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.

Gene structure, chromosomal localization and immunolocalization of chicken centromere proteins CENP-C and ZW10.

We determined the genomic structures and complete sequences of the coding regions of the chicken CENP-C and ZW10 genes. These two genes encode proteins that are thought to be involved in maintaining the fidelity of chromosome segregation. The chicken CENP-C gene is 30 kb in length and contains 19 exons. The chicken ZW10 gene spans 10 kb and contains 15 exons. The 5'-untranslated regions of these genes contain several binding sites for transcription factors such as Sp-1, E2F, p300, and members of the GATA family. By fluorescence in situ hybridization (FISH) analysis, the CENP-C was mapped to chromosome 4 and the ZW10 gene was mapped to a microchromosome. Antibodies against the chicken ZW10 protein revealed a cell cycle-dependent staining pattern in DT40 cells. ZW10 protein was distributed throughout the cytoplasm of DT40 cells during interphase. In most metaphase cells, ZW10 proteins appeared equally divided between the centromere and the spindle apparatus. During anaphase, chicken ZW10 proteins were no longer localized near chromosomes or the mitotic apparatus but were present diffusely in the cytoplasm.

Distribution and transcription activity of nucleolar DNA in higher plant cells.

By using the NAMA-Ur DNA selective staining method, we have observed in situ the location of nucleolar DNA in onion cells and found it at the boundary between fibrillar centres (FC) and dense fibrillar component (DFC) in transcriptionally active nucleolus. We have also used anti-NOR serum, which is identified as the RNA Polymerase I transcription factor (UBF) antibody, to study its reactivity with higher plant cells and demonstrated this factor associated to the DFC but not present at the interior of FC. Finally, by employing anti-DNA/RNA hybrid antibodies, we labeled the transcriptionally active rRNA genes in active nucleolus and testified that at the boundary between FC and DFC. The results provide the evidence that the boundary between FC and DFC is the genuine transcription site of rRNA genes in nucleolus.

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.

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.

The DNA binding domains of UBF represent major human autoepitopes conserved in vertebrates species.

A human autoantigen (NOR-90) previously shown to be the upstream binding factor UBF, binds to the ribosomal RNA genes clustered at the nucleolus organizer regions (NORs). Truncated recombinant forms of hamster UBF were expressed in E. coli and it served to demonstrate that the DNA binding domains of UBF are major autoepitopes as shown by immunoblots analyses with a human autoimmune anti-NOR serum. Several monospecific antibodies to those recombinant truncated UBF polypeptides were generated as shown by immunofluorescence (IF) and Western blots. Immunoblots and IF studies using these anti-UBF sera in cell cultures of various vertebrates species from fish to mammals, indicate the conservation of the DNA binding domains of the ribosomal transcription factor during evolution.

The fragile-X-related gene FXR1 is a human autoantigen processed during apoptosis.

We describe a new human autoimmune antigen in a patient suffering from scleroderma with high levels of antibodies to nucleolus and cytoplasmic antigens. Using a Chinese hamster ovary cell expression library, we have shown that this antigen corresponds to the autosomal Fragile-X-related gene FXR1. The deduced amino acid sequence from the hamster cDNA is 97, 98, and 58% homologous to the human, mouse, and Xenopus laevis FXR1 genes, respectively. Expression of the hamster cDNA clone in Escherichia coli and antibody production indicates unequivocally the location of the FXR1 protein in the cytoplasm of hamster cells. Affinity chromatography followed by immunofluorescence microscopy analysis and immunoblots demonstrated the presence of autoimmune IgGs to FXR1 in the scleroderma patient. Immunolabeling studies in Jurkat cells, induced to apoptosis by anti-Fas/APO1 serum, indicated that the FXR1 antigens were clearly displaced from their original cytoplasmic location to several punctuated foci, resembling the bleb-like membranous structures characteristic of cells at certain stages of apoptosis. This phenomenon could be part of a putative mechanism in which the FXR1 protein is presented as a target for the autoimmune response in humans.

Recombinant somatolactin as a stable and bioactive protein in a cell culture bioassay: development and validation of a sensitive and reproducible radioimmunoassay.

A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA. In contrast to native proteins, which tend to dimerize and aggregate immediately after pituitary isolation, the majority of recombinant sole SL (rsSL) remained as a monomeric protein after long-term storage, as shown by size exclusion chromatography and Western blot. Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED50) of the assay were 0.15 and 1.8-2.1 ng/ml respectively. Intra-and interassay coefficients of variation were 4.3% and 6.5% at ED50 levels. Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism between rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed. In order to demonstrate some biological activity of rsSL, the ability of this recombinant product to prime gilthead sea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay. A bell-shape dose-response curve was obtained with a maximum at 50 nM (1.2 micrograms/ml), similar to that reported previously for GH. Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which retains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.

Immunohistochemical detection of ribosomal transcription factor UBF: diagnostic value in malignant specimens.

The nucleolar organizer regions (NORs) of human chromosome can be identified in interphase and mitotic cells by localization of some intrinsic components such as the associated enzyme RNA polymerase I. A new sensitive staining method for NORs is described using a specific antibody to the ribosomal transcription factor UBF. By indirect immunofluorescence and enzyme-labelling methods, NORs stained in benign and malignant cells from a variety of tissues with monospecific anti-UBF serum showed significant morphological differences which correlated well with histopathological evaluation. The number of NORs per cell in malignant preparations increased significantly. Furthermore, the staining of a NOR protein component such as UBF appears to be as sensitive as the silver-staining technique (AgNOR) and might be a better alternative for detecting ribosomal activity in malignant tissues.

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.

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

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.

Molecular cloning of gilthead seabream (Sparus aurata) pituitary transcription factor GHF-1/Pit-1.

We report here the complete nucleotide sequence of a cDNA clone encoding Sparus aurata GHF-1/Pit-1 isolated from an expression library prepared from gilthead seabream pituitary gland poly(A)+ RNA. The cDNA sequence (saGHF-1/Pit-1) encodes a protein of 371 amino acids (aa) containing a POU domain (aa 194-343) and a transactivation, STA domain (aa 1-128). Northern blot hybridization of pituitary RNA detected a single 3.0 kb band and a rat GHF-1/Pit-1 antiserum was found to immunoreact with pituitary protein species of 42 kDa by Western blot analysis. When compared with mammalian GHF-1/Pit-1 aa sequence, the POU and STA domains of saGHF-1/Pit-1 protein show 83% and 48% aa identity, respectively. In spite of the low homology of the transactivation domain, saGHF-1/Pit-1 is able to activate the transcription of the human growth hormone promoter.

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.

Growth hormone as a function of age and dietary protein: energy ratio in a marine teleost, the gilthead sea bream (Sparus aurata).

We examined in a factorial design the effect of dietary protein (45%, 52% and 60%) and lipids (8%, 12%, 17%) on growth performance and circulating growth hormone (GH) levels of fingerling sea bream (5-month-old) fed to satiation with self-feeders. Daily weight gain (2.6-2.9%) and feed gain ratio (1.1-1.3) of fish fed high protein-low lipid diets were comparable to those found in fast growing strains of rainbow trout. However, increasing hyperphagia in association with the decrease of daily weight gain and feed conversion efficiency were found with the decrease of dietary protein:energy ratio. This growth impairment was linked to increased concentrations of circulating GH, which would exacerbate glucose and lipid intolerance. We consider the elevated concentration of circulating GH to be a risk factor leading to some state of metabolic starvation, in which feeding behavior and feed conversion efficiency are largely altered. From our results, it can be also concluded that circulating and pituitary GH availability decreases progressively from 1- to 3-year-old fish. This blunted GH synthesis and release is discussed in relation to age decrease in the optimum dietary protein:energy ratio.

Cloning and sequencing of the genes encoding the hamster ribosomal transcription factors UBF1 and UBF2.

The RNA polymerase I transcription factor, UBF, belongs to a family of high-mobility-group DNA-binding proteins. Here, a human autoantibody reactive with the nucleolus organizer regions (NOR) was used to select cDNA clones encoding the hamster transcription factors, UBF1 and UBF2. Comparison at the nucleotide level showed a high degree of homology with other mammalian upstream binding factors (UBF) already identified. The deduced amino acid sequences are identical for both UBF1 and UBF2, except for a 37 amino acid insertion found in UBF1. This insertion is completely conserved among mammalian UBF1 which indicates a putative role of this region on the function of this transcript.

Bacterial production and purification of the fish pituitary hormone somatolactin.

Somatolactin, a pituitary hormone belonging to the growth hormone/prolactin family, is produced in the intermediate lobe of teleost pituitary. To date, the functions of this new hormone and the target tissues are unknown. A Solea senegalensis somatolactin (ssSL) cDNA has previously been cloned and isolated. Here we have inserted this cDNA into a pET-3a plasmid in order to produce recombinant ssSL in E. coli BL21 (DE3) cells. The protein induced was isolated from inclusion bodies by a solubilization-renaturation procedure originally developed to generate native disulfide bonds, to get putative active proteins. The recombinant somatolactin was further purified to homogeneity by gel filtration on FPLC. The estimated molecular weight of 26 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis agrees well with the molecular mass calculated from the translated cDNA sequence and with native somatolactin (SL). The recombinant protein showed electrophoretic mobility identical to that of one of the native forms of SL secreted in vitro by cultured pituitaries from sole. Another native SL expressed in S. senegalensis represented a glycosylated modified hormone as shown by N-glycosidase treatment. Further, recombinant SL was recognized by an anti-native SL antibody and used to generate polyclonal sera reactive with the native pituitary hormone. To date, this represents the first recombinant SL protein isolated in sufficient quantities for biophysical and biochemical investigation and for studies on its physiological actions.