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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Manganous superoxide dismutase (MnSOD) gene expression is stimulated by endotoxin, tumor necrosis factor, and interleukin-1, agents thought to cause cellular damage through intracellular generation of reactive oxygen species. To study the molecular mechanisms underlying the induction of MnSOD mRNA by these stimuli, we cloned a bovine MnSOD cDNA and used it to isolate the promoter region of the bovine MnSOD gene. A 14 kb genomic DNA fragment (lambda BS1) containing the first and second exons and 5' flanking region of the gene was characterized. The transcription start site was determined by primer extension and S1 nuclease protection assays and found to be 88 bp upstream of the translation initiation codon. The sequence of approximately 1 kb of DNA upstream of the start site was determined and examined for potential regulatory elements. DNA immediately upstream of the transcription start site was GC-rich and contained two AP-2 and eight Sp-1 consensus sequences. It did not contain either a CCAAT or TATA box. A 956 bp fragment of this DNA fragment was transcriptionally active when fused to a luciferase reporter gene and transfected into both bovine pulmonary artery endothelial and hamster insulinoma tumor cells. Transfection analysis of three additional deletion mutants, whose 5' end-points were -317, -182, and -70 bp, respectively, showed a step-like reduction in transfection efficiency, suggesting the presence of regulatory elements throughout this DNA fragment that contribute to transcriptional activity of the MnSOD promoter. Despite the high homology of the bovine MnSOD cDNA to other mammalian MnSODs, the promoter sequences of bovine and rat MnSOD genes showed a virtual lack of similarity.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1994 Jan
PMID:Identification and functional characterization of the bovine manganous superoxide dismutase promoter. 829 76

Glucagon-like peptide-1(7-36)amide (GLP-1(7-36) amide) and gastric inhibitory polypeptide (GIP), peptides of the glucagon family, stimulate insulin secretion in vitro and in vivo. They possess high N-terminal sequence homology. Binding studies with 125I-labelled GIP and 125I-labelled GLP-1(7-36)amide were performed in RINm5F insulinoma cells to investigate receptor specificity and to compare both receptors directly. Both binding sites were highly ligand-specific: GIP did not bind to the GLP-1(7-36)amide receptor and vice versa. Both peptides increased intracellular cyclic AMP levels; GLP-1(7-36)amide was 100-fold more potent in stimulating cyclic AMP production when compared with GIP. At ranges of 1-10 nmol GLP-1(7-36)amide/l and 0.1-10 nmol GIP/l, corresponding to submaximal binding concentrations, the hormones showed an additive effect on cyclic AMP production. The N-terminal portion of GIP was important for binding, as GIP(1-30) showed almost full binding and biological activity. GIP(17-42) bound in a concentration-dependent manner with approximately 500-fold lower potency than GIP. At concentrations of up to 10 mumol GIP(17-42)/l no stimulation of cyclic AMP was observed.
J Mol Endocrinol 1993 Jun
PMID:Binding specificity and signal transduction of receptors for glucagon-like peptide-1(7-36)amide and gastric inhibitory polypeptide on RINm5F insulinoma cells. 839 43

In order to study the role of arachidonic acid (AA) in depolarization-induced insulin secretion rat insulinoma cells (RINm5F) were depleted of AA by cultivation in essential fatty acid-free medium. Within 2 weeks AA content of these cells was decreased to a non-detectable level as assessed by gas chromatography (GC). Different cell lines were obtained by supplementation of the defatted medium with oleic acid or the AA precursor linoleic acid (7 and 70 microM, each). The AA content varied in dependence from the precursor availability from 0 to about 14% of long chain fatty acids. Variation in AA content or the depletion of AA to a non-detectable level did not modulate insulin synthesis, basal and potassium-induced insulin release, cell growth (cell number and protein), membrane depolarization and increases in cytosolic Ca2+. In AA containing cells no eicosanoids was produced in the course of stimulated hormone release. The data suggest that in RINm5F cells release of AA and/or formation of oxidized metabolites from AA are not essential for functional integrity.
Mol Cell Endocrinol 1993 Feb
PMID:Insulin secretion without the participation of arachidonic acid. 847 46

We have cloned the 5' region of human aromatic L-amino acid decarboxylase (AADC) gene in a cosmid and an overlapping lambda clone, and sequenced the first five exons. A 61 base pair (bp) non-coding, first exon containing for the 5' end of a human pheochromocytoma AADC cDNA was localized 16 kb upstream of exon 2, in which translation is initiated. The transcription start site was localized by RNAse mapping, primer extension and reverse transcription-PCR. The non-conventional cap site was preceded by a modified TATA box at position -29. A strong promoter was characterized in the 560 bp region upstream of the cap site by linkage to the reporter gene LacZ, and transfection in human neuroblastoma SK-N-BE and SK-N-BE-K2 cells. Using a series of constructs bearing a varying length of 5' flanking region, three positive regulatory elements have been localized in the -560 to -394, -244 to -200 and -147 to -1 regions. Negative regulatory elements were localized in the -9000 to -560 and -394 to -316 regions. Surprisingly, constructs comprising all or the major part of intron 1 were inactive, suggesting the presence of a silencer in the first intron, or incorrect splicing events. The construct containing 560 bp of 5' flanking sequence did not express in human cholinergic neuroepithelioma cells MC-I-XC, and in three non-neuronal cell lines which displayed high AADC activities: human pancreatic carcinoma cells AsPC-1, rat insulinoma cells RINm5F and mouse anterior pituitary cells AtT20. These data suggest that we have identified a neuron-specific AADC promoter. An extensive search for a second promoter responsible for AADC gene expression in non-neuronal cells only gave negative results.
Brain Res Mol Brain Res 1993 Mar
PMID:Identification of a neuron-specific promoter of human aromatic L-amino acid decarboxylase gene. 851 Apr 97

The post-translational processing and intracellular sorting of the proinsulin-converting enzyme carboxypeptidase H (CPH) was studied in isolated rat islets of Langerhans. Pulse-chase-radiolabelling experiments using sequence-specific antisera showed that CPH was synthesized initially as a 57-kDa glycoprotein which was processed to a 54-kDa mature form by proteolytic processing at the N-terminus. Processing of the CPH precursor occurred rapidly (t(1/2) = 30) after an initial delay of 15-30 min and the enzyme was secreted in parallel with the insulin-related peptides in response to glucose-stimulation within 1 h after radiolabelling. This indicated that the proteins were packaged into nascent secretory granules at approximately the same rate following synthesis. Conversion of proinsulin and the 57-kDa form was inhibited markedly by chase incubation of islets at 20 degrees C, indicating that maturation of both proteins occurs in a post-Golgi compartment. Affinity purification of the enzyme from insulinoma subcellular fractions showed that the 57-kDa form was associated with endoplasmic reticulum or Golgi elements, and the 54-kDa form was present in secretory granules. Structural analysis showed that the granule form of the enzyme had an N-terminal amino acid sequence beginning at residue 42 of rat CPH, thereby implicating cleavage of the precursor after the fourth Arg in a site containing five consecutive Arg residues. These findings indicate that post-translational processing of CPH is mediated by an endoprotease which cleaves at sites containing multiple basic amino acid residues upon segregation of the enzyme to the secretory granules.
Mol Cell Endocrinol 1995 Aug 30
PMID:The post-translational processing and intracellular sorting of carboxypeptidase H in the islets of Langerhans. 867 18

In MIN6 insulinoma cells, transforming growth factor-beta (TGF-beta) induced the oscillatory elevation of the cytoplasmic free calcium concentration, [Ca2+]c, in the presence of 5.5 mM glucose. The increase in [Ca2+]c induced by TGF-beta was totally dependent on calcium entry and attenuated by nifedipine or nickel chloride. In contrast, carbachol elevated [Ca2+]c in the presence of nickel chloride. When the plasma membrane was hyperpolarized by diazoxide, TGF-beta did not raise [Ca2+]c, whereas both carbachol and depolarizing concentration of potassium elevated [Ca2+]c under the same conditions. TGF-beta did not affect either the cellular cyclic AMP or inositol trisphosphate levels. In the presence of 5.5 mM glucose, TGF-beta induced a 3-fold increase in insulin secretion and the effect of TGF-beta was blocked by either nifedipine or nickel chloride. TGF-beta did not stimulate insulin secretion in the presence of 100 microM diazoxide, whereas both carbachol and 40 mM potassium chloride significantly increased insulin secretion. These results suggest that TGF-beta induces the oscillatory elevation of [Ca2+]c in MIN6 cells by stimulating calcium entry via voltage-dependent calcium channels. Calcium is an intracellular messenger of the action of TGF-beta on insulin secretion.
Mol Cell Endocrinol 1996 Mar 01
PMID:Calcium as a second messenger of the action of transforming growth factor-beta on insulin secretion. 873 68

Galanin is a ubiquitous neuropeptide that regulates a wide array of physiological processes via interaction with specific G protein-coupled receptors. A rat galanin receptor cDNA was cloned from the Rin14B insulinoma cell line. The isolated cDNA encodes a 346 amino acid G protein-coupled receptor that is 92% identical to the recently reported human GALR1 galanin receptor. [125I]Galanin binds with high affinity to two receptor states in COS1 cell membranes containing the rat GALR1 receptor, consistent with coupling of the receptor to a G protein in these membranes. N-terminal galanin fragments and the putative galanin receptor antagonists galantide, C7, M35 and M40 bind with high affinity to the rat GALR1 receptor. In contrast, C-terminal galanin fragments do not bind to this receptor. Galanin inhibits basal and forskolin-stimulated cAMP formation in CHO cells expressing the rat GALR1 receptor via a pertussis toxin-sensitive G protein. The GALR1 receptor is expressed in rat spinal cord, small intestine, Rin14B insulinoma cells and several brain regions, particularly ventral hippocampus, amygdala, supraoptic nucleus, hypothalamus, thalamus, lateral parabrachial nucleus and locus coeruleus. Cloning of the rat GALR1 galanin receptor cDNA will permit many new experimental strategies to be applied to studies of the structure and function of galanin receptors.
Brain Res Mol Brain Res 1995 Dec 28
PMID:Cloning and characterization of the rat GALR1 galanin receptor from Rin14B insulinoma cells. 875 Aug 21

GH and PRL stimulate both proliferation and insulin production in pancreatic beta-cells as well as in the rat insulinoma cell line RIN-5AH, We report here that human GH increases insulin mRNA levels in RIN-5AH cells via both somatogenic and lactogenic receptors. GH stimulated the rat insulin 1 promoter activity 2-fold, and this stimulation was abolished by introduction of a block mutation in a gamma-interferon-activated sequence (GAS)-like element (GLE) with the sequence 5'-TTCTGGGAA-3' located in the rat insulin 1 enhancer at position -330 to -322. This element, termed Ins-GLE, was able to confer GH responsiveness to a heterologous promoter. GH induced the binding of two protein complexes to the Ins-GLE. An antibody directed against the transcription factor STAT5 (signal transducer and activator of transcription) supershifted the GH-induced complexes. Furthermore, in COS7 cells transiently transfected with STAT5 and GH receptor cDNAs, it was found that expression of STAT5 was necessary for GH induction of these two DNA-binding complexes. These results suggest that GH stimulates insulin 1 promoter activity by inducing the binding of STAT5 to Ins-GLE.
Mol Endocrinol 1996 Jun
PMID:Identification of a growth hormone-responsive STAT5-binding element in the rat insulin 1 gene. 877 25

The upstream glucokinase (GK) promoter is expressed specifically in several different neural/neuroendocrine (NE) cell types, including the pancreatic beta-cell and pituitary corticotrope. Previously, a mutational and evolutionary analysis of this promoter identified two identical 9-bp motifs (TGGTCACCA) termed Pal-1 and Pal-2 that are essential for high level expression in HIT M2.2.2 cells, an insulinoma cell line. Here we show that these motifs are also necessary for efficient expression in AtT-20 cells, a corticotrope-derived cell line, and that proteins from both NE and non-NE cells bind to the Pal motifs, although the DNA-protein complexes differ by cell type. Complexes formed using nuclear extracts from NE cells contained an extra NE cell-specific band and differed in the relative abundance of two other bands when compared with non-NE cells, UV laser cross-linking experiments further supported the cell-specific binding of two proteins, 110 and 150 kDa in size, to these motifs. The presence or absence of the NE-specific band correlates with transcription of GK promoter fusion gene constructs, suggesting a key role for this protein in determining the cell-specific expression of GK. The Pal motifs themselves do not function as enhancers but seem to be essential components of a larger transcriptional regulatory domain that is active only in certain NE cells. Together, these studies suggest that the NE cell-specific expression of the upstream GK promoter involves the formation of a distinct protein complex on the two Pal motifs.
Mol Endocrinol 1996 Jun
PMID:Characterization of the Pal motifs in the upstream glucokinase promoter: binding of a cell type-specific protein complex correlates with transcriptional activation. 877 32

Activin A stimulates insulin secretion in pancreatic beta-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of 125I-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more 125I-labeled activin A-binding sites than parental cells and the apparent Kd was 1.15 nM, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with 125I-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5.5 mM for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but this decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in beta-cells by both calcium-dependent and -independent mechanisms.
J Mol Endocrinol 1996 Jun
PMID:Two distinct signaling pathways activated by activin A in glucose-responsive pancreatic beta-cell lines. 878 83


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