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)

Recombinant HIV-1 p66 (rp66, a subunit of reverse transcriptase (RT), a heterodimer of p66 and p51) was produced in Escherichia coli in three different ways. First, rp66 was produced as a part of the fusion protein of lacZ protein and HIV-1 pol protein consisting of three components: protease (p10), RT (p51/p66), and integrase (p31), and was released from the fusion protein by the protease (pol-rp66). Second, rp66 with Ser-Ser at the N-terminus was produced as a fusion protein with maltose-binding protein containing a factor Xa site between the two proteins (MBP-Ser-Ser-rp66) and was released from the fusion protein by factor Xa (Ser-Ser-rp66). Third, rp66 with Met-Gly at the N-terminus was produced in transformed cells (Met-Gly-rp66). The recombinant proteins were purified from sonic extracts of transformed cells by ammonium sulfate fractionation and various column chromatographies. MBP-Ser-Ser-rp66 and Met-Gly-rp66 were readily purified in sufficient amounts for labeling with 2, 4-dinitrophenyl groups and beta-D-galactosidase from E. coli, but pol-rp66 and Ser-Ser-rp66 were not for enzyme-labeling. Ser-Ser-rp66 was not only polymerized but also degraded to considerable extents. The purified preparations were labeled with 2,4-dinitrophenyl groups and beta-D-galactosidase and were tested in immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 RT using serum samples from 600 HIV-1 seronegative and 30 HIV-1 seropositive subjects. Among various combined uses of the two labeled preparations, the uses of 2,4-dinitrophenylated MBP-Ser-Ser-rp66 and pol-rp66 with beta-D-galactosidase-labeled Met-Gly-rp66 showed the highest (99.8%) and the second highest (99.5%) specificities, which were higher than that with the labeled preparations used in the previous study (98. 0%).
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PMID:Preparations of recombinant HIV-1 p66 antigen to improve the specificity of immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 reverse transcriptase. 1090 70

From the roots of the Chinese ginseng Panax ginseng a protein designated panaxagin with ribonuclease activity, but possessing a sequence distinct from ribonucleases previously reported from ginseng calluses, was isolated. The purification protocol employed comprised extraction with cold saline, (NH4)2SO4 precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and gel filtration on Superdex 75 by fast protein liquid chromatography. The purified protein was composed of two identical subunits each with a molecular weight of 26 kDa. Its N-terminal amino acid sequence exhibits sites of similarity with the sequences of plant ribosome inactivating proteins and fungal ribonucleases. The spectrum of biological activities of panaxagin encompassed ribonuclease activity toward yeast transfer RNA, translation-inhibitory activity in a rabbit reticulocyte lysate system, and antifungal activity against fungi including Coprinus comatus and Fusarium oxysporum, but not against Rhizoctonia solani. In addition it displayed an inhibitory activity against human immunodeficiency virus reverse transcriptase and succinylation augmented this activity.
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PMID:Panaxagin, a new protein from Chinese ginseng possesses anti-fungal, anti-viral, translation-inhibiting and ribonuclease activities. 1120 66

The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a sigma(54)-type promoter. Shorter transcripts sequentially missing genes of the 3' part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% +/- 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid "switch-off" of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.
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PMID:Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp. strain BH72. 1137 40

Transgenic plants of Arabidopsis bearing the spinach (Spinacia oleracea) nitrite reductase (NiR, EC 1.7.7.1) gene that catalyzes the six-electron reduction of nitrite to ammonium in the second step of the nitrate assimilation pathway were produced by use of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator. Integration of the gene was confirmed by a genomic polymerase chain reaction (PCR) and Southern-blot analysis; its expression by a reverse transcriptase-PCR and two-dimensional polyacrylamide gel electrophoresis western-blot analysis; total (spinach + Arabidopsis) NiR mRNA content by a competitive reverse transcriptase-PCR; localization of NiR activity (NiRA) in the chloroplast by fractionation analysis; and NO(2) assimilation by analysis of the reduced nitrogen derived from NO(2) (NO(2)-RN). Twelve independent transgenic plant lines were characterized in depth. Three positive correlations were found for NiR gene expression; between the total NiR mRNA and total NiR protein contents (r = 0.74), between the total NiR protein and NiRA (r = 0.71), and between NiRA and NO(2)-RN (r = 0.65). Of these twelve lines, four had significantly higher NiRA than the wild-type control (P < 0.01), and three had significantly higher NO(2)-RN (P < 0.01). Each of the latter three had one to two copies of spinach NiR cDNA per haploid genome. The NiR flux control coefficient for NO(2) assimilation was estimated to be about 0.4. A similar value was obtained for an NiR antisense tobacco (Nicotiana tabacum cv Xanthi XHFD8). The flux control coefficients of nitrate reductase and glutamine synthetase were much smaller than this value. Together, these findings indicate that NiR is a controlling enzyme in NO(2) assimilation by plants.
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PMID:Nitrite reductase gene enrichment improves assimilation of NO(2) in Arabidopsis. 1140 1

The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.
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PMID:Asialofetuin liposome-mediated human alpha1-antitrypsin gene transfer in vivo results in stationary long-term gene expression. 1140 12

Thai bitter gourd protein (MRK29) was isolated from Momordica charantia ripe fruit and seed. The purification was performed by ammonium sulfate fractionation and gel filtration chromatography. MRK29 possessed one isoelectric point of (pI) > or = 9, and the time of flight mass spectrum (TOFMS) indicated its molecular weight at 28.6 kD. The twenty amino acid sequence from the N-terminus was in the following order: 1Asp Val Asn Phe Arg Leu Ser Gly Ala 10Asp Pro Arg X Tyr Gly Met Phe Ile Glu 20Asp. MRK29 inhibited the HIV-1 reverse transcriptase with 50% IR at the concentration of 18 micrograms/ml. MRK29 was concentrated in the 30-60% salt precipitated fraction, at which the concentration of 0.175 microgram/ml exerted 82% reduction of viral core protein p24 expression in HIV-infected cells. MRK29 might have modulatory role on immune cells, because it increased 3-fold TNF activity.
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PMID:HIV inhibitor from Thai bitter gourd. 1145 53

The chemokine receptors CXCR4 and CCR5 are used as co-receptors by the T cell-tropic (X4) and macrophage-tropic (R5) HIV-1 strains, respectively, for entering their host cells. Viral entry can be inhibited by the natural ligands for CXCR4, the CXC chemokine SDF-1 and CCR5, the CC chemokines RANTES, MIP-1alpha and MIP-1beta. Several peptidic compounds, T22 (an 18-mer), T134 (a 14-mer), ALX40-4C (a 9-mer) and CGP 64222 (also a 9-mer), have been identified as CXCR4 antagonists and show anti-HIV activity. Also, the HIV-1 tat protein has been described as a 'natural' CXCR4 antagonist with anti-HIV-1 activity. The most potent and specific CXCR4 antagonists are the bicyclam derivatives, which also potently block X4 HIV replication. AMD3100 has proved to be a highly specific CXCR4 antagonist, which consistently blocks the outgrowth of all X4 HIV and dual-tropic (R5/X4) variants that use CXCR4 for entering the cells (cell lines, CXCR4-transfected cell lines, lymphocytes or monocytes/ macrophages). From the bicyclam analogues, AMD3100 was selected as the clinical drug candidate, which, after initial Phase I (safety) studies, has proceeded to Phase II (efficacy) trials. The first non-peptidic compound that interacts with CCR5, and not with CXCR4, is a quaternary ammonium derivative, called TAK-779, which also has potent but variable anti-HIV activity. We believe that HIV entry/fusion inhibitors will become important new antiviral agents to combat AIDS. However, like the current clinically approved agents, they will need to be used in combinations consisting of antivirals that target other aspects of the HIV replication cycle, such as reverse transcriptase and protease, to obtain optimum therapeutic effects.
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PMID:Inhibition of HIV infection by CXCR4 and CCR5 chemokine receptor antagonists. 1159 85

A mannose-binding lectin was isolated from the inner shoots of the chive Allium tuberosum. The procedure involved aqueous extraction, (NH4)2SO4 precipitation, dialysis to remove (NH4)2SO4, affinity chromatography on mannose-agarose, ion exchange chromatography on SP-Sepharose, gel filtration on Superdex 75, and ion exchange chromatography on Mono S. Lectin activity was adsorbed on mannose-agarose, SP-Sepharose, and Mono S. The lectin demonstrated a molecular weight of 13 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, indicating that it is a single-chain protein. N-terminal sequence analysis revealed its remarkable homology to Allium cepa lectin and similarity to a lesser extent to lectins from members of the Amaryllidaceae, Orchidaceae, and Liliaceae. The lectin manifested mitogenic activity in murine splenocytes and inhibitory activity against human immunodeficiency virus type 1 reverse transcriptase.
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PMID:A monomeric mannose-binding lectin from inner shoots of the edible chive (Allium tuberosum). 1173 87

NifA, the transcriptional activator of nitrogenase (nif) genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the gamma-subgroup of the PROTEOBACTERIA: This paper reports a functionally similar NifL-like protein outside this group in Azoarcus sp. strain BH72, a diazotrophic grass endophyte belonging to the beta-subgroup of the PROTEOBACTERIA: Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O(2) and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an Azoarcus sp. BH72 genomic library was used to select cosmids that complemented a nifA mutation in Azotobacter vinelandii. Sequence analysis of the 3.4 kb genomic region complementing nifA showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of Azotobacter vinelandii. According to Northern blot and reverse transcriptase PCR analysis, the nifLA transcript was more abundant at low combined nitrogen and O(2) levels, results which were corroborated by GUS (beta-glucuronidase) assays using a transcriptional nifL::gusA fusion. N(2) fixation was abolished in a NifLA(-) and a NifA(-) mutant, wild-type fixation being restored by nifLA in trans. The NifLA(-) mutant also failed to activate nifH::gus expression, indicating that NifA is the obligate transcriptional activator for nifHDK. A nifL mutant was diazotrophic and did not show repression of nifH::gusA by ammonium or O(2), suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O(2) and combined nitrogen as NifL in bacteria of the gamma-PROTEOBACTERIA:
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PMID:Identification of a NifL-like protein in a diazotroph of the beta-subgroup of the Proteobacteria, Azoarcus sp. strain BH72. 1236 54

HIV entry within the cell involves the presence of at least two chemokine co-receptors, the CCR5 and CXCR4 receptors. Viral entry can be inhibited by the natural ligands for CXCR4, the CXC chemokine SDF-1 and CCR5, the CC chemokines RANTES, MIP-1alpha and MIP-1beta, respectively. Much research has been devoted ultimately to the development of small molecule chemokine antagonists that inhibit virus entry within the cell, and constitute in this way novel antiviral medications. The most potent and specific CXCR4 antagonists reported up to now are the bicyclam derivatives, which also potently block X4 HIV replication. One such compound, AMD3100 has proved to be a highly specific CXCR4 antagonist, which consistently blocks the outgrowth of all X4 HIV and dual-tropic (R5/X4) variants that use CXCR4 for entering the cells. From such bicyclam analogues, AMD3100 was selected as the clinical candidate, which, after initial Phase I studies, proceeded to Phase II trials, but unfortunately showed significant cardiac side effects which lead to its withdrawal from further development. The first nonpeptidic compound that interacts with CCR5, but not with CXCR4, is a quaternary ammonium derivative, TAK-779, which also shows potent but variable anti-HIV activity. A large number of potent CCR5 antagonists from several classes of polycyclic derivatives have been recently disclosed. Many such derivatives showed nanomolar binding affinity to the receptor, and at least one of them, the oxime-piperidine derivative SCH-351125 has progressed to clinical evaluation. The development of such agents for clinical use may constitute an additional approach for the treatment of HIV infection, in addition to the classical one involving reverse transcriptase and protease inhibitors.
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PMID:Non-peptidic chemokine receptors antagonists as emerging anti-HIV agents. 1242 Jul 52


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