Small Structural Differences in Proline-Rich Decapeptides Have Specific Effects on Oxidative Stress-Induced Neurotoxicity and L-Arginine Generation by Arginosuccinate Synthase.

INTRODUCTION: The proline-rich decapeptide 10c (Bj-PRO-10c; ENWPHPQIPP) from the Bothrops jararaca snake modulates argininosuccinate synthetase (AsS) activity to stimulate L-arginine metabolite production and neuroprotection in the SH-SY5Y cell line. The relationships between structure, interactions with AsS, and neuroprotection are little known. We evaluated the neuroprotective effects of Bj-PRO-10c and three other PROs (Bn-PRO-10a, Bn-PRO-10c > Bn-PRO-10a-MK > Bn-PRO-10a. The structure of PROs and their correlations with enzyme activity revealed that histidine (H5) and glutamine (Q7) in Bj-PRO-10c potentiated their affinity for AsS. CONCLUSIONS: Our investigation provides the first insights into the structure and molecular interactions of PROs with AsS, which could possibly further their neuropharmacological applications.

Evaluation of the Inhibitory Potential of Synthetic Peptides Homologous to CDR3 Regions of a Monoclonal Antibody against Bothropic Venom Serine Proteases.

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.

Comparing Traditional and Toxin-Oriented Approaches towards Antivenom Production against Bitis arietans Snake Venom.

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom ("traditional approach") and immunization with selected key toxins isolated from the snake venom ("toxin oriented" approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

Effects of Mauritia flexuosa L. f. buriti oil on symptoms induced by Bothrops moojeni snake envenomation.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, there are species of snakes that become involved in accidents and cause serious health problems to the inhabitants, highlighting the genus Bothrops for being responsible for approximately 90% of accidents reported annually. In the northern region of the country, this genus is responsible for the largest number of accidents, especially among rural dwellers. These populations invest in alternative treatments for with the purpose of improving the symptoms caused by snakebites. The species Mauritia flexuosa L. f., known as buriti, is traditionally used for the treatment of envenomation by snakes. AIM OF THE STUDY: This study aimed to evaluate the antiophidic potential of the oil of Mauritia flexuosa L. f. for Bothrops moojeni H. venom, confronting cultural and scientific knowledge. MATERIALS AND METHODS: The physicochemical properties were determined, and the components present in the oil, extracted from fruit pulp, were analyzed by Gas Chromatography Coupled with Mass Spectrometry. The in vitro inhibitory capacity of the oil for phospholipase, metalloprotease and serine protease activities was investigated. In the in vivo studies, male Swiss mice were used to evaluate the effect of oil on lethality and toxicity, and hemorrhagic, myotoxic and edematogenic activities were assessed. RESULTS: GC‒MS analysis identification of 90.95% of the constituents of the oil, with the main components being 9-eicosenoic acid, (Z)- (34.54%), n-hexadecanoic acid (25.55%) and (E)-9-octadecenoic acid ethyl ester (12.43%). For the substrates, the outcomes indicate that the oil inhibited the activity of the main classes of toxins present in Bothrops moojeni H. venom (VBm) at the highest dose tested (0.5 µL), with inhibition of 84% for the hydrolysis of the selective substrate for serine protease and inhibition of 60% for the hydrolysis of substrates for PLA2 and metalloproteases. The antiophidic activity in vivo was evaluated with two concentrations of the oil: 1.5 mg, the dosage the population, diluted in mineral oil to a volume of 1 tablespoon and 15 mg, administered by gavage 30 min before poisoning and at time zero (concomitant to poisoning), and both concentrations administered by gavage in combination with topical use at time zero. The bleeding time in the group treated with oil at a concentration of 15 mg administered at time zero was significantly lower than that in the control group (p < 0.05). However, a greater inhibition of bleeding time was observed when local application was combined with the gavage treatment at both concentrations tested at time zero (p < 0.05). In the myotoxicity test, oil was efficient in reducing the myotoxic effects induced by the venom at the two concentrations tested, with gavage administration at time zero and gavage plus topical administration at time zero (p < 0.05). CONCLUSIONS: The data obtained show that the oil is safe to use at the concentrations studied and contains fatty acids that may collaborate for cellular-level repair of the injuries caused by Bm poisoning. The in vitro and in vivo experiments showed that oil inhibits the main proteolytic enzymes present in the venom and that it has important activities to control the local effects caused by bothropic venom.

Anti-Metalloproteases: Production and Characterization of Polyclonal IgG Anti-F2 Fraction Antibodies Purified from the Venom of the Snake Bitis arietans.

Bitis arietans is a medically important snake found in Sub-Saharan Africa. The envenomation is characterized by local and systemic effects, and the lack of antivenoms aggravates the treatment. This study aimed to identify venom toxins and develop antitoxins. The F2 fraction obtained from Bitis arietans venom (BaV) demonstrated the presence of several proteins in its composition, including metalloproteases. Titration assays carried out together with the immunization of mice demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only BaV had peptides recognized by anti-F2 fraction antibodies. In vivo analyses demonstrated the hemorrhagic capacity of the venom and the effectiveness of the antibodies in inhibiting up to 80% of the hemorrhage and 0% of the lethality caused by BaV. Together, the data indicate: (1) the prevalence of proteins that influence hemostasis and envenomation; (2) the effectiveness of antibodies in inhibiting specific activities of BaV; and (3) isolation and characterization of toxins can become crucial steps in the development of new alternative treatments. Thus, the results obtained help in understanding the envenoming mechanism and may be useful for the study of new complementary therapies.

Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity.

Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

Antibodies as Snakebite Antivenoms: Past and Future.

Snakebite envenomation is considered a neglected tropical disease, affecting tens of thousands of people each year. The recommended treatment is the use of antivenom, which is composed of immunoglobulins or immunoglobulin fragments obtained from the plasma of animals hyperimmunized with one (monospecific) or several (polyspecific) venoms. In this review, the efforts made in the improvement of the already available antivenoms and the development of new antivenoms, focusing on snakes of medical importance from sub-Saharan Africa and Latin America, are described. Some antivenoms currently used are composed of whole IgGs, whereas others use F(ab')2 fragments. The classic methods of attaining snake antivenoms are presented, in addition to new strategies to improve their effectiveness. Punctual changes in immunization protocols, in addition to the use of cross-reactivity between venoms from different snakes for the manufacture of more potent and widely used antivenoms, are presented. It is known that venoms are a complex mixture of components; however, advances in the field of antivenoms have shown that there are key toxins that, if effectively blocked, are capable of reversing the condition of in vivo envenomation. These studies provide an opportunity for the use of monoclonal antibodies in the development of new-generation antivenoms. Thus, monoclonal antibodies and their fragments are described as a possible alternative for the production of antivenoms, regardless of the venom. This review also highlights the challenges associated with their development.

New Insights into the Hypotensins from Tityus serrulatus Venom: Pro-Inflammatory and Vasopeptidases Modulation Activities.

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 µM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice's naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-I.

Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization.

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims' death or permanent disability. To better characterize the inflammatory process induced by this snake's venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1ß in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Novel neuroprotective peptides in the venom of the solitary scoliid wasp Scolia decorata ventralis.

BACKGROUND: Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. METHODS: A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. RESULTS: Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and ß-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, ß-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, ß-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. CONCLUSION: Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. ß-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.

Novel neuroprotective peptides in the venom of the solitary scoliid wasp Scolia decorata ventralis

Background Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. Methods A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. Results Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. Conclusion Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.(AU)

A Kunitz-type peptide from Dendroaspis polylepis venom as a simultaneous inhibitor of serine and cysteine proteases.

BACKGROUND: Proteases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated with several pathologies. Therefore, proteases have become potential therapeutic targets regarding the search for inhibitors. Snake venoms are complex mixtures of molecules that can feature a variety of functions, including peptidase inhibition. Considering this, the present study reports the purification and characterization of a Kunitz-type peptide present in the Dendroaspis polylepis venom as a simultaneous inhibitor of elastase-1 and cathepsin L. METHODS: The low molecular weight pool from D. polylepis venom was fractionated in reverse phase HPLC and all peaks were tested in fluorimetric assays. The selected fraction that presented inhibitory activity over both proteases was submitted to mass spectrometry analysis, and the obtained sequence was determined as a Kunitz-type serine protease inhibitor homolog dendrotoxin I. The molecular docking of the Kunitz peptide on the elastase was carried out in the program Z-DOCK, and the program RosettaDock was used to add hydrogens to the models, which were re-ranked using ZRANK program. RESULTS: The fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 µM. The docking analysis led us to synthesize two peptides: PEP1, which was substrate for both elastase-1 and cathepsin L, and PEP2, a 30-mer cyclic peptide, which showed to be a cathepsin L competitive inhibitor, with a Ki of 1.96 µM, and an elastase-1 substrate. CONCLUSION: This work describes a Kunitz-type peptide toxin presenting inhibitory potential over serine and cysteine proteases, and this could contribute to further understand the envenomation process by D. polylepis. In addition, the PEP2 inhibits the cathepsin L activity with a low inhibition constant.

A Kunitz-type peptide from Dendroaspis polylepis venom as a simultaneous inhibitor of serine and cysteine proteases

Proteases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated with several pathologies. Therefore, proteases have become potential therapeutic targets regarding the search for inhibitors. Snake venoms are complex mixtures of molecules that can feature a variety of functions, including peptidase inhibition. Considering this, the present study reports the purification and characterization of a Kunitz-type peptide present in the Dendroaspis polylepis venom as a simultaneous inhibitor of elastase-1 and cathepsin L. Methods: The low molecular weight pool from D. polylepis venom was fractionated in reverse phase HPLC and all peaks were tested in fluorimetric assays. The selected fraction that presented inhibitory activity over both proteases was submitted to mass spectrometry analysis, and the obtained sequence was determined as a Kunitz-type serine protease inhibitor homolog dendrotoxin I. The molecular docking of the Kunitz peptide on the elastase was carried out in the program Z-DOCK, and the program RosettaDock was used to add hydrogens to the models, which were re-ranked using ZRANK program. Results: The fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 μM. The docking analysis led us to synthesize two peptides: PEP1, which was substrate for both elastase-1 and cathepsin L, and PEP2, a 30-mer cyclic peptide, which showed to be a cathepsin L competitive inhibitor, with a Ki of 1.96 µM, and an elastase-1 substrate. Conclusion: This work describes a Kunitz-type peptide toxin presenting inhibitory potential over serine and cysteine proteases, and this could contribute to further understand the envenomation process by D. polylepis. In addition, the PEP2 inhibits the cathepsin L activity with a low inhibition constant.(AU)

Edema and Nociception Induced by Philodryas patagoniensis Venom in Mice: A Pharmacological Evaluation with Implications for the Accident Treatment.

We have investigated the mechanisms involved in the genesis of edema and nociception induced by Philodryas patagoniensis venom (PpV) injected into the footpad of mice. PpV induced dose-related edema and nociceptive effects. Pretreatment of mice with cyclooxygenase inhibitor (indomethacin), but not with cyclooxygenase 2 inhibitor (celecoxib) markedly inhibited both effects. Pretreatments with H1 receptor antagonist (promethazine) or with dual histamine-serotonin inhibitor (cyproheptadine) failed in inhibiting both effects. In groups pretreated with captopril (angiotensin-converting enzyme inhibitor) the edema was unaltered, but nociception was clearly increased, suggesting the participation of kinins in the pathophysiology of the nociception but not of the edema-forming effect of PpV. When PpV was treated with EDTA, the nociception was similar to the one induced by untreated venom, but edema was markedly reduced. We concluded that PpV-induced edema and nociception have cyclooxygenase eicosanoids as the main mediators and no participation of vasoactive amines. Kinins seem to participate in nociception but not in edema induced by PpV. The results also suggest that metalloproteinases are the main compounds responsible for the edema, but not for the nociception induced by this venom.

Intrahippocampal injection of TsTX-I increases the levels of INF-γ in the cerebral tissue but not the levels of glutamate.

TsTX-I, isolated from Tityus serrulatus scorpion venom, causes epileptic-like discharges when injected into the central nervous system. The involvement of excitatory amino acids and cytokines in this activity was investigated. Our results have demonstrated that TsTX-I increases the release of IFN-γ but does not alter the intracerebral concentration of the excitatory amino acids in rats. Thus, this cytokine seems to be more important in the convulsive process than glutamate.

Angiotensin-degrading serine peptidase: a new chymotrypsin-like activity in the venom of Bothrops jararaca partially blocked by the commercial antivenom.

Snakebite envenomation is considered a highly relevant public health hazard in South America, having an impact in terms of mortality and morbidity. In Brazil, Bothrops (sensu latu) poisoning is responsible for 90% of the snakebites and in patients treated at the Vital Brazil Hospital (Butantan Institute) this index reaches 97.5%. The objective of the present study was to analyze more specifically the ability of the antibothropic antivenom, produced by the Butantan Institute, São Paulo, Brazil, to neutralize metallo-and serine peptidases, known as the major toxins present in Bothrops jararaca venom. A set of Fret peptides (Free Ressonance Energy Transfer) was studied using the BjV (B. jararaca venom) and site-directed inhibitors PMSF, EDTA and 1,10-phenanthroline. Two substrates were reached to be used as specific tools for studies with metallo peptidases, Abz-FASSAQ-EDDnp, and the serine peptidases, Abz-RPPGFSPFRQ-EDDnp. In disagreement with the literature, the use of both substrates and the antibothropic serum showed a weak neutralization of the serine peptidases present in this venom and a strong neutralization of the metallo peptidases. In order to investigate possible mechanisms of action that have not yet been described for the serine peptidases from the BjV, the present study shows for the first time a new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine peptidase activity, that was partially blocked by the antibothropic serum. In conclusion, the antivenom presented a good neutralization of metallo peptidases but not of serine peptidases, indicating that further studies about serine peptidases immunogenicity are necessary to improve the antibothropic serum.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae.

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40 degrees C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Purification and characterization of loxnecrogin, a dermonecrotic toxin from Loxosceles gaucho brown spider venom.

The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bite site. Dermonecrotic toxins from Loxosceles gaucho venom were purified and characterized by mass spectrometry (capillary liquid chromatography followed by mass spectrometry detection). Two components were purified: a major one of 31,444 Da, called loxnecrogin A, and a minor one of 31,626 Da, called loxnecrogin B, being probably two isoforms of the toxin. The N-terminal sequence of loxnecrogin A showed similarity with N termini of other sphingomyelinolytic dermonecrotic toxins isolated from venoms of different Loxosceles species. The internal sequences did not present any statistically significant hits in sequence databases searches. However, loxnecrogin A partial sequence showed high similarity to regions of L. intermedia LiD1 recombinant protein sequence, recently described in the literature but not yet deposited in databanks.