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PubMed1996

Restoration of tRNA3Lys-primed(-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs. Implications for retroviral replication.

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Delta13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA3Lys, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA3Lys. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA3Lys with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA3Lys extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA3Lys and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.

Base Sequence↗
PubMed2001

Estimation of binding affinities for HEPT and nevirapine analogues with HIV-1 reverse transcriptase via Monte Carlo simulations.

The interactions and energetics associated with the binding of 20 HEPT and 20 nevirapine nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) have been explored in an effort to establish simulation protocols and methods that can be used in the development of more effective anti-HIV drugs. Using crystallographic structures as starting points, all 40 inhibitors were modeled in the bound and unbound states via Monte Carlo (MC) statistical mechanics methods. Potentially useful descriptors of binding affinity were configurationally averaged for each inhibitor during the MC simulations, and correlations were sought with reported experimental activities. A viable regression equation was obtained using only four descriptors to correlate the 40 experimental activities with an r(2)() of 0.75 and cross-validated q(2)() of 0.69. The computed activities show a rmsd of 0.94 kcal/mol in comparison with experiment and an average unsigned error of 0.69 kcal/mol. The MC results reveal three physically reasonable parameters that control the binding affinities: (1) loss of hydrogen bonds with the inhibitor is unfavorable, (2) burial of hydrophobic surface area is favorable, and (3) a good geometrical fit without steric clashes is needed for the protein-inhibitor complex. It is gratifying that the corresponding descriptors are statistically the most important quantities for determining the anti-HIVRT activity for the 40 compounds. Representative examples are also given in which structural and thermodynamic information from the MC simulations is used to help understand binding differences for related compounds. A key pi-type hydrogen bond has been identified between secondary-amide nevirapine analogues and Tyr188A of HIVRT that explains their otherwise surprising activity and the ineffectiveness of nevirapine against the Y188C mutant.

Anti-HIV Agents↗
PubMed2004

Emtricitabine: a once-daily nucleoside reverse transcriptase inhibitor.

OBJECTIVE: To review the pharmacology, virology, pharmacokinetics, safety, and efficacy of the nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine. DATA SOURCES: English-language reports were accessed using MEDLINE (1966-June 2003) and the Iowa Drug Information Service database (1966-June 2003) using emtricitabine and Coviracil as key words. (Coviracil was the proposed trade name for the product prior to approval.) The Internet was also searched using the terms HIV/AIDS conferences, then emtricitabine within the conference proceedings. STUDY SELECTION AND DATA EXTRACTION: Abstracts, posters, and oral presentations from scientific conferences, both published and unpublished, were included. Preference was given to published controlled trials. Studies providing a description of the pharmacology, virology, effectiveness, safety, or pharmacokinetics of emtricitabine were used in this review. DATA SYNTHESIS: Emtricitabine is an NRTI used to treat HIV-1 infection. Once-daily administration can decrease pill burden and potentially increase adherence to multidrug HIV therapy. Further, emtricitabine has shown equivalent or improved outcomes compared with lamivudine and stavudine. CONCLUSIONS: Emtricitabine is a safe and effective option for HIV-1 infection in adults as part of a multidrug regimen. It may be a better alternative than lamivudine for once-daily therapy because of its extended intracellular half-life and better than lamivudine and stavudine because of a possibly decreased potential for drug resistance.

Clinical Trials as Topic↗
PubMed1997

Attenuated replication of human immunodeficiency virus type 1 with a didanosine-selected reverse transcriptase mutation.

The Leu-74 to Val (Leu74Val) mutation in human immunodeficiency virus type 1 reverse transcriptase (RT) develops as a consequence of didanosine (ddI) therapy and is associated with a decreased susceptibility to ddI. In this report, we provide evidence that the ddI-associated Leu74Val mutation confers a replication disadvantage to the virus. In a series of experiments, we have shown that (i) a cloned virus with an engineered Leu74Val mutation in RT was attenuated for replication; (ii) a Val-to-Leu revertant of Leu74Val in the pNL4-3 background replicated with an efficiency similar to that of the wild-type virus; (iii) when two isolates from the same patient were compared, a clinical isolate containing mutations Leu74Val and Thr215Tyr was attenuated for replication compared to one in which the Thr215Tyr mutation alone was present; and (iv) the viruses with the Leu74Val mutation showed an 11% loss of fitness in a single passage compared to the wild-type and a mutant virus containing a Lys70Arg mutation. The loss of fitness for viruses grown in drug-free medium could result in an inability to detect a Leu74Val mutant in clinical isolates obtained post-ddI therapy. The decreased replication ability of the Leu74Val mutant virus selected by ddI therapy provides a strong rationale for the lower viral RNA levels observed with ddI therapy compared to zidovudine therapy in clinical trials.

Amino Acid Substitution↗
PubMed2005

The L74V mutation in human immunodeficiency virus type 1 reverse transcriptase counteracts enhanced excision of zidovudine monophosphate associated with thymidine analog resistance mutations.

Thymidine analog mutations (TAMs) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) confer resistance to zidovudine (AZT) by increasing the rate of ATP-dependent phosphorolysis of the terminal nucleotide monophosphate (primer unblocking). By contrast, the L74V mutation, which confers resistance to didanosine, sensitizes HIV-1 to AZT and partially restores AZT susceptibility when present together with one or more TAMs. To compare rates of primer unblocking in RTs carrying different clusters of TAMs and to explore the biochemical mechanism by which L74V affects AZT susceptibility, ATP-mediated rescue of AZT-blocked DNA synthesis was assayed using a series of purified recombinant RTs. Rates of primer unblocking were higher in the 67N/70R/219Q RT than in the 41L/210W/215Y enzyme and were similar to rates observed with an RT carrying six TAMs (41L/67N/70R/210W/215Y/219Q). The presence of 74V in an otherwise wild-type RT reduced the rate of primer unblocking to a degree similar to that observed with the M184V mutation for lamivudine resistance, which also sensitizes HIV-1 to AZT. Introduction of 74V into RTs carrying TAMs partially counteracted the effect of TAMs on the rate of primer unblocking. The effect of 74V was less marked than that of the 184V mutation in the 67N/70R/219Q and 41L/210W/215Y RTs but similar in the RT carrying six TAMs. These results demonstrate that L74V enhances AZT susceptibility by reducing the extent of its removal by ATP-dependent phosphorolysis and provides further evidence for a common mechanism by which mutations conferring resistance to didanosine and lamivudine sensitize HIV-1 to AZT.

Adenosine Triphosphate↗
PubMed1985

Formycin 3' end modified tRNATrp. Recognition by avian myeloblastosis virus reverse transcriptase and primer function.

Primer tRNATrp has been modified at the 3' end by adenosine analogues: 2'deoxyadenosine, 3'deoxyadenosine, 3' amino-3' deoxyadenosine and formycin. Aminoacylation of modified tRNATrp with cognate aminoacyl-tRNA synthetase and primer function for DNA synthesis catalyzed by AMV reverse transcriptase have been studied. The tRNATrp was able to accept tryptophan but did not initiate the DNA synthesis directed by 35S AMV RNA. Recognition of modified tRNATrp by AMV reverse transcriptase was not affected as followed by enzyme-tRNA complex formation. The functional consequences of these effects are discussed.

Adenosine↗
PubMed1999

Mutations in the primer grip region of HIV reverse transcriptase can increase replication fidelity.

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3'-ends of primers. Point mutations were created by alanine substitution at positions 224-235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses.

Base Pair Mismatch↗
PubMed1992

Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease.

Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.

Amino Acid Sequence↗
PubMed1997

Metabolism of the human immunodeficiency virus type 1 reverse transcriptase inhibitor delavirdine in rats.

Delavirdine mesylate (U-90152T) is a highly specific nonnucleoside reverse transcriptase inhibitor currently under development for the treatment of AIDS. The excretion, disposition, and metabolism of delavirdine were investigated in Sprague-Dawley rats after oral administration of [14C]delavirdine mesylate at single doses ranging from 10 to 250 mg/kg and multiple doses ranging from 20 to 250 mg/kg/day. Excretion studies showed that feces was the major route of elimination, delavirdine was well absorbed (>80%) after a 10 mg/kg single dose, and excretion was dose-dependent. The metabolism of delavirdine in the rat was extensive. The following metabolites were identified (% of dose in rats given 10 and 100 mg/kg, respectively): 6'-hydroxy delavirdine (7.1% and 15.6%) and its glucuronide (12.2% and 6.2%) and sulfate (5.5% and 3.2%) conjugates, despyridinyl delavirdine (12.1% and 11.7%) and its conjugate (13.0% and 11.7%), desalkyl delavirdine (16.5% and 13.4%), and its N-sulfamate, 6'- and 4'-sulfate conjugates (2.9% and 3.9%). Cleavage of the amide bond in delavirdine to give N-isopropylpyridinepiperazine and indole carboxylic acid constituted a minor pathway. Degradation of 6'-hydroxy delavirdine generated despyridinyl delavirdine and the pyridine-ring opened MET-14. The metabolic pathway of delavirdine involved N-desalkylation, pyridine ring hydroxylation, pyridine ring cleavage, and amide bond cleavage.

Administration, Oral↗
PubMed2004

Real-time determination of human telomerase reverse transcriptase mRNA in gastric cancer.

AIM: To set up a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay, to detect human telomerase reverse transcriptase (hTERT) messenger RNA in gastric carcinomas, and to evaluate quantitative determination of hTERT mRNA in the diagnostic value of gastric carcinomas, and to analyze the correlation between the expression level of hTERT mRNA and clinicopathological parameters in patients with gastric cancer. METHODS: A real-time quantitative RT-PCR (RQ-PCR) based on TaqMan fluorescence methodology and the LightCycler system was used to quantify the full range of hTERT mRNA copy numbers in 35 samples of gastric carcinomas and corresponding adjacent non-cancerous tissues. The normalized hTERT (NhTERT) was standardized by quantifying the number of GAPDH transcripts as internal control and expressed as 100X (hTERT/GAPDH) ratio. Variables were analyzed by the Student's t-test, chi2 test and Fisher's exact test. RESULTS: NhTERT from gastric carcinomas and corresponding adjacent non-cancerous tissues was 6.27+/-0.89 and 0.93+/-0.18, respectively (t = 12.76, P<0.001). There was no significant association between gastric cancer hTERT mRNA expression level and patient's age, gender, tumor size, location and stage (pTNM), but a significant correlation was found between hTERT mRNA expression level in gastric carcinomas and the degree of differentiation. CONCLUSION: Quantitative determination of hTERT mRNA by RQ-PCR is a rapid and sensitive method. hTERT might be a potential biomarker for the early detection of gastric cancer.

Adult↗
PubMed1996

A method for combined immunoaffinity purification and assay of HIV-1 reverse transcriptase activity useful for crude samples.

Detection of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity in crude specimens was greatly enhanced using a novel capture RT assay. Eighteen different monoclonal antibodies (Mabs) raised against purified HIV-1 RT were tested for their ability to bind to HIV-1 RT without affecting its activity. The anti-HIV-1 RT Mabs were immobilized on plastic macrobeads and used as solid carriers in the capture RT assay. The assay system first involved RT's adherence to the immobilized Mabs. Nonspecific enzymes and other impurities were removed by a simple wash after which the RT reaction mixture was added. Substrate and product were finally separated by a wash of the beads. Practically all radioactivity incorporated into DNA (>98%) was recovered on the bead. The Michaelis-Menten constants and the saturation velocity values for the nucleotide substrate were similar for free and immobilized RT. The reaction mechanism for the immobilized RT is discussed. When comparing the function of this assay with more conventional soluble RT assays for samples consisting of recombinant HIV-1 RT mixed with an extract of peripheral blood lymphocytes (PBL), an almost 100-fold higher sensitivity was found. The capture RT assay had the capacity to recover approximately 80% of the RT activity added to an extract of 1 x 10(7) PBL cells/ ml. A strong correlation (r = 0.947) between the results obtained with this assay and a HIV-1 p24 enzyme-linked immunosorbent assay was found, when samples from a collection of 16 HIV strains propagated in cell culture were analyzed.

Antibodies, Monoclonal↗
PubMed2005

Detection of bovine central nervous system tissue in liver sausages using a reverse transcriptase PCR technique and a commercial enzyme-linked immunosorbent assay.

The suitability of a reverse transcriptase (RT) PCR assay was evaluated for the detection of bovine central nervous system (CNS) tissue specifically in liver sausages. Because of its emulsifying effect, CNS tissue was frequently added to this kind of meat product in the past. On standard samples, the RT-PCR technique reliably detected a concentration of 0.25% bovine CNS tissue in liver sausages stored for up to 28 days. Following the successful application of RT-PCR for the detection of bovine CNS tissue in these specially prepared samples, a field study was performed with a total of 258 liver sausages purchased in retail markets. All sausages were tested with both an RT-PCR assay and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Nine (3.5%) of the retail liver sausage samples were positive for CNS tissue in the ELISA, but none were positive for this tissue in the RT-PCR assay. All positive ELISA results indicated the presence of 0.23 to 0.30% CNS tissue. Recent studies have indicated that the RT-PCR assay is not as sensitive for porcine CNS tissue as for bovine CNS tissue, which this assay can detect at 0.25%. Although the ELISA is not species specific, the CNS tissue detected by the ELISA is assumed to stem from a nonbovine species. The RT-PCR technique is a sensitive tool for the detection of bovine CNS tissues in a problematic matrix such as liver sausage. ELISA screening followed by a species-specific RT-PCR assay for bovine CNS tissue is a practical approach for monitoring meat products for compliance with European food regulations.

Animals↗
PubMed1999

Highly specific recognition of primer RNA structures for 2'-OH priming reaction by bacterial reverse transcriptases.

A minor population of Escherichia coli contains retro-elements called retrons, which encode reverse transcriptases (RT) to synthesize peculiar satellite DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize specific RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). The resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific RNA structure in its primer-template RNA. On the basis of structural comparison with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86 and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec73 (316 residues) share only 71 identical residues (22%). From the analysis of 10 such constructs, the C-terminal 91-residue sequence of RT-Ec86 was found to be essential for the recognition of the unique stem-loop structure and the branching G residue in the primer-template RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by exponential enrichment) method with RT-Ec86 and primer RNAs containing random sequences, the identical stem-loop structure (including the 3-U loop) to that found in the retron-Ec86 primer-template RNA was enriched. In addition, the highly conserved 4-base sequence (UAGC), including the branching G residue, was also enriched. These results indicate that the highly diverse C-terminal region recognizes specific stem-loop structures and the branching G residue located upstream of the stem-loop structure. The present results with seemingly primitive RNA-dependent DNA polymerases provide insight into the mechanisms for specific protein RNA recognition.

Amino Acid Sequence↗
PubMed2003

Mutagenesis of cysteine 280 of the reverse transcriptase of human immunodeficiency virus type-1: the effects on the ribonuclease H activity.

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RT of human immunodeficiency virus type-1 (HIV-1) is composed of two subunits. The p51, which is the smaller subunit, shares with the larger p66 subunit the same amino-terminal part (which encompasses the DNA polymerase domain) and lacks the carboxyl-terminal segment of the p66 (which is the RNase H domain). The structure of the polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains) linked to the RNase H domain. Chemical modifications by thiol-specific reagents of cysteine 280, located in alpha helix I in the thumb subdomain of the polymerase domain, affect substantially only the RNase H activity. Also, the substitution of a serine for C280 did not alter any of the RT activities. Here we have systematically modified the C280 residue to either of the following residues: W, P, H, L, M, Y, Q, E or R. Only the first two mutations lead to a marked reduction in the RNase H activity, whereas none of the mutations affected the polymerase function to a significant extent. As expected, due to their impaired RNase H, the C280W and C280P mutants also had a very low DNA strand-transfer activity. It is also apparent from subunit-directed mutagenesis that each of the RT subunits contributes to the level of RNase H activity, yet the contribution of the p51 subunit to this activity is somewhat higher than that of the p66. Steady-state kinetic analyses have indicated that the RNase H activity was reduced mainly due to the sharp increase in the K(m) rather than changes in the k(cat) values. This suggests that the modifications of C280 lead to an impaired affinity of HIV-1 RT towards the RNA-DNA substrate.

Catalysis↗
PubMed1992

The binding of a novel bisheteroarylpiperazine mediates inhibition of human immunodeficiency virus type 1 reverse transcriptase.

The bisheteroarylpiperazines (BHAPs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and specifically block HIV-1 replication (Romero, D. L., Busso, M., Tan, C.-K., Reusser, F., Palmer, J. R., Poppe, S. M., Aristoff, P. A., Downey, K. M., So, A. G., Resnick, L., and Tarpley, W. G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8806-8810). Here we show that the radiolabeled BHAP [3H]U-88204 binds specifically to HIV-1 RT with high affinity (KD of 50 nM) and a stoichiometry of 1 mol of U-88204 per 1 mol of p66/p51 RT heterodimer. Binding of [3H]U-88204 to RT is unaffected by the presence of saturating poly(rC).oligo (dG)12-18 template-primer. Direct measurement of competition between [3H]U-88204 and other RT inhibitors for binding to RT reveals mutually exclusive competition between [3H]U-88204 and the non-nucleoside RT inhibitor BI-RG-587 (Kopp, E. B., Miglietta, J. J., Shrutkowski, A. G., Shih, C.-K., Grob, P. M. and Skoog, M.T. (1991) Nucleic Acids Res. 19, 3035-3039), indicating that both share the same binding site. Phosphonoformate in concentrations up to 50 microM shows no competition with [3H]U-88204 for binding to RT either alone or in the presence of template-primer. Dideoxynucleotide RT inhibitors affect the binding of [3H]U-88204 to RT when complementary template-primer is present. [3H]U-88204 and the dideoxynucleotide ddGTP can bind RT simultaneously, but the presence of one ligand decreases the affinity of RT for the second. Inasmuch as ddGTP approximates the nucleotide substrate of RT, the direct demonstration of an RT-dideoxynucleotide-[3H]U-88204 complex validates the use of indirect kinetic methods to assess the strength of BHAP interaction with RT and suggests that RT inhibition by U-88204 is achieved via effects on nucleotide substrate binding.

Benzodiazepines↗
PubMed2003

Role of residues in the tryptophan repeat motif for HIV-1 reverse transcriptase dimerization.

The tryptophan repeat motif of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) is comprised of a cluster of six tryptophan residues at codons 398, 401, 402, 406, 410 and 414 that are highly conserved amongst primate lentiviral RTs. To determine the contributions of each of these residues for HIV-1 RT dimerization, we introduced changes into cloned DNA and tested the mutant subunits for their capacity to mediate heterodimerization in the yeast two-hybrid system. Changes of residue 401 to either leucine or alanine (but not phenylalanine) and residue 414 to leucine resulted in major reductions in beta-galactosidase activity produced from the reporter gene as compared to yeast expressing wild-type p66 bait and p51 prey fusions. Subunit selective mutagenesis revealed that the effect of these mutations was mediated mainly through the p66 subunit. Introduction of tryptophan mutants into the bacterial expression vector pRT6H/NB-PROT showed that RTs containing W401A or W401L substitutions (but not W401F) and W414L were defective for dimerization in vitro. Consistent with their dimerization defect, the W401A, W401L and W414L mutants were devoid of RT activity. Using the yeast two-hybrid system, we identified several second-site suppressors in p66 that restored interaction of the p66W401A bait to the p51W401A prey. The suppressors (T409I, D110G, V372A and I393M) also restored heterodimerization of bacterially expressed W401A subunits. When introduced into the W401A mutant, T409I was able to restore RT activity to 50% of the wild-type level. Examination of the RT structures revealed that K331 in p51 makes multiple hydrogen bond contacts with residues in the p66 loop spanned by W401 and W414. Consistent with this observation, the K331A RT mutant was dimerization-defective. We conclude that mutations at codons 401 and 414 in p66 impair dimerization by altering the proper positioning of structural elements in between these residues that make important contacts with p51.

Binding Sites↗
PubMedDate not supplied

Safety profile of nevirapine, a nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus infection.

Nevirapine (NVP) is a nonnucleoside reverse transcriptase inhibitor widely used in combination with other antiretroviral agents for the treatment of human immunodeficiency virus disease. To establish its safety profile, we conducted a review of data from prospective US and international clinical trials involving a total of 906 adult patients and 468 pediatric patients treated with NVP. Drug-related adverse events were similar in adults and children, with rash and nausea most frequently reported in adults and rash and granulocytopenia most frequently reported in children. A separate analysis of rash based on data from adult patients in controlled trials demonstrated a 16% rate of NVP-attributable rash in these patients. Of patients with NVP-associated rash, 65% developed rash within the first 6 weeks of therapy, and it has been shown that a lower lead-in dose (200 mg/d vs the standard 400 mg/d) for the first 2 weeks of NVP treatment reduces the frequency of drug-associated rash. Serious rash (Stevens-Johnson syndrome [SJS] or SJS/toxic epidermal necrolysis transition syndrome) occurred with an incidence of 0.3% and clinical hepatitis with an incidence of 1.0% among NVP-treated patients in clinical trials. Adverse event data from long-term clinical trials demonstrated a lower incidence of NVP-related adverse events than in short-term trials of NVP therapy. An analysis of abnormal laboratory findings using thresholds similar to those found in the prescribing information for other commonly used antiretroviral agents and data from controlled trials in adults showed that the most frequently observed laboratory abnormalities were elevations in liver function test results. Approximately 50,000 patients in the United States had been treated with marketed NVP at the time of writing, and postmarketing surveillance has supported the overall safety profile observed in clinical trials. NVP has been shown to be well tolerated in both adult and pediatric patients.

Adolescent↗
PubMed2004

Quantitative nested reverse transcriptase PCR vs. real-time PCR for measuring AML1/ETO (MTG8) transcripts.

A quantitative nested reverse transcriptase polymerase chain reaction (QN-RT-PCR) method was developed using a plasmid cDNA containing the AML1/ETO (MTG8) fusion transcript from Kasumi-1 cells, an acute-myelogenous leukemia cell line with the t(8;21) translocation. In this method, the plasmid was detectable at a concentration of 10(-17) m. The fusion transcript in a mixture of 10(7) Rice94 (Burkitt lymphoma cell line) cells containing two Kasumi-1 cells was detectable at 10(-17) m. In a previously published real-time PCR method, the plasmid containing the fusion transcript was detectable at 10(-16) m or higher, and 20 or more Kasumi-1 cells were detectable in 10(7) Rice94 cells. Thus, this QN-RT-PCR method is more sensitive than the real-time PCR. When the same samples were examined by real-time PCR and our QN-RT-PCR method, in one patient in clinical remission after chemotherapy and allogeneic-bone marrow transplantation (BMT), the transcript was detected by QN-RT-PCR 60 days prior to hematological relapse, in contrast to 10 days before hematological relapse by real-time PCR. The transcript level was below 10(-17) m (undetectable) with this QN-RT-PCR in patients in clinical remission after chemotherapy and BMT, while it was 10(-15)-10(-16) m in patients in clinical remission after chemotherapy alone. The quantitative difference of the transcript level in minimal residual disease (MRD) between these two different types of clinical remission was estimated to be at least 10(2)-fold. This QN-RT-PCR method is useful for predicting hematological relapse and for quantitatively estimating MRD in different types of clinical remission.

Cell Line, Tumor↗