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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The previous reports of inhibition of alcohol dehydrogenase and lactate dehydrogenase by the vitamin folic acid and its analogues are in error. The high absorbance of solutions containing folate causes distortion of the measurements of reaction velocities, leading to apparent inhibitions. When cuvettes of sufficiently short optical path length are used, no inhibition by folate can be observed. Similarly, the reported inhibition of ribonuclease by folate is an artifact. Glutamate dehydrogenase and dihydropterin reductase actually are inhibited by folate. The reported nonspecific inhibitions of over a dozen enzymes by folate, though, must be regarded as erroneous.
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PMID:Nonspecific inhibition of dehydrogenases by folates: an artifact. 50 60

A novel anti-tumour amphibian oocyte RNase, ONCONASER (ONC), previously known as P-30 Protein, is in the clinical trials. The effect of ONC alone and in combination with lovastatin (LVT), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme of mevalonate (MVA) and cholesterol synthesis pathway, in three human tumour cell lines ASPC-1 pancreatic, A-549 lung, and HT-520 lung carcinomas, has been presently studied. A synergism between ONC and LVT in inducing the cytostatic and cytotoxic effects was observed. The cytostatic effect, seen during the early phase of the treatment with this combination of drugs was manifested as prolongation of the cell cycle duration, especially of the G1 phase; cell death was apparent after 72 h of treatment. The synergistic effect of ONC and LVT was also evident in the clonogenicity assays. Both LVT lactone and its in vitro activated beta-hydroxy acid form, alone and in respective combinations with ONC, exerted similar degree of growth suppression. The effects of both forms of LVT (used alone or in combination with ONC) were reversed by MVA, which suggests that HMG-CoA reductase inhibition is a primary mechanism of LVT action. The data indicate that the LVT lactone can be activated intracellularly by tumour cells studied, and that the combination of ONC with LVT can produce significantly enhanced anti-tumour activities.
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PMID:Synergism between a novel amphibian oocyte ribonuclease and lovastatin in inducing cytostatic and cytotoxic effects in human lung and pancreatic carcinoma cell lines. 150 3

Inability to culture the disease-producing amastigote form of Leishmania has greatly hampered its study. We have biochemically characterized an axenically cultured amastigote-like form of Leishmania pifanoi. This form closely resembles amastigotes in proteinase, ribonuclease, adenine deaminase and peroxidase activity. It also exhibits comparable rates of growth, transformation, synthesis of DNA, RNA and protein, and metabolism of glucose and linoleic acid. It is distinct from promastigotes in these characteristics. The expression of the genes for beta-tubulin and the P100/11E reductase is developmentally regulated in this axenic form as in amastigotes. These results, combined with previous demonstrations of amastigote morphology and antigenicity in the culture form, confirm that Leishmania amastigotes have been successfully propagated in axenic media. This strain should serve as an excellent model for the study of amastigote biochemistry, pharmacology and immunology, and the molecular genetics of the transformation between amastigote and promastigote forms.
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PMID:Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture. 177 52

Previous studies in which investigators have induced the rate of polyamine uptake in vitro have used either inhibitors of polyamine biosynthesis or growth factors that induce cell proliferation. Recently, however, we have described the induction of putrescine uptake in cultured adult mouse hepatocytes and have shown that uptake is independent of both intracellular polyamine levels and proliferation. Although proliferation was not apparent in those studies, data suggested that, after isolation, the cells entered G1 of the cell cycle. In this study, we have examined whether the induction of putrescine uptake is a function of entry into the cell cycle and whether uptake activity is essential for optimal progression into the S phase. Using ribonuclease reductase subunit M1 as a marker of entry into the cell cycle, we have shown that hepatocytes enter G1 during the first 4 hr of culture. Both putrescine uptake and ornithine decarboxylase activity increased as the cells entered G1. Treatment of the cells with retinoic acid (10 to 33 mumol/L) prevented them from entering G1 and also inhibited the induction of the putrescine transporter by up to 90%. In contrast, initiation of G1 to S phase transition markedly down-regulated the activity of the transporter. Thus induction of the putrescine transporter in isolated hepatocytes appears to be a G1-specific event. Culturing the hepatocytes in the presence of 1,1'-bis[3-(1'-methyl-[4,4'-bipyridinium]-1-yl)-propyl]- 4,4'-bipyridinium, a potent competitive inhibitor of putrescine uptake, resulted in a 47% decrease in intracellular putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cell cycle-dependent uptake of putrescine and its importance in regulating cell cycle phase transition in cultured adult mouse hepatocytes. 195 75

A cDNA expression library in lambda gt11 was screened with affinity-purified polyclonal anti-rat cytochrome b5 reductase antibodies. One positive clone out of 450,000 clones was isolated and found to be incomplete. This clone was used to rescreen the library, and a second, overlapping clone that contained the entire coding sequence was isolated. RNA gel blots showed that the two overlapping clones contained approximately 90% of the reductase mRNA sequence. Sequencing data showed (i) that rat reductase has a 93% sequence similarity with bovine and human reductase and (ii) that reductase is not synthesized as a high molecular weight precursor. Results of Southern blot analysis were consistent with the hypothesis that a single gene codes for the soluble and membrane-bound (microsomal and mitochondrial) forms of the reductase, present in erythrocytes and liver, respectively. The cloned cDNA was used to study reductase transcripts in liver and reticulocytes. Two antisense RNA probes that together covered the entire coding region and part of the noncoding region of reductase mRNA were used in RNase A protection experiments. These probes detected only one transcript in liver, suggesting that endoplasmic reticulum and mitochondrial reductase are translated from the same mRNA. In contrast, two transcripts were detected in reticulocytes, one of which mismatched the liver probe approximately 30 nucleotides downstream from the initiation codon. Since the soluble and membrane form of the reductase are known to differ at the N terminus, we suggest that this second transcript encodes soluble reductase.
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PMID:Two transcripts encode rat cytochrome b5 reductase. 317 30

Protoplasts of Listeria monocytogenes strain 42 were fractionated after control lysis on a Ficoll (a polysucrose) density gradient. Visually, five zones could be recognized in the gradient. The first one was composed of amorphous cytoplasmic solutes (fraction 1a) and a mixture of particles (fraction 1b). These were: (i) light particles that were lipase-sensitive and composed of six subunits and (ii) heavy particles, sensitive to ribonuclease and devoid of fine structure. The second zone consisted of tubules and vesicles still harboring cytoplasmic components (fraction 2), whereas the third zone contained only empty vesicles and protoplast ghosts (fraction 3). The material congregating into the fourth zone was morphologically identical to that of the third (fraction 3a). The fifth and heaviest zone contained a mixture of (i) particles without any substructure and (ii) partly lysed protoplasts (fraction 4). Fractions 1b and 4 were the richest in nucleic acids (ribonucleic acid, 11.4 and 9.4%, respectively; deoxyribonucleic acid, 5.1 and 4.8%, respectively), whereas fraction 1b had the highest protein contents (74.6%). Phospholipids were mainly found in fractions 2 and 3. Except for fraction 1, all materials contained significant amounts of protein-bound phosphorus. The main concentrations of four enzymes were: glucose-6-phosphate dehydrogenase (fraction 1a); adenosine triphosphatase and reduced nicotinamide adenine diphosphate oxidase (fraction 3); nitro blue tetrazolium chloride reductase (fraction 2). Fractionation of strain 42 after addition of (32)P during the mid-log phase of growth revealed that the radio-activity was mainly detected in fraction 1b, when growth in the presence of the marker was allowed for 10 min, and in fraction 2, when growth was allowed for 90 min. The vesicles of fraction 2, often tubular, are probably of mesosomal origin, whereas those of fraction 3, which are always spherical, represent, most likely, the bulk of the cell plasma membrane. Our data showed slight chemical differences between these two fractions, but the differences in enzymatic activities and lipid-phosphorus incorporation during long pulse experiments were most dramatic.
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PMID:Fractionation and characterization of the plasma and mesosome membrane of Listeria monocytogenes. 430 41

1. Treatment of washed rat liver microsomes in a medium containing 0.12m-sucrose, 12.5mm-potassium chloride, 2.5mm-magnesium chloride and 25mm-tris-hydrochloric acid buffer, pH7.6, with 2m-lithium chloride at 5 degrees for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting (14)C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH-2,6-dichlorophenol-indophenol reductase, NADH-neo-tetrazolium reductase, NADH-cytochrome c reductase and ribonuclease activities. 5. (3)H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of (3)H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.
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PMID:Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride. 431 14

A retinol dehydrogenase, RoDH(1), which recognizes holo-cellular retinol-binding protein (CRBP) as substrate, has been cloned, expressed, and identified as a short-chain dehydrogenase/reductase (Chai, X., Boerman, M. H. E. M., Zhai, Y., and Napoli, J. L. (1995) J. Biol. Chem. 270, 3900-3904). This work reports the cloning and expression of a cDNA encoding a RoDH isozyme, RoDH(II). The predicted amino acid sequence verifies RoDH(II) as a short-chain dehydrogenase/reductase, 82% identical with RoDH(I). RoDH(II) recognized the physiological form of retinol as substrate, CRBP, with a Km of 2 mM. Similar to microsomal RoDH and RoDH(I), RoDH(II) had higher activity with NADP rather than NAD, was stimulated by ethanol and phosphatidyl choline, was not inhibited by the medium-chain alcohol dehydrogenase inhibitor 4-methylpyrazole, but was inhibited by phenylarsine oxide and the short-chain dehydrogenase/reductase inhibitor carbenoxolone. Northern blot analysis detected RoDH(I) and RoDH(II) mRNA only in rat liver, but RNase protection assays revealed RoDH(I) and RoHD(II) mRNA in kidney, lung, testis, and brain. These data indicate that short-chain dehydrogenases/reductase isozymes expressed tissue-distinctively catalyze the first step of retinoic acid biogenesis from the physiologically most abundant substrate, CRBP.
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PMID:Cloning of a cDNA for a second retinol dehydrogenase type II. Expression of its mRNA relative to type I. 749 45

Transcripts for hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are heterogeneous in length. This heterogeneity is due to variations in the length of 5'-untranslated leader (UTL) sequences, which are generated by both alternate splicing within the first exon as well as alternate transcription start sites. Because mRNA 5'-UTL sequences have a role in regulating translational efficiency, the level and distribution of HMG-CoA reductase transcripts were measured in both total cellular RNA and polysomes from the Syrian hamster cell line C100. Cells were treated with either lovastatin alone, lovastatin and 25-hydroxycholesterol (25-OH C), or lovastatin, 25-OH C, and mevalonate, three treatment regimens used in an earlier study to demonstrate nonsterol-mediated translational control of HMG-CoA reductase synthesis [D. M. Peffley (1992) Somat. Cell Mol. Genet. 18, 19-32]. When reductase mRNA was measured by 5'-extension analysis under the same conditions, levels of transcripts with 5'-UTL regions ranging from 41 to 81 bases were reduced approximately four- to eightfold. In contrast, transcripts with 5'-UTL regions 93 to 100 bases in length were not reduced, and transcripts with 5'-UTL regions approximately 300-400 bases in length increased twofold. The addition of 25-OH C alone or both 25-OH C and mevalonate to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels fivefold in total cellular RNA as determined by RNase protection assay. No comparable change was observed with control ribosomal protein S17 mRNA. Postmitochondrial supernatants representing both translationally inactive monosomes and translationally active polysomes were prepared by sucrose gradient fractionation from cells incubated with the standard three treatments. Because 5'-UTL sequences of many mRNAs have a role in regulating translational efficiency we isolated RNA from each fraction and measured levels of reductase transcripts by 5'-extension analysis. Under all three conditions, transcripts with 5'-UTL sequences 41-103 bases in length were primarily associated with dense sucrose fractions that contain polysomes. In contrast, reductase transcripts with leader sequences 300 to 400 bases were almost exclusively associated with the less dense sucrose fractions containing monosomes. These results indicate that both the level and polysome distribution of individual reductase transcripts are influenced by the length of 5'-UTL sequences.
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PMID:The length of 5'-untranslated leader sequences influences distribution of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in polysomes: effects of lovastatin, oxysterols, and mevalonate. 757 24

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist. 758 48


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