Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

Microbial biomass production from rice straw by fermentation with Penicillium janthinellum

Production of microbial biomass through fermentation of pretreated rice straw using Penicillium janthinellum (St-F-3B) is reported, with emphasis on the use of non-effluent generating pretreatment procedures. The fungus readily metabolized a rice straw substrate that had been subjected to alkali pretreatment by steaming at atmospheric pressure followed by the neutralization of the alkali. The crude protein content of the microbial biomass averaged 15–20%. The fermentation could be carried out in aerated-agitated fermenters using fertilizer grade nutrient salts to produce a biomass with 17.5% crude protein. When operated on a semicontinuous basis using 20% of the previous batch as inoculum, successive batches produced a biomass product with 12–19% crude protein content in 48 h. The problems of developing a technology for protein from agricultural residues with particular reference to developing countries are discussed.     

Decreased Benzodiazepine Receptor Density in Rat Cerebellum Following Neurotoxic Doses of Phenytoin

Abstract: The effect of acute and chronic administration of phenytoin on [³H]-flunitrazepam binding was examined in the rat cerebellum. There was no significant effect of phenytoin on [³H]flunitrazepam binding in the rat cerebellum 1 and 6 h after a single i.p. injection of 200 mg/kg of phenytoin. However, after 14 days and 28 days of chronic phenytoin administration, significant de-creases in [³H]flunitrazepam receptor density were observed, with no changes in apparent affinity constants in the rat cerebellum. This effect of phenytoin was dose-dependent, as lower doses of phenytoin (100 mg/kg/day) for 14 or 28 days produced no alterations in [³H]flunitrazepam binding in the rat cerebellum. Light-microscopic examination of the rat cerebellum treated with 200 mg/kg/day of phenytoin for 14 days showed degeneration of the Purkinje cells, with edematous Bergmann astrocytes. These data provide evidence for the neuronal localization of benzodiazepine receptors on cerebellar Purkinje cells.     

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.     

Capsules for Frozen Sectioning of Small Specimens

It was necessary to make sections of small unfixed specimens which had been frozen while still immersed in their normal culture medium. The principal difficulty stemmed from the poor sectioning quality of the frozen culture medium. A capsule is described which has a narrow well in which the tissue specimen fits snugly within a small amount of culture medium. After freezing, the whole capsule is sectioned and the resulting sections, being nearly devoid of culture medium, are of good quality.     

Effect of fatty acids on cellulase production by Penicillium funiculosum and its mutants

Summary Penicillium funiculosum and its mutants namely BU-36 and N-4 responded differently to the addition of fatty acids. Addition of 0.1% oleic, linoleic, and linolenic acids resulted in significant increase in extracellular exo-glucanase and -glucosidase in wild and N-4 strains, whereas no appreciable increase was noticed in BU-36. However, BU-36 showed positive response with 0.1% palmitic and stearic acids. In all the strains, the addition of different fatty acids did not have any effect on endoglucanase activity. Our results indicate that fatty acids do have a role in the release of the cell-bound cellulolytic enzymes.NCL Communication No. : 4113     

Increases in coronary vein CO2 during cardiac resuscitation

We investigated the aortic, mixed venous, and great cardiac vein acid-base changes in eight domestic pigs during cardiac arrest produced by ventricular fibrillation and during cardiopulmonary resuscitation (CPR). The great cardiac vein PCO2 increased from a control value of 52 +/- 2 to 132 +/- 28 (SD) Torr during CPR, whereas the arterial PCO2 was unchanged (39 +/- 4 vs. 38 +/- 4). The coronary venoarterial PCO2 gradient, therefore, increased remarkably from 13 +/- 2 to 94 +/- 29 Torr. The simultaneously measured great cardiac vein lactate concentrations increased from 0.24 +/- 0.06 to 7.3 +/- 2.34 mmol/l. Much more moderate increases in the lactate content of aortic blood from 0.64 +/- 0.25 to 2.56 +/- 0.27 mmol/l were observed. Increases in great cardiac vein PCO2 and lactate were highly correlated during CPR (r = 0.91). After successful CPR, the coronary venoarterial PCO2 gradient returned to normal levels within 2 min after restoration of spontaneous circulation. Lactate content was rapidly reduced and lactate extraction was reestablished within 30 min after CPR. These studies demonstrate marked but reversible acidosis predominantly as the result of myocardial CO2 production during CPR.     

Effect of phenobarbital on metabolism of polyphosphoinositides of rat brain synaptosomes

Synthesis and degradation of polyphosphoinositides in a rat brain synaptosome preparation were depressed by phenobarbital. Phosphatidylinositol-4-phosphate kinase (PIP-kinase), the enzyme which synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2) was most strongly affected (50% inhibition at 3 mM phenobarbital); phosphatidylinositol (PI-kinase) followed (50% at 15 mM). The phosphoesterases were less sensitive: PIP-monoesterase (50% at 39 mM), PIP2-monoesterase (at 47 mM), and, least inhibited, PIP-diesterase (50% at 65 mM) and PIP2-diesterase (at 68 mM). Phenobarbital by inhibiting PIP-kinase may reduce the membrane concentration of PIP2 and thus dampen the stimulus-response which leads to the hydrolysis of PIP2 and the formation of the second messenger, inositol-1,4,5-trisphosphate (IP3), involved in mobilization of intracellular Ca2+.     

Potential of DNA markers in detecting divergence and in analysing heterosis in Indian elite chickpea cultivars

 Molecular markers such as RAPDs and microsatellites were used to study genetic diversity in 29 elite Indian chickpea genotypes. In general, microsatellites were more efficient than the RAPD markers in detecting polymorphism in these genotypes. Among the various microsatellites, (AAC)₅, (ACT)_5, (AAG)₅ and (GATA)₄ were able to differentiate all 29 chickpea cultivars. The mean value of probability of identical match by chance was 2.32×10^-25 using DraI-(ACT)₅, TaqI-(AAC)₅, TaqI-(AAG)₅ and TaqI-(GATA)₄ enzyme-probe combinations. The dendrogram, constructed on the basis of similarity index values, grouped the chickpea genotypes into five main clusters with 8 cultivars genetically distant and outgrouped from the main clusters. To investigate if DNA markers are useful in predicting F₁ performance and heterosis in chickpea, we crossed 8 genotypes having important agronomic characters in a diallel set. The F₁s and their parents in the diallel set were analysed for agronomic traits for better parent and midparent heterosis. Heterosis was found to be much higher for yield than for yield components that fit a multiplicative model. The analysis of genetic divergence using D² statistics clustered the 8 cultivars into two groups. Although molecular marker-based genetic distance did not linearly correlate to heterosis, two heterotic groups could be identified on the basis of the general marker heterozygosity. Received: 1 August 1998 / Accepted: 29 September 1998