Regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C through the transcriptional repression of upstream activating sequence inositol containing genes

The regulation of phospholipid biosynthesis in Saccharomyces cerevisiae through cis-acting upstream activating sequence inositol (UAS(ino)) and trans-acting elements, such as the INO2-INO4 complex and OPI1 by inositol supplementation in growth is thoroughly studied. In this study, we provide evidence for the regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C (PLC) through UAS(ino) and the trans-acting elements. Gene expression analysis and radiolabelling experiments demonstrated that the overexpression of rice PLC in yeast cells altered phospholipid biosynthesis at the levels of transcriptional and enzyme activity. This is the first report implicating PLC in the direct regulation of lipid biosynthesis. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

In vitro exposure of tobacco specific nitrosamines decreases the rat lung phospholipids by enhanced phospholipase A(2) activity

Tobacco-specific nitrosamines (TSNA) have implications in the pathogenesis of various lung diseases and conditions are prevalent even in non-smokers. N-nitrosonornicotine (NNN) and 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are potent pulmonary carcinogens present in tobacco product and are mainly responsible for lung cancer. TSNA reacts with pulmonary surfactants, and alters the surfactant phospholipid. The present study was undertaken to investigate the in vitro exposure of rat lung tissue slices to NNK or NNN and to monitor the phospholipid alteration by P-32]orthophosphate labeling. Phospholipid content decreased significantly in the presence of either NNK or NNN with concentration and time dependent manner. Phosphatidylcholine (PC) is the main phospholipid of lung and significant reduction was observed in PC similar to 61%, followed by phosphatidylglycerol (PG) with 100 mu M of NNK, whereas NNN treated tissues showed a reduction in phosphatidylserine (PS) similar to 60% and PC at 250 mu M concentration. The phospholipase A(2) assays and expression studies reveal that both compounds enhanced phospholipid hydrolysis, thereby reducing the phospholipid content. Collectively, our data demonstrated that both NNK and NNN significantly influenced the surfactant phospholipid level by enhanced phospholipase A(2) activity. (C) 2014 Elsevier Ltd. All rights reserved.

A phospholipase A1 platelet activator from the wasp venom of Vespa magnifica (Smith)

Wasp is an impor tant venomous animal that can induce human fatalities. Aortic thrombosis and cerebral infarction are major clinical symptoms after massive wasp stings but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnifin, contains phospholipase-like activity and induces platelet aggregation. The cDNA encoding magnifin is cloned from the venom sac cDNA library of the wasp. The predicted protein was deduced from the cDNA with a sequence composed of 337 amino acid residues. Magnifin is very similar to other phospholipase A(1) (PLA(1)), especially to other wasp allergen PLA(1). Magnifin can activate platelet aggregation and induce thrombosis in vivo. The current results proved that PLA(1) in wasp venom could be contributable to aortic thrombosis after massive wasp stings. (c) 2007 Elsevier Ltd. All rights reserved.

Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake

A phospholipase A(2) (PLA(2)) called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 mumol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 mug/ml enzyme was incubated for 90 min in the presence of PC and Ca2+. No detectable hemolysis was noticed when PC was not added. Ca2+ was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca2+ was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC50 of 1.0 muM. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D49 PLA(2)s. Also, the residues concerned to Ca2+ binding were conserved. This is the first report of cDNA sequence of T jerdonii venom PLA(2). (C) 2002 Elsevier Science Ltd. All rights reserved.

Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A(2)

Several biochemical and biological activities such as phospholipase A(2), arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA(2)) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA(2) had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA(2) was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops muerosquamatus by chromatography. It

N49 phospholipase A(2), a unique subgroup of snake venom group II phospholipase A(2)

A novel phospholipase A(2) (PLA(2)) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C-18 chromatography and designated as TM-N49

Fluorescence analysis of phospholipase A(2) isolated from Chinese agkistrodon blomhoffii Ussurensis venom

The general and synchronous spectra of phospholipase A(2) (PLA(2)) isolated from Chinese agkistrodon blomhoffii Ussurensis snake venom were studied. The chromophores of PLA(2) were mainly contributed by tyrosine and tryptophane residues when the intervals between the excitation wavelength and the emssion waveleagth (Delta lambda) were 20nm and 75nm, respectively. The pH of buffers could change the fluorescence intensities of PLA(2) by changing the charge distribution of its amino acid chain. Ca2+ can not only increase the emission fluorescence intensity of PLA(2) but also improve the reaction rate of PLA(2) with its corresponding substrate DPPC.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A(2) and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase AZ and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained, In addition to the dominant protein [M+H](+) ions, multimeric and multiply charged ions were also observed in the mass spectra, The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected, Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes, Mass accuracies of 0.1 and 0.3 % were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-palyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants, Copyright (C) 1999 John Wiley & Sons, Ltd.

A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.

Ca2+-independent phospholipase A2-dependent gating of TRPM8 by lysophospholipids

TRPM8 represents an ion channel activated by cold temperatures and cooling agents, such as menthol, that underlies the cold-induced excitation of sensory neurons. Interestingly, the only human tissue outside the peripheral nervous system, in which the expression of TRPM8 transcripts has been detected at high levels, is the prostate, a tissue not exposed to any essential temperature variations. Here we show that the TRPM8 cloned from human prostate and heterologously expressed in HEK-293 cells is regulated by the Ca(2+)-independent phospholipase A(2) (iPLA(2)) signaling pathway with its end products, lysophospholipids (LPLs), acting as its endogenous ligands. LPLs induce prominent prolongation of TRPM8 channel openings that are hardly detectable with other stimuli (e.g. cold, menthol, and depolarization) and that account for more than 90% of the total channel open time. Down-regulation of iPLA(2) resulted in a strong inhibition of TRPM8-mediated functional responses and abolished channel activation. The action of LPLs on TRPM8 channels involved either changes in the local lipid bilayer tension or interaction with the critical determinant(s) in the transmembrane channel core. Based on this, we propose a novel concept of TRPM8 regulation with the involvement of iPLA(2) stimulation. This mechanism employs chemical rather than physical (temperature change) signaling and thus may be the main regulator of TRPM8 activation in organs not exposed to any essential temperature variations, as in the prostate gland.