Penetration of phospholipases A2 and C into the squid (Loligo pealii) giant axon

Summary Following 1 h exposure, the level of phospholipase A₂ penetration into the axoplasm of the squid giant axon was 107 to 350% of that in the external media; corresponding values for phospholipase C were 18 to 31%. Phospholipases can therefore be used to study phospholipid function in axons since they can penetrate through connective tissue and Schwann cell to reach the axolemma.I thank Dr.Toshio Narahashi for allowing me to use his laboratory facilities at the Marine Biological Laboratory, Woods Hole, Mass. for the dissection of squid axons. Expert technical assistance in these studies were provided by Mr.Steven Trudel and Mr.Stuart Owen Rosenberg. This work was supported in part by a grant from the University of Connecticut Research Foundation.     

Adaptation of lipase and phospholipase A activities in pancreas and pancreatic juice in rats with diets rich in triglycerides and phospholipids]

Lipid rich diets containing about 20% of triglycerides or phospholipids given to Rats during 2 months were observed to increase lipase and phospholipase A2 activities in pancrease and pancreatic juice. The phospholipase and lipase activities are higher, respectively, on the phospholipid and triglyceride diet. Lower effects are observed after a 7-day administration of diet containing 40% of total lipid.     

Spontaneous phospholipase A activity of rat pancreatic homogenates]

Rat pancreas presents a spontaneous phospholipase A activity which appears before trypsin activation at optimal pH 6.5. The responsible enzyme is independent of pancreatic prophospholipase A, as can be seen through experiments done in the presence of trypsin inhibitors. On the other hand, this enzyme is distinct from excretory phospholipase which is more active and whose optimal pH is 8.8. Thermostability and insensibility of spontaneously active phospholipase A to DFP differentiate it from lipase, carboxyl-esterhydrolase and lysophospholipase, respectively.     

Serienbestimmung der Phospholipase-A-Aktivität in chromatographischen Fraktionen

Summary A simple assay is described for phospholipase A in chromatographic fractions. 0.01 ml of the fraction is added to 1 ml of a 1% solution of purified lecithin in ether (100 vol) — picoline (10 vol) — 4.5 mmol CaCl₂ (1 vol). The presence of phospholipase A is indicated by the appearance of a turbidity due to precipitation of lysolecithin. The lag period until precipitation occurs gives an indication of the relative concentration of enzyme present.     

Multiple phospholipase activation by OX1 orexin/hypocretin receptors

We investigated coupling of OX(1) receptors to phospholipase activation and diacylglycerol generation in Chinese hamster ovary (CHO) cells using both biochemical and fluorescence "real-time" methods. The results indicate that at lowest orexin-A concentrations (highest potency), diacylglycerol generated results from phospholipase D activity. At 10-100-fold higher orexin-A concentrations, phospholipase C is activated, likely hydrolyzing phosphatidylinositol (PI) or phosphatidylinositol monophosphate (PIP) but not phosphatidylinositol bisphosphate (PIP(2)). At further 7-fold higher orexin-A concentrations, PIP(2) is hydrolyzed, releasing both diacylglycerol and inositol-1,4,5-trisphosphate. Thus, OX(1) orexin receptors connect to multiple phospholipase activities, apparently composed of at least one phospholipase D and two different phospholipase C activities. At low agonist concentrations, diacylglycerol and phosphatidic acid are the preferred products, and interestingly, it seems that even the primarily activated phospholipase C mainly works to increase diacylglycerol and not inositol-1,4,5-trisphosphate.     

Effect of starvation on enzymes related to lipid metabolism in guinea-pig lungs

The changes in activities of acetyl CoA carboxylase, microsomal fatty acid elongation enzyme, choline phosphotransferase, triglyceride lipase, phospholipase A1 and phospholipase A2 were followed in guinea-pig lungs at 24, 48 and 72 h after food deprivation. Triglyceride lipase was elevated and phospholipase A1 and phospholipase A2 were unaffected, while the other activities decreased. The significance of these findings in relation to food deprivation is discussed.     

Specificity of action of phospholipases of rat intestinal mucosa]

The total homogenate of rat intestine is devoid of C or D phospholipase activity. At pH 6,5 in the presence of phosphatidylcholines, it exhibits a B phospholipase activity and in the presence of exogen lysophosphatidylcholines an A1 lysophospholipase activity. At pH 8,5 the intestinal mucous membrane of a rat shows an isolated A2 phospholipase activity.     

Arachidonic acid mobilization in platelets: the possible role of protein kinase C and G-proteins

A major route for the release of arachidonic acid from platelet phospholipids appears to be catalyzed by a phospholipase A2 that can be stimulated by a rise of cytosolic Ca2+. This paper discusses certain other mechanisms for regulation of this process. Release of arachidonic acid by calcium ionophores is potentiated by pretreatment with stimulators of protein kinase C; e.g. diglyceride, phorbol esters and the terpene diester mezerein. This effect appears to be coincident with phosphorylation of a certain group of proteins (not 47 KDa protein), and is sensitive to depletion of ATP, activation of Ca2+ dependent phosphatase, and the kinase C inhibitor H-7, but is unaffected by Na+/H+ exchange inhibitors. Recent results in other cell types strongly indicate that phospholipase A2 is also directly under control of certain GTP-binding proteins.     

The generation and identification of the hemolysin of Trypanosoma congolense

Summary The hemolytic activity of Trypanosoma congolense appears to be due to the presence of free fatty acids generated by the action of phospholipase A on endogenous phosphatidyl choline. Some lysolecithin also contributes to the lytic activity. Trypanosoma lewisi, being devoid of phospholipase A does not generate free fatty acids and is therefore non-hemolytic.This research was supported by the International Development Research Center.     

Transduction of the chemotactic cAMP signal across the plasma membrane ofDictyostelium cells

AggregatingDictyostelium cells secrete cAMP during cell aggregation. cAMP induces two fast responses, the production of more cAMP (relay) and directed cell locomotion (chemotaxis). Extracellular cAMP binds to G-protein-coupled receptors leading to the activation of second messenger pathways, including the activation of adenylyl cyclase, guanylyl cyclase, phospholipase C and the opening of plasma membrane Ca²⁺ channels. Many genes encoding these sensory transduction proteins have been cloned and null mutants of nearly all components have been characterized in detail. Undoubtedly, activation of adenylyl cyclase is the most complex, involving G-proteins, a soluble protein called CRAC and components of the MAP kinase pathway. Null mutants in this pathway do not aggregate, but can exhibit chemotaxis and develop normally when supplied with exogenous cAMP. The pathways leading to the activation of phospholipase C were identified, but unexpectedly, deletion of the phospholipase C gene has no effect on chemotaxis and development, nor on intracellular Ins(1,4,5)P₃ levels; the metabolism of this second messenger will be discussed in some detail. Activation of guanylyl cyclase is G-protein-dependent and essential for chemotaxis. Analysis of a collection of chemotactic mutants reveals that most mutants are defective in either the production or intracellular detection of cGMP, thereby placing this second messenger at the center of chemotactic signal transduction. Analysis of the cAMP-mediated opening of plasma membrane calcium channels in signal transduction mutants suggests that it has two components, one that depends on G-proteins and intracellular cGMP and one that is G-protein-independent.     

Basic phospholipase of Naja nigricollis venom]

It is confirmed that N. nigricollis venom contains several phospholipases one of these is a basic phospholipase A. This enzyme is toxic for mice when injected intravenously. In vitro it reacts on egg yolk lecithin producing lysolecithin and prevents the phenomenon of blood clotting. An immunological identity has been established between this basic phospholipase and two acidic phospholipases present in the same venom.     

DOCA administration increases renal phospholipase activity in the rat

Summary The phospholipase activity of renal tissue has been evaluated in controls and in DOCA treated rats. DOCA treated animal showed a higher than normal enzyme activity. Since a phospholipase is the key step in prostaglandin biosynthesis, it is suggested that the increased prostaglandin release promoted by mineraloactive steroids is mediated by an activation of this key enzyme.This work has been partly supported by a grant from Consiglio Nazionale delle Ricerche.To whom reprint requests should be addressed.     

Activity of phospholipases A and some lysosomal enzymes in rat testes at different stages of hypervitaminosis A

Summary Excess of vitamin A induces decrease of neutral phospholipase A₁ and A₂ activity in rat testes homogenates on the 4th day, and increase of -galactosidase activity on the 8th day of treatment. It is suggested that phospholipase A activity decrease is of great importance in development of testicular disorders, caused by disbalance of vitamin A.     

Functional expression of mammalian opioid receptors in insect cells and high-throughput screening platforms for receptor ligand mimetics

Lepidopteran cell lines have been engineered to constitutively express high levels of mouse opioid receptors either alone or in combination with human G16 protein. Biochemical and pharmacological studies demonstrate that these lines contain all the mediator G proteins and downstream effectors required for opioid receptor function, including phospholipase C, and that expression of exogenous G16 does not contribute significantly to increased receptor responses upon activation. The activation of the phospholipase C pathway in the transformed cells upon stimulation with known receptor ligands results in easily and quantitatively measurable increases in free intracellular calcium, which can be monitored by automated fluorescent methods, while the addition of specific antagonists blocks the agonist-induced responses. Therefore, the transformed lepidopteran cell lines can be used as sensitive high-throughput screening platforms for fast detection of opioid receptor ligand mimetics (agonists and antagonists) in collections of natural products and synthetic compounds.Received 3 December 2004; received after revision 3 February 2005; accepted 10 February 2005L. Swevers and E. Morou contributed equally to this work.     

Effect of piperazine on the level of phospholipids and on the activities of certain enzymes of phospholipid metabolism in human Ascaris lumbricoides.

The phospholipid level in the human parasitic nematode Ascaris lumbricoides is decreased by piperazine, by partially stimulating catabolic enzymes such as phospholipase C and partially inhibiting anabolic enzymes such as choline kinase.     

Isolation of a hexaprenylhydroquinone sulfate from the marine sponge Dysidea sp. as an H,K-ATPase inhibitor

A hexaprenylhydroquinone sulfate has been isolated as an H,K-ATPase inhibitor from a marine sponge Dysidea sp. It also inhibited phospholipase A2 as well as secretion of gastric acid in rats.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

Lipase activity of anaerobic bacteria on glycerol-tributyrate determined by gas-liquid chromatography]

Anaerobic bacteria are classified among other criteria by the presence or absence of phospholipase and lipase. The liquid gas chromatographic method detects with great sensibility the lipasic activity of the anaerobic bacteria. A liidolytic action has been demonstrated in Cl. perfringens.     

The generation of toxic activity from trypanosoma congolense.

Trypanosoma congolense organism, on incubation at 20 degrees C for 91/2h, were found to generate phospholipase like activity which was capable of mediating lysis of both nucleated cells and erythrocytes as well as acute inflammatory response on intradermal inoculation.     

Regulation of SNARE fusion machinery by fatty acids

Vesicle fusion is a ubiquitous biological process involved in membrane trafficking and a variety of specialised events such as exocytosis and neurite outgrowth. The energy to drive biological membrane fusion is provided by fusion proteins called SNAREs. Indeed, SNARE proteins play critical roles in neuronal development as well as neurotransmitter and hormone release. SNARE proteins form a very tight alpha-helical bundle that can pull two membranes together, thereby initiating fusion. Whereas a great deal of attention has been paid to partner proteins that can affect SNARE function, recent genetic and biochemical evidence suggests that local lipid environment may be as important in SNARE regulation. Direct lipid modification of SNARE fusion proteins and their regulation by fatty acids following phospholipase action will be discussed here in detail. Our analysis highlights the fact that lipids are not a passive platform in vesicle fusion but intimately regulate SNARE function. Received 20 December 2006; received after revision 6 February 2007; accepted 15 March 2007