Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Insulin-like growth factors, IGF-I and IGF-II, are potent regulators of oligodendrocyte development. Most of the IGF present in vivo is bound to members of a family of six high-affinity IGF-binding proteins (IGFBPs), which can either potentiate or inhibit IGF action, depending on other conditions. Additionally, serum contains a structurally unrelated protein, acid-labile sub-unit (ALS), which forms a ternary complex with IGF and IGFBP3. In this study, we used reverse-transcriptase polymerase chain reaction (RT-PCR) to examine the expression of mRNAs for IGFBP 1-6 and ALS in purified populations of oligodendroglial cells and astrocytes. We found that astrocytes express all six IGFBPs. A2B5+/O4- oligodendrocyte precursors, O4+/O1- intermediate precursors, and O1+ oligodendrocytes express IGFBP3, 5, and 6, while IGFBP4 is expressed in oligodendrocyte precursors but not at more mature stages. We were unable to detect ALS mRNA in whole brain or in cultured oligodendroglial cells. The presence of differentially expressed IGFBPs in developing oligodendrocytes and astrocytes could significantly affect the biological activity of IGF-I and IGF-II in the central nervous system and the IGF-responsiveness of the IGFBP-expressing cells.
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PMID:Expression of insulin-like growth factor-binding protein messenger RNAs in developing rat oligodendrocytes and astrocytes. 941 60

Insulin-like growth factor-I (IGF-I) is implicated in the development, survival and maintenance of function of sympathetic and sensory neurons. These neurons are affected at an early stage during the course of diabetes. Reverse transcriptase polymerase chain reaction (RT-PCR) based assay revealed that rat superior cervical ganglia (SCG) express mRNA transcripts for IGF-I and its receptor. Moreover, specific membrane protein binding sites for IGF-I within the SCG have also been demonstrated using competition-inhibition and affinity cross-linking techniques. An induction of diabetes with streptozotocin (STZ, 55 mg/kg, i.v.) produced a marked decrease in the SCG levels of mRNA transcripts for IGF-I and its receptor. Concentrations of circulating IGF-I and its receptor protein within the SCG were also reduced in this disease state. Insulin treatment partially prevented diabetes-related alterations in circulating IGF-I and the SCG-IGF-I system. Overall, the data described in this study may be of value in understanding the pathogenetic mechanism(s) responsible for the development of diabetic sympathetic neuropathy.
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PMID:Diabetes-induced suppression of IGF-1 and its receptor mRNA levels in rat superior cervical ganglia. 948 70

The present study assessed the feasibility of transferring the insulin gene into liver cells of diabetic individuals using a defective adenoassociated viral (AAV) vehicle. AAV offers several advantages over other viral vectors, since this vehicle can facilitate transfection in vivo without cell division and without any viral coding sequences (thus minimizing inflammation). The rat insulin gene and lacZ were each packed into a defective AAV vehicle (AAV-INS and AAV-lacZ, respectively). Successful AAV-mediated transfection and expression of lacZ into hepatocytes in primary cell culture were demonstrated by chemiluminescent assay of beta-galactosidase. Similarly, AAV-mediated transfection and expression of the insulin gene into hepatocytes was demonstrated by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). After AAV-mediated transfection of the insulin gene into hepatocytes, glucose in the medium was significantly reduced for up to 5 days. After direct injection of AAV-INS into liver parenchyma of diabetic mice, successful transfection was demonstrated by RT-PCR, and blood glucose was significantly decreased for at least 6 days. These studies suggest that the AAV vector may be used to transfer the insulin gene into liver cells in vitro and in vivo.
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PMID:Defective adenoassociated viral-mediated transfection of insulin gene by direct injection into liver parenchyma decreases blood glucose of diabetic mice. 949 94

Resident macrophages have been suggested to participate in the initiation of beta cell damage during the development of autoimmune diabetes. The purpose of this study was to determine if the endogenous production and release of interleukin 1 (IL-1) in human islets of Langerhans by resident macrophages results in the inhibition of beta cell function. Treatment of human islets with a combination of tumor necrosis factor (TNF) + lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) stimulates inducible nitric oxide synthase (iNOS) expression, nitric oxide production, and inhibits glucose-stimulated insulin secretion. The IL-1 receptor antagonist protein (IRAP) prevents TNF + LPS + IFN-gamma-induced iNOS expression and nitrite production, and attenuates the inhibitory effects on glucose-stimulated insulin secretion by human islets. Inhibition of iNOS activity by aminoguanidine also attenuates TNF + LPS + IFN-gamma-induced inhibition of insulin secretion by human islets. These results indicate that the inhibitory effects of TNF + LPS + IFN-gamma are mediated by nitric oxide, produced by the actions of IL-1 released endogenously within human islets. Reverse transcriptase polymerase chain reaction was used to confirm that TNF + LPS + IFN-gamma stimulates the expression of both IL-1alpha and IL-1beta in human islets. Two forms of evidence indicate that resident macrophages are the human islet cellular source of IL-1: culture conditions that deplete islet lymphoid cells prevent TNF + LPS + IFN-gamma-induced iNOS expression, nitric oxide production, and IL-1 mRNA expression by human islets; and IL-1 and the macrophage surface marker CD69 colocalize in human islets treated with TNF + LPS + IFN-gamma as determined by immunohistochemical analysis. Lastly, nitric oxide production is not required for TNF + LPS + IFN-gamma-induced IL-1 release in human islets. However, cellular damage stimulates IL-1 release by islet macrophages. These findings support the hypothesis that activated islet macrophages may mediate beta cell damage during the development of insulin-dependent diabetes by releasing IL-1 in human islets followed by cytokine-induced iNOS expression by beta cells.
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PMID:IL-1 produced and released endogenously within human islets inhibits beta cell function. 969 Oct 88

Reverse transcriptase- polymerase chain reaction (RT-PCR) enhances the probability of detecting rare transcripts in complex mixtures of mRNA. Using thyroid autoantigens and the controversy about the role of the TSH-receptor (TSH-R) in thyroid-associated ophthalmopathy as an example, this study demonstrates the problems of interpreting RT-PCR results in typically non-expressing tissues resulting from the extremely high sensitivity of the method. Unexpected transcripts for thyroperoxidase, thyroglobulin, TSH-R (exon 1-4, 354 bp), FSH-receptor, or insulin fragments were demonstrated in a number of thyroid or orbit-derived as well as unrelated tissues or cell types. Unexpected transcripts were most prevalent in fibroblasts, irrespective of the tissue of origin and most likely caused by ectopic transcription. To establish a physiological significance of rare transcripts such as the TSH-R in orbital tissues, demonstration of the protein in addition to the positive RT-PCR results is needed.
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PMID:Transcription of thyroid autoantigens in non-expressing tissues. 979 65

Recent reports using immunohistochemistry have shown that Galphaolf which shares 88% homology with Galphas was expressed in pancreatic islets. To test the specificity of the expression of this G protein isotype in rat islet cells, B and non-B cells were separated by flow cytometry. The expression of Galphaolf and adenylyl cyclases (AC) of types II, III, V, and VI was evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). Since alterations in the expression of AC III were recently reported in the GK rat (a model of non-insulin-dependent diabetes mellitus, NIDDM), we also have analyzed the mRNA expression of Galphaolf and AC isoforms in pancreatic islets from GK rats and from adult rats neonatally treated by streptozotocin (nSTZ rats), another model of NIDDM. Southern blots of amplicons generated with specific primers of Galphaolf revealed the presence of a 540-bp band only in B cells. AC of types II, III, V, and VI were expressed both in B and non-B cells. However, AC III mRNA was clearly more abundant in non-B than in B cells. Moreover, in B cells the expression of AC VI was higher than that of AC V, whereas equal expressions of AC V and AC VI were found in non-B cells. In GK rat islets, the mRNA expressions of Galphaolf, AC II, and AC III were clearly increased and no change in AC V and AC VI was found. In nSTZ rat islets, Galphaolf expression was barely detectable, but AC II and AC III mRNA levels were higher than those observed in controls. In conclusion, Galphaolf mRNA appeared specifically expressed in islet B cells and was increased in GK islets. The steady-state mRNA levels of AC II and AC III were clearly increased in the islets of the two rat models of NIDDM. Thus, alterations in the expression of G protein isotypes and AC isoforms could contribute to the diabetic phenotype.
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PMID:Galphaolf identification by RT-PCR in purified normal pancreatic B cells and in islets from rat models of non-insulin-dependent diabetes. 992 Jul 69

The function of soluble N-ethylmaleimide-sensitive attachment protein-alpha (alpha-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of alpha-SNAP in the secretion of insulin and gamma-aminobutryric acid (GABA) from pancreatic beta cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed alpha-SNAP, but not beta-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that alpha-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. alpha-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. An in vitro binding study showed that both wild-type alpha-SNAP and C-terminal-deleted alpha-SNAP mutant (1-285) can bind to syntaxin 1A. alpha-SNAP mutant (1-285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of alpha-SNAP mutant (1-285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous alpha-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if alpha-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic beta cells, the functional role of alpha-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of alpha-SNAP mutant (1-285) overexpression on GABA release. We conclude that 1) alpha-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic beta cells; and 2) the interaction of alpha-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.
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PMID:alpha-soluble N-ethylmaleimide-sensitive factor attachment protein is expressed in pancreatic beta cells and functions in insulin but not gamma-aminobutyric acid secretion. 1007 5

The presence and functional significance of the extracellular calcium-sensing receptor (CaR) on human pancreatic beta-cells were investigated. Reverse transcriptase-polymerase chain reaction with primers for the extracellular domain of the CaR expressed in human parathyroid-secreting cells identified a product of the expected size in human pancreatic mRNA. Immunocytochemistry using an antibody against the extracellular region of CaR showed extensive immunoreactivity in insulin- and glucagon-containing cells but not in somatostatin-containing cells. In perifusion experiments, elevations in extracellular Ca2+ produced initial transient increases in insulin secretion, followed by a concentration-dependent and prolonged, but reversible, inhibition of secretion. Microfluorometric measurements of intracellular Ca2+ ([Ca2+]i) in isolated human beta-cells demonstrated that elevations in extracellular Ca2+ (0.5-10 mmol/l) caused rapid elevations in [Ca2+]i. Increases in extracellular Ca2+ caused small increases in the cyclic AMP content of whole human islets. These studies demonstrated that human beta-cells express an extracellular CaR and that activation of the receptor inhibits basal and nutrient-stimulated insulin secretion. The transduction mechanism that mediates this inhibitory effect is unknown, but our results suggest that it is unlikely to be through the adenylate cyclase-cyclic AMP pathway or through the phospholipase C-IP3 pathway. This CaR-mediated inhibitory mechanism may be an important autoregulatory mechanism in the control of insulin secretion.
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PMID:The extracellular calcium-sensing receptor on human beta-cells negatively modulates insulin secretion. 1086 62

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (beta-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTPgammaS alone, the C-terminal octapeptide cholecystokinin (CCK-8), or gastrin. All compounds caused a significant 2- to 4.4-fold stimulation of PLC activity in the different cell lines, which was diminished by the non-hydrolyzable GDP analogue GDPbetaS. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCK(A) receptor antagonist L-364,718 (K(d) = 0.24 nM) and tritiated CCK(B) receptor antagonist L-365,260 (K(d) = 0.13 nM) were only present in Rin 1027-B2 cells. High-affinity binding sites for both ligands were not found in beta-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCK(A) receptor-selective) and CR 2945 (CCK(B) receptor-selective), as well as microphysiometry experiments, resulted in the same receptor distribution. Reverse transcriptase-polymerase chain reaction confirmed the CCK receptor distribution pattern for Rin 1027-B2 cells, but in addition showed the existence of CCK(B) receptors in beta-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein alpha-subunits demonstrated inhibition of CCK-stimulated PLC activity in beta-TC3 cells by G(q/11)alpha antiserum (70%), in Rin 1027-B2 cells by G(q/11)alpha antiserum (70%) and G(i)-3alpha antiserum (23%), and in INR1-G9 cells by G(q/11)alpha antiserum (60%) and G(o)alpha antiserum (45%). We conclude that CCK receptor subtypes with different G-protein-coupling specificities to PLC are present in the different hormone-secreting cells of the endocrine pancreas.
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PMID:Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines. 1093 May 42

Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.
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PMID:Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet beta -cells. 1093 83


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