Potential therapeutic efficacy of pachymic acid in chronic kidney disease induced in rats: role of Wnt/ß-catenin/renin-angiotensin axis.

OBJECTIVES: Chronic kidney disease (CKD) is a major public health problem associated with high mortality. The therapeutic effects of pachymic in CKD management and its underlying mechanisms have not been studied. Therefore, we aimed to investigate the possible inhibitory effect of PA on renal Wnt/ß-catenin signalling in CKD. METHODS: CKD was induced in rats by doxorubicin (DOX; 3.5 mg/kg i.p., twice weekly for 3 weeks). Rats were treated orally with PA (10 mg/kg/day), LOS (10 mg/kg/day) or their combination (PA + LOS) for 4 weeks starting after the last dose of DOX. KEY FINDINGS: DOX-induced renal injury was characterized by high serum cystatin-C, and urine albumin/creatinine ratio, renal content of podocin and klotho were decreased. Tumour necrosis factor-α, interleukin-6, interleukin-1ß, Wnt1, active ß-catenin/total ß-catenin ratio and fibronectin along with mRNA expression of RENIN, ACE and AT1 were increased in renal tissues. Treatment with either PA or LOS ameliorated all DOX-induced changes. The combined treatment was more effective in improving all changes than monotherapy. CONCLUSIONS: These results suggest a new therapeutic benefit of PA in ameliorating CKD in rats through its up-regulatory effect on renal klotho thereby preventing Wnt/ß-catenin reactivation and RAS gene expression. PA/LOS combination provided an additional inhibition of Wnt/ß-catenin signalling and its downstream targets.

Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA2, a few spots corresponding to acidic PLA2 were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. BIOLOGICAL SIGNIFICANCE: Local tissue damage induced by myotoxic PLA2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein.

Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin.

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Pachymic Acid Induces Apoptosis of EJ Bladder Cancer Cells by DR5 Up-Regulation, ROS Generation, Modulation of Bcl-2 and IAP Family Members.

Pachymic acid (PA) is a lanostane-type triterpenoid derived from Poria cocos mushroom that possess various biological effects such as anti-cancer, antiinflammatory and anti-metastasis effects. In this study, we investigated the anti-cancer effects of PA in EJ bladder cancer cells. The results showed that PA significantly inhibited proliferation of EJ cells in a dose-dependent manner. PA induced accumulation of sub-G1 DNA content (apoptotic cell population), apoptotic bodies and chromatin condensation and DNA fragmentation in EJ cells in a dose-dependent manner. PA also induces activation of caspase-3, -8 and -9, and subsequent cleavage of poly (ADP-ribose) polymerase, and significantly suppressed the inhibitor of apoptosis protein family proteins in a dose-dependent manner. Furthermore, PA activates Bid and induced the loss of mitochondrial membrane potential (ΔΨm ) with up-regulated pro-apoptotic proteins (Bax and Bad), down-regulated anti-apoptotic proteins (Bcl-2 and Bcl-xL) and cytochrome c release. In turn, PA increased the generation of reactive oxygen species (ROS); also, the ROS production was blocked by N-acetyl-L-cysteine. The expressions of TNF-related apoptosis inducing ligand and death receptor 5 were up-regulated by PA in a dose-dependent manner, suggesting extrinsic pathway also involved in PA-induced apoptosis. This study provides evidence that PA might be useful in the treatment of human bladder cancer.

Correlação e associação de renda e escolaridade com condições de saúde e nutrição em obesos graves / Correlation and association of income and educational level with health and nutritional conditions among the morbidly obese

O objetivo deste artigo é investigar relações entre renda e escolaridade com condições de saúde e nutrição em obesos graves. Estudo transversal ambulatorial com 79 pacientes de primeira consulta, com Índice de Massa Corporal (IMC) ≥ 35 kg/m2 e idade ≥ 20 anos. Coletaram-se dados: sociodemográficos, antropométricos, estilo de vida, exames bioquímicos e consumo alimentar. O IMC médio foi 48,3 ± 6,9 kg/m2. Observou-se correlação negativa significante de escolaridade com variáveis peso (r = -0,234) e IMC (r = -0,364) e de renda familiar per capita com consumo diário de vegetal A (r = -0,263). Após análise multivariada maior renda familiar per capita se associou à ausência de cardiopatia (RP: 0,51, IC95%: 0,32-0,81), maior consumo diário de vegetal A (RP: 1,79, IC95%: 1,16-2,75) e doces (RP: 3,12, IC95%: 1,21-8,04). Em obesos graves a maior renda familiar per capita se associou à ausência de cardiopatia e maior consumo de vegetais folhosos e doces. Já a escolaridade não se manteve associada às condições de saúde e nutrição.

This article seeks to investigate the relationship between income and educational level and health and nutritional conditions among the morbidly obese. A cross-sectional study was conducted with 79 patients at first appointment, with Body Mass Index (BMI) ≥ 35 kg/m2 and age ≥ 20 years. The following data was collected: demographic, socioeconomic, anthropometric, lifestyle, biochemical and food intake data. Average BMI was 48.3 ± 6.9 kg/m2. There was a significant negative correlation between education level and the variables of weight (r = -0.234) and BMI (r = -0.364) and per capita family income with daily consumption of leafy vegetables (r = -0.263). After multivariate analysis, higher per capita family income was associated with the absence of heart disease (PR: 0.51, CI95%: 0.32-0.81), higher daily consumption of leafy vegetables (PR: 1.79, CI95%: 1.16-2.75) and candy (PR: 3.12, CI95%: 1.21-8.04). In the morbidly obese, per capita household income was associated with absence of heart disease and higher consumption of leafy vegetables and candy. On the other hand, education level was not associated with health and nutrition conditions.

[Role of secreted and lipoprotein-associated phospholipase A2 in cardiovascular risk]. / Ruolo della fosfolipasi A2 secretoria e della fosfolipasi associata alle lipoproteine nel rischio cardiovascolare.

Phospholipase A(2) (PLA(2)) are enzymes that hydrolyze the ester bond of glycerophospholipids releasing free fatty acids and lysophospholipids, including the arachidonic acid, the precursor of the eicosanoids and the inflammatory cascades. PLA(2) are present in the atherosclerotic plaques and their direct involvement in the proatherogenic inflammatory response is well documented. Epidemiological and genetic studies have demonstrated the correlation of the PLA(2) mass and enzymatic activity with the incidence of cardiovascular diseases. The potential pro-atherogenic role of PLA(2) led to the development of two small molecules, varespladib, a reversible sPLA(2) inhibitor, and darapladib, a selective Lp-PLA(2) inhibitor. Both molecules have demonstrated antiatherosclerotic properties in animal models, and positive effects on atherosclerotic plaque composition evaluated in phase 2 clinical trials. On these grounds, the results of three phase 3 studies have recently been published: the VISTA-16 study with varespladib in patients with acute coronary syndrome, and the STABILITY and SOLID-TIMI 52 studies with darapladib in patients with stable coronary heart disease and acute coronary syndrome, respectively. Unexpectedly, both studies did not demonstrate an additional protective action of PLA 2 inhibitors over the standard of care treatment with statins, antiplatelet drugs, and coronary revascularization. In the present article, the enzymatic properties and the involvement of sPLA(2) and Lp-PLA(2) in atherogenesis are reviewed, with a focus on the results of experimental studies and clinical studies with both varespladib and darapladib inhibitors.

Interaction of phospholipase A of the E. coli outer membrane with the inhibitors of eucaryotic phospholipases A2 and their effect on the Ca²âº-induced permeabilization of the bacterial membrane.

Phospholipase A of the bacterial outer membrane (OMPLA) is a ß-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2--palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)--with OMPLA and considers a possible involvement of the enzyme in the Ca²âº-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca²âº-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²âº and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.

Acute coronary syndromes: targeting inflammation-what has the VISTA-16 trial taught us?

The VISTA-16 trial of varespladib, a secretory phospholipase A2 (sPLA2) inhibitor, in patients with an acute coronary syndrome was terminated prematurely owing to futility and a signal towards harm. Despite these discouraging results, therapies that target inflammation to modify pathways in atherogenesis remain an area of active investigation.

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial.

IMPORTANCE: Secretory phospholipase A2 (sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2 inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2 inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measure was a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95% CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95% CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2 inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246.

Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins.

Protobothrops flavoviridis serum proteins precipitated with ammonium sulfate were chromatographed on a DEAE-Toyopearl 650M column at pH 7.5 with stepwise increase or with linear gradient of NaCl concentration. Peaks 3 and 4 serum proteins, obtained by linear gradient elution and named Fr(de3) and Fr(de4), contained Habu serum factors (HSF) and phospholipase A2 (PLA2) inhibitors (PfPLI), respectively. The serum proteins eluted at 0.2 M NaCl by stepwise elution, named Fr(0.2NaCl), effectively suppressed myonecrosis and hemorrhage caused by P. flavoviridis venom in rat or mouse thigh muscles. The Fr(0.2NaCl) were fractionated by HPLC and the fractions, after SDS-PAGE, underwent far-western blot analysis with PLA2 ([Asp(49)]PLA2) and BPI ([Lys(49)]PLA2) as the probes. Four PfPLIs, namely, PfαPLI-A, PfαPLI-B, PfγPLI-A and PfγPLI-B, were identified together with their selective binding specificities to PLA2 species. In addition, a new 9 kDa protein, which is specifically bound to BPI, was found. Suppression of P. flavoviridis venom-induced severe lesions, such as myonecrosis, hemorrhage and edema, with its serum proteins was histopathologically observed in the present work for the first time. The cooperative use of P. flavoviridis antivenom and its serum proteins as medication for P. flavoviridis snake bites is discussed.

Involvement of phospholipase A/acyltransferase-1 in N-acylphosphatidylethanolamine generation.

Anandamide and other bioactive N-acylethanolamines (NAEs) are a class of lipid mediators and are produced from glycerophospholipids via N-acylphosphatidylethanolamines (NAPEs). Although the generation of NAPE by N-acylation of phosphatidylethanolamine is thought to be the rate-limiting step of NAE biosynthesis, the enzyme responsible, N-acyltransferase, remains poorly characterized. Recently, we found that five members of the HRAS-like suppressor (HRASLS) family, which were originally discovered as tumor suppressors, possess phospholipid-metabolizing activities including NAPE-forming N-acyltransferase activity, and proposed to call HRASLS1-5 phospholipase A/acyltransferase (PLA/AT)-1-5, respectively. Among the five members, PLA/AT-1 attracts attention because of its relatively high N-acyltransferase activity and predominant expression in testis, skeletal muscle, brain and heart of human, mouse and rat. Here, we examined the formation of NAPE by PLA/AT-1 in living cells. As analyzed by metabolic labeling with [(14)C]ethanolamine or [(14)C]palmitic acid, the transient expression of human, mouse and rat PLA/AT-1s in COS-7 cells as well as the stable expression of human PLA/AT-1 in HEK293 cells significantly increased the generation of NAPE and NAE. Liquid chromatography-tandem mass spectrometry also exhibited that the stable expression of PLA/AT-1 enhanced endogenous levels of NAPE, N-acylplasmenylethanolamine, NAE and glycerophospho-NAE. Furthermore, the knockdown of endogenous PLA/AT-1 in mouse ATDC5 cells lowered NAPE levels. Interestingly, the dysfunction of peroxisomes, which was caused by PLA/AT-2 and -3, was not observed in the PLA/AT-1-expressing HEK293 cells. Altogether, these results suggest that PLA/AT-1 is at least partly responsible for the generation of NAPE in mammalian cells.

Antioxidant protection by PECAM-targeted delivery of a novel NADPH-oxidase inhibitor to the endothelium in vitro and in vivo.

Oxidant stress caused by pathological elevation of reactive oxygen species (ROS) production in the endothelial cells lining the vascular lumen is an important component of many vascular and pulmonary disease conditions. NADPH oxidase (NOX) activated by pathological mediators including angiotensin and cytokines is a major source of endothelial ROS. In order to intercept this pathological pathway, we have encapsulated an indirect NOX inhibitor, MJ33, into immunoliposomes (Ab-MJ33/IL) targeted to endothelial marker platelet endothelial cell adhesion molecule (PECAM-1). Ab-MJ33/IL, but not control IgG-MJ33/IL are specifically bound to endothelium and attenuated angiotensin-induced ROS production in vitro and in vivo. Additionally, Ab-MJ33/IL inhibited endothelial expression of the inflammatory marker vascular cell adhesion molecule (VCAM) in cells and animals challenged with the cytokine TNF. Furthermore, Ab-MJ33/IL alleviated pathological disruption of endothelial permeability barrier function in cells exposed to vascular endothelial growth factor (VEGF) and in the lungs of mice challenged with lipopolysaccharide (LPS). Of note, the latter beneficial effect has been achieved both by prophylactic and therapeutic injection of Ab-MJ33/IL in animals. Therefore, specific suppression of ROS production by NOX in endothelium, attainable by Ab-MJ33/IL targeting, may help deciphering mechanisms of vascular oxidative stress and inflammation, and potentially improve treatment of these conditions.

Inhibition of breast cancer metastasis via PITPNM3 by pachymic acid.

Breast cancer metastasis is the most common cause of cancer-related death in women. Thus, seeking targets of breast tumor cells is an attractive goal towards improving clinical treatment. The present study showed that CCL18 from tumor-associated macrophages could promote breast cancer metastasis via PITPNM3. In addition, we found that pachymic acid (PA) could dose-dependently inhibit migration and invasion of MDA-MB-231 cells, with or without rCCL18 stimulation. Furthermore, evidence was obtained that PA could suppress the phosphorylation of PITPNM3 and the combination of CCL18 and PITPNM3. Therefore, we speculate that PA could inhibit breast cancer metastasis via PITPNM3.

Phospholipase A2 inhibitors in bacterial culture broth enhance pathogenicity of a fungus Nomuraea rileyi.

An entomopathogenic fungus, Nomuraea rileyi, was isolated and its identity was confirmed by its internal transcribed spacer DNA sequence. The isolated N. rileyi exhibited a specific pathogenicity to lepidopteran species. This study was focused on enhancing the fungal pathogenicity by using immunosuppressive agents. In response to infection of N. rileyi, Spodoptera exigua larvae significantly induced catalytic activity of phospholipase A(2) (PLA(2)) in three immune-associated tissues, namely hemocytes, fat body, and hemolymph plasma. Furthermore, the infected S. exigua larvae induced transcription of several antimicrobial peptide (AMP) genes. Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata subsp. temperata (Ptt), possessed specific PLA(2)-inhibitory activities and their culture broths significantly inhibited the enzyme activities in hemocytes, fat body, and plasma of S. exigua. In addition, the bacterial metabolites inhibited transcription of AMP genes in S. exigua that would normally respond to the immune challenge by N. rileyi. The immunosuppressive effect of Xn or Ptt bacterial broth resulted in significant enhancement of the fungal pathogenicity against late instar larvae of S. exigua and Plutella xylostella. The effect of such a mixture was confirmed by field assay against two lepidopteran species. These results suggest that the bacterial and fungal mixture can be applied to develop a novel biopesticide to control lepidopteran species.

Modulation of proteasome machinery by natural and synthetic analogues of the marine bioactive compound petrosaspongiolide M.

Natural or synthetic? Several petrosaspongiolide M natural and synthetic analogues have been tested as proteasome inhibitors and apoptosis modulators. The natural petrosaspongiolide M congeners gave a consistent decrease in activity. Among the synthetic analogues, the introduction of the benzothiophene ring resulted in a bioequivalent alternative of the petrosaspongiolide M terpenoid system.

Morelloflavone, a biflavonoid inhibitor of migration-related kinases, ameliorates atherosclerosis in mice.

While macrophages take up modified LDL to form foam cells and multiply to develop fatty streaks, vascular smooth muscle cells (VSMC) migrate from the media to intima, secrete extracellular matrix, and increase the volume of atherosclerotic lesions. A medicinal plant Garcinia dulcis has been used in traditional Thai medicine for centuries to treat various chronic human diseases. Morelloflavone, a biflavonoid and an active ingredient of the plant, has been shown to inhibit VSMC migration through its inhibition of multiple migration-related kinases such as focal adhesion kinase, c-Src, ERK, and RhoA. However, the exact role of morelloflavone in atherosclerogenesis was unknown. We fed Ldlr(-/-)Apobec1(-/-) mice with either normal chow or chow containing 0.003% morelloflavone for 8 mo and assessed the extent of atherosclerosis by the en face and cross-sectional analyses. A cell composition analysis of atherosclerotic tissue was carried out using immunohistochemical staining. Oral morelloflavone therapy significantly reduced the atherosclerotic areas of the mouse aortas (a 26% reduction), without changing plasma lipid profiles or weights. Immunohistochemical analyses showed that morelloflavone reduced the number of VSMC in the atherosclerotic lesion while it did not change the density of macrophages in the lesion or the percentages of proliferating and apoptotic cells. Oral, low-dose, morelloflavone therapy retards atherosclerogenesis by limiting the migration of VSMC into the intima in the mouse model of human atherosclerosis. Upon further investigation, morelloflavone may be found to be a novel oral antiatherosclerotic agent and a viable addition to the conventional therapies such as statins in humans.

A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

Neutralization of haemorrhagic activity of viper venoms by 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-3-oxo-1,3-dihydroisobenzofuran-5-carbonitrile.

Viper envenomation undeniably induces brutal local manifestations such as haemorrhage, oedema and necrosis involving massive degradation of extracellular matrix at the bitten region and many a times results in dangerous systemic haemorrhage including pulmonary shock. Snake venom metalloproteases (SVMPs) are being considered to be the primary culprits for the venom-induced haemorrhage. As a consequence, the venom researchers and medical practitioners are in deliberate quest of SVMP inhibitors. In this study, we evaluated the inhibitory effect of 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-3-oxo-1,3-dihydroisobenzofuran-5-carbonitrile (DFD) on viper venom-induced haemorrhagic and PLA(2) activities. DFD effectively neutralized the haemorrhagic activity of the medically important viper venoms such as Echis carinatus, Echis ocelatus, Echis carinatus sochureki, Echis carinatus leakeyi and Crotalus atrox in a dose-dependent manner. The histological examinations revealed that the compound DFD effectively neutralizes the basement membrane degradation, and accumulation of inflammatory leucocytes at the site of Echis carinatus venom injection further confirms the inhibition of haemorrhagic activity. In addition, DFD dose dependently inhibited the PLA(2) activities of Crotalus atrox and E. c. leakeyi venoms. According to the docking studies, DFD binds to hydrophobic pocket of SVMP with the ki of 19.26 × 10(-9) (kcal/mol) without chelating Zn(2+) in the active site. It is concluded that the clinically approved inhibitors of haemorrhagins could be used as a potent first-aid agent in snakebite management. Furthermore, a high degree of structural and functional homology between SVMPs and their relatives, the MMPs, suggests that DFD analogues may find immense value in the regulation of multifactorial pathological conditions like inflammation, cancer and wound healing.

Identification of a novel and potent inhibitor of phospholipase A(2) in a medicinal plant: crystal structure at 1.93Å and Surface Plasmon Resonance analysis of phospholipase A(2) complexed with berberine.

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93Å and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).

Benzylideneacetone, an eicosanoid biosynthesis inhibitor enhances baculovirus pathogenicity in the diamondback moth, Plutella xylostella.

Benzylideneacetone (BZA) is a monoterpenoid compound produced by an entomopathogenic bacterium, Xenorhabdus nematophila. BZA inhibits phospholipase A(2) to suppress biosynthesis of eicosanoids that mediate immune responses in insects. In response to per os infection of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), the diamondback moth, Plutella xylostella, developed red spots on the midgut epithelium. The midgut exhibiting red spot formation suffered abnormal cell integrity, such as genomic DNA fragmentation and condensed spots in the nucleoplasm. The number of red spots increased with viral dose and incubation time after the viral treatment. BZA inhibited the formation of the midgut red spots in a dose-dependent manner. However, the inhibitory effect of BZA on the red spot formation was reversed by addition of arachidonic acid, suggesting that the red spot response may be mediated by eicosanoids. BZA treatment resulted in significant enhancement of AcMNPV occlusion body (OB) pathogenicity to P. xylostella.