Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

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 50 mM Tris-HCl buffer, pH 9.5, for 20 min 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 10 mM sodium citrate buffer, pH 6.0, for 15 min 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. (C) 2011 Elsevier B.V. All rights reserved.

Insulin Affects Tissue Organization and the Kinetics of Epithelial Cell Death in the Rat Ventral Prostate After Castration

Prostate growth and physiology are regulated by steroid hormones and modulated by multiple endocrine factors We investigated the action of insulin on the tissue organization and kinetics of epithelial cells in the rat ventral prostate (VP) in response to castration up to 120 hours after surgery by using an acute protocol of alloxan induced diabetes Diabetes caused a reduction in volume density (Vv(o)/) and volume of the epithelium The effects of castration on the epithelium were accelerated in the diabetic animals as determined by changes in V(o)/, and volume The smooth muscle cells became atrophic and apparently relaxed in response to castration in contrast to the spinous aspect observed in nondiabetic castrated rats Counting of apoptotic nuclei in the epithelium showed the classical apoptosis peak at 72 hours in nondiabetic rats and an advance of the apoptosis peak to 48 hours after castration in diabetic rats Insulin restored the time of the peak to 72 hours These results were confirmed after immunostaining for cleaved caspase 3 and suggest a survival and antiapoptotic effect on VP epithelial cells in both the presence and absence of androgen stimulation This idea is supported by the observation that insulin also reduced the overall rate of apoptosis at all experimental points analyzed before and after castration

HOME RANGE AND SPATIAL ORGANIZATION OF MANED WOLVES IN THE BRAZILIAN GRASSLANDS

The maned wolf (Chrysocyon brachyurus) is the largest canid in South America, weighing up to 30 kg, and exhibits an omnivorous diet based on fruits and small vertebrates. Maned wolves are considered to live in monogamous pairs defending a common territory, with mates living a largely solitary life, but these conclusions come from few studies with small samples. We captured maned wolves in Emas National Park, central Brazil, and monitored their use of space using radiotelemetry. Home-range size and overlap of 45 adults, and interactions between members of 5 pairs, were investigated. Home-range sizes of resident adults averaged 80.18 km(2) using the fixed kernel with 95% of the locations, and averaged 13.78 km(2) with 50% of the locations. Overlap of 95% ranges between male-male, female-female, or mixed dyads was similar, approximately 0.20, whereas 50% ranges of maned wolves showed less overlap overall but more tolerance for overlap with the opposite sex. Members of a pair were located alone more often than together, and even when located simultaneously maintained a mean distance of >0.5 km apart, independent of time of day. Results are in agreement with a spatial organization based on monogamous mating pairs with little intrapair sociality, but the latter needs to be investigated in more detail.

Somatotopic organization and cortical projections of the ventrobasal complex of the flying fox: an "inverted" wing representation in the thalamus

The present study investigates the somatotopic representation in the somatosensory thalamus of a megachiropteran bat. Using standard microelectrode mapping techniques, representational maps were generated for the ventrobasal (Vb) and posterior (Po) thalamic complexes of the Grey-headed flying fox. Anatomical tracing from neocortical injections provided additional data confirming the somatotopy found physiologically. A full representation of the body surface innervated by the trigeminal and spinal nerves was found. However, in contrast with other mammals, the representations of the forelimb and adjacent thoracic trunk within the thalamus were inverted. This means that the distal portions of the wing membrane and the tips of the digits were represented dorsally in Vb, and the thoracic trunk was represented ventrally In Po the digit tips were represented in the ventral most portion and the thoracic trunk in the dorsal portion of the nucleus. These results are discussed in relation to similarities of megachiropteran somatosensory thalamic nuclei to those of other mammalian species and in relation to the formation of thalamic somatotopic maps and fiber sorting.

Genomic organization of the CC chemokine MIP-3 alpha/CCL20/LARC/EXODUS/SCYA20, showing gene structure, splice variants, and chromosome localization

We describe the genomic organization of a recently identified CC chemokine, MIP3 alpha /CCL20 (HGMW-approved symbol SCYA20). The MIP-3 alpha /CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISK analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3 alpha /CCL20, Ala MLP-3 alpha /CCL20 and Ser MIP-3 alpha /CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ata MIP-3 alpha /CCL20 or Ser MIP-3 alpha /CCL20. Both forms of MIP-3cr/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-a-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3 alpha /CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3 alpha /CCL20 and Ala MIP-3 alpha /CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known. (C) 2001 Academic Press.

Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system

Current serotyping methods classify Pasteurella multocida into five capsular serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serotypes 1 to 16). In the present study, we have developed a multiplex PCR assay as a rapid alternative to the conventional capsular serotyping system. The serogroup-specific primers used in this assay were designed following identification, sequence determination, and analysis of the capsular biosynthetic loci of each capsular serogroup. The entire capsular biosynthetic loci of P. multocida A:1 (X-73) and B:2 (M1404) have been cloned and sequenced previously (J. Y. Chung, Y. M. Zhang, and B. Adler, FEMS Microbiol. Lett. 166:289-296, 1998; J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). Nucleotide sequence analysis of the biosynthetic region (region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the capsular polysaccharide composition. The multiplex capsular PCR assay was highly specific, and its results, with the exception of those for some serogroup F strains, correlated well with conventional serotyping results. Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The multiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification of P. multocida capsular types

Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L. (Platyhelminthes; Trematoda)

The complete nucleotide sequence of the mitochondrial (mt) DNA molecule of the liverfluke, Fasciola hepatica (phylum Platyhelminthes, class Trematoda, family Fasciolidae), was determined, It comprises 14462 bp, contains 12 protein-encoding, 2 ribosomal and 22 transfer RNA genes, and is the second complete flatworm (and the first trematode) mitochondrial sequence to be described in detail. All of the genes are transcribed from the same strand. Of the genes typically found in mitochondrial genomes of eumetazoans, only atp8 is absent. The nad4L and nad4 genes overlap by 40 nt. Most intergenic sequences are very short. Two larger non-coding regions are present. The longer one (817 nt) is located between trnG and cox3 and consists of 8 identical tandem repeats of 85 nt, rich in G and C, followed by 1 imperfect repeat. The shorter non-coding region (187 nt) exhibits no special features and is separated from the longer region by trnG. The gene arrangement resembles that of some other trematodes including the eastern Asian Schistosoma species (and cyclophyllidean cestode species) but it is strikingly different from that of the African schistosomes, represented by Schistosoma mansoni. The genetic code is as inferred previously for flatworms. Transfer RNA genes range in length from 58 to 70 nt, their products producing characteristic 'clover leaf' structures, except for tRNA(S-VON) and tRNA(S-AGN) lacking the DHU arm.

Demarcações conceituais dos princípios científicos da Arquivologia e da Ciência da Informação : contribuições para a configuração científica das disciplinas no campo da informação

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciência da Informação, Programa de Pós-Graduação em Ciência da Informação, 2016.

The tale of a Trojan horse or the quest for market access? China and the World Trade Organization

In 2001, China finally joined the WTO. The accession of China was looked forward to by many WTO members and China itself. However, observers had some fears that the Chinese accession would prove to be a Trojan horse, disrupting the working of the WTO. This paper looks into the Chinese accession and its involvement in the WTO Dispute Settlement and argues that these fears seem so far to be unfounded.