New Insights into Immunopathology Associated to Bothrops lanceolatus Snake Envenomation: Focus on PLA2 Toxin.

The systemic increase in inflammatory mediator levels can induce diverse pathological disorders, including potentially thrombus formation, which may be lethal. Among the clinical conditions in which the formation of thrombi dictates the patient's prognosis, envenomation by Bothrops lanceolatus should be emphasized, as it can evolve to stroke, myocardial infarction and pulmonary embolism. Despite their life-threatening potential, the immunopathological events and toxins involved in these reactions remain poorly explored. Therefore, in the present study, we examined the immunopathological events triggered by a PLA2 purified from B. lanceolatus venom, using an ex vivo human blood model of inflammation. Our results showed that the purified PLA2 from the venom of B. lanceolatus damages human erythrocytes in a dose dependent way. The cell injury was associated with a decrease in the levels of CD55 and CD59 complement regulators on the cell surface. Moreover, the generation of anaphylatoxins (C3a and C5a) and the soluble terminal complement complex (sTCC) indicates that human blood exposure to the toxin activates the complement system. Increased production of TNF-α, CXCL8, CCL2 and CCL5 followed complement activation. The venom PLA2 also triggered the generation of lipid mediators, as evidenced by the detected high levels of LTB4, PGE2 and TXB2. The scenario here observed of red blood cell damage, dysfunctions of the complement regulatory proteins, accompanied by an inflammatory mediator storm, suggests that B. lanceolatus venom PLA2 contributes to the thrombotic disorders present in the envenomed individuals.

Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms.

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Snake and arthropod venoms: Search for inflammatory activity in human cells involved in joint diseases.

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1ß, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and ß-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Integrative multiomics analysis of Premolis semirufa caterpillar venom in the search for molecules leading to a joint disease.

The joint disease called pararamosis is an occupational disease caused by accidental contact with bristles of the caterpillar Premolis semirufa. The chronic inflammatory process narrows the joint space and causes alterations in bone structure and cartilage degeneration, leading to joint stiffness. Aiming to determine the bristle components that could be responsible for this peculiar envenomation, in this work we have examined the toxin composition of the caterpillar bristles extract and compared it with the differentially expressed genes (DEGs) in synovial biopsies of patients affected with rheumatoid arthritis (RA) and osteoarthritis (OA). Among the proteins identified, 129 presented an average of 63% homology with human proteins and shared important conserved domains. Among the human homologous proteins, we identified seven DEGs upregulated in synovial biopsies from RA or OA patients using meta-analysis. This approach allowed us to suggest possible toxins from the pararama bristles that could be responsible for starting the joint disease observed in pararamosis. Moreover, the study of pararamosis, in turn, may lead to the discovery of specific pharmacological targets related to the early stages of articular diseases.

Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Snake and arthropod venoms: search for inflammatory activity in human cells involved in joint diseases

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1β, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and β-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Skin gland concentrations adapted to different evolutionary pressures in the head and posterior regions of the caecilian Siphonops annulatus.

Amphibian skin is rich in mucous glands and poison glands, secreting substances important for gas exchange and playing a fundamental role in chemical defense against predators and microorganisms. In the caecilian Siphonops annulatus (Mikan, 1920) we observed a concentration of enlarged mucous glands in the head region. In the posterior region of the body a similar concentration is made up of enlarged poison glands. These accumulations of glands structurally resemble the macroglands previously reported in anurans and salamanders. The skin glands in these regions are each surrounded by collagen walls forming a honeycomb-like structure. The collagen network in the head region firmly attaches to tiny pits in the bones of the skull. The two extremities of the body produce different secretions, containing exclusive molecules. Considering the fossorial lifestyle of caecilians, it seems evident that the secretions of the head and caudal region serve different functions. The anterior macrogland of mucous glands, rich in mucous/lipid secretion, in conjunction with the funnel-shaped head, may act to lubricate the body and penetrate the soil, thus facilitating locomotion underground. The blunt posterior end bearing an internalized macrogland of poison glands in the dermis may act in chemical defense and/or by blocking invasion of tunnels.

New insights into immunopathology associated to Bothrops lanceolatus snake envenomation: focus on PLA2 toxin

The systemic increase in inflammatory mediator levels can induce diverse pathological disorders, including potentially thrombus formation, which may be lethal. Among the clinical conditions in which the formation of thrombi dictates the patient’s prognosis, envenomation by Bothrops lanceolatus should be emphasized, as it can evolve to stroke, myocardial infarction and pulmonary embolism. Despite their life-threatening potential, the immunopathological events and toxins involved in these reactions remain poorly explored. Therefore, in the present study, we examined the immunopathological events triggered by a PLA2 purified from B. lanceolatus venom, using an ex vivo human blood model of inflammation. Our results showed that the purified PLA2 from the venom of B. lanceolatus damages human erythrocytes in a dose dependent way. The cell injury was associated with a decrease in the levels of CD55 and CD59 complement regulators on the cell surface. Moreover, the generation of anaphylatoxins (C3a and C5a) and the soluble terminal complement complex (sTCC) indicates that human blood exposure to the toxin activates the complement system. Increased production of TNF-α, CXCL8, CCL2 and CCL5 followed complement activation. The venom PLA2 also triggered the generation of lipid mediators, as evidenced by the detected high levels of LTB4, PGE2 and TXB2. The scenario here observed of red blood cell damage, dysfunctions of the complement regulatory proteins, accompanied by an inflammatory mediator storm, suggests that B. lanceolatus venom PLA2 contributes to the thrombotic disorders present in the envenomed individuals.

Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Revisiting antithrombotic therapeutics; sculptin, a novel specific, competitive, reversible, scissile and tight binding inhibitor of thrombin.

Thrombin is a multifunctional enzyme with a key role in the coagulation cascade. Its functional modulation can culminate into normal blood coagulation or thrombosis. Thus, the identification of novel potent inhibitors of thrombin are of immense importance. Sculptin is the first specific thrombin inhibitor identified in the transcriptomics analysis of tick's salivary glands. It consists of 168 residues having four similar repeats and evolutionary diverged from hirudin. Sculptin is a competitive, specific and reversible inhibitor of thrombin with a Ki of 18.3 ± 1.9 pM (k on 4.04 ± 0.03 × 107 M-1 s-1 and k off 0.65 ± 0.04 × 10-3 s-1). It is slowly consumed by thrombin eventually losing its activity. Contrary, sculptin is hydrolyzed by factor Xa and each polypeptide fragment is able to inhibit thrombin independently. A single domain of sculptin alone retains ~45% of inhibitory activity, which could bind thrombin in a bivalent fashion. The formation of a small turn/helical-like structure by active site binding residues of sculptin might have made it a more potent thrombin inhibitor. In addition, sculptin prolongs global coagulation parameters. In conclusion, sculptin and its independent domain(s) have strong potential to become novel antithrombotic therapeutics.

Integrative multiomics analysis of Premolis semirufa caterpillar venom in the search for molecules leading to a joint disease

The joint disease called pararamosis is an occupational disease caused by accidental contact with bristles of the caterpillar Premolis semirufa. The chronic inflammatory process narrows the joint space and causes alterations in bone structure and cartilage degeneration, leading to joint stiffness. Aiming to determine the bristle components that could be responsible for this peculiar envenomation, in this work we have examined the toxin composition of the caterpillar bristles extract and compared it with the differentially expressed genes (DEGs) in synovial biopsies of patients affected with rheumatoid arthritis (RA) and osteoarthritis (OA). Among the proteins identified, 129 presented an average of 63% homology with human proteins and shared important conserved domains. Among the human homologous proteins, we identified seven DEGs upregulated in synovial biopsies from RA or OA patients using meta-analysis. This approach allowed us to suggest possible toxins from the pararama bristles that could be responsible for starting the joint disease observed in pararamosis. Moreover, the study of pararamosis, in turn, may lead to the discovery of specific pharmacological targets related to the early stages of articular diseases.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Histone H1 is phosphorylated in non-replicating and infective forms of Trypanosoma cruzi.

The nuclear structure changes during the differentiation from growing to infective stages of Trypanosoma cruzi. As histone modifications have been correlated with structural and functional changes of chromatin, we investigated whether histones in T. cruzi are modified during the life cycle of this protozoan parasite. We found that histone H1 isolated from proliferating forms (epimastigotes) and from differentiated/infective forms (trypomastigotes) have a distinct migrating pattern in Triton-acetic acid-urea gel electrophoresis. While epimastigotes contain predominantly a fast migrating form, a slow migrating band is prominent in trypomastigotes. By metabolically labeling the cells with radioactive phosphate, we demonstrated that the slow migrating histone H1 band is phosphorylated, and that after alkaline phosphatase treatment, it migrates as the fast form. Parasites arrested at the onset of the S phase of the cell cycle with hydroxyurea (HU) also predominantly have the phosphorylated form of histone H1, suggesting that phosphorylation occurs in non-replicating stages of T. cruzi. We also found that the phosphorylated histone H1 is more weakly associated with the chromatin, being preferentially released at 150 mM NaCl. Therefore, histone H1 phosphorylation varies during the life cycle of T. cruzi, and might be related to changes in the chromatin structure.

Identification of five new bradykinin potentiating peptides (BPPs) from Bothrops jararaca crude venom by using electrospray ionization tandem mass spectrometry after a two-step liquid chromatography.

Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were described in the middle of 1960s and were the first natural inhibitors of the angiotensin-converting enzyme displaying strong anti-hypertensive effects in human subjects. The BPPs can be recognized by their typical pyroglutamyl proline-rich oligopeptide sequences presenting invariably a proline residue at the C-terminus. In the present study, we identified 18 BPPs, most of them already described for the B. jararaca venom. We isolated and sequenced new peptides ranging from 5 to 14 amino acid residues exhibiting similar amino acid sequence features. The applied methodology consisted of a strait two-step liquid chromatography, followed by mass spectrometry analysis. Besides the amino acid sequence homology, the corresponding synthetic peptides were able to potentiate bradykinin on the isolated guinea-pig ileum.

Snake venom metalloproteinases: structure/function relationships studies using monoclonal antibodies.

Snake Venom Metalloproteinases (SVMPs) are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP, isolated from the venom of Bothrops jararaca, comprising metalloproteinase, disintegrin-like and cysteine-rich domains. The catalytic domain is responsible for the hemorrhagic activity. The disintegrin-like/cysteine-rich domains block alpha2beta1 integrin binding to collagen and apparently enhance the hemorrhagic activity of SVMPs. The relevance of disintegrin-like domain is described in this paper using a series of mouse anti-jararhagin monoclonal antibodies (MAJar 1-7). MAJar 3 was the only antibody able to completely neutralize jararhagin hemorrhagic activity. Neutralization of catalytic activity was partial by incubation with MAJar 1. MAJars 1 and 3 efficiently neutralized jararhagin binding to collagen with IC50 of 330 and 8.4 nM, respectively. MAJars 1 and 3 recognized the C-terminal portion of the disintegrin domain, which is apparently in conformational proximity with the catalytic domain according to additivity tests. These data suggest that disintegrin-like domain epitopes are in close contact with catalytic site or functionally modulate the expression of hemorrhagic activity in SVMPs.

Skin gland concentrations adapted to different evolutionary pressures in the head and posterior regions of the caecilian Siphonops annulatus

Amphibian skin is rich in mucous glands and poison glands, secreting substances important for gas exchange and playing a fundamental role in chemical defense against predators and microorganisms. In the caecilian Siphonops annulatus (Mikan, 1920) we observed a concentration of enlarged mucous glands in the head region. In the posterior region of the body a similar concentration is made up of enlarged poison glands. These accumulations of glands structurally resemble the macroglands previously reported in anurans and salamanders. The skin glands in these regions are each surrounded by collagen walls forming a honeycomb-like structure. The collagen network in the head region firmly attaches to tiny pits in the bones of the skull. The two extremities of the body produce different secretions, containing exclusive molecules. Considering the fossorial lifestyle of caecilians, it seems evident that the secretions of the head and caudal region serve different functions. The anterior macrogland of mucous glands, rich in mucous/lipid secretion, in conjunction with the funnel-shaped head, may act to lubricate the body and penetrate the soil, thus facilitating locomotion underground. The blunt posterior end bearing an internalized macrogland of poison glands in the dermis may act in chemical defense and/or by blocking invasion of tunnels.

Crystal structures of a plant trypsin inhibitor from Enterolobium contortisiliquum (EcTI) and of its complex with bovine trypsin.

A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1' pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.

Skin gland concentrations adapted to different evolutionary pressures in the head and posterior regions of the caecilian Siphonops annulatus

Amphibian skin is rich in mucous glands and poison glands, secreting substances important for gas exchange and playing a fundamental role in chemical defense against predators and microorganisms. In the caecilian Siphonops annulatus (Mikan, 1920) we observed a concentration of enlarged mucous glands in the head region. In the posterior region of the body a similar concentration is made up of enlarged poison glands. These accumulations of glands structurally resemble the macroglands previously reported in anurans and salamanders. The skin glands in these regions are each surrounded by collagen walls forming a honeycomb-like structure. The collagen network in the head region firmly attaches to tiny pits in the bones of the skull. The two extremities of the body produce different secretions, containing exclusive molecules. Considering the fossorial lifestyle of caecilians, it seems evident that the secretions of the head and caudal region serve different functions. The anterior macrogland of mucous glands, rich in mucous/lipid secretion, in conjunction with the funnel-shaped head, may act to lubricate the body and penetrate the soil, thus facilitating locomotion underground. The blunt posterior end bearing an internalized macrogland of poison glands in the dermis may act in chemical defense and/or by blocking invasion of tunnels.