Toxicon : official journal of the International Society on Toxinology, Jan 25, 2015
A potent insecticidal toxin, β/δ-PrIT1, molecular mass of 5598.86[M+H](+), was characterized from... more A potent insecticidal toxin, β/δ-PrIT1, molecular mass of 5598.86[M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. β/δ-PrIT1 was very toxic (LD50=4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-β/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1=34.7 pM and Kd2=35.1 pM). Its association is rather fast (t1/2(1)=1.4 min, t1/2(2)=8.5 min) and its dissociation is a slower process (t1/2(1)=5.4 min, t1/2(2)=32.8 min). On rat brain synaptosomes β/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, β/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50=80 pM) with the "alpha-...
International journal of biological macromolecules, Jan 18, 2015
There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans.... more There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone ...
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was puri... more A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka.
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was puri... more A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from ...
The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri... more The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri venom in both in vivo and in vitro models. In anesthetized rats, doses of the venom (14-216 mg protein/kg) induced a transient increase in the mean arterial pressure. However at higher dose (338 mg protein/kg) this effect was followed by a sudden hypotension and the animal evolved to death. The heart rate was temporarily increased and followed by bradycardia using doses !108 mg/kg. In isolated rat hearts the crude venom (5-80 mg protein) produced dose-dependent positive ventricular chronotropic, inotropic, lusitropic and coronary vasoconstriction responses. Partial purification of an active fraction (CF, cardiovascular fraction) which reproduced the cardiovascular effects induced by crude venom on isolated hearts was achieved by conventional gel filtration chromatography. Adrenergic blockades, prazosin and propranolol, significantly attenuated these responses. The coronary vasoconstriction response to CF was also attenuated by chemical endothelium denudation. In conclusion, the data showed that S. plumieri fish venom induces disorders in the cardiovascular system. It also suggests that a 1 and b-adrenergic receptors, and the vascular endothelium, are involved at least partially, in these cardiac effects.
The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in exper... more The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF-TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial. Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.
A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity ... more A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an M r of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 M) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Tri-meresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.
Poisoning by organophosphorus insecticides is often accompanied by cardiac complications which ma... more Poisoning by organophosphorus insecticides is often accompanied by cardiac complications which may be serious and even fatal. However, the effects of these compounds on the cardiovascular mechanisms involved in blood pressure regulation are not known. The aim of this study was to evaluate the effects of a sublethal dose (8 mg/kg, i.p.) of the organophosphorus methamidophos on chemoreceptor (CR) and Bezold-Jarisch (BJR) cardiovascular reflexes. Male Wistar rats were treated with single intraperitoneal injections of methamidophos in saline (n¼23) or saline (0.9 percent, n¼20) and underwent catheterization of femoral artery and vein one day after the injections. Cardiovascular recordings were performed 24 h after the catheterization procedure. Plasma cholinesterase (ChE) activity was measured 24 h after similar treatments in separate groups (n¼10/group). The bradycardic component of CR and BJR was significantly attenuated in animals treated with methamidophos. The ChE activity was 80 percent reduced in the methamidophos-treated animals. Methamidophos impairment of the bradycardic component of two important cardiovascular reflexes may contribute to the cardiovascular toxicity associated with acute organophosphorus insecticides exposure.
Toxicon : official journal of the International Society on Toxinology, Jan 25, 2015
A potent insecticidal toxin, β/δ-PrIT1, molecular mass of 5598.86[M+H](+), was characterized from... more A potent insecticidal toxin, β/δ-PrIT1, molecular mass of 5598.86[M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. β/δ-PrIT1 was very toxic (LD50=4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-β/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1=34.7 pM and Kd2=35.1 pM). Its association is rather fast (t1/2(1)=1.4 min, t1/2(2)=8.5 min) and its dissociation is a slower process (t1/2(1)=5.4 min, t1/2(2)=32.8 min). On rat brain synaptosomes β/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, β/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50=80 pM) with the "alpha-...
International journal of biological macromolecules, Jan 18, 2015
There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans.... more There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone ...
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was puri... more A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka.
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was puri... more A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from ...
The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri... more The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri venom in both in vivo and in vitro models. In anesthetized rats, doses of the venom (14-216 mg protein/kg) induced a transient increase in the mean arterial pressure. However at higher dose (338 mg protein/kg) this effect was followed by a sudden hypotension and the animal evolved to death. The heart rate was temporarily increased and followed by bradycardia using doses !108 mg/kg. In isolated rat hearts the crude venom (5-80 mg protein) produced dose-dependent positive ventricular chronotropic, inotropic, lusitropic and coronary vasoconstriction responses. Partial purification of an active fraction (CF, cardiovascular fraction) which reproduced the cardiovascular effects induced by crude venom on isolated hearts was achieved by conventional gel filtration chromatography. Adrenergic blockades, prazosin and propranolol, significantly attenuated these responses. The coronary vasoconstriction response to CF was also attenuated by chemical endothelium denudation. In conclusion, the data showed that S. plumieri fish venom induces disorders in the cardiovascular system. It also suggests that a 1 and b-adrenergic receptors, and the vascular endothelium, are involved at least partially, in these cardiac effects.
The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in exper... more The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF-TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial. Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.
A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity ... more A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an M r of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 M) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Tri-meresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.
Poisoning by organophosphorus insecticides is often accompanied by cardiac complications which ma... more Poisoning by organophosphorus insecticides is often accompanied by cardiac complications which may be serious and even fatal. However, the effects of these compounds on the cardiovascular mechanisms involved in blood pressure regulation are not known. The aim of this study was to evaluate the effects of a sublethal dose (8 mg/kg, i.p.) of the organophosphorus methamidophos on chemoreceptor (CR) and Bezold-Jarisch (BJR) cardiovascular reflexes. Male Wistar rats were treated with single intraperitoneal injections of methamidophos in saline (n¼23) or saline (0.9 percent, n¼20) and underwent catheterization of femoral artery and vein one day after the injections. Cardiovascular recordings were performed 24 h after the catheterization procedure. Plasma cholinesterase (ChE) activity was measured 24 h after similar treatments in separate groups (n¼10/group). The bradycardic component of CR and BJR was significantly attenuated in animals treated with methamidophos. The ChE activity was 80 percent reduced in the methamidophos-treated animals. Methamidophos impairment of the bradycardic component of two important cardiovascular reflexes may contribute to the cardiovascular toxicity associated with acute organophosphorus insecticides exposure.
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