Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line.

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion. It is currently under therapeutic evaluation because it enhances insulin secretion in type 2 diabetes. Previous studies using the GLP-1 secreting cell line GLUTag have shown that the cells are electrically active, and that the action potential frequency is regulated by nutrients. In this study we characterize voltage gated currents underlying this electrical activity and correlate the electrophysiological findings with gene expression determined by microarrays. Whole cell voltage clamp experiments designed to separate different ionic components revealed rapidly inactivating sodium currents sensitive to tetrodotoxin, calcium currents sensitive to nifedipine and omega-conotoxin GVIA, and sustained as well as rapidly inactivating potassium currents, which were sensitive to TEA and 4-AP, respectively. In perforated patch experiments we also observed hyperpolarization-activated currents which were inhibited by ZD7288. The amplitude of the sodium current was approximately 10 times that of the other depolarizing currents and tetrodotoxin abolished action potential firing. In secretion experiments, however, nifedipine, but not tetrodotoxin, omega-conotoxin GVIA or ZD7288, inhibited glucose-induced GLP-1 release. Consistent with this finding, the intracellular Ca2+ response to glucose was impaired by nifedipine but not by tetrodotoxin. Thus, in GLUTag cells, GLP-1 release is not dependent on the firing of Na+-carrying action potentials but requires membrane depolarization and Ca2+ entry through L-type Ca2+ channels. Understanding the characteristics of the currents and the molecular identification of the underlying channels in GLP-1 secreting cells might facilitate the development of agents to enhance GLP-1 secretion in vivo.

Molecular cloning and expression analysis of a mouse UDP-GlcNAc:Gal(beta1-4)Glc(NAc)-R beta1,3-N-acetylglucosaminyltransferase homologous to Drosophila melanogaster Brainiac and the beta1,3-galactosyltransferase family.

We have isolated a murine cDNA coding for a beta1,3-N-acetylglucosaminyltransferase enzyme ( beta3GnT). This enzyme is similar in sequence to Drosophila melanogaster Brainiac and to the murine and human beta1,3-galactosyltransferase family of proteins. The mouse beta 3GnT protein is 397 amino acids in length and contains 7 cysteine residues that are conserved in the human orthologue. beta 3GnT is a type II membrane protein localized to the Golgi apparatus. Enzyme assays with recombinant mouse beta 3GnT reveal that it has a preference for acceptors with Gal(beta1-4)Glc(NAc) at the non-reducing termini. Proton NMR analysis of product showed incorporation of GlcNAc in beta1,3 linkage to the terminal Gal of Gal(beta1-4)Glc(beta1-O-benzyl). Northern blot analysis revealed the presence of a single 3.0[emsp4 ]kb transcript in all adult mouse and human organs tested, with highest levels in the kidney, liver, heart and placenta. The beta 3GnT gene is also expressed in a number of tumor cell lines. The human orthologue of beta 3GnT is located on chromosome 2pl5.

Modulation of the retinoic acid and retinoid X receptor signaling pathways in P19 embryonal carcinoma cells by calreticulin.

Calreticulin is a widely expressed calcium binding protein that can bind to an amino acid sequence motif, KXGFFKR, which is present in the cytoplasmic domain of all integrin alpha-subunits. Closely related sequences, KXFFKR and KXFFRR, are encoded in the DNA-binding domain of all members of the steroid/thyroid/retinoid receptor superfamily and it has recently been demonstrated that calreticulin inhibits their activity both in vitro and in vivo. Here we present novel evidence that calreticulin can interfere directly with the retinoic acid (RARs) and retinoid X (RXRs) receptor pathways. Calreticulin exhibits the ability to inhibit DNA-binding activity of both heterodimeric RAR/RXR and homodimeric RXR complexes in vitro. Inhibition of RXR binding to DNA is achieved with a concentration of calreticulin that is approximately fourfold lower than that required for inhibition of RAR/RXR binding to a cognate binding site. Coprecipitation experiments suggest a direct protein:protein interaction between calreticulin and retinoid receptors. Stable overexpression of calreticulin in P19 embryonal carcinoma cells significantly decreases the rapid activation of the endogenous RA-responsive RARbeta gene, abrogates the ability of endogenous RAR/RXR complexes to bind to DNA, and inhibits the emergence of the RA-induced differentiated phenotype. These data demonstrate that calreticulin can interfere with the two distinct retinoid signaling pathways through a mechanism likely involving direct protein:protein interactions and that disruption of the retinoid signal alters biological processes in vivo.

Determinants of target gene specificity for ROR alpha 1: monomeric DNA binding by an orphan nuclear receptor.

The ROR alpha isoforms are orphan members of the steroid/thyroid/retinoid receptor superfamily. Previous DNA-binding studies indicated that ROR alpha isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence and that the distinct amino-terminal domains of each isoform influence DNA-binding specificity. In this report, we have investigated in detail the protein determinants of target gene specificity for the ROR alpha 1 isoform and have now identified the minimal sequence both in its amino- and carboxy-terminal domains required for high-affinity DNA binding. High-resolution methylation and ethylation interference analyses and mixing of truncated proteins in a DNA-binding assay show that ROR alpha 1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR alpha 1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR alpha 1 and NGFI-B demonstrated that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR alpha 1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors.

The nonconserved hinge region and distinct amino-terminal domains of the ROR alpha orphan nuclear receptor isoforms are required for proper DNA bending and ROR alpha-DNA interactions.

ROR alpha 1 and ROR alpha 2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3' nuclear receptor core half-site AGGTCA preceded by a 5' AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR alpha 1 and ROR alpha 2 to the RORE induces a large DNA bend of approximately 130 degrees which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3' AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR alpha is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR alpha protein does not alter the DNA bend angle but shifts the DNA bend center 5' relative to the bend induced by intact ROR alpha. Methylation interference studies using the NTD-deleted ROR alpha 1 mutant indicate that some DNA contacts in the 5' AT-rich half of the RORE are also shifted 5', while those in the 3' AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR alpha NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR alpha DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.

Identification of RVR, a novel orphan nuclear receptor that acts as a negative transcriptional regulator.

A novel member of the steroid/thyroid/retinoid superfamily of nuclear receptors has been isolated as part of a screen to identify genes related to the recently characterized orphan receptor ROR alpha. This new orphan receptor, cloned from a mouse brain cDNA library, is closely related to the rat Rev-ErbA alpha gene product (97% and 68% identity in the DNA- and ligand-binding domains, respectively) and referred to as RVR. Northern blot analysis reveals that two RVR mRNA species are expressed during mouse embryogenesis and widely expressed in adult tissues. Studies with in vitro translated RVR protein show that it binds the DNA sequence ATAACTAGGTCA, a hormone response element composed of a 6-base pair AT-rich sequence preceding a single nuclear receptor recognition half-site core motif PuGGTCA. We show that RVR recognizes this hormone response element with a specificity similar to that of the orphan receptor ROR alpha 2. However, cotransfection studies indicate that RVR does not activate transcription when this hormone response element is linked to a reporter gene but rather acts as a potent competitive repressor of ROR alpha function. These results indicate the existence of an orphan nuclear receptor-based signaling pathway with the intrinsic ability to regulate the expression of specific gene networks through competition between transcriptional activators and repressors for the same recognition site.

Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors.

Three isoforms of a novel member of the steroid hormone nuclear receptor superfamily related to the retinoic acid receptors have been identified. The three isoforms, referred to as ROR alpha 1, ROR alpha 2, and ROR alpha 3, share common DNA- and putative ligand-binding domains but are characterized by distinct amino-terminal domains generated by alternative RNA processing. An exon encoding a functionally important subregion of the amino-terminal domain of the ROR alpha 2 isoform resides on the opposite strand of a cytochrome c-processed pseudogene. Binding site selection using in vitro-synthesized proteins reveals that the ROR alpha 1 and ROR alpha 2 isoforms bind DNA as monomers to hormone response elements composed of a 6-bp AT-rich sequence preceding a half-site core motif PuGGTCA (RORE). However, ROR alpha 1 and ROR alpha 2 display different binding specificities: ROR alpha 1 binds to and constitutively activates transcription from a large subset of ROREs, whereas ROR alpha 2 recognizes ROREs with strict specificity and displays weaker transcriptional activity. The differential DNA-binding activity of each isoform maps to their respective amino-terminal domains. Whereas truncation of the amino-terminal domain diminishes the ability of ROR alpha 1 to bind DNA, a similar deletion relaxes ROR alpha 2-binding specificity to that displayed by ROR alpha 1. Remarkably, transfer of the entire amino-terminal region of ROR alpha 1 or amino-terminal deletion of ROR alpha 2 confers RORE-binding specificities to heterologous receptors. These results demonstrate that the amino-terminal domain and the zinc finger region work in concert to confer high affinity and specific DNA-binding properties to the ROR isoforms and suggest a novel strategy to control DNA-binding activity of nuclear receptors.

Efecto tóxico del óxido de etileno en individuos expuestos / Toxic effect of the ethylene oxide in exposed individuals

El óxido de etileno es un potente inductor de daño al material genético ensayado en sistemas tales como vegetales, Drosophila, rata y líneas celulares humanas. Al ser usado como esterilizante en instrumental de cirugía, se realiza el estudio del personal expuesto al mismo, detectándose un aumento significativo en la frecuencia de aberraciones cromosómicas hepático en los mencionados individuos (AU)

Efecto tóxico del óxido de etileno en individuos expuestos / Toxic effect of the ethylene oxide in exposed individuals

El óxido de etileno es un potente inductor de daño al material genético ensayado en sistemas tales como vegetales, Drosophila, rata y líneas celulares humanas. Al ser usado como esterilizante en instrumental de cirugía, se realiza el estudio del personal expuesto al mismo, detectándose un aumento significativo en la frecuencia de aberraciones cromosómicas hepático en los mencionados individuos