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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate, a committed step in triacylglycerol and phospholipid biosynthesis. We have previously reported the cDNA cloning and transcriptional regulation of the murine mitochondrial GPAT (mGPAT). We now report the cloning of the 5'-flanking region of the murine mitochondrial GPAT. The transcription start site was identified by primer extension and RNase protection assays. A TATA box-like motif (TTATTAT) was located between -34 and -29 and a reverse CCAAT box (ATTGG) was located between -78 and -74, relative to the transcription start site. To begin studying mechanisms underlying transcriptional regulation of the mGPAT gene, chimeric luciferase (LUC) plasmids containing serial deletions, from -1447 to -38, of the 5'-flanking region of the murine mGPAT gene were prepared and transfected into 3T3-L1 cells. The fusion construct -1447 GPAT.LUC showed high promoter activity and deletions to -1353, -747, -322, and -86 did not markedly change the promoter activity. With all constructs, luciferase activity was 2-fold higher when plasmids were transfected into 3T3-L1 adipocytes. However, deletion of sequences between -86 and -55 resulted in a 9-fold decrease in LUC activity in both preadipocytes and adipocytes. Deletion of sequences between -55 and -38 did not alter promoter activity. DNase I footprint analysis revealed a protected region between -95 and -65 which included the putative CTF/NF1 binding site. Electrophoretic mobility shift assays demonstrated a single protein-DNA complex formation. Oligonucleotides synthesized according to the CTF/NF1 consensus sequence or the adenovirus NF-1 site showed a different and more complex pattern of protein-DNA interaction and were not able to compete away the mGPAT promoter-protein complex, indicating that a distinct protein was bound to -86/-55, a region important for the basal promoter activity in 3T3-L1 cells. Luciferase activity was increased 2.8- and 8-fold when adipocytes stably transfected with -322 GPAT.LUC were treated with 5 and 25 mM glucose, respectively, in the presence of 10 nM insulin. These results indicate that carbohydrate-responsive sequences are located within -322 base pairs of the mGPAT promoter.
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PMID:Characterization of the murine mitochondrial glycerol-3-phosphate acyltransferase promoter. 783 9

Insulin-dependent diabetes mellitus (IDDM) is associated with elevated plasma triglyceride levels that normalize after insulin administration. The observation that overexpression of the apoC-III gene in transgenic mice can cause hypertriglyceridemia and other evidence implicating apoC-III in the regulation of triglyceride levels prompted us to examine whether apoC-III might be involved in the hypertriglyceridemia associated with IDDM. To this end, the regulation of apoC-III gene expression was studied in the streptozotocin-treated mouse model of IDDM. In the insulin-deficient diabetic state, these mice have elevated glucose and triglyceride levels and a 1.4- to 1.5-fold increase in hepatic apoC-III mRNA levels, by Northern analysis as well as quantitative solution hybridization RNase protection assay. Insulin treatment normalized the glucose and triglyceride levels and diminished hepatic apoC-III mRNA levels by 59%. Analysis of transcription rates using the nuclear run-on technique demonstrated that the changes in hepatic apoC-III mRNA levels were the results of changes in the transcriptional activity of the gene. To determine the role of insulin in the regulation of apoC-III transcription, HepG2 cells were transfected with an apoC-III reporter construct, and treated with different insulin concentrations. The results demonstrated that insulin treatment induced a dose-dependent down-regulation of apoC-III transcriptional activity. These data suggest that the apoC-III transcriptional changes seen in animals are caused by differences in insulin concentrations. Assuming that apoC-III mRNA levels reflect the synthesis and secretion of the protein, these results present the possibility that overexpression of the apoC-III gene could contribute to the hypertriglyceridemia observed in IDDM.
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PMID:Transcriptional regulation of the apoC-III gene by insulin in diabetic mice: correlation with changes in plasma triglyceride levels. 786 70

Previous studies have indicated that insulin secretion in response to glucose diminishes with age but insulin synthesis and gene transcription do not. To determine whether expression of genes other than those that encode insulin are subject to age-related changes that could alter pancreatic islet function, mRNAs for insulins I and II, amylin, glucose transporter 2 (GluT2), glucagon, and glucokinase were quantified in 2-, 6-, 12-, and 24-month-old Fischer 344 rats using species-specific ribonuclease (RNase) protection assays. There was only a modest (1.2- to 1.3-fold) increase in insulin I and insulin II mRNAs between ages 2 and 12 months. There were no statistically significant changes in levels of glucokinase mRNA with age. In contrast, the abundances of amylin, GluT2, and glucagon mRNAs all doubled during the same period. Variance in values from 24-month-old rats was too great to allow conclusions, except that the ratio of insulin II mRNA to insulin I mRNA increased with age. This change was not related to islet mass or total insulin mRNA abundance because it persisted at age 24 months, when total mRNA abundance had decreased. These results indicate that aging is associated with significant alterations in the relative proportion of expression of pancreatic islet cell genes implicated in insulin secretion and in intraislet glucose metabolism.
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PMID:Age-related changes in pancreatic islet cell gene expression. 788 76

Glycation and oxidation reactions contribute to protein modification in aging and diabetes. Formation of dicarbonyl sugars during autoxidation of glucose is the hypothetical first step in the autoxidative glycosylation and subsequent browning of proteins by glucose [Wolff, S. P., & Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identify the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (pH 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen with transition metal chelators (antioxidative conditions). Dicarbonyl compounds were analyzed spectrophotometrically and by HPLC after reaction with Girard-T reagent. Carbohydrates were analyzed by gas chromatography-mass spectrometry. Both dicarbonyl sugar and arabinose concentrations increased with time and glucose concentration in incubations conducted under oxidative conditions; only trace amounts of these products were detected in glucose incubated under antioxidative conditions. HPLC analysis of adducts formed with Girard-T reagent indicated that glyoxal was the only alpha-dicarbonyl sugar formed on autoxidation of glucose. Glyoxal and arabinose accounted for > or = 50% of the glucose lost during a 21 day incubation. Neither glucosone nor its degradation product, ribulose, was detectable. Reaction of glyoxal with RNase yielded the glycoxidation product, N epsilon-(carboxymethyl)lysine, while arabinose is a source of pentosidine. Our results implicate glyoxal and arabinose as intermediates in the browning and crosslinking of proteins by glucose under oxidative conditions. They also provide a mechanism by which antioxidants and dicarbonyl trapping reagents, such as aminoguanidine, limit glycoxidation reactions and support further evaluation of these types of compounds for inhibition of chemical modification and crosslinking of proteins during aging and diabetes.
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PMID:Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose. 789 66

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.
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PMID:Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line. 792 31

Matrix-assisted laser desorption/ionization mass spectrometry has been successfully applied in the study of non-enzymatic glycation of different proteins. In the case of bovine serum albumin, glycated by in vitro experiments performed under pseudophysiological conditions, a clear increase in molecular weight is observed with respect to both glucose concentrations and incubation time. The in vitro glycation of ribonuclease with glucose and fructose shows some peculiar differences either in terms of the number of condensed sugar molecules or in terms of the reaction kinetics. The same approach, applied to plasma proteins of healthy and diabetic subjects, provides evidence for the occurrence of glycation of human serum albumin for the latter subjects.
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PMID:Matrix-assisted laser desorption/ionization capabilities in the study of non-enzymatic protein glycation. 794 31

Diabetes alters the level of insulin-like growth factor I (IGF-I) mRNA in tissues of postnatal animals, but the impact of maternal diabetes or gestational diabetes on IGF-I mRNA abundance in fetal tissues has not been examined. Pregnant pigs were injected with either buffer or alloxan (50 mg/kg) at day 75 of gestation to induce diabetes. Fetal tissue samples were collected at day 105 of gestation, and IGF-I mRNA abundance (densitometric units/10 micrograms total RNA) were estimated by specific ribonuclease protection assay. Fetal glucose and IGF-I concentrations were increased 166 and 34%, respectively, by maternal diabetes. Maternal diabetes induced an increase in abundance of IGF-I mRNA in fetal skeletal muscle, liver, heart, kidney, and placenta. IGF-I mRNA levels were depressed by maternal diabetes in fetal adipose tissue and brain compared with the respective tissues from fetuses of control pigs. These data indicate that circulating levels of IGF-I and the steady-state levels of IGF-I mRNA in fetal tissues can respond to the metabolic and endocrine alterations occurring during maternal diabetes. The large variation in expression and degree of response among fetal tissues indicates that the fetus experiences tissue-specific regulation of IGF-I expression during development.
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PMID:Alteration in IGF-I mRNA content of fetal swine tissues in response to maternal diabetes. 797 70

The developmental expression of the glucose-dependent insulinotropic polypeptide (GIP) gene was investigated in rat intestine. Steady state levels of GIP mRNA were determined in the intestine during fetal and postnatal development by double ribonuclease protection assays. GIP mRNA could be detected as early as day 20 of embryonic development and very low levels remained until postnatal day 3. The GIP mRNA levels increased markedly in the period between days 3 and 5 of postnatal life and then gradually increased toward adult levels. Since intron 1 of the GIP gene contains putative TATA and CCAAT boxes, and some potential cis-acting promoter elements, we examined whether or not another transcript starting from exon 2 of the GIP gene is expressed during development of rat intestine. Ribonuclease protection assays suggested that although an abbreviated transcript might exist starting from exon 2, it appears to be minor and its relative abundance is unchanged during development or following intraduodenal glucose stimulation. These observations suggest that GIP may play an important role in early postnatal development probably associated with suckling.
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PMID:Developmental expression of the glucose-dependent insulinotropic polypeptide gene in rat intestine. 800 38

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype. Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.
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PMID:Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae. 824 8

Methylprednisolone stimulates rabbit ileal neutral NaCl absorption; and aminoglutethimide, which decreases glucocorticoid levels, decreases NaCl absorption. Studies were carried out to determine the mechanism of these effects and to determine which members of the gene family of mammalian Na+/H+ exchangers were involved. Rabbits were treated subcutaneously with methylprednisolone (40 mg daily for 24 or 72 h), aminoglutethimide (100 mg twice daily for 72 h), or saline as a control. Ileal brush border membranes were prepared by magnesium precipitation, and brush border Na+/H+ exchange was determined by 22Na+ uptake over 3-8 s. The 22Na+ uptake experiments were performed in the presence of a voltage clamp using either valinomycin/potassium or tetramethylammonium/nitrate to eliminate potential contributions by other electrogenic transport processes. Methylprednisolone treatment approximately doubled ileal brush border Na+/H+ exchange, whereas aminoglutethimide led to a 50% decrease in Na+/H+ exchange. These effects were specifically on Na+ uptake with an acid inside pH gradient, whereas diffusive Na+ uptake (no pH gradient), glucose-dependent Na+ uptake, and glucose and Na+ equilibrium volumes were not affected. To determine if the increase in Na+/H+ exchange was associated with an increase in message expression, mRNA levels were measured by ribonuclease protection assay. Methylprednisolone stimulated the NHE-3 mRNA level by 4-6-fold at 24 h, which remained increased at 72 h. In contrast, messages for NHE-1 and NHE-2 were not affected by methylprednisolone. In summary, 1) methylprednisolone stimulation of rabbit ileal Na+ absorption is due to stimulation of ileal villus cell brush border Na+/H+ exchange; 2) basal ileal brush border Na+/H+ exchange is dependent on glucocorticoid levels; and 3) an increase in NHE-3 message, but not in NHE-1 or NHE-2 message, correlates with the stimulation of ileal brush border Na+/H+ exchange. It is likely that NHE-3 is an Na+/H+ exchanger that is involved in ileal Na+ absorption.
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PMID:Glucocorticoid stimulation of ileal Na+ absorptive cell brush border Na+/H+ exchange and association with an increase in message for NHE-3, an epithelial Na+/H+ exchanger isoform. 838 Jan 55


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