes and nymphs of each species were collected in Florida. The chromatographic profiles of peaks were distinct and easily recognizable for each species, regardless of sex or developmental stage. Distinct sexual differences were found in S. acletus and S. abbreviatus. Specimens of S. abbreviatus from Puerto Rico and S. vicinus from Bolivia produced gas chromatography (GC) traces very similar to those of conspecifics collected in Florida. Evidence is presented to illustrate the potential importance of volatile cuticular lipid analysis as a tool for mole cricket identification. Cuticular extracts of an undescribed short-winged species of Scapteriscus from Bolivia were also examined and produced GC traces unlike those of any other species analyzed to date.     

The amount and nature of glutathione transferases in rat liver microsomes determined by immunochemical methods

The amount and nature of glutathione transferases in rat liver microsomes were determined using immunological techniques. It was shown that cytosolic glutathione transferase subunits A plus C, and B plus L were present at levels of 2.4 ± 0.6 and 1.5 ± 0.1 μg/mg microsomal protein, respectively. These levels are 10-times higher than those for non-specific binding of cytosolic components judging from the distribution of lactate dehydrogenase, a cytosolic marker. The possibility that a portion of these glutathione transferases is functionally localized on the endoplasmic reticulum is discussed. A previously described microsomal glutathione transferase which is distinct from the cytosolic enzymes is present in an amount of 31 ± 6 μg/mg microsomal protein.     

Kinetics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate

The herbicide glyphosate (N-phosphonomethyl glycine) is a potent reversible inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity of the purified arom multienzyme complex from Neurospora crassa. Inhibition of the EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate, with K(i) 1.1 microM, and uncompetitive with respect to shikimate-3-phosphate. The kinetic patterns are consistent with a compulsory order sequential mechanism in which either PEP or glyphosate can bind to an enzyme: shikimate-3-phosphate complex.     

The experimental herbicide UKJ72J is an inhibitor of succinate oxidation in plant mitochondria

Fragments derived from human plasma fibronectin by enzymatic degradation were tested in the Boyden chamber for chemotactic activity towards various fibroblast strains. The results provide clear evidence that the chemotactic activity is restricted to a defined region of the fibronectin molecule which is the same for various fibroblast strains. The active domain is localized between the collagen binding site and the major heparin binding site, about 170 kDa apart from the N-terminal and about 70 kDa from the C-terminal ends of the two subunit peptide chains.     

A rapid one-step purification of NADPH-cytochrome c (P-450) reductase from rat liver microsomes

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.