Sepsis is associated with an upregulation of functional beta3 adrenoceptors in the myocardium.

OBJECTIVE: To analyze the implication of the beta3-adrenoceptor (beta3-AR) pathway in human septic myocardium and a murine model of sepsis, a condition associated with myocardial depression. METHODS AND RESULTS: beta3-AR and eNOS protein abundance were increased (332+/-66.4% and 218+/-39.3; P<0.05) in hearts from septic patients. The effect of BRL37344, a beta3-AR-preferential agonist, was analyzed by videomicroscopy on the contractility of neonatal mouse ventricular myocytes (NMVM) incubated with conditioned medium from LPS-stimulated cultured macrophages (Mc-LPS+ medium). Stimulation of untreated NMVM with BRL37344 dose-dependently decreased the amplitude of contractile shortening (P<0.05). This response was abolished by L-NAME (NOS inhibitor). Incubation in Mc-LPS+ medium potentiated the depressing effect of BRL37344 (P<0.05) as well as of SR58611A (P<0.05) in wild-type myocytes. Importantly, the contractile depression was abrogated in cardiomyocytes from beta3-AR KO mice. CONCLUSIONS: beta3-AR are upregulated during sepsis in the human myocardium and by cytokines in murine cardiomyocytes, where they mediate an increased negative inotropic response to beta3 agonists. Activation of the beta3-AR pathway by catecholamines may contribute to the myocardial dysfunction in sepsis.

Human HMG box transcription factor HBP1: a role in hCD2 LCR function.

The locus control region (LCR) of the human CD2 gene (hCD2) confers T cell-specific, copy-dependent and position-independent gene expression in transgenic mice. This LCR consists of a strong T cell-specific enhancer and an element without enhancer activity (designated HSS3), which is required for prevention of position effect variegation (PEV) in transgenic mice. Here, we identified the HMG box containing protein-1 (HBP1) as a factor binding to HSS3 of the hCD2 LCR. Within the LCR, HBP1 binds to a novel TTCATTCATTCA sequence that is higher in affinity than other recently reported HBP1-binding sites. Mice transgenic for a hCD2 LCR construct carrying a deletion of the HBP1-binding sequences show a propensity for PEV if the transgene integrates in a heterochromatic region of the chromosome such as the centromere or telomere. We propose that HBP1 plays an important role in chromatin opening and remodelling activities by binding to and bending the DNA, thus allowing DNA-protein and/or protein-protein interactions, which increase the probability of establishing an active locus.

Pit-1 mediates cell-specific and cAMP-induced transcription of the tilapia GH gene.

Expression of the tilapia growth hormone (tiGH) gene is pituitary-specific and controlled by intracellular cAMP levels. DNaseI protection experiments allowed us to identify four Pit-1 binding sites in the tiGH - 465/ + 19 region. Deletion and mutagenesis analysis revealed that the - 131/+ 19 region, containing two Pit-1 sites, or four copies of the most proximal site tiGHF1 fused to the heterologous Tk promoter, confer high level expression in rat pituitary cells and direct transcription in non-pituitary cells only after expression of rat Pit-1. We show that a tilapia pituitary factor specifically binds to site tiGHF1 and obtained a partial cDNA sequence coding for tilapia Pit-1. The cAMP stimulation is mediated by the proximal (- 131/- 31) promoter region. It is Pit-1-dependent and requires the tiGHF1 site. In addition, four copies of this site confer cAMP inducibility to the Tk promoter in GC cells.

Structure and functional analysis of a tilapia (Oreochromis mossambicus) growth hormone gene: activation and repression by pituitary transcription factor Pit-1.

A gene encoding the Tilapia mossambica (Oreochromis mossambicus) growth hormone (tiGH) was isolated and sequenced. The gene spans 5.6 kb, including 3.7 kb of 5' and 0.2 kb of 3' flanking sequences and a 1.7-kb transcription unit comprised of six exons and five introns. The gene and the 5' flanking region contain several potential binding sites for Pit-1, a key transcription activator of mammalian GH genes. One of these (-57/-42) is highly conserved in fish GH genes. It activates transcription in pituitary cells and binds Pit-1. Transfection of luciferase reporter plasmids containing either the -3602/+19 tiGH sequence or one of its 5' deletion mutants (-2863/, -1292/, and -463/+19) resulted in strong activity in Pit-1-producing rat pituitary GC cells. A dose-dependent activation of the tiGH promoter was achieved in nonpituitary fish EPC and monkey COS cells cotransfected with a rat Pit-1 expression vector, demonstrating the crucial role played by Pit-1 as an activator of the tiGH gene. Fusion of the tiGH promoter with the beta-galactosidase gene led to transient expression specifically in the nervous system of microinjected zebrafish embryos. The activity of the tiGH promoter in GC and EPC cells was strongly repressed by extending its 3' end from +19 to +40, a sequence in which a Pit-1-binding site was identified using gel retardation assays. Point mutations of the site that suppressed Pit-1 binding in vitro restored full tiGH promoter activity. Thus, a Pit-1-binding site located in the 5' untranslated region mediates Pit-1-dependent repression of the tiGH gene.

Replication, expression, and fate of foreign DNA during embryonic and larval development of the African catfish (Clarias gariepinus).

The transfer of exogenous DNA in fish represents a powerful strategy to study the regulation of gene expression in vivo. The African catfish (Clarias gariepinus) was chosen for this study because of its scientific and economic importance due to its easy husbandry, its short developmental period, and its value as a protein source in Africa and Asia. Fertilized eggs (1- and 2-cell stage) were cytoplasmatically injected with either supercoiled or linearized plasmids harboring the fusion genes encoding beta-galactosidase (lacZ) or luciferase (Luc) without a promoter or fused to the promoter/enhancer of human cytomegalovirus (CMV). Replication of the exogenous DNA peaked at 4 hours (early gastrula) and again at 2 days (which corresponds to the developmental stage of yolksac resorption). Foreign DNA persisted during embryogenesis, and it was still detectable 8 months after injection. In vivo transient expression of both CMV fusion genes was mosaic and peaked within 24 hours after DNA injection. Transient expression of the luciferase reporter gene could be detected with a much higher sensitivity than the lacZ gene. These data establish African catfish as a suitable in vivo assay system, and they confirm the luciferase reporter gene as a high quality reporter gene in fish.

Structure of the tilapia (Oreochromis mossambicus) prolactin I gene.

The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its transcript (3,677 bases long) begins with a guanine and is organized in five exons and four introns like the other known prolactin genes. Analysis of the 1,555-bp 5'-flanking region suggests that pituitary-specific expression of the gene could be regulated through a trans-factor related to the mammalian pituitary-specific factor Pit-1. Two potential binding sites for such a factor were found in the first intron, suggesting a possible regulatory role for this region. Moreover, two potential Z-DNA regions are located at positions -837 to -812 and -246 to -179 from the transcription start site. These two regions could play an important role in the regulation of PRL gene expression.