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

---

Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The current pandemic of sexually transmitted human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide. In a systematic effort to identify a non-detergent-type antiviral spermicide, we have rationally designed and synthesized a series of cyclohexenyl thiourea (CHET) nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) with sperm-immobilizing activity (SIA). To gain further insight into the structural requirements for the optimal activity of these dual-function NNIs, we compared the effects of thiazolyl, benzothiazolyl, and pyridyl ring substitutions and functionalization with electron-donating and electron-withdrawing groups as well as the importance of thiourea and urea moieties of 15 heterocyclic ring-substituted NNIs. RT activity and p24 antigen production in HIV-infected peripheral blood mononuclear cells were used as markers of viral replication. Computer-assisted sperm analysis was used for evaluating SIA of CHET compounds. The rabbit model was used for evaluation of in vivo mucosal toxicity and contraceptive activity of the lead NNIs. Three CHET-NNIs with a bromo, chloro, or methyl substitution at the 5 position of the pyridyl ring exhibited potent anti-HIV activity at nanomolar concentrations (IC(50) = 3-5 nM) and SIA at micromolar concentrations (EC(50) = 45-96 micro M). The dual-function CHET-NNIs were potent inhibitors of drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance. Upon substitution of the sulfur atom of the thiourea moiety with an oxygen atom, the most striking difference noted was a 38-fold reduction in time required for 50% sperm immobilization (T(1/2)). A quantitative structure-activity relationship (QSAR) analysis was used in deriving regression equations between 20 physicochemical properties and SIA of NNIs. QSAR analysis showed that the T(1/2) values positively correlated with values for molecular refractivity (r = 0.88), hydrophobicity (r = 0.72), atomic polarizability (r = 0.70), and principal moment of inertia (r = 0.63) of spermicidal NNIs. A stepwise multiple regression model to describe the relationship of T(1/2) values with these four regressors provided excellent predictability (r = 0.93). Exposure of semen to thiourea/urea NNIs either alone or in combination at the time of artificial insemination led to marked or complete inhibition of pregnancy in rabbits as assessed by the number of embryo implants versus corpora lutea on Day 8 of pregnancy. Repeated intravaginal application of a gel-microemulsion with and without 0.5%, 1%, and 2% CHET-NNI or its urea analog either alone or in combination did not induce mucosal toxicity. We hypothesize that the gain of spermicidal function by CHET-NNIs is due to their metabolic oxidation to urea analogs by sperm. Three reaction pathways are discussed. The extremely rapid SIA of the urea analog as well as the broad-spectrum anti-HIV activity of spermicidal CHET-NNIs together with their lack of mucosal toxicity and the marked ability to reduce in vivo fertility is particularly useful for the clinical development of a dual-function spermicidal microbicide. The cyclohexenyl pyridyl NNIs, especially N-[2-(1-cyclohexenyl)ethyl] N'-[2-(5-bromopyridyl)]-thiourea in combination with the urea analog, show unique clinical potential as anti-HIV spermicides aimed at curbing the sexual transmission of multidrug-resistant HIV-1 while providing effective fertility control for women.
...
PMID:Structural requirements for potent anti-human immunodeficiency virus (HIV) and sperm-immobilizing activities of cyclohexenyl thiourea and urea non-nucleoside inhibitors of HIV-1 reverse transcriptase. 1244 75

A potential safety concern in biotechnology purification schemes that employ re-use of column media, often for large numbers of chromatography runs, is loss of the virus removal capacity of the chromatographic purification operation over time. To define chromatography performance attributes that best predict retrovirus clearance during extended re-use of protein A media, small-scale protein A columns were cycled 150 to 460 times using concentrates of murine hybridoma cell culture supernatants, standard low pH elution buffers and different cleaning solutions (6 M urea, 6 M guanidine, 100 mM NaOH or 500 mM NaOH). Load, flow-through and eluate samples were taken periodically and assayed for reverse transcriptase (RT, an enzyme component of retroviruses) activity, bovine IgG (a component of the culture media), genomic DNA, leached protein A, and mouse IgG. Under all cleaning conditions tested, the log,10 reduction value (LRV) of RT activity did not decrease and impurity co-elution did not increase during the 150 to 460 purification/cleaning cycles. In the two studies in which the columns were cleaned with NaOH, the chromatography performance attribute that best predicted the column media lifespan was column capacity, as measured by antibody (Ab) step yield and breakthrough. In both studies, Ab capture decayed in a biphasic manner starting at cycle 200 (100 mM NaOH) or cycle 50 (500 mM NaOH). For media cycled 300+ times using 6 M urea or 6 M guanidine cleaning buffers, column performance, including RT activity LRV, was more stable, although small upward trends in Ab breakthrough were evident. In summary, our studies identify Ab step yield and breakthrough as performance attributes that decay prior to retrovirus LRV when protein A media is multiply-cycled. Thus, we propose that virus removal validation studies should be performed on new media only and these attributes can be monitored during protein A unit operations in lieu of performing virus removal validation studies with cycled protein A media.
...
PMID:Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use. 1264 Dec 91

Soybean (Glycine max [L.] Merrill) mutant aj6 carries a single recessive lesion, aj6, that eliminates ubiquitous urease activity in leaves and callus while retaining normal embryo-specific urease activity. Consistently, aj6/aj6 plants accumulated urea in leaves. In crosses of aj6/aj6 by urease mutants at the Eu1, Eu2, and Eu3 loci, F(1) individuals exhibited wild-type leaf urease activity, and the F(2) segregated urease-negative individuals, demonstrating that aj6 is not an allele at these loci. F(2) of aj6/aj6 crossed with a null mutant lacking the Eu1-encoded embryo-specific urease showed that ubiquitous urease was also inactive in seeds of aj6/aj6. The cross of aj6/aj6 to eu4/eu4, a mutant previously assigned to the ubiquitous urease structural gene (R.S. Torisky, J.D. Griffin, R.L. Yenofsky, J.C. Polacco [1994] Mol Gen Genet 242: 404-414), yielded an F(1) having 22% +/- 11% of wild-type leaf urease activity. Coding sequences for ubiquitous urease were cloned by reverse transcriptase-polymerase chain reaction from wild-type, aj6/aj6, and eu4/eu4 leaf RNA. The ubiquitous urease had an 837-amino acid open reading frame (ORF), 87% identical to the embryo-specific urease. The aj6/aj6 ORF showed an R201C change that cosegregated with the lack of leaf urease activity in a cross against a urease-positive line, whereas the eu4/eu4 ORF showed a G468E change. Heteroallelic interaction in F(2) progeny of aj6/aj6 x eu4/eu4 resulted in partially restored leaf urease activity. These results confirm that aj6/aj6 and eu4/eu4 are mutants affected in the ubiquitous urease structural gene. They also indicate that radical amino acid changes in distinct domains can be partially compensated in the urease heterotrimer.
...
PMID:Interallelic complementation at the ubiquitous urease coding locus of soybean. 1291 38

Cirrhosis is considered to be the precursor of most hepatocellular carcinomas. To gain insight into the early molecular mechanisms of liver carcinogenesis, this study compared, using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), the expression levels of 31 selected genes in normal livers, cirrhotic nodules, and hepatocellular carcinomas. Since cirrhosis is composed of a mixture of polyclonal and monoclonal nodules, gene expression levels were also compared according to the clonal status of the cirrhotic nodules. The expression of eight of the 31 genes studied was significantly increased (NEGF2, ANGPT1, ARF, KRT19, SFN, CLDN4, MMP7, and ETV4) in cirrhotic nodules compared with normal liver, while only one was decreased (LYVE1). The same trend of variation was observed in cirrhosis and hepatocellular carcinomas for all of these genes except KRT19. When gene expression variation was compared according to the clonal status of cirrhotic nodules, only the LYVE1 expression level was significantly different. The LYVE1 gene expression level decreased progressively from polyclonal cirrhotic nodules to monoclonal cirrhotic nodules (polyclonal nodules 0.39 +/- 0.25; monoclonal nodules 0.20 +/- 0.14; p < 0.05) and to hepatocellular carcinoma (0.07 +/- 0.1). In conclusion, this study highlights the fact that among genes strongly dysregulated in hepatocellular carcinoma, some are already abnormally expressed in cirrhosis. The decrease in the expression level of one of these genes, LYVE1, was associated with monoclonality in cirrhotic nodules.
...
PMID:Quantitative RT-PCR in cirrhotic nodules reveals gene expression changes associated with liver carcinogenesis. 1451 43

Aquaporin 9 (AQP9) is a recently cloned water channel that is permeable to monocarboxylate, glycerol and urea. In rat, AQP9 has been found in testis and liver as well as in brain where its expression has been initially shown in glial cells in forebrain. However, the expression of AQP9 has not been investigated in the brainstem. The purpose of this study is to describe the distribution of AQP9-immunoreactive cells throughout the adult rat brain using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. We performed immunolabeling on brain from animals perfused with fixative and we show that AQP9 is expressed (i) in astrocytes in the glia limitans, in the white matter and in glial cells of the cerebellum, (ii) in the endothelial cells of pial vessels, and (iii) in specific groups of neurons. The neuronal AQP9 expression was almost exclusively observed in catecholaminergic cells including the adrenergic, noradrenergic and dopaminergic groups, but not in other monoaminergic neurons such as serotonergic or histaminergic cells. A slight labeling was also observed in non-catecholaminergic neurons localized in the paraventricular nucleus of the hypothalamus. These results indicate that AQP9 has a unique brain distribution with a preferential localization in catecholaminergic nuclei known to be involved in many cerebral functions. While the presence of AQP9 in glia limitans and in endothelial cells of the pial vessels could be related to water transport through the blood-brain barrier, its expression in neuronal cells, not directly involved in the osmoregulation, suggests that brain AQP9 could also be used as a metabolite channel since lactate and glycerol can be energy substrates for neurons.
...
PMID:Distribution of Aquaporin 9 in the adult rat brain: preferential expression in catecholaminergic neurons and in glial cells. 1545 Mar 51

Hemizygous deletions in genomic DNA appear to play an important role in tumorigenesis. The loss or inactivation of tumour suppressor genes (TSGs) is of critical importance in most malignancies, and has been shown to affect response to therapy. Here, we report a quantitative real-time polymerase chain reaction (qPCR) designed to detect two TSGs at the CDKN2A locus, p16(INK4A) and p14(ARF) that allows the detection of hemizygous deletions. Testing by qPCR of 18 bone marrow specimens from paediatric acute lymphoblastic leukaemia (ALL) patients at diagnosis revealed nine to be GG, six to be GD and three to be DD for exon 2 of p14(ARF)/p16(INK4A), concordant with Southern blotting analysis. A panel of 13 ALL cell lines was investigated for deletions at the CDKN2A locus and one of the lines, typed as GD for all exons, was further assessed by fluorescence in situ hybridisation, confirming the qPCR findings. The expression levels of p16(INK4A) and p14(ARF) were measured in all cell lines and these quantitative reverse transcriptase PCR results also agreed with the typing by qPCR. The qPCR method described is suitable for detection of hemizygous loss in primary patient material and the accuracy of the method was verified by three independent techniques.
...
PMID:Detection of hemizygous deletions in genomic DNA from leukaemia specimens for the diagnosis of patients. 1560 65

The molecular mechanisms of acute hepatitis C virus (HCV) infection, end-stage hepatitis (cirrhosis), and hepatocellular carcinoma have been extensively studied, but little is known of the changes in liver gene expression during the early stages of liver fibrosis associated with chronic HCV infection, that is, the transition from normal liver (NL) of uninfected patients to the first stage of liver fibrosis (F1-CH-C). To obtain insight into the molecular pathogenesis of F1-CH-C, we used real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to study the mRNA expression of 240 selected genes in liver tissue with F1-CH-C, in comparison with NL. The expression of 54 (22.5%) of the 240 genes was significantly different between F1-CH-C and NL; 46 genes were upregulated and 8 were downregulated in F1-CH-C. The most noteworthy changes in gene expression mainly affected the transcriptional network regulated by interferons (IFNs), including both IFN-alpha/beta-inducible genes (STAT1, STAT2, ISGF3G/IRF9, IFI27, G1P3, G1P2, OAS2, MX1) and IFN-gamma-inducible genes (CXCL9, CXCL10, CXCL11). Interesting, upregulation of IFN-alpha/beta-inducible genes (but not IFN-gamma-inducible genes) was independent of histological scores (grade and stage of fibrosis) and HCV characteristics (hepatic HCV mRNA levels and the HCV genotype), and was specific to HCV (as compared to hepatitis B virus (HBV)). Other genes dysregulated in F1-CH-C, albeit less markedly than IFN-alpha/beta- and IFN-gamma-inducible genes, were mainly involved in the activation of lymphocytes infiltrating the liver (IFNG, TNF, CXCL6, IL6, CCL8, CXCR3, CXCR4, CCR2), cell proliferation (p16/CDKN2A, MKI67, p14/ARF), extracellular matrix remodeling (MMP9, ITGA2), lymphangiogenesis (XLKD1/LYVE), oxidative stress (CYP2E1), and cytoskeleton microtubule organization (STMN2/SCG10). Thus, a limited number of signaling pathways, and particularly the transcriptional network regulated by interferons, are dysregulated in the first stage of HCV-induced liver fibrosis. Some of the genes identified here could form the basis for new approaches aimed at refining IFN-based therapies for chronic HCV infection.
...
PMID:Molecular profiling of early stage liver fibrosis in patients with chronic hepatitis C virus infection. 1566 Nov 46

The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by second-generation NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the beta7-beta8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.
...
PMID:The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors. 1583 34

Amino acids N137 and P140 in the p51 subunit of HIV-1 reverse transcriptase (RT) are part of the beta7-beta8-loop that contributes to the formation of the base of the non-nucleoside RT inhibitor (NNRTI)-binding pocket and makes up a substantial part of the dimerization interface. Amino acid P95 in p66 also markedly contributes to the dimerization binding energy. Nine RT mutants at amino acid 137 were constructed bearing the mutations Y, K, T, D, A, Q, S, H or E. The prolines at amino acid positions 95 and 140 were replaced by alanine in separate enzymes. We found that all mutant RT enzymes showed a dramatically decreased RNA-dependent DNA polymerase activity. None of the mutant RT enzymes showed marked resistance against any of the clinically used NNRTIs but they surprisingly lost significant sensitivity for NRTIs such as ddGTP. The denaturation analyses of the mutant RTs by urea are suggestive for a relevant role of N137 in the stability of the RT heterodimer and support the view that the beta7-beta8 loop in p51 is a hot spot for RT dimerization and instrumental for efficient polymerase catalytic activity. Consequently, N137 and P140 in p51 and P95 in p66 should be attractive targets in the design of new structural classes of RT inhibitors aimed at compromising the optimal interaction of the beta7-beta8 loop in p51 at the p66/p51 dimerization interface.
...
PMID:The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti-HIV-1 drugs. 1584 61

Our project is designed to clone a 1.3kb gene fragment of telomerase catalytic subunit gene which contains seven reverse transcriptase motifs and specific region with conserved sequence termed "T motif". The gene fragment was amplified by PCR and was inserted into expression vector pET28-b. The recombinant plasmid was induced by IPTG for 4h and a 52KD recombinant protein was produced. Amount of hTRT recombinant protein expression was 20% of total bacterial protein in the form of inclusion. Inclusion was dissolved in 8 mol/L urea and 10 mmol/L DTT and carried out affinity purification under denaturing condition. The purified hTRT recombinant protein was conformed by Western-blot successfully.
...
PMID:[Expression and purification of telomerase resever transcriptase motifs in E.coli]. 1597 68


<< Previous 1 2 3 4 5 6 7 Next >>

---