975 resultados para 41 kDa protein


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Both soluble (SfTre1) and membrane-bound (SfTre2) trehalases occur along the midgut of Spodoptera frugiperda larvae. Released SfTre2 was purified as a 67 kDa protein. Its K(m) (1.6 mM) and thermal stability (half life 10 min at 62 degrees C) are different from the previously isolated soluble trehalase (K(m) = 0.47 mM; 100% stable at 62 degrees C). Two cDNAs coding for S. frugiperda trehalases have been cloned using primers based on consensus sequences of trehalases and having as templates a cDNA library prepared from total polyA-containing RNA extracted from midguts. One cDNA codes for a trehalase that has a predicted transmembrane sequence and was defined as SfTre2. The other, after being cloned and expressed, results in a recombinant trehalase with a K(m) value and thermal stability like those of native soluble trehalase. This enzyme was defined as SfTre1 and, after it was used to generate antibodies, it was immunolocalized at the secretory vesicles and at the glycocalyx of columnar cells. Escherichia coli trehalase 3D structure and sequence alignment with SfTre1 support a proposal regarding the residue modulating the pKa value of the proton donor.

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The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

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Toxocara vitulorum is a nematode parasite of the small intestine of cattle and water buffalo, particularly buffalo calves between one and three months of age, causing high morbidity and mortality. The purpose of this research was to characterize the excretory/secretory (ES) antigens of T vitulorum larvae by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot (WB), using immune sera and colostrum of buffalo naturally infected by T vitulorum. The parasitological status of the buffalo calves was also evaluated using sequential fecal examinations. The results showed that the ES antigen revealed eight (190, 150, 110, 90, 64, 56, 48, and 19 kDa) protein bands by SDS-PAGE. The majority of these bands were recognized in the sera and colostrum of infected buffalo with T vitulorum when analyzed by WB. However, particularly fractions of high molecular weight (190, 150, 110, and 90 kDa) were represented in more prominent bands and persisted in the groups of buffalo calves at the peak of egg output, as well as during the period of rejection of T vitulorum by the feces of the calves. During the period of post-rejection of the worms (between the day 118 and 210 of age) the serum antibodies did not react with any protein bands. on the other hand, sera from buffalo calves at one day of age (after suckling the colostrum and at the beginning of infection) reacted with the same bands detected in the serum and colostrum of the buffalo cows.

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

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BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 Angstrom. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 Angstrom.

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SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

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The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5' regions of the two genomic RNAs contain a 'cap' structure and poly(A) tails were identified in the 3'-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.

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Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-γ responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-γ secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-γ synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-γ. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM. © 2002 Elsevier Science Ltd. All rights reserved.

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BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system. © 2007 Wiley-Liss, Inc.

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Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15. kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins. © 2012.

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The aim of this study was to investigate the acute phase response (APR) in 15 horses by quantifying physiological venous blood variables and serum acute phase proteins (APP) at 5 minutes and 6 and 12 hours after a training match of high-goal polo. The horses were divided into three experimental groups based on their team positions, including defense (n = 6), midfield (n = 5), and attack (n = 4). Serum proteinograms were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Data were evaluated using analysis of variance for repeated measures. The match represented a high-intensity stimulus for all positions. Defenders appeared to use the anaerobic pathway more than the other positions, as shown by their lower pH and greater lactatemia. Alterations in muscle membrane permeability were observed in all horses, as seen by the increase in serum creatine kinase activity without a correlation with APR. Significant elevations in total serum protein, albumin, ceruloplasmin, haptoglobin, alpha-1 antitrypsin, and 23-kDa protein were seen only during the course of the physical exertion of the match, although there were no differences in these values among positions of the team. After 6 hours of the match, the concentration of transferrin declined, whereas that of alpha-1 acid glycoprotein remained unaltered at all assessed times. These results demonstrated that the defenders required the most use of the anaerobic pathway during the match, and that equestrian polo exercise triggers an acute phase response of relatively short duration; this APR is characterized as noninflammatory, as APR appears to be a physiological alteration related to the stress inherent in physical exercise. © 2013 Elsevier Inc. All rights reserved.

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Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains. ©2013, American Society for Microbiology.