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PubMed1996

Differential labelings suggest two specific phospholipid subclass hydrolysis promoted by PDGF-BB in vascular smooth muscle cells.

We have used differential phospholipid subclass labelings performed with [3H]lyso PAF and [3H] myristic acid into vascular smooth muscle cells (vSMC) to characterize the subclasses of phospholipid substrates upon different stimulation times with platelet-derived growth factor (PDGF-BB). In cells labeled with [3H]lyso PAF, PDGF-BB induced a sustained hydrolysis of alkyl-PE. In contrast, in [3H]myristic acid-labeled cells, PDGF-BB promoted a rapid and transitory hydrolysis of diacyl-PC. This hydrolysis was concomitant with an synthesis of diglyceride (DG) and phosphatidic acid (PA). Thus, both diacyl-PC and alkyl-PE appear to be major targets in PDGF-BB stimulation of SMC. These results suggest that agonists could induce the hydrolysis of precise phospholipid subclasses leading to a new specificity into the signal transduction cascade.

Animals↗

PubMed1996

Aberrations in normal systemic lipid metabolism in ovarian cancer patients.

Numerous investigations have demonstrated altered systemic lipid metabolism in cancer patients, as well as aberrant lipid utilization by tumor cells. The most common measure of altered systemic lipid metabolism in these individuals has been hyperlipidemia. Although cachexia is not generally considered to be associated with gynecologic cancers, this study demonstrates the presence of lipolysis-promoting activity, detectable in sera and ascites of ovarian cancer patients and indices of altered systemic lipid metabolism. Elevated lipolysis promoting activity was detectable in the sera of 7/9 patients and in the ascites of 5/5 patients. Since previous studies have indicated that cancer patients exhibit a 2.5-fold enhancement in hormone-sensitive lipase (HSL) versus normal controls, as a potential mechanism for elevated lipolysis, the ability of ascites-derived factors to induce HSl was examined. The addition of three of four ascites fluids increased the level of HSL in normal adipocytes. All of the patients' samples exhibited elevated lipid levels versus normal peritoneal fluid. Isolation and analysis of lipids from three ovarian cancer patients revealed four consistent altered lipid parameters compared to normal peritoneal fluid: elevated monoglycerides, diglycerides, and free fatty acids and decreased triacylglycerides. While "classical" cachexia is not a common feature of ovarian cancer, the presence of circulating lipolysis-promoting activity and altered lipid metabolism, generally observed in cachectic individuals, can be demonstrated in these ovarian cancer patients. Based on recent evidence indicating a role of lipids in carcinogenic initiation or promotion, the presence of tumor-derived lipolysis-promoting factor and lipid metabolism alterations may provide a mechanism for the epidemiologically observed association between lipids and certain cancers, including ovarian cancer.

Adult↗

PubMed2002

Rheological characterization, crystallization, and gelation behavior of monoglyceride gels.

Saturated monoglycerides can form firm gels in water. These gels are networks of stiff plate-like beta-crystals of monoglycerides (a "cardhouse"), grown from a space-filling lamellar liquid-crystalline phase. The molecular mechanism of crystallization is discussed in the light of network formation. The concentration dependence of gel development of (shear-cooled) monoglyceride gels has been studied by rheology. A gelation mechanism has been proposed, consisting of two steps: (i) After formation of a nucleus, rapid crystallization in a lateral direction occurs (probably within one bilayer) by which the first space-filling network is formed. (ii) This is followed by reinforcement of the network by which stacks of crystalline bilayers are formed. The plate-like crystals are linked in connective domains or junction zones, probably containing all the material (cosurfactants, diglycerides, etc.) that does not fit in the crystalline array. Small deformation rheology shows that above about 2 wt% monoglyceride a percolating network is formed. The large deformation rheology is typical for a particle gel with a relatively small strain at failure (both in shear deformation and compression). The connective domains or junction zones already fail when relatively small deformations are put on the system.

Journal Article↗

PubMed1999

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Acetylation↗

PubMed1996

Distinct signal transduction pathways for activation of rabbit alveolar macrophages in vitro by cotton bract tannin.

These experiments were designed to study signal transduction pathways in alveolar macrophages stimulated by condensed tannin or zymosan. Condensed tannins, present in cotton mill dust, alter the host-defense function of alveolar macrophages and may contribute to the pathogenesis of byssinosis. We tried to determine the early steps in signal transduction mechanisms of cell activation by tannin. With the quantification of 51Cr release, we determined that tannin was cytotoxic for the cells after 30 min activation with 130 micrograms for 2 x 10(6) cells. 51Cr release was similar for control cells and zymosan- or 30 micrograms tannin-activated cells. Using the luciferine luciferase reaction, we showed that tannin markedly depleted ATP cell content. In inositol-labeled cells, tannin increased inositolphosphate release in a dose-dependent manner. In lysoPAF-labeled cells, tannin induced synthesis of phosphatidic acid and diglycerides. In the presence of ethanol, the level of tannin-induced phosphatidic acid was slightly reduced, and phosphatidylethanol was synthesized. No phosphatidylethanol was found in alveolar macrophages stimulated by zymosan in the presence of ethanol. GF 109203X, a specific inhibitor of protein kinase C decreased only tannin-induced phosphatidylethanol synthesis. In conclusion, tannin (at 30 or 130 micrograms/ml) activated an inositol phospholipase C in alveolar membranes. Phosphatidylcholine phospholipases C and D were found only at the higher concentration of tannin.

Adenosine Triphosphate↗

PubMed1992

Regulation of arachidonic acid metabolism in the perinatal brain during development and under ischemic stress.

Oxygen deprivation following cessation of blood flow to vital organs such as brain, heart, and kidney is a ubiquitous human disease, invariably leading to devastating consequences. Studies in experimental models support the contention that membrane permeability is altered, ion fluxes impaired, and energy stores depleted under these circumstances. Certain lipids such as diglycerides (DG) and arachidonic acid (AA), both of which are important cellular second messengers, appear to increase during ischemia. At this point, the contribution of these and other lipids to cell deregulation, loss of function, and ultimate death has not been clarified because no precise link between lipid alterations as detected in ischemia and subsequent cellular processes has been made. In this report we examine the origin of lipid-derived second messengers in fetal rat brain prelabeled with [3H]AA and study the fate of various lipids upon obstruction of the fetal-maternal circulation. The data support the possibility of a phospholipase A2-mediated deacylation of poly-phosphoinositides (poly-PI) to form free AA and a phospholipase C-mediated hydrolysis of PC to form DG during ischemia.

Animals↗

PubMed1980

The specificity of beta-galactosidase in the degradation of gangliosides.

Available evidence indicates that a least two genetically distinct acidic lysosomal beta-galactosidases are present in mammalian tissues. One of them, galactosylceramidase, is primarily responsible for degradation of galactosylceramide, galactosylsphingosine, and monogalactosyl-diglyceride, while the other, GM1-ganglioside beta-galactosidase, degrades GM1-ganglioside and asialo GM1-ganglioside. Lactosylceramide can be hydrolyzed by either of the two enzymes. These substrate specificities of the two beta-galactosidases can adequately explain the known findings in the two genetic beta-galactosidase deficiency diseases. The possibilities of the specific lactosylceramidase have not yet received the necessary independent confirmation.

Brain↗

PubMed1987

Synthesis, storage and degradation of myocardial triglycerides.

In the mammalian myocardium, an active triglyceride synthesis pathway is operating, (re)esterifying activated fatty acids from endogenous or exogenous sources, with the glycolytically derived three-carbon intermediates dihydroxyacetone-phosphate and glycerol-3-phosphate by the so-called Kennedy pathway. The seven enzymes of triglyceride synthesis are membrane bound and located at the sarcoplasmic reticulum. The first enzyme in the glycerol-3-phosphate pathway, glycerol-3-phosphate acyltransferase, is proposed to be rate limiting for triglyceride formation. This microsomal enzyme is regulated by phosphorylation (inactiycation)-dephosphorylation (activation) coupled to the beta-receptor--adenyl cyclase--protein kinase system. Additional regulatory steps in triglyceride formation are the reactions catalyzed by the microsomal phosphatidic acid phosphatase and diglyceride acyltransferase. Intracellular triglycerides occur as free floating cytosolic droplets, membrane-bound particles and lipid-filled lysosomes. No consensus exists about the metabolically active portion of myocardial triglycerides. Various lipases have been proposed to be involved in endogenous lipolysis: the lysosomal acid, microsomal and soluble neutral triglyceride, intracellular lipoprotein lipases and the microsomal di- and monoglyceridase. It has been acknowledged that the bulk of the intracellular neutral lipase represents the precursor of vascular lipoprotein lipase. The presence of a neutral lipase, as distinct from lipoprotein lipase, in the rat heart was recently advocated. Endogenous lipolysis is a hormone-sensitive process. Hormone-sensitivity may involve direct alteration of enzyme activity by protein phosphorylation-dephosphorylation but is also dependent on the removal rate of product fatty acids, since feedback inhibition is a common property of all lipases in the heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1977

Effects of isolation and transplantation of the corpus cardiacum on hormone release from its glandular cells after flight in Locusta migratoria. A quantitative electron microscopical study.

The glandular lobe of the corpus cardiacum (CC) of Locusta migratoria contains secretory cells and axon terminals. It is generally accepted that the glandular cells produce an adipokinetic hormone which stimulates the release of diglycerides from the fat body. The axon terminals are supposed to be involved in the regulation of the secretory activity of these cells. CC were transplanted into locust hosts. The secretory activity of the glandular cells and axons was studied in the transplants as well as the intact host CC. Quantitative electron microscopical examination of the glandular cells of the CC in animals both at rest and after flight shows that only in the host CC the secretory activity of the glandular cells is stimulated by flight, as indicated by an increase in the activity of the Golgi zones and in the number of exocytotic pits. In the axon terminals release of secretory products, indicated by synaptic vesicles and membrane indentations, is reduced as a result of the transplantation. It is concluded that the glandular cells need excitatory input from outside the CC for stimulation of their secretory activity. Furthermore, it is proposed that the transplanted glandular cells decrease the level of activity of the intact host cells by way of a negative feedback mechanism.

Animals↗

PubMedDate not supplied

Biochemistry of radioiodinated free fatty acids.

Radioiodinated free fatty acids have been developed to study myocardial metabolism non-invasively in man. In the present study the distribution of radiolabeled lipids in the myocardium and in arterial and coronary sinus blood was evaluated following injection of three commonly used iodinated fatty acids in fasted (n = 5) and lactate loaded (n = 3) dogs. Five minutes after simultaneous i.v. injection of radioiodinated 17-I-heptadecanoic acid (IHDA), 15-(p-I-phenyl) pentadecanoic acid (IPPA) and 15-(p-I-phenyl)-3,3-dimethyl-pentadecanoic acid (DMIPPA) a biopsy specimen and samples of arterial and coronary sinus blood were taken. After extraction and TLC the relative distribution of radioactivity in the aqueous phase (containing the oxidation products), pellet and organic phase was calculated. The organic phase was further divided into phospholipids, diglycerides, free fatty acids, triglycerides and cholesterol-esters. Seventy two percent of IHDA was oxidized, 36% of IPPA and 7% of DMIPPA. The organic phase consisted primarily of triglycerides and phospholipids. The ratios of triglycerides to phospholipids were about the same for IHDA, IPPA and DMIPPA (0.58, 0.65 and 0.50, respectively). Free IHDA in tissue samples was low (4%) and elevated for IPPA and DMIPPA, (17% and 37%). During lactate loading triglycerides were higher for all three fatty acids. For IHDA and IPPA this increase was paralleled by a decrease in the aqueous phase, in case of DMIPPA the aqueous phase remained the same. Five minutes after injection most of the organic phase of both arterial and coronary sinus blood consisted of the injected fatty acids, the aqueous phase contained oxidation products. There were only minor differences during lactate loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes.

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5 microM CaCl2, 5 mM cholate and 100 nM [3H-]Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5) P2) resulted in the formation of [3H-]InsP3. GTP-gamma-S (125 microM) stimulated the production of [3H-]InsP3. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [3H-]PtdIns(4,5)P2 hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca2+. Addition of phorbol ester (100 nM PMA) enhanced the 32P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca2+, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomal GTP-gamma-S-stimulated PtdIns(4,5)P2-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate alpha 1-adrenoceptor mediated PtdIns(4,5)P2 hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.

Animals↗

PubMed1977

Isolation and characterization of lipid globules from the zoospores of Blastocladiella emersonii.

Lipid globules were isolated and characterized both chemically and morphologically. They were composed mainly of triglyceride and free sterol, which accounted for over 90% of the total globule content. Smaller amounts of diglyceride, carotenoid, free fatty acid, phospholipid and protein were found. No sterol esters or monoglycerides were detected. Morphologically, the isolated lipid globules resembled the lipid globules in situ. They were spherical, 0.4-1.5 mum in diameter and lacked a trilaminar membrane.

Blastocladiella↗

PubMed1988

Control of exocytosis from adrenal chromaffin cells.

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.

Adrenal Medulla↗

PubMed1990

Characterization of neurofilament-associated protein kinase activities from bovine spinal cord.

1. A neurofilament-enriched preparation from bovine spinal cord contains endogenous protein kinases that phosphorylate high, middle, and low molecular weight neurofilament subunits (NF-H, NF-M, and NF-L), as well as certain other endogenous and exogenous substrates. 2. Most of this associated kinase activity can be separated from the neurofilament subunits and the bulk of the protein by extraction of the neurofilament preparation with 0.8 M KCl. Assays using specific exogenous substrates, activators, and inhibitors for known kinases reveal significant levels of Ca2(+)-calmodulin-dependent, cyclic nucleotide-dependent, Ca2(+)-phosphatidylserine diglyceride-dependent, and regulator-independent kinase activities in the high-salt extract. 3. Fractionation of the salt extract on a gel filtration column resolves a regulator-independent kinase activity identified by its ability to phosphorylate purified NF-M. This preparation can phosphorylate all three neurofilament proteins either in purified form or in the assembled form, as well as alpha-casein. Only the regulator-independent kinase activity in this fraction is responsible for the phosphorylation of neurofilament proteins. 4. While this partially purified kinase activity does not show a strong substrate specificity between the three neurofilament subunits, the phosphorylation pattern it produces upon incubation with salt-extracted neurofilaments is similar to the regulator-independent phosphorylation pattern found in the original neurofilament preparation and, thus, represents a useful starting point for the further purification of this neurofilament-associated kinase activity.

Animals↗

PubMed1983

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain.

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.

Animals↗

PubMed1991

Stimulation of mono- and diacylglycerol lipase activities by gangliosides in chicken neuronal cultures.

Chicken neurons in culture display measurable activities of mono- and diacylglycerol lipases. Treatment of chicken neuronal cultures with gangliosides (10(-8)M to 10(-5)M) resulted in a time and dose dependent increase in monoacylglycerol lipase activity. The diacylglycerol lipase showed significant increase in specific activity before that of monoacylglycerol lipase. The increase was observed only up to 24 hours and no differences between diacylglycerol lipase activity of control and ganglioside treated cells were observed after 48 hours. The data indicate that the treatment of neurons with exogenous gangliosides affect the diglyceride metabolism in stimulating not only the enzymes catalyzing their production but also those involved in their catabolism.

Animals↗

PubMed1989

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate, and [14C]linolenate.

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [14C]linoleate, [14C]linolenate, or [3H]myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [3H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With 14C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC greater than PI greater than PE greater than PS with [14C]linoleate, and PE greater than PC greater than PI = PS with [14C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with 14C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic [( 14C]linoleate) or a tetraenoic [( 14C]linolenate) fatty acid. MBP was not labeled with [3H]myristate, but was slightly labeled with both 14C-essential fatty acids. The signification of the latter result is discussed.

Animals↗

PubMed1988

The incorporation of intracranially injected glycerol into brain glycerides of young rats born to normal and alcohol-fed mothers.

Growth alters the ability of rat brain to incorporate [2-3H]glycerol into glycerides; indeed, 12 min after the intracranial administration of the precursor, diglyceride becomes more radioactive in newborn than in 19-day-old brain, the reverse being true for total glycerophospholipid and triglyceride. The ratio between the labeling of phospholipid and that of neutral lipid in the experimental conditions described in this paper is proposed as a marker of brain maturity. The distribution of labeling among phospholipid classes also varies with age, and the increase of labeling in total phospholipid occurring with increasing age is almost entirely due to phosphatidylethanolamine and phosphatidylcholine. The metabolism of myelin lipids might be responsible for these age-dependent variations. The administration of ethanol to dams during pregnancy and lactation alters the distribution of the label among neutral glycerolipid and total glycerophospholipid in an age-dependent manner. The labeling distribution among phospholipid classes is also affected.

Aging↗