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Biomedical subjects

G M Hatch

Publications and source records attributed to G M Hatch.

At least 19 recordsLinked to original sources
PubMed1999

N-Acetylsphingosine stimulates phosphatidylglycerolphosphate synthase activity in H9c2 cardiac cells.

Cardiolipin and phosphatidylglycerol biosynthesis were examined in H9c2 cells incubated with short-chain ceramides. Incubation of cells with N-acetylsphingosine or N-hexanoylsphingosine stimulated [1, 3-3H]glycerol incorporation into phosphatidylglycerol and cardiolipin, with N-acetylsphingosine having the greater effect. The mechanism for the ceramide-mediated stimulation of de novo phosphatidylglycerol and cardiolipin biosynthesis appeared to be an increase in the activity of phosphatidylglycerolphosphate synthase, the committed step of phosphatidylglycerol and cardiolipin biosynthesis. The presence of the potent protein phosphatase inhibitors calyculin A or okadaic acid attenuated the N-acetylsphingosine-mediated stimulation of phosphatidylglycerolphosphate synthase activity and of phosphatidylglycerol and cardiolipin biosynthesis, indicating the involvement of a ceramide-activated protein phosphatase(s). The presence of 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated enzyme activity and [1,3-3H]glycerol incorporation into phosphatidylglycerol and cardiolipin. The effects of CPT-cAMP and N-acetylsphingosine on phosphatidylglycerol and cardiolipin biosynthesis and on phosphatidylglycerolphosphate synthase activity were additive. Phosphatidylglycerol biosynthesis from sn-[14C]glycerol 3-phosphate in permeabilized H9c2 cells was stimulated by preincubation with N-acetylsphingosine, and this was attenuated by okadaic acid. N-Acetylsphingosine treatment of cells elevated mitochondrial phospholipase A2 activity. Since the pool sizes of phosphatidylglycerol and cardiolipin were unaltered in these cells, the observed increase in phosphatidylglycerolphosphate synthase activity may be a compensatory mechanism for the N-acetylsphingosine-mediated elevation of mitochondrial phospholipase A2 activity. Finally, addition of tumour necrosis factor alpha to H9c2 cells resulted in an elevation of both phosphatidylglycerolphosphate synthase and phospholipase A2 activities. The results suggest that phosphatidylglycerol and cardiolipin metabolism in H9c2 cells may be regulated by intracellular ceramide signalling.

Animals↗
PubMed1998

Thyroxine stimulates the acylation of lysophosphatidylethanolamine in rat heart.

The acylation of cardiac lysophosphatidylethanolamine (LPE) was examined in rats treated with thyroid hormone. Rats were treated for five consecutive days with thyroxine (250 microg/kg) and controls were treated with saline. On the sixth day after an overnight fast, the hearts were removed and perfused in the Langendorff mode with 0.1 mM [1-14C]oleic acid. Radioactivity incorporated into phosphatidylethanolamine (PE) was increased 1.5-fold (P < 0.025) compared to controls. Radioactivity incorporated into phosphatidylcholine was not effected. The pool size of phosphatidylethanolamine and de novo biosynthesis of this phospholipid from [3H(G)]serine or [1,2-14C]ethanolamine were unaltered by thyroxine treatment. Treatment of rats with thyroxine resulted in a 1.5-fold (P < 0.025) increase in the relative percent of oleic acid in cardiac phosphatidylethanolamine. Thyroxine treatment resulted in a 1.8-fold (P < 0.025) increase in cardiac microsomal acyl-coenzyme A:1-acyl glycerophosphorylethanolamine acyltransferase activity compared to controls whereas, phospholipase A, acyl-coenzyme A hydrolase and fatty acyl-coenzyme A synthase activities were unaltered. The results demonstrate that the reacylation of cardiac LPE is regulated by thyroid hormone.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1998

Cardiolipin remodeling in eukaryotic cells infected with Chlamydia trachomatis is linked to elevated mitochondrial metabolism.

Cardiolipin remodeling in mammalian eukaryotic cells was examined subsequent to infection with Chlamydia trachomatis, an intracellular parasite of eukaryotic cells. HeLa cells were labeled for 6 h with [1-14C]myristate or [1-14C]palmitate or [1-14C]oleate 20 h post infection with C. trachomatis and the radioactivity incorporated into glycerophospholipids examined. Chlamydia infection resulted in a 2-4 fold elevation of radioactive myristate, palmitate or oleate incorporation into phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositiol compared to mock-infected cells. However, a 4-10 fold elevation in radioactivity incorporated into the mitochondrial glycerophospholipids phosphatidylglycerol and cardiolipin was observed in chlamydia-infected cells compared to mock-infected controls. Glycerophospholipid remodeling in CCL16-B2 cells, a mitochondrial respiration mutant with elevated glycerophospholipid metabolism, was compared to its parental cell line CCL16-B1 infected with C. trachomatis. Infection of the wild type CCL16-B1 cells with C. trachomatis resulted in an almost identical pattern of [1-14C]-palmitate labeling of glycerophospholipids compared to the uninfected mitochondrial mutant CCL16-B2 cells. The results suggest that cardiolipin, and glycerophospholipid, fatty acid molecular remodeling in eukaryotic cells infected with C. trachomatis may be linked to an elevation in mitochondrial metabolism.

Animals↗
PubMed1998

Lysophosphatidylcholine inhibits cardiolipin biosynthesis in H9c2 cardiac myoblast cells.

The effect of phospholipase A2 treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with Naja mocambique mocambique phospholipase A2 reduced the pool sizes of phosphatidylcholine and phosphatidylethanolamine compared with controls. The pool sizes of lysophosphatidylcholine and lysophosphatidylethanolamine were elevated, whereas the pool sizes of cardiolipin and other phospholipids were unaffected by phospholipase A2 treatment. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or preincubated in the absence or presence of phospholipase A2. In all experiments, radioactivity incorporated into cardiolipin was reduced in phospholipase A2-treated cells with time compared with controls, indicating attenuated de novo biosynthesis of cardiolipin. The mechanism for the reduction in cardiolipin biosynthesis in phospholipase A2-treated cells was a decrease in the activity of phosphatidic acid:cytidine-5'-triphosphate cytidylyltransferase, the rate-limiting enzyme of cardiolipin biosynthesis, mediated by elevated cellular lysophosphatidylcholine levels. The results suggest that de novo cardiolipin biosynthesis in H9c2 cells may be regulated by the cellular level of lysophosphatidylcholine.

Animals↗
PubMed1998

Phospholipid composition of purified Chlamydia trachomatis mimics that of the eucaryotic host cell.

Chlamydia trachomatis is an obligate intracellular eubacterial parasite capable of infecting a wide range of eucaryotic host cells. Purified chlamydiae contain several lipids typically found in eucaryotes, and it has been established that eucaryotic lipids are transported from the host cell to the parasite. In this report, we examine the phospholipid composition of C. trachomatis purified from host cells grown under a variety of conditions in which the cellular phospholipid composition was altered. A mutant CHO cell line, with a thermolabile CDP-choline synthetase, was used to show that decreased host cell phosphatidylcholine levels had no significant effect on C. trachomatis growth. However, less phosphatidylcholine was transported to the parasite and purified elementary bodies contained decreased levels of phosphatidylcholine. Brefeldin A, fumonisin B1, and exogenous sphingomyelinase were used to alter levels of host cell sphingomyelin. None of the agents had a significant effect on C. trachomatis replication. Treatment with fumonisin B1 and exogenous sphingomyelinase resulted in decreased levels of host cell sphingomyelin. This had no effect on glycerophospholipid trafficking to chlamydiae; however, sphingomyelin trafficking was reduced and elementary bodies purified from treated cells had reduced sphingomyelin content. Exposure to brefeldin A, which had no adverse effect on chlamydia growth, resulted in an increase in cellular levels of sphingomyelin and a concomitant increase in the amount of sphingomyelin in purified chlamydiae. Under the experimental conditions used, brefeldin A treatment had only a small effect on sphingomyelin trafficking to the host cell surface or to C. trachomatis. Thus, the final phospholipid composition of purified C. trachomatis mimics that of the host cell in which it is grown.

Animals↗
PubMed1998

Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian cells (Review).

Cardiolipin is the principal polyglycerophospholipid found in the heart and most mammalian tissues. This phospholipid is the only phospholipid localized exclusively to the mitochondria of mammalian cells. Cardiolipin appears to be involved, either directly or indirectly, in the modulation of a number of cellular processes including the activation of mitochondrial enzymes and hence production of energy by oxidative phosphorylation. The regulatory properties which govern cardiolipin biosynthesis, its remodeling and trafficking are beginning to emerge. Studies in the isolated perfused rat heart and H9c2 cardiac myoblast cells have indicated that the rate-limiting step of cardiolipin biosynthesis, via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway, is the conversion of phosphatidic acid and cytidine-5'-triphosphate to cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol. The cellular level of cytidine-5'-triphosphate appears to control the production of cardiolipin in H9c2 cells. The activities of the other enzymes of the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol pathway of cardiolipin biosynthesis in the heart may be modulated by thyroid hormone and unsaturated fatty acids. In addition, extra-mitochondrial cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol and phosphatidylglycerol may be utilized for cardiolipin biosynthesis in the heart and permeabilized cells. Cardiolipin may be readily hydrolyzed by phospholipases and may be remodeled by a deacylation-reacylation pathway. Studies with a Chinese hamster lung fibroblast cell line CCL16-B2 have indicated that the remodeling of cardiolipin is markedly altered in the mitochondria of these cells and that this alteration in remodeling may be one of the underlying mechanisms for the mutation in oxidative energy production in these cells. Host cell cardiolipin may be trafficked from the mitochondria to an intracellular bacterial parasite Chlamydia trachomatis. The purpose of this review is to briefly discuss some of the more recent findings in cardiolipin metabolism in the heart and mammalian cells and to provide insight into their possible implications in the regulation of some cellular functions in mammalian tissues and cells.

Animals↗
PubMed1998

On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells.

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-3H]glycerol indicating the involvement of 1,2-diacyl-sn glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5'triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacylsn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-3H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.

Animals↗
PubMed1997

On the mechanism of the losartan-mediated inhibition of phosphatidylcholine biosynthesis in H9c2 cells.

Phosphatidylcholine is the major phospholipid in mammalian tissues and the biosynthesis of phosphatidylcholine in H9c2 cells was previously shown to be stimulated by angiotensin II. In this study, we used the potent AT1 receptor antagonist, losartan, to determine if the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis was mediated by AT1 receptors. H9c2 cells were incubated with angiotensin II in the absence or presence of various concentrations of losartan. The cells were then incubated with [methyl-3H]choline for an additional 60 min and the radioactivity incorporated into phosphatidylcholine and its choline-containing metabolites determined. Losartan at concentrations which block AT1 receptors did not effect phosphatidylcholine biosynthesis mediated by angiotensin II. In contrast, higher concentrations of losartan inhibited radioactivity incorporated into phosphatidylcholine and its metabolites and this was due to a losartan-mediated reduction in choline uptake. Kinetic studies revealed that the losartan-mediated inhibition of choline uptake was competitive. High concentrations of losartan caused a translocation of CTP:phosphocholine cytidylyltransferase from the cytosolic (inactive) to the membrane (active) fraction likely as a compensatory mechanism for the losartan-mediated reduction in new phosphatidylcholine biosynthesis. Incubation of cells with PD123319, a potent AT2-receptor antagonist, did not block the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis. The results suggest that angiotensin II stimulates phosphatidylcholine biosynthesis independent of AT1- and AT2-receptor activation and losartan inhibits phosphatidylcholine biosynthesis by reducing choline uptake in H9c2 cells.

Angiotensin II↗
PubMed1997

Induction of free radicals in hepatocytes, mitochondria and microsomes of rats by ochratoxin A and its analogs.

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and O alpha; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe3+ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe3+. The spin adduct hyperfine splitting constants indicated the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.

Animals↗
PubMed1997

Cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol import into mitochondria through mitochondrial membrane contact sites in permeabilized rat liver hepatocytes.

The location of cytidine-5'-diphosphate-1,2-diacylsn-glycerol import into mictochondria was examined in permeabilized rat liver hepatocytes. The model utilized the ability of intact mitochondria to freely import [14C]glycerol-3-phosphate. Import of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol was measured by the synthesis of phosphatidyl[14C]glycerol. Phosphatidyl [14C]glycerol was synthesized in a time- and concentration-dependent manner in the presence of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol. The presence of ATP in the incubations stimulated phosphatidyl[14C]glycerol formation. The presence of adriamycin, at concentrations that block import of proteins into mitochondria, inhibited the formation of phosphatidyl [14C]glycerol. In addition, adriamycin, inhibited in vitro biosynthesis of phosphatidyl[14C]glycerol in mitochondrial but not microsomal fractions. 1,4-Dinitrophenol, which may decrease the number of mitochondrial inner and outer membrane contact sites, inhibited formation of phosphatidyl[14C]glycerol in permeabilized hepatocytes. The results demonstrate that cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol may be imported into mitochondria through inner and outer mitochondrial membrane contact sites in hepatocytes.

Animals↗
PubMed1997

Cardiolipin remodeling in a Chinese hamster lung fibroblast cell line deficient in oxidative energy production.

The metabolism of cardiolipin was investigated in a Chinese hamster lung fibroblast cell line CCL16-B2 deficient in oxidative energy metabolism and its parental cell line CCL16-B1. Mitochondrial enzyme activities involved in de novo cardiolipin biosynthesis were elevated in CCL16-B2 cells compared with CCL16-B1 cells, indicating initially an elevation in cardiolipin biosynthesis. Content of all phospholipids, including cardiolipin and its precursors, and high energy nucleotides were unaltered in CCL16-B2 cells compared to CCL16-B1 cells. When cells were incubated with [1,3-(3)H]glycerol for up to 4 h radioactivity incorporated into cardiolipin in CCL16-B2 cells did not differ compared with CCL16-B1 cells. In contrast, radioactivity incorporated into phosphatidylglycerol, the immediate precursor of cardiolipin, was elevated over 2-fold in CCL16-B2 cells compared with CCL16-B1 cells. Analysis of the fatty acid molecular species in cardiolipin revealed alterations in the level of unsaturated but not saturated fatty acids in B2 compared with B1 cells. In vivo cardiolipin remodeling, that is, the deacylation of cardiolipin to monolysocardiolipin followed by reacylation back to cardiolipin, with [1-(14)C]palmitate and [1-(14)C]oleate and in vitro mitochondrial phospholipid remodeling with [1-(14)C]linoleate were altered in CCL16-B2 cells compared to CCL16-B1 cells. Since both the appropriate content and molecular composition of cardiolipin is required for optimum mitochondrial oxidative phosphorylation, we suggest that the difference in CL molecular species composition observed in CCL16-B2 cells, mediated by alterations in in vivo cardiolipin remodeling, may be one of the underlying mechanisms for the reduction in oxidative energy production in CCL16-B2 cells.

Animals↗
PubMed1997

Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis.

There is little information on the trafficking of eukaryotic lipids from a host cell to either the cytoplasmic membrane of or the vacuolar membrane surrounding intracellular pathogens. Purified Chlamydia trachomatis, an obligate intracellular bacterial parasite, contains several eukaryotic glycerophospholipids, yet attempts to demonstrate transfer of these lipids to the chlamydial cell membrane have not been successful. In this report, we demonstrate that eukaryotic glycerophospholipids are trafficked from the host cell to C. trachomatis. Phospholipid trafficking was assessed by monitoring the incorporation of radiolabelled isoleucine, a precursor of C. trachomatis specific branched-chain fatty acids, into host-derived glycerophospholipids and by monitoring the transfer of host phosphatidylserine to chlamydiae and its subsequent decarboxylation to form phosphatidylethanolamine. Phospholipid trafficking to chlamydiae was unaffected by brefeldin A, an inhibitor of Golgi function. Furthermore, no changes in trafficking were observed when C. trachomatis was grown in a mutant cell line with a nonfunctional, nonspecific phospholipid transfer protein. Host glycerophospholipids are modified by C. trachomatis, such that a host-synthesized straight-chain fatty acid is replaced with a chlamydia-synthesized branched-chain fatty acid. We also demonstrate that despite the acquisition of host-derived phospholipids, C. trachomatis is capable of de novo synthesis of phospholipids typically synthesized by prokaryotic cells. Our results provide novel information on chlamydial phospholipid metabolism and eukaryotic cell lipid trafficking, and they increase our understanding of the evolutionary steps leading to the establishment of an intimate metabolic association between an obligate intracellular bacterial parasite and a eukaryotic host cell.

Animals↗
PubMed1997

Oxidative modification of low density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation.

The elevated level of plasma low density lipoprotein (LDL) in hyperlipidemic patients is an important risk factor for the production of atherosclerosis. Plasma LDL must be modified before it can produce an impairment of endothelium-dependent relaxation in aortic rings or enhancement of uptake by macrophages. The dramatic increase in lysophosphatidylcholine (lysoPC) content in oxidatively modified LDL has been touted as an important biochemical factor for the impairment of endothelium-dependent relaxation. The present study was designed to examine the lysoPC composition of oxidized LDL samples from normal and hyperlipidemic subjects, and their effects on the impairment of endothelium-dependent relaxation. Oxidatively modified LDL from hyperlipidemic patients contained a slightly higher level (17%) of lysoPC, but produced a disproportionately greater impairment of endothelium-dependent relaxation than that from normal subjects. As lysoPC is composed of many molecular species, its composition in oxidized LDL samples was analyzed. In hyperlipidemic patients, lysoPC samples were found to contain a higher proportion of long-chain acyl groups. Subsequent studies revealed that only long-chain lysoPC (C > 16:0) were effective in impairing endothelium-dependent relaxation. Experimental loading of oxidized LDL from normal subjects with long chain lysoPC to mimic levels observed in oxidized LDL from hyperlipidemic patients resulted in further impairment of endothelium-dependent relaxation. We conclude that the greater proportion of long-chain lysoPC found in the oxidized LDL of hyperlipidemic subjects is responsible for the increased impairment of endothelium-dependent vascular relaxation. We propose that the high level of LDL found in the plasma of hyperlipidemic patients, coupled with its enhanced ability to generate long chain species of lysoPC during oxidative modification, are important factors for the development of atherosclerosis in these patients.

Adult↗