Association of Bartonella spp bacteremia with Chagas cardiomyopathy, endocarditis and arrythmias in patients from South America

Infection with Bartonella spp may cause cardiac arrhythmias, myocarditis and endocarditis in humans. The aim of the present study was to evaluate a possible association between Bartonella spp bacteremia and endocarditis, arrhythmia and Chagas cardiomyopathy in patients from Brazil and Argentina. We screened for the presence of bacterial 16S rRNA in human blood by PCR using oligonucleotides to amplify a 185-bp bacterial DNA fragment. Blood samples were taken from four groups of subjects in Brazil and Argentina: i) control patients without clinical disease, ii) patients with negative blood-culture endocarditis, iii) patients with arrhythmias, and iv) patients with chronic Chagas cardiomyopathy. PCR products were analyzed on 1.5% agarose gel to visualize the 185-bp fragment and then sequenced to confirm the identity of DNA. Sixty of 148 patients (40.5%) with cardiac disease and 1 of 56 subjects (1.8%) from the control group presented positive PCR amplification for Bartonella spp, suggesting a positive association of the bacteria with these diseases. Separate analysis of the four groups showed that the risk of a Brazilian patient with endocarditis being infected with Bartonella was 22 times higher than in the controls. In arrhythmic patients, the prevalence of infection was 45 times higher when compared to the same controls and 40 times higher for patients with Chagas cardiomyopathy. To the best of our knowledge this is the first report of the association between Bartonella spp bacteremia and Chagas disease. The present data may be useful for epidemiological and prevention studies in Brazil and Argentina.

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces alfa2beta1 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.

rACLF, a recombinant snake venom metalloprotease, activates endothelial cells in vitro

Snake venom metalloproteases (SVMPs) comprise a family of snake venom toxins responsible for most of local and systemic effects observed during envenomation by snakes from the Viperidae family. The vascular system and more specifically the endothelium seem to be the preferential targets of these proteins. This work describes the effects of rACLF, a recombinant SVMP from Agkistrodon contortrix laticinctus on human umbilical vein endothelial cells (HUVECs) in vitro. Our results showed that rACLF activates HUVECs by the release of mediators involved in inflammation and hemostasis such as prostacyclin and interleukin-8. We also demonstrated that rACLF increased the expression of ICAM-I and decay accelerating factor (DAF). Moreover, rACLF protects the HUVECs against apoptosis induced by serum deprivation. These results suggest that the endothelial cell activation induced by SVMPs may have a significant role in the development of the local inflammatory lesion observed in Viperidae envenomation.

Early remodeling of rat cardiac muscle induced by swimming training

The aim of the present investigation was to study the effect of acute swimming training with an anaerobic component on matrix metallopeptidase (MMP) activity and myosin heavy chain gene expression in the rat myocardium. Animals (male Wistar rats, weighing approximately 180 g) were trained for 6 h/day in 3 sessions of 2 h each for 1 to 5 consecutive days (N = 5 rats per group). Rats swam in basins 47 cm in diameter and 60 cm deep filled with water at 33 to 35°C. After the training period a significant increase (P < 0.05) was observed in the heart weight normalized to body weight by about 22 and 35 percent in the groups that trained for 96 and 120 h, respectively. Blood lactate levels were significantly increased (P < 0.05) in all groups after all training sessions, confirming an anaerobic component. However, lactate levels decreased (P < 0.05) with days of training, suggesting that the animals became adapted to this protocol. Myosin heavy chain-ß gene expression, analyzed by real time PCR and normalized with GAPDH gene expression, showed a significant two-fold increase (P < 0.01) after 5 days of training. Zymography analysis of myocardium extracts indicated a single ~60-kDa activity band that was significantly increased (P < 0.05) after 72, 96, and 120 h, indicating an increased expression of MMP-2 and suggesting precocious remodeling. Furthermore, the presence of MMP-2 was confirmed by Western blot analysis, but not the presence of MMP-1 and MMP-3. Taken together, our results indicate that in these training conditions, the rat heart undergoes early biochemical and functional changes required for the adaptation to the new physiological condition by tissue remodeling.

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.

Remodelamento muscular induzido por treinamento: expressao de miosina em musculo esqueletico de rato: uma anlise qualitativa / Effects of training in muscular remodeling: myosin expression in rat skeletal muscle - a qualitative analysis

O musculo possui uma habiliade inerente de adpatacao diante de variadas condicoes, como tipo de inervacao, atuacao de hormonios, atividade contratil (treinamento), condicao de alongamento, o proprio crescimento pos-natal, entre outros. Ha grande correlacao entre a isoforma de cadeia pesada de miosina (CPM) expressa e a funcao muscular. O presente estudo teve por objetivo estudar o remodelamento muscular por meioda analise da expressao das diferentes isoformas de CPM de musculo esqueletico em ratos submetidos a treinamento fisico. A metodologia utilizada consistiu em treinamento de ratos albinos (n=10), machos, em protocolo de natacao, em um periodo de 6 horas/dia, em 3 sessoes de 2 horas, com intervalos de 30 minutos entre cadasessao, totalizando 5 dias de treinamento. Ao termino do treinamento, os animais foram sacrificados para extracao do musculo soleo. Foi feita extracao de RNA total e posterior reacao de RT-PCR utilizando oligonucleotideos iniciadores especificos para as diferentes isoformas de CPM. Tambem foi realizada, a partir de extratos proteicos do musculo retirado, a separacao eletroforetica das isiformas de CPM, utilizando SDS-PAGE com gradiente de (7 - 10 por cento). Os resultados obtidos com a tecnica de RT-PCR demonstrarm expressao de todas as isoformas de CPM, tanto em ratos sedentarios como nos treinados. Apesar de a analise nao ter sido feita de forma quantitativa, parece haver tendencia de aumento especialmente das isiformas IIa e IIx com a evolucao do treinamento. A separacao eletroforetica das isoformas mostrou que nao houve alteracao na expressao das isoformas de CPM (I, IIa, IIb e IIx) com o treinamento

Identification of metalloprotease gene families in sugarcane

Metaloproteases exercem papéis importantes em muitos processos fisiológicos em mamíferos tais como migraçäo celular, remodelamento tecidual e processamento de fatores de crescimento. Estas enzimas estäo envolvidas também na pato-fisiologia de um grande número de doenças humanas como hipertensäo e câncer. Muitas bactérias patogênicas dependem de proteases para infectar o hospedeiro. Diversas classes de metaloproteases foram descritas em seres humanos, bactérias, venenos de serpentes e insetos. No entanto, a presença e a caracterizaçäo de metaloproteases em plantas estäo pouco descritas na literatura. Neste trabalho, foi pesquisada a biblioteca de cDNA de etiquetas de seqüências expressas da cana-de-açúcar (SUCEST) para identificar, por homologia com seqüências depositadas em outros bancos de dados, famílias gênicas de metaloproteases expressas em diferentes condições. Foram utilizadas seqüências protéicas de Arabidopis thaliana e Glycine max e seqüências de nucleotídeos de Sorghum bicolor. Regiões conservadas correspondentes aos diferentes domínios e motivos de seqüência de metaloproteases foram identificadas nos cDNAs de cana-de-açúcar para caracterizar cada grupo de enzimas. Pelo menos quatro classes de metaloproteases foram identificadas na cana-de-açúcar, a saber, metaloproteases de matriz extracelular, zincinas, inverzincinas e metaloproteases dependentes de ATP. Cada uma destas classes foi analisada quanto a sua expressäo nas diferentes condições e tecidos utilizados na construçäo das bibliotecas de cDNA.