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)

Eight male cynomolgus monkeys (Macaca fascicularis) on a normal chow diet were orally administered gemfibrozil daily using a weekly rising dose protocol for 3 weeks (50, 125, and 200 mg/kg per day). At these drug doses, Lp[a] levels were reduced: 83.7% +/- 3.2 (SEM), (P < 0.024); 63.7% +/- 4.1 (P < 0.013); and 36.2% +/- 1.1 (P < 0.002), respectively, of pretreatment values. Lp[a] reduction was directly related to blood gemfibrozil concentration (range 36-428 microM, r = 0.969) and occurred without concomitant changes in apolipoprotein B. Three weeks posttreatment Lp[a] levels returned to pretreatment values. A specific ribonuclease protection assay demonstrated that liver apolipoprotein[a] (apo[a]) mRNA expression was decreased in all animals to an average of 19.1% +/- 3.0 (P < 0.0026), of pretreatment values after the 200 mg/kg treatment, whereas, albumin, apolipoprotein A-I, apolipoprotein E, and glyceraldehyde-3-phosphate dehydrogenase mRNAs were unchanged. Lp[a] levels were unaffected by gemfibrozil in HepG2 cells permanently transfected with an apo[a] 10-kringle cDNA construct containing partial 5'- and 3'-untranslated sequences and under control of a constitutive CMV promoter. However, both Lp[a] and apo[a] mRNA in primary cynomolgus monkey hepatocytes were coordinately lowered in a dose-dependent fashion by gemfibrozil. Thus, Lp[a] can be regulated by gemfibrozil at the level of apo[a] mRNA expression.
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PMID:Gemfibrozil significantly lowers cynomolgus monkey plasma lipoprotein[a]-protein and liver apolipoprotein[a] mRNA levels. 766 7

Previous studies have suggested that oleic acid (OA), by increasing availability of triglyceride (TG) and/or cholesteryl ester (CE), increases the secretion of apolipoprotein B (apoB) from HepG2 cells. The present studies were conducted to determine the effect of exogenous very low density lipoproteins (VLDL), which can provide TG and cholesterol to cells and be returned to the liver after secretion, in the regulation of hepatic apoB secretion. Addition of exogenous VLDL (50 micrograms protein/ml) to culture media was found to significantly stimulate apoB secretion from HepG2 cells. This effect was observed consistently with VLDL isolated from either normal or hyperlipidemic subjects. The effects of addition of VLDL were compared to that of OA, which was previously shown to stimulate apoB secretion by a posttranslational mechanism. VLDL appeared to increase apoB secretion by two mechanisms; the dominant one being posttranslational. Thus, VLDL protected newly synthesized apoB from rapid intracellular degradation in a manner similar to OA. Although treatment with VLDL increased the mass of both TG and CE, 3-fold and 2.6-fold, respectively, it appeared that the increase in TG was the critical factor associated with increased apoB secretion. Triacsin D, which is a potent inhibitor of TG synthesis, significantly inhibited the VLDL-induced stimulation of apoB secretion. Inhibition of apoB secretion by Triacsin D was associated with the loss of the protective effect of VLDL on newly synthesized apoB. In addition to its posttranslational effects, exogenous VLDL also regulated apoB secretion at the pretranslational level. Thus, we also observed that VLDL treatment consistently increased synthesis of apoB protein by 20-30%, an effect that is not observed after treatment of HepG2 cells with OA. A sensitive solution hybridization/RNase protection assay indicated that the increased apoB synthesis was associated with a 20-30% increase in apoB mRNA in VLDL-treated HepG2 cells. OA treatment had no effect on apoB mRNA levels. We conclude that VLDL treatment stimulates apoB secretion in HepG2 cells primarily by supplying fatty acids for TG synthesis. However, 20-30% of the stimulatory effect was due to a second mechanism that appeared to be pretranslational. Based on studies with OA, it appears that some component of VLDL other than TG-derived fatty acid was responsible for this effect.
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PMID:Exogenous VLDL stimulates apolipoprotein B secretion from HepG2 cells by both pre- and post-translational mechanisms. 796 81

Rats and mice are frequently used in studies of the regulation of lipoprotein metabolism. Although the species are closely related, they differ dramatically in the responses of their lipoproteins to estrogen administration. In rats, estrogens produce profound decreases in the levels of all plasma lipoproteins and this is attributed largely to estrogen-induced increases of hepatic low-density lipoprotein receptor (LDL-receptor) activity. Estrogens affect mouse plasma lipoproteins to a much lesser extent. Therefore, one of our aims was to compare the regulation of LDL-receptor gene expression in rats and mice at several potential loci of regulation. To assess the specificity of the estrogen effect, we also compared the responses of apolipoprotein AI (apoAI), apolipoprotein B (apoB), and beta-actin to the response of the LDL-receptor. In male Sprague Dawley rats given 17 beta-estradiol or 17 alpha-ethinyl estradiol at supraphysiological doses of 5 micrograms/g body mass/day, plasma total cholesterol and triacylglycerols fell to approximately 5% and approximately 50%, and, plasma apoAI and apoB fell to approximately 12% and approximately 16% of controls, respectively. By contrast, in male C3H/HeJ mice the above parameters dropped only to approximately 65% of controls and apoB concentrations rose to approximately 200% of controls. In rats, relative rates of LDL-receptor mRNA transcription (nuclear 'run-off' assay) and total hepatic, nuclear and polysomal LDL-receptor mRNA levels (RNase protection assay) increased by 1.5-2-fold, while synthesis of LDL-receptor protein on hepatic polysomes (in a wheat-germ translation system) increased 8-fold and LDL-receptor protein mass in hepatic plasma membranes increased 10-fold (by immunoblotting). In mouse liver, too, LDL-receptor mRNA levels increased 1.5-fold and the LDL-receptor mRNA transcription start sites in rat and mouse were found to be the same, but mouse LDL-receptor protein mass did not change, i.e. LDL-receptors of mice were similar to rat with respect to transcriptional regulation, but differed in their post-transcriptional control mechanisms. In rats, estrogen administration increased apoAI mRNA transcription rates 1.6-fold and also apoAI mRNA levels in total liver homogenates, nuclei and polysomes, (2-fold for each) consistent with transcriptional regulation. However, apoAI synthesis on total RNA increased less than apoAI mRNA, indicating that apoAI translational control mechanisms, at least in part, also regulate hepatic rates of apoAI production. ApoB mRNA transcription rates and levels showed small increases following estrogen administration. Hepatic beta-actin mRNA transcription and levels did not change.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vivo regulation of low-density lipoprotein receptors by estrogen differs at the post-transcriptional level in rat and mouse. 837 91

In the present study, we investigated the mechanisms that regulate apolipoprotein B-100 (apoB) secretion in response to the change of intracellular cholesteryl ester contents by adding low density lipoprotein (LDL) and an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (pravastatin) in rabbit hepatocyte culture system. LDL caused a significant dose-dependent increase in apoB secretion. On the other hand, the addition of pravastatin decreased apoB secretion significantly. LDL caused a dose-dependent increase in cellular cholesteryl ester, while cellular cholesteryl ester was decreased by pravastatin significantly. Therefore, these results indicate that the change of apoB secretion was in parallel with the change of cellular cholesteryl ester contents. Cellular contents of free cholesterol, triglyceride, and phospholipid did not change. To investigate intracellular degradation of apoB prior to secretion, pulse-chase experiments were performed. It was shown that the addition of pravastatin accelerated intracellular degradation of apoB, while LDL slowed the apoB intracellular degradation rate. We also investigated whether the change of cellular cholesteryl ester could affect the apoB mRNA level. Northern blot analysis and solution hybridization RNase protection assay demonstrated that neither LDL nor pravastatin caused a significant change in cellular apoB mRNA level. We conclude that intracellular cholesteryl ester contents play a critical role in apoB secretion, and the intracellular apoB degradation rate could be the main mechanism that regulates apoB secretion in response to the change of intracellular cholesteryl ester level.
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PMID:Regulation of apolipoprotein B production and secretion in response to the change of intracellular cholesteryl ester contents in rabbit hepatocytes. 850 6

The purpose of this study was to characterize intestinal apolipoprotein B (apoB) metabolism in subjects with familial hypobetalipoproteinemia (FHBL), where segregation analysis supports linkage to the apoB gene but no apoB truncations are present. We investigated cholesterol and fat absorption, intestinal apoB mRNA synthesis and editing, as well as apoB-48 synthesis. Plasma triglycerides (TG) and retinyl palmitate in the chylomicron fractions were analyzed after 12 hours of fasting and then repeatedly for 14 hours after ingestion of a vitamin A-containing high-fat meal. Cholesterol absorption was assessed using a dual stable-isotope method. Mean peak times and concentrations and areas under the curve (AUCs) for fat absorption and mean percentages of cholesterol absorption were comparable in affected and nonaffected family members. Intestinal biopsies were extracted for total RNA and also incubated with 35S-methionine for measurements of apoB synthesis. Similar quantities of apoB mRNA were found to be expressed in the intestine in affected and control subjects by RNase protection assay. ApoB mRNA editing assay showed that the majority of apoB-100 mRNA was edited to the apoB-48 form to a similar extent in both groups. Virtually no apoB-100 protein was synthesized by the intestine in any subject, and apoB-48 protein synthesis was not significantly different in the affected individuals. These data are consistent with in vivo metabolism data that show normal production rates for liver-derived apoB-100 but increased apoB-100 fractional catabolic rates in affected members of this family. Thus, the molecular defect probably does not affect transcription, translation, or secretion of apoB-containing lipoproteins, but may instead affect their clearance.
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PMID:Normal intestinal dietary fat and cholesterol absorption, intestinal apolipoprotein B (ApoB) mRNA levels, and ApoB-48 synthesis in a hypobetalipoproteinemic kindred without any ApoB truncation. 928 3

In the present studies, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor atorvastatin was used to test the hypothesis that inhibition of cholesterol biosynthesis in vivo with a consequent reduction in the availability of hepatic cholesterol for lipoprotein synthesis, would (1) reduce very low density lipoprotein (VLDL) apolipoprotein B (apoB) secretion into the plasma, (2) reduce the conversion of VLDL apoB to LDL apoB, and (3) reduce LDL apoB direct synthesis. ApoB kinetic studies were carried out in six control miniature pigs and in six animals after 21 days of administration of atorvastatin (3 mg/kg per day). Pigs were fed a fat- (34% of calories; polyunsaturated to monounsaturated to saturated ratio, 1:1:1) and cholesterol- (400 mg/d cholesterol; 0.1%; 0.2 mg/kcal) containing pig chow-based diet. Atorvastatin treatment significantly reduced plasma total cholesterol, LDL cholesterol, total triglyceride, and VLDL triglyceride concentrations by 16%, 31%, 19%, and 28%, respectively (P < .01). Autologous 131I-VLDL, 125I-LDL, and [3H]leucine were injected simultaneously into each pig, and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). The VLDL apoB pool size decreased by 29% (0.46 versus 0.65 mg/kg; P = .002), which was entirely due to a 34% reduction in the VLDL apoB production rate (PR) (1.43 versus 2.19 mg/kg per hour; P = .027). The fractional catabolic rate (FCR) was unchanged. The LDL apoB pool size decreased by 30% (4.74 versus 6.75 mg/kg; P = .0004), which was due to a 22% reduction in the LDL apoB PR (0.236 versus 0.301 mg/kg per hour; P = .004), since the FCR was unchanged. The reduction in LDL apoB PR was primarily due to a 34% decrease in conversion of VLDL apoB to LDL apoB; however, this reduction was not statistically significant (P = .114). Hepatic apoB mRNA abundance quantitated by RNase protection assay was decreased by 13% in the atorvastatin-treated animals (P = .003). Hepatic and intestinal LDL receptor mRNA abundances were not affected. We conclude that inhibition of hepatic HMG-CoA reductase by atorvastatin reduces both VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion into the plasma and not by an increase in hepatic LDL receptor expression. This decrease in apoB secretion may, in part, be due to a reduction in apoB mRNA abundance.
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PMID:Inhibition of HMG-CoA reductase by atorvastatin decreases both VLDL and LDL apolipoprotein B production in miniature pigs. 940 31

The C to U editing of apolipoprotein B (apoB) mRNA converts a glutamine codon in apoB100 mRNA into a stop translation codon thereby generating apoB48. The catalytic subunit of the editing enzyme, APOBEC-1, is an RNA-binding cytidine deaminase that requires auxiliary factors for the editing of apoB mRNA. Computer modeling and ribonuclease probing of the wild-type and mutant apoB RNA substrates reveal a stem loop at the editing site. This structure incorporates the essential sequence motifs required for editing. The localization of the edited cytidine within the loop suggests how it could be presented to the active site of APOBEC-1 for deamination. We have identified 43/45 kDa proteins from chick enterocytes and show evidence for their involvement in auxiliary editing activity. p43/45 demonstrates preferential binding to AU-rich RNA and to the Caauuug motif that forms the loop and proximal stem of the apoB mRNA.
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PMID:Secondary structure for the apolipoprotein B mRNA editing site. Au-binding proteins interact with a stem loop. 982 32

Target substrate-specific hammerhead ribozyme cleaves the specific mRNA and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To modulate apoB gene expression, we designed hammerhead ribozymes targeted at AUA(6665) and GUA(6679) of apoB mRNA, designated RB16 and RB15, respectively, and investigated their effects on apoB mRNA in HepG2 cells. The results demonstrated that RB15 and RB16 ribozyme RNAs cleaved apoB RNA efficiently in vitro. Both ribozymes, RB15 and RB16, were used to construct recombinant adenoviral vectors, designated AvRB15 and AvRB16, respectively, for in vivo gene transfer. HepG2 cells were infected with 2 x 10(5) plaque-forming units of AvRB15 for 5, 10, 15, and 24 h. An RNase protection assay showed that the expression of the RB15 transcript was time-dependent; it increased approximately 300-fold from 5 to 24 h. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site of apoB mRNA was confirmed by sequencing. Importantly, the levels of apoB mRNA in HepG2 cells decreased approximately 80% after AvRB15 infection. Pulse/chase experiments on HepG2 cells treated with AvRB15 and AvRB16 demonstrated that ribozyme cleavage produced a truncated protein that was secreted at a density of 1. 063-1.210 g/ml. The cleavage activity of RB15 on apoB mRNA was more efficient than that of RB16. Moreover, pulse/chase experiments in HepG2 cells treated with AvRB15 revealed that most of the truncated apoB protein was degraded intracellularly. We conclude that hammerhead ribozyme targeted at GUA(6679) of apoB mRNA cleaves apoB mRNA, results in decreased apoB mRNA levels, and generates a truncated apoB of the expected size in vivo. Thus, the therapeutic application of ribozyme in regulating apoB production holds promise.
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PMID:Hammerhead ribozyme cleavage of apolipoprotein B mRNA generates a truncated protein. 1044 90

In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we used adenovirus-mediated vector to target hammerhead ribozyme at GUA(6679) downward arrow of apoB mRNA (designated AvRB15) in the liver of a dyslipidemic mouse model that is deficient in apoB mRNA editing enzyme and overexpresses human apoB100. In this study, we delivered approximately 4 x 10(11) virus particles of AvRB15 (active ribozyme) or AvRB15-mutant (inactive ribozyme) to the animals. Using Southern blot analysis, we readily detected RB15 DNA in the mouse liver as long as day 35 after injection. This result was correlated with the RNA expression of RB15 by RNase protection assay. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site was confirmed by sequencing. Importantly, the levels of human and mouse apoB mRNA decreased approximately 80% after AvRB15 transduction. There was a marked decrease in plasma cholesterol, triglyceride, and human apoB of 42, 51, and 62%, respectively, when compared with the inactive ribozyme-treated group. Moreover, ribozyme cleavage of apoB mRNA generated a truncated protein of the expected size (apoB48.1), which was associated with lipoprotein particles in the very low density, low density, and high density lipoprotein fractions. Taken together, these results indicate that apoB mRNA-specific hammerhead ribozyme can be used as a potential therapeutic agent to modulate apoB gene expression and to treat hyperlipidemia.
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PMID:Hammerhead ribozyme as a therapeutic agent for hyperlipidemia: production of truncated apolipoprotein B and hypolipidemic effects in a dyslipidemia murine model. 1109 45

The expression of activation-induced cytidine deaminase (AID) is prerequisite to a "trifecta" of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA-DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC --> dU deamination activity most avidly on double-stranded DNA substrates containing a small "transcription-like" single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.
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PMID:Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. 1265 44


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