Digenetic trematodes infection in a Bothrops moojeni (Viperidae) population from a fauna rescue in Porto Primavera, São Paulo State

No presente estudo é descrita a infecção por trematódeos digenéticos parasitas da cavidade oral e esôfago em uma população de serpentes Bothrops moojeni provenientes de resgate de fauna em Porto Primavera, Estado de São Paulo. Foi observada prevalência de infecção de 68%. O grau de infecção (número de trematódeos por serpente) variou de 2 a 51 helmintos. Os trematódeos encontrados foram Ophisthogonimus spp. e Sticholecitha serpentis. A alta prevalência de infecção foi associada com a drástica alteração ambiental e o estresse multi-fatorial aos quais os animais foram submetidos, que poderiam ter favorecido o ciclo dos parasitas.

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA(2) activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 mug/ml) plus AE (400 mug/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 mug/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 mug/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 mug/ml, I h) did not reveal any change in muscle fibers, but severe necrosis induced by -BV or toxins (20 mug/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA(2), activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 mug of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 mug), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with I h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors. (C) 2004 Elsevier GmbH. All rights reserved.

Treatment of venous ulcers with fibrin sealant derived from snake venom

Venous ulcers of the lower limbs complicated by infection or chronicity represent a serious public health problem. The elevated number of those afflicted burdens the health services, interferes in quality of life and causes absenteeism. Although there are 2,500 items on the market, ranging from the simplest dressing up to the most complex types of dressing, treatment remains a challenge. Among the substances used, fibrin sealant is the one that promotes diminution of bacterial colonization and of edema, controls hemorrhaging, alters the pain threshold by protecting the nerve endings, hydrates the wound bed and forms granulation tissue that favors healing. Its disadvantages include higher cost and utilization of human fibrinogen that can transmit infectious diseases. The Center for the Study of Venoms and Venomous Animals (CEVAP) at São Paulo State University (UNESP) developed a new sealant made up of fibrinogen extracted from large animals and from an enzyme obtained from snake venom. The present study, developed in the Health Education Clinic (CEPS) of Sacred Heart University (USC) aimed to evaluate the effect of the new sealant on the healing process of venous ulcers in 24 adult patients, seven of whom were male and 17 female. Two study groups were formed as follows: Group 1 (G1) - control group of 11 patients treated with essential fatty acid (EFA) and Unna's boot, and Group 2 (G2) - 13 patients treated with essential fatty acid (EFA), fibrin sealant and Unna's boot. The follow-up lasted eight weeks and the sealant was applied at only the first and fourth weeks. The results showed that Group 2 presented worse lesion conditions as to healing, but, when comparing the two groups, it was noteworthy that the the sealant was effective in healing venous ulcers. There is evidence that the new sealant is recommended for leg ulcers with the following advantages: ease of application, preparation of the wound bed, diminution of pain and a higher number of discharges in the eighth week. More important, other positive characteristics are non-transmission of infectious diseases, absence of adverse reactions, and economic advantage of being produced by Brazilian technology. Finally, it is suggested that the weekly application of sealant, for at least eight weeks, could improve the healing process and consequently life quality.

Treatment of venous ulcers with surgical adhesives derived from snake venom

A Brazilian dictionary states that "ulcer", from the Latin word ulcuserise, is a superficial lesion on skin or mucous membrane characterized by an inflammatory process and loss of tissue. Ulcers are a relatively frequent problem that may be worsen by infection or long-time duration. In these cases, they can represent a serious public health issue, since the increased case number contributes to the burden on the public health service. In recent decades, wound treatment has advanced in technological and scientific aspects regarding both development of new products and techniques. Ongoing research in this area is providing new products for wound treatment. Among newly tested substances that have presented successful results is fibrin glue, a snake venom derivative that promotes reduction of infection and edema as well as bleeding control and pain decrease. Thus, the present study aimed to evaluate fibrin glue effects on ulcers of venous origin in 24 patients that were divided into two groups. The first one (G1) consisted of 11 patients treated without the adhesive while the second group (G2) was composed of 13 persons who were under therapy with the adhesive. The results of this study revealed that patients treated with fibrin glue evolved to a satisfactory healing when compared to the other group. Therefore, fibrin glue is an appropriate alternative for treating leg ulcers with several advantages: easy application, less pain, early hospital discharge, not transmiting diseases, lower cost than the adhesive currently available in Brazil and, finally, it is a Brazilian technology.

In Vivo Effects of Bothrops jararaca Venom on Metabolic Profile and on Muscle Protein Metabolism in Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of snake-necked turtle Hydromedusa tectifera (Cope, 1870) (Testudines: Chelidae) plasma protein concentrations by refractometry and the biuret method.

The purpose of this study was to evaluate the accuracy of hand-held refractometer in determining plasma protein concentrations in South American snake-necked turtle (Hydromedusa tectifera) as compared with the standard biuret method. The results indicated that plasma protein values may be accurately determined in snake- necked turtle with a hand-held refractometer.

Paralyzing and myotoxic effects of a recombinant bothropstoxin-1 (BthTX-I) on mouse neuromuscular preparations

As a first step to investigate the structure-function relationship of bothropstoxin-1 (BthTX-1), a myotoxin from Bothrops jararacussu snake venom, Our group previously cloned a recombinant toxin (rBthTX-1) in Escherichia coli. The aim or this work was to characterize the biological activities of this rBthTX-1 (1.0 mu M) in both phrenic-diaphragm and extensor digitorum longus preparations in vitro, by means of myographic and morphologic techniques. Native BthTX-1 (1.0 mu M) was used as a standard. The influence of heparin (27.5 mu g/ml) upon the biological activities of both toxins was also investigated. rBthTX-1 had similar effects to the native toxin inducing blockage of both directly and indirectly evoked contractions in phrenic-diaphragm preparations, and muscle damage characterized by edema, round fibers, and cell areas devoid of myofibrils. Interestingly the paralyzing activity of rBthTX-1 was slightly more potent than the native toxin. Heparin prevented paralyzing and myotoxic effects of both the native and recombinant toxins. This work shows that rBthTX-1 was expressed in a fully active form, and presents a biological profile similar to the native toxin. (c) 2005 Elsevier GmbH All rights reserved.

Antagonism of myotoxic and paralyzing activities of bothropstoxin-I by surarnin

Polyanionic substances are known to inhibit the myotoxic effects of some crotalide snake venoms. Bothropstoxin-I (BthTX-I), a basic Lys49 phospholipase (PLA(2)) homologue from Bothrops jararacussu venom, besides inducing muscle damage, also promotes the blockade of both directly and indirectly evoked contractions in mouse neuromuscular preparation. In this work, we evaluated the ability of suramin, a polysulfonated naphtylurea derivative, to antagonize the myotoxic and the paralyzing activities of BthTX-I on mice neuromuscular junction in vitro. Myotoxicity was assessed by light and electronic microscopic analysis of extensor digitorum longus (EDL) muscles; paralyzing activity was evaluated through the recording of both directly and indirectly evoked contractions of phrenic-diaphragm (PD) preparations. BthTX-I (1 muM) alone, or pre-incubated with suramin (10 muM) at 37degreesC for 15 min was added to the preparations for 120 min. BthTX-I induced histological alterations typical of myonecrosis in 14.6 +/- 1.0% of EDL muscle fibers. In addition, BthTX-I blocked 50% of both directly and indirectly evoked contractions in PD preparations in 72.1 +/- 9.1 and 21.1 +/- 2.0 min, respectively. Pre-incubation with suramin abolished both the muscle-damaging and muscle-paralyzing activities of BthTX-I. Since suramin is a polyanionic substance, we suggested that its effects result from the formation of inactive acid-base complexes with BthTX-I. (C) 2003 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9-and 1.45-A resolution

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-Angstrom resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred. (C) 2004 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of a myotoxic Lys49-PLA(2) from Bothrops jararacussu venom complexed with p-bromophenacyl bromide

For the first time, a non-catalytic and myotoxic Lys49-PLA(2) (BthTX-I from Bothrops jararacussu venom) has been crystallized with BPB inhibitor. X-ray diffraction data were collected and electron-density calculations showed that the ligand is bound to the His48 residue. BthTX-I with His48 chemically modified by BPB shows strongly reduced myotoxic and cytotoxic activities. This suggests a biological correlation between the modification of His48, which is associated with catalytic activity of PLA(2)s, and other toxicological activities of Lys49-PLA(2)s.

Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A(2) with low catalytic activity from Bothrops jararacussu venom

For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A(2) (Asp49-PLA(2)) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA(2) PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA(2)s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA(2) from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA(2)s.

Crystallization and preliminary X-ray analysis of S-III-SPIII, a phospholipase A2 isolated from the venom of Bothrops jararacussu.

Large single crystals have been obtained of S-III-SPIII, a phospholipase A(2) from the venom of Bothrops jararacussu. The crystals belong to the orthorhombic system space group C222, and diffract X-rays to a resolution of 1.9 Angstrom. Preliminary analysis reveals the presence of one molecule in the crystallographic asymmetric unit. The crystal structure is currently being determined using molecular replacement techniques.

Structural insights for fatty acid binding in a Lys49-phospholipase A(2): crystal structure of myotoxin II from Bothrops molojeni complexed with stearic acid

The crystal structure of dimeric Lys49-phospholipase A2 myotoxin-II from Bothrops moojeni (MjTX-II) co-crystallized with stearic acid (C18H36O2) has been determined at a resolution of 1.8 angstrom. The electron density maps permitted the unambiguous inclusion of six stearic acid molecules in the refinement. Two stearic acid molecules could be located in the substrate-binding cleft of each monomer in positions, which favor the interaction of their carboxyl groups with active site residues. The way of binding of stearic acids to this Lys49-PLA(2)s is analogous to phospholipids and transition state analogues to catalytically active PLA(2)s. Two additional stearic acid molecules were located at the dimer interface region, defining a hitherto unidentified acyl-binding site on the protein surface. The strictly conserved Lys122 for Lys49-PLA(2)s may play a fundamental role for stabilization of legend-protein complex. The comparison of MjTX-II/satiric acid complex with other Lys-PLA(2)s structures whose putative fatty acids were located at their active site is also analysed. Molecular details of the stearic acid/protein interactions provide insights to binding in croup I/II PLA(2)s and to the possible interactions of Lys49-PLA(2)s with target membranes. (c) 2004 Elsevier SAS. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of two myotoxic Lys49-phospholipases A(2) complexed with alpha-tocopherol

BnSP-7 and BnSP-6, two Lys49-phospholipase A(2) isolated from Bothrops neuwiedi pauloensis snake venom, were co-crystallized with a-tocopherol and X-ray diffraction data were collected for both complexes (2.2 and 2.6 angstrom). A new alternative quaternary conformation for these two complexes compared with all other dimeric Lys49-PLA(2) has been observed.