Methylamine does not inhibit rates of endogenous lipolysis in isolated myocardial cells from rat heart

Triacylglycerol lipase activity with a pH optimum of 5 was present in homogenates of myocardial cells from rat heart. Acid lipase activity was inhibited by serum, heparin, and increased ionic strength. Methylamine, a lysosomotropic agent, did not inhibit the basal or isoproterenol-stimulated rate of endogenous lipolysis as measured by glycerol output from control myocytes. Similarly, accelerated rates of glycerol output that are a consequence of an elevation in the intracellular stores of triacylglycerols in myocytes from diabetic rat hearts and from myocytes prepared with free fatty acids in the isolation solutions were not reduced by methylamine. Therefore, the acid lysosomal triacylglycerol lipase must not be involved in the mobilization of endogenous triacylglycerols in myocardial cells from rat heart.     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

Long term incubation of cardiac myocytes with oleic acid and very-low density lipoprotein reduces heparin-releasable lipoprotein lipase activity

An exogenous [³H]triolein emulsion was hydrolyzed by intact cardiac myocytes with functional LPL located on the cell surface. This surface-bound LPL could be released into the medium when cardiac myocytes were incubated with heparin. Incubation of cardiac myocytes with VLDL, or the products of TG breakdown, oleic acid or 2-monoolein, did not increase LPL activity in the medium. However, incubation of cardiac myocytes with either VLDL or oleic acid for > 60 min did reduce heparin-releasable LPL activity. In the heart, this inhibitory effect of FFA could regulate the translocation of LPL from its site of synthesis in the cardiac myocyte to its functional site at the capillary endothelium.Abbreviations LPL lipoprotein lipase - TG triacylglycerol - FFA free fatty acids - VLDL very-low density lipoprotein     

Diacylglycerol lipase and kinase activities in rat brain microvessels

Diacylglycerols can accumulate transiently in intact cells as a consequence of the degradation of phosphatidylinositol by phospholipase C, but little information is available concerning their metabolic fate in the vascular endothelium. Diacylglycerol lipase and kinase activities were measured in rat brain microvessel preparations. Lipase activity, measured by the release of free fatty acids, was much greater at pH 4.5 than at pH 7. The acid lipase was predominantly particulate and likely originated in lysosomes, whereas the neutral lipase was mainly soluble. The fatty acid at the sn-1 position of the diacylglycerol substrate was hydrolyzed faster than that at the sn-2 position at both pH 4.5 and 7. The 2-monoacylglycerol accumulated at pH 4.5 but not at 7 due to the presence of a monoacylglycerol lipase activity with a neutral pH optimum. The formation of phosphatidic acid (kinase activity) was also measured in microvessels. When lipase and kinase activities were measured simultaneously, the formation of phosphatidic acid from a 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol substrate was 4-fold greater than the release of fatty acid (oleate) from the sn-2 position. Introduction of arachidonic acid to the sn-2 position of the diacylglycerol substrate increased kinase activity but reduced lipase activity. The release of fatty acids from the sn-2 position of phosphatidic acid could not be detected.     

Treatment of cardiac myocytes with 8-(4-chlorophenylthio)-adenosine 3'',5''-cyclic monophosphate, forskolin or cholera toxin does not stimulate cellular or heparin-releasable lipoprotein lipase activities.

Incubation of isolated cardiac myocytes with 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) or 100 microM-forskolin for 2 1/2 h did not increase the heparin-induced release of lipoprotein lipase (LPL) into the medium. When LPL activity in cardiac myocytes was depleted by treatment of rats with cycloheximide (2 mg/kg; 2.5 h) and inclusion of the protein-synthesis inhibitor in the isolation solutions, incubation with CPT-cAMP or forskolin did not influence the rate of repletion of LPL activity in cells or the recovery of heparin-releasable LPL activity. Although the administration of cholera toxin (0.5 mg/kg; 16-17 h) to rats increased LPL activity in a low-speed supernatant fraction from heparin-perfused hearts, LPL activity was not increased in cardiac myocytes from cholera-toxin-treated rat hearts, and the heparin-induced release of LPL was unchanged. Incubation of cultured ventricular myocytes with 1 microgram of cholera toxin/ml or 500 microM-CPT-cAMP for 24 h did not increase cellular LPL activity or LPL released into the culture medium after a 40 min incubation with heparin. Therefore interventions that stimulate adenylate cyclase activity (forskolin, cholera toxin) or incubation with CPT-cAMP do not increase cellular LPL activity or promote the translocation of LPL to a heparin-releasable fraction in cardiac myocytes.     

Restriction Fragment Length Polymorphism Mapping of Quantitative Trait Loci for Malaria Parasite Susceptibility in the Mosquito Aedes Aegypti

Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F(2) populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs[2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filarial nematode Brugia malayi and the yellow fever virus.     

Effect of hyperinsulinemia on acid cholesterol ester hydrolase activity in liver, heart and epididymal fat pad preparations from rats and mice

The effect of insulin on lysosomal acid cholesterol ester hydrolase activity was studied in liver, heart and fat pad preparations from rats and mice. Hyperinsulinemia was induced for a period of 6 days in rats by the subcutaneous administration of exogenous insulin by an osmotic minipump. The effect of more chronic endogenous hyperinsulinemia was studied using genetic strains of diabetic (db/db) mice at 12 weeks of age. Mouse liver and heart preparations were characterized as having an acid pH optimum of 4.5-5 for cholesterol ester hydrolase activity; a smaller but distinct pH optimum could also be observed at pH 7. In contrast, hydrolase activity in mouse fat pad preparations had only one distinct pH optimum of 6.5. Hyperinsulinemia in rats and mice resulted in a significant decrease in acid cholesterol ester hydrolase activity in heart preparations, but had no consistent effect on acid hydrolase activity observed in liver and fat pad preparations. This decrease in lysosomal acid cholesterol ester hydrolase activity in cardiac tissue due to hyperinsulinemia cannot be related to any changes in lipoprotein turnover caused by insulin or diabetes.     

Bicluster pattern of codon context usages between flavivirus and vector mosquito Aedes aegypti: relevance to infection and transcriptional response of mosquito genes

The mosquito Aedes aegypti is the primary vector of dengue virus (DENV) infection in most of the subtropical and tropical countries. Besides DENV, yellow fever virus (YFV) is also transmitted by A. aegypti. Susceptibility of A. aegypti to West Nile virus (WNV) has also been confirmed. Although studies have indicated correlation of codon bias between flaviviridae and their animal/insect hosts, it is not clear if codon sequences have any relation to susceptibility of A. aegypti to DENV, YFV and WNV. In the current study, usages of codon context sequences (codon pairs for neighboring amino acids) of the vector (A. aegypti) genome as well as the flaviviral genomes are investigated. We used bioinformatics methods to quantify codon context bias in a genome-wide manner of A. aegypti as well as DENV, WNV and YFV sequences. Mutual information statistics was applied to perform bicluster analysis of codon context bias between vector and flaviviral sequences. Functional relevance of the bicluster pattern was inferred from published microarray data. Our study shows that codon context bias of DENV, WNV and YFV sequences varies in a bicluster manner with that of specific sets of genes of A. aegypti. Many of these mosquito genes are known to be differentially expressed in response to flaviviral infection suggesting that codon context sequences of A. aegypti and the flaviviruses may play a role in the susceptible interaction between flaviviruses and this mosquito. The bias in usages of codon context sequences likely has a functional association with susceptibility of A. aegypti to flaviviral infection. The results from this study will allow us to conduct hypothesis-driven tests to examine the role of codon context bias in evolution of vector–virus interactions at the molecular level.