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At least 253 records · Page 14Linked to original sources
PubMed1991

Chimeric human immunodeficiency virus type 1/type 2 reverse transcriptases display reversed sensitivity to nonnucleoside analog inhibitors.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), an important therapeutic target in the treatment of AIDS, is effectively inhibited by a class of nonnucleoside analog compounds that includes nevirapine (BI-RG-587) and tetrahydroimidazo[4,5,1-jk]-[1,4]benzodiazepin-2(1H)-one and -thione. We show that both tyrosine residues at positions 181 and 188 flanking the putative catalytic site of HIV-1 RT are required for sensitivity of the enzyme to these compounds. HIV-2 RT, which does not have tyrosines at these positions, is resistant to these nonnucleoside analog inhibitors. Substitution of the HIV-2 RT amino acid residues at position 181 or 188 into HIV-1 RT results in an enzyme that is resistant to these compounds while retaining sensitivity to 3'-azido-2',3'-dideoxythymidine triphosphate. HIV-2 RT substituted with amino acids 176-190 from HIV-1 RT acquires sensitivity to these nonnucleoside analog inhibitors.

Affinity Labels↗
PubMed2002

Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography.

A rapid (less than 30 min), sensitive, and specific liquid chromatography method for simultaneous assay of nine antiretroviral drugs in human plasma is described. This technique allows therapeutic drug monitoring of six approved protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and two approved non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). Assays were performed after diethyl ether liquid-liquid extraction from 250-microL plasma samples. Chromatographic separation was achieved on an X-TERRA (Waters; Saint Quentin, France) column using a 58% water (with 3 mmol/L pyrrolidine) and 42% acetonitrile mobile phase. Three ultraviolet wavelengths were used for detection with a diode array detector. This method allowed quantitative assay of all nine antiretroviral drugs within a concentration range of 25 ng/mL to 9000 ng/mL. The method has been validated extensively and has been in routine use in our laboratory for several months for drug monitoring in plasma samples from patients treated with antiretroviral drugs.

Chromatography, High Pressure Liquid↗
PubMed2002

Detection procedures for neuroblastoma cells metastatic to blood and bone marrow: blinded comparison of chromogranin A heminested reverse transcription polymerase chain reaction to tyrosine hydroxylase nested reverse transcription polymerase chain reaction and to anti-GD2 immunocytology.

Specific and sensitive tumor cell detection is becoming increasingly important for diagnosing and staging as well as for the therapeutic management of neuroblastoma patients. We propose a chromogranin A heminested reverse transcription polymerase chain reaction (CgA hn RT-PCR) procedure for the detection of neuroblastoma minimal residual disease in peripheral blood and bone marrow samples. The results were checked in comparison with the presently available procedures (i.e., with the tyrosine hydroxylase nested RT-PCR [TH n RT-PCR] and with the immunocytochemical approach using anti-GD2 antibodies). Controls from healthy patients or from people with unrelated disease (12 samples of bone marrow and 23 samples of peripheral blood) and serial dilution experiments using neuroblastoma cell lines (SKNLP, SKNFI, STA6, STA8) showed CgA hn RT-PCR full specificity and sensitivity ranging from 10(3) to 10(6) (depending on the cell line). The results compared favorably with those obtained using TH n RT-PCR. Preliminary data obtained analyzing bone marrow and peripheral blood specimens from stage IV neuroblastomas showed substantially overlapping results between CgA and TH n RT-PCR procedures. Our data support the potential usefulness of CgA heminested RT-PCR as a specific and sensitive procedure for minimal disease detection in neuroblastoma. A prospective evaluation of this tool in clinical studies might be warranted.

Adolescent↗
PubMed2004

Phenotypic impact of HIV reverse transcriptase M184I/V mutations in combination with single thymidine analog mutations on nucleoside reverse transcriptase inhibitor resistance.

OBJECTIVES: To analyse the impact of the M184I/V mutation and individual thymidine-associated mutations (TAM) on nucleoside reverse transcriptase inhibitor (NRTI) phenotypic susceptibility and compare these results with those obtained using commercial and public algorithms. DESIGN: An HIV genotypic/phenotypic database with over 27 000 samples was used to obtain the median fold change (5-95th percentile) in NRTI phenotypic susceptibility for viruses from patients containing individual TAM with or without the M184I or V mutation and for wild-type patient viruses. RESULTS: The resulting data indicated that in vitro, individual TAM do not have an equivalent impact on NRTI resistance, with some individual TAM having little or no impact on NRTI resistance (e.g. M41L or K219Q/E/H/R). In the presence of the M184I/V mutation, re-sensitization to some drugs, including zidovudine, stavudine and tenofovir was observed despite the presence of a TAM. For didanosine and abacavir, the presence of the M184V mutation and a single TAM did not result in a fold-change increase associated with decreased drug susceptibility. Analysis of public and commercial algorithms revealed a lack of concordance regarding the impact of these mutations, and with the observed phenotypic data. CONCLUSION: These analyses should assist in the creation of rules for genotypic drug resistance algorithms for a better reflection of the impact of individual TAM and also the impact of M184I/V on resistance. These data provide additional evidence that retaining lamivudine in those treatment regimens in which TAM can be selected may provide some therapeutic benefit by maintaining the M184V mutation.

Adenine↗
PubMed2002

Pressure of zidovudine accelerates the reversion of lamivudine resistance-conferring M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1.

We cultured lamivudine-resistant human immunodeficiency virus type 1 (HIV-1) variants over an extended period of time in the presence of zidovudine and observed a premature reversion of the resistance-conferring M184V mutation. These data suggest that the presence of ZDV amplifies differences in replication capacity between wild-type HIV-1 and the mutant variant.

Anti-HIV Agents↗
PubMed1999

Second-site reversion of a human immunodeficiency virus type 1 reverse transcriptase mutant that restores enzyme function and replication capacity.

Nonconservative substitutions for Tyr-115 in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) lead to enzymes displaying lower affinity for deoxynucleoside triphosphates (dNTPs) (A. M. Martín-Hernández, E. Domingo, and L. Menéndez-Arias, EMBO J. 15:4434-4442, 1996). Several mutations at this position (Y115W, Y115L, Y115A, and Y115D) were introduced in an infectious HIV-1 clone, and the replicative capacity of the mutant viruses was monitored. Y115W was the only mutant able to replicate in MT-4 cells, albeit very poorly. Nucleotide sequence analysis of the progeny virus recovered from supernatants of four independent transfection experiments showed that the Y115W mutation was maintained. However, in all cases an additional substitution in the primer grip of the RT (M230I) emerged when the virus increased its replication capacity. Using recombinant HIV-1 RT, we demonstrate that M230I mitigates the polymerase activity defect of the Y115W mutant, by increasing the dNTP binding affinity of the enzyme. The second-site suppressor effects observed were mediated by mutations in the 66-kDa subunit of the RT, as demonstrated with chimeric heterodimers. Examination of available crystal structures of HIV-1 RT suggests a possible mechanism for restoration of enzyme activity by the second-site revertant.

Animals↗
PubMed2005

Mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer.

The specific impact of mutations that abrogate human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) dimerization on virus replication is not known, as mutations shown previously to inhibit RT dimerization also impact Gag-Pol stability, resulting in pleiotropic effects on HIV-1 replication. We have previously characterized mutations at codon 401 in the HIV-1 RT tryptophan repeat motif that abrogate RT dimerization in vitro, leading to a loss in polymerase activity. The introduction of the RT dimerization-inhibiting mutations W401L and W401A into HIV-1 resulted in the formation of noninfectious viruses with reduced levels of both virion-associated and intracellular RT activity compared to the wild-type virus and the W401F mutant, which does not inhibit RT dimerization in vitro. Steady-state levels of the p66 and p51 RT subunits in viral lysates of the W401L and W401A mutants were reduced, but no significant decrease in Gag-Pol was observed compared to the wild type. In contrast, there was a decrease in processing of p66 to p51 in cell lysates for the dimerization-defective mutants compared to the wild type. The treatment of transfected cells with indinavir suggested that the HIV-1 protease contributed to the degradation of virion-associated RT subunits. These data demonstrate that mutations near the RT dimer interface that abrogate RT dimerization in vitro result in the production of replication-impaired viruses without detectable effects on Gag-Pol stability or virion incorporation. The inhibition of RT activity is most likely due to a defect in RT maturation, suggesting that RT dimerization represents a valid drug target for chemotherapeutic intervention.

Codon↗
PubMed2005

Letter. In vitro phenotypic susceptibility to nucleoside reverse transcriptase inhibitors of HIV-2 isolates with the Q151M mutation in the reverse transcriptase gene.

In HIV-2 infection, many studies have reported a high frequency of selection of the Q151M mutation, but its impact on phenotypic susceptibility of HIV-2 isolates remains unclear. Four HIV-2 infected patients from the French ANRS HIV-2 cohort, with evidence of Q151M mutation in both plasma and available peripheral blood mononuclear cells (PBMCs) co-cultivated supernatants, were selected. In vitro phenotypic susceptibilities to different nucleoside reverse transcriptase inhibitors (NRTIs) were determined using a PBMC assay. In HIV-2 isolates, the Q151M mutation alone impacts only the phenotypic susceptibility to stavudine and abacavir. A decrease in susceptibility to all NRTIs was observed when Q151M was selected with V111I, a mutation of unknown impact on HIV-1 resistance. Clinical relevance of these phenotypic susceptibility results needs to be evaluated in HIV-2 treated patients.

Drug Resistance, Multiple, Viral↗
PubMed1994

Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse transcriptase gene to prevent dimerization of the expressed chimaeric protein: purification and characterization of a monomeric HIV-1 reverse transcriptase.

We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant reverse transcriptase (RT) engineered to contain a 26-amino-acid linker insertion from the tether domain of feline leukaemia virus (FLV) RT. The chimaeric protein was expressed in Escherichia coli and migrated on SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT has been prepared by the coordinated applications of immobilized-metal-affinity chromatography and gel filtration on Superose 12 columns. The monomeric nature of this chimaeric HIV-I RT was further characterized by cross-linking studies using disuccinimidyl suberate. The RNA-dependent DNA polymerase activity of the monomeric chimaeric HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These results support our recent studies on the monomeric polymerase domain (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy, Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The inability of the monomeric chimaeric HIV-1 RT to display polymerase activity like that of the heterodimeric HIV-1 RT is attributed to a decrease in the processive rate of DNA synthesis (75%) and DNA binding (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT. These results suggest that the linker insertion from FLV RT does not interfere with the RNAase H activity associated with the monomeric HIV-1 RT.

Amino Acid Sequence↗
PubMed1980

Parameters of unbalanced growth and reversible inhibition of deoxyribnucleic acid synthesis in Brevibacterium ammoniagenes ATCC 6872 induced by depletion of Mn2+. Inhibitor studies on the reversibility of deoxyribonucleic acid synthesis.

Unbalanced growth induced by depletion of manganese ions was a prerequisite for production of ribonucleotides in a high salt mineral medium with the wildtype strain Brevibacterium ammoniagenes ATCC 6872. The concentration of manganese strictly controlled the overall deoxyribonucleic acid (DNA) synthesis, whereas ribonucleic acid (RNA), protein and cell wall synthesis remained essentially unimpaired in the manganese-lacking cells. The reversibility of inhibition of overall DNA synthesis was shown by enhanced incorporation (up to threefold compared to the cultures supplied with sufficient manganese) of [8-14C] adenine into alkali-stable, trichloroacetic acid-insoluble material after subsequent addition of 10 microM MnCl2 to 15 h-old depleted cultures. The results of inhibitor studies on the restoration of overall DNA synthesis due to subsequent addition of manganese ions to depleted cultures suggest that ribonucleotide reduction is the primary target of the manganese starvation during nucleotide fermentation with Brevibacterium ammoniagenes ATCC 6872.

Bacterial Proteins↗
PubMed1996

Drug-induced reversion of progression phenotype is accompanied by reversion of AP-1 phenotype in JB6 cells.

Transformed JB6 cells can be stably reverted to nontransformed phenotype by AP-1 inhibiting gluccorticoid fluocinolone (FA) and cAMP elevator forskolin (FN), yielding stable revertants of promotion resistant (P-) and promotion sensitive (P+) phenotypes. AP-1 activity of nontransformed P- and P+ revertant clones was decreased under a variety of experimental conditions compared with their transformed counterparts. Moreover, AP-1 activity in P+ cells under anchorage-independent conditions was induced by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) while AP-1 activity in the reverted P- cells was not induced, just as observed for the original P+ and P- variants. Taken together these data suggest that changes in AP-1 activity may be one key mediator not only of forward progression but also of reversion of tumor cells to nontransformed phenotype. In addition, the higher transfection efficiency of the new reverted P- and P+ cells renders them useful for studying the role of transcription factors in tumor promotion.

3T3 Cells↗
PubMed1997

Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA.

Reverse transcriptase (RT) is the key enzyme required for conversion of RNA to DNA. Cloning of Moloney murine leukemia virus (MMLV) RT has enable engineering an RT that lacks endogenous RNase H activity. RT catalyzes cDNA synthesis more efficiently in the absence of RNase H. We describe here a number of properties of MMLV RT and RNase H-minus MMLV RT not summarized in a single location elsewhere, providing a basis for best use of these enzymes in cDNA synthesis. In addition, general guidelines and detailed protocols are provided for use of MMLV RTs in one tube double-stranded cDNA synthesis, in [32P]cDNA synthesis, and in RT-PCR and long RT-PCR.

DNA, Complementary↗
PubMed1989

Experimental reversal of the normal dorsal-ventral timing of blastopore formation does not reverse axis polarity in Xenopus laevis embryos.

During gastrulation in Xenopus laevis, the dorsal lip of the blastopore normally appears before the ventral lip. Metabolic gradient models propose that the dorsal lip develops from the region of highest metabolic activity and somehow dominates other regions to prevent them from becoming dorsal. To test these ideas, I applied a temperature gradient of 12 degrees C across the embryo. Localized heating of the prospective ventral vegetal region from early in the first cleavage period until gastrulation causes the blastopore lip to form first by 2 hr at the prospective ventral meridian rather than at the prospective dorsal meridian. Despite this reversal of the timing of blastopore formation, gastrulation is completed, and the neural plate forms at its usual position on the prospective dorsal meridian. This demonstrates that the earliest gastrulating regions of the blastopore do not necessarily become dorsal, nor do they inhibit dorsal development by other regions. It is unlikely that axis polarity is based on regional differences in energy metabolism.

Animals↗
PubMed1993

The Mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor.

We show that the reverse transcriptase (RT) encoded by the Mauriceville mitochondrial plasmid of Neurospora closely resembles viral RNA-dependent RNA polymerases in initiating cDNA synthesis opposite the penultimate C residue of a 3' tRNA-like structure and has the unprecedented ability for a DNA polymerase to initiate DNA synthesis at a specific site in a natural template without a primer. The Mauriceville plasmid enzyme can also use DNA or RNA primers in a manner suggesting how a primitive RT could have evolved from an RNA-dependent RNA polymerase into retroviral and other types of RTs. The characteristics of the Mauriceville plasmid RT suggest that it may be related to the progenitor of present-day RTs and DNA polymerases.

Base Sequence↗
PubMed1990

Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase.

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.

Chromatography, Affinity↗
PubMed2006

Static and serial assessments of coronary arterial remodeling are discordant: an intravascular ultrasound analysis from the Reversal of Atherosclerosis with Aggressive Lipid Lowering (REVERSAL) trial.

BACKGROUND: Arterial remodeling is a major determinant of the clinical manifestations of coronary artery disease. Assessment of arterial remodeling with intravascular ultrasound (IVUS) used to rely on comparing the external elastic membrane (EEM) areas of lesion and reference sites at a single time point (static assessment). Recently, performance of serial IVUS provided the opportunity for direct assessment of remodeling. Our aim was to study the concordance of the static and serial methods. METHODS: We identified 210 focal coronary lesions on the baseline IVUS interrogations of the REVERSAL trial. A follow-up IVUS was performed at 18 months. Static assessment was performed by calculating the remodeling index (RI) (lesion EEM area / reference EEM area) and serial assessment by comparing the changes in atheroma and EEM areas at follow-up. RESULTS: Using the serial method, there was a 1.24 mm2 increase in EEM area for every 1 mm2 increase in atheroma area at the lesion site, which was indicative of expansive (overcompensatory) remodeling. By using the static method, the RI was paradoxically lower at follow-up (1.062 +/- 0.15 at baseline vs 1.027 +/- 0.14 at follow-up, P < .001), indicating a trend toward constrictive remodeling. The reason for decreased RI at follow-up was the relatively larger expansion of the EEM areas at the reference sites. CONCLUSIONS: Static and serial assessments of arterial remodeling are discordant. This is due to concomitant remodeling of the reference sites used in the static assessment. Intravascular ultrasound performed at a single point in time does not reflect the dynamic remodeling response.

Aged↗
PubMed2003

Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase.

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is a potentially useful diagnostic marker for cancers. There have been few studies in which immunological detection of hTERT has been attempted and its subcellular localization has not been precisely defined. In the present study, we re-evaluated expression and localization of hTERT in cancer and normal cells using a newly developed antibody. Immunohistochemistry revealed that hTERT is expressed in approximately 80% of gynecological cancers, but some premalignant lesions exhibited weak expression of hTERT. Interestingly, not only nuclei but also cytoplasm of cancer cells were positive for hTERT staining. This finding was supported by the results of Western blot analysis of cell lines, in which both nuclear and cytoplasmic extracts exhibited significant hTERT bands. Cytoplasmic hTERT in cancer cells may be functional because the telomeric repeat amplification protocol assay of cytoplasmic extracts showed high levels of telomerase activity. Unexpectedly, not all normal primary cells and telomerase-negative cancer cell lines lacked hTERT expression; some exhibited weak TERT signals. In Western analysis, hTERT signals did not always correlate with telomerase activity of the various cell types. These findings suggest that functional hTERT is expressed in both the nucleus and cytoplasm of cancer cells and that hTERT expression does not strictly reflect telomerase activity. Further analysis is needed to clarify the biological significance of cytoplasmic hTERT.

Blotting, Western↗