Pituitary gene and protein expression under experimental variation on salinity and temperature in gilthead sea bream Sparus aurata.

Temperature and salinity are important factors that affect several physiological processes in aquatic organisms, which could be produced by variation of certain hormones. In this study, the expression of pituitary hormones involved in the acclimation to different temperatures and salinities was examined in Sparus aurata, a euryhaline and eurytherm species, by Q-Real Time RT-PCR and Western blot analyses for mRNA and protein expression, respectively. Three different experimental conditions were designed with specimens (10 per treatment) acclimated to: a) low salinity water; b) sea water; and c) high salinity water. Additionally, fish under different salinities were acclimated to three different temperatures: 12, 19 and 26 degrees C. Animals were maintained seven weeks before sampling pituitary glands. Our results provided enough evidence for a differential expression of PRL, GH and SL in the pituitary of gilthead sea bream, under different temperature and salinity regimes.

Gene and protein expression for prolactin, growth hormone and somatolactin in Sparus aurata: seasonal variations.

The seasonal variation of PRL, GH and SL gene and protein expression has been analyzed in gilthead sea bream (Sparus aurata) pituitaries using Real-Time Q-PCR and Western Blots, respectively. Animals were cultured in earthen ponds under natural photoperiod, temperature and salinity conditions. Samples were taken during winter 2005 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Beta-actin, used as the housekeeping gene both for Q-RT-PCR and Western analysis, did not present significant differences among seasons. Higher expression was observed during spring and autumn for PRL, summer and winter for GH, and spring for SL. Expression of PRI, GH and SL, presented seasonal variation, suggesting that these hormones could play a role in the molecular signal transduction of environmental factors (especially of photoperiod and temperature) in eurythermal fish.

Characterization of a partial alpha-amylase clone from red porgy (Pagrus pagrus): expression during larval development.

A partial alpha-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about alpha-amylase gene expression.

[Based on one case of dissociative disorder: a conceptual review]. / A propósito de un caso de trastorno disociativo: una revisión conceptual.

Based on the description of a clinical case treated by the authors, and diagnosed of dissociative disorder, a review of the diagnosis of dissociative disorder and its polemics is carried out. The authors discuss concepts such as dissociation and hysteria, their historic evolution and their relationships. Some modern cognitive theories on dissociative disorders and their relationship or opposition to psychodynamic theories are presented. The differences between dissociation and repression with these two different approaches are also mentioned. The authors conclude that at the present time important questions must be solved in the area of dissociative disorders in order to progress in the psychiatric knowledge of dissociative processes.

A propósito de un caso de trastorno disociativo: una revisión conceptual / Based on one case of dissociative disorder: a conceptual review

A partir de la descripción de un caso clínico visto por los autores, con diagnóstico de trastorno disociativo, se realiza una revisión del tema de estos trastornos y de las polémicas que lo acompañan. Se presta atención a los conceptos de disociación e histeria, su evolución histórica y sus relaciones. Se mencionan también algunas teorías explicativas actuales desde el modelo cognitivo y su posible relación u oposición con las teorías psicodinámicas, resaltando la diferencia, desde estos distintos enfoques, entre disociación y represión. En las conclusiones se señala que en el campo de lo disociativo hay todavía problemas importantes a los que enfrentarse para avanzar en el conocimiento psiquiátrico de este grupo de trastornos (AU)

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Somatotropic regulation of fish growth and adiposity: growth hormone (GH) and somatolactin (SL) relationship.

Growth hormone (GH) and insulin-like growth factors (IGFs) play a major role in fish development and metabolism, and several studies have allowed discernment of a complex and tissue-specific collection of salmonid IGF-I transcripts (Ea-4, Ea-3, Ea-2, Ea-1), which are the result of the alternative splicing of the E-domain region. However, the pattern of IGF-I expression is different in non-salmonid fish, and only one or two transcripts (Ea-4, Ea-2) have been detected in hepatic and extrahepatic tissues of common carp, barramundi, black sea bream and gilthead sea bream. Despite this, when comparisons are made within Mediterranean fish species (European sea bass, common dentex and gilthead sea bream), plasma IGF-I levels are consistent with fish species differences in growth rates. Changes of growth rates, and plasma IGF-I and GH levels are also found in response to changes in diet composition and ration size, which may serve to assess the suitability of feeding regimes in aquaculture practice. Regulation of plasma somatolactin (SL) levels is also examined in gilthead sea bream, and the resulting plasma SL profile differs from that of GH. Thus, in contrast to GH, plasma SL levels augment with the increase of ration size and fish size (advancement of age). A transient increase in plasma SL levels is also found in short-term fasted fish, and this fish peptide may act as an anti-obesity hormone helping to expedite growth-reproductive processes following replenishment of fat stores, and/or mediate the adaptation to fasting until the lipolytic action of GH and/or other endocrine factors is fully accomplished. This agrees with the known increase of plasma SL levels during acute stress and exhaustive exercise. However, a causal link between SL and energy mobilisation (lipid metabolism) remains to be established, and further research is needed to determine the extent to which SL and GH act in a complementary manner to make available metabolic fuels and to regulate body fat mass and feeding behaviour.

Genomic structure and chromosome location of the human gene encoding the zinc finger autoantigen ZNF330.

We have recently described a novel zinc finger cDNA, ZNF330, which was immunologically characterized as a new human autoantigen, highly conserved during evolution from nematodes to humans. The protein was found at the nucleolus and the cytoplasm in interphase and transiently associates with centromeres in mitosis as determined by immunofluorescence analysis. We now describe that the association of ZNF330 with the nucleolus but not with the cytoplasm is RNA-dependent as shown by RNAse treatment of fixed culture cells, since ZNF330 localization was unaffected by DNAse treatment. We also report the cloning, structural organization and chromosome location of the human ZNF330 gene. The gene is comprised of 10 exons and spans approximately 16 kb of genomic DNA. The conserved residues forming nine CXXC motifs are contained in exons 3 to 9. Several major transcription initiation sites were located 126, 124 and 121 bp upstream of the translation initiation codon ATG, as determined by primer extension analysis. The human ZNF330 gene was mapped by FISH to chromosome 4q31.1-->q31.2, the site of the FRA4C locus previously described as a common fragile site for acquired chromosome instability in humans.

Molecular analysis of the 5' region of human ribosomal transcription factor UBF.

Upstream binding factor, UBF, is a nucleolar autoantigen involved in the transcription of ribosomal DNA genes. Previously, human genomic clones served to demonstrate that an alternative pre-mRNA splicing of a single gene is used to form UBF1 and UBF2. Here, to complete characterizing the 5'end genomic organization of this nucleolar transcription factor, lambda clones containing the human UBF gene were isolated from a human placenta genomic library using a hamster UBF cDNA as a probe. An additional PCR product was isolated from HeLa genomic DNA to cover the first translated 60 nt of the gene containing the ATG initiation codon. We have also determined the transcription start site of the gene by primer extension analysis at nt 188 upstream from the start ATG codon. It served first, to identify an untranslated initial exon on the UBF gene covering the first 121 nt of human UBF cDNA, and also to establish the sequence of the proximal promoter. The human UBF promoter lacks a TATA and CAAT boxes but contains multiple binding sites for SP1, AP1, AP2, TFIID, NF-1 and a single site for NFAT-1. Consequently we have defined the first five exons of the human UBF gene covering 7.5kb. The complete gene now consists of 20 exons with intervening sequences and spans approximately 15kb of DNA.

Immunohistochemical detection of ribosomal transcription factor UBF and AgNOR staining identify apoptotic events in neoplastic cells of Hodgkin's disease and in other lymphoid cells.

Ribosomal RNA synthesis is a key molecular process for understanding the mechanisms that drive cell proliferation. In this process, the upstream binding factor (UBF) is involved in regulating rDNA transcription at the nucleolus, together with RNA polymerase I. Recently, UBF was demonstrated to be a substrate for selective cleavage by specific proteases during apoptosis. Here we studied the expression of UBF in several cases of Hodgkin's disease (HD) by immunostaining and found it to be absent or clearly diminished in a high proportion of Reed-Sternberg cells and Hodgkin cells compared to small reactive lymphocytes. This result contrasted with labeling of those cells by the AgNOR technique, a marker of cell proliferation dependent on increased amounts of several proteins related to ribosome assembly. Disappearance of UBF and preservation of other NOR proteins is consistent with the pattern of selective proteolysis by caspases described in early stages of apoptosis. This correlates well with our results observed on induction of apoptosis in Jurkat cells treated with anti-FAS/APO-1 serum and with those in aged germinal center B-cells, in which UBF was no longer seen although the staining signal of other NOR proteins was maintained. These results support the concept that the rate of apoptosis is higher in neoplastic cells of HD than in the benign reactive lymphocyte population. Differential proteolysis of NOR proteins, as revealed by double staining of UBF and AgNOR, may prove valuable for identification of early stages of apoptosis in cytological and histopathological samples.

Cloning and expression of somatolactin, a pituitary hormone related to growth hormone and prolactin from gilthead seabream, Sparus aurata.

A pituitary hormone, somatolactin (SL), belonging to the GH/PRL family, is produced in the intermediate lobe of the teleost pituitary. The function of this protein is uncertain. Clones coding for SL were isolated and sequenced from a gilthead seabream pituitary cDNA expression library. The nucleotide sequence of the larger cDNA isolated was 1.5 kb containing a 0.8-kb 3'-untranslated region and two potential polyadenylation signals (AATAAA). The mature polypeptide is composed of 207 amino acids, and a signal peptide of 24 residues was also found in the SL precursor. A potential N-glycosylation site Asn-Lys-Thr was identified in gilthead seabream SL. A comparison of the SL amino acid sequences of several fishes indicated that seven cysteine residues are characteristically present in all the SLs so far isolated. Six of those residues are present in homologous positions in SL and GH Sparus aurata proteins. SL and GH from S. aurata showed a 43% homology at the nucleotide level and 22% identity at the amino acid level. Expression of recombinant SL (rSL) in Escherichia coli and isolation from inclusion bodies led to a monomeric form of SL identical in electrophoretic mobility to one of the two forms of the native SL secreted from gilthead seabream pituitaries cultured in vitro. Further, a native glycosylated modified SL secreted in vitro as shown by N-glycosidase treatment was identified. Specific anti-SL antibodies that discriminate well against gilthead sea-bream GH and PRL in immunoblotting were also raised against rSL.