Liver-specific expression of the human factor VII gene.

Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to +1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G + C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic-specific factors as well as the transcription factor Sp1. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays.

Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells.

The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound.

Free amino acids exhibit anthozoan "host factor" activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro.

Reef-building corals and other tropical anthozoans harbor endosymbiotic dinoflagellates. It is now recognized that the dinoflagellates are fundamental to the biology of their hosts, and their carbon and nitrogen metabolisms are linked in important ways. Unlike free living species, growth of symbiotic dinoflagellates is unbalanced and a substantial fraction of the carbon fixed daily by symbiont photosynthesis is released and used by the host for respiration and growth. Release of fixed carbon as low molecular weight compounds by freshly isolated symbiotic dinoflagellates is evoked by a factor (i.e., a chemical agent) present in a homogenate of host tissue. We have identified this "host factor" in the Hawaiian coral Pocillopora damicornis as a set of free amino acids. Synthetic amino acid mixtures, based on the measured free amino acid pools of P. damicornis tissues, not only elicit the selective release of 14C-labeled photosynthetic products from isolated symbiotic dinoflagellates but also enhance total 14CO2 fixation.

Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone.

Augmentation of vertebrate growth by growth hormone (GH) is primarily due to its regulation of insulin-like growth factor I (IGF I) and IGF II levels. To characterize the effect of GH on the levels of IGF I and IGF II mRNA in a teleost, 10 micrograms of bovine GH (bGH) per g of body weight was administered to juvenile rainbow trout (Oncorhynchus mykiss) through i.p. injection. The levels of IGF I and IGF II mRNA were determined simultaneously, by using RNase protection assays, in the liver, pyloric ceca, kidney, and gill at 0, 1, 3, 6, 12, 24, 48, and 72 hr after injection. In the liver, IGF I mRNA levels were significantly elevated at 6 and 12 hr (approximately 2- to 3-fold, P < or = 0.01), while IGF II mRNA levels were significantly elevated at 3 and 6 hr (approximately 3-fold, P < or = 0.01). In the pyloric ceca, IGF II mRNA levels were significantly elevated at 12, 24, and 48 hr (approximately 3-fold, P < or = 0.01), while IGF I mRNA was below the limits of assay accuracy. GH-dependent IGF mRNA appearance was not detected in the gill and kidney. Serum bGH levels, determined by using a radioimmunoassay, were significantly elevated at 3 and 6 hr (P < 0.005). In primary hepatocyte culture, IGF I and IGF II mRNA levels increased in a bGH dose-dependent fashion, with ED50 values of approximately 45 and approximately 6 ng of bGH per ml, respectively. The GH-dependent appearance of IGF II mRNA in the liver and pyloric ceca suggests important roles for this peptide hormone exclusive of IGF I.

Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis.

DNA replication of the adenovirus genome complexed with viral core proteins is dependent on the host factor designated template activating factor I (TAF-I) in addition to factors required for replication of the naked genome. Recently, we have purified TAF-I as 39- and 41-kDa polypeptides from HeLa cells. Here we describe the cloning of two human cDNAs encoding TAF-I. Nucleotide sequence analysis revealed that the 39-kDa polypeptide corresponds to the protein encoded by the set gene, which is the part of the putative oncogene associated with acute undifferentiated leukemia when translocated to the can gene. The 41-kDa protein contains the same amino acid sequence as the 39-kDa protein except that short N-terminal regions differ in both proteins. Recombinant proteins, which were purified from extracts of Escherichia coli, expressing the proteins from cloned cDNAs, possessed TAF-I activities in the in vitro replication assay. A particular feature of TAF-I proteins is the presence of a long acidic tail in the C-terminal region, which is thought to be an essential part of the SET-CAN fusion protein. Studies with mutant TAF-I proteins devoid of this acidic region indicated that the acidic region is essential for TAF-I activity.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

The 12-Item General Health Questionnaire (GHQ-12): Reliability,external validity and factor structure in the Spanish population

The purpose of this study was to analyze the internal consistency and the external and structure validity of the 12-Item General Health Questionnaire (GHQ-12) in the Spanish general population. A stratified sample of 1001 subjects, ages between 25 and 65 years, taken from the general Spanish population was employed. The GHQ-12 and the Inventory of Situations and Responses of Anxiety-ISRA were administered. A Cronbach’s alpha of .76 (Standardized Alpha: .78) and a 3-factor structure (with oblique rotation and maximum likelihood procedure) were obtained. External validity of Factor I (Successful Coping) with the ISRA is very robust (.82; Factor II, .70; Factor III, .75). The GHQ-12 shows adequate reliability and validity in the Spanish population. Therefore, the GHQ-12 can be used with efficacy to assess people’s overall psychological well-being and to detect non-psychotic psychiatric problems. Additionally, our results confirm that the GHQ-12 can best be thought of as a multidimensional scale that assesses several distinct aspects of distress, rather than just a unitary screening measure.

Probing a single nuclear spin in a silicon single electron transistor

We study single electron transport across a single Bi dopant in a silicon nanotransistor to assess how the strong hyperfine coupling with the Bi nuclear spin I = 9/2 affects the transport characteristics of the device. In the sequential tunneling regime we find that at, temperatures in the range of 100 mK, dI/dV curves reflect the zero field hyperfine splitting as well as its evolution under an applied magnetic field. Our non-equilibrium quantum simulations show that nuclear spins can be partially polarized parallel or antiparallel to the electronic spin just tuning the applied bias.

A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse

The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fms transcripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5' flanking sequence and the down-stream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms-positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location Was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (the Fms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function. (C) 2003 by The American Society of Hematology.

A novel screen for nuclear mitochondrial gene associations with Parkinson's disease

Genetic factors play an important role in the aetiology of Parkinson's disease (PD). We have screened nuclear genes encoding subunits of mitochondrial complex I for associations between single nucleotide polymorphisms (SNPs) and PD. Abnormal functioning of complex I is well documented in human PD. Moreover, toxicological inhibition of complex I can lead to parkinsonism in animals. Thus, commonly occurring variants in these genes could potentially influence complex I function and the risk of developing PD. A sub-set of 70 potential SNPs in 31 nuclear complex I genes were selected and association analysis was performed on 306 PD patients plus 321 unaffected control subjects. Genotyping was performed using the DASH method. There was no evidence that the examined SNPs were significant genetic risk factors for PD, although this initial screen could not exclude the possibility that other disease-influencing variations exist within these genes.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

Regulation of Amh during sex determination in chickens: Sox gene expression in male and female gonads

During mammalian sexual development, the SOX9 transcription factor up-regulates expression of the gene encoding anti-Mullerian hormone (AMH), but in chickens, Sox9 gene expression reportedly occurs after the onset of Amh expression. Here, we examined expression of the related gene Sox8 in chicken embryonic gonads during the sex-determining period. We found that cSox8 is expressed at similar levels in both sexes at embryonic day 6 and 7, and only at the anterior tip of the gonad, suggesting that SOX8 is not responsible for the sex-specific increase in cAmh gene expression at these stages. We also found that several other chicken Sox genes (cSox3, cSox4 and cSox11) are expressed in embryonic gonads, but at similar levels in both sexes. Our data suggest that the molecular mechanisms involved in the regulation of Amh genes of mouse and chicken are not conserved, despite similar patterns of Amh expression in both species.

Growth hormone as a cytokine

1. The growth hormone (GH) receptor was the first of the class 1 cytokine receptors to be cloned. It shares a number of structural characteristics with other family members and common signalling mechanisms based on common usage of the Janus kinase 2 (JAK2). 2. Growth hormone receptor activation is initiated by GH-induced homodimerization of receptor molecules. This has enabled the creation of specific hormone antagonists that block receptor dimerization. 3. The details of the transcription factors used by the activated receptor are being revealed as a result of promoter analyses and electrophoretic mobility gelshift analysis. 4. Growth hormone receptors are widespread and their discovery in certain tissues has led to the assignment of new physiological roles for GH, Some of these involve local or paracrine roles for GH, as befits its cytokine status. 5. Four examples of such novel roles are discussed, These are: (i) the brain GH axis; (ii) GH and the vitamin B-12 axis; (iii) GH in early pre-implantation development; and (iv) GH in development of the tooth. 6. We propose that the view that GH acts through the intermediacy of insulin-like growth factor-1 is simplistic; rather, GH acts to induce an array of growth factors and their receptors and the composition of this array varies with tissue type and, probably, stage of development.