Potential role of famciclovir for prevention of herpetic whitlow in the health care setting.
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Oral famciclovir was initiated in a health care worker immediately after an accidental percutaneous injury involving a needle freshly removed from a patient's herpes labialis vesicles. In follow-up, the health care worker remained seronegative for herpes simplex I and II antibodies (IgG and IgM) and did not develop herpetic whitlow, supporting the potential role of famciclovir in the prevention of herpetic whitlow in a health care setting. (+info)
Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics.
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The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to approximately 200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A-->G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (tau approximately 200 ms). Addition of Mg(2+) ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg(2+) are very similar to those induced by Ca(2+). The relaxation data do not provide any evidence for formation of Mg(2+)-inner sphere complexes in hammerhead ribozymes, because a Mg(2+)-specific relaxation effect was not visible. However, a Mg(2+)-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere complexation. Amplitudes and time constants show that the equilibrium constant of inner sphere complexation is 1.2, corresponding to 55% inner sphere state of the Mg(2+) complexes; the rate constant 6.6 x 10(3) s(-1) for inner sphere complexation is relatively low and shows the existence of some barrier(s) on the way to inner sphere complexes. (+info)
Femtosecond direct observation of charge transfer between bases in DNA.
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Charge transfer in supramolecular assemblies of DNA is unique because of the notion that the pi-stacked bases within the duplex may mediate the transport, possibly leading to damage and/or repair. The phenomenon of transport through pi-stacked arrays over a long distance has an analogy to conduction in molecular electronics, but the mechanism still needs to be determined. To decipher the elementary steps and the mechanism, one has to directly measure the dynamics in real time and in suitably designed, structurally well characterized DNA assemblies. Here, we report our first observation of the femtosecond dynamics of charge transport processes occurring between bases within duplex DNA. By monitoring the population of an initially excited 2-aminopurine, an isomer of adenine, we can follow the charge transfer process and measure its rate. We then study the effect of different bases next to the donor (acceptor), the base sequence, and the distance dependence between the donor and acceptor. We find that the charge injection to a nearest neighbor base is crucial and the time scale is vastly different: 10 ps for guanine and up to 512 ps for inosine. Depending on the base sequence the transfer can be slowed down or inhibited, and the distance dependence is dramatic over the range of 14 A. These observations provide the time scale, and the range and efficiency of the transfer. The results suggest the invalidity of an efficient wire-type behavior and indicate that long-range transport is a slow process of a different mechanism. (+info)
2-Aminopurine fluorescence quenching and lifetimes: role of base stacking.
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2-Aminopurine (2AP) is a fluorescent analog of guanosine and adenosine and has been used to probe nucleic acid structure and dynamics. Its spectral features in nucleic acids have been interpreted phenomenologically, in the absence of a rigorous electronic description of the context-dependence of 2AP fluorescence. Now, by using time-dependent density functional theory, we describe the excited-state properties of 2AP in a B-form dinucleotide stacked with guanosine, adenosine, cytosine, or thymine. Calculations predict that 2AP fluorescence is quenched statically when stacked with purines, because of mixing of the molecular orbitals in the ground state. In contrast, quenching is predicted to be dynamic when 2AP is stacked with pyrimidines, because of formation of a low-lying dark excited state. The different quenching mechanisms will result in different experimentally measured fluorescence lifetimes and quantum yields. (+info)
Real time fluorescence analysis of the RecA filament: implications of base pair fluidity in repeat realignment.
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During recombination, when Escherichia coli RecA mediates annealing across DNA repeats, Watson-Crick chemistry can only specify the complementarity of pairing, but not the most optimal frame of alignment. We describe that although stochastic alignments across poly(dA) and poly(dT) can lead to sub-optimally annealed duplexes containing ssDNA gaps/overhangs, the same are realigned into an optimal frame by a putative motor activity of RecA [Sen et al. (2000) Biochemistry 39, 10196-10206]. In the present study, we analyze the nature of realignment intermediates in real time, by employing a fluorescent probe, 2-aminopurine (2AP), which can not only report the status of RecA on the unstacked duplex, but also the fluidity of bases in such a filament. Although known to display a lower affinity for duplex DNA, RecA seems to remain functionally associated with these sub-optimally aligned repeat duplexes, until the realignment approaches completion. Moreover, a comparison of 2AP fluorescence in repeat versus mixed sequences indicates that bases in a RecA repetitive DNA filament exhibit higher degrees of freedom that might mediate a 'non-planar hydrogen bonding cross talk' across the bases on either strand. We discuss a model to explain the mechanistic basis of realignment and its implications in signaling the end of homology maximization, which triggers RecA fall off. (+info)
Peculiar 2-aminopurine fluorescence monitors the dynamics of open complex formation by bacteriophage T7 RNA polymerase.
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The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at -4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase. Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at -4 from the guanine at -5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, ED(c) is formed with a K(d) of 1.8 microm. This complex isomerizes to an open complex, ED(o1), in an energetically unfavorable reaction with an equilibrium constant of 0.12. The ED(o1) further isomerizes to a more stable open complex, ED(o2), with a rate constant around 300 s(-1). Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s(-1). (+info)
Famciclovir for ophthalmic zoster: a randomised aciclovir controlled study.
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AIMS: To compare the efficacy and safety of famciclovir with aciclovir for the treatment of ophthalmic zoster. METHODS: Randomised, double masked, aciclovir controlled, parallel group in 87 centres worldwide including 454 patients with ophthalmic zoster of trigeminal nerve (V(1)) comprised the intent to treat population. Oral famciclovir 500 mg three times daily or oral aciclovir 800 mg five times daily for 7 days. Assessments included day 0 (screening), days 3 and 7 (during treatment), days 10, 14, 21, 28 and monthly thereafter, up to 6 months (follow up). Proportion of patients who experienced ocular manifestations, severe manifestations and non-severe manifestations; loss of visual acuity was the main outcome measure. RESULTS: The percentage of patients who experienced one or more ocular manifestations was similar for famciclovir (142/245, 58.0%) and aciclovir (114/196, 58.2%) recipients, with no significant difference between groups (OR 0.99; 95% CI 0.68, 1.45). The percentage of patients who experienced severe and non-severe manifestations was similar between groups, with no significant difference. The prevalence of individual ocular manifestations was comparable between groups. There was no significant difference between groups for visual acuity loss. CONCLUSION: Famciclovir 500 mg three times daily was well tolerated and demonstrated efficacy similar to aciclovir 800 mg five times daily. (+info)
Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus.
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Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains. (+info)