Biomedical subjects

Russolina B Zingali

Publications and source records attributed to Russolina B Zingali.

16 recordsLinked to original sources

PubMed2006

Molecular diversity of disintegrin-like domains within metalloproteinase precursors of Bothrops jararaca.

Disintegrins are small peptides isolated from the venom of several snake families which act as integrin-antagonists or agonists, interacting with a variety of biological processes mediated by integrins. In this work we describe five new disintegrin-like domains within metalloproteinase precursor sequences, obtained from a Bothrops jararaca venom gland cDNA library. Among the new disintegrin-like domains, four were contained in PIII metalloproteinase precursors, with three of them presenting ECD-motifs and one presenting a new KCD-motif. Moreover, we found three disintegrin-like domains within PII metalloproteinase precursors. Two of them are similar to the already described disintegrins jarastatin and jararacin. The third molecule is unusual, presenting some typical PIII metalloproteinase characteristics but lacking the cysteine-rich domain being, thus, classified as a PII metalloproteinase. Only few reports presented molecules with these characteristics. Sequence analysis suggests that these molecules are intermediate steps between the more ancient PIII and the more recent PII metalloproteinases. We also investigated disintegrin N-terminus diversity in B. jararaca crude venom by purifying jarastatin and jararacin and analyzing them by mass spectrometry.

Amino Acid Sequence↗

PubMed2006

Bothrops jararaca venom gland transcriptome: analysis of the gene expression pattern.

Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization.

Animals↗

PubMed2006

Counteracting effect of glycyrrhizin on the hemostatic abnormalities induced by Bothrops jararaca snake venom.

1. Envenomation by the snake Bothrops jararaca is typically associated with hemostatic abnormalities including pro- and anticoagulant disturbances. Glycyrrhizin (GL) is a plant-derived thrombin inhibitor that also exhibits in vivo antithrombotic properties. Here, we evaluated the ability of GL to counteract the hemostatic abnormalities promoted by B. jararaca venom. 2. GL inhibited the human fibrinogen clotting (IC50 = approximately 1.0 mg ml(-1); 1.2 mM), H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride hydrolysis (IC50 = approximately 0.4 mg ml(-1); 0.47 mM) and platelet aggregation (IC50 = approximately 0.28 mg ml(-1); 0.33 mM) induced by B. jararaca venom, in vitro. 3. The in vivo effect of GL was tested in rats using a model of venous thrombosis in which intravenous (i.v.) administration of B. jararaca venom (100 microg kg(-1)) produced in all animals a thrombus with a mean weight of 10.6+/-1.7 mg. 4. Prior administration of GL (180 mg kg(-1)) or antibothropic serum (27 microl kg(-1)) inhibited thrombus formation by 86 and 67%, respectively. Remarkably, co-administration of ineffective doses of GL and antibothropic serum markedly decreased thrombus weight, suggesting a synergistic effect. 5. Co-administration of GL with antibothropic serum abolished venom-induced bleeding. Ex vivo clotting times showed that rat plasma was non-clotting after i.v. administration of B. jararaca venom. Treatment with GL, antibothropic serum or both before venom administration efficiently prevented this abnormality. 6. Altogether, we demonstrate here that GL prevents both in vitro and in vivo venom-induced changes in hemostasis, suggesting a potential antiophidic activity.

Animals↗

PubMed2006

Ecotin modulates thrombin activity through exosite-2 interactions.

Ecotin is a Escherichia coli-derived protein that has been characterized as a potent inhibitor of serine-proteases. This protein is highly effective against several mammalian enzymes, which includes pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. In this work we showed that ecotin binds to human alpha-thrombin via its secondary binding site, and modulates thrombin catalytic activity. Formation of wild type ecotin-alpha-thrombin complex was observed by native PAGE and remarkably, gel filtration chromatography showed an unusual 2:1 ecotin:enzyme stoichiometry. Analysis of the protease inhibitor effects on thrombin biological activities showed that (i) it decreases the inhibition of thrombin by heparin/antithrombin complex (IC50=3.2 microM); (ii) it produces a two-fold increase in the thrombin-induced fibrinogen clotting; and (iii) it inhibits thrombin-induced platelet aggregation (IC50=4.5 microM). Allosteric changes on thrombin structure were then evaluated. Complex formation with ecotin caused a three-fold increase in the rate of thrombin inhibition by BPTI, suggesting a displacement of the enzyme's 60-loop. In addition, ecotin modulated the enzyme's catalytic site, as demonstrated by changes in the fluorescence emission of fluorescein-FPRCK-alpha-thrombin (EC50=3.5 microM). Finally, solid phase competition assays demonstrated that heparin and prothrombin fragment 2 prevents thrombin interaction with ecotin. Altogether, these observations strongly support an ecotin interaction with thrombin anion-binding exosite-2, resulting in modulation of its biological activities. At this point, ecotin might be useful as a new tool for studying thrombin allosteric modulation.

Binding Sites↗

PubMed2005

Fast analysis of low molecular mass compounds present in snake venom: identification of ten new pyroglutamate-containing peptides.

Characterization of the peptide content in snake venoms can be an important tool for the investigation of new pharmacological lead compounds. For this purpose, single-step analysis of crude venoms has recently been demonstrated using mass spectrometry (MS) techniques. Reproducible profiles of ions in MS and MS/MS spectra may also be used to compare venoms from different species. In this work matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to obtain mass patterns of the major peptides (<8 kDa) found in pooled venoms from the genera Bothrops and Crotalus. Venoms from five different Bothrops species (B. jararaca, B. insularis, B. alternatus, B. jararacussu, and B. neuwiedi) and three Crotalus species (C. viridis, C. adamanteus and C. durissus terrificus) were analyzed. In agreement with other reports, venoms from Bothrops species contained a variety of peptides in the range m/z 1000-1500, and in some samples larger components (m/z 7000-8000) were detected. In the Crotalus species venoms were rich in peptides ranging from m/z 1000-1500 and 4000-5500. MS/MS experiments on the low molecular mass peptides (m/z 1000-1500) confirmed the presence of ten new bradykinin-potentiating peptides among venoms from genera Bothrops and Crotalus. In order to determine whether additional peptides could be identified after partial purification, B. jararaca venom was subjected to size-exclusion chromatography on Sephacryl S-200, and two distinct low molecular mass pools were analyzed further by MALDI-TOFMS. No additional peptides were detected from the pool with masses below 2000 Da but a substantial improvement with better resolution was observed for the pool with masses above 7000 Da, indicating that complex samples such as crude snake venoms can be analyzed for low molecular mass peptides using a single-step procedure.

Amino Acid Sequence↗

PubMed2005

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom.

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.

Amino Acid Sequence↗

PubMed2005

Bothrojaracin, a Bothrops jararaca snake venom-derived (pro)thrombin inhibitor, as an anti-thrombotic molecule.

Bothrojaracin (BJC) is a selective and potent thrombin inhibitor (KD = 0.6 nM) which also binds to prothrombin on proexosite I (KD = 175 nM). Incubation of BJC with human or rat plasma produced a band that co-migrates with purified prothrombin-BJC complex. We further analyzed the in vivo anti-thrombotic effect of BJC on a venous thrombosis model in rats that combines stasis and hypercoagulability. The administration of 1 mg/kg (i.v.) doses of BJC decreased thrombus weight by approximately 95%. Evaluation of the in vivo effect of BJC in mice using a pulmonary thromboembolism model induced by thrombin showed that BJC protects 100% of mice from death. Altogether, our data show that BJC is a potent anti-thrombotic agent that could further help the development of new prothrombin-directed drugs.

Animals↗

PubMed2004

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom.

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.

Amino Acid Sequence↗

PubMed2004

RGD- and MLD-disintegrins, jarastatin and EC3, activate integrin-mediated signaling modulating the human neutrophils chemotaxis, apoptosis and IL-8 gene expression.

The effects of jarastatin (JT), a monomeric RGD-disintegrin, were compared with those of the heterodimeric MLD-disintegrin, EC3, on human neutrophil activation and functions. Both disintegrins inhibited neutrophil chemotaxis induced by fMet-Leu-Phe and were also potent chemotactic agents. These effects were accompanied by an increase in actin polymerization, and both were inhibited by genistein, a tyrosine kinase inhibitor. While JT, but not other RGD-disintegrins, inhibited EC3-induced chemotaxis, EC3 was not able to inhibit JT effect. The chemotactic effect of JT was blocked by anti-alpha(M) antibody whereas anti-alpha(9)beta(1) inhibited EC3 effect. Both JT and EC3 induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Accordingly, LY294002, a PI3K inhibitor, impaired their chemotactic effect on neutrophils. JT induced Erk-2 translocation to nucleus and a delay of the spontaneous apoptosis of neutrophils in vitro. In contrast, EC3 inhibited Erk-2 activation and had a proapoptotic effect. These effects were reverted by PD98059, an MEK 1/2 inhibitor and blocked by z-VAD-FMK, a caspase inhibitor. In addition, JT, but not EC3, increased the IL-8 mRNA levels in neutrophils. The data indicate that JT and EC3 directly activate an integrin-coupled signaling and modulate the MAPK pathway in different ways, leading the neutrophils to express different functional response.

Active Transport, Cell Nucleus↗

PubMed2004

New insights into conformational and functional stability of human alpha-thrombin probed by high hydrostatic pressure.

The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of human alpha-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm(-1). HHP (270 MPa) shifted the centre of mass by only 370 cm(-1). HHP combined with a subdenaturing urea concentration (1.5 m) displaced the centre of mass by approximately 750 cm(-1). The binding of the fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to thrombin was increased by 1.8-, 4.0-, and 2.7-fold after treatment with high urea concentration, HHP or HHP combined with urea, respectively, thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand, treatment of thrombin with urea (but not HHP) combined with dithiothreitol progressively displaced the fluorescent probe, thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein-d-Phe-Pro-Arg-cloromethylketone-alpha-thrombin: addition of urea up to 2 m gradually decreased the fluorescence emission of the probe to 65% of the initial intensity, whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was enhanced (35%) in 2 m urea and gradually abolished at higher concentrations, while HHP (270 MPa) inhibited the enzyme's catalytic activity by 45% and abolished it when 1.5 m urea was also present. Altogether, analysis of urea and HHP effects on thrombin structure and activity indicates the formation of dissimilar intermediate states during denaturation by these agents.

Anilino Naphthalenesulfonates↗

PubMed2003

Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives.

This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC(50)=24+/-0.5 micro M), from which emerge this new series 2. These new compounds 2a-p were readily synthesized, characterized and tested on platelet aggregation assays induced by collagen (5 micro g/mL), ADP (5 micro M) and arachidonic acid (100 micro M) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and 2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect on collagen (IC(50)=21.6+/-0.4 micro M) and arachidonic acid-induced platelet aggregation (IC(50)=2.2+/-0.06 micro M), suggesting that the nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.

Animals↗

PubMed2003

Subunit dissociation, unfolding, and inactivation of bothrojaracin, a C-type lectin-like protein from snake venom.

Snake venoms contain a large number of hemostatically active proteins that are structurally related to Ca(2+)-dependent animal lectins. These proteins, called C-type lectin-like proteins (CLPs), are generally found as heterodimers composed of two homologous subunits linked by a disulfide bond. Here, bothrojaracin (BJC), a CLP from Bothrops jararaca venom that is also a thrombin inhibitor, has been used as a model to study the subunit dissociation and unfolding of CLPs from snake venom. Dithiothreitol (DTT) up to 10 mM produces minor effects on the tertiary structure and activity of BJC. On the other hand, chromatographic studies and fluorescence polarization measurements indicate that the interchain disulfide bond is disrupted by DTT, although the dimeric association is maintained. Treatment of BJC with urea produces a progressive red shift in the emission spectra of the tryptophan residues, and circular dichroism measurements show that BJC retains significant secondary structure in the presence of 8 M urea, suggesting only partial unfolding. The effects of urea are fully reversible, as there is complete recovery of BJC activity after removal of the denaturing agent. Addition of DTT to a protein sample previously treated with 8 M urea produces a slightly larger spectral shift than that observed with urea alone. Furthermore, in this condition BJC loses its secondary structure, and its subunits are dissociated. After removal of urea and DTT, BJC is inactive toward thrombin, suggesting the irreversibility of their combined action. Altogether, our data show that (i) BJC is highly resistant to urea or DTT effects, requiring the simultaneous action of both agents to fully denature the protein, and (ii) BJC monomers are tightly associated, and the presence of DTT combined with high urea concentrations is necessary to disrupt them. On the basis of these results we propose the first denaturation model for a CLP from snake venom.

Animals↗

PubMed2003

Antithrombotic effect of Glycyrrhizin, a plant-derived thrombin inhibitor.

Glycyrrhizin (GL), an anti-inflammatory compound isolated from licorice (Glycyrrhiza glabra), has been previously identified as a thrombin inhibitor (Francischetti et al., Biochem Biophys Res Commun 1997;235:259-63). Here we report the in vivo effects of GL upon two experimental models of induced thrombosis in rats. Intravenous administration of GL caused a dose-dependent reduction in thrombus size on a venous thrombosis model that combines stasis and hypercoagulability. It was observed that GL doses of 180 mg/kg body weight produced 93% decrease on thrombus weight. This effect showed a time-dependent pattern being significantly reduced when the thrombogenic stimulus was applied 60 min after drug administration. GL was also able to prevent thrombosis using an arteriovenous shunt model. GL doses of 180 and 360 mg/kg decreased the thrombus weight by 35 and 90%, respectively. Accordingly, the APTT ex vivo was enhanced by 1.5- and 4.3-fold at GL doses of 180 and 360 mg/kg, respectively. In addition, GL doses above 90 mg/kg caused significant hemorrhagic effect. In contrast with heparin, GL did not potentiate the inhibitory activity of antithrombin III or heparin cofactor II towards thrombin. Altogether, data indicate that GL is an effective thrombin inhibitor in vivo, which may account for its other known pharmacological properties.

Animals↗

PubMed2003

Changes in salivary nitrophorin profile during the life cycle of the blood-sucking bug Rhodnius prolixus.

The insect Rhodnius prolixus is a hematophagous hemipteran that has five nymphal instars. Fifth instar nymphs contain, in their salivary glands, four nitrophorins which have already been described in the literature (NP1, NP2, NP3 and NP4). Two new hemeproteins were isolated and partially characterized from first instar nymphs. NP2, that shows an anticoagulant activity, was also identified, but NP1, NP3 and NP4 were not found. As these new hemeproteins have amino-terminal sequences clearly homologous to already described nitrophorins and were capable of binding nitric oxide, they were named nitrophorins 5 and 6, although they showed an unusual Soret band at 412 nm. In each subsequent nymphal stage, a new nitrophorin emerges. In the second instar, NP4 comes into view, in the third instar NP1 appears, and NP3 is only found in fifth instar nymphs and adults, showing that the nitrophorin profile of R. prolixus saliva is stage-specific.

Amino Acid Sequence↗

PubMed2002

Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom.

A new phospholipase A2 (PLA2) isoenzyme was isolated from Lachesis muta crude venom, and was named LM-PLA2-II. This enzyme was purified by gel filtration on a Sephacryl S-200 HR column followed by reverse-phase chromatography on a C2/C18 column. LM-PLA2-II consists of a single polypeptide chain with an apparent molecular mass of 18 kDa and an isoelectric point at pH 5.4. The amino terminal sequence of the enzyme revealed a high degree of homology with other PLA2s from several sources. LM-PLA2-II has a high indirect hemolytic activity and a potent inhibitory effect on platelet aggregation induced by ADP and collagen. It also produces a significant paw edema reaction in rats. The edematous response in rats was abolished by pretreatment with either indomethacin or dexamethasone, suggesting the involvement of cyclo-oxygenase. Pretreatment of LM-PLA2-II with p-bromophenacyl bromide abolished all of these actions, clearly indicating that the biological activities, including the edematogenic effect, are dependent entirely on its enzymatic activity.

Adenosine Diphosphate↗

PubMed2002

Bothrojaracin, a proexosite I ligand, inhibits factor Va-accelerated prothrombin activation.

Bothrojaracin (BJC) is a 27 kDa snake venom protein from Bothrops jararaca that has been characterized as a potent ligand (KD = 75 nM) of human prothrombin (Monteiro RQ, Bock PE, Bianconi ML, Zingali RB, Protein Sci 2001; 10: 1897-904). BJC binds to the partially exposed anion-binding exosite I (proexosite I) forming a stable 1:1, non-covalent complex with the zymogen whereas no interaction with fragment 1 or 2 domains is observed. In addition, BJC interacts with thrombin through exosites I and II (KD = 0.7 nM), and influences but does not block the proteinase catalytic site. In the present work we studied the effect of BJC on human prothrombin activation by factor Xa in the absence or in the presence of its cofactors, factor Va and phospholipids. In the absence of phospholipids, BJC strongly inhibited (80%) the zymogen activation by factor Xa in the presence but not in the absence of factor Va, suggesting a specific interference in the cofactor activity. In the presence of phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine), BJC also inhibited (35%) prothrombin activation by factor Xa in the presence but not in the absence of factor Va. BJC showed a higher inhibitory effect (70%) towards thrombin formation by prothrombinase complex assembled on phospholipid vesicles composed by 95% phosphatidylcholine, 5% phosphatidylserine. Activation of prothrombin by platelet-assembled prothrombinase complex (factor Xa, factor Va and thrombin-activated platelets) showed that hirudin (SO3-) and BJC efficiently inhibit the thrombin formation (43% and 84%, respectively). Taken together, our results suggest that proexosite I blockage decreases the productive recognition of prothrombin as substrate by factor Xa-factor Va complex and prothrombinase complex. Furthermore, data obtained with human platelets suggest that proexosite I may play an important role in the physiological activation of prothrombin.

Binding Sites↗