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)

Regulation of the gene expression of type-1 angiotensin II receptor (AT1) by treatment with manidipine, a calcium channel blocker, or delapril, an angiotensin converting enzyme inhibitor, for one week was assessed in the adrenal gland, heart, kidney, and brain from spontaneously hypertensive rats (SHR). Tissue AT1 receptor messenger RNA (mRNA) content was measured by reverse transcriptase-polymerase chain reaction. Treatment with manidipine (3 mg/kg/day) or delapril (30 mg/kg/day) lowered systolic blood pressure (SBP) significantly (p < 0.01) (delta SBP; -73 mmHg or -67 mmHg, respectively). Although delapril markedly increased plasma renin activity (PRA), manidipine did not alter PRA. AT1 receptor mRNA content in the adrenal gland was significantly (p < 0.01) decreased by treatment with manidipine or delapril. In contrast, cardiac AT1 receptor mRNA content was significantly (p < 0.01) increased by treatment with either agent. There was no significant change in renal and brain AT1 receptor mRNA contents. These findings suggest that although the expression of AT1 receptor gene depends on the circulating renin-angiotensin system (RAS), it is regulated independently in a tissue-specific manner via the local RAS in each tissue of SHR.
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PMID:Regulation of the gene expression of type-1 angiotensin II receptor in spontaneously hypertensive rats. 134 80

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.
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PMID:On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli. 138 56

Retroviruses encode proteinases necessary for the proteolytic processing of the viral gag and gag-pol precursor proteins. These enzymes have been shown to be structurally and functionally related to aspartyl proteinases such as pepsin and renin. Cerulenin is a naturally occurring antibiotic, commonly used as an inhibitor of fatty acid synthesis. Cerulenin has been observed to inhibit production of Rous sarcoma virus and murine leukaemia virus by infected cells, possibly by interfering with proteolytic processing of viral precursor proteins. We show here that cerulenin inhibits the action of the HIV-1 proteinase in vitro, using 3 substrates: a synthetic heptapeptide (SQNYPIV) which corresponds to the sequence at the HIV-1 gag p17/p24 junction, a bacterially expressed gag precursor, and purified 66 kDa reverse transcriptase. Inhibition of cleavage by HIV-1 proteinase required preincubation with cerulenin. Cerulenin also inactivates endothiapepsin, a well-characterised fungal aspartyl proteinase, suggesting that the action of cerulenin is a function of the common active site structure of the retroviral and aspartic proteinases. Molecular modelling suggests that cerulenin possesses several of the necessary structural features of an inhibitor of aspartyl proteinases and retroviral proteinases. Although cerulenin itself is cytotoxic and inappropriate for clinical use, it may provide leads for the rational design of inhibitors of the HIV proteinase which could have application in the chemotherapy of AIDS.
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PMID:In vitro inhibition of HIV-1 proteinase by cerulenin. 169 Jan 52

A metal binding peptide, hexahistidine, preceding a renin cleavage sequence (Pro-Phe-His-Leu-Val-Ile-His-) was engineered on to the N-terminus of HIV-1 reverse transcriptase (RT). The chimeric protein was expressed in Escherichia coli and characterized after purification by DEAE chromatography and HPLC. Amino-terminal sequencing confirmed the presence of the first 15 amino acids of the chimeric protein. The chimeric exhibited RT activity like that of HIV-1 RT and was cleaved by human renin at the expected site. The potential of a hexa-histidine fusion in the purification of recombinant HIV-1 RT by immobilized metal affinity chromatography (IMAC) on the commonly used resin (IDA-Ni2+) was investigated. The chimeric gene product from a crude E. coli extract was strongly retarded on a immobilized nickel column, while most of the contaminating E. coli proteins were eliminated after elution with 20-35 mM imidazole. The bound chimeric protein was eluted with 300 mM imidazole and appeared predominantly as a single band on an SDS-polyacrylamide gel. The remarkable specificity of this affinity tail was further demonstrated by separating the chimeric protein from HIV-1 RT in a crude extract prepared by mixing extracts from cells expressing HIV-1 RT and the hexahistidine recombinant chimeric protein. The usefulness of a enzymatically cleavable metal binding peptide in the rapid purification and production of HIV-1 RT without proteolysis to a heterodimer is discussed.
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PMID:Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain. 171 13

A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro-Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography.
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PMID:Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage. 172 60

Inbred strains of mice can be categorized into two groups based on the absence or presence of a duplicated copy of the renin structural gene; one-gene strains carry a single renin gene (Ren-1), whereas two-gene strains carry two renin genes (Ren-1 and Ren-2). To investigate the contribution that each locus makes to the composite levels of renin mRNA observed to accumulate in different tissues of two-gene strains, we have developed two assays capable of distinguishing the highly homologous Ren-1 and Ren-2 transcripts. Both methods take advantage of established base sequence differences between Ren-1 and Ren-2 coding regions by using reverse transcriptase-mediated primer extension of oligonucleotide primer/mRNA hybrids in the presence of appropriate deoxy-and dideoxynucleotide phosphates. Using these techniques we found that Ren-1 and Ren-2 mRNAs accumulate in the kidney of two-gene strains to approximately equal levels. These observations are discussed in light of potential mechanisms regulating the tissue-specific expression of the Ren-1 and Ren-2 loci.
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PMID:Ren-1 and Ren-2 loci are expressed in mouse kidney. 389 81

Activation of the renin-angiotensin system by sodium deficiency is associated with reciprocal changes in the expression of angiotensin II receptors in adrenal glomerulosa and vascular smooth muscle cells. The effects of dietary sodium changes on the expression of brain angiotensin receptor subtype 1 (AT1) mRNAs were examined in rats maintained on normal, low, and high sodium intake for 3 weeks. Plasma aldosterone and renin activity were elevated in rats maintained on a low salt diet compared with normal rats and were reduced in rats maintained on a high salt diet. These results are consistent with previous findings on the effects of altered dietary sodium on the renin-angiotensin system. The expression of AT1A and AT1B receptor subtype mRNAs was determined by quantitative reverse transcriptase-polymerase chain reaction during changes in sodium intake. The results revealed that sodium deprivation enhanced the expression of AT1B receptors in decorticated brains by 164% compared with high sodium intake. Conversely, high sodium diet increased the expression of AT1A receptors by 155% in the brain compared with low sodium intake. These data suggest that AT1A and AT1B receptors play reciprocal roles in central mechanisms for the control of fluid homeostasis. Further analysis of the molecular biology of angiotensin II receptor regulation in the brain may provide new insights into the interplay between the renin-angiotensin system and blood pressure regulation and also into the role of angiotensin II in the pathogenesis of essential hypertension.
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PMID:Regulation of angiotensin II receptors in rat brain during dietary sodium changes. 750 98

Nitric oxide (NO) has effects on renal blood flow, glomerular filtration rate, renin secretion, and renal sodium excretion. Four isoforms of nitric oxide synthase (NOS) have been cloned to date. However, the molecular identity of NOS present in the renal vasculature is unknown. Endothelial NOS (NOS-III) is regulated both acutely by cell calcium and chronically by shear stress. To determine if renal blood vessels and the glomerulus express NOS-III mRNA, we used degenerate polymerase chain reaction (PCR) to clone a portion of rat NOS-III. We then assayed NOS-III mRNA in microdissected renal structures by reverse transcriptase-PCR. NOS-III mRNA was expressed at high levels in glomeruli, arcuate vessels, and interlobular artery/afferent arterioles. NOS-III mRNA was detected inconsistently in proximal tubules, thick ascending limbs, and cortical and inner medullary collecting ducts. Previous studies have shown that chronic oral treatment with the NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) decreases NO synthesis and causes hypertension. To determine if the systemic blockade occurs only by competitive inhibition, we determined the effect of L-NAME on glomerular NOS-III mRNA. L-NAME administration (5 days) decreased NOS-III mRNA in the glomerulus to 25 +/- 12% of control levels. We conclude that endothelial NOS-III mRNA is preferentially expressed in the glomerulus and renal vasculature, where it can modulate renal blood flow and glomerular filtration rate. Furthermore, glomerular NOS-III may be modulated at the level of mRNA abundance in vivo by systemic L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization and regulation of endothelial NO synthase mRNA expression in rat kidney. 752 Jun 68

A quantitative reverse transcriptase-polymerase chain reaction for mouse renin mRNA was utilized to study the influence of classic second messenger molecules on renin mRNA levels in primary cultures of juxtaglomerular (JG) cells isolated from the kidneys of C57/B16 mice. We found that forskolin (3 microM), an activator of adenylate cyclase led to proportional increases of renin secretion and renin mRNA levels. The nitric oxide (NO) donor, sodium nitroprusside (100 microM), stimulated both renin secretion and renin gene expression, the effect on secretion being stronger than that on renin mRNA levels. An increase of the extracellular concentration of calcium from 0.5 to 3 mM led to a transient inhibition of renin secretion, followed by a marked stimulation of secretion and to a continuous suppression of renin mRNA levels. These were also decreased by the calcium ionophore A 23187 (1 microM). The membrane permeable 8-bromo-cyclic GMP (100 microM) inhibited basal renin secretion without an effect on renin mRNA levels. The phorbol ester phorbol-12-myristate-13-acetate (1 to 100 nM), which was used to stimulate protein kinase C activity, had no significant effects on renin secretion and renin mRNA levels, neither alone nor in combination with forskolin. These findings suggest that cAMP, NO and calcium are effective regulators of renin gene expression in renal JG cells, in a way that cAMP and NO are stimulators and calcium acts as an inhibitor. Moreover, in these acute experiments there appears to be no obligatory link between the secretion and the expression of renin, suggesting that both parameters are separately regulated.
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PMID:Opposite regulation of renin gene expression by cyclic AMP and calcium in isolated mouse juxtaglomerular cells. 763 56

Recent reports have shown the existence of renin in Leydig cells of the rat testis and immunoreactive renin activity in the human testis. But we could not rule out the possibility that the renin found in the testis is contamination of plasma components by endocytosis. In the present study we tried to demonstrate the renin production in the human testis by the following experiments. At first, we measured the plasma renin activity and testosterone in the internal spermatic vein simultaneously after treatment with hCG and obtained the results that suggested hCG-induced renin secretion in the human testis. Next, we showed the hCG-dependent renin production from the cultured human Leydig cell tumors in vitro. Thirdly, we demonstrated the existence of renin mRNA in the human testisusing reverse transcriptase-polymerase chain reaction (RT-PCR). Fourthly, we detected significant signals of human renin mRNA in the human testicular tissues by in situ hybridization. From the above results, it was demonstrated that the human renin gene was hCG-dependently expressed in the human testis.
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PMID:[Studies on the existence of renin in the human testis]. 841 97


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