Explore the vast range of titles available.

Group IIA secreted phospholipase A 2 (group IIA sPLA 2 ) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of native stingray and dromedary groups V, IIA, and IB sPLA 2 s on several Gram-positive and Gram-negative strains. The rank order potency among both marine and mammal sPLA 2 s against Gram-positive bacteria is group IIA > V > IB, whereas Gram-negative bacteria exhibited a much higher resistance. There is a synergic action of the sPLA 2 with lysozyme when added to the bacteria culture prior to sPLA 2 .The bactericidal efficiency of groups V and IIA sPLA 2 s was shown to be dependent upon the presence of calcium ions and to a less extent Mg 2+ ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray and dromedary groups V, IIA, and IB sPLA 2 s present no cytotoxicity after their incubation with MDA-MB-231cells. stingray groups V and IIA sPLA 2 s, like mammal ones, may be considered as future therapeutic agents against bacterial infections.

Human parvovirus B19 (B19V) is the causative agent of erythema infectiosum in humans. B19 infection also causes severe disease manifestations, such as chronic anemia in immunocompromised patients, aplastic crisis in patients with a high turnover rate of red blood cells, and hydrops fetalis in pregnant women. Although a secreted phospholipase A2 (PLA2) motif has been identified in the unique region of the B19V minor capsid protein VP1(VP1u), the determinants for its enzyme activity and its influences on host cells are not well understood. The purpose of this study was to investigate the contribution of the PLA2 motif and other regions of the VP1u to the PLA2 activity, to determine the cellular localization of the VP1u protein, and to examine the effects of VP1u on cellular cytokines. We found that in addition to the critical conserved and non-conserved amino acids within the VP1u PLA2 motif, amino acid residues outside the VP1u PLA2 motif are also important for the PLA2 activity. VP1u and various mutants all revealed a nucleo-cytoplasmic distribution. UT7-Epo cells treated with prokaryotic expressed VP1u or mutant proteins with PLA2 activity released a large amount of free fatty acid (FFA), and the cell morphological change occurred dramatically. However, neither free fatty acid nor cell morphology change occurred for cells treated with the mutants without PLA2 activity. The wild type and the VP1u mutants with the PLA2 activity also activated TNF-α promoter and upregulated the transcription activity of NF-κB in transfected cells. In addition, we found that the amino acids outside the PLA2 domain are critical for the viral PLA2 activity, and that these tested VP1u mutants did not affect the localization of the VP1u protein.

Lancehead pitvipers, snakes, or, popularly, \"jararacas\", are common and broadly distributed in the Americas, especially in Brazil, where they are responsible for causing a high number of snakebite accidents. Their venoms are able to induce local and systemic effects, such as hemorrhaging, acute kidney failure, and shock, that can be fatal. Among the compounds of the venom are phospholipases A (PLA s), which are abundant in some species. PLA s can perform different activities during envenoming, such as neurotoxicity, myotoxicity, and cytotoxicity, among others, through the hydrolysis of the ester bond at the sn-2 position of phospholipids, producing free fatty acids and lysophospholipids. Although different PLA s can be classified into different PLA groups and subgroups, according to structure, function, size, localization and Ca dependence, they converge to be available in biotechnological and therapeutic applications, such as antiviral and antitumor, among others, being relevant molecules to be deeply studied. Here, we provide the state of the art of PLA s, found in snake venoms, focusing on venoms, as well as their potential applications, beyond their inhibitors, that also receive attention due their importance in PLA studies and diverse applications.

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

Drugs such as necopidem, saripidem, alpidem, zolpidem, and olprinone contain nitrogen-containing bicyclic, condensed-imidazo[1,2-α]pyridines as bioactive scaffolds. In this work, we report a high-yield one pot synthesis of 1-(2-methyl-8-aryl-substitued-imidazo[1,2-α]pyridin-3-yl)ethan-1-onefor the first-time. Subsequently, we performed in silico mode-of-action analysis and predicted that the synthesized imidazopyridines targets Phospholipase A2 (PLA2). In vitro analysis confirmed the predicted target PLA2 for the novel imidazopyridine derivative1-(2-Methyl-8-naphthalen-1-yl-imidazo [1,2-α]pyridine-3-yl)-ethanone (compound 3f) showing significant inhibitory activity towards snake venom PLA2 with an IC50 value of 14.3 μM. Evidently, the molecular docking analysis suggested that imidazopyridine compound was able to bind to the active site of the PLA2 with strong affinity, whose affinity values are comparable to nimesulide. Furthermore, we estimated the potential for oral bioavailability by Lipinski's Rule of Five. Hence, it is concluded that the compound 3f could be a lead molecule against snake venom PLA2.

Integrins are essential in the complex multistep process of angiogenesis and are thus attractive targets for the development of antiangiogenic therapies. Integrins are antagonized by disintegrins and C-type lectin-like proteins, two protein families from snake venom. Here, we report that CC-PLA2-1 and CC-PLA2-2, two novel secreted phospholipases A2 (PLA2) isolated from Cerastes cerastes venom, also showed anti-integrin activity. Indeed, both PLA2s efficiently inhibited human brain microvascular endothelial cell adhesion and migration to fibrinogen and fibronectin in a dose-dependent manner. Interestingly, we show that this anti-adhesive effect was mediated by α5β1 and αv-containing integrins. CC-PLA2s also impaired in vitro human brain microvascular endothelial cell tubulogenesis on Matrigel and showed antiangiogenic activity in vivo in chicken chorioallantoic membrane assay. The complete PLA2 cDNAs were cloned from a venom gland cDNA library. Mature CC-PLA2-1 and CC-PLA2-2 contain 121 and 120 amino acids, respectively, including 14 cysteines each and showed 83% identity. Tertiary model structures of CC-PLA2-1 and CC-PLA2-2 were generated by homology modeling. This is thus the first study describing an antiangiogenic effect for snake venom PLA2s and reporting first clues to their mechanism of action on endothelial cells.

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25–37∘C. Maximum PLA2 activity required Ca2+ and in the presence of Cd2+, Zn2+, Mn2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.

Human phospholipase A2 (hPLA2) of the IIA group (HGIIA) catalyzes the hydrolysis of membrane phospholipids, producing arachidonic acid and originating potent inflammatory mediators. Therefore, molecules that can inhibit this enzyme are a source of potential anti-inflammatory drugs, with different action mechanisms of known anti-inflammatory agents. For the study and development of new anti-inflammatory drugs with this action mechanism, snake venom PLA2 (svPLA2) can be employed, since the svPLA2 has high similarity with the human PLA2 HGIIA. Despite the high similarity between these secretory PLA2s, it is still not clear if these toxins can really be employed as an experimental model to predict the interactions that occur with the human PLA2 HGIIA and its inhibitors. Thus, the present study aims to compare and evaluate, by means of theoretical calculations, docking and molecular dynamics simulations, as well as experimental studies, the interactions of human PLA2 HGIIA and two svPLA2s, Bothrops toxin II and Crotoxin B (BthTX-II and CB, respectively). Our theoretical findings corroborate experimental data and point out that the human PLA2 HGIIA and svPLA2 BthTX-II lead to similar interactions with the studied compounds. From our results, the svPLA2 BthTX-II can be used as an experimental model for the development of anti-inflammatory drugs for therapy in humans.

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA 2 ) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA 2 and the Gly80Ser mutation with function. sPLA 2 with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70 ns. Structural analysis revealed the enzymes to comprise three helices (H1–H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1–L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1 Å. The active site within the substrate binding channel shows a catalytic triad of water–His67–Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD.