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Abstract: Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50mM Tris–HCl buffer, pH 9.5, for 20min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10mM sodium citrate buffer, pH 6.0, for 15min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. [Copyright &y& Elsevier]

Abstract: Phospholipase A2 inhibitors (PLIs) are glycoproteins secreted by snake liver into the circulating blood aiming the self-protection against toxic venom phospholipases A2. In the present study, we describe the first complete nucleotide sequence of a βPLI from venom glands of a New World snake, Lachesis muta. The deduced primary structure was compared to other known βPLIs and recent literature findings of other possible roles of PLIs in snakes are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase highperformance liquid chromatography using a C18 column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins. [ABSTRACT FROM AUTHOR]

Abstract: Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization. [Copyright &y& Elsevier]

Abstract: Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3days and produced a similar amount of PsaA protein (150–250ng per 109 CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments. [Copyright &y& Elsevier]