Host genetic requirements for DNA release of lactococcal phage TP901-1

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.     

Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.     

Urinary pS2/TFF1 levels in the management of hormonodependent breast carcinomas

pS2/TFF1 overexpression in breast carcinomas correlates with response to hormonotherapy. We evaluated the clinical relevance of urinary pS2/TFF1 in breast cancer patients. In healthy controls (100 cases), it represents an individual and relatively stable parameter. Although 24 out 83 pre-operative breast cancer patients showed elevated levels, both the sensitivity and specificity of the test were too low for breast cancer screening. However, neoadjuvant hormonotherapy decreased pS2/TFF1 levels in nine out of 20 patients. Furthermore, among 22 patients receiving long-term adjuvant hormonotherapy, four exhibited elevated levels, two of them at the time of relapse. Thus, urinary pS2/TFF1 quantification might be suitable as an in vivo diagnosis for tumor hormonodependency, and disease follow-up during hormonotherapy.     

Double chip protein arrays using recombinant single-chain Fv antibody fragments

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.     

An in vitro dual model of mycobacterial granulomas to investigate the molecular interactions between mycobacteria and human host cells

In the majority of individuals infected with Mycobacterium tuberculosis, the bacilli cause a long-term asymptomatic infection called latent tuberculosis, a state during which the bacilli reside within granulomas. Latently infected individuals have around 10% risk of progression to clinical disease at a later stage. Determining the state of the mycobacteria and the host cells during this latent phase, i.e. within the granulomas, would greatly improve our understanding of the physiopathology of tuberculosis, and thus enable the development of new therapeutic means to treat the one-third of the world's population who are latently infected. We have developed an in vitro model of human mycobacterial granulomas, enabling the cellular and molecular analysis of the very first steps in the host granulomatous response to either mycobacterial compounds or live mycobacterial species. In vitro mycobacterial granulomas mimic natural granulomas very well, with the progressive recruitment of macrophages around live bacilli or mycobacterial antigen-coated beads, their differentiation into multinucleated giant cells and epithelioid cells, and the final recruitment of a ring of activated lymphocytes. Besides morphological similarities, in vitro granulomas also functionally resemble natural ones, with the development of intense cellular co-operation and intracellular mycobactericidal activities.     

Phytosterols in low- and nonfat beverages as part of a controlled diet fail to lower plasma lipid levels

Dietary phytosterols have been shown to reduce plasma cholesterol concentrations when consumed in different food matrices, but their effectiveness in nonfat or low-fat beverages has not been established. The objective of this study was to examine whether phytosterols alter plasma lipid levels when incorporated into nonfat or low-fat beverages. Fifteen moderately hypercholesterolemic men and women consumed three precisely controlled diets for periods of 21 days each in random order. Diets contained either a nonfat placebo beverage (NF), a beverage that is nonfat with added phytosterols (NFPS), or a beverage that is low in fat with added phytosterols (LFPS). Total cholesterol concentrations were not different between groups at endpoint, decreasing (P < 0.05) equally by 8.5%, 11.6%, and 10.1% with NF, NFPS, and LFPS consumption, respectively. There was no effect of dietary treatment on LDL cholesterol concentrations, which decreased over time (P < 0.05) by 5%, 10.4%, and 8.5% with NF, NFPS, and LFPS, respectively. HDL cholesterol and triacylglycerol concentrations were unaffected by the diets. Provision of phytosterols as part of nonfat and low-fat beverages did not exert any greater hypocholesterolemic effect than a nonfat placebo beverage. These results show that intake of phytosterols in a low-fat beverage format is not efficacious for lipid level modification.     

Elastin: a representative ideal protein elastomer

During the last half century, identification of an ideal (predominantly entropic) protein elastomer was generally thought to require that the ideal protein elastomer be a random chain network. Here, we report two new sets of data and review previous data. The first set of new data utilizes atomic force microscopy to report single-chain force-extension curves for (GVGVP)(251) and (GVGIP)(260), and provides evidence for single-chain ideal elasticity. The second class of new data provides a direct contrast between low-frequency sound absorption (0.1-10 kHz) exhibited by random-chain network elastomers and by elastin protein-based polymers. Earlier composition, dielectric relaxation (1-1000 MHz), thermoelasticity, molecular mechanics and dynamics calculations and thermodynamic and statistical mechanical analyses are presented, that combine with the new data to contrast with random-chain network rubbers and to detail the presence of regular non-random structural elements of the elastin-based systems that lose entropic elastomeric force upon thermal denaturation. The data and analyses affirm an earlier contrary argument that components of elastin, the elastic protein of the mammalian elastic fibre, and purified elastin fibre itself contain dynamic, non-random, regularly repeating structures that exhibit dominantly entropic elasticity by means of a damping of internal chain dynamics on extension.     

Methods for homocysteine analysis and biological relevance of the results

It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B(2), B(6), B(9), B(12)) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.