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Graduated in Biology (1992), Master (1995) and Ph.D. (2000) in biological sciences by the State University of Campina... moreGraduated in Biology (1992), Master (1995) and Ph.D. (2000) in biological sciences by the State University of Campinas. Pos Ph.D. in endocrine physiology with a grant from FAPESP 2000-2003. In 2010 obtained the title of Professor of Biochemistry at UNESP, Campus Experimental do Litoral Paulista. He is currently a professor of Biochemistry of Universidade Estadul Paulista (UNESP) -Campus Experimental do Litoral Paulista at Sao Vicente, Sao Paulo. At the present time is to develop activities of study and research in secretory Phospholipase A2 (sPLA2), mainly from the venoms and interaction of these protein with several polyphenolic compound and other natural compounds. This new line of research aims to evaluate the potential anti PLA2, anti venom and anti inflammatory. edit
[No abstract available]5806/07/1561
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Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of... more
Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BcPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BcPLA(2)1 (20 microg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.
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Biology, Medicine, Apis mellifera, Glucose, Kidney, and 15 moreInsulin, Insulin Secretion, Animals, Male, Honey bee, Coastal waters, Biological activity, Amino Acid Sequence, Enzymatic Activity, Mesenteric Arteries, Chemical Treatment, Glomerular Filtration Rate, Molecular Sequence Data, In Vitro Techniques, and biological effect
Scorpion neurotoxin Ts-gamma was isolated from Tityus serrulatus venom and purified to apparent homogeneity by ion-exchange HPLC. Crystals of the toxin were grown using polyethylene glycol 6000 as precipitant and were found to belong to... more
Scorpion neurotoxin Ts-gamma was isolated from Tityus serrulatus venom and purified to apparent homogeneity by ion-exchange HPLC. Crystals of the toxin were grown using polyethylene glycol 6000 as precipitant and were found to belong to the monoclinic space group P21 with cell parameters a = 22.20, b = 36.90, c = 31.57 A, beta = 100.85 degrees. The crystals diffract beyond 1.73 A resolution at a synchrotron beamline, being notably stable during X-ray exposure. The structure has been solved by molecular replacement using the very high resolution structure of Sahara scorpion Androctonus australis Hector (PDB code 1AHO) as a search model.
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This study describes the purification of an N-acetylglucosamine-binding lectin from Koelreuteria paniculata seeds and its effects on the larval development of Callobruchus maculatus and Anagasta kuehniella. The lectin (KpLec) was... more
This study describes the purification of an N-acetylglucosamine-binding lectin from Koelreuteria paniculata seeds and its effects on the larval development of Callobruchus maculatus and Anagasta kuehniella. The lectin (KpLec) was characterized and isolated by gel filtration, affinity column, and reverse phase chromatography. SDS-PAGE indicated that this lectin is a dimer composed of subunits of 22 and 44 kDa. The N terminus exhibited 40% similarity with Urtiga dioica agglutinin. KpLec was tested for anti-insect activity against C. maculatus and A. kuehniella. With regard to C. maculatus, an artificial diet containing 0.7 and 1% KpLec produced LD(50) and ED(50) value, respectively. However, for A. kuenhiella, an artificial diet containing 0.65% KpLec produced an LD(50), whereas 0.2% KpLec produced an ED(50). The transformation of genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.
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Biochemistry, Organic Chemistry, Plant Biology, Biology, Macrophages, and 15 moreApoptosis, Medicine, Platelet aggregation, Animals, Candida albicans, Proteins, High Pressure Liquid Chromatography, Bothrops, Microbial Sensitivity Tests, Amino Acid Profile, Amino Acid Sequence, Enzymatic Activity, Molecular Sequence Data, Pharmacology and pharmaceutical sciences, and Functional Characterization
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Microbiology, Zoology, Biology, Leishmania, Medicine, and 4 moreVirulence, In Vitro, In Vivo, and Immune system
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Envenoming by Crotalus durissus subspecies leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure. The most serious systemic alteration and primary cause of death after snakebite is acute renal failure. In this... more
Envenoming by Crotalus durissus subspecies leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure. The most serious systemic alteration and primary cause of death after snakebite is acute renal failure. In this work, we isolated crotapotin, an acid component (Crtp) of crotoxin from Crotalus durissus cascavella venom and we investigated its bactericidal and pro-inflammatory activities as well as its
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ABSTRACT Acacia farnesiana lectin-like protein (AFAL) showed bacterioestatic effects against Xanthomonas axonopodis pv. passiflorae (Gram-negative) and Clavibacter michiganensis michiganensis (Gram-positive), with the latter being more... more
ABSTRACT Acacia farnesiana lectin-like protein (AFAL) showed bacterioestatic effects against Xanthomonas axonopodis pv. passiflorae (Gram-negative) and Clavibacter michiganensis michiganensis (Gram-positive), with the latter being more sensitive. This effect is probably due to the ability of AFAL to interact with the bacterial cell wall where we observed that AFAL induced macroscopic change. The maximum bacterial growth inhibition was approximately 78% when incubated with Gram-negative strains, and as high as 92% percent for the Gram-positive one. The antibacterial effect of flavonoids (rutin, quercetin and morin) was also observed using low concentrations against both bacterial strains. Prior incubation of both with AFAL at high concentrations increases the inhibitory effect of flavonoids on bacterial growth. The potential use of AFAL as a control agent against the root-knot nematode Meloidogyne incognita was investigated as well, showing anti-nematode properties involving both egg hatching and motility. In the juvenile second-stage, AFAL showed reduction in larval mobility when measured against a control group. The results suggest that AFAL is effective against M. incognita and could be used as a component of integrated pest management programs. These data also suggest that lectins probably play a role in plant defense not only against invertebrate phytopathogens, herbivores and fungi but also against bacteria.
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In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A2 (PLA2) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122... more
In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A2 (PLA2) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80 with crotalic
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The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2s) and crotapotins showed antimicrobial activity... more
The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2s) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA2 also revealed significant anticoagulant activity, althrough for this to
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A lectin was purified from Crotalaria paulina seeds by ion-exchange and FPLC molecular exclusion chromatography. CrpL had an apparent molecular mass of 30 kDa, as determined by SDS-PAGE under non-reducing and reducing conditions. CrpL... more
A lectin was purified from Crotalaria paulina seeds by ion-exchange and FPLC molecular exclusion chromatography. CrpL had an apparent molecular mass of 30 kDa, as determined by SDS-PAGE under non-reducing and reducing conditions. CrpL effectively agglutinated human and cow erythrocytes, and this activity was not affected by 20 mM EDTA, showing no dependence of divalent cations. Hemagglutination was inhibited by N-acetyl- D-galactosamine,
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Biochemistry, Biology, Protein, Medicine, In Vitro, and 15 moreProteins, Plant Lectins, Sequence, In Vivo, Phaseolus vulgaris, Molecular Mass, Colocasia esculenta, Lectin, Ion Exchange, Amino Acid Sequence, Bacterial Infection, Biochemistry and cell biology, Crotalaria, Amino acid composition, and Medical biochemistry and metabolomics
: Molecular exclusion HPLC chromatography on a Protein Pak SW 300 column and reversed phase HPLC on a µ-Bondapack Cl8 column were used to purify four novel neurotoxins (rom the crotoxin complex, and five crotoxin subunits (PLA2 and... more
: Molecular exclusion HPLC chromatography on a Protein Pak SW 300 column and reversed phase HPLC on a µ-Bondapack Cl8 column were used to purify four novel neurotoxins (rom the crotoxin complex, and five crotoxin subunits (PLA2 and crotapotin) as well as two crotamine isoforms. A single preparative reversed phase HPLC steps yielded a major basic myotoxin followed by the subunits of crotoxin (three PLA2 isoforms and two crotapotin isoforms). This modified procedure may be useful for the rapid, large-scale purification of toxins from Crotalid venoms.
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A disciplina de bioquímica no geral é considerada de alto grau de dificuldade. Contudo, a aplicação de jogos lúdicos como metodologia de ensino vem se disseminando em várias disciplinas. “METABOLIC RIDE” é uma ferramenta de avaliação... more
A disciplina de bioquímica no geral é considerada de alto grau de dificuldade. Contudo, a aplicação de jogos lúdicos como metodologia de ensino vem se disseminando em várias disciplinas. “METABOLIC RIDE” é uma ferramenta de avaliação conceitual e de percepção para o ensino de bioquímica, visando rever conceitos difundidos em sala de aula, promovendo um desafio competitivo aos estudantes sem negar as ferramentas que estão a sua disposição, estimulando diversas aptidões dos mesmos, como a sua criatividade. Ainda, possibilita correlacionar a importância das rotas metabólicas e suas interligações a fim de sedimentar que os caminhos metabólicos não estão separados, como um mapa ferroviário. Ademais, este jogo se mostrou uma excelente ferramenta de avaliação complementar, mostrando que quando devidamente estimulados alguns grupos foram capazes de mostrar uma capacidade produtiva e criativa. Além disso, mostrou-se novas formas de abordar temas complexos em bioquímica com práticas lúdicas.
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Two myotoxins, MP-I and MP-II, from Bothrops pirajai snake venom, have been purified by a quick high performance liquid chromatography (HPLC) procedure. Based on the HPLC coelution profile, amino acid composition, N-terminal sequence,... more
Two myotoxins, MP-I and MP-II, from Bothrops pirajai snake venom, have been purified by a quick high performance liquid chromatography (HPLC) procedure. Based on the HPLC coelution profile, amino acid composition, N-terminal sequence, polyacrylamide gel electrophoresis (PAGE) migration, as well as lack of phospholipase-A2 (PLA2) and proteolytic activities, MP-I and MP-II were identified as piratoxin-I (PrTX-I) and II (PrTX-II), respectively. This procedure affords, aside the reduced operation time, a high yield (35% of the applied sample in terms of A280nm) of MP-I, which is the major myotoxin of the venom. The N-terminal sequences of MP-I, MP-II, PrTX-I and PrTX-II, up to the 51st, 41st, 46th and 39th residues, respectively, have been determined, revealing MP-I (and hence PrTX-I) as a Lys-49 PLA2-like myotoxin. Both MP-I and MP-II have been shown, by SDS-PAGE, to occur in dimeric isoforms.
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Biochemistry, Chemistry, Chromatography, Medicine, Pubmed, and 14 moreVenom, Gel Permeation Chromatography, Neurotoxins, High Pressure Liquid Chromatography, Amino Acids, Snake Venom, Time Factors, Fractionation, Biochemistry and molecular biology, Biological activity, Amino Acid Sequence, Biochemistry and cell biology, Myotoxin A, and Molecular Sequence Data
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Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite... more
Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa(+)) at 120 min and of chloride (%TCl(-)) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC(50) of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca(2+) in a concentration dependent manner, indicating that Ca(2+) may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism.
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ABSTRACT Glycation, a chemical modification of proteins with reducing sugars, indicating a possible explanation for the association between hyperglycemia and the wide variety of tissue pathologies. A good example is glycosiled hemoglobin... more
ABSTRACT Glycation, a chemical modification of proteins with reducing sugars, indicating a possible explanation for the association between hyperglycemia and the wide variety of tissue pathologies. A good example is glycosiled hemoglobin that can be used as a molecular tool to characterize the severity of diabetes. In this in vitro study, we investigated the effect of glucose, galactose and lactose on the structure and function of BthTX-I, which was chosen as the structural model of sPLA2. Glucose is known and well characterized as one of the most important glycacanting agent of protein. Galactose is another monosaccharide with the same functional groups found on glucose and lactose which is the dimer of galactose and glucose were also evaluated in this study.
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sPLA2 from Crotalus durissus terrificus venom, free of crotapotin (Cdt sPLA2), purified and isolated sPLA2, was able to significantly increase lipid peroxidation, which occurred simultaneously with increased arachidonic acid (AA)... more
sPLA2 from Crotalus durissus terrificus venom, free of crotapotin (Cdt sPLA2), purified and isolated sPLA2, was able to significantly increase lipid peroxidation, which occurred simultaneously with increased arachidonic acid (AA) metabolism. In addition, MDA and AA levels were elevated at 15 min after Cdt sPLA2 injection and after peak edema (negative control). Thus, oxidative stress and ROS play important roles in the inflammation induced by Cdt sPLA2. On the other hand, edema induced by sPLA2 involves the direct and indirect mobilization of arachidonic acid by the involvement of phosphokinase C (PKC) and phospholipase C (PLC), which indirectly stimulates cytosolic PLA2 (cPLA2). We also observed that the specific antivenin against Cdt venom had no significant effect on the neutralization of induced edema compared to the natural products 5-caffeine-linoleic acid (5CQA) and dexamethasone (AACOCF3). Our results also indicate that there was improvement in the inhibition of edema of nat...
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Biochemistry, Chemistry, Biology, Medicine, High Frequency, and 15 moreMice, Animals, Anticoagulants, Ion Exchange Chromatography, High Pressure Liquid Chromatography, Bothrops, Amino Acid Profile, Asper, Ion Exchange, Biological activity, Amino Acid Sequence, Clinical Signs, Isozymes, Biochemistry and cell biology, and Molecular Sequence Data
A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column.... more
A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60 degrees C. The lectin required Mn2+ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.
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Biochemistry, Chemistry, Chromatography, Humans, Animals, and 15 moreBlood groups, Lectins, Ion Exchange Chromatography, High Performance Liquid Chromatography, Amino Acids, Fabaceae, Lectin, Gel electrophoresis, Amino Acid Sequence, Complex, Hemagglutination, Biochemistry and cell biology, Delonix regia, Edman degradation, and Amino acid composition
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Sea anemones contain a variety of interesting biologically active compounds, including some potent toxins. PLA2 from Bunodosoma caissarum, a sea anemone endemic in the Brazilian southern coast, has shown renal alterations on isolated... more
Sea anemones contain a variety of interesting biologically active compounds, including some potent toxins. PLA2 from Bunodosoma caissarum, a sea anemone endemic in the Brazilian southern coast, has shown renal alterations on isolated kidney. The aim of this study was to evaluate the renal and vascular effects of B. caissarum crude extract (BcE) on isolated perfused kidney and arteriolar mesenteric bed, as well the involvement of prostaglandins and endothelin. BcE did not show any effect on arteriolar mesenteric bed, but increased perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and decreased the percentage of sodium tubular transport on isolated perfused kidney. Indomethacin blocked the renal effects induced by BcE and tezosentan only partially blocked these effects. These results demonstrate the effects of BcE on kidney in situ, suggesting the involvement of prostaglandins and endothelin.
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Snake envenomation (Bothrops genus) is common in tropical countries and acute kidney injury is one of the complications observed in Bothrops snakebite with relevant morbidity and mortality. Here, we showed that Bothropoides pauloensis... more
Snake envenomation (Bothrops genus) is common in tropical countries and acute kidney injury is one of the complications observed in Bothrops snakebite with relevant morbidity and mortality. Here, we showed that Bothropoides pauloensis venom (BpV) decreased cell viability (IC50 of 7.5 μg/mL). Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by apoptosis and late apoptosis, through caspases 3 and 7 activation, mitochondrial membrane potential collapse and ROS overproduction. BpV reduced perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, percentage of sodium, chloride or potassium tubular transportation. These findings demonstrated that BpV cytotoxicity on renal epithelial cells might be responsible for the nephrotoxicity observed in isolated kidney.
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Flow Cytometry, Biology, Apoptosis, Medicine, Dogs, and 15 moreKidney, Reactive Oxygen Species, Animals, Male, Caspase, Epithelial cells, Bothrops, Toxicity Tests, Cell Survival, Toxicon, Propidium Iodide, Glomerular Filtration Rate, Annexin, Pharmacology and pharmaceutical sciences, and In Vitro Techniques
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Biochemistry, Chemistry, Electron Microscopy, Biology, Medicine, and 15 moreLeishmaniasis, Gram Positive, Animals, Bacteria, Catalase, Ion Exchange Chromatography, Antimicrobial, Enzyme, Hydrogen Peroxide, Crotalus, Bacterial growth, Amino Acid Profile, Molecular Mass, Amino Acid Sequence, and Enzymatic Activity
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Mass Spectrometry, Biology, Calcium, Medicine, In Vitro, and 15 moreMuscle, HPLC, Mice, Animals, High Pressure Liquid Chromatography, Muscle contraction, Molecular Mass, Protein isoforms, Amino Acid Sequence, Phrenic Nerve, Crotamine, Myotoxin A, Molecular Sequence Data, Pharmacology and pharmaceutical sciences, and diaphragm
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Biology, Inflammation, Lung Inflammation, Medicine, Elasticity, and 15 moreMice, Animals, Pneumonia, Male, Acute Lung Injury, Blood Coagulation, Lung, Rats, Lethal Dose, Toxicon, Respiratory Mechanics, Control Group, Biomechanical Phenomena, Pharmacology and pharmaceutical sciences, and mononuclear cell
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Biology, Animals, Brush Border, Enzyme, Clearance, and 15 moreHigh Pressure Liquid Chromatography, Hydrogen Peroxide, Activation, Isolation, Crotalus, Cells, Blood Flow, Bothrops, Amino Acid Profile, Biological activity, Amino Acid Sequence, Enzymatic Activity, L, Glomerular Filtration Rate, and biological effect
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Biology, Medicine, Animals, Viperidae, P-glycoprotein, and 14 moreVenom, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Ion Exchange Chromatography, Bothrops, Molecular weight, Amino Acid Sequence, Opossum, Opossums, Toxicon, Blood Proteins, Didelphis, Molecular Sequence Data, Antivenins, and Pharmacology and pharmaceutical sciences
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Immunology, Medical Microbiology, Biology, Leishmania, Medicine, and 15 moreLeishmaniasis, Mice, Animals, Parasites, Male, CD, High Pressure Liquid Chromatography, Immune system, Public health systems and services research, Protozoan Proteins, Cell Proliferation, Antigen, immunoglobulin G, Interleukin, and Interferon gamma
Secretory phospholipases A2(sPLA2) exert proinflammatory actions through lipid mediators. These enzymes have been found to be elevated in many inflammatory disorders such as rheumatoid arthritis, sepsis, and atherosclerosis. The aim of... more
Secretory phospholipases A2(sPLA2) exert proinflammatory actions through lipid mediators. These enzymes have been found to be elevated in many inflammatory disorders such as rheumatoid arthritis, sepsis, and atherosclerosis. The aim of this study was to evaluate the effect of harpalycin 2 (Har2), an isoflavone isolated fromHarpalyce brasilianaBenth., in the enzymatic, edematogenic, and myotoxic activities of sPLA2fromBothrops pirajai, Crotalus durissus terrificus, Apis mellifera,andNaja najavenoms. Har2 inhibits all sPLA2tested. PrTX-III (B. pirajaivenom) was inhibited at about 58.7%, Cdt F15 (C. d. terrificusvenom) at 78.8%, Apis (from bee venom) at 87.7%, and Naja (N. najavenom) at 88.1%. Edema induced by exogenous sPLA2administration performed in mice paws showed significant inhibition by Har2 at the initial step. In addition, Har2 also inhibited the myotoxic activity of these sPLA2s. In order to understand how Har2 interacts with these enzymes, docking calculations were made, in...
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Human neutrophil migration in vitro induced by secretory phospholipases A2: a role for cell surface glycosaminoglycans11Abbreviations: PLA2s, phospholipases A2; sPLA2, secretory PLA2; MEM, Eagle’s Minimum Essential Medium; HPF, high-power field; LTB4, leukotriene B4; PAF, platelet-activating fact...more
The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory... more
The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.
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Chemistry, Medicine, Humans, Apis mellifera, Heparin, and 15 moreFlavobacterium, Chemotaxis, Biochemical, Glycosaminoglycan, Neutrophils, Glycosaminoglycans, BIOCHEMICAL PHARMACOLOGY, Chondroitin Sulfate, Neutrophil chemotaxis, Cell Surface Markers, Binding Site, Benzoquinones, Heparan Sulfate, Dermatan sulfate, and In Vitro Techniques
A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4 x... more
A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4 x 10(-10) and 1.6 x 10(-10) M were obtained with bovine and porcine trypsin, respectively. This constant was lower (2.6 x 10(-7) M) for chymotrypsin. The inhibitory activity was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the P. dubium inhibitor showed a high degree of homology with other Kunitz-type inhibitors. When fed to the insect Anagasta kuehniella, in an artificial diet (inhibitor concentration 1.6%), the inhibitor produced approximately 56% and delayed the development of this lepidopteran. The concentration of inhibitor in the diet necessary to cause a 50% reduction in the weight (ED50) of fourth instar larvae was approximately 1%. The action of the P. dubium trypsin inhibitor (PDTI) on A. kuehniella may involve inhibition of the trypsin-like activity present in the larval midgut, resistance of the inhibitor to digestion by midgut enzymes and bovine trypsin, and association of the inhibitor with a chitin column and chitinous structures in the peritrophic membrane and/or midgut of the insect.