Cloning of a novel acidic phospholipase A2 from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

The phospholipase A2 superfamily encompasses 15 groups that are classified into: secreted PLA2 (sPLA2); cytosolic PLA2 (cPLA2); Ca2+-independent intracellular PLA2 (iPLA2); platelet-activating factor acetylhydrolase (PAF-AH); and lysosomal PLA2. Currently, approximately 700 PLA2 sequences are known, of which 200 are obtained from the venom gland of Crotalinae snakes. However, thus far, little information is available on cloning, purification and structural characterization of PLA2 from Crotalus durisssus cascavela venom gland. In the present work, we report the molecular cloning of a novel svPLA2 from C. d. cascavella (Cdc), a predominant rattlesnake subspecies in northeastern Brazil. The Cdc svPLA2 cDNA precursor is 689 nucleotides long and encodes a protein of 138 amino acid residues, with a calculated molecular mass of approximately 13,847 Da and an estimated isoelectric point of 5.14. Phylogenetic analysis of Crotalinae PLA2 reveals that Cdc PLA2 clustered with other acidic type IIA PLA2 homologues is also present in the venom of North American rattlesnakes. Hitherto, this study presents a novel PLA2 cDNA precursor from C. d. cascavella and data reported herein will be useful for further steps in svPLA2 purification and analysis.

Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma

Mammalian seminal plasma contains among others, proteins called spermadhesins, which are the major proteins of boar and stallion seminal plasma. These proteins appear to be involved in capacitation and sperm-egg interaction. Previously, we reported the presence of a protein related to spermadhesins in goat seminal plasma. In the present study, we have further characterized this protein, and we propose ion-exchange chromatography to isolate this seminal protein. Semen was obtained from four adult Saanen bucks. Seminal plasma was pooled, dialyzed against distilled water and freeze-dried. Lyophilized proteins were loaded onto an ion-exchange chromatography column. Dialyzed-lyophilized proteins from the main peak of DEAE-Sephacel were applied to a C2/C18 column coupled to an RP-HPLC system, and the eluted proteins were lyophilized for electrophoresis. The N-terminal was sequenced and amino acid sequence similarity was determined using CLUSTAL W. Additionally, proteins from DEAE-Sephacel chromatography step were dialyzed and submitted to a heparin-Sepharose high-performance liquid chromatography. Goat seminal plasma after ion-exchange chromatography yielded 6.47 +/- 0.63 mg (mean +/- SEM) of the major retained fraction. The protein was designated BSFP (buck seminal fluid protein). BSFP exhibited N-terminal sequence homology to boar, stallion and bull spermadhesins. BSFP showed no heparin-binding capabilities. These results together with our previous data indicate that goat seminal plasma contains a protein that is structurally related to proteins of the spermadhesin family. Finally, this protein can be efficiently isolated by ion-exchange and reverse-phase chromatography.

Crotacetin, a novel snake venom C-type lectin, is homolog of convulxin

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD). A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX), from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXa, 13.9 kDa) and beta (CVXb, 12.6 kDa), joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric a4b4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin b subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria

Integrins, cancer and snake toxins (mini-review)

Integrins encompass a family of transmembrane heterodimeric proteins of adhesion that maintain cells attached to other cells and to the extracellular matrix (ECM). Integrins work as bi-directional mechanotransducers, conveying mechanical signal from outside to inside the cell through a cascade of phosphorylation signals. On the other hand, the signal from inside to outside controls the strength and affinity of integrin adhesion. As proteins of focal contact, integrins are involved in diverse cell functions, such as cell activation, migration, growth, and survival. In the development of neoplastic disease and metastatic tumor, integrins can influence cancer invasiveness and progression, as well as mediate the formation of new blood vessels (angiogenesis). Diverse snake venom toxins have the ability to interact with multiple integrins, what results in inhibition of cell attachment, inhibition of angiogenesis, and induction of apoptotic death of tumor and vascular endothelial cells. The aim of this review is to present data about snake venom toxins that bind to integrins and evoke antiangiogenesis and antitumoral effects

Presencia de inhibidores del tiempo tromboplastina parcial activada (TTPA): repercusión clínica en pacientes obstétricas / Presence of thromboplastin time partial activated (TTPA): clinical repercussion in obstetrical patients

El tiempo de tromboplasina parcial activado con caolin (TTPA) es un ensayo cualitativo utilizado para evidenciar defectos de algún factor de la vida intrínseca de la coagulación o para descartar la presencia de algún inhibidor circulante. El anticoagulane lúpico (AL) forma parte de un grupo de varios autoanticuerpos con potencial patogénico en distintas ramas médicas, principalmente reumatología, hematología y geinecoobstericia. En esta última área, el AL se ha asociado a diferentes repercusiones obstétricas. El objetivo de este estudio fue determinar los principales eventos obstétricos asociados a pacientes con anticoagulantes circulantes identificados por TTPA con caolin. Noventa y seis pacientes fueron incluidas, en el estudio de casos-control. El grupo I (n=48) casos y el grupo II (n=48) controles, fueron seleccionadas de una misma población y fuente de información. Los casos fueron incluidos por presentar prolongación de más de 4 segundos del TTPA activado con caolin con respecto a un testigo, con falta de corección con plasma normal. Una hoja-registro de captación de datos fue diseñada especialmente para el estudio. La comparabilidad de ambos grupos fue establecida al no encontrarse diferencias significativas entre variables consideradas basales. El grupo de casos presentó una frecuencia mayor de aborto habiatual, muerte neonatal y fenómenos trombóticos. Esta relación de cuadros autoinmunológicos con pérdida fetal recurrente y accidentes tromboembólicos ha sido descrita consistentemente en la literatura. De han descrito al respecto una gama de anormalidades inmunológicas incluyendo anticuerpos alticardiolipina positivos y VDRL falsamente positivo. La incidencia en nuestro país de etas entidades es desconocida. Estas observaiones inciden sobre la necesidad de una superación metodológica. Se concluye que el reconocimiento de estos procesos autoimunes en relación al campo de la ginecología y obstetricia puede abrir nuevas perspectivas en eldiagnóstico y tratamiento de entidades como aborto habitual, tromboembolias y retardo del crecimiento intrauterino.

Características fisiológicas de levaduras del género Malassezia / Physiological characteristic of yeasts of the genera Malassezia

Cultivos de Malassezia furfur de origen humano y de M. pachydermatis aislados de perros fueron identificados por las pruebas de asimilación de fuentes de carbono, presentaron ambas un patrón individual y uniforme. El medio de agar Sabouraud dextrosado con bilis de buey al 0,1% es importante en el mantenimiento de los cultivos y en la obtención de los inóculos para pruebas fisiológicas. Los halos de asimilación de fuentes carbonadas fueron más evidentes después de adicionar lugol sobre la superficie del medio

Estudo micologico e clinico de 102 casos de onicopatias. / Mycological and clinical study in 102 patients with abnormal nails

Sao apresentados os resultados do estudo clinico e micologico realizado em 102 pacientes com onicopatias. Maior frequencia foi verificada em adultos do sexo feminino Candidiase foi diagnosticada em 19 pacientes. C. albicans (13) e C. guilliermondii (1) foram as leveduras identificadas.A micosa foi mais comum em mulheres de afazeres domesticos que tinham o habito de molhar as maos. Tricofitose foi reconhecida em 29 pacientes. T. rubrum (11), T. mentagrophytes (1) e T. tonsurans (1) foram as especies isoladas. Traumatismo provocado por sapatos e uso de meias, fabricadas em fibras sinteticas,podem favorecer o desenvolvimento dos fungos e explicar a maior incidencia de dermatofitos em unhas dos pes. As preparacoes microscopicas diretas revelaram maior positividade do que os cultivos. Na opiniao dos autores, a ceratose subungueal nao deve ser sempre interpretada omo infeccao por fungos filamentosos. Em nossa pesquisa, nenhum agente fungico foi identificado, em 54 dos 83 casos nos quais os pacientes apresentavam esse sinal clinico, apesar da repeticao dos exames laboratoriais, por uma dezena de vezes

Microrganismos difteroides: agentes de onicopatias? / Diphtheroid microrganisms: onicopathies' agents?

Foram estudados 30 casos de pacientes adultos com onicopatias, dos quais 20 (15 mulheres - 5 homens) apresentavam lesoes nas unhas dos pes e sete (2 mulheres - 5 homens) nas unhas das maos. Unhas das maos e dos pes lesionadas foram observadas em tres pacientes (homens). Em repetidos exames laboratoriais, nenhum agente fungico foi identificado. No entanto, bacterias em grande quantidade foram visualizadas nessas preparacoes. E ressaltada a identificacao de microrganismos difteroides, em cultivo, e a sua possivel participacao em patologia ungueal

Tinea nigra contracted in Spain.

Se relata un caso de tinea nigra diagnosticado en Sao Paulo (Brasil) en una paciente espanola de 4 anos de edad. Por sus caracteristicas morfologicas el agente fue clasificado com Exophiala werneckii. De aucerdo con la revision de la literatura europea, probablemente sea este el primer caso de tinea nigra de Espana comprobado micologicamente