: The present study explores conditions to activate the carboxyl group before the coupling of C-t... more : The present study explores conditions to activate the carboxyl group before the coupling of C-terrninus-AA with TMS-ITC. N-tert-butoxy-carbonyl amino acids (Boc-AAs) and Leucine Enkephalin were used as models and isobutyl chloroformate was used as a C-terminal activator. The reaction coupling activated AA with TMS ITC was temperature dependent. We also·report here a new, efficient, economical and ecological technique to separate the mixture of amino acids thiohydantoins (TH-AA).
: The present study explores conditions to activate the carboxyl group before the coupling of C-t... more : The present study explores conditions to activate the carboxyl group before the coupling of C-terrninal amino acid with Trimethylsilyl -isothiocyanate (TMS-ITC). Protected amino acids and different peptides were used as model and 1,3'-Dicyclohexylcarbodiimide (DCC) plus 1-Hydroxybenzotriazole (HOBt) and 2-(lH-Benzotriazol-l-y)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) plus HOBt were used as carboxyl activators. The coupling reaction of activated AA with TMS-ITC was temperature dependent and the best yield (100 % of coupling) was obtained at 70°C, when DCC or TBTU both with HOBt were used as activating reagents.
a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom. Toxicon 31, 941-94... more a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom. Toxicon 31, 941-947, 1993 .-A bradykinin-potentiating peptide was isolated and characterized from venom of the scorpion Tityus serrulatus by chromatographic techniques followed by biological assays. The complete amino acid sequence (13 residues) of peptide is presented. The peptide potentiated the contractile activity of bradykinin on the isolated guinea-pig ileum, and inhibited the hydrolysis of bradykinin by angiotensin-converting enzyme from B. jararaca plasma and the conversion of angiotensin I to angiotensin II by kininase II from guinea-pig ileum tissue. The peptide also increased the depressor effect of bradykinin on arterial blood pressure in the anaesthetized rat.
Snake venom can affect the growth of Try-panosoma cruzi and Leishmania spp. As new classes of the... more Snake venom can affect the growth of Try-panosoma cruzi and Leishmania spp. As new classes of therapeutic drugs against protozoan parasites could be derived from snake venom, alterations in the ultra-structure and growth of the epimastigotes, trypom-astigotes and amastigotes of T. cruzi, as well as the promastigotes of Leishmania major, were analyzed after treatment with crude venom from Bothrops jararaca. Parasite growth (epimastigotes and promastigotes) of venom treated cultures showed a negative correlation between cell growth and venom concentration. No growth occurred at a dose of 100 lg/ml of venom, while 50 % growth inhibition was obtained in the range 0.1– 0.3 lg/ml. Ultrastructural observations of treated bloodstream trypomastigotes, intracellular amastigotes, as well as axenic cultures of epimastigotes and prom-astigotes, demonstrated mitochondrial swelling and kinetoplast disorganization. Our data show that B. jararaca venom effectively inhibited the growth of T. cruzi an...
Ti-based ceramic coatings on Ti6Al4V substrates were successfully prepared through a laser claddi... more Ti-based ceramic coatings on Ti6Al4V substrates were successfully prepared through a laser cladding process using pre-placed starting materials of TiCN + SiO2 mixed powder without or with adding a 3 wt % CeO2 nanoparticles additive, aiming at improving the wear resistance of the Ti6Al4V alloy for biological applications. The effects of the CeO2 nanoparticles additive on the microstructure, microhardness, and wear performance of the coatings were analyzed in detail. The observations showed that the main compositions of the cladding coating were TiCN and TiN phase. Compared to the coatings without CeO2, the coatings modified with CeO2 nanoparticles led to more excellent mechanical properties. The average microhardness of the coatings modified with CeO2 nanoparticles was approximately 1230 HV0.2, and the wear volume loss of the coatings modified with CeO2 nanoparticles was approximately 14% less than that of the coatings without CeO2 under a simulated body fluid (SBF) lubrication environment. The major reasons included that the microstructure of the coatings modified with CeO2 nanoparticles was refined and compact granular crystalline. The wear mechanisms of the coatings were investigated from the worn surface of the coatings, wear debris, and the worn surface of the counter-body balls. The wear mechanisms of the coatings without CeO2 included abrasive wear, adhesive wear, and fatigue wear, while the wear mechanisms of the coatings modified with CeO2 nanoparticles included only abrasive wear and adhesive wear, because the fine microstructure of the coatings had an excellent resistance to fatigue wear.
Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex ... more Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex G-10 column followed by high-performance liquid chromatography, from the venom of the spider Scaptocosa raptoria. The isolated peptides (peptide-S and peptide-R) were shown to cause contraction on the isolated guinea-pig ileum at amounts equivalent to those shown by bradykinin. Both peptides relaxed the isolated rat duodenum, increased the capillary permeability, caused decreasing and biphasic effect of the arterial blood pressure in conscious rats and induced oedema in the rat paw. The peptides had activity and structural similarities to other peptides (kinin-like) isolated from venoms. The complete amino acid analysis gave peptide-S a structure with 36 amino acid residues and peptide-R 22 amino acid residues. The mol. wts were estimated to be in the range of 4000 and 2870, respectively.
Brazilian Journal of Medical and Biological Research
The Ca2+ release mechanism that triggers muscle contraction is still not completely understood. W... more The Ca2+ release mechanism that triggers muscle contraction is still not completely understood. We compared Ca2+ accumulation and acetyl phosphate hydrolysis by the Ca(2+)-ATPases present in the longitudinal and junctional membrane of the sarcoplasmic reticulum of rabbit skeletal muscle and found that Ca(2+)-ATPase is more sensitive to ADP inhibition when the enzyme is located on the junctional membrane than when the enzyme is located on the longitudinal membrane (K0.5 = 144 microM for the junctional enzyme vs K0.5 = 415 microM for the longitudinal enzyme). When the enzyme was solubilized in non-ionic detergent (2% v/v Triton X-100) and tested again using 2 mM AcP as substrate, the difference in ADP sensitivity observed with native preparations disappeared. We conclude that the enzyme is regulated differently depending on its localization on the membrane of the sarcoplasmic reticulum.
Journal of submicroscopic cytology and pathology, 2000
It has been suggested that proteins produced in specific regions of the epididymis, mostly androg... more It has been suggested that proteins produced in specific regions of the epididymis, mostly androgen dependent glycoproteins, are involved in the sperm maturation process. In the present work, the glycoconjugated distribution pattern and the electrophoretic characteristics of the stallion epididymal proteins were examined using lectin probes. The identification in the luminal fluid of some new proteins, probably synthesized and secreted by the epididymis, is an important initial step to investigate their interaction with the stallion sperm membrane. The binding of FITC-lectins (ConA, WGA, LPA, UEA, RCA, HPA) confirmed the presence of macromolecules containing carbohydrate residues in the epithelial cells with a distribution and relative density that was dependent on the epididymal region analyzed. The epithelium displayed affinity for more than one lectin, indicating diversity in the exposed sugar residues. The electrophoretic pattern of proteins obtained from ductus efferentes, corp...
Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activitie... more Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources. LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was st...
Different peptides have been isolated from a wide range of animal species. It is has become incre... more Different peptides have been isolated from a wide range of animal species. It is has become increasingly clear that due to the development of antibiotic-resistant microbes, antibacterial and antifungal peptides have attracted the attention in recent years, in order to find new therapeutic agents. In this work, a novel peptide with high inhibitory activity against fungi growth have been isolated from the venom of the Brazilian snake Bothrops jararaca. A Sephacryl S-100 gel filtration column was employed for further separation of proteins. The FV fraction with high antifungal activity was named Pep5Bj, pooled and submitted to reverse-phase chromatography in HPLC. The fraction containing the isolated peptide inhibited the growth of different phytopathogenic fungi (Fusarium oxysporum and Colletotrichum lindemuthianum) and yeast (Candida albicans and Saccharomyces cerevisiae). The peptide minimal inhibitory concentration is comparable to other known antifungal peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. We investigate F. oxysporum cells membrane permeabilization using SYTOX Green uptake, an organic compound that fluoresces upon interaction with nucleic acids after penetration in cell with compromised plasma membranes. When viewed under fluorescence optical microscopy, F. oxysporum cells exposed to Pep5Bj display strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. The SYTOX Green data suggested that this effect is related to membrane permeabilization. The molecular masses of this peptide was obtained by MALDI-TOF spectrometry and corresponded to 1370Da.
Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respec... more Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, timedependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.
... HEMORRHAGIN FROM B. atrox VENOM Jorge H. Petretski 1, Milton M. Kanashiro 1, Flavia G. Rodriq... more ... HEMORRHAGIN FROM B. atrox VENOM Jorge H. Petretski 1, Milton M. Kanashiro 1, Flavia G. Rodriques 1, Elias W. Alves 2,Olga LT Machado2, and Tereza L. Kipnis 1 * ... [10] De Luca, M., Ward, CM, Ohmori, K., Amdrews, RK, and Berndt, MC (1995) Biochem. Biophys. Res. ...
Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here... more Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here we fractionated the venom and observed that the anti-T. cruzi activity was associated with fractions that present L-amino acid oxidase (L-AAO) activity. L-AAO produces H(2)O(2), which is highly toxic. The addition of catalase to the medium, a H(2)O(2) scavenger, reverted the killing capacity of venom fractions. The anti-T. cruzi activity was also abolished when parasites were cultured in a medium without hydrophobic amino acids that are essential for L-AAO activity. These results were confirmed with a commercial purified L-AAO. Treatment for 24 h with fractions that present L-AAO activity induced parasites cytoplasmic retraction, mitochondrial swelling and DNA fragmentation, all morphological characteristics of programmed cell death. Similar changes were also observed when parasites were treated with H(2)O(2). These results indicate that H(2)O(2), the product of L-AAO reaction, induces programmed cell death explaining the anti-T. cruzi activity of B. jararaca venom.
Effects of canatoxin on the Cat+-ATPase of sarcoplasmic reticulum membranes. Toxicon 30, 1411-141... more Effects of canatoxin on the Cat+-ATPase of sarcoplasmic reticulum membranes. Toxicon 30, 1411-1418, 1992 .-Canatoxin, a toxic protein isolated from the seeds of Canavalia ensiformis, was studied for its effects on the sarcoplasmic reticulum vesicles obtained from rabbit skeletal muscle. Canatoxin inhibited Ca2+ accumulation catalysed by the Cat+-ATPase, without affecting the hydrolytic activity of this enzyme or membrane permeability to Cat+. The effects of canatoxin were dose dependent, but not time dependent. It is concluded that Canatoxin interacts with the Cal+ pump and uncouples Ca 21 uptake from Ca2+-dependent ATP hydrolysis.
The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skel... more The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skeletal muscle of sarcoplasmic reticulum was studied. With the use of native vesicles it was found that arsenate accelerates the rate of ITP hydrolysis and inhibits both Ca2+ or Sr2+ uptake. These effects were not observed when ATP was used as substrate or, with the use of ITP, when leaky vesicles were assayed. Activation of ITP hydrolysis is related to an increase of the enzyme's apparent affinity for ITP. Arsenate increases the steady-state level of the phosphoenzyme formed from ITP. This depends on the concentration of both Pi and Ca2+, in the medium. Ca2+ and Sr2+ efflux were accelerated by arsenate. The fast Ca2+ efflux promoted by arsenate is impaired by external Ca2+. Arsenate competes with Pi for the phosphorylating site of the enzyme.
Neste trabalho, foi purificada uma proteína macho específica com peso molecular de 19 Kda, a part... more Neste trabalho, foi purificada uma proteína macho específica com peso molecular de 19 Kda, a partir de espermatozóides de bovinos, por intermédio de cromatografia de gel filtração. O anticorpo monoclonal (Mab) desenvolvido (C11F) contra esta proteína discriminou duas populações de espermatozóides, como revelado por imunofluorescência em microscopia e citometria de fluxo. O epitopo reconhecido pelo Mab C11F esteve presente em 49,7% da população de espermatozóides. Quando adicionado ao sêmen bovino fresco, o Mab C11F reduziu a motilidade e promoveu a aglutinação dos espermatozóides.
Snake venoms hydrolyze several phosphorylated substrates. However, not is clearly understood whic... more Snake venoms hydrolyze several phosphorylated substrates. However, not is clearly understood which enzyme(s) is (are) involved in these process. Here, we propose the existence of an independent ADPase activity. In addition, we studied the reactions mechanism of nucleotide hydrolysis. This system resembles membrane ecto-nucleotidases and acts with a multi enzymatic complex transforming ATP in adenosine without the accumulation of intermediates.
: The present study explores conditions to activate the carboxyl group before the coupling of C-t... more : The present study explores conditions to activate the carboxyl group before the coupling of C-terrninus-AA with TMS-ITC. N-tert-butoxy-carbonyl amino acids (Boc-AAs) and Leucine Enkephalin were used as models and isobutyl chloroformate was used as a C-terminal activator. The reaction coupling activated AA with TMS ITC was temperature dependent. We also·report here a new, efficient, economical and ecological technique to separate the mixture of amino acids thiohydantoins (TH-AA).
: The present study explores conditions to activate the carboxyl group before the coupling of C-t... more : The present study explores conditions to activate the carboxyl group before the coupling of C-terrninal amino acid with Trimethylsilyl -isothiocyanate (TMS-ITC). Protected amino acids and different peptides were used as model and 1,3'-Dicyclohexylcarbodiimide (DCC) plus 1-Hydroxybenzotriazole (HOBt) and 2-(lH-Benzotriazol-l-y)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) plus HOBt were used as carboxyl activators. The coupling reaction of activated AA with TMS-ITC was temperature dependent and the best yield (100 % of coupling) was obtained at 70°C, when DCC or TBTU both with HOBt were used as activating reagents.
a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom. Toxicon 31, 941-94... more a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom. Toxicon 31, 941-947, 1993 .-A bradykinin-potentiating peptide was isolated and characterized from venom of the scorpion Tityus serrulatus by chromatographic techniques followed by biological assays. The complete amino acid sequence (13 residues) of peptide is presented. The peptide potentiated the contractile activity of bradykinin on the isolated guinea-pig ileum, and inhibited the hydrolysis of bradykinin by angiotensin-converting enzyme from B. jararaca plasma and the conversion of angiotensin I to angiotensin II by kininase II from guinea-pig ileum tissue. The peptide also increased the depressor effect of bradykinin on arterial blood pressure in the anaesthetized rat.
Snake venom can affect the growth of Try-panosoma cruzi and Leishmania spp. As new classes of the... more Snake venom can affect the growth of Try-panosoma cruzi and Leishmania spp. As new classes of therapeutic drugs against protozoan parasites could be derived from snake venom, alterations in the ultra-structure and growth of the epimastigotes, trypom-astigotes and amastigotes of T. cruzi, as well as the promastigotes of Leishmania major, were analyzed after treatment with crude venom from Bothrops jararaca. Parasite growth (epimastigotes and promastigotes) of venom treated cultures showed a negative correlation between cell growth and venom concentration. No growth occurred at a dose of 100 lg/ml of venom, while 50 % growth inhibition was obtained in the range 0.1– 0.3 lg/ml. Ultrastructural observations of treated bloodstream trypomastigotes, intracellular amastigotes, as well as axenic cultures of epimastigotes and prom-astigotes, demonstrated mitochondrial swelling and kinetoplast disorganization. Our data show that B. jararaca venom effectively inhibited the growth of T. cruzi an...
Ti-based ceramic coatings on Ti6Al4V substrates were successfully prepared through a laser claddi... more Ti-based ceramic coatings on Ti6Al4V substrates were successfully prepared through a laser cladding process using pre-placed starting materials of TiCN + SiO2 mixed powder without or with adding a 3 wt % CeO2 nanoparticles additive, aiming at improving the wear resistance of the Ti6Al4V alloy for biological applications. The effects of the CeO2 nanoparticles additive on the microstructure, microhardness, and wear performance of the coatings were analyzed in detail. The observations showed that the main compositions of the cladding coating were TiCN and TiN phase. Compared to the coatings without CeO2, the coatings modified with CeO2 nanoparticles led to more excellent mechanical properties. The average microhardness of the coatings modified with CeO2 nanoparticles was approximately 1230 HV0.2, and the wear volume loss of the coatings modified with CeO2 nanoparticles was approximately 14% less than that of the coatings without CeO2 under a simulated body fluid (SBF) lubrication environment. The major reasons included that the microstructure of the coatings modified with CeO2 nanoparticles was refined and compact granular crystalline. The wear mechanisms of the coatings were investigated from the worn surface of the coatings, wear debris, and the worn surface of the counter-body balls. The wear mechanisms of the coatings without CeO2 included abrasive wear, adhesive wear, and fatigue wear, while the wear mechanisms of the coatings modified with CeO2 nanoparticles included only abrasive wear and adhesive wear, because the fine microstructure of the coatings had an excellent resistance to fatigue wear.
Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex ... more Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex G-10 column followed by high-performance liquid chromatography, from the venom of the spider Scaptocosa raptoria. The isolated peptides (peptide-S and peptide-R) were shown to cause contraction on the isolated guinea-pig ileum at amounts equivalent to those shown by bradykinin. Both peptides relaxed the isolated rat duodenum, increased the capillary permeability, caused decreasing and biphasic effect of the arterial blood pressure in conscious rats and induced oedema in the rat paw. The peptides had activity and structural similarities to other peptides (kinin-like) isolated from venoms. The complete amino acid analysis gave peptide-S a structure with 36 amino acid residues and peptide-R 22 amino acid residues. The mol. wts were estimated to be in the range of 4000 and 2870, respectively.
Brazilian Journal of Medical and Biological Research
The Ca2+ release mechanism that triggers muscle contraction is still not completely understood. W... more The Ca2+ release mechanism that triggers muscle contraction is still not completely understood. We compared Ca2+ accumulation and acetyl phosphate hydrolysis by the Ca(2+)-ATPases present in the longitudinal and junctional membrane of the sarcoplasmic reticulum of rabbit skeletal muscle and found that Ca(2+)-ATPase is more sensitive to ADP inhibition when the enzyme is located on the junctional membrane than when the enzyme is located on the longitudinal membrane (K0.5 = 144 microM for the junctional enzyme vs K0.5 = 415 microM for the longitudinal enzyme). When the enzyme was solubilized in non-ionic detergent (2% v/v Triton X-100) and tested again using 2 mM AcP as substrate, the difference in ADP sensitivity observed with native preparations disappeared. We conclude that the enzyme is regulated differently depending on its localization on the membrane of the sarcoplasmic reticulum.
Journal of submicroscopic cytology and pathology, 2000
It has been suggested that proteins produced in specific regions of the epididymis, mostly androg... more It has been suggested that proteins produced in specific regions of the epididymis, mostly androgen dependent glycoproteins, are involved in the sperm maturation process. In the present work, the glycoconjugated distribution pattern and the electrophoretic characteristics of the stallion epididymal proteins were examined using lectin probes. The identification in the luminal fluid of some new proteins, probably synthesized and secreted by the epididymis, is an important initial step to investigate their interaction with the stallion sperm membrane. The binding of FITC-lectins (ConA, WGA, LPA, UEA, RCA, HPA) confirmed the presence of macromolecules containing carbohydrate residues in the epithelial cells with a distribution and relative density that was dependent on the epididymal region analyzed. The epithelium displayed affinity for more than one lectin, indicating diversity in the exposed sugar residues. The electrophoretic pattern of proteins obtained from ductus efferentes, corp...
Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activitie... more Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources. LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was st...
Different peptides have been isolated from a wide range of animal species. It is has become incre... more Different peptides have been isolated from a wide range of animal species. It is has become increasingly clear that due to the development of antibiotic-resistant microbes, antibacterial and antifungal peptides have attracted the attention in recent years, in order to find new therapeutic agents. In this work, a novel peptide with high inhibitory activity against fungi growth have been isolated from the venom of the Brazilian snake Bothrops jararaca. A Sephacryl S-100 gel filtration column was employed for further separation of proteins. The FV fraction with high antifungal activity was named Pep5Bj, pooled and submitted to reverse-phase chromatography in HPLC. The fraction containing the isolated peptide inhibited the growth of different phytopathogenic fungi (Fusarium oxysporum and Colletotrichum lindemuthianum) and yeast (Candida albicans and Saccharomyces cerevisiae). The peptide minimal inhibitory concentration is comparable to other known antifungal peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. We investigate F. oxysporum cells membrane permeabilization using SYTOX Green uptake, an organic compound that fluoresces upon interaction with nucleic acids after penetration in cell with compromised plasma membranes. When viewed under fluorescence optical microscopy, F. oxysporum cells exposed to Pep5Bj display strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. The SYTOX Green data suggested that this effect is related to membrane permeabilization. The molecular masses of this peptide was obtained by MALDI-TOF spectrometry and corresponded to 1370Da.
Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respec... more Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, timedependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.
... HEMORRHAGIN FROM B. atrox VENOM Jorge H. Petretski 1, Milton M. Kanashiro 1, Flavia G. Rodriq... more ... HEMORRHAGIN FROM B. atrox VENOM Jorge H. Petretski 1, Milton M. Kanashiro 1, Flavia G. Rodriques 1, Elias W. Alves 2,Olga LT Machado2, and Tereza L. Kipnis 1 * ... [10] De Luca, M., Ward, CM, Ohmori, K., Amdrews, RK, and Berndt, MC (1995) Biochem. Biophys. Res. ...
Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here... more Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here we fractionated the venom and observed that the anti-T. cruzi activity was associated with fractions that present L-amino acid oxidase (L-AAO) activity. L-AAO produces H(2)O(2), which is highly toxic. The addition of catalase to the medium, a H(2)O(2) scavenger, reverted the killing capacity of venom fractions. The anti-T. cruzi activity was also abolished when parasites were cultured in a medium without hydrophobic amino acids that are essential for L-AAO activity. These results were confirmed with a commercial purified L-AAO. Treatment for 24 h with fractions that present L-AAO activity induced parasites cytoplasmic retraction, mitochondrial swelling and DNA fragmentation, all morphological characteristics of programmed cell death. Similar changes were also observed when parasites were treated with H(2)O(2). These results indicate that H(2)O(2), the product of L-AAO reaction, induces programmed cell death explaining the anti-T. cruzi activity of B. jararaca venom.
Effects of canatoxin on the Cat+-ATPase of sarcoplasmic reticulum membranes. Toxicon 30, 1411-141... more Effects of canatoxin on the Cat+-ATPase of sarcoplasmic reticulum membranes. Toxicon 30, 1411-1418, 1992 .-Canatoxin, a toxic protein isolated from the seeds of Canavalia ensiformis, was studied for its effects on the sarcoplasmic reticulum vesicles obtained from rabbit skeletal muscle. Canatoxin inhibited Ca2+ accumulation catalysed by the Cat+-ATPase, without affecting the hydrolytic activity of this enzyme or membrane permeability to Cat+. The effects of canatoxin were dose dependent, but not time dependent. It is concluded that Canatoxin interacts with the Cal+ pump and uncouples Ca 21 uptake from Ca2+-dependent ATP hydrolysis.
The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skel... more The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skeletal muscle of sarcoplasmic reticulum was studied. With the use of native vesicles it was found that arsenate accelerates the rate of ITP hydrolysis and inhibits both Ca2+ or Sr2+ uptake. These effects were not observed when ATP was used as substrate or, with the use of ITP, when leaky vesicles were assayed. Activation of ITP hydrolysis is related to an increase of the enzyme's apparent affinity for ITP. Arsenate increases the steady-state level of the phosphoenzyme formed from ITP. This depends on the concentration of both Pi and Ca2+, in the medium. Ca2+ and Sr2+ efflux were accelerated by arsenate. The fast Ca2+ efflux promoted by arsenate is impaired by external Ca2+. Arsenate competes with Pi for the phosphorylating site of the enzyme.
Neste trabalho, foi purificada uma proteína macho específica com peso molecular de 19 Kda, a part... more Neste trabalho, foi purificada uma proteína macho específica com peso molecular de 19 Kda, a partir de espermatozóides de bovinos, por intermédio de cromatografia de gel filtração. O anticorpo monoclonal (Mab) desenvolvido (C11F) contra esta proteína discriminou duas populações de espermatozóides, como revelado por imunofluorescência em microscopia e citometria de fluxo. O epitopo reconhecido pelo Mab C11F esteve presente em 49,7% da população de espermatozóides. Quando adicionado ao sêmen bovino fresco, o Mab C11F reduziu a motilidade e promoveu a aglutinação dos espermatozóides.
Snake venoms hydrolyze several phosphorylated substrates. However, not is clearly understood whic... more Snake venoms hydrolyze several phosphorylated substrates. However, not is clearly understood which enzyme(s) is (are) involved in these process. Here, we propose the existence of an independent ADPase activity. In addition, we studied the reactions mechanism of nucleotide hydrolysis. This system resembles membrane ecto-nucleotidases and acts with a multi enzymatic complex transforming ATP in adenosine without the accumulation of intermediates.
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Papers by Elias Alves