Group VIB Ca2+-independent Phospholipase A2γ Promotes Cellular Membrane Hydrolysis and Prostaglandin Production in a Manner Distinct from Other Intracellular Phospholipases A2

Envenoming due to snakebites is an important public health problem in many tropical and subtropical countries which, in addition to mortality, may result in permanent sequelae as a consequence of local tissue damage, i.e. necrosis and hemorrhage. Toxins that play a leading role in the complex pathogenesis of these feared venom actions have been identified as members of the phospholipase A2 (PLA2) and metalloproteinase protein families. Phospholipases A2 are enzymes responsible for cellular membrane disruption through Ca2+-dependent hydrolysis of phospholipids. A class of these proteins (Lys49-PLA2s) does not show catalytic activity but can exert a pronounced local myotoxic effect that is not neutralized by serum therapy. After more than 20 years of structural, biochemical and biological studies with this class of proteins, its biological mechanism still remain not totally understood. Here, based in a comprehensive study including over than 30 crystallographic structures, Small Angle X-ray Scattering, Dynamic Light Scattering, Isothermal Titration Calorimetry, Biochemical, Bioinformatics, Phylogenic and Myografic experiments, we proposed a complete myotoxic mechanism. This work confirms the biological dimer indicated by recent studies in which both C-termini are in the dimeric interface. In this configuration, we propose that the myotoxic site of these toxins is composed by the Lys 20, Lys115 and Arg118 residues. The extensive structural analysis also include: (i) the function of hydrophobic long-chain molecules as Lys49-PLA2s inhibitors, (ii) the role of Lys122, previously indicated as being responsible for Lys49-PLA2s catalytic inactivity, (iii) a structural comparison of the Ca2+-binding loop region between Lys49 and Asp49-PLA2s, (iv) the importance of Tyr119 residue, (v) the role of different classes of inhibitors and (vi) the role of hydrophobic knuckle. Taking into account all these issues we were able to propose a complete mechanism of action of these proteins and also proposed the different ways to inhibit them. These results may be useful to guide new experiments that can definitively clarify the action mechanism of snake venom PLA2s and lead to the design of structure-based inhibitors to complement the serum therapy.

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Cytochemical analysis of mast cell granules after poly-dl-lysine action

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008225x ◽

1978 ◽

Vol 36 (2) ◽

pp. 278-279

Author(s):

R. Courtoy ◽

L.J. Simar ◽

J. Christophe

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Chemical Compounds ◽

Cellular Membrane ◽

Thin Sections ◽

Organic Bases ◽

Ferric Thiocyanate ◽

Cytochemical Analysis ◽

Mast Cell Granule ◽

Amine Liberation

Several chemical compounds induce amine liberation from mast cells but do not necessarily provoque the granule expulsion. For example, poly-dl-lysine induces modifications of the cellular membrane permeability which promotes ion exchange at the level of mast cell granules. Few of them are expulsed but the majority remains in the cytoplasm and appears less dense to the electrons. A cytochemical analysis has been performed to determine the composition of these granules after the polylysine action.We have previously reported that it was possible to demonstrate polyanions on epon thin sections using a cetylpyridinium ferric thiocyanate method. Organic bases are selectively stained with cobalt thiocyanate and the sulfhydryle groups are characterized with a silver methenamine reaction. These techniques permit to reveal the mast cell granule constituents, i.e. heparin, biogenic amines and basic proteins.

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The Effect of Mitomycin C on Platelet Aggregation and Adenosine 3′, 5′-Monophosphate Metabolism

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646667 ◽

1978 ◽

Vol 39 (01) ◽

pp. 177-185 ◽

Cited By ~ 3

Author(s):

Shuichi Hashimoto ◽

Sachiko Shibata ◽

Bonro Kobayashi

Keyword(s):

Platelet Aggregation ◽

Cyclic Amp ◽

Mitomycin C ◽

Blood Platelets ◽

Adenosine Diphosphate ◽

Cellular Membrane ◽

Adenyl Cyclase ◽

Cyclic Amp Phosphodiesterase ◽

Membrane Structure And Function ◽

And Function

SummaryThe effect of Mitomycin C on aggregation, adenosine 3′, 5′-monophosphate (cyclic AMP) metabolism and reactions induced by thrombin was studied in rabbit platelets. Mitomycin C inhibited the platelet aggregation induced by adenosine diphosphate or thrombin. The level of radioactive cyclic AMP derived from 8-14C adenine or 8-14C adenosine increased after incubating intact platelets with Mitomycin G. Formation of radioactive adenosine triphosphate also increased though mitochondrial oxidation was not stimulated. Similar effect was observed also in rabbit liver. Mitomycin C failed to stimulate platelet adenyl cyclase but inhibited cyclic AMP phosphodiesterase in the absence of theophylline. In the platelets preincubated with Mitomycin C, thrombin-induced inhibition of adenyl cyclase, stimulation of membrane-bound cyclic AMP phosphodiesterase, and release of 250,000 dalton protein from platelet membranes were prevented. These results suggest that Mitomycin C will affect cellular membrane structure and function, and this extranuclear effect of Mitomycin C will lead to inhibition of aggregation in blood platelets.

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Daunorubicin and Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650125 ◽

1981 ◽

Vol 45 (01) ◽

pp. 038-042 ◽

Cited By ~ 9

Author(s):

M E Pogliani ◽

R Fantasia ◽

G Lambertenghi-Deliliers ◽

E Cofrancesco

Keyword(s):

Electron Microscope ◽

Cellular Membrane ◽

Hemorrhagic Diathesis ◽

Clot Retraction ◽

Freezing And Thawing ◽

Platelet Factor ◽

High Doses ◽

Very High ◽

Platelet Functions

SummaryThe influence of Daunorubicin on some platelet functions in vitro was investigated, using different concentrations of the drug (0.01-0.02-0.04 μg/ml). Daunorubicin was shown to inhibit Collagen and Thrombin induced platelet aggregation and the intensity of inhibition depended on both drug concentration and the time of preincubation.Daunorubicin was also shown to inhibit the release reaction, the platelet prostaglandin pathway and the availability platelet factor 3; the drug at concentrations for clinical use does not damage the platelet membrane, as is the case with the freezing and thawing test, in platelet uptake of 14C-serotonin and as confirmed by the electron microscope. When very high doses (0.16 mg) of Daunorubicin are used, lysis of the platelets can be observed and this is confirmed under the electron microscope by the presence of empty platelets with fractures at the level of the cytoplasmic membrane.Finally, Daunorubicin causes irreversible inhibition of reptilase clot-retraction, even if this is less severe than with Vincristine. Working with gel-filtered platelets, it would appear that the inhibition exercised by the drug on platelet reactions is not caused through modifications in Ca++ metabolism.The authors suggest that Daunorubicin, at the dosages used clinically, induces in vitro thrombocytopathy without damaging the cellular membrane as confirmed by the electron microscope.This impairment of platelet functions could play a part in hemorrhagic diathesis observed during Daunorubicin therapy.

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EFFECT OF CELLULAR MEMBRANE RESISTIVITY INHOMOGENEITY ON THE THERMAL NOISE-LIMIT

JOURNAL OF ADVANCES IN PHYSICS ◽

10.24297/jap.v11i3.6859 ◽

2015 ◽

Vol 11 (3) ◽

pp. 3171-3183

Author(s):

Gyula Vincze

Keyword(s):

Field Strength ◽

Thermal Noise ◽

Cellular Membrane ◽

Thermal Limit ◽

Low Frequencies ◽

Membrane Resistivity ◽

Noise Limit ◽

The Asymmetry ◽

Characteristic Field

Our objective is to generalize the Weaver-Astumian (WA) and Kaune (KA) models of thermal noise limit to the case ofcellular membrane resistivity asymmetry. The asymmetry of resistivity causes different effects in the two models. In the KAmodel, asymmetry decreases the characteristic field strength of the thermal limit over and increases it below the breakingfrequency (10ï· ï€½ï´ mï€), while asymmetry decreases the spectral field strength of the thermal noise limit at all frequencies.We show that asymmetry does not change the character of the models, showing the absence of thermal noise limit at highand low frequencies in WA and KA models, respectively.

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Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190725102812 ◽

2019 ◽

Vol 19 (22) ◽

pp. 1952-1961 ◽

Cited By ~ 1

Author(s):

J.C. Sobrinho ◽

A.F. Francisco ◽

R. Simões-Silva ◽

A.M. Kayano ◽

J.J. Alfonso Ruiz Diaz ◽

...

Keyword(s):

Amino Acids ◽

Molecular Docking ◽

Synthetic Peptides ◽

Cell Damage ◽

Neutralization Assay ◽

Phospholipase Activity ◽

Phospholipases A2 ◽

Catalytically Active ◽

Bothrops Atrox

Background: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. Objective: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. Methods: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. Results: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. Conclusion: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

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Characterization of Venoms of Deinagkistrodon acutus and Bungarus multicinctus Using Proteomics and Peptidomics

Current Proteomics ◽

10.2174/1570164617666191121112319 ◽

2020 ◽

Vol 17 (3) ◽

pp. 241-254

Author(s):

Yaqiong Zhang ◽

Zhiping Jia ◽

Yunyang Liu ◽

Xinwen Zhou ◽

Yi Kong

Keyword(s):

Snake Venom ◽

Protein Families ◽

Traditional Medicines ◽

Phospholipases A2 ◽

Inhibitory Peptides ◽

Sds Page ◽

Deinagkistrodon Acutus ◽

Venom Proteins ◽

Proteins And Peptides

Background: Deinagkistrodon acutus (D. acutus) and Bungarus multicinctus (B. multicinctus) as traditional medicines have been used for hundreds of years in China. The venoms of these two species have strong toxicity on the victims. Objective: The objective of this study is to reveal the profile of venom proteins and peptides of D. acutus and B. multicinctus. Method: Ultrafiltration, SDS-PAGE coupled with in-gel tryptic digestion and Liquid Chromatography- Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS) were used to characterize proteins and peptides of venoms of D. acutus and B. multicinctus. Results: In the D. acutus venom, 67 proteins (16 protein families) were identified, and snake venom metalloproteinases (SVMPs, 38.0%) and snake venom C-type lectins (snaclecs, 36.7%) were dominated proteins. In the B. multicinctus venom, 47 proteins (15 protein families) were identified, and three-finger toxins (3FTxs, 36.3%) and Kunitz-type Serine Protease Inhibitors (KSPIs, 32.8%) were major components. In addition, both venoms contained small amounts of other proteins, such as Snake Venom Serine Proteinases (SVSPs), Phospholipases A2 (PLA2s), Cysteine-Rich Secreted Proteins (CRISPs), 5'nucleotidases (5'NUCs), Phospholipases B (PLBs), Phosphodiesterases (PDEs), Phospholipase A2 Inhibitors (PLIs), Dipeptidyl Peptidases IV (DPP IVs), L-amino Acid Oxidases (LAAOs) and Angiotensin-Converting Enzymes (ACEs). Each venom also had its unique proteins, Nerve Growth Factors (NGFs) and Hyaluronidases (HYs) in D. acutus, and Cobra Venom Factors (CVFs) in B. multicinctus. In the peptidomics, 1543 and 250 peptides were identified in the venoms of D. acutus and B. multicinctus, respectively. Some peptides showed high similarity with neuropeptides, ACE inhibitory peptides, Bradykinin- Potentiating Peptides (BPPs), LAAOs and movement related peptides. Conclusion: Characterization of venom proteins and peptides of D. acutus and B. multicinctus will be helpful for the treatment of envenomation and drug discovery.

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Differential effects of calmodulin antagonists on phospholipases A2 and C in thrombin-stimulated platelets.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43306-6 ◽

1981 ◽

Vol 256 (23) ◽

pp. 12523-12528 ◽

Cited By ~ 11

Author(s):

R.W. Walenga ◽

E.E. Opas ◽

M.B. Feinstein

Keyword(s):

Calmodulin Antagonists ◽

Differential Effects ◽

Phospholipases A2

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Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach

Toxins ◽

10.3390/toxins13070453 ◽

2021 ◽

Vol 13 (7) ◽

pp. 453

Author(s):

Sebastian Estrada-Gómez ◽

Leidy Johana Vargas-Muñoz ◽

Cesar Segura Latorre ◽

Monica Maria Saldarriaga-Cordoba ◽

Claudia Marcela Arenas-Gómez

Keyword(s):

Enzymatic Activity ◽

Molecular Mass ◽

Evolutionary Relationship ◽

Venom Gland ◽

Duplication Event ◽

High Molecular Mass ◽

Amino Acid Sequences ◽

Secretory Proteins ◽

Phospholipases A2 ◽

Kunitz Type

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

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