The Experts below are selected from a list of 507 Experts worldwide ranked by ideXlab platform
Long-sen Chang - One of the best experts on this subject based on the ideXlab platform.
-
Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus Notexin
Toxicon : official journal of the International Society on Toxinology, 2014Co-Authors: Yiling Chiou, Shinneren Lin, Long-sen ChangAbstract:Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus Notexin induced apoptosis of human cancer cells. However, NnaPLA2 and Notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and Notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and Notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and Notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and Notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of Notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of Notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and Notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and Notexin, and membrane compositions determine the effect of PS on PLA2 activity.
-
guanidination of Notexin promotes its phospholipase a2 activity independent fusogenicity on vesicles with lipid supplied negative curvature
Toxicon, 2012Co-Authors: Peihsiu Kao, Yiling Chiou, Yingjung Chen, Shinneren Lin, Long-sen ChangAbstract:To address the requirement of phospholipase A2 (PLA2) activity in membrane fusion events and membrane perturbation activity of Notexin and guanidinated Notexin (Gu-Notexin), the present study was conducted. Notexin and Gu-Notexin did not show PLA2 activity after the removal of Ca2+ with EDTA. Metal-free Notexin and Gu-Notexin were found to induce membrane leakage and fusion of phospholipid vesicles. Fusogenic activity of native and modified Notexin correlated positively with their membrane-damaging activity underlying the deprivation of PLA2 activity. Compared with Ca2+-bound Gu-Notexin, fusogenicity of metal-free Gu-Notexin was notably increased by incorporation of cholesterol, cholesterol sulfate, phosphatidylethanolamine, α-tocopherol and phosphatidic acid that supplied negative curvature into phospholipid bilayer. The ability of Gu-Notexin to induce membrane fusion of vesicles with lipid-supplied negative curvature was higher than that of Notexin regardless of the absence or presence of Ca2+. Consistently, metal-free Gu-Notexin markedly induced membrane fusion of red blood cells (RBCs) compared with metal-free Notexin, and fusion activity of metal-free Gu-Notexin on cholesterol-depleted RBCs notably reduced. Compared with Notexin, Gu-Notexin highly induced uptake of calcein-loaded phosphatidylcholine (PC)/cholesterol and PC/cholesterol sulfate vesicles by K562 cells in the presence of EDTA. Taken together, our data suggest that Notexin and Gu-Notexin could induce vesicle leakage and fusion via a PLA2 activity-independent mechanism, and guanidination promotes PLA2 activity-independent fusogenicity of Notexin on vesicles with lipid-supplied negative curvature.
-
guanidination of Notexin alters its membrane damaging activity in response to sphingomyelin and cholesterol
Journal of Biosciences, 2010Co-Authors: Peihsiu Kao, Yiling Chiou, Shinneren Lin, Long-sen ChangAbstract:To elucidate the contribution of phospholipase A 2 (PLA 2 ) activity of Notexin to its ability to perturb membranes, comparative studies on the interaction of Notexin and guanidinated Notexin (Gu-Notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of Notexin against EYPC vesicles, but had an insignifi cant infl uence on that of Gu-Notexin. Unlike the effects noted with Notexin, inactivation of PLA 2 activity by EDTA led to a reduction in the ability of Gu-Notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-Notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of Notexin and Gu-Notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of Notexin and Gu-Notexin differed in either Ca 2+ -bound or metal-free states. These results indicate that Notexin and Gu-Notexin could induce membrane permeability without the involvement of PLA 2 activity, and suggest that guanidination alters the membrane-bound mode of Notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.
-
Notexin upregulates fas and fasl protein expression of human neuroblastoma sk n sh cells through p38 mapk atf 2 and jnk c jun pathways
Toxicon, 2010Co-Authors: Ku Chung Chen, Long-sen ChangAbstract:Abstract Notechis scutatus scutatus Notexin induced an increase in Fas and FasL protein expression of human neuroblastoma SK-N-SH cells in a dose- and time-dependent manner. Moreover, Notexin treatment upregulated transcription of Fas/FasL mRNA. Downregulation of FADD blocked Notexin-induced procaspase-8 degradation and cleavage of Bid and rescued viability of Notexin-treated cells. Upon exposure to Notexin, activation of JNK and p38 MAPK was observed in SK-N-SH cells. Notexin-induced upregulation of Fas and FasL was suppressed by SB202190 (p38 MAPK inhibitor) and S600125 (JNK inhibitor). Downregulation of p38α MAPK and JNK1 by siRNA proved that upregulation of Fas/FasL was related to p38α MAPK and JNK1 activation. Notexin treatment evoked p38α MAPK-mediated ATF-2 phosphorylation and JNK1-mediated c-Jun phosphorylation. Knockdown of c-Jun and ATF-2 by siRNA or overexpression of dominant-negative c-Jun and ATF-2 revealed that both c-Jun and ATF-2 were crucial for Fas/FasL upregulation. Taken together, our data indicate that Notexin-induced upregulation of Fas and FasL is triggered by p38 MAPK/ATF-2 and JNK/c-Jun signaling pathways in SK-N-SH cells.
-
calcium stimulated mitogen activated protein kinase activation elicits bcl xl downregulation and bak upregulation in Notexin treated human neuroblastoma sk n sh cells
Journal of Cellular Physiology, 2010Co-Authors: Ku Chung Chen, Peihsiu Kao, Wen Hsin Liu, Long-sen ChangAbstract:Notechis scutatus scutatus Notexin induced apoptotic death of SK-N-SH cells accompanied with downregulation of Bcl-xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to Notexin, Ca(2+)-mediated JNK and p38 MAPK activation were observed in SK-N-SH cells. Production of ROS was a downstream event followed by Ca(2+)-mediated mitochondrial alteration. Notexin-induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho-p38 MAPK and phospho-JNK were proved to be involved in Bcl-xL degradation, and overexpression of Bcl-xL attenuated the cytotoxic effect of Notexin. Bak upregulation was elicited by p38 MAPK-mediated ATF-2 activation and JNK-mediated c-Jun activation. Suppression of Bak upregulation by ATF-2 siRNA or c-Jun siRNA attenuated Notexin-evoked mitochondrial depolarization and rescued viability of Notexin-treated cells. Taken together, our data indicate that Notexin-induced apoptotic death of SK-N-SH cells is mediated through mitochondrial alteration triggering by Ca(2+)-evoked p38 MAPK/ATF-2 and JNK/c-Jun signaling pathways.
John Harris - One of the best experts on this subject based on the ideXlab platform.
-
the neurotoxicity of the venom phospholipases a2 Notexin and taipoxin
Experimental Neurology, 2000Co-Authors: John Harris, B D Grubb, C A Maltin, R DixonAbstract:The presynaptically active, toxic phospholipases known as Notexin and taipoxin are principal components of the venom of the Australian tiger snake and the Australian taipan respectively. The inoculation of the toxins into one hind limb of rats caused, within 1 h, the depletion of transmitter from the motor nerve terminals of the soleus muscle. This was followed by the degeneration of the motor nerve terminals and of the axonal cytoskeleton. By 24 h 70% of muscle fibers were completely denervated. Regeneration and functional reinnervation were almost fully restored by 5 days, but collateral innervation was common in the regenerated muscles, and this abnormality persisted for at least 9 months. The data provide an explanation for both the severity of neuromuscular paralysis that can accompany envenoming bites by tiger snakes and taipans and the difficulty experienced by physicians in managing the envenomed subjects.
-
myotoxic activity of the toxic phospholipase Notexin from the venom of the australian tiger snake
Journal of Neuropathology and Experimental Neurology, 1996Co-Authors: Rupert W Dixon, John HarrisAbstract:Notexin is a neurotoxic and myotoxic phospholipase A 2 derived from the venom of the Australian tiger snake, Notechis scutatus. Though the phospholipase activity has been implicated in the toxicity of Notexin, little is understood of its site and mode of action. In this study we investigated the myotoxicity of Notexin on the skeletal muscle of the rat. Using immunogold labeling both in vivo and in vitro, we demonstrated that Notexin bound exclusively to the sarcolemma. At the early stages when Notexin was injected into the muscle there was no evidence of internalization, though at more advanced degeneration when muscle fibers were necrotic, the toxin was able to penetrate the interior of the fibers to exhibit nonspecific labeling. We also showed the toxin was able to bind to glycolytic muscle fibers, which are known to be resistant to the myotoxic effects of Notexin. Electron microscopy confirmed the localization of the binding site to the sarcolemma. Scanning electron microscopy showed that the primary pathological changes associated with exposure to Notexin were the appearance of areas of hypercontraction along the muscle fibers associated with small lesions in the sarcolemma. At more advanced stages large tears appeared in the sarcolemma between adjacent areas of hypercontraction, revealing apparently intact myofibrils below. We conclude that the sarcolemma is the binding site for the toxin. We propose that the hydrolytic activity causes the appearance of small lesions in the membrane, the loss of ion gradients, and hypercontraction. The weakened area between sites of hypercontraction is then ruptured, leading ultimately to the degeneration of the muscle fibers.
J B Harris - One of the best experts on this subject based on the ideXlab platform.
-
the early expression of myotoxicity and localization of the binding sites of Notexin in the soleus muscle of the rat
Advances in Experimental Medicine and Biology, 1996Co-Authors: R W Dixon, J B HarrisAbstract:Notexin is a major toxic component of the venom of the Australian tiger snake, Notechis scutatus scutatus . It is a peptide of 119 residues with 7 disulphide bridges and a relative molecular mass of 13.6 KDa. Notexin is one of a group of neurotoxic myotoxins that exhibit phospholipase A2 activity and are homologous with mammalian pancreatic phospholipases (Harris, 1991). The pathological responses of skeletal muscle to Notexin and related toxins have been well documented (Harris and Cullen, 1990) but little is known of the precise mechanism of action of the toxins. Based on a great deal of descriptive data, Harris (1984) suggested that the hydrolysis and fragmentation of the the plasma membrane was the primary event, leading to the loss of ionic gradients, influx of Ca2+, hypercontraction and degeneration of major muscle fibre proteins. It was conceded, however, that the physical disintegration of the membrane was not a necessary prerequisite for the onset of degeneration, and that the hydrolysis of membrane lipids could lead to a “leaky” membrane without evidence of structural damage.
Paola Caccin - One of the best experts on this subject based on the ideXlab platform.
-
production in escherichia coli folding purification and characterization of Notexin with wild type sequence and with n terminal and catalytic site mutations
Toxicon, 2014Co-Authors: Morena Simonato, Paola Caccin, Laura Morbiato, Veronica Zorzi, Julian Fernandez, Maria Lina Massimino, Patrizia Polverino De Laureto, Fiorella TonelloAbstract:Notexin (Ntx) is a group I phospholipase A2 (PLA2) protein, main component of the Australian snake Notechis scutatus scutatus venom. It is both a presynaptic neurotoxin and a myotoxin. In this work, for the first time, a method for the production and folding of recombinant Ntx was developed. Ntx was produced with wild type sequence (rNtx), with an extra peptide (T7-Ntx) or a methionine (M-Ntx) before Asn-1, and with Asn-1 substituted by alanine (Ntx-A1) or by serine (Ntx-S1). The proteins were analyzed for their catalytic and toxic activities. rNtx activity resulted to be comparable to that of the venom extracted protein. The Ntx N-terminus was found to have a major influence on both the catalytic and toxic activities of the protein. The first amino acid of snake venom PLA2s is highly conserved: it is an asparagine in about all group I PLA2s, while in most (>70%) of group II PLA2s it is a serine or an asparagine. Interestingly, Ntx-S1 resulted to be, for both enzymatic and toxic activities, the mutant most similar to the wild type protein. The role of the catalytic activity of Ntx in its toxicity was investigated by replacing the aspartic acid 49, involved in the coordination of the cofactor calcium ion, by a lysine. The obtained mutant (Ntx-K49) is deprived of catalytic activity but possesses a residual toxicity.
-
calcium overload in nerve terminals of cultured neurons intoxicated by alpha latrotoxin and snake pla2 neurotoxins
Toxicon, 2009Co-Authors: Erik Tedesco, Michela Rigoni, Paola Caccin, Eugene V Grishin, Ornella Rossetto, Cesare MontecuccoAbstract:Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca(2+) which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of beta-bungarotoxin, taipoxin, Notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca(2+) influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca(2+) overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins.
-
snake presynaptic neurotoxins with phospholipase a2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles
Journal of Cell Science, 2004Co-Authors: Michela Rigoni, Paola Caccin, Ornella Rossetto, Cesare Montecucco, Giampietro Schiavo, Anne E Weston, Federica Allegrini, Maria Pennuto, Flavia ValtortaAbstract:The mechanisms of action of four snake presynaptic phospholipase A2 neurotoxins were investigated in cultured neurons isolated from various parts of the rat brain. Strikingly, physiological concentrations of Notexin, β-bungarotoxin, taipoxin or textilotoxin induced a dose-dependent formation of discrete bulges at various sites of neuronal projections. Neuronal bulging was paralleled by the redistribution of the two synaptic vesicle markers synaptophysin I (SypI) and vesicle-attached membrane protein 2 (VAMP2) to the bulges, and by the exposure of the luminal domain of synaptotagmin on the cell surface. These neurotoxins induced glutamate release from cultured neurons similarly to the known evoked release of acetylcholine from neuromuscular junctions. In addition, partial fragmentation of F-actin and neurofilaments was observed in neurons, but not in astrocytes. These findings indicate that these snake presynaptic neurotoxins act with by same mechanism and that the observed phenotype results from the fusion of synaptic vesicles with the plasma membrane not balanced by an adequate membrane retrieval. These changes closely resemble those occurring at neuromuscular junctions of intoxicated animals and fully qualify these primary neuronal cultures as pertinent models for studying the molecular mode of action of these neurotoxins.
Peihsiu Kao - One of the best experts on this subject based on the ideXlab platform.
-
guanidination of Notexin promotes its phospholipase a2 activity independent fusogenicity on vesicles with lipid supplied negative curvature
Toxicon, 2012Co-Authors: Peihsiu Kao, Yiling Chiou, Yingjung Chen, Shinneren Lin, Long-sen ChangAbstract:To address the requirement of phospholipase A2 (PLA2) activity in membrane fusion events and membrane perturbation activity of Notexin and guanidinated Notexin (Gu-Notexin), the present study was conducted. Notexin and Gu-Notexin did not show PLA2 activity after the removal of Ca2+ with EDTA. Metal-free Notexin and Gu-Notexin were found to induce membrane leakage and fusion of phospholipid vesicles. Fusogenic activity of native and modified Notexin correlated positively with their membrane-damaging activity underlying the deprivation of PLA2 activity. Compared with Ca2+-bound Gu-Notexin, fusogenicity of metal-free Gu-Notexin was notably increased by incorporation of cholesterol, cholesterol sulfate, phosphatidylethanolamine, α-tocopherol and phosphatidic acid that supplied negative curvature into phospholipid bilayer. The ability of Gu-Notexin to induce membrane fusion of vesicles with lipid-supplied negative curvature was higher than that of Notexin regardless of the absence or presence of Ca2+. Consistently, metal-free Gu-Notexin markedly induced membrane fusion of red blood cells (RBCs) compared with metal-free Notexin, and fusion activity of metal-free Gu-Notexin on cholesterol-depleted RBCs notably reduced. Compared with Notexin, Gu-Notexin highly induced uptake of calcein-loaded phosphatidylcholine (PC)/cholesterol and PC/cholesterol sulfate vesicles by K562 cells in the presence of EDTA. Taken together, our data suggest that Notexin and Gu-Notexin could induce vesicle leakage and fusion via a PLA2 activity-independent mechanism, and guanidination promotes PLA2 activity-independent fusogenicity of Notexin on vesicles with lipid-supplied negative curvature.
-
guanidination of Notexin alters its membrane damaging activity in response to sphingomyelin and cholesterol
Journal of Biosciences, 2010Co-Authors: Peihsiu Kao, Yiling Chiou, Shinneren Lin, Long-sen ChangAbstract:To elucidate the contribution of phospholipase A 2 (PLA 2 ) activity of Notexin to its ability to perturb membranes, comparative studies on the interaction of Notexin and guanidinated Notexin (Gu-Notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of Notexin against EYPC vesicles, but had an insignifi cant infl uence on that of Gu-Notexin. Unlike the effects noted with Notexin, inactivation of PLA 2 activity by EDTA led to a reduction in the ability of Gu-Notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-Notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of Notexin and Gu-Notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of Notexin and Gu-Notexin differed in either Ca 2+ -bound or metal-free states. These results indicate that Notexin and Gu-Notexin could induce membrane permeability without the involvement of PLA 2 activity, and suggest that guanidination alters the membrane-bound mode of Notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.
-
calcium stimulated mitogen activated protein kinase activation elicits bcl xl downregulation and bak upregulation in Notexin treated human neuroblastoma sk n sh cells
Journal of Cellular Physiology, 2010Co-Authors: Ku Chung Chen, Peihsiu Kao, Wen Hsin Liu, Long-sen ChangAbstract:Notechis scutatus scutatus Notexin induced apoptotic death of SK-N-SH cells accompanied with downregulation of Bcl-xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to Notexin, Ca(2+)-mediated JNK and p38 MAPK activation were observed in SK-N-SH cells. Production of ROS was a downstream event followed by Ca(2+)-mediated mitochondrial alteration. Notexin-induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho-p38 MAPK and phospho-JNK were proved to be involved in Bcl-xL degradation, and overexpression of Bcl-xL attenuated the cytotoxic effect of Notexin. Bak upregulation was elicited by p38 MAPK-mediated ATF-2 activation and JNK-mediated c-Jun activation. Suppression of Bak upregulation by ATF-2 siRNA or c-Jun siRNA attenuated Notexin-evoked mitochondrial depolarization and rescued viability of Notexin-treated cells. Taken together, our data indicate that Notexin-induced apoptotic death of SK-N-SH cells is mediated through mitochondrial alteration triggering by Ca(2+)-evoked p38 MAPK/ATF-2 and JNK/c-Jun signaling pathways.
-
phospholipase a2 activity independent membrane damaging effect of Notexin
Toxicon, 2007Co-Authors: Peihsiu Kao, Shinneren Lin, Long-sen ChangAbstract:Abstract To elucidate whether the phospholipase A2 (PLA2) activity of Notexin was exclusively associated with the manifestation of its pharmacological activities, the interaction of Notexin with phospholipid liposomes was explored by fluorescence and CD measurement underlying the conditions of depriving its PLA2 activity. Although a higher membrane-damaging activity was noted with Ca2+-bound Notexin, abolishment of PLA2 activity by EDTA and Sr2+ could not diminish the membrane-damaging activity of Notexin. Fluorescence-quenching studies and CD measurement indicated that Ca2+-bound, Sr2+-bound or metal-free Notexin did not adopt the same conformation upon binding with phospholipids. Regardless of the presence of Ca2+, Sr2+ or EDTA, self-quenching assay with rhodamine-labeled Notexin revealed that the toxin pertained to form oligomer when it bound with liposomes. Although Lys-modified Notexin retained full PLA2 activity, a notable decrease in membrane-damaging activity was observed. These results indicate that Notexin could directly cause a leakage of membrane via a PLA2 activity-independent manner, and implicate that another biological event contributes to the activity of Notexin in vivo.
