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)

An azidothymidine (AZT)-resistant virus strain (HIV-1/AZT) (containing the 67 Asp --> Asn, 70 Lys --> Arg, 215 Thr --> Phe and 219 Lys --> Gln mutations into its reverse transcriptase) was grown in the combined presence of 2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) and the nonnucleoside reverse transcriptase inhibitor (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dih ydroquinoxaine-2(1H)-thione (quinoxaline HBY 097). Replication of HIV-1/AZT was inhibited to a significantly greater extent by the combination of 3TC and quinoxaline HBY 097 than by either drug alone. Virus breakthrough was markedly delayed in the combined presence of 3TC and HBY 097 at drug concentrations as low as 0.05 microg/mL and 0.0025 microg/mL, respectively. The virus that was recovered after exposure to the compounds (3TC and HBY 097) individually had acquired, in the genetic AZT-resistance background of HIV-1/AZT, 103 Lys --> Glu and 106 Val --> Ala mutations. The 103 Lys --> Glu mutation had not been observed before. However, both virus mutants retained marked sensitivity to HBY 097. In all cases, the genotypic AZT-resistance mutations were maintained in the mutant virus RT genomes, and the viruses also remained phenotypically resistant to AZT. Given the exquisite potency of a concomitant combination of 3TC and HBY 097 in suppressing virus replication, this drug combination should be further pursued in clinical trials in HIV-1-infected individuals.
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PMID:Retention of marked sensitivity to (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-di hydroquin oxaline-2(1H)-thione (HBY 097) by an azidothymidine (AZT)-resistant human immunodeficiency virus type 1 (HIV-1) strain subcultured in the combined presence of quinoxaline HBY 097 and 2',3'-dideoxy-3'-thiacytidine (lamivudine). 951 72

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.
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PMID:Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke. 953 62

Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase.
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PMID:Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis. 966 81

Human neutrophils in whole blood become bipolar in shape after exposure to chemokinetic stimuli. In normal blood, the proportion of non-spherical neutrophils was 1.2 +/- 0.07% (n = 101). After incubation of blood samples with corticotropin-releasing hormone (CRF, 1 to 20 microM) 36 of 101 subjects exhibited a > or = 10% bipolar-shape ellipsoid response. This ellipsoid response was more frequent in female than in male subjects (32/75 vs. 4/26, p < 0.01). Female Caucasian subjects were more sensitive to CRF than female East Asian subjects (25/48 vs. 2/15, p < 0.01). Age was not a factor in sensitivity to CRF. In young female East Asian subjects (23 +/- 0.4 years, n = 8) that did not manifest the ellipsoid response to CRF, formyl-Met-Leu-Phe (fMLP), a chemotactic peptide, 10(-9) M increased non-spherical neutrophils to 31 +/- 0.8%. In these individuals, the fMLP response was inhibited in a dose-dependent manner by CRF. The pharmacological profile of the stimulatory and fMLP-inhibitory actions of CRF on neutrophil shape was consistent with that of a CRF1-receptor mediated response. Expression of mRNA for the CRF1-receptor was detected in hematopoietic cell lines (e.g., HL-60) using a reverse transcriptase polymerase chain-reaction method. The bipolar-shape response of human neutrophils to CRF has the potential to be a useful indicator of the functional state of this hormone-receptor system in inflammation.
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PMID:Bipolar-shape response of human neutrophils to corticotropin-releasing factor. 967 Nov 11

Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36-59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.
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PMID:Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity. 967 43

Nucleotide sequences of the reverse transcriptase (RT) coding region have been compared in four new human immunodeficiency virus type 1 (HIV-1) group O isolates. Phylogenetic analysis of this pol region highlights a cluster of these four HIV-1 group O sequences with seven other group O isolates (5% intracluster nucleotide sequence diversity) similar to clusters classified as subtypes in HIV-1 group M (an average of 4.9% intrasubtype sequence diversity). Based on these analyses, this group O cluster has been designated subtype A-O. A longitudinal study of a heterosexual couple infected with group O (ESP1 and ESP2) allowed a detailed analysis of RT sequences (amino acids 28 to 219). Directed evolution and a slightly higher mutation frequency was observed in the RT sequences of patient ESP2, treated with antiretroviral drugs, than that from the untreated patient ESP1. Antiretroviral treatment also selected for specific substitutions, M184V and T215Y in the RT coding region, conferring resistance to 3'-dideoxy-3'-thiacytidine and zidovudine, respectively. A Gly98 to Glu RT substitution identified in the treated patient suggests a possible reversion of a nonnucleoside RT inhibitor-resistant phenotype. Using RT clones from this longitudinal study, both heteroduplex tracking assay and cloning-sequencing techniques were employed for an extensive genetic analysis of pol gene quasispecies. Amino acid substitutions (i.e., Phe-77 to Leu, Lys-101 to Glu, and Val-106 to Iso) associated with antiretroviral resistance were identified in RT clones from HIV-1 group O-infected patients not subjected to drug therapy or treated with unrelated drugs. Finally, phylogenetic relationships between RT clones of the treated ESP2 patient and those of the untreated ESP1 patient show how drug pressure can direct evolution of viral pol gene quasispecies independently of direct drug-resistant substitutions.
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PMID:Analysis of pol gene heterogeneity, viral quasispecies, and drug resistance in individuals infected with group O strains of human immunodeficiency virus type 1. 976 45

To clarify the roles of alpha-thrombin and prostaglandin E2 (PGE2) in the healing and inflammatory processes of dental pulp, their effects on the DNA synthesis of human pulp cells were investigated by measurement of [3H]thymidine incorporation. At a concentration range of 1 to 25 units/ml, alpha-thrombin stimulated DNA synthesis of the pulp cells by 1.5 to 2.6-fold. On the contrary, PGE2 (> 0.05 microgram/ml) suppressed DNA synthesis by 24 to 39%. Using reverse transcriptase-polymerase chain reaction, thrombin receptor mRNA expression was identified in the pulp cells. Furthermore, alpha-thrombin-induced DNA synthesis could be inhibited by antithrombin III (2 units/ml) with heparin (2 units/ml) or D-Phe-Pro-ArgCH2Cl (50 micrograms/ml). PGE2 (0.1 to 0.5 microgram/ml) also inhibited the thrombin-induced DNA synthesis by 39 to 64%. These results imply that pulp cells express the thrombin receptor that is activated by the serine protease activity of thrombin. Interactions of thrombin and PGE2 are important in modulating the inflammatory and healing processes of the pulp.
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PMID:Thrombin-induced DNA synthesis of cultured human dental pulp cells is dependent on its proteolytic activity and modulated by prostaglandin E2. 985 18

The structure of the gene encoding the apoprotein of phytochrome B (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcription start site was found by 5' RACE at -116. Tomato PHYB1 spans 7 kb starting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino acids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% and 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse transcriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the coding region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily red-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each mutation arose from a different, single-base substitution. Allele tri1 is presumably a null because the mutation introduces a stop at codon 92. In tri3, val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri1. Alleles tri2 and tri4 encode proteins truncated at their C-termini. The former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lacks 225.
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PMID:Characterization of tomato PHYB1 and identification of molecular defects in four mutant alleles. 986 19

Tyrosine 222 of MuLV RT is an invariant residue of the highly conserved YXDD motif in the reverse transcriptase class of enzymes. The residue X is Met 184 in HIV-1 RT and Val 223 in MuLV RT. This residue has been implicated in the fidelity of DNA synthesis, whereas the role of the preceding tyrosine in this aspect, as well as in the catalytic mechanism of MuLV RT, remains to be elucidated. We have substituted Tyr 222 with Phe, Ser, and Ala by site-directed mutagenesis and have characterized the properties of the individual mutant enzymes. The results show that Tyr-->Phe substitution did not affect the polymerase activity of the enzyme, while Tyr-->Ser and Tyr-->Ala substitutions significantly reduced the polymerase activity. The pyrophosphorolysis activities of these mutants showed the same trend as the polymerase activities, suggesting an essential role for Y222 in the catalytic mechanism of MuLV RT. One of the most interesting observations of Y-->F substitution was the significantly increased fidelity of DNA synthesis on RNA templates. In addition, a limited extent of ribonucleotide incorporation on RNA template that was consistently noted with the wild-type enzyme was reduced with the Y222F mutant. The resistance to all four ddNTPs, however, persisted in the wild type and Y222 mutants on the RNA template. A ternary complex model of MuLV RT shows that (a) the aromatic ring of Tyr/Phe is positioned between the terminal and penultimate primer bases and (b) the phenolic OH group is seen within hydrogen bonding distance with the base moieties of two template and penultimate primer nucleotides. We propose that the base stacking interaction of Tyr 222 stabilizes the primer terminus position which is essential for the catalytic reaction. However, the weaker stacking interaction of Y compared to F, due to polarization of the pi-charge toward the phenoxyl-OH as well as the resonating character of its H-bond center, may provide slight flexibility to the position of the template base which may be responsible for the error-proneness of MuLV RT.
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PMID:Tyrosine 222, a member of the YXDD motif of MuLV RT, is catalytically essential and is a major component of the fidelity center. 1005 31

Cathepsin K is a cysteine protease involved in degradation of human type I collagen and plays a primary role in bone resorption. We have cloned rhesus monkey cathepsin K by reverse transcriptase-polymerase chain reaction (RT-PCR) from rhesus ovary poly A+ RNA. The sequence for the rhesus enzyme is 98% identical to that of the human with 100% identity within the mature active form of cathepsin K. Rhesus monkey cathepsin K was transiently expressed in Chinese hamster ovary (CHO) cells and found to be secreted as the proenzyme in the culture media and 50% activated to the mature form intracellularly. The substrate specificity preference of aminomethylcoumarin and rhodamine peptide substrates was Leu > Phe > Pro in the P2 position when tested with constant arginine at P1. The enzyme activity expressed in CHO cell extracts was sensitive to inhibition by E-64 and cystatin with IC50s of 3.5 nmol/L and 13 ng/mL, respectively. The apparent second order rate constants of inactivation by E-64 were 66,000 M(-1) s(-1) and 130,000 M(-1) s(-1) for the recombinantly expressed rhesus monkey and human cathepsin K, respectively. The high similarity between the sequences and the kinetic properties of rhesus monkey and human cathepsin K establishes this monkey species as a suitable animal model for development of novel cathepsin K inhibitors as antiresorptive agents.
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PMID:Cloning and expression of rhesus monkey cathepsin K. 1045 86


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