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)

Resistance to antimitotic chemotherapeutics in pathogenic nematodes, fungi and mammalian cells is closely associated with structural changes in cytoskeletal beta-tubulin. We investigated the possibility of using the well-characterised free-living nematode Caenorhabditis elegans as a model for studying the mechanism of resistance against benzimidazole (BZ) drugs in the parasitic nematode Haemonchus contortus. Functional analysis of a conserved beta-tubulin isotype (tub-1) mutation near GTP-binding domain II, which is linked to BZ resistance, was carried out in C. elegans by heterologous expression of: (1) parasite BZ-sensitive alleles; (2) BZ-resistant alleles; and (3) in vitro mutagenised beta-tubulin gene constructs. The injected heterologous gene constructs were not only stably maintained, but also expressed as shown by reverse transcriptase-polymerase chain reaction analysis. The degree of BZ drug susceptibility of the transformants was assayed and quantified by incubation with both benomyl and thiabendazol. All H. contortus tub-1 constructs, which encoded Phe at position 200, conferred susceptibility to thiabendazole in BZ-resistant C. elegans ben-1 mutants. In contrast, constructs carrying Tyr200 did not alter the BZ drug phenotype. From these experiments we conclude that: (1) C. elegans can be used as an expression host, since injected parasite genes were biologically active; and (2) the single Phe to Tyr mutation at position 200 in beta-tubulin isotype 1 is the cause of BZ resistance in H. contortus.
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PMID:Beta-tubulin genes from the parasitic nematode Haemonchus contortus modulate drug resistance in Caenorhabditis elegans. 787 71

The ouabain-resistant cell line H1C1 displays a 30-fold differential of reduced sensitivity to the structurally related cardiac glycosides digoxin and digitoxin (Baker, R. M. (1976) in Biogenesis and Turnover of Membrane Macromolecules (Cook, J.S., ed) pp. 93-103, Raven Press, New York). Since these ligand congeners differ only by the presence of a hydroxyl group at C-12 of digoxin we predicted that the H1C1 phenotype must reflect a mutation which alters the binding site of the cardiac glycoside receptor (Na,K-ATPase). Complementary DNA encoding the alpha 1 Na,K-ATPase was prepared from H1C1 cell total RNA by reverse transcription-coupled polymerase chain reaction and these cDNAs were cloned. Sequence analysis of the reverse transcriptase-polymerase chain reaction clones revealed several independent isolates containing a G > A transition at nucleotide 332 of the propeptide coding sequence, generating the amino acid substitution C108Y. The ability of this substitution to confer differential sensitivity for digoxin and digitoxin was tested and confirmed by expressing a human alpha 1 C108Y-Na,K-ATPase in wild type HeLa cells and assaying for inhibition of cell growth and inhibition of Na,K-ATPase activity. Phenylalanine or alanine substitutions of this cysteine also confer this pattern of ligand discrimination. Ouabain-resistant Na,K-ATPase substitutions, at positions other than Cys-108 failed to exhibit differential sensitivity indicating that this ligand discrimination is unique to Cys-108 substitutions rather than a general property of cardiac glycoside-resistant mutants. It is proposed that differential resistance of the C108Y receptor for these ligands is a consequence of altering two features of the ligand-receptor interaction; one, a disruption of a common hydrogen bond resulting in general loss of affinity for cardiac glycosides and the other, formation of a new H-bond between the C-12 hydroxyl of digoxin and the receptor, specifically augmenting the stability of this ligand-receptor complex.
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PMID:Identification of an amino acid substitution in human alpha 1 Na,K-ATPase which confers differentially reduced affinity for two related cardiac glycosides. 792 66

The bombesin-like peptides were originally characterized in frog skin, then later found to have a wide distribution and range of actions in mammals. The bombesin-like peptides have classically been divided into three subfamilies, the bombesin subfamily, of which gastrin-releasing peptide (GRP) is the mammalian form; the ranatensin subfamily, of which neuromedin-B (NMB) is the mammalian form; and the phyllolitorin subfamily, which to date has only been characterized in amphibians. As a first step in characterizing mammalian phyllolitorin-like peptides, we have cloned complementary DNAs (cDNAs) encoding Leu8 and Phe8 phyllolitorin from Phyllomedusa sauvagei. Sequence analysis revealed that the amphibian phyllolitorin messenger RNA (mRNA) encodes a precursor of 90 amino acids containing a signal peptide sequence, an amino-terminal extension peptide, the phyllolitorin peptide of nine amino acids, and a carboxy-terminal extension peptide. Northern blot, reverse transcriptase-polymerase chain reaction (PCR), and in situ hybridization analysis showed that the mRNA was present at highest levels in skin, at lower levels in brain, and at lowest levels in gut. Phylogenetic analysis of bombesin-like peptide prohormone sequences showed that the phyllolitorin prohormones are much more closely related to the bombesin and ranatensin prohormones than to the GRP and NMB prohormones. This analysis suggests that the bombesin-like peptides should be reclassified into the GRP subfamily, the NMB subfamily, and the skin peptide subfamily. Surprisingly, the cDNAs encoding Phe8 and Leu8 phyllolitorins were identical except for a single T to C difference in the codon coding for the Phe or Leu residue of phyllolitorin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cloning of complementary DNAs encoding the amphibian bombesin-like peptides Phe8 and Leu8 phyllolitorin from Phyllomedusa sauvagei: potential role of U to C RNA editing in generating neuropeptide diversity. 799 36

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.
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PMID:Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase. 817 19

Prostaglandin F2 alpha (PGF2 alpha) stimulates protein synthesis of skeletal and smooth muscle cells in culture and is elevated in the heart during compensatory growth. We hypothesized that PGF2 alpha stimulates hypertrophic growth of neonatal rat cardiac myocytes. Prostaglandin F2 alpha increased [3H]phenylalanine incorporation by cultured ventricular myocytes in a dose-dependent manner (EC50 = 11 nM), suggesting action through a PGF-specific receptor. Semiquantitative reverse transcriptase polymerase chain reaction revealed that PGF receptor mRNA is expressed in ventricular myocytes > A7R5 vascular smooth muscle cells >> cardiac fibroblast-like cells. The protein content of cardiomyocyte cultures was increased by 10 nM PGF2 alpha and 11 beta-PGF2 alpha but was unchanged by 10 nM PGD2, PGE2, PGF1 alpha, carbaprostacyclin, U-46619, or 12- or 15-hydroxyeicosatrienoic acid. Stimulation of myofibrillar gene expression by PGF2 alpha was demonstrated by Northern and Western blot analysis for myosin light chain-2 (MLC-2) and by transient transfection experiments with MLC-2 luciferase expression plasmids. In addition, myofibrillogenesis was increased by PGF2 alpha as assessed by immunocytochemical staining with MLC-2 antisera. Prostaglandin F2 alpha did not affect myocyte proliferation or [3H]thymidine incorporation, thus myocyte growth occurred by hypertrophy. Proliferative and hypertrophic growth of cardiac fibroblast-like cells were unaffected by PGF2 alpha. We conclude that PFG2 alpha stimulates hypertrophic growth of neonatal rat ventricular myocytes in culture and speculate that PGF2 alpha plays a role in myocardial adaptation to chronic hypertrophic stimuli, recovery from injury, and cardiac ontogeny.
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PMID:Prostaglandin F2 alpha stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes. 855 48

A cDNA encoding the mouse delta opioid receptor was expressed stably in a Rat 1 fibroblast cell line. Expression of this receptor was demonstrated both in ligand binding studies and by reverse transcriptase-PCR. In membranes of clone D2 cells the opioid peptide [D-Ala(2)]-leucine enkephalin (DADLE) produced a robust, concentration-dependent, stimulation of basal high-affinity GTPase activity; the prototypic opioid antagonist naloxone and the highly selective and potent delta opioid ligands H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Tic[Ch2-NH]Phe-Phe-OH (TIPP[psi]) had little effect but N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI174864) caused a marked dose-dependent inhibition of this activity (Tic, 1,2,3,4-tetrahydroisoquinolin-2-yl-carbonyl]; Aib, alpha-aminobutyric acid). This effect of ICI174864 was reversed by TIPP[psi] and attenuated after treatment of the cells with pertussis toxin. No stimulation by DADLE or inhibition by ICI174864 was observed in Rat 1 fibroblasts that did not express the delta opioid receptor. Basal binding of [(35)S]guanosine 5'-O-(3-thio-triphosphate) to membranes of clone D2 cells was also stimulated by DADLE and inhibited by ICI174864; both of these effects were reversed by co-incubation with TIPP[psi]. When cholera toxin-catalysed [(32)P]ADP-ribosylation was performed on membranes of clone D2 cells in the absence of guanine nucleotides, a 40 kDa G1-family polypeptide was labelled in addition to both the long and short isoforms of Gsalpha. Labelling of the 40 kDa polypeptide was enhanced by addition of DADLE and fully attenuated by addition of ICI174864. In contrast, labelling of the isoforms of Gsalpha was unaffected by either opioid ligand. Again, both the positive effect of DADLE and the inhibitory effect of ICI174864 were prevented by co-incubation with TIPP[psi] which, in isolation, had little effect on cholera toxin-catalysed [(32)P]ADP-ribosylation of either Gs or Gi. These data demonstrate that the delta opioid receptor displays a spontaneous activity when expressed in this genetic background. Attenuation of this activity is produced by ICI174864, which by acting as an 'inverse agonist' in this system, functionally uncouples the expressed receptor from the cellular G-protein population. The complete attenuation of agonist-independent cholera toxin-catalysed [(32)P]ADP-ribosylation of Gi demonstrated that ICI174864 acts as an inverse agonist with high intrinsic activity at this receptor.
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PMID:Analysis of inverse agonism at the delta opioid receptor after expression in Rat 1 fibroblasts. 867 Jan 11

Among human immunodeficiency type 1 viruses (HIV-1) isolated during long-term 3'-azido-3'-deoxythymidine (AZT) therapy, 4 condoms (Asp67, Lys70, Thr215, and Lys219) in the HIV-1-reverse transcriptase (RT)-coding region have been considered to be related to the AZT resistance of HIV-1. Therefore we determined these mutation patterns in HIV-1 isolates from patients undergoing long-term AZT treatment. In 41 clones of HIV-1 from 7 patients, the Thr215 mutation was the most predominant (97.6%), and more frequent than Asp67 (48.8%), Lys70 (31.7%) and Lys219 (9.8%) mutations. All 22 clones from isolates cultured in the presence of 1 microM AZT Thr215 mutation; such a high frequency was not found for the other 3 codon mutations. In a clinical follow-up study, Thr215 mutation appeared in the late stage of AZT treatment parallel with the emergence of AZT insusceptibility. It is worth noting that this Thr215 mutation to Tyr or Phe is a 2-nucleotide mutation in contrast to the 1-nucleotide mutations seen in the other 3 codons. Isolates with the single amino acid change of Thr215 of the RT-coding region obtained after long-term AZT treatment grew in the presence of 1 microM AZT. This single amino acid mutation in the Thr215 codon is the most important factor in AZT resistance.
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PMID:Predominance of codon 215 mutation in reverse transcriptase-coding region of 3'-azido-3'-deoxythymidine (AZT)-resistant HIV-1 isolates after long-term AZT therapy. 870 9

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184-->Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183-->Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.
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PMID:Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis. 876 85

In the past few years, five different somatostatin (SRIF) receptor subtypes (sst1.5) have been identified, which form a distinct group in the superfamily of G-protein-coupled receptors. The naturally occurring somatostatins SRIF-28, SRIF-25, and SRIF-14 all reveal high-affinity binding for sst1.5. In contrast, short synthetic analogs that are in clinical use, such as SMS 201-995, RC-160, or BIM 23014, primarily interact with the sst2 subtype. Some SRIF analogs were previously reported to be selective for one SRIF receptor subtype, eg, the sst2 (MK 678), the sst3 (BIM 23056), or the sst5 (BIM 23052, L362-855) subtype. However, when we studied the binding affinities of these SRIF analogs for human (h) sst1.5 expressed in either CHO or COS-1 cells, we were unable to confirm these previously reported selectivities. The absence of sst antagonists is a major drawback for investigating the functional role of each sst subtype. We used site-directed mutagenesis to identify amino acids that determine ligand specificity for sst2. A single Ser305 to Phe mutation in TM VII increased the affinity of hsst1, for SMS 201-995 nearly 100-fold, and when Gln291 was also exchanged to Asn in TM VII of hsst1, almost full sst2-like binding of SMS 201-995 was obtained. These data may aid in the design and synthesis of new selective type sst ligands. We have identified the expression of sst subtypes in nonclassical SRIF target tissue such as the lung. The pKi values for SRIF and various SRIF analogs in rat lung tissue preparations were in close correlation with those obtained for CHO cells expressing the sst4 subtype. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the predominant expression of mRNA specific for sst4 in mouse, rat, and human lung tissue, confirmed by autoradiographies of rat lung. No specific binding for [125I]Tyr3-SMS 201-995 was detected, since SMS 201-995 has low affinity for sst4. In contrast, specific binding of [125I]SRIF-28 to rat lung sections was demonstrated, which could be displaced by unlabelled SRIF-14 and SRIF-28, indicating specific, high affinity binding of this radioligand to sst4 receptors.
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PMID:Binding properties of somatostatin receptor subtypes. 876 72

Tyr115 is located in the vicinity of the polymerase catalytic site of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Site-directed mutagenesis was used to generate variant enzymes having Phe, Trp, Ala, Ser, Asp or Lys instead of Tyr115. The substitution of Tyr115 by Phe renders a fully active polymerase, displaying similar kinetic parameters, processivity and misinsertion fidelity of DNA synthesis as the wild-type enzyme. In contrast, the replacement of Tyr by Asp or Lys produced enzymes with a very low polymerase activity. The activity of the variant enzymes having Trp, Ala or Ser instead of Tyr115 was reduced significantly, particularly when poly(rA)484 was used as template. This effect was caused by a dramatic increase in the Km value for dTTP, and was detected using a DNA template mimicking a proviral HIV-1 gag sequence. Misinsertion fidelity assays revealed that mutants Y115W, Y115A and Y115S had a higher misinsertion efficiency than the wild-type reverse transcriptase. The low fidelity of these mutants appears to be related to nucleotide recognition rather than altered DNA-DNA template-primer interactions. The effects observed on the steady state kinetic constants, processivity and fidelity were mediated by the 66 kDa subunit, as demonstrated using chimeric heterodimers with the Y115A substitution in either p66 or p51.
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PMID:Human immunodeficiency virus type 1 reverse transcriptase: role of Tyr115 in deoxynucleotide binding and misinsertion fidelity of DNA synthesis. 886 70


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