Manuel M. Valdivia

Endocrine mediators of seasonal growth in gilthead sea bream (Sparus aurata): the growth hormone and somatolactin paradigm

Regulation of somatolactin (SL) and the somatotropic axis was examined year-around at three different stocking times (spring, summer, and autumn) in a Mediterranean fish, the gilthead sea bream (Sparus aurata). The overall timing of plasma growth hormone (GH) increase was similar among trials (late spring-early summer), but the range of variation year-around was different and followed changes in food intake. Total plasma insulin-like growth factor-I primarily followed changes on growth rates, and a close positive correlation between IGF-I and thermal-unit growth coefficient (TGC) was found irrespective of fish stocking time. Thus, the activation of the somatotropic axis preceded always warm growth spurts, whereas the rise of SL in concurrence with low plasma cortisol levels was found at late autumn. This up-regulation of circulating SL titres preceded the winter inhibition of feeding, and it was more severe in big fish (spring and summer stocking times) than in small fish (autumn stocking time), growing with a relative high efficiency during the cold season despite of a severe hypertriglyceridemia and a high hepatosomatic index. These new insights provide good evidence for a different timing of GH and SL increases, and it is likely that the dominant role of SL in energy homeostasis is to be a mediator of the adaptation to fasting after replenishment of body fat stores, whereas GH and IGF-I are perceived as growth-promoting signals in times of food intake and increasing temperature and day-length.

Co‐localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X α‐satellite array

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIα cleavage activity; and mitotic stability. We have determined that the human X major α‐satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored ∼150 kb in from the Xp‐edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topo isomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology.

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.

Hierarchical Inactivation of a Synthetic Human Kinetochore by a Chromatin Modifier

We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes. Instead it reflects a hierarchical disruption of the outer kinetochore, with CENP-C levels falling before CENP-A levels and, in certain instances, CENP-H being lost more readily than CENP-C. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore.

Overview of fish growth hormone family. New insights in genomic organization and heterogeneity of growth hormone receptors

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) are single chain proteins structurally and functionally related. Fish PRL and GH receptors (PRLR, GHR) have been characterized in several fish species. There is limited evidence of fish PRLR isoforms, but emerging data support the existence of different GHR variants. In gilthead sea bream, black sea bream, turbot and fugu, but not in zebrafish, GHR has retained an exclusive fish intron (10/10A). In gilthead sea bream and turbot, this intron is not alternatively spliced, but the black sea bream intron is either removed or retained during mRNA processing, resulting in a long GHR isoform with a 31 amino acid insertion that does not alter the open reading frame. This or any other GHR variant are not found in gilthead sea bream, but a truncated anchored form has been reported in turbot. The latter GHR isoform comprises extracellular and trans-membrane domains, the first 28 amino acids of the intracellular domain and 21 divergent amino acids before a stop codon. This GHR variant is the result of alternative splicing, being the 3′ UTR and the divergent sequence identical to the sequence of the 5′ end of the 9/10 intron. The physiological significance of different fish GHR isoforms remains unclear, but emerging data provide suitable evidence for season and nutrition related changes in the somatototropic axis activity. The up-regulation of circulating GH together with the decrease of plasma titres of insulin-like growth factor-I (IGF-I), an altered pattern of serum IGF binding proteins and a reduced expression of hepatic IGF-I and GHRs represent a mechanism conserved through vertebrate evolution. It secures the preferential utilization of mobilized substrates to maintain energy homeostasis rather than tissue growth. Somatolactin also changes as a function of season, ration size, dietary amino acid profile and dietary protein source creating opposite plasma GH and SL profiles. There is now direct evidence for a lipolytic effect of fish SL, acting at the same time as an inhibitory factor of voluntary food intake. Indeed, long-term feeding restriction results in the enlargement of the summer GH peak, whereas the SL rise coincident with shortened day length is delayed in juvenile fish until late autumn. These findings agree with the idea that SL may act as a marker of energy surplus, priming some particular process such as puberty onset. However, it remains unclear whether SL works through specific receptors and/or dimers or heterodimers of GH and PRL receptors.

Microinjection of antibodies to centromere protein CENP-A arrests cells in interphase but does not prevent mitosis

Abstract. Centromere protein CENP-A is a histone H3-like protein associated specifically with the centromere and represents one of the human autoantigens identified by sera taken from patients with the CREST variant of progressive systemic sclerosis. Injection of whole human autoimmune serum to the centromere into interphase cells disrupts some mitotic events. It has been assumed that this effect is due to CENP-E and CENP-C autoantigens, because of the effects of injecting monospecific sera to those proteins into culture cells. Here we have used an antibody raised against an N-terminal peptide of the human autoantigen CENP-A to determine its function in mitosis and during cell cycle progression. Affinity-purified anti-CENP-A antibodies injected into the nucleus during the early replication stages of the cell cycle caused cells to arrest in interphase before mitosis. These cells showed highly condensed small nuclei, a granular cytoplasm and loss of their division capability. On the other hand, microinjection of nocodazole-blocked HeLa cells in mitosis resulted in the typical punctate staining pattern of CENP-A for centromeres during different stages of mitosis and apparently normal cell division. This was corroborated by time-lapse imaging microscopy analysis of mid-interphase-injected cells, revealing that they undergo mitosis and divide properly. However, a significant delay throughout the progression of mitotic stages was observed. These results suggest that CENP-A is involved predominantly in an essential interphase event at the centromere before mitosis. This may include chromatin assembly at the kinetochore coordinate with late replication of satellite DNA to form an active centromere.

A novel centromere monospecific serum to a human autoepitope on the histone H3-like protein CENP-A

Centromere autoantibodies are commonly found in the serum of patients with some systemic autoimmune diseases. Previous studies have shown that a major human centromere autoantigen is the histone H3‐like protein CENP‐A. Although the human cDNA has been cloned, native CENP‐A has been neither isolated nor expressed in Escherichia coli, and specific antibodies to this chromatin‐associated centromere protein are not available yet. In this report, a highly charged peptide on CENP‐A (residues 3–17) was used to generate a monospecific antibody that reacts by immunoblots with the 17 kDa centromeric protein. Immunofluorescence analysis showed reactivity of this anti‐CENP‐A serum in several but not all mammalian culture cells analyzed, suggesting that the sequence of this histone‐like centromere protein could be more variable throughout evolution than originally thought. Selective extractions of human placenta nuclear proteins and immunoblot analysis indicated that CENP‐A behaves in a similar way to the core histone polypeptides after nuclease digestion of chromatin. Also, immunoblot analysis demonstrated that the CENP‐A peptide used as immunogen is a target region on the CENP‐A molecule in several but not all CREST patients analyzed with high titers of autoantibodies to the centromere. Lastly, we found that in Jurkat cells induced to apoptosis, CENP‐A remains associated with the centromere, in contrast to other human autoantigens studied during apoptosis.

The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.

A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.

Cloning of a somatolactin-encoding cDNA from sole (Solea senegalensis)

From a Solea senegalensis cDNA expression library, clones encoding somatolactin (SL), a new pituitary hormone belonging to the growth hormone/prolactin family, were isolated and analyzed. Northern blot analysis showed a unique 1.0-kb mRNA species. The sole SL 778-bp cDNA encoded full-size S. senegalensis SL (ssSL) (230 amino acids), including seven Cys and two potential glycosylation sites. A consensus polyadenylation signal, AATAAA, was found. Protein homology and DNA sequence alignment of SL cDNAs from other evolutionarily distant marine fishes suggest that the SL sequence is highly conserved.

Molecular Cloning of a Zinc Finger Autoantigen Transiently Associated with Interphase Nucleolus and Mitotic Centromeres and Midbodies ORTHOLOGOUS PROTEINS WITH NINE CXXC MOTIFS HIGHLY CONSERVED FROM NEMATODES TO HUMANS

We have cloned a novel human autoimmune antigen in a patient suffering from rheumatoid arthritis with high levels of antibodies to the nucleolus organizer regions. Initially the human autoimmune serum was used to select a cDNA of 317 amino acids from a hamster expression library. Using the hamster DNA as a probe, we isolated the human homologous cDNA of 320 amino acids. Human and hamster polypeptides share a 95% amino acid homology. The deduced 36-kDa protein contains a putative amino-terminal NLS signal, nine cysteine-X-X-cysteine motifs highly conserved, and a carboxyl-terminal poly acidic region. Several homologous expressed sequence tags have been identified in data bases suggesting that orthologous proteins are present throughout evolution from worms to humans. A Drosophila expressed sequence tag was further completely sequenced for a full-length protein with 60% amino acid identity to the human homologue. Northern blot analysis revealed that this novel protein is widely distributed in human tissues with significantly higher expression levels in heart and skeletal muscle. Specific antibodies to the recombinant protein and transfection experiments demonstrated by immunofluorescence the localization of the protein predominantly but not exclusively to the nucleolus of interphase mammalian cells. In actinomycin D-treated cells the protein remains associated with the nucleolus but is not segregated, like other ribosomal factors such as upstream binding factor. In mitosis the protein was found to be associated with centromeres and concentrated at the midbody in cytokinesis. Transient distribution of this evolutionarily conserved zinc finger nucleolar autoantigen to the mitotic centromeres may provide the means for several aspects of cell cycle control and transcriptional regulation.