Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle

The Presynaptic Activity Of Bothropstoxin-i, A Myotoxin From Bothrops Jararacussu Snake Venom.

Bothropstoxin-I from Bothrops jararacussu snake venom is a lysine-49 phospholipase A(2) with myotoxic and neurotoxic activities. In this study, we used mouse phrenic nerve-diaphragm preparations in the absence and presence of manganese (Mn(2+)), a presynaptic blocker, to investigate a possible presynaptic action of bothropstoxin-I. At concentrations of 0.9 mM and 1.8 mM, Mn(2+) produced 50% neuromuscular blockade in less than 4 min., which was spontaneously reversible at the lower concentration. Bothropstoxin-I (1.4 microM) irreversibly inhibited neuromuscular blockade by 50% in 31+/-4 min. (mean+/-S.E.M., n = 9). Pretreating preparations with 0.9 mM Mn(2+) prevented the blockade by bothropstoxin-I. When added after bothropstoxin-I, Mn(2+) produced its characteristic blockade and, after washing, the twitch tension returned to pre-Mn(2+) levels, indicating that bothropstoxin-I caused irreversible damage before the addition of Mn(2+). Electrophysiological measurements showed that a concentration of bothropstoxin-I (0.35 microM), which did not produce neuromuscular blockade, caused the appearance of giant miniature end-plate potentials with no change in the membrane resting potential but increased the quantal content. Preparations preincubated with Mn(2+) (0.9 mM, 30 min.) were protected against the depolarizing action of bothropstoxin-I (0.7 microM). These results show that, in addition to its well-known myotoxic effect, bothropstoxin-I also has a presynaptic action.95175-8

Pharmacological Properties of Vochysia Haenkeana (Vochysiaceae) Extract to Neutralize the Neuromuscular Blockade Induced by Bothropstoxin-I (Lys49 Phospholipase A2) Myotoxin

Purpose: Bothrops snakes are responsible for more than 70 % of snakebites every year in Brazil and their venoms cause severe local and systemic damages. The pharmacological properties of medicinal plants have been widely investigated in order to discover new alternative treatments for different classes of diseases including neglected tropical diseases as envenomation by snakebites. In this work, we have investigated the ability of Vochysia haenkeana stem barks extract (VhE) to neutralize the neuromuscular effects caused by Bothropstoxin-I (BthTX-I), the major phospholipase A2 (PLA2) myotoxin from B. jararacussu venom. Methods: The biological compounds of VhE were analysed under thin layer chromatography (TLC) and its neutralizing ability against BthTX-I was assessed through twitch-tension recordings and histological analysis in mouse phrenic nerve-diaphragm (PND) preparations. The antimicrobial activity of VhE was assessed against S. aureus, E. coli and P. aeruginosa strains. The aggregation activity of VhE was analysed under protein precipitation assay. Results: VhE showed the presence of phenolic compound visualized by blue trace under TLC. VhE abolished the neuromuscular blockade caused by BthTX-I applying the pre-toxin incubation treatment and partially neutralized the BthTX-I action under post-toxin incubation treatment; VhE contributed slightly to decrease the myotoxicity induced by BthTX-I. The neutralizing mechanism of VhE may be related to protein aggregation. VhE showed no antimicrobial activity. Conclusion: V. haenkeana extract which has no antimicrobial activity exhibited neutralizing ability against the neuromuscular blockade caused by BthTX-I and also contributed to decrease its myotoxicity. Protein aggregation involving phenolic compounds may be related in these protective effects

Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle.

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle

Amino acid substitutions in the DNA-binding domain of the human androgen receptor are a frequei.t cause of receptor binding positive androgen resistance

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMP alpha-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPa-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S-2-S '(2) subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMP alpha-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S-2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the Si subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process. (c) 2014 Elsevier B.V. All rights reserved

Effect of toxins on lipids droplets.

<p>Huh-7.5 cell line stably expressing SGR-luc-JFH-1 were treated with 10 μg/mL of CX, CP or PLA₂-CB at 37° for 48 hours. Cell were fixed and nuclei, Lipid droplets (LDs) and viral protein NS5A were labelled with DAPi (blue), BODYPI 493/503 (green) and antibodies against NS5A (red), respectively. PBS was used as untreated control. Scale bars, 200 nm.</p

Effect of the toxins on HCV infectivity.

<p>Infectious supernatant and toxins were added in different times to the cells and intracellular virus was titrated 48 h post-infection by analyzing focus-forming units per milliliters (Ffu/mL). For entry assay, Huh-7.5 cells were infected with JFH-1 HCVcc and toxins were immediately added. After 4 h, the supernatant was replaced by fresh medium after repeated washes with PBS to remove completely the inoculum (A). For virucidal assay, JFH-1 HCVcc particles were incubated with toxins for 1 h prior to the infection. After that, the inoculum was used to infect naïve Huh-7.5 cells for 4 h. Cells were extensively washed and medium was added (B). In the pre-treatment assay, cells were previously treated with toxins for 1 h, washed to completely remove toxins and infected with JFH-1 virus for 4 h. Cells were then washed to virus removal and replaced with fresh media for up to 48 h post-infection (C). PBS was used as negative control and EGCG as control of entry blockage. Mean values of three independent experiments each measured in triplicate including the standard deviation are shown. P < 0.001 was considered significant.</p

Multiple effects of toxins isolated from <i>Crotalus durissus terrificus</i> on the hepatitis C virus life cycle

<div><p><i>Hepatitis C virus</i> (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as <i>Dengue virus</i>, <i>Yellow fever virus</i> and <i>Measles virus</i>. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A₂ (PLA₂-CB) isolated from the venom of <i>Crotalus durissus terrificus</i> on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA₂-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle.</p></div