KSHV gB associated RGD interactions promote attachment of cells by inhibiting the potential migratory signals induced by the disintegrin-like domain

Background: Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is not only expressed on the envelope of mature virions but also on the surfaces of cells undergoing lytic replication. Among herpesviruses, KSHV gB is the only glycoprotein known to possess the RGD (Arg-Gly-Asp) binding integrin domain critical to mediating cell attachment. Recent studies described gB to also possess a disintegrin-like domain (DLD) said to interact with non-RGD binding integrins. We wanted to decipher the roles of two individually distinct integrin binding domains (RGD versus DLD) within KSHV gB in regulating attachment of cells over cell migration

A transcriptomic analysis of gene expression in the venom gland of the snake Bothrops alternatus (urutu)

<p>Abstract</p> <p>Background</p> <p>The genus <it>Bothrops </it>is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of <it>Bothrops alternatus</it>, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay.</p> <p>Results</p> <p>A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A₂(5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A₂were essentially acidic; no basic PLA₂were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed.</p> <p>Conclusions</p> <p><it>Bothrops alternatus </it>venom gland contains the major toxin classes described for other <it>Bothrops </it>venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA₂agrees with the lower myotoxicity of this venom compared to other <it>Bothrops </it>species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.</p

Metastasis inhibition in breast cancer by targeting cancer cell extravasation

M&aacute;rcia R Cominetti,1 Wanessa F Altei,2 Heloisa Sobreiro Selistre-de-Araujo21Department of Gerontology, Federal University of S&atilde;o Carlos, S&atilde;o Carlos, SP, Brazil; 2Department of Physiological Sciences, Federal University of S&atilde;o Carlos, S&atilde;o Carlos, SP, BrazilAbstract: The spread of cells from primary tumors toward distant tissues and organs, also known as metastasis, is responsible for most cancer-associated deaths. The metastasis cascade comprises a series of events, characterized by the displacement of tumor cells (TCs) from the primary tumor to distant organs by traveling through the bloodstream, and their subsequent colonization. The first step in metastasis involves loss of cell-cell and cell-matrix adhesions, increased invasiveness and migratory abilities, leading to intravasation of TCs into the blood or lymphatic vessels. Stationary TCs must undergo the process of epithelial-mesenchymal transition in order to achieve this migratory and invasive phenotype. Circulating tumor cells that have survived in the circulation and left the blood or lymphatic vessels will reach distant sites where they may stay dormant for many years or grow to form secondary tumors. To do this, cells need to go through the mesenchymal-epithelial transition to revert the phenotype in order to regain epithelial cell-to-cell junctions, grow and become a clinically relevant and detectable tumor mass. This work will review the main steps of the metastatic cascade and describe some strategies to inhibit metastasis by reducing cancer cell extravasation presenting recent studies in the context of breast cancer.Keywords: breast cancer, metastasis, extravasation, circulating tumor cell

Matrix metallopeptidase 2 activity in tendon regions: effects of mechanical loading exercise associated to anabolic-androgenic steroids

Matrix metallopeptidases (MMPs) are responsible for degradation of the extracellular matrix components and tissue remodeling. To achieve a better understanding of AAS effects in rat tendon, MMP-2 activity in the proximal and distal regions of the calcanear tendon (CT) and proximal, intermediate and distal region of superficial (SFT) and deep flexor tendons (DFT) after mechanical load exercise associated with AAS was investigated. Animals were grouped into four groups: sedentary animals (S); sedentary animals with ASS supplementation (S + A); trained animals (T) and trained animals with AAS supplementation (T + A). Analysis of MMP activity in tendon extracts was done by gelatin zymography. Both proximal and distal regions of the calcanear tendon showed the lowest MMP-2 concentration and the highest proportion in MMP-2 active form. The intermediate region of the SFT differed (P < 0.01) from the proximal and distal regions with higher % of active MMP-2 in the sedentary group. The proportion of active MMP-2 decreased in the proximal region of the CT. AAS treatment strongly decreased both MMP-2 concentration and active form in the three regions of the SFT and on the proximal region of the CT, but not on the DFT. The differences in the response to exercise and AAS treatment are a result of distinct metabolism and recruitment of these tendon regions in the exercise program employed in this study.10461087109

Gene expression in distinct regions of rat tendons in response to jump training combined with anabolic androgenic steroid administration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The aim of this study was to evaluate the expression of key genes responsible for tendon remodeling of the proximal and distal regions of calcaneal tendon (CT), intermediate and distal region of superficial flexor tendon (SFT) and proximal, intermediate and distal region of deep flexor tendon (DFT) submitted to 7 weeks of jumping water load exercise in combination with AAS administration. Wistar male rats were grouped as follows: sedentary (S), trained (jumping water load exercise) (T), sedentary animals treated with AAS (5 mg/kg, twice a week) and animals treated with AAS and trained (AAST). mRNA levels of COL1A1, COL3A1, TIMP-1, TIMP-2, MMP-2, IGF-IEa, GAPDH, CTGF and TGF-beta-1 were evaluated by quantitative PCR. Our main results indicated that mRNA levels alter in different regions in each tendon of sedentary animals. The training did not alter the expression of COL1A1, COL3A, IGF-IEa and MMP-2 genes, while AAS administration or its combination with training reduced their expression. This study indicated that exercise did not alter the expression of collagen and related growth factors in different regions of rat tendon. Moreover, the pattern of gene expression was distinct in the different tendon regions of sedentary animals. Although, the RNA yield levels of CT, SFT and DFT were not distinct in each region, these regions possess not only the structural and biochemical difference, but also divergence in the expression of key genes involved in tendon adaptation.112415051515Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Danish Medical Research Council [271-07-0742]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [06/50986-6]Danish Medical Research Council [271-07-0742

Overexpression, purification, and biochemical characterization of GumC, an enzyme involved in the biosynthesis of exopolysaccharide by Xylella fastidiosa

GumC is one of nine enzymes involved in the biosynthesis of fastidian gum, an exopolysaccharide produced by Xylella fastidiosa that may be linked directly to the pathogenicity of the microorganism. GumC may be responsible for gum polymerization or secretion through the membrane of X. fastidiosa. To perform structure and functions studies, we developed an expression system for the production of GumC as a fusion protein with maltose binding protein (MBP) using pMAL-c2x vector. The GumC-MBP fusion protein was expressed as a 94 kDa protein, which strongly reacts with anti-MBP antibodies. GumC-MBP was isolated by affinity chromatography through an amylose column and used to produce antibodies against the fusion protein. After the enzymatic cleavage of MBP, GumC was purified on a Q Sepharose Fast Flow column. GumC showed a molecular weight corresponding to the expected one (52 kDa) and its N-terminal sequence was identical to that deduced from the DNA. The shape of the circular dichroism spectrum was compatible with a folded protein that contains alpha-helical regions in its structure. Therefore, in this study we describe, for the first time, the production of GumC recombinant protein. (C) 2003 Elsevier Inc. All rights reserved.34222322

Biomechanical responses of different rat tendons to nandrolone decanoate and load exercise

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Androgenic-anabolic steroids (AAS) have been associated with an increased incidence of tendon rupture. The aim of this study was to compare the biomechanical properties of the rat calcaneal tendon (CT), superficial flexor tendon (SFT), and deep flexor tendon (DFT), and to determine the effect of jump training in association with AAS. Animals were separated into four groups: sedentary, trained, AAS-treated sedentary rats (AAS), and AAS-treated and trained animals. Mechanical testing showed that the CT differed from the DFT and SFT, which showed similar mechanical properties. Jump caused the CT to exhibit an extended toe region, an increased resistance to tensional load, and a decreased elastic modulus, characteristics of an elastic tendon capable of storing energy. AAS caused the tendons to be less compliant, and the effects were reinforced by simultaneous training. The DFT was the most affected by training, AAS, and the interaction of both, likely because of its involvement in the toe-off step of jumping, which we suggest is related to the rapid transmission of force as opposed to energy storage. In conclusion, tendons are differently adapted to exercise, but responded equally to AAS, showing reduced flexibility, which is suggested to increase the risk of tendon rupture in AAS consumers.216E91E99Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [06/50986-6

Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa

The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (GIc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX7E motif that can be involved in the catalysis of GumH. (C) 2004 Elsevier Inc. All rights reserved.315248549