VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.     

NK cells and HIV infection: lessons from other viruses

Although the means by which NK cells may contribute to anti viral defense are still incompletely understood, various studies merge to a better comprehension of pathways that mediate NK cell activation (NK cell mediated cytotoxic activity and cytokine production) and their implications during the immune response towards a variety of viruses. Characterization of a specific expression pattern of ligands for NK receptors on virally infected cells and consequent modulation of NK cell activity have provided new insights in the field. A major break through to a direct evidence of a role for NK cells and NK cell receptors in immune protection against viral infection, was the recent implication of the murine activating Ly49H receptors in immune protection against MCMV infection. Although much remains to be learned concerning implication of NK cells in HIV infection, various reports have documented alteration in NK cell function and numbers during the course of HIV infection or treatment of AIDS. This review will focus on the current knowledge about the factors which might influence NK cell activation during various viral challenge and an emerging view of their alteration during HIV infection.     

Multi-compartmental modelling and experimental design for glucose transport studies in insulin-resistant rats

Many pathologies are associated with abnormalities of glucose metabolism or with perturbations of its transport (type 2 diabetes or insulin-resistance). The pre-diabetic state is characterised by a state of insulin-resistance, in others words a defect of glucose transport in insulin-sensible tissues, such as muscles and adipose tissues. The mathematical modelling of experimental data can be an excellent method to explore the mechanisms implied in the studied biological phenomenon. Thus, starting from a symbolic formulation like the compartmental modelling, it can be possible to develop a theoretical basis for the observation and to consider the best-adapted experiments for the study. We showed with mathematical models that [123I]-6-deoxy-6-iodo-D-glucose (6-DIG), shown as a tracer of glucose transport in vitro, could point out this transport abnormality. To quantify the insulin resistance, we estimated the fractional transfer coefficients of 6-DIG from the blood to the organs. We realised many studies to lead to a satisfying model; special attention has been paid to the precision of the parameter to select the best model. The results showed that by associating experimental data obtained with 6-DIG activities and an adapted mathematical model, discriminating parameters (in and out fractional transfer coefficients) between the two groups (control and insulin-resistant rats) could be pointed out.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.     

PIP(2)-PDZ domain binding controls the association of syntenin with the plasma membrane

PDZ proteins organize multiprotein signaling complexes. According to current views, PDZ domains engage in protein-protein interactions. Here we show that the PDZ domains of several proteins bind phosphatidylinositol 4,5-bisphosphate (PIP(2)). High-affinity binding of syntenin to PIP(2)-containing lipid layers requires both PDZ domains of this protein. Competition and mutagenesis experiments reveal that the protein and the PIP(2) binding sites in the PDZ domains overlap. Overlay assays indicate that the two PDZ domains of syntenin cooperate in binding to cognate peptides and PIP(2). Experiments on living cells demonstrate PIP(2)-dependent and peptide-dependent modes of plasma membrane association of the PDZ domains of syntenin. These observations suggest that local changes in phosphoinositide concentration control the association of PDZ proteins with their target receptors at the plasma membrane.     

A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector

The stimulatory effect of VIP on intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimeric VPAC(1)/VPAC(2), or mutated receptors. The VIP-induced [Ca(2+)](i) increase was linearly correlated with receptor density and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar VPAC(2) receptor density. The study was performed to establish the receptor sequence responsible for that difference. VPAC(1)/VPAC(2) chimeric receptors were first used for a broad positioning: those having the third intracellular loop (IC(3)) of the VPAC(1) or of the VPAC(2) receptor behaved, in that respect, phenotypically like VPAC(1) and VPAC(2) receptor, respectively. Replacement in the VPAC(2) receptor of the sequence 315-318 (VGGN) within the IC(3) by its VPAC(1) receptor counterpart 328-331 (IRKS) and the introduction of VGGN in state of IRKS in VPAC(1) was sufficient to mimic the VPAC(1) and VPAC(2) receptor characteristics, respectively. Thus, a small sequence in the IC(3) of the VPAC(1) receptor, probably through interaction with G(alphai) and G(alphaq) proteins, is responsible for the efficient agonist-stimulated [Ca(2+)](i) increase.