Chronic viral hepatitis C: management update

The management of chronic viral hepatitis C is evolving rapidly. Monotherapy with interferon, the accepted standard of treatment until recently, achieves only a modest sustained virological response rate of 15%. Combination treatment with alpha-2b interferon and ribavirin has been shown to increase sustained response rates to 40% in patients who have never been treated with interferon and to 50% in those who have relapsed following monotherapy with interferon. However, side effects, which have led to the discontinuation of combination treatment in a significant proportion of patients, must be carefully monitored. Treatment with interferon alpha-2b and ribavirin has now been approved in Canada, but the selection and monitoring of patients suitable for combination treatment requires special expertise. Although improvements in current therapeutic options may be possible with more frequent, higher doses or long-acting forms of interferon together with ribavirin, low sustained response rates (i.e., below 30%) for patients with hepatitis C virus genotype 1 emphasize the need for novel antiviral medications that will target the functional sites of the HCV genome.     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.     

The catalytic-centre activity and kinetic properties of bovine milk alkaline phosphatase

1. The phosphorylation of milk alkaline phosphatase was studied under various conditions: maximum incorporation occurred at pH5.0 and 50% incorporation at pH6.6-7.0. 2. The phosphorylation was shown to be specific and the results suggest that the active centre of the enzyme is involved in the process. 3. Phosphoryl-enzyme is rapidly hydrolysed at alkaline pH. at pH7.0 the results suggest that a phosphoryl-enzyme could occur as a transient intermediate in the hydrolysis of phosphate esters by the phosphatase. 4. The catalytic-centre activity of the enzyme was found to be 2700sec.(-1) at pH10.0 and 25 degrees with p-nitrophenyl phosphate as substrate.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.     

Physiological routes from intra-uterine seminal contents to advancement of ovulation

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.     

Predicting nucleic acid binding interfaces from structural models of proteins

The function of DNA‐ and RNA‐binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure‐based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high‐resolution three‐dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I‐TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high‐resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I‐TASSER produces high‐quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low‐resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Proteins 2012. © 2011 Wiley Periodicals, Inc.