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PubMed2000

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities.

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.

Amino Acid Sequence↗

PubMed1995

[Endogenous cellular reverse transcriptase in the rat brain. A comparative analysis of the authentic enzyme and recombinant reverse transcriptase coded by a mobile genetic element of the LINE class].

A purified preparation of endogenous RNA-dependent DNA-polymerase (reverse transcriptase) earlier identified in rat brain (Ivanov, V.A., Pakhotin, P.I., Bobkova, N.V., and Ilyin, Yu.V. parallel Dokl. RAN (1992). V. 323, P. 173-177) has been obtained. A comparative analysis of the enzyme and recombinant reverse transcriptase coded by the mobile genome element jockey earlier expressed in a heterological cell system (Ivanov V.V., Melnikov A.A., Siunov A.V., Fodor I.I., Ilyin, Yu.V. parallel EMBO J. (1991), V. 10, P. 2489-2495) has been carried out. Like retroviral RNA-dependent DNA-polymerases, these enzymes show preference for polyribonucleotides and can use poly(rCm) as template. Besides they are inhibited by SH-reagents and require bivalent cations (Mg2+ or Mn2+) and detergent and/or KCl as ionic strength carrier. The enzymes differ drastically from retrovirus reverse transcriptases by a number of catalytic properties (low optima of concentration of requisite cations and ionic strength, strong preference for Mn2+, highly efficiency in using poly(rCm), lack of associated RNase H activity) but exhibit a high degree of similarity among themselves with regard to the above properties. It is suggested that endogenous reverse transcriptase from rat brain is a product of expression of the mobile genome element of the LINE family.

Animals↗

PubMed1995

Single-step electrotransfer of reverse-stained proteins from sodium dodecyl sulfate-polyacrylamide gel onto reversed-phase minicartridge and subsequent desalting and elution with a conventional high-performance liquid chromatography gradient system for analysis.

Isolation of proteins from polyacrylamide electrophoresis gels by a novel combination of techniques is described. A given protein band from a reverse stained (imidazol-sodium dodecyl sulfate--zinc salts) gel can be directly electrotransferred onto a reversed-phase chromatographic support, packed in a self-made minicartridge (2 mm in thickness, 8 mm in internal diameter, made of inert polymeric materials). The minicartridge is then connected to a high-performance liquid chromatography system and the electrotransferred protein eluted by applying an acetonitrile gradient. Proteins elute in a small volume ( < 700 microL) of high-purity volatile solvents (water, trifluoroacetic acid, acetonitrile) and are free of contaminants (gel contaminants, salts, etc). Electrotransferred proteins were efficiently retained, e.g., up to 90% for radioiodinated alpha-lactalbumin, by the octadecyl matrix, and their recovery on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73% for alpha-lactalbumin. The technique was successfully applied to a variety of proteins in the molecular mass range 6-68 kDa, and with amounts between 50 and 2000 pmol. The good mechanical and chemical stability of the developed minicartridges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single-step separation of two proteins unresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to their different elution from the reversed-phase support. The isolated proteins were amenable to analysis by N-terminal sequencing, enzymic digestion and mass spectrometry of their proteolytic fragments. Chromatographic elution of proteins from the reversed-phase mini-cartridge was apparently independent of the specific loading mode employed, i.e., loading by conventional loop injection or by electrotransfer.

Animals↗

PubMed1994

Proteolyses of a fluorogenic insulin derivative and native insulin in reversed micelles monitored by fluorescence emission, reversed-phase high-performance liquid chromatography, and capillary zone electrophoresis.

The preparation and substrate properties of the fluorogenic insulin derivative N alpha A1-aminobenzoyl-N epilson B29-Tyr(NO2)- insulin are described. This semisynthetic protein intramolecularly quenched by long-range resonance energy transfer between the donor/acceptor pair 2-aminobenzoic acid and 3-nitrotyrosine was used to prove the activity of serine proteases toward substrates of high molecular weight after incorporation in reversed micelles. The proteases investigated, trypsin and alpha-chymotrypsin, were shown to be hydrolytically active in reversed micellar solvent systems stabilized by cetyltrimethylammonium bromide or sodium-1,2-bis(2-ethylhexylcarbonyl)-1- ethane sulfonate. Apart from fluorometric enzyme assays, methods for monitoring proteolyses in reversed micelles were elaborated using either reversed-phase high-performance liquid chromatography or capillary zone electrophoresis. Enzymatic digestions of native insulin by the specific protease trypsin and the less specific protease alpha-chymotrypsin were performed. In contrast to aqueous solution, high but still variable specificity of alpha-chymotrypsin which was dependent on the micellar environment was observed. The results promise further insight into the influence of interfacial environments on enzyme action and a novel approach to enzyme-mediated protein modifications by the use of microstructured solvent systems.

Chromatography, High Pressure Liquid↗

PubMed1987

Osmotic reversal induces assembly of tight junction strands at the basal pole of toad bladder epithelial cells but does not reverse cell polarity.

This paper reports the effect of reversing the osmotic environment between luminal and serosal compartments of a toad urinary bladder on the polarity of assembly of tight junction strands. Toad bladders were filled with Ringer's solution (220 mOsm) and were immersed in distilled water at room temperature or at 37 degrees C. Within two minutes, new tight junction strands are assembled. The new tight junctional strands unite the basal pole of epithelial cells with the apical side of basal cells. Physiological studies show that oxytocin, a synthetic analog of antidiuretic hormone, is still capable of inducing increases in water transport in epithelia which were osmotically reversed. This capacity decreases significantly for longer periods of osmotic reversal. Osmotic reversal does not alter the original polarity of epithelial cells: the apical tight junction belt, at the apical pole, is not displaced; the freeze-fracture morphology typical of apical plasma membrane (particle-rich E faces; particle-poor P faces) is not altered; oxytocin and cyclic AMP induce aggregates which are observed only at the apical plasma membrane. Massive assembly of junctional elements occurs even in epithelia preincubated in the presence of cycloheximide (an inhibitor of protein synthesis) or of cytoskeleton perturbers. Our experiments show that the polarity of assembly of tight junction strands depends on the vectorial orientation of the osmotic environment of the epithelium.

Animals↗

PubMed1982

Reversibility of the transformed and neoplastic phenotype. III. Long-term treatment with electrophoretically pure mouse interferon leads to the progressive reversion of the phenotype of X-ray transformed C3H/10T1/2 cells.

Electrophoretically pure mouse interferon induced the progressive reversion of the transformed phenotype of a clone of X-ray transformed C3H/10T1/2 cells. Cells of this clone did not harbor C-type particles and reverse transcriptase activity was not detected. Interferon-treated transformed cells were aligned without cellular overlapping and attained low cell densities. Morphologic changes were associated with the appearance of a thick layer of submembranous microfilaments. The tumorigenicity of interferon-treated cells was markedly reduced. Back reversion to the transformed phenotype occurred progressively when these cells were passaged in the absence of interferon. These results suggest that interferon may induce the reversion of the transformed phenotype by a mechanism other than by its antiviral activity.

Animals↗

PubMed1989

Basic considerations in the reversal of atherosclerosis: significance of high-density lipoprotein in stimulating reverse cholesterol transport.

Atherosclerotic cardiovascular disease in the elderly is the result of several decades of cholesterol accretion. Advanced lesions may not be amenable to treatment, but a reversal of cholesterol accumulation may be possible. High-density lipoproteins (HDL) could serve an important function in this reversal through their role in the process of reverse cholesterol transport, which removes cholesterol from the body. Reverse cholesterol transport could be stimulated by raising plasma HDL level, but the efficacy of the process may be determined by the way in which HDL level is elevated. The increase of HDL synthesis rate may be the best approach. The antiatherosclerotic effects of gemfibrozil, a lipid-lowering agent that appears to raise HDL synthesis rate, may be mediated through this mechanism.

Age Factors↗

PubMed1991

Phenotype reversal in induced mutants of CHO cells: analysis of the reversed cell lines.

We have previously shown that descendants of CHO-derived hprt or aprt mutants induced by ethyl methanesulphonate usually undergo a rapid loss of the mutant phenotype during the 10 generations or so of culture in non-selective medium immediately following mutagenesis (Bradley, 1980; Bradley and Laviolette, 1989). We now present an analysis of several mutants and their descendants which have lost the mutant phenotype, or 'reversed'. The drug-resistance properties of reversed cells were generally intermediate between W.T. and mutant, and message level and enzyme-specific activity were also intermediate, correlating with the phenotype. Although this was consistent with a model of inactivation-reactivation of the target gene to explain the reversal phenomenon, the model was ruled out by Northern blot analysis of several induced mutants, which showed no correlation between level of message and tendency of the mutant to lose its phenotype. Karyotype analysis showed that three out of four reversed lines were near-tetraploid and the fourth had a substantial proportion of near-tetraploid cells. This suggests cell fusion between a mutant and a W.T. cell may explain the phenomenon. A prediction of this model, namely that mutagen treatment increases cell hybrid formation, was tested and found to be true.

Adenine Phosphoribosyltransferase↗

PubMed1985

Naloxone-reversible and non-reversible shock-induced analgesia during development.

We evaluated the development of naloxone-reversible and naloxone-non-reversible analgesia induced by footshock in rats of different ages and correlated it with the concentrations of beta-endorphin and dynorphin in brain areas and the spinal cord. We observed that naloxone-non-reversible shock-induced analgesia appeared first and its appearance might be related to the early presence of high dynorphin concentrations in the spinal cord. Naloxone-reversible analgesia appeared later together with the reaching of adult concentrations of cerebral beta-endorphin.

Analgesia↗

PubMed2002

Prolonged reversal of morphine tolerance with no reversal of dependence by protein kinase C inhibitors.

The phosphatidylinositol (PI) cascade plays a pivotal role in mediating behavioral tolerance to the antinociceptive effects of morphine. Earlier we reported that antinociceptive tolerance was completely reversed 30 min after the administration of inhibitors of each step in the PI cascade. The aim of this study was to determine whether injection of a single dose of protein kinase C (PKC) inhibitor would elicit a prolonged reversal of morphine tolerance for up to 24 h. Three days after implantation of placebo- or 75-mg morphine pellets, mice received intracerebroventricular (i.c.v.) injections of vehicle or PKC inhibitor drug. Morphine challenge doses were then administered 4, 8 and 24 h later to test for tolerance reversal. In non-tolerant mice, Gö-7874 and sangivamycin had no effect on the potency of morphine. However, Gö-7874 and sangivamycin significantly reversed morphine tolerance at 4, 8 and 24 h. In addition, the role of PKC in morphine physical dependence was determined. Gö-7874 and sangivamycin by themselves did not precipitate spontaneous morphine withdrawal. Therefore, experiments were conducted to determine whether the PKC inhibitors would block naloxone-precipitated withdrawal. However, neither a 30-min nor a 24-h pretreatment with Gö-7874 or sangivamycin blocked naloxone withdrawal. Our results along with other publications indicate that PKC is a pivotal kinase essential for maintaining animals in an opioid tolerant state. Finally, the use of persistent PKC inhibitors that lasted for 24 h demonstrated that the neuronal systems in these animals did not adapt by increasing the activity of other protein kinase cascades to re-establish morphine tolerance.

Animals↗

PubMed2002

Electrophoretic analysis of amines using reversed-phase, reversed-polarity, head-column field-amplified sample stacking and laser-induced fluorescence detection.

This paper describes the use of reversed-phase, reversed-polarity head-column field-amplified sample stacking (HCFASS) for on-line sample concentration in conventional capillary electrophoresis. The effective stacking efficiency was determined as a function of sodium hydroxide concentration in the sample matrix. Results concur with theoretical predictions where stacking efficiency depends on the conductivity (electric field strength) and electrophoretic mobility in the sample matrix solution. Fluorescein isothiocyanate-derivatized aniline and 2,4-dimethylaniline were dissolved in sodium hydroxide (800 microM), separated in a phosphate running buffer (0.05 M, pH 9.0) and detected utilising laser-induced fluorescence. The use of reversed-phase, reversed-polarity HCFASS with laser-induced fluorescence detection yielded sensitivity improvements with respect to normal injection schemes in excess of three orders of magnitude, and a limit of detection as low as 10(-13) M.

Amines↗

PubMed2003

The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions.

BACKGROUND: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. OBJECTIVES: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. STUDY DESIGN: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. RESULTS AND CONCLUSIONS: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo.

Cell Line↗

PubMed2003

Comprehensive coupled reversed-phase reversed-phase separations of a complex isomeric mixture.

Using predictions based on results obtained from Information Theory and Factor Analysis for the two-dimensional separation of a complex isomeric mixture, a practical experimental comprehensive coupled reversed phase-reversed phase chromatographic system was developed. In total four reversed phase-reversed phase systems were studied, each of which theory predicted would be able to resolve essentially equal numbers of components. However, in practice only one of these coupled systems realised the theoretical potential. This system employed as the first dimension, a C18 stationary phase with methanol as the mobile phase and as the second dimension, carbon clad zirconia as the stationary phase and acetonitrile as the mobile phase. In this system, 27 of the 32 isomers of a mixture of oligostyrenes were resolved. Failure of the remaining coupled systems to achieve the theoretical potential was attributed to high solute crowding, low efficiency of separation space utilisation and long analysis times in the second dimension.

Journal Article↗

PubMedDate not supplied

Determination of free and reversibly bound sulphite in foods by reverse-phase, ion-pairing high-performance liquid chromatography.

The reaction of sulphite with formaldehyde to form hydroxymethylsulphonate (HMS), which is very stable under the controlled conditions of this assay, was used as the first step in an analytical procedure to determine foodborne sulphite. The effect of mobile-phase pH on the stability of HMS during high-performance liquid chromatography was studied. It was found that on-column HMS dissociation to formaldehyde and bisulphite increased with the pH of the mobile phase; therefore the relatively low pH 4.7, at which the dissociation of HMS was approximately 2%, was selected for the analysis. In addition, the release of sulphite from its reversibly bound forms in wine and other foods was examined as a function of the pH of the extraction medium by following the appearance of HMS formed from the reaction of the freed sulphite with formaldehyde. The rate of dissociation of the reversibly bound sulphite was relatively slow at pH 3 but very rapid at pH 7. This difference in kinetics was exploited to develop a procedure to determine free and reversibly bound sulphite in food. The method was challenged by post-reagent spiking studies, i.e. adding the sulphite spike after the food has been blended with the sulphite-protective formaldehyde solution but before proceeding with the remainder of the assay. An average recovery of 100% with a standard deviation of 5.2% (n = 45) was realized at levels of 5, 10 and 20 ppm by weight as sulphur dioxide. Recovery of the sulphite added as the bisulphite addition product of acetaldehyde, a model compound for reversibly bound sulphite, was 95%.

Animals↗

PubMed2000

A comparison of mutation spectra detected by the Escherichia coli lac(+) reversion assay and the Salmonella typhimurium his(+) reversion assay.

Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A.T-->T.A, G.C-->A.T and G.C-->T.A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the S.TYPHIMURIUM: strains to G.C-->A.T transitions induced by N(4)-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G.C-->A.T transitions induced by 2-aminopurine and phosmet. Escherichia coli strain WP3104P, which detects G.C-->T.A transversions, was superior to the S.TYPHIMURIUM: strains in detecting transversions induced by N(4)-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Escherichia coli WP3105P was also more sensitive than S. TYPHIMURIUM: to A.T-->T.A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine. The present results indicate that the E.COLI: Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the S.TYPHIMURIUM: His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens.

2-Aminopurine↗

PubMed1998

The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain.

Heterologous expression of human LINE-1 ORF2 in yeast yielded a single polypeptide (Mr145 000) which reacted with specific antibodies and co-purified with a reverse transcriptase activity not present in the host cells. Various deletion derivatives of the ORF2 polypeptide were also synthesized. Reverse transcriptase assays using synthetic polynucleotides as template and primer revealed that ORF2 protein missing a significant portion of the N-terminal endonuclease domain still retains some activity. Deletion of the C-terminal cysteine-rich motif reduces activity only a small amount. Three non-overlapping deletions spanning 144 amino acids just N-terminal to the common polymerase domain of the ORF2 protein were analyzed for their effect on reverse transcriptase activity; this region contains the previously-noted conserved Z motif. The two deletions most proximal to the polymerase domain eliminate activity while the third, most-distal deletion had no effect. An inactive enzyme was also produced by substitution of two different amino acids in a highly-conserved octapeptide sequence, Z8, located within the region removed to make the deletion most proximal to the polymerase domain; substitution of a third had no effect. We conclude that the octapeptide sequence and neighboring amino acids in the Z region are essential for reverse transcriptase activity, while the endonuclease and cysteine-rich domains are not absolutely required.

Animals↗

PubMed2000

Reversal of repolarization gradient does not reverse the chirality of shock-induced reentry in the rabbit heart.

INTRODUCTION: Two hypotheses have been proposed to explain the mechanisms of vulnerability and related failure of defibrillation therapy: the cross-field-induced critical point hypothesis and the virtual electrode-induced phase singularity hypothesis. These two hypotheses predict the opposite effect of preshock repolarization on the chirality (direction of rotation) of shock-induced reentry. The former suggests its reversal upon reversal of repolarization, whereas the latter suggests its preservation. The aim of this study was to determine, by reversing the repolarization sequence, which of the mechanisms is responsible for internal shock-induced arrhythmia in the Langendorff-perfused rabbit heart. METHODS AND RESULTS: We used high-resolution optical mapping to assess the chirality of postshock reentry in 11 hearts. Hearts were paced at a coupling interval of 300 msec at various sites around the field of view (13.5 x 13.5 to 16.5 x 16.5 mm). Cathodal monophasic implantable cardioverter defibrillator shocks (-100 V, 8 msec) were applied during the T wave from a 10-mm coil electrode placed into the right ventricular cavity. We used 3.5 +/- 0.8 different pacing sites per heart. Change in direction of repolarization did not result in change of chirality. Chirality was constant in all 11 hearts despite the complete reversal of activation and repolarization patterns. However, the position of resulting vortices depended on transmembrane polarization gradient inverted delta Vm and amplitude of negative polarization Vm (deexcitation). Stronger gradients and deexcitation produced earlier epicardial break excitation (P = 0.04 and P < 0.0001, respectively). CONCLUSION: Virtual electrode-induced phase singularity mechanism underlies internal shock-induced arrhythmia in this model.

Animals↗

PubMed1991

Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function.

The reverse transcriptase enzymes of retroviruses are multifunctional proteins containing both DNA polymerase activity and a nuclease activity, termed RNase H, specific for RNA in RNA-DNA hybrid form. To determine the role of RNase H activity in retroviral replication, we constructed a series of mutant genomes of Moloney murine leukemia virus that encoded reverse transcriptase enzymes that were specifically altered to retain polymerase function but lack RNase H activity. The mutant genomes were all replication defective. Analysis of in vitro reverse transcription reactions carried out by mutant virions showed that minus-strand strong-stop DNA was formed but did not efficiently translocate to the 3' end of the genome; rather, the DNA was stably retained in RNA-DNA hybrid form. Plus-strand strong-stop DNA was not detected. These results suggest that RNase H normally promotes strong-stop translocation, perhaps by exposing single-stranded DNA sequences for base pairing. Four new DNA species were also detected among the reaction products. Analysis of these DNAs suggested that they were minus-strand DNAs formed from VL30 RNAs encoded by the mouse genome. We suggest that reverse transcriptase can initiate DNA synthesis at any one of four alternate tRNA primer-binding sites near the 5' ends of VL30 RNAs.

Animals↗