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PubMedDate not supplied

Task-specific reversal of visual hemineglect following bilateral reversible deactivation of posterior parietal cortex: a comparison with deactivation of the superior colliculus.

The purpose of the present study was to compare and contrast behavioral performance on three different tasks of spatial cognition during unilateral and bilateral reversible deactivation of posterior parietal cortex. Specifically, we examined posterior middle suprasylvian (pMS) sulcal cortex in adult cats during temporary and reversible cooling deactivation. In Task 1, the cats oriented to a high-contrast, black visual stimulus moved into the visual field periphery. In Task 2, the cats oriented to a static light-emitting diode (LED). Task 3 examined the cats' ability to determine whether a black-and-white checkered, landmark box was closer to the right or left side of the testing apparatus. Following training on all tasks, cryoloops were implanted bilaterally within the pMS sulcus. Unilateral deactivation of pMS sulcal cortex resulted in virtually no responses to either moved or static stimuli and virtually no responses to landmarks presented in the contralateral hemifield, and a profound contralateral hemifield neglect was induced. Responses to stimuli and landmarks presented in the ipsilateral hemifield were unimpaired. Additive, bilateral cooling of the homotopic region in the contralateral hemisphere, but not an adjacent region, resulted in reversal of the initial hemineglect for the moved stimulus, yet induced a complete failure to orient to peripheral static LED stimuli. Bilateral cooling also reversed the contralateral neglect of the landmark, but then cats could not accurately determine position of the landmark anywhere in the visual field because performance was reduced to chance levels for all landmark loci in both hemifields. In this instance, as the contralateral neglect disappeared during bilateral cooling of pMS cortex, a new spatial discrimination deficit was revealed across the entire visual field. We conclude that pMS cortex contributes in multiple ways to the analyses of space, and that these contributions cannot be safely predicted from analyses of unilateral deactivations or from one task to another. Moreover, it is clear that other structures are capable of guiding orienting to high contrast, moved targets when pMS cortex is eliminated from brain circuitry. However, these same structures are incapable of supporting either orienting to static stimuli or analyses of spatial relations as tested with the landmark task. The impact of reversible deactivation of the superior colliculus on these same tasks is discussed.

Animals↗
PubMed1996

Reversible thermal unfolding of ribonuclease T1 in reverse micelles.

The reverse micellar system formed by the negatively charged surfactant AOT and the organic solvent isooctane is used to solubilize the protein RNase T1. The physicochemical properties of the entrapped protein have been studied using intrinsic tryptophan fluorescence and far-and near-UV CD. These studies indicate a similar structure for the protein in reverse micelles and in pH 7.0 buffer. Thermal unfolding has been studied as a function of W0, the molar ratio of water to AOT, in the solution. Measuring the change in fluorescence intensity as a function of temperature, we observe a reversible transition for W0 in the range 5-12. Heating rate dependencies carried out on these transitions (0.6-3.0 degrees C/min) indicate that the transition temperature and the apparent van't Hoff enthalpy change depend on the scanning rate as well as on W0. The values of the transition temperature, T(m) and the enthalpy change, delta H degrees(un), extrapolated to an infinitely slow scanning rate, are analyzed considering the electrostatic interaction of the charged residues of the protein with the charges of the surfactant molecules forming reverse micelles, the variation of the size of the reverse micelles, and the relative rates of unfolding, refolding, and irreversible denaturation.

Calorimetry, Differential Scanning↗
PubMed1981

Adaptation to the reversal of binocular depth cues: effects of wearing left-right reversing spectacles on stereoscopic depth perception.

The principle of stereopsis, that crossed disparity causes a convex perception and uncrossed disparity a concave one, has for a long time been considered to depend on a very rigid neural mechanism not affected by experience. Experiments are reported here which show that this relationship between disparity and perceived depth can be reversed by experience. An observer wore a pair of left-right reversing spectacles continuously for nine days. The spectacles also reversed the relation between the direction of perceived depth and the direction of binocular depth cues, ie disparity and vergence. For a period starting two days before wearing the spectacles and continuing until seventy-nine days after their removal the observer was examined with a haploscope and an electrooculograph. All the stereoscopic experiments were carried out without spectacles in order to examine some aftereffects of wearing spectacles. For the stereograms with linear contours not only the adaptive reversal of the relation between disparity and perceived depth, but also some abnormal depth perceptions and long-lasting aftereffects were found. For Julesz's random-dot stereograms, however, in which contours can be seen only after binocular combination, no adaptive change or reversal occurred. These results suggest that the process of stereopsis consists of two concurrent subprocesses.

Adaptation, Ocular↗
PubMed1997

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors.

Reverse transcriptase must perform two specialized template switches during retroviral DNA synthesis. Here, we used Moloney murine leukemia virus-based vectors to examine the site of one of these switches during intracellular reverse transcription. Consistent with original models for reverse transcription, but in contrast to previous experimental data, we observed that this first strand transfer nearly always occurred precisely at the 5' end of genomic RNA. This finding allowed us to use first strand transfer to study the classes of errors that reverse transcriptase can and/or does make when it switches templates at a defined position during viral DNA synthesis. We found that errors occurred at the site of first strand transfer approximately 1000-fold more frequently than reported average reverse transcriptase error rates for template-internal positions. We then analyzed replication products of specialized vectors that were designed to test possible origins for the switch-associated errors. Our results suggest that at least some errors arose via non-templated nucleotide addition followed by mismatch extension at the point of strand transfer. We discuss the significance of our findings as they relate to the possible contribution that template switch-associated errors may make to retroviral mutation rates.

Animals↗
PubMed1986

Identification of genes for reverse transcriptase-like enzymes in two Drosophila retrotransposons, 412 and gypsy; a rapid detection method of reverse transcriptase genes using YXDD box probes.

Using synthetic oligonucleotides corresponding to the amino acid sequences best conserved among retroviral reverse transcriptases, we developed a rapid method to detect cloned DNA fragments with the genes for reverse transcriptases. By this technique followed by nucleotide sequence determination, nucleotide sequences coding for reverse transcriptase-like enzymes were identified in two Drosophila retrotransposons, gypsy and 412. Our sequence analysis suggested that there are at least two major groups of retrotransposons in Drosophila with respect to putative reverse transcriptases and both gypsy and 412 belong to a category of retrotransposons which have putative reverse transcriptases very similar in amino acid sequence to the counterpart of Moloney murine leukaemia virus, a typical mammalian retrovirus.

Amino Acid Sequence↗
PubMed1987

Comparative study of the Jordan left-right reversal test, the reversals frequency test, and teachers' observations.

The intertest pass/fail reliability of the Jordan left-right reversal test (JRT) and reversals frequency test (RFT) was compared on 510 "regular classroom" and 126 "learning handicapped" children. Children were divided into two age groups, 5 to 8 years and 9 to 13 years. Teachers completed a questionnaire assessing whether they felt a reversal problem existed. Statistical analysis showed low to moderate agreement between the pass/fail rate of the two tests (p less than 0.001) for both age groups of normal children; however, no statistical agreement was found for either age group of learning handicapped children. There was only very low agreement between the teacher's assessment of reversals and the pass/fail rates for both age groups of normals and the 9- to 13-year-old learning handicapped group. No agreement was found for the 5- to 8-year-old learning handicapped group. The effect of test design and recommendations for the clinical investigation of reversals are discussed.

Adolescent↗
PubMed1991

Reverse-transcriptase-associated RNaseH activity mediates template switching during reverse transcription in vitro.

During the first steps of reverse transcription of the retroviral genome, sequences present at the extremities of the RNA are used to reconstitute a host cell PolII promoter. The assembly of the promoter occurs by template switching, which takes advantage of a direct repeat at the ends of the RNA molecule. These steps are catalysed by the viral reverse transcriptase, which carries an intrinsic RNaseH activity that is probably also involved therein. To study the role of the RNaseH activity in this first template-switching event, an in vitro system has been developed based on primer extensions of synthetic RNAs. When an RNA was reverse transcribed with wild-type reverse transcriptase in the presence of a second RNA the 3' part of which was repeated at the 5' end of the first one, extension products could be observed corresponding to a chimeric cDNA comprising both RNA species. This template switching could not be detected when a mutant reverse transcriptase lacking the RNaseH activity was used. The results show that the RNaseH activity is needed to remove the 5' RNA sequences from the cDNA:RNA hybrid thereby enabling its translocation to another RNA containing an appropriate complementary target sequence.

Endoribonucleases↗
PubMed1994

Initiation of reverse transcription during cell-to-cell transmission of human immunodeficiency virus infection uses pre-existing reverse transcriptase.

H3B cells, a laboratory clone of H9 cells persistently infected with the HTLV-IIIB strain of human immunodeficiency virus (HIV), contained significant levels of cell-associated reverse transcriptase (RT) activity measured by in vitro assays using either exogenous or endogenous templates. The cell-associated RT activity detected using exogenous template was almost wholly in a soluble (non-sedimentable) form whereas endogenous activity sedimented as a particulate structure associated with viral RNA. Despite this, H3B cells did not contain episomal HIV DNA detectable by Southern blot, indicating that in vivo reverse transcription was not occurring to any significant extent in these cells. However, when susceptible HUT 78 cells were infected by co-cultivation with H3B cells, dramatic synthesis of episomal HIV DNA occurred. Concurrently with this de novo initiation of reverse transcription, however, we found no detectable change in intracellular levels or cleavage profiles of immunoprecipitable RT polypeptides. Finally, actinomycin D pre-treatment of H3B cells to prevent de novo transcription from donor cell proviral DNA after co-cultivation did not affect the initiation of in vivo reverse transcription following cell-to-cell HIV infection. These results demonstrated that cells persistently infected with HIV contained significant fully cleaved cell-associated RT in a form that was active in vitro but not in vivo and that following cell-to-cell transmission of HIV infection to susceptible cells, de novo reverse transcription was initiated without detectable evidence of further synthesis or proteolytic processing of HIV RT. The nature of this initiation process requires further study.

Biological Transport↗
PubMed1997

Transcomplementation of nucleotide priming and reverse transcription between independently expressed TP and RT domains of the hepatitis B virus reverse transcriptase.

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. We recently expressed human hepatitis B virus (HBV) reverse transcriptase (pol) in insect cells by using the recombinant baculovirus system. The purified protein is active in nucleotide priming and reverse transcription reactions. In this report, we demonstrate that the tyrosine residue at amino acid number 63 within the TP (terminal protein) domain of the polymerase is the site of covalent linkage of the first nucleotide of minus-strand DNA. Analysis of pol polypeptides with mutations in the TP and RT (reverse transcriptase) domains indicated that both domains were required for in vitro nucleotide priming activity. Polymerase proteins with mutations in the TP and RT domains were not capable of complementing each other in the nucleotide priming reaction, suggesting that transcomplementation between full-length polypeptides was not possible. However, when the TP and RT domains were expressed as separate polypeptides, they formed a highly stable complex that was active in nucleotide priming and reverse transcription. The presence of an epsilon stem-loop dramatically increased the nucleotide priming activity in transcomplementation assays, even though full-length pol displayed similar activities in the absence and presence of epsilon. These data raise the possibility that in the transcomplementation assay, epsilon may play a role in the formation of a functional complex between TP and RT, rather than being required only as the template for nucleotide priming. The results indicate that using the baculovirus system, it is possible to dissect the protein-protein and protein-RNA interactions required for HBV genome replication.

Animals↗
PubMed1999

To reverse or not to reverse: when is an ambiguous figure not ambiguous?

The role of bottom-up processes in our perception of reversible figures was examined. In Experiment 1 the overlapping squares figure and nonsense reversible figures were used. The effects of adapting subjects for differing durations to an unambiguous version of the figure before presentation of the traditional reversible figure were determined under conditions of varying precision of fixation. In Experiment 2 the research was expanded to other examples of reversible figures. In both experiments, results with two dependent measures (the subject's first percept and the number of reversals reported) were generally consistent with the interpretation of bottom-up processes underlying the adaptation effects. However, the crucial role of stimulus and procedural variables and the differential sensitivity of the two dependent measures was demonstrated.

Adult↗
PubMed2006

HIV-1 reverse transcriptase plus-strand initiation exhibits preferential sensitivity to non-nucleoside reverse transcriptase inhibitors in vitro.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are highly specific and potent allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. NNRTIs inhibit reverse transcription in a substrate length-dependent manner in biochemical assays and in cell-based HIV-1 replication assays, suggesting a stochastic inhibitory mechanism. Surprisingly, we observed that NNRTIs potently inhibited plus-strand initiation in vitro under conditions in which little or no inhibition of minus-strand DNA synthesis was observed. In assays that recapitulated the initiation of plus-strand DNA synthesis, greater inhibition was observed with an RNA PPT primer than with a DNA primer of corresponding sequence and with wild-type reverse transcriptase but not with NNRTI-resistant enzymes. Structural elements that dictate sensitivity to NNRTIs were revealed using modified plus-strand initiation substrates. The data presented here suggest that specific inhibition of plus-strand initiation may be an important mechanism by which NNRTIs block HIV-1 replication.

Alkynes↗
PubMed2000

Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture.

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three- to fourfold slowing of both DNA 3'-end-directed and RNA 5'-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803-5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3'-end- and RNA 5'-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.

Cells, Cultured↗
PubMed2001

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription.

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Anti-HIV Agents↗
PubMed2006

Rationale and design of a randomized controlled trial to assess the safety and efficacy of cardiac resynchronization therapy in patients with asymptomatic left ventricular dysfunction with previous symptoms or mild heart failure--the REsynchronization reVErses Remodeling in Systolic left vEntricular dysfunction (REVERSE) study.

BACKGROUND: Cardiac resynchronization therapy (CRT) improves symptoms, reduces heart failure (HF)-related hospitalizations, and reverses left ventricular remodeling in some patients with moderate to severe HF and ventricular dyssynchrony defined by a prolonged QRS duration. The effects of CRT on HF outcomes in patients with asymptomatic left ventricular dysfunction (ALVD) or mild HF remain to be determined. METHODS: The REsynchronization reVErses Remodeling in Systolic left vEntricular dysfunction (REVERSE) study is a prospective, multicenter, randomized, double-blind, parallel, controlled clinical trial designed to establish whether CRT combined with optimal medical treatment can attenuate HF disease progression compared with optimal medical treatment alone in patients with ALVD +/- New York Heart Association class I American College of Cardiology/American Heart Association stage C or New York Heart Association class II HF, QRS duration > or =120 milliseconds, left ventricular ejection fraction < or =0.40, and left ventricular end-diastolic diameter > or =55 mm. The primary end point is the HF clinical composite response and left ventricular end-systolic volume index is the first-order secondary end point. Approximately 500 patients from 100 centers in the United States, Canada, and Europe will be randomized to CRT versus no CRT. The follow-up is 5 years in total with the primary and first secondary end points reported at 12 months. Enrollment began in September 2004 and is expected to be completed in 2006. CONCLUSION: REVERSE will assess the safety and efficacy of CRT in patients with ALVD or mild HF and electrocardiographic evidence of ventricular dyssynchrony.

Cardiac Output, Low↗
PubMed1998

Initiation of in vitro reverse transcription from tRNA(Lys3) on HIV-1 or HIV-2 RNAs by both type 1 and 2 reverse transcriptases.

HIV reverse transcription is initiated from a cellular tRNA partially associated with the retroviral genome. Here we studied homologous HIV-2 cDNA synthesis using natural or synthetic primers. With natural tRNA(Lys3), synthesis of early products comprising nucleotides +5 to +7 preceded the elongation step leading to synthesis of (-) strong-stop cDNA. In the presence of a poly(A) x oligo(dT) trap, no full-length product was observed while early products were still present, showing a transition between initiation and elongation. With DNA primers only an unspecific elongation was found. Our data show a similar mechanism of reverse transcription initiation by HIV-1 and HIV-2 reverse transcriptases. Furthermore, using a heterologous system we found that HIV-1 RNA, in contrast to data reported in the literature, was an excellent template for HIV-2 reverse transcriptase.

Base Sequence↗
PubMed2005

Mutagenic properties of ribonucleotide analogues in reverse transcription with HIV and AMV reverse transcriptases.

Pyrimidine analogues, N4-hydroxycytosine (C(oh)), N4-methoxycytosine (C(mo)) and 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P) can form base pairs with both adenine and guanine. We examined the mutagenic properties of these ribonucleotide analogues in RNA in reverse transcription with HIV and AMV reverse transcriptases. Both reverse transcriptases incorporated dATP and dGTP opposite these analogues in RNA. The incorporation ratio of dGTP to dATP opposite each analogue was measured to estimate the potential for inducing U-to-C mutations. The potentials may be rC(oh) > rC(mo) > rP for both reverse transcriptases. It might be possible to induce mutations in the retroviral genomes and to develop a new antiviral therapy, if these analogues are incorporated by a human RNA polymerase.

Avian Myeloblastosis Virus↗
PubMed2004

Requirement for integrase during reverse transcription of human immunodeficiency virus type 1 and the effect of cysteine mutations of integrase on its interactions with reverse transcriptase.

Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4(+)-T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT.

Cell Line↗
PubMed2004

Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: involvement of viral RNA sequences and the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 uses the tRNA(3)(Lys) molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNA(Pro) or tRNA(1,2)(Lys). These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA(1,2)(Lys)-using virus.

Amino Acid Substitution↗