Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

Algorithm for multi-curve-fitting with shared parameters and a possible application in evoked compound action potential measurements

BACKGROUND: Experimental results are commonly fitted by determining parameter values of suitable mathematical expressions. In case a relation exists between different data sets, the accuracy of the parameters obtained can be increased by incorporating this relationship in the fitting process instead of fitting the recordings separately. METHODS: An algorithm to fit multiple measured curves simultaneously was developed. The method accounts for parameters that are shared by some curves. It can be applied to either linear or nonlinear equations. Simulated noisy "measurement results" were created to compare the introduced method to the "straight forward" way of fitting the curves separately. RESULTS: The analysis of the simulated measurements confirm, that the introduced method yields more accurate parameters compared to the ones gained by fitting the measurements separately. Therefore it needs more computer time. As an example, the new fitting algorithm is applied to the measurements of the evoked compound action potentials (ECAP) of the auditory nerve: This leads to promising ideas to reduce artefacts generated by the measuring process. CONCLUSION: The introduced fitting algorithm uses the relationship between multiple measurement results to increase the accuracy of the parameters. Its application in the field of ECAP measurements is promising and should be further investigated

Electron excitation and energy transfer rates for H2O in the upper atmosphere

Recent measurements of the cross sections for electronic state excitations in H2O have made it possible to calculate rates applicable to these excitation processes. We thus present here calculations of electron energy transfer rates for electronic and vibrational state excitations in H2O, as well as rates for excitation of some of these states by atmospheric thermal and auroral secondary electrons. The calculation of these latter rates is an important first step towards our aim of including water into a statistical equilibrium model of the atmosphere under auroral conditions.Comment: 15 pages, 8 figure

On computational approaches for size-and-shape distributions from sedimentation velocity analytical ultracentrifugation

Sedimentation velocity analytical ultracentrifugation has become a very popular technique to study size distributions and interactions of macromolecules. Recently, a method termed two-dimensional spectrum analysis (2DSA) for the determination of size-and-shape distributions was described by Demeler and colleagues (Eur Biophys J 2009). It is based on novel ideas conceived for fitting the integral equations of the size-and-shape distribution to experimental data, illustrated with an example but provided without proof of the principle of the algorithm. In the present work, we examine the 2DSA algorithm by comparison with the mathematical reference frame and simple well-known numerical concepts for solving Fredholm integral equations, and test the key assumptions underlying the 2DSA method in an example application. While the 2DSA appears computationally excessively wasteful, key elements also appear to be in conflict with mathematical results. This raises doubts about the correctness of the results from 2DSA analysis

Asteroseismology and Interferometry

Asteroseismology provides us with a unique opportunity to improve our understanding of stellar structure and evolution. Recent developments, including the first systematic studies of solar-like pulsators, have boosted the impact of this field of research within Astrophysics and have led to a significant increase in the size of the research community. In the present paper we start by reviewing the basic observational and theoretical properties of classical and solar-like pulsators and present results from some of the most recent and outstanding studies of these stars. We centre our review on those classes of pulsators for which interferometric studies are expected to provide a significant input. We discuss current limitations to asteroseismic studies, including difficulties in mode identification and in the accurate determination of global parameters of pulsating stars, and, after a brief review of those aspects of interferometry that are most relevant in this context, anticipate how interferometric observations may contribute to overcome these limitations. Moreover, we present results of recent pilot studies of pulsating stars involving both asteroseismic and interferometric constraints and look into the future, summarizing ongoing efforts concerning the development of future instruments and satellite missions which are expected to have an impact in this field of research.Comment: Version as published in The Astronomy and Astrophysics Review, Volume 14, Issue 3-4, pp. 217-36

Total Hadronic Cross Section Data and the Froissart-Martin Bound

The energy dependence of the total hadronic cross section at high energies is investigated with focus on the recent experimental result by the TOTEM Collaboration at 7 TeV and the Froissart-Martin bound. On the basis of a class of analytical parametrization with the exponent $\gamma$ in the leading logarithm contribution as a free parameter, different variants of fits to $pp$ and $\bar{p}p$ total cross section data above 5 GeV are developed. Two ensembles are considered, the first comprising data up to 1.8 TeV, the second also including the data collected at 7 TeV. We shown that in all fit variants applied to the first ensemble the exponent is statistically consistent with $\gamma$ = 2. Applied to the second ensemble, however, the same variants yield $\gamma$'s above 2, a result already obtained in two other analysis, by U. Amaldi \textit{et al}. and by the UA4/2 Collaboration. As recently discussed by Ya. I. Azimov, this faster-than-squared-logarithm rise does not necessarily violate unitarity. Our results suggest that the energy dependence of the hadronic total cross section at high energies still constitute an open problem.Comment: 20 pages, 10 figures, introduction extended and general references added to match editorial style, to appear in the Brazilian Journal of Physic

Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved

A quantitative analysis of the effect of cycle length on arrhythmogenicity in hypokalaemic Langendorff-perfused murine hearts

The clinically established proarrhythmic effect of bradycardia and antiarrhythmic effect of lidocaine (10 μM) were reproduced in hypokalaemic (3.0 mM K+) Langendorff-perfused murine hearts paced over a range (80–180 ms) of baseline cycle lengths (BCLs). Action potential durations (at 90% repolarization, APD90s), transmural conduction times and ventricular effective refractory periods (VERPs) were then determined from monophasic action potential records obtained during a programmed electrical stimulation procedure in which extrasystolic stimuli were interposed following regular stimuli at successively decreasing coupling intervals. A novel graphical analysis of epicardial and endocardial, local and transmural relationships between APD90, corrected for transmural conduction time where appropriate, and VERP yielded predictions in precise agreement with the arrhythmogenic findings obtained over the entire range of BCLs studied. Thus, in normokalaemic (5.2 mM K+) hearts a statistical analysis confirmed that all four relationships were described by straight lines of gradients not significantly (P > 0.05) different from unity that passed through the origin and thus subtended constant critical angles, θ with the abscissa (45.8° ± 0.9°, 46.6° ± 0.5°, 47.6° ± 0.5° and 44.9° ± 0.8°, respectively). Hypokalaemia shifted all points to the left of these reference lines, significantly (P < 0.05) increasing θ at BCLs of 80–120 ms where arrhythmic activity was not observed (∼63°, ∼54°, ∼55° and ∼58°, respectively) and further significantly (P < 0.05) increasing θ at BCLs of 140–180 ms where arrhythmic activity was observed (∼68°, ∼60°, ∼61° and ∼65°, respectively). In contrast, the antiarrhythmic effect of lidocaine treatment was accompanied by a significant (P < 0.05) disruption of this linear relationship and decreases in θ in both normokalaemic (∼40°, ∼33°, ∼39° and ∼41°, respectively) and hypokalaemic (∼40°, ∼44°, ∼50° and ∼48°, respectively) hearts. This extended a previous approach that had correlated alterations in transmural repolarization gradients with arrhythmogenicity in murine models of the congenital long QT syndrome type 3 and hypokalaemia at a single BCL. Thus, the analysis in terms of APD90 and VERP provided a more sensitive indication of the effect of lidocaine than one only considering transmural repolarization gradients and may be particularly applicable in physiological and pharmacological situations in which these parameters diverge

Stereo-Selectivity of Human Serum Albumin to Enantiomeric and Isoelectronic Pollutants Dissected by Spectroscopy, Calorimetry and Bioinformatics

1–naphthol (1N), 2–naphthol (2N) and 8–quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (Kb) of these pollutants to HSA were moderate (104–105 M−1). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39–5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy–entropy compensation (EEC). The difference observed between ΔCpexp and ΔCpcalc are suggested to be caused by binding–induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants