Pseudechis australis Venomics: Adaptation for a Defense against Microbial Pathogens and Recruitment of Body Transferrin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A(2) from Bothrops pirajai snake venom

Piratoxins (PrTX) I and III are phospholipases A(2) (PLA(2)s) or PLA(2) homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA(2), while PrTX-I is a Lys49 PLA, homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities. (C) 2001 Academic Press.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Calcium-Independent Membrane Damage by Venom Phospholipases A(2)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

COX-2 inhibition decreases VEGF expression and alveolar bone loss during the progression of experimental periodontitis in rats

Background: Vascular endothelial growth factor (VEGF) is a macromolecule of importance in inflammation that has been implicated in periodontitis. The aims of this study were to investigate VEGF expression during the progression of periodontal disease and to evaluate the effect of a preferential cyclooxygenase (COX)-2 inhibitor meloxicam on VEGF expression and alveolar bone loss in experimentally induced periodontitis.Methods: A total of 120 Wistar rats were randomly separated into groups 1 (control) and 2 (meloxicam, 3 mg/kg/day, intraperitoneally, for 3, 7, 14, or 30 days). Silk ligatures were placed at the gingival margin level of the lower right first molar of all rats. VEGF expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemical (IHC) analyses. The hemiarcades were processed for histopathologic analysis. RT-PCR and WB results were submitted to analysis of variance, the Tukey test, and Pearson correlation analysis (P<0.05).Results: A reduction in alveolar bone resorption was observed in the meloxicam-treated group compared to the control group at all periods studied. There was a positive correlation between COX-2 mRNA and VEGF mRNA in the gingival tissues and periodontal disease (R = 0.80; P = 0.026). Meloxicam significantly reduced the increased mRNA VEGF expression in diseased tissues after 14 days of treatment (P = 0.023). Some alterations in VEGF receptor I mRNA expression were observed, but these were not statistically significant. VEGF protein expression in WB experiments was significantly higher in diseased sites compared to healthy sites (P<0.05). After 14 days of treatment with meloxicam, an important decrease in VEGF protein expression was detected in diseased tissues (P = 0.08). Qualitative IHC analysis revealed that VEGF protein expression was higher in diseased tissues and decreased in tissues from rats treated with meloxicam.Conclusions: The present data suggest an important role for VEGF in the progression of periodontal disease. Systemic therapy with meloxicam can modify the progression of experimentally induced periodontitis in rats by reducing VEGF expression and alveolar bone loss.

Síntese e modelagem molecular do novo derivado indolinônico como candidato a antiinflamatório COX-2 seletivo

Anti-inflammatory drugs are known to be the most widely-marketed drugs in the world, despite their serious side effects, mainly on the gastrointestinal tract. Thus, there are constant efforts to discover new prototypes with improved therapeutic activity and safety for the patient. Since the advent of the computational chemistry, the theoretical study of the physiological behavior of a new compound and hence an understanding of its supposed mechanism of action have been made a lot more accessible. Thus, molecule-receptor mathematical modeling was applied to compound I (1-(2,6-dichlorophenyl)indolin-2-one), to predict theoretically its ability to inhibit, selectively, the COX-2 isoform of prostaglandin endoperoxide synthase (PGHS-2), and the best positions to introduce chemical groups and to make molecular modifications.

Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A(2)-like from Bothrops brazili venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L.) venom on rat aorta

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

An Evaluation of 3-Rhamnosylquercetin, a Glycosylated Form of Quercetin, against the Myotoxic and Edematogenic Effects of sPLA(2) from Crotalus durissus terrificus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biological characterization of the Amazon coral Micrurus spixii snake venom: isolation of a new neurotoxic phospholipase A2

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The adipokine chemerin augments vascular reactivity to contractile stimuli via activation of the MEK-ERK1/2 pathway

Aims: Cytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. Main methods: Male, 10-12 week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5 ng/mL or 5 ng/mL, for 1 or 24 h), and isometric contraction was recorded. Protein expression was determined by Western blotting. Key findings: Constrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1 h with chemerin. Chemerin incubation for 24 h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24 h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. Significance: Chemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases. (c) 2012 Elsevier Inc. All rights reserved.

Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats

Martins JM, Longhi-Balbinot DT, Soares DM, Figueiredo MJ, Malvar D do C, de Melo MC, Rae GA, Souza GE. Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats. J Appl Physiol 113: 1456-1465, 2012. First published August 30, 2012; doi:10.1152/japplphysiol.00936.2011.-This study investigated the involvement of prostaglandins and regulated on activation, normal T cell expressed and secreted (RANTES), in fever induced by live Staphylococcus aureus (no. 25923, American Type Culture Collection) injection in rats. S. aureus was injected intraperitoneally at 10(9), 10(10), and 2 x 10(10) colony-forming units (CFU)/cavity, and body temperature (T-b) was measured by radiotelemetry. The lowest dose of S. aureus induced a modest transient increase in T-b, whereas the two higher doses promoted similar long-lasting and sustained T-b increases. Thus, the 10(10) CFU/cavity dose was chosen for the remaining experiments. The T-b increase induced by S. aureus was accompanied by significant decreases in tail skin temperature and increases in PGE(2) levels in the cerebrospinal fluid (CSF) and hypothalamus but not in the venous plasma. Celecoxib (selective cyclooxygenase-2 inhibitor, 2.5 mg/kg po) inhibited the fever and the increases in PGE(2) concentration in the CSF and hypothalamus induced by S. aureus. Dipyrone (120 mg/kg ip) reduced the fever from 2.5 to 4 h and the PGE(2) increase in the CSF but not in the hypothalamus. S. aureus increased RANTES in the peritoneal exudate but not in the CSF or hypothalamus. Met-RANTES (100 mu g/kg iv), a chemokine (C-C motif) receptor (CCR)1/CCR5 antagonist, reduced the first 6 h of fever induced by S. aureus. This study suggests that peripheral (local) RANTES and central PGE(2) production are key events in the febrile response to live S. aureus injection. As dipyrone does not reduce PGE(2) synthesis in the hypothalamus, it is plausible that S. aureus induces fever, in part, via a dipyrone-sensitive PGE(2)-independent pathway.

Inhibition of cPLA(2) and sPLA(2) Activities in Primary Cultures of Rat Cortical Neurons by Centella asiatica Water Extract

Leaf extract of Centella asiatica has been used as an alternative medicine for memory improvement in the Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of Alzheimer's disease, the molecular mechanism of C. asiatica on neuroprotection still remains unexplained. In this study, we investigated the effects of C. asiatica water extract on activity of subtypes of phospholipase A(2) (PLA(2)) in primary cultures of rat cortical neurons and quantified by HPLC a possible molecule responsible for the activity. The cPLA(2) and sPLA(2) activities were inhibited in vitro by asiaticoside present in the water extract of C. asiatica. This extract may be a candidate for the treatment of neurodegenerative processes because of its pharmacological activity in the brain and its low toxicity, as attested by its long popular use as a natural product.

Ethanol induces vascular relaxation via redox-sensitive and nitric oxide-dependent pathways

We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with L-NAME (non selective NOS inhibitor, 100 mu mol/L), 7-nitroindazole (selective nNOS inhibitor, 100 mu mol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, I mu mol/L), glibenclamide (selective blocker of ATP-sensitive K+ channels, 3 mu mol/L) and 4-aminopyridine (selective blocker of voltage-dependent K+ channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O-2(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H2O2) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 mu mol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 mu mol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by L-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca2+ concentration ([Ca2+]c), O-2(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca-2]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity. (C) 2011 Elsevier Inc. All rights reserved.

Ginger phenylpropanoids inhibit IL-1beta and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases A2

The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A(2) (iPLA(2))-triggered maturation and the cytosolic phospholipase A(2) (cPLA(2))-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA(2) assay, most major ginger phenylpropanoids directly inhibited i/cPLA(2) from U937 macrophages, but not hog pancreas secretory phospholipase A(2). The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA(2) and methyl arachidonyl fluorophosphonate for cPLA(2), with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE(2) and thromboxane B2 (>50%) and partially also leukotriene B(4) in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA(2) and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA(2), the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.