Protein--protein interaction maps: a lead towards cellular functions

The availability of complete genome sequences now permits the development of tools for functional biology on a proteomic scale. Several experimental approaches or in silico algorithms aim at clustering proteins into networks with biological significance. Among those, the yeast two-hybrid system is the technology of choice to detect protein-protein interactions. Recently, optimized versions were applied at a genomic scale, leading to databases on the web. However, as with any other 'genetic' assay, yeast two-hybrid assays are prone to false positives and false negatives. Here we discuss these various technologies, their general limitations and the potential advances they make possible, especially when in combination with other functional genomics or bioinformatics analyses.     

Circadian fluctuations in the muscular efficiency of athletes: with sleep versus sleep deprivation

The influence of time of day on muscular performance was studied. From part of the results of two different studies (EAS et EPS), the effects of sleep deprivation were appreciated. Seven times over the 24-h period, developed torque and myoelectric activity were estimated during maximal isometric voluntary contractions using an isokinetic dynamometer: elbow flexion for EAS in standardised sleep, and knee extension for EPS in complete sleep deprivation. The results showed nycthemeral changes in torque in both conditions (p < 0.005), with maximal values recorded at the beginning of night. Although during sleep deprivation (EPS) the rhythm followed neurophysiological factors, during EAS, this rhythm was accounted for by the variations in the contractile state of muscle.     

Two-liquid-phase slurry bioreactors to enhance the degradation of high-molecular-weight polycyclic aromatic hydrocarbons in soil

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. The addition of a water-immiscible, nonbiodegradable, and biocompatible liquid, silicone oil, to a soil slurry was studied to promote the desorption of PAHs from soil and to increase their bioavailability. First, the transfer into silicone oil of phenanthrene, pyrene, chrysene, and benzo[a]pyrene added to a sterilized soil (sandy soil with 0.65% total volatile solids) was measured for 4 days in three two-liquid-phase (TLP) slurry systems each containing 30% (w/v) soil but different volumes of silicone oil (2.5%, 7.5%, and 15% [v/v]). Except for chrysene, a high percentage of these PAHs was transferred from soil to silicone oil in the TLP slurry system containing 15% silicone oil. Rapid PAH transfer occurred during the first 8 h, probably resulting from the extraction of nonsolubilized and of poorly sorbed PAHs. This was followed by a period in which a slower but constant transfer occurred, suggesting extraction of more tightly bound PAHs. Second, a HMW PAH-degrading consortium was enriched in a TLP slurry system with a microbial population isolated from a creosote-contaminated soil. This consortium was then added to three other TLP slurry systems each containing 30% (w/v) sterilized soil that had been artificially contaminated with pyrene, chrysene, and benzo[a]pyrene, but different volumes of silicone oil (10%, 20%, and 30% [v/v]). The resulting TLP slurry bioreactors were much more efficient than the control slurry bioreactor containing the same contaminated soil but no oil phase. In the TLP slurry bioreactor containing 30% silicone oil, the rate of pyrene degradation was 19 mg L(-)(1) day(-)(1) and no pyrene was detected after 4 days. The degradation rates of chrysene and benzo[a]pyrene in the 30% TLP slurry bioreactor were, respectively, 3.5 and 0.94 mg L(-)(1) day(-)(1). Low degradation of pyrene and no significant degradation of chrysene and benzo[a]pyrene occurred in the slurry bioreactor. This is the first report in which a TLP system was combined with a slurry system to improve the biodegradation of PAHs in soil.     

Kinetics of T-cell receptor binding by bivalent HLA-DR. Peptide complexes that activate antigen-specific human T-cells

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at >100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.     

The glycosylphosphatidyl inositol-anchored adhesion molecule F3/contactin is required for surface transport of paranodin/contactin-associated protein (caspr)

Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100-insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.     

Haemolytic activity,cytotoxicity and membrane cell permeabilization of semi-synthetic and natural lupane- and oleanane-type saponins

The haemolysis of red blood cells inducing toxicity in most animals including humans is a major drawback for the clinical development of saponins as antitumour agents. In this study, the haemolytic and cytotoxic activities as well as the membrane cell permeabilization property of a library of 31 semi-synthetic and natural lupane- and oleanane-type saponins were evaluated and the structure–activity relationships were established. It was shown that lupane-type saponins do not exhibit any haemolytic activity and membrane cell permeabilization property at the maximum concentration tested (100 μM) independently of the nature of the sugar moieties. While oleanane-type saponins such as β-hederin (25) and hederacolchiside A₁ (27) cause the death of cancer cell lines by permeabilizing the cellular membranes, lupane-type saponins seem to proceed via another mechanism, which could be related to the induction of apoptosis. Altogether, the results indicate that the cytotoxic lupane-type glycosides 10 and 22 bearing an α-l-rhamnopyranose moiety at the C-3 position represent promising antitumour agents for further studies on tumour-bearing mice since they are devoid of toxicity associated with the haemolysis of red blood cells.     

Prediction of bone density around orthopedic implants delivering bisphosphonate

The fixation of an orthopedic implant depends strongly upon its initial stability. Peri-implant bone may resorb shortly after the surgery. This resorption is directly followed by new bone formation and implants fixation strengthening, the so-called secondary fixation. If the initial stability is not reached, the resorption continues and the implant fixation weakens, which leads to implant loosening. Studies with rats and dogs have shown that a solution to prevent peri-implant resorption is to deliver bisphosphonate from the implant surface.The aims of the study were, first, to develop a model of bone remodeling around an implant delivering bisphosphonate, second, to predict the bisphosphonate dose that would induce the maximal peri-implant bone density, and third to verify in vivo that peri-implant bone density is maximal with the calculated dose.The model consists of a bone remodeling equation and a drug diffusion equation. The change in bone density is driven by a mechanical stimulus and a drug stimulus. The drug stimulus function and the other numerical parameters were identified from experimental data. The model predicted that a dose of 0.3 μg of zoledronate on the implant would induce a maximal bone density. Implants with 0.3 μg of zoledronate were then implanted in rat femurs for 3, 6 and 9 weeks. We measured that peri-implant bone density was 4% greater with the calculated dose compared to the dose empirically described as best.The approach presented in this paper could be used in the design and analysis processes of experiments in local delivery of drug such as bisphosphonate.