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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously showed that the processing of proparathyroid hormone (proPTH) to PTH was accomplished most efficiently by furin (17). Colocalization studies demonstrated that furin is expressed in the parathyroid, whereas proprotein convertase (PC)1 and PC2 are not. Since that time, another member of the PC family, called PC7, has been identified. Here we show, using coinfection studies, that PC7, as well as furin, can appropriately cleave PTH from proPTH. ProPTH and PTH were purified from cell extracts by reversed-phase HPLC and were identified by Western blot analysis and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization studies, using Northern blot and reverse transcriptase-PCR analyses, showed that PC7 messenger RNA (mRNA) is expressed in the parathyroid gland. Therefore, PC7, like furin, has the potential to be involved in the physiological processing of proPTH to PTH. The two major regulators of parathyroid cell synthetic and secretory activity are the extracellular fluid calcium and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. We investigated whether either of these agents might modulate processing of proPTH to PTH by altering parathyroid convertase gene expression. In both in vitro and in vivo systems in which regulation of PTH mRNA levels were clearly apparent, there was no effect of either calcium or 1,25(OH)2D3 on parathyroid furin or PC7 mRNA levels. This is in contrast to the processing of proinsulin to insulin in the pancreatic beta-cell, which is up-regulated by glucose stimulation of PC1 and PC2 synthesis.
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PMID:Proparathyroid hormone processing by the proprotein convertase-7: comparison with furin and assessment of modulation of parathyroid convertase messenger ribonucleic acid levels by calcium and 1,25-dihydroxyvitamin D3. 1043 21

Nitric oxide synthase (NOS) containing nerve regeneration can be seen six months after unilateral cavernous nerve neurotomy in rats. However, its molecular mechanism is still unknown. It is believed that growth factors are involved in this phenomenon. In this study we investigated the change of NOS containing nerve fibers and the RNA expression of insulin like growth factor (IGF)-I, nerve growth factor (NGF), transforming growth factor (TGF)-alpha, TGF-beta 1, TGF-beta 2. TGF-beta 3 and NOS on the penis after cavernous nerve neurotomy in rats. Male rats were divided into three groups: (1) sham operation (N = 10); (2) unilateral neurotomy of a 5 mm segment of the cavernous nerve (N = 15); and (3) bilateral neurotomy (n = 15). Electrostimulation of the intact cavernous nerve or pelvic ganglion was performed at one, three and six months. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining was used to identify NOS in the penile nerve fibers. The gene expression for growth factors and bNOS was investigated in corporal tissue by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific oligonucleotide primers. One month after neurotomy, both unilateral and bilateral neurotomy groups showed a significant decrease in NOS-containing nerve fibers on the dorsal and intracavernosal nerves on the side of neurotomy, and a significantly lower mRNA expression of bNOS, IGF-I and TGF-beta 2. At three months, the number of NOS-containing nerve fibers in the unilateral neurotomy group increased only slightly but at six months those in the intracavernosal nerve increased in a significant amount (P < 0.0001), however mRNA expression of bNOS, IGF-I and TGF-beta 2 showed a significant increase as early as at three months. After bilateral neurotomy, the NOS-positive nerve fibers in the dorsal and intracavernosal nerve were significantly decreased at one month and remained so at six months; no erectile response could be elicited by pelvic ganglion stimulation. In the unilateral neurotomy group at six months, more NOS-positive neurons in the pelvic ganglia were found on the intact side than on the side of the neurotomy (P < 0.003), indicating that the regeneration derives from pelvic ganglion neurons on the intact side. Furthermore, electrostimulation in the unilateral neurotomy group revealed a greater maximal intracavernosal pressure and a shorter latency period at six months than at one month (P < 0.014, P < 0.001, respectively). These data suggest that IGF-I and TGF-beta 2 may play a key role in regeneration of NOS-containing nerve fibers in the dorsal and intracavernosal nerves after unilateral cavernous nerve injury.
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PMID:The role of growth factor on regeneration of nitric oxide synthase (NOS)--containing nerves after cavernous neurotomy in the rats. 1046 23

The induction of cytochrome P450 (CYP450) and Phase II conjugating enzymes by prototypical hepatic enzyme inducers was studied in adult male rat hepatocytes. Hepatocytes were suspended and cultured in diluted Matrigel in a basal serum-free Dulbecco's modified Eagle medium and exposed to the prototypical liver enzyme inducers, 3-methylcholanthrene, phenobarbital, hydrocortisone, and clofibrate for 48 h. Total RNA and microsomes were isolated and prepared, respectively, at 72 h. The expression of CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A1, CYP3A2, CYP4A1, fatty acyl-CoA oxidase, uridine diphosphate-glucuronosyltransferase, glutathione-S-transferase, and sulfotransferase was determined at the mRNA level with reverse transcriptase polymerase chain reaction (RT-PCR). The expression of CYP1A1, CYP2B1, CYP2C11, CYP2E1, and CYP4A1 was also measured at the apoprotein level by Western immunoblotting. Using these culture and expression analysis techniques, we have found that the expression of these metabolic enzymes can be maintained in culture for up to 7 d at the mRNA and apoprotein levels. In addition, hepatocytes were found to respond to chemical enzyme inducers with marked increases in enzyme expression at either the mRNA or protein level and in a concentration-related fashion. Cells were responsive to enzyme induction as early as 24 h after initial plating. The results obtained from this investigation indicate that the presence of diluted Matrigel (at a concentration of 0.35 mg/ml), the use of low concentrations of insulin (1 microM), hydrocortisone (0.1 microM), and serum-free culture medium can maintain the differentiated phenotype and responsiveness of cultured hepatocytes to chemical-induced metabolic enzyme expression. Under the conditions used in this study, enzyme induction in adult male rat hepatocytes shows close agreement with enzyme induction observed in the livers of rats exposed to these or similar prototypical enzyme inducers. Rat hepatocytes cultured in the presence of diluted Matrigel coupled with enzyme mRNA expression analysis with RT-PCR are proven to be a valuable and important in vitro toxicological approach to assess the chemical-induced changes in expression of liver CYP450 and Phase II conjugating enzymes.
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PMID:Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes. 1047 7

Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in stimulating insulin release in the pancreas as well as inhibiting gastric acid secretion in the stomach. GIP has been found in specific endocrine cells located in the mucosal layer of the small intestine and in the submandibular salivary gland. In this study, the tissue-specific expression of GIP guided by 1.2 kb of the human GIP (hGIP) gene 5' flanking region was investigated by a transgenic mouse approach. A chimeric promoter-reporter gene construct linking the 5'-flanking region of the hGIP gene with the thymidine kinase gene of the herpes simplex virus was introduced into the genomes of mice by microinjection. By reverse transcriptase-PCR (RT-PCR) and thymidine kinase assays, transgene expression was found in the stomach and pancreas. The enzyme activity detected in the stomach was about 6-fold higher than that found in the pancreas, suggesting that GIP may be expressed in the stomach. This observation is supported by RT-PCR studies since both human and mouse GIP transcripts are detected in the stomach and small intestine. In addition, distinct GIP-producing cells were identified in both tissues in mouse by in situ hybridization and immunohistochemical staining. Taken together, our data demonstrate for the first time that GIP is expressed in human and mouse stomach.
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PMID:Glucose-dependent insulinotropic polypeptide gene expression in the stomach: revealed by a transgenic mouse study, in situ hybridization and immunohistochemical staining. 1050 10

The insulin-like growth factors (IGFs) are important in the regulation of normal fetal musculoskeletal growth and development, and their actions have been shown to be modulated by IGF binding proteins (IGFBPs). Because the anatomical distribution of IGFBPs is likely to dictate IGF bioavailability, we determined the cellular distribution and expression of IGF-I, IGF-II, and IGFBP-1 to IGFBP-6 in epiphyseal growth plates of the fetal sheep, using immunocytochemistry and in situ hybridization. Little mRNA for IGF-I was detectable within the growth plates, but mRNA for IGF-II was abundant in germinal and proliferative chondrocytes, although absent from some differentiating chondrocytes and hypertrophic cells. Immunohistochemistry for IGF-I and IGF-II showed a presence of both peptides in all chondrocyte zones, including hypertrophic cells. Immunoreactive IGFBP-2 to -5 were localized within the germinal and proliferative zones of chondrocytes, but little immunoreactivity was present within the columns of differentiating cells. IGFBP immunoreactivity again appeared in hypertrophic chondrocytes. IGFBP mRNA in chondrocytes of the epiphyseal growth plate was below the detectable limit of in situ hybridization. However, low levels of mRNAs for IGFBP-2 to -6 were detected by the reverse transcriptase polymerase chain reaction. A co-localization of IGFBPs with IGF peptides in intact cartilage suggests that they may regulate IGF bioavailability and action locally. To test this hypothesis, monolayer cultures of chondrocytes were established from the proliferative zone of the growth plate, and were found to release immunoreactive IGF-II and to express mRNAs encoding IGFBP-2 to -6. Exogenous IGFBP-3, -4, and -5 had an inhibitory action on IGF-II-dependent DNA synthesis. IGFBP-2 had a biphasic effect, potentiating IGF-II action at low concentrations but inhibiting DNA synthesis at equimolar or greater concentrations relative to IGF-II. Long R3 IGF-I, which has a reduced binding affinity for many IGFBPs, was more potent than native IGF-I in promoting DNA synthesis by chondrocytes. Our findings suggest that locally produced IGF-II and IGF-I derived from the circulation can influence fetal epiphyseal chondrogenesis, and that this may be modulated locally by multiple IGFBP expression.
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PMID:Cellular localization and expression of insulin-like growth factors (IGFs) and IGF binding proteins within the epiphyseal growth plate of the ovine fetus: possible functional implications. 1053 72

Exposure of pancreatic islet B-cells to D-glucose and many other insulinotropic agents results in an increase of cytoplasmic calcium concentration, which triggers the exocytosis of secretroy granules. Previous studies have demonstrated that calcium itself, at concentrations ranging from 2 to 18 mM, is able to induce a dose-related stimulation of insulin secretion, even in the absence of any other secretagogue. It was recently demonstrated that parathyroid cells and several other cell types, whether involved or not in calcium homeostasis, sense extracellular calcium through a G-protein coupled calcium-sensing receptor (CaSR). In the present study, the presence of the receptor in islet pancreatic B-cells was scrutinized. Using reverse transcriptase-polymerase chain reaction and Northern blot analysis, we demonstrate the expression of the CaSR in purified rat pancreatic islet B-cells. The nucleotide sequences of the rt-PCR products demonstrated more than 99% homology with the rat kidney CaSR complementary DNA. A specific 5.3 kb transcript of the CaSR was expressed in normal pancreatic B-cells as well as in tumoral insulin-secreting cells. In pancreatic islets, the physiological role of the CaSR in the regulation of insulin release could involve the sensing of endogenous ligands other than calcium.
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PMID:Expression of the calcium-sensing receptor in pancreatic islet B-cells. 1054 80

Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low K(m) hexokinases (HK) exhibit a leftward shift of the normal dose-response curve for glucose-induced insulin release. Furthermore, HK catalyzes roughly 50% of total glucose phosphorylation measured in extracts from freshly isolated rodent islets, suggesting that HK participates in the process of glucose sensing in beta cells. We previously observed that HK activity represents 20% of total glucose phosphorylation in purified rat beta cell preparations and that HK is not homogenously distributed over these cells. The present study provides several arguments for the idea that HK detected in freshly isolated rat islets or islet cell preparations originates mainly from contaminating exocrine cells. First, reverse transcriptase-polymerase chain reaction using isoform-specific primers allowed detection of hexokinase I and IV mRNA in rat beta cells, whereas the messenger levels encoding the hexokinase II and III isoforms were undetectably low. However, immunoblots indicated that hexokinase I protein was 10-fold more abundant in freshly isolated islets and flow-sorted exocrine cells than in purified rat beta cell preparations. Second, comparison of HK activity in the different pancreatic cell types resulted in 15-25-fold higher values in exocrine than in endocrine cells (acinar cells: 21 +/- 3 pmol of glucose 6-phosphate formed/h/ng of DNA; duct cells: 30 +/- 8 pmol/h/ng of DNA; islet beta cells: 1.2 +/- 0.2 pmol/h/ng DNA; alpha cells: 0.9 +/- 0.4 pmol/h/ng of DNA). Since freshly purified beta cell preparations contain 3 +/- 1% exocrine cells, at least 50% of their HK activity can be accounted for by exocrine contamination. Third, after 5 days of culture of purified islet beta cells, both HK activity and the proportion of exocrine cells decreased by more than 1 order of magnitude, while the ratio of glucokinase over hexokinase activity increased more than 10-fold. Finally, preincubating the cells with 50 mmol/liter 2-deoxyglucose did not affect glucose stimulation of insulin biosynthesis and release. In conclusion, the observation that pancreatic exocrine cells are responsible for a major part of HK activity in islet cell preparations cautions against the use of HK measurements in islet extracts in the study of these enzymes in glucose sensing by pancreatic beta cells.
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PMID:Cellular origin of hexokinase in pancreatic islets. 1055 41

The molecular mechanism of nitric oxide synthase (NOS)-containing nerve regeneration is still unknown. It is believed that growth factors are involved in this phenomenon. We investigated the change of NOS containing nerve fibers and the mRNA expression of insulin like growth factor (IGF)-I, nerve growth factor (NGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, TGF-beta3, vascular endothelial growth factor (VEGF), endothelial NOS (eNOS) and neuronal NOS (nNOS) on the penis after cavernous nerve neurotomy in rats. Male rats were divided into four groups: (1) sham operation (n = 14); (2) unilateral neurotomy of a 5 mm segment of the cavernous nerve (n = 21); (3) unilateral neurotomy with growth hormone (n = 14); and (4) bilateral neurotomy (n = 21). Electrostimulation of the intact cavernous nerve or pelvic ganglion were performed at one, three and six months. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining and immunohistochemistry were used to identify NOS in the penis. The gene expression for growth factors, eNOS and nNOS were investigated in corporal tissue by reverse transcriptase-polymerase chain reaction (RT-PCR). One month after neurotomy, both unilateral and bilateral neurotomy groups showed significant decreases in NOS-containing nerve fibers on the dorsal and intracavernosal nerves on the side of neurotomy. Significantly lower mRNA expression of nNOS, IGF-I and TGF-beta2, higher mRNA expression of eNOS and VEGF189 were shown in these groups. At three months, the number of NOS-containing nerve fibers in the unilateral neurotomy group increased only slightly, while the GH-treated group showed a significant increase. At six months, those in the intracavernosal nerve only increased in a significant amount (P < 0.0001), however mRNA expression of nNOS, IGF-I and TGF-beta2 showed a significant increase as early as at three months. After bilateral neurotomy, the NOS-positive nerve fibers in the dorsal and intracavernosal nerve were significantly decreased at one month and remained so at six months; no erectile response could be elicited by pelvic ganglion stimulation. In the unilateral neurotomy group at six months, more NOS-positive neurons in the pelvic ganglia were found on the intact side than on the side of the neurotomy (P < 0.003), indicating that the regeneration derived from pelvic ganglion neurons on the intact side. Furthermore, electrostimulation in the unilateral neurotomy group revealed a greater maximal intracavernosal pressure and a shorter latency period at six months than at one month (P < 0.014, P < 0.001, respectively). These data suggest that IGF-I and TGF-beta2 may play a key role in the regeneration of nNOS-containing nerve fibers in the dorsal and intracavernosal nerves, and eNOS increases temporarily in the intracavernous involving VEGF189 after unilateral cavernous nerve injury.
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PMID:IGF-I and TGF-beta2 have a key role on regeneration of nitric oxide synthase (NOS)-containing nerves after cavernous neurotomy in rats. 1055 3

The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides. The paired half of this consensus sequence has similarities to the PAX6 paired domain consensus binding site, and the two proteins bind to common sequences in several islet genes, although with different relative affinities. When expressed in an alpha-cell line, PAX4 represses transcription through the glucagon or insulin promoters or through an isolated PAX4 binding site. This repression is not simply due to competition with the PAX6 transcriptional activator for the same binding site, since PAX4 fused to the unrelated yeast GAL4 DNA binding domain also represses transcription through the GAL4 binding site in the alpha-cell line and to a lesser degree in beta-cell lines and NIH 3T3 cells. Repressor activity maps to more than one domain within the molecule, although the homeodomain and carboxyl terminus give the strongest repression. PAX4 transcriptional regulation apparently plays a role only early in islet development, since Pax4 mRNA as determined by reverse transcriptase PCR peaks at embryonic day 13.5 in the fetal mouse pancreas and is undetectable in adult islets. In summary, PAX4 can function as a transcriptional repressor and is expressed early in pancreatic development, which may allow it to suppress alpha-cell differentiation and permit beta-cell differentiation.
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PMID:Paired-homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development. 1056 52

Protease inhibitor treatment has dramatically improved rates of morbidity and mortality in HIV-infected patients. However, it has recently been shown that this medication is associated with long-term side effects characterized by metabolic, clinical and biological alterations. These modifications have been described in patients treated with highly active antiretroviral therapy (HAART), including nucleoside analogue reverse transcriptase inhibitors (NRTI) and generally (but not always) protease inhibitors (PI). Clinical alterations are characterised by a body fat redistribution syndrome or lipodystrophy, with peripheral lipoatrophy and/or central fat accumulation. They are often associated with biological alterations, i.e. insulin resistance, hyperglycaemia and dyslipidaemia, which can also be observed alone. The pathophysiology of these alterations is presently unknown. The deleterious effect of PI on adipose tissue could be direct or indirect, and is probably modulated by genetic or environmental factors. NRTI could also be involved because of their mitochondrial toxicity. The purpose of the treatment is to control metabolic disturbances in order to prevent immediate complications such as acute pancreatitis and limit possible cardiovascular and diabetic complications at longer term. Studies are in progress to evaluate the possibility of therapeutic alternatives to PI when major metabolic disturbances are present.
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PMID:Adverse metabolic disorders during highly active antiretroviral treatments (HAART) of HIV disease. 1059 60


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