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)

We isolated two novel actin filament (F-actin)-binding proteins from rat brain and rat 3Y1 fibroblast. They were splicing variants, and we named brain big one b-nexilin and fibroblast small one s-nexilin. b-Nexilin purified from rat brain was a protein of 656 amino acids (aa) with a calculated molecular weight of 78,392, whereas s-nexilin, encoded by the cDNA isolated from rat 3Y1 cells by the reverse transcriptase-PCR method, was a protein of 606 aa with a calculated molecular weight of 71,942. b-Nexilin had two F-actin- binding domains (ABDs) at the NH2-terminal and middle regions, whereas s-nexilin had one ABD at the middle region because 64 aa residues were deleted and 14 aa residues were inserted in the first NH2-terminal ABD of b-nexilin, and thereby the first ABD lost its activity. b- and s-nexilins bound along the sides of F-actin, but only b-nexilin showed F-actin cross-linking activity. b-Nexilin was mainly expressed in brain and testis, whereas s-nexilin was mainly expressed in testis, spleen, and fibroblasts, such as rat 3Y1 and mouse Swiss 3T3 cells, but neither b- nor s-nexilin was detected in liver, kidney, or cultured epithelial cells. An immunofluorescence microscopic study revealed that s-nexilin was colocalized with vinculin, talin, and paxillin at cell- matrix adherens junction (AJ) and focal contacts, but not at cell-cell AJ, in 3Y1 cells. Overexpressed b- and s-nexilins were localized at focal contacts but not at cell-cell AJ. These results indicate that nexilin is a novel F-actin-binding protein localized at cell-matrix AJ.
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PMID:Nexilin: a novel actin filament-binding protein localized at cell-matrix adherens junction. 983 51

In an attempt to combine the human immunodeficiency virus type 1 (HIV-1)-inhibitory capacity of 2',3'-dideoxy-2',3'-didehydronucleoside analogues [nucleoside reverse transcriptase (RT) inhibitors; NRTI] and non-nucleoside RT inhibitors (NNRTI), we have designed, synthesized and evaluated for their anti-HIV activity several heterodimers of the general formula [d4T]-NH-(CH2)n-NH-[imidazo[1,5-b]pyridazine]. The synthesis of these heterodimers was conducted in three parts. The first part focused on the synthesis of the NRTI. The second part was devoted to the NNRTI and the NNRTI linked to appropriate spacers: [NNRTI]-NH-(CH2)n-NH2. In the third part, the condensation between the NRTI and the [NNRTI]-NH-(CH2)n-NH2 was performed. The in vitro inhibitory activities against HIV-1 of the [d4T]-NH-(CH2)n-NH-[imidazo[1,5-b]pyridazine] heterodimers were found to be comparable to that of d4T (stavudine) in HIV-infected cells. Moreover, the heterodimers were endowed with anti-HIV-2 activity and with anti-nevirapine-resistant HIV-1 activity. None of the heterodimers proved markedly cytotoxic to CEM-SS or MT-4 cells. There was not a clear trend toward antiviral potency on lengthening the methylene spacer in the [d4T]-NH-(CH2)n-NH-[imidazo[1,5-b]pyridazine] heterodimers.
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PMID:Imidazo[1,5-b]pyridazine-d4T conjugates: synthesis and anti-human immunodeficiency virus evaluation. 987

A 44 nucleotide DNA template containing a single site-specifically placed cisplatin adduct (cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]]) was annealed with a primer, positioning its 3'-end four bases before the adduct in the template strand. DNA polymerization in the presence of all four nucleotides revealed that both HIV-1 reverse transcriptase (RT) and T7 DNA polymerase strongly paused at one nucleotide preceding the first platinated guanine and at the positions opposite the two platinated guanines. Analysis of single nucleotide incorporation at each pause site showed that polymerization occurs with biphasic kinetics. A small percentage of DNA was bound productively, providing a small amplitude (1-3%) of a fast phase of polymerization, whereas most of the bound DNA (1-34%) was positioned at the pause site in a nonproductive manner and therefore elongated slowly (0.04-0.06 s-1). DNA substrates annealed to the cisplatin-modified template bind to HIV-1 RT with an affinity (10-20 nM) similar to that of unmodified substrates (6-9 nM). The cisplatin-DNA cross-link moderately weakened DNA binding to T7 DNA polymerase (12-115 nM) but significantly slowed the rate of incorporation of the next nucleotide (2-7 s-1 ), with larger effects closer to the cisplatin-DNA adduct. The crystal structure of the same cisplatin-DNA adduct [Takahara, P. M., Frederick, C. A., and Lippard, S. J. (1996) J. Am. Chem. Soc. 118, 12309-12321] reveals not only the bent DNA duplex but also the propeller twisted base pairs near the cisplatin-DNA adduct. The twisted base pairs may cause misalignment of the cisplatin-modified DNA at the binding cleft of T7 DNA polymerase and significantly slow the rate of the protein conformational change preceding polymerization, leading to the slight accumulation of intermediates within five base pairs of the adduct. The ground-state binding of the next correct nucleotide to the enzyme.DNA complex was weakened by the adduct with T7 DNA polymerase but unchanged with HIV-1 RT at sites other than the three strong pause sites. Nucleotide binding to both enzymes at the three strong pause sites was significantly weaker and less selective.
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PMID:Single d(GpG)/cis-diammineplatinum(II) adduct-induced inhibition of DNA polymerization. 988 12

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the beta subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms.
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PMID:Molecular analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in compost and composted materials. 992 59

The identification and study of genes expressed in hematopoietic stem/progenitor cells should further our understanding of hematopoiesis. Transcription factors in particular are likely to play important roles in maintaining the set of genes that define the stem/progenitor cell. We report here the identification of a putative KRAB-zinc finger gene (SZF1) from a cDNA library prepared from human bone marrow CD34+ cells. Characterization of SZF1 implicates its role in hematopoiesis. The predicted protein contains a highly conserved KRAB domain at the NH2 terminus and four zinc fingers of the C2H2 type at the COOH terminus. Two alternatively spliced products of SZF1 were isolated, which predict proteins of 421 (SZF1-1) and 361 (SZF1-2) amino acids, differing from each other only at the carboxy terminus. The two transcripts of SZF1 have different expression patterns. SZF1-2 is ubiquitously expressed, as indicated by Northern blot, RNase protection, and reverse transcriptase polymerase chain reaction. SZF1-1 expression, in contrast, was detected only in CD34+ cells. We recently isolated the promoter region for the stem/progenitor cell expressed FLT3/FLK-2/STK-1 gene and used this region to generate a reporter construct to test the effect of SZF1 expression. Cotransfection of the reporter construct with SZF1 constructs showed that SZF1-2 repressed transcription three- to fourfold, whereas SZF1-1 showed a lower level of repression. The expression pattern of SZF1 transcripts and the transcriptional repression of a CD34+-specific promoter demonstrate a possible role for SZF1 in hematopoietic stem/progenitor cell differentiation.
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PMID:SZF1: a novel KRAB-zinc finger gene expressed in CD34+ stem/progenitor cells. 1002 71

The polycystic kidney disease 2 (PKD2) gene, encoding a 968-amino acid integral membrane protein with six predicted membrane-spanning domains and intracellular NH2 and COOH termini, is mutated in approximately 15% of the cases of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease frequently resulting in renal failure. For a better understanding of the cause of this disorder, we searched for mutations in the PKD2 gene in two PKD2-linked families characterized by different clinical phenotypes. A common polymorphism, a nonsense mutation, and a frameshift mutation were found. Both mutations are predicted to produce truncated proteins of 314 and 386 amino acids, arrested at the first extracellular loop of the protein. Restriction enzyme analysis of polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR products, respectively, showed that mutations cosegregated with the disease and mutated alleles were expressed at the messenger RNA level in lymphoblastoid cell lines. However, in these cells, Western blot analysis showed only PKD2 normal protein, and it was expressed at a lower level than that found in cells without the PKD2 mutation. These findings suggest that in lymphoblastoid cells, the truncated protein product of the mutant allele may not be stable.
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PMID:Mutations in autosomal dominant polycystic kidney disease 2 gene: Reduced expression of PKD2 protein in lymphoblastoid cells. 1021 43

Ammonia is a neurotoxic substance which accumulates in brain in liver failure and it has been suggested that ammonia plays a key role in contributing to the astrocytic dysfunction characteristic of hepatic encephalopathy. In particular, the effects of ammonia may be responsible for the reduced astrocytic uptake of neuronally-released glutamate and high extracellular glutamate levels consistently seen in experimental models of hepatic encephalopathy. To further address this issue, [(3)H]-D-aspartate uptake was examined in primary rat cortical astrocyte cultures exposed to 5 mM ammonium chloride for a period of 7 days. In addition, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot studies were performed to examine the mRNA and protein expression respectively of the glutamate transporter GLAST in ammonia-treated cells. Studies revealed a 57% (p<0.05) decrease in [(3)H]-D-aspartate uptake and a concomitant significant decrease in GLAST transporter protein (43%, p<0.05) and mRNA (32%, p<0.05) expression. The reduced capacity of astrocytes to reuptake glutamate following ammonia exposure may result in compromised neuron-astrocyte trafficking of glutamate and could thus contribute to the pathogenesis of the cerebral dysfunction characteristic of hyperammonemic syndromes such as hepatic encephalopathy.
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PMID:Effects of ammonia on glutamate transporter (GLAST) protein and mRNA in cultured rat cortical astrocytes. 1081 9

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.
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PMID:Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase. 1135 68

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction.
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PMID:Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle. 1167 22

The reverse transcriptase inhibition of HIV-1, the most common form of HIV, by non-nucleoside 2-arylsulfonyl-6-substituted benzonitriles is analysed through Fujita-Ban and Hansch approaches. The analyses have helped to ascertain the role of different substituents in explaining the observed inhibitory actions of these compounds. From both approaches it appeared that SO2 instead of SO or S at X; and NH2 instead of F at Y (see Figure 1) are advantageous to improving the activity of a compound against HIV-1. This in turn leads to the suggestion that the 2-arylsulfonyl-6-aminobenzonitrile scaffold is the only appropriate structural entity that may further result into potential compounds. Further, the compounds having a OMe substituent at the orthoposition, the bulkier substituents at meta-positions and "no" substituent at para-position of 2-arylsulfonyl moiety are beneficial in raising the activity. The two quantitative structure-activity relationship (QSAR) analyses, differing in parametric approach, therefore, provided the grounds for rationalizing the substituent selection in designing more potent compounds of the series.
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PMID:Quantitative structure-activity relationship study of 2-arylsulfonyl-6-substituted benzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1. 1253 Apr 74


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