The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C virus Helicase NS3h is Dependent on Both the Sugar and Base Moieties

The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single-stranded nucleic acid with bases rU, dU and dT and have found that the macroscopic rate of translocation is dependent upon both the base and sugar moieties of the nucleic acid, with approximate macroscopic translocation rates of 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein to the respective substrates such that weaker affinity corresponded to faster translocation. The values of K0.5 for NS3h translocation at a saturating ATP concentration are: (3.3 ± 0.4) μM nucleotide (poly-dT), (27 ± 2) μM nucleotide (poly-dU), and (36 ± 2) μM nucleotide (poly-rU). Furthermore, the results of isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS° are the principal source of the differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation was identical for all three substrates, ~0.5 ATP molecules consumed per nucleotide translocated. This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates

Commentaires préliminaires

Dans les parties de cette histoire qui me sont personnelles, j’ai, jusqu’à présent, divisé mon sujet en leçons, afin que ces parties pussent être vendues et envoyées aux souscripteurs par livraisons, comme l’avait été le second volume. Mais aujourd’hui que la vente par volume est substituée à la vente par livraison, cette division de mon sujet en leçons est inutile, et je l’abandonne dans les volumes complémentaires que je commence de publier. À la vérité, elle aurait donné à la forme de tou..

Prefatory Comments

In the sections of the history that are my own, I have up until now divided the subject into lessons so that these parts could be sent to subscribers in installments, as it was done for the second volume. Nowadays, sale is done by volume rather than by installment, so dividing the subject into lessons is therefore unnecessary and will be discontinued in the subsequent volumes I will publish. In truth, such division [if continued] would have given a unified structure [to all five volumes] but..

Banana meal for feeding pigs: digestive utilization, growth performance and feeding behavior

The main objective of the present work was to determine the nutritional value and the strategies of using green banana meal (BM) in growing pigs. Two trials involving a total of 96 growing pigs were designed to study the effect of the harvest stage on the nutritional and energy values of BM (trial 1) and to evaluate the consequence of feeding gradual levels of BM on growth performance and feeding behavior in growing pigs (trial 2). In trial 1, the digestive utilization of three diets including 40% BM were compared with a control (C) soybean meal-corn diet in two batches of 12 pigs. BM was obtained from fruits harvested at 750 degrees-days (DD; early harvesting stage), 900 DD (normal harvesting stage) and 1150 DD (late harvesting stage). In trial 2, 72 Large White pigs were grouped in pens of nine animals and were given ad libitum access to one of the four dietary treatments (two pens/diet) differing from the rate of inclusion of 900 DD BM (0%, 20%, 40%, 60%). The estimated energy apparent digestibility coefficients of BM increased with the harvest stage (75.5%, 80.7% and 83.2% for BM at 750, 900 and 1150 DD, respectively). Digestible energy and metabolizable energy values were higher for BM at 1150 DD (13.56 and 13.05 MJ/kg DM, respectively) than at 900 DD (13.11 and 12.75 MJ/kg DM, respectively) or at 750 DD (12.00 and 11.75 MJ/kg DM, respectively). In trial 2, average daily gain and feed conversion ratio were not affected (P>0.05) by the rate of BM inclusion (822 g/day and 2.75 kg/kg on average, respectively). Feed intake and feeding behavior parameters were not significantly influenced by the dietary treatments except for the rate of feed ingestion with a lower value for the diet with 40% of BM (27.4 v. 32.2 g/min on average; P<0.01) when compared with the other diets. Results of this study indicate that the energy value of BM increases with the harvest stage and that BM can be incorporated up to 60% in growing finishing pig diets

L'apport de la spectrométrie de masse haute-résolution à l'obtention d'empreintes moléculaires d'échantillons d'urine. Applications potentielles au dopage.

National audienc

Large-Scale Functional Purification of Recombinant HIV-1 Capsid

<div><p>During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of <i>Escherichia coli</i> expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.</p> </div

Polymerization kinetics of (A) full-length wild type capsid (WT CA) and (C) mutant capsid [A14C,E45C,W184A,M185A] monomer (CA 4Mu).

<p>Assembly reactions were induced by 2-fold dilution of capsid protein into buffer containing 50 mM sodium phosphate (pH 7.5), 4 M NaCl at 25°C. The increase in samples turbidity (absorbance at 350 nm) due to the formation of large oligomeric structures was plotted as a function of time. Final concentration of capsid protein in reaction is depicted on the right side of the kinetic traces. The data points represent an average of three determinations with standard deviation shown as grey bars. The dependence of the initial rate of capsid polymerization is plotted against the concentration of (<b>B</b>) WT CA and (<b>D</b>) CA 4Mu monomer. The initial rate was calculated from the slope of the linear phase of the polymerization curve.</p