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)

High glucose (HG) is the underlying factor contributing to long term complications of diabetes mellitus. The molecular mechanisms transforming the glomerular mesangial cell phenotype to cause nephropathy including diacylglycerol-sensitive protein kinase C (PKC) are still being defined. Reactive oxygen species (ROS) have been postulated as a unifying mechanism for HG-induced complications. We hypothesized that in HG an interaction between ROS generation, from NADPH oxidase, and PKC suppresses mesangial Ca2+ signaling in response to endothelin-1 (ET-1). In primary rat mesangial cells, growth-arrested (48 h) in 5.6 mM (NG) or 30 mm (HG) glucose, the total cell peak [Ca2+]i response to ET-1 (50 nM) was 630 +/- 102 nM in NG and was reduced to 159 +/- 15 nM in HG, measured by confocal imaging. Inhibition of PKC with phorbol ester down-regulation in HG normalized the ET-1-stimulated [Ca2+]i response to 541 +/- 74 nM. Conversely, an inhibitory peptide specific for PKC-zeta did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-beta or novel PKC-delta in NG diminished the [Ca2+]i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47phox antisense oligonucleotide normalized the [Ca2+]i response to ET-1 in HG to 521 +/- 58 nM and 514 +/- 48 nM, respectively. Pretreatment with carbonyl cyanide m-chlorophenylhydrazone or rotenone did not restore Ca2+ signaling in HG. Detection of increased intracellular ROS in HG by dichlorofluorescein was inhibited by catalase, diphenyleneiodonium, or p47phox antisense oligonucleotide. HG increased p47phox mRNA by 1.7 +/- 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-beta and -delta, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-delta visualized by confocal imaging was attenuated by diphenyleneiodium chloride. Thus, mesangial cell [Ca2+]i signaling in response to ET-1 in HG is attenuated through an interaction mechanism between NADPH oxidase ROS production and diacylglycerol-sensitive PKC.
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PMID:High glucose-suppressed endothelin-1 Ca2+ signaling via NADPH oxidase and diacylglycerol-sensitive protein kinase C isozymes in mesangial cells. 1282 78

Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated beta-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with L-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-L-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with L-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress.
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PMID:Inhibition of c-Myc oncoprotein limits the growth of human melanoma cells by inducing cellular crisis. 1282 59

Eosinophil recruitment to airway tissue is a key feature of asthma, and release of a wide variety of toxic mediators from eosinophils leads to the tissue damage that is a hallmark of asthma pathology. Factors that control the release of these toxic mediators are targets for potential therapeutic intervention. Protease-activated receptors (PARs) are a novel class of receptors that are activated by cleavage of the N terminus of the receptor by proteases such as thrombin or trypsin-like enzymes. To date, PAR1-4 have been identified, and there are several studies that have demonstrated the expression of PARs in airway tissue, particularly the respiratory epithelium. We have investigated whether eosinophils express PARs and if activation of these receptors will then trigger a functional response. Using a combination of reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometry analysis, we have demonstrated that eosinophils express PAR1 and PAR2. FACS analysis showed that PAR1 could be clearly detected on the surface of the cells, whereas PAR2 appeared to be primarily intracellular. Trypsin and the PAR2 agonist peptide were seen in trigger shape change, release of cysteinyl leukotrienes, and most obviously, generation of reactive oxygen species. In contrast, thrombin had no effect on eosinophil function. The PAR1 agonist peptide did have a minor effect on eosinophil function, but this was most likely down to its ability to activate PAR1 and PAR2. These results demonstrate that PAR2 is the major PAR receptor that is capable of modulating eosinophil function.
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PMID:Expression of and functional responses to protease-activated receptors on human eosinophils. 1283 43

The thymidine 5'-triphosphate analogue containing a methylene group in place of the 5' oxygen atom can be prepared using modifications of published procedures and can substitute for the natural thymidine triphosphate in chain extension reactions catalyzed by Moloney-MLV reverse transcriptase. Using rabbit beta-globin mRNA as the template together with an appropriate primer, the enzyme readily makes full-length DNA transcripts in which all thymidine 5' oxygen atoms have been replaced with methylene groups. In sequence analyses using the partial depurination procedure, the analogue DNA transcript produces electrophoretic gel patterns identical with those of the corresponding natural DNA transcript. Experiments on second strand synthesis using the four regular triphosphates show that the analogue DNA transcript, like the natural transcript, can serve as a template. The two DNA duplexes (natural/natural and analogue/natural) formed by these reactions produce similar electrophoretic cleavage patterns when treated with either of the endonucleases HaeIII and EcoRI. However, further studies on template properties indicate that, while the enzyme makes a full-length product when using the analogue substrate with a natural DNA strand as template, it appears unable to use the analogue transcript as template with the analogue triphosphate as substrate during second strand synthesis. Preliminary experiments have also been carried out with a DNA polymerase. No products are detected reactions using Taq polymerase with PCR protocols containing the analogue triphosphate as the only source of thymidine.
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PMID:The analogue of thymidine triphosphate containing a methylene group in place of the 5' oxygen can serve as a substrate for reverse transcriptase. 1288 22

The exact mechanism of lipoatrophy remains unclear. One hypothesized mechanism is accumulation of reactive oxygen free radicals, which is possibly related to dysfunctional mitochondria. We evaluated plasma levels of F2-isoprostanes-the most accurate method to measure oxidant stress in vivo-in a group of 59 nucleoside reverse transcriptase inhibitor-treated HIV-1-infected subjects. All had serial measurements of venous lactate levels as well as clinical evaluations for assessment of lipoatrophy and symptoms of mitochondrial toxicity. Overall, 16 subjects had sustained hyperlactatemia (4 of whom were symptomatic) and 43 had serial normal lactate levels. We found a significant increase in circulating products of lipid peroxidation, F2-isoprostanes (nanograms per milliliter), in subjects with lipoatrophy when compared with subjects without lipoatrophy (0.060 +/- 0.025 vs. 0.0420 +/- 0.02, respectively; P = 0.02). Interestingly, there was no significant difference in F2-isoprostane levels (nanograms per milliliter) between patients with persistently normal lactate and those who exhibited a sustained asymptomatic hyperlactatemia (0.053 +/- 0.027 vs. 0.053 +/- 0.021, respectively; P > 0.05). This could be explained by the yet unclear significance of asymptomatic hyperlactatemia, even in a setting like ours, where lactate levels were measured with close attention to the method of collection and processing. In contrast, the 4 subjects with symptomatic hyperlactatemia/lactic acidosis had a significant increase in their F2-isoprostanes compared with subjects with asymptomatic sustained hyperlactatemia (0.082 +/- 0.021 vs. 0.053 +/- 0.021, respectively; P < 0.05).
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PMID:Lipid oxidative markers are significantly increased in lipoatrophy but not in sustained asymptomatic hyperlactatemia. 1450 92

Two different mechanisms for Mg-protoporphyrin monomethyl ester (MgPMe) cyclization are shown to coexist in Rubrivivax gelatinosus and are proposed to be conserved in all facultative aerobic phototrophs: an anaerobic mechanism active under photosynthesis or low oxygenation, and an aerobic mechanism active only under high oxygenation conditions. This was confirmed by analyzing the bacteriochlorophyll accumulation in the wild type and in three mutant strains grown under low or high aeration. A mutant lacking the acsF gene is photosynthetic, exhibits normal bacteriochlorophyll accumulation under low oxygenation and anaerobiosis, and accumulates MgPMe under high oxygenation. The photosynthesis-deficient bchE mutant produces bacteriochlorophyll only under high oxygenation and accumulates MgPMe under low oxygenation and anaerobiosis. The double knockout mutant is devoid of photosystem and accumulates MgPMe under both conditions indicating the involvement of the two enzymes at the same step of the biosynthesis pathway. Oxygen-mediated expression of bchE was studied in the wild type and in a regulatory mutant. The reverse transcriptase-PCR and the bchE promoter activity results demonstrate that the expression of the bchE gene is oxygen-independent and suggest that it is rather the enzyme activity that should be oxygen-sensitive. No obvious sequence similarities were found between oxygen-dependent AcsF and the oxygen-independent anaerobic Mg-protoporphyrin monomethylester cyclase (BchE) enzymes. However, common to all BchE proteins is the conserved CXXX-CXXC sequence. This motif is essential for 4Fe-4S cluster formation in many anaerobic enzymes. Expression and purification of BchE were achieved, and the UV-visible spectral analyses confirmed the presence of an active 4Fe-4S cluster in this protein. The use of different classes of enzymes catalyzing the same reaction under different oxygen growth conditions appears to be a common feature of different biosynthetic pathways, and the benefit of possessing both aerobic and anaerobic systems is discussed.
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PMID:Aerobic and anaerobic Mg-protoporphyrin monomethyl ester cyclases in purple bacteria: a strategy adopted to bypass the repressive oxygen control system. 1461 30

Retroviral conversion of single-stranded RNA into double-stranded DNA requires priming for each strand. While host cellular t-RNA serves as primer for the first strand, the viral polypurine tract (PPT) is primer for the second. Therefore, polypurine tracts of retroviruses are essential for viral replication by reverse transcriptase (RT). These purine tracts are resistant to cleavage during first strand synthesis. In obtaining the primer for second strand synthesis, the RNase H function of RT must cleave the PPT exactly for in vivo transcription to proceed efficiently and proper integration to occur. At the RNase H active site the protein makes contacts primarily along the backbone, with hydrogen bonds to the sugar-phosphate oxygen atoms. A high-resolution structure (1.10A) of the first ten base-pairs of the RNA/DNA hybrid PPT, r-(c-a-a-a-g-a-a-a-a-g)/d-(C-T-T-T-T-C-T-T-T-G), contains the highly deformable r-(a-g-a) steps found in retroviral polypurine tracts. This r-(a-g-a) motif is utilized in the "unzipping" or unpairing of bases that occurs when RT binds a malleable PPT. Another unusual feature found in our high-resolution PPT structure is the sugar switch at RNA adenine 2. All the RNA sugars are the expected C3'-endo, except sugar 2, which is C2'-endo, characteristic of B-form sugars. This local A-to-B conversion adversely affects the pattern of hydrogen bonds from protein to sugar-phosphate backbone, disrupting the catalytic site. Disruption could cause the enzyme to pause at the 5'-end of the PPT, leaving it intact. Pyrimidine-purine (YR) steps are most deformable and the T-A step especially can undergo A-to-B transitions readily.
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PMID:An unusual sugar conformation in the structure of an RNA/DNA decamer of the polypurine tract may affect recognition by RNase H. 1463 94

Hemoglobin II from the clam Lucina pectinata is an oxygen-reactive protein with a unique structural organization in the heme pocket involving residues Gln65 (E7), Tyr30 (B10), Phe44 (CD1), and Phe69 (E11). We employed the reverse transcriptase-polymerase chain reaction (RT-PCR) and methods to synthesize various cDNA(HbII). An initial 300-bp cDNA clone was amplified from total RNA by RT-PCR using degenerate oligonucleotides. Gene-specific primers derived from the HbII-partial cDNA sequence were used to obtain the 5' and 3' ends of the cDNA by RACE. The length of the HbII cDNA, estimated from overlapping clones, was approximately 2114 bases. Northern blot analysis revealed that the mRNA size of HbII agrees with the estimated size using cDNA data. The coding region of the full-length HbII cDNA codes for 151 amino acids. The calculated molecular weight of HbII, including the heme group and acetylated N-terminal residue, is 17,654.07 Da.
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PMID:The cDNA-derived amino acid sequence of hemoglobin II from Lucina pectinata. 1471 36

The release of the complete genome sequence of Arabidopsis enabled the largest sucrose synthase family described to date, comprising six distinct members, for which expression profiles were not yet available, to be identified. Aimed at understanding the precise function of each AtSUS member among the family, a comparative study of protein structure was performed, together with an expression profiling of the whole gene family using the technique of real-time quantitative reverse transcriptase-polymerase chain reaction. Transcript levels were analysed in several plant organs, including both developing and germinating seeds. A series of treatments such as oxygen deprivation, dehydration, cold treatment, or various sugar feedings were then carried out to characterize the members of the family further. The AtSUS genes exhibit distinct but partially redundant expression profiles. Under anaerobic conditions, for instance, both AtSUS1 and AtSUS4 mRNA levels increase, but in a distinct manner. AtSUS2 is specifically and highly induced in seeds at 12 d after flowering and appears as a marker of seed maturation. AtSUS3 seems to be induced in various organs under dehydration conditions including leaves deprived of water or submitted to osmotic stress as well as late-maturing seeds. AtSUS5 and AtSUS6 are expressed in nearly all plant organs and do not exhibit any transcriptional response to stresses. These results add new insights on the expression of SUS genes and are discussed in relation to distinct functions for each member of the AtSUS family.
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PMID:Structure and expression profile of the sucrose synthase multigene family in Arabidopsis. 1473 63

A newly isolated denitrifying bacterium, Thauera sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (tod) and benzylsuccinate synthase (bss), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the tod genes were induced under aerobic conditions, whereas the bss genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.
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PMID:Aerobic and anaerobic toluene degradation by a newly isolated denitrifying bacterium, Thauera sp. strain DNT-1. 1500 57


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