Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venom

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 Angstrom resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.

Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 Angstrom resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 Angstrom. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation. (C) 3001 Elsevier B.V. B.V. All rights reserved.

Compartment Syndrome After South American Rattlesnake (crotalus Durissus Terrificus) Envenomation.

In order to report the outcome of a patient who developed compartment syndrome after South American rattlesnake (Crotalus durissus terrificus) envenomation, confirmed by subfascial pressure measurement and magnetic resonance imaging (MRI). A 63-year-old male was admitted 1 h after being bitten on the right elbow by a large snake, which was not brought for identification. Physical and laboratory features upon admission revealed two fang marks, local tense swelling, paresthesia, intense local pain, hypertension, coagulopathy, and CK = 1530 U/L (RV < 170 U/L). The case was initially treated with bothropic antivenom (80 mL, intravenously), with no improvement. Evolution within 13-14 h post-bite revealed generalized myalgia, muscle weakness, palpebral ptosis, and severe rhabdomyolysis (CK = 126,160 U/L) compatible with envenoming by C. d. terrificus. The patient was then treated with crotalic antivenom (200 mL, intravenously), fluid replacement, and urine alkalinization. Twenty-four-hour post-bite MRI showed marked muscular edema in the anterior compartment of the right forearm, with a high subfascial pressure (40 mmHg) being detected 1 h later. ELISA of a blood sample obtained upon admission, before antivenom infusion, revealed a high serum concentration of C. d. terrificus venom. No fasciotomy was performed and the patient was discharged seven days later without sequelae. Snakebite by C. d. terrificus with subfascial venom injection may lead to increased intracompartmental pressure.

Intrahippocampal Infusion Of Crotamine Isolated From Crotalus Durissus Terrificus Alters Plasma And Brain Biochemical Parameters.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. Here we sought to investigate the inflammatory and toxicological effects induced by the intrahippocampal administration of crotamine isolated from Crotalus whole venom. Adult rats received an intrahippocampal infusion of crotamine or vehicle and were euthanized 24 h or 21 days after infusion. Plasma and brain tissue were collected for biochemical analysis. Complete blood count, creatinine, urea, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine-kinase (CK), creatine kinase-muscle B (CK-MB) and oxidative parameters (assessed by DNA damage and micronucleus frequency in leukocytes, lipid peroxidation and protein carbonyls in plasma and brain) were quantified. Unpaired and paired t-tests were used for comparisons between saline and crotamine groups, and within groups (24 h vs. 21 days), respectively. After 24 h crotamine infusion promoted an increase of urea, GOT, GPT, CK, and platelets values (p ≤ 0.01), while red blood cells, hematocrit and leukocytes values decreased (p ≤ 0.01). Additionally, 21 days after infusion crotamine group showed increased creatinine, leukocytes, TBARS (plasma and brain), carbonyl (plasma and brain) and micronucleus compared to the saline-group (p ≤ 0.01). Our findings show that crotamine infusion alter hematological parameters and cardiac markers, as well as oxidative parameters, not only in the brain, but also in the blood, indicating a systemic pro-inflammatory and toxicological activity. A further scientific attempt in terms of preserving the beneficial activity over toxicity is required.

Herpetofauna do Parque Estadual Carlos Botelho, São Paulo, Brasil

O presente trabalho consiste em um inventário da herpetofauna do Parque Estadual Carlos Botelho (PECB), localizado na região da Serra de Paranapiacaba, Estado de São Paulo. Os dados foram obtidos por meio de coletas em seis áreas dentro do PECB durante um período de 76 dias distribuídos em um ano, e também por meio de consulta a coleções científicas para obtenção de dados secundários. São apresentados resultados sobre a biologia e ocorrência das espécies no PECB e no Brasil, além de fotografias das diferentes espécies que compõem a herpetofauna do PECB. A herpetofauna do PECB pode ser considerada uma das mais diversificadas de São Paulo, com 65 espécies de anfíbios confirmadas e 59 espécies de répteis registrados neste trabalho. Das 65 espécies de anfíbios, 84% (55 spp.) são endêmicas das formações florestais da Mata Atlântica. Devido às características do relevo do PECB, foram encontrados diferentes padrões altitudinais para os anfíbios: 46% das espécies foram registradas apenas em altitudes acima de 500 m, enquanto que 9% são exclusivas das regiões abaixo de 400 m e 45% ocorrem em todas as áreas amostradas do Parque. Das 59 espécies de répteis do PECB, foram registradas 10 espécies de lagartos, 48 de serpentes e um quelônio. Dentre as serpentes coletadas no PECB, a jararaca Bothrops jararaca foi a mais frequente, com 26,9% (N = 14) do total registrado. Espécies de difícil amostragem, como Echinanthera cephalostriata (13,5%; N = 7) e Taeniophallus affinis (7,7%; N = 4), também foram numerosas no PECB. Dentre os lagartos, Enyalius iheringii foi a espécie mais abundante, com 50% (N = 16) de registros. Uma análise de agrupamento entre 25 taxocenoses de anfíbios brasileiras, incluindo o PECB, resultou na formação de quatro agrupamentos principais. A anurofauna do PECB é mais relacionada com as taxocenoses do Parque Estadual Jacupiranga (0,68) e do Parque Estadual Intervales (0,66). Estes Parques se encontram geograficamente próximos e constituem um dos maiores fragmentos preservados de Mata Atlântica no Brasil. Este trabalho é o primeiro a apresentar a lista de répteis do PECB, alem de complementar o conhecimento em relação a fauna de anfíbios do PECB.

Differential gene expression profiling in mucus glands of honey bee (Apis mellifera) drones during sexual maturation

The mating sign that each drone leaves when mating with a queen essentially consists of mucus gland proteins. We employed a Representational Difference Analysis (RDA) methodology to identify genes that are differentially expressed in mucus glands during sexual maturation of drones. The RDA library for mucus glands of newly emerged drones was more complex than that of 8 day-old drones, with matches to 20 predicted genes. Another 26 reads matched to the Apis genome but not to any predicted gene. Since these ESTs were located within ORFs they may represent novel honey bee genes, possibly fast evolving mucus gland proteins. In the RDA library for mucus glands of 8 day-old drones, most reads corresponded to a capsid protein of deformed wing virus, indicating high viral loads in these glands. The expression of two genes encoding venom allergens, acid phosphatase-1 and hyaluronidase, in drone mucus glands argues for their homology with the female venom glands, both associated with the reproductive system.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured Muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom. (C) 2008 Elsevier Ltd. All rights reserved.

Biochemistry of the Envenomation Response-a Generator Theme for Interdisciplinary Integration

The understanding of complex physiological processes requires information from many different areas of knowledge. To meet this interdisciplinary scenario, the ability of integrating and articulating information is demanded. The difficulty of such approach arises because, more often than not, information is fragmented through under graduation education in Health Sciences. Shifting from a fragmentary and deep view of many topics to joining them horizontally in a global view is not a trivial task for teachers to implement. To attain that objective we proposed a course herein described Biochemistry of the envenomation response aimed at integrating previous contents of Health Sciences courses, following international recommendations of interdisciplinary model. The contents were organized by modules with increasing topic complexity. The full understanding of the envenoming pathophysiology of each module would be attained by the integration of knowledge from different disciplines. Active-learning strategy was employed focusing concept map drawing. Evaluation was obtained by a 30-item Likert-type survey answered by ninety students; 84% of the students considered that the number of relations that they were able to establish as seen by concept maps increased throughout the course. Similarly, 98% considered that both the theme and the strategy adopted in the course contributed to develop an interdisciplinary view.

Influence of Urea, Isopropanol, and Propylene Glycol on Rutin In Vitro Release from Cosmetic Semisolid Systems Estimated by Factorial Design

Rutin, one of the major flavonoids found in an assortment of plants, was reported to act as a sun protection factor booster with high anti-UVA defense, antioxidant, antiaging, and anticellulite, by improvement of the cutaneous microcirculation. This research work aimed at evaluating the rutin in vitro release from semisolid systems, in vertical diffusion cells, containing urea, isopropanol and propylene glycol, associated or not, according to the factorial design with two levels with center point. Urea (alone and in association with isopropanol and propylene glycol) and isopropanol (alone and in association with propylene glycol) influenced significant and negatively rutin liberation in diverse parameters: flux (g/cm2.h); apparent permeability coefficient (cm/h); rutin amount released (g/cm2); and liberation enhancement factor. In accordance with the results, the presence of propylene glycol 5.0% (wt/wt) presented statistically favorable to promote rutin release from this semisolid system with flux = 105.12 8.59 g/cm2.h; apparent permeability coefficient = 7.01 0.572 cm/h; rutin amount released = 648.80 53.01 g/cm2; and liberation enhancement factor = 1.21 0.07.

Stability and in vitro penetration study of rutin incorporated in a cosmetic emulsion through an alternative model biomembrane

Rutin is employed as antioxidant and to prevent the capillary fragility and, when incorporated in cosmetic emulsions, it must target the action site. In vitro cutaneous penetration studies through human skin is the ideal situation, however, there are difficulties to obtain and to maintain this tissue viability. Among the membrane models, shed snake skin presents itself as pure stratum corneum, providing barrier function similar to human and it is obtained without the animal sacrifice. The objectives of this research were the development and stability evaluation of a cosmetic emulsion containing rutin and propylene glycol (penetration enhancer) and the evaluation or rutin in vitro cutaneous penetration and retention from the emulsion, employing an alternative model biomembrane. Emulsion was developed with rutin and propylene glycol, both at 5.0% w/w. Active substance presented on the formulation was quantified by a validated spectrophotometric method at 361.0 nm. Rutin Rutin cutaneous penetration and retention was performed in vertical diffusion cells with shed snake skin of Crotalus durissus, as alternative model biomembrane, and distilled water and ethanol 99.5% (1:1), as receptor fluid. The experiment was conducted for six hours, at 37.0 +/- 0.5 degrees C with constant stirring of 300 rpm. Spectrophotometry at 410.0 nm, previously validated, determined the active substance after cutaneous penetration/ retention. Emulsion did not promote rutin cutaneous penetration through C. durissus skin, retaining 0.931 +/- 0.0391 mu g rutin/mg shed snake skin. The referred formulation was chemically stable for 30 days after stored at 25.0 +/- 2.0 degrees C, 5.0 +/- 0.5 degrees C and 45.0 +/- 0.5 degrees C. In conclusion, it has not been verified the active cutaneous penetration through the model biomembrane, but only its retention on the Crotalus durissus stratum corneum, condition considered stable for 30 days.

Biophysical techniques used to evaluate the stratum corneum

Assortments of biophysical methods are used to the study the stratum corneum morphology and dynamic with the objective to elucidate the correlation between its structure and functions. Among these methods, there are: X-ray diffraction, electron paramagnetic resonance, differential scanning calorimetry, Raman spectroscopy with Fourrier transform, infrared spectroscopy and photoacustic spectroscopy. In this manuscript, methods are presented and discussed in relation to the use indication, interpretation of results and advantages and limitations to the stratum corneum analysis.

Antinociceptive and anti-inflammatory properties of 7-hydroxycoumarin in experimental animal models: potential therapeutic for the control of inflammatory chronic pain

Objectives In the present study we investigated the anti nociceptive, anti-inflammatory and antipyretic effects of 7-hydroxycoumarin (7-HC) in animal models. Methods The effects of oral 7-HC were tested against acetic acid-induced writhing, formalin test, tail flick test, complete Freund`s adjuvant (CFA)-induced hypemociception, carrageenan-induced paw oedema, lipopolysaccharide-induced fever and the rota rod test. Key findings 7-HC (3-60 mg/kg) produced a dose-related antinociception against acetic acid-induced writhing in mice and in the formalin test. In contrast, treatment with 7-HC did not prevent thermal nociception in the tail flick test. A single treatment with 7-HC, 60 mg/kg, produced a long-lasting antinociceptive effect against CFA-induced hypernociception, a chronic inflammatory pain stimulus. Notably, at 60 mg/kg per day over 4 days the administration of 7-HC produced a continuous antinociceptive effect against CFA-induced hypernociception. 7-HC (30-120 mg/kg) produced anti-inflammatory and antipyretic effects against carrageenan-induced inflammation and lipopolysaccharide-induced fever, respectively. Moreover, 7-HC was found to be safe with respect to ulcer induction. In the rota rod test, 7-HC-treated mice did not show any motor performance alterations. Conclusions The prolonged antinociceptive and anti-inflammatory effects of 7-HC, in association with its low ulcerogenic activity, indicate that this molecule might be a good candidate for development of new drugs for the control of chronic inflammatory pain and fever.

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for similar to 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (similar to 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed. (C) 2010 Elsevier B.V. All rights reserved.

Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D H-1 NMR data, Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern), The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues, Both of these structural features may play a role in its binding to the voltage-gated sodium channel. (C) 1997 Federation of European Biochemical Societies.