Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.

We evaluated in CHO cells expressing the VPAC₁ receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y²²⁴, N²²⁹, F²³⁰, W²³², E²³⁶, G²³⁷, Y²³⁹, L²⁴⁰. N²²⁹A VPAC₁ mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca²⁺]_i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N²²⁹D mutant was not expressed at the membrane, and the N²²⁹Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N²²⁹A and N²²⁹Q VPAC₁ were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.

Mutation of that conserved amino acid in VPAC₂ could be investigated only by a conservative mutation (N²¹⁶Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.     

MAP kinase-dependent degradation of p27Kip1 by calpains in choroidal melanoma cells. Requirement of p27Kip1 nuclear export

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.     

Molecular modeling of glycosyltransferases involved in the biosynthesis of blood group A,blood group B,Forssman, and iGb3 antigens and their interaction with substrates

A terminal alpha1-3 linked Gal or GalNAc sugar residue is the common structure found in several oligosaccharide antigens, such as blood groups A and B, the xeno-antigen, the Forssman antigen, and the isogloboside 3 (iGb3) glycolipid. The enzymes involved in the addition of this residue display strong amino acid sequence similarities, suggesting a common fold. From a recently solved crystal structure of the bovine alpha3-galactosyltransferase complexed with UDP, homology modeling methods were used to build the four other enzymes of this family in their locked conformation. Nucleotide-sugars, the Mn2+ ion, and oligosaccharide acceptors were docked in the models. Nine different amino acid regions are involved in the substrate binding sites. After geometry optimization of the complexes and analysis of the predicted structures, the basis of the specificities can be rationalized. In the nucleotide-sugar binding site, the specificity between Gal or GalNAc transferase activity is due to the relative size of two clue amino acids. In the acceptor site, the presence of up to three tryptophan residues define the complexity of the oligosaccharide that can be specifically recognized. The modeling study helps in rationalizing the crystallographic data obtained in this family and provides insights on the basis of substrate and donor recognition.     

Organotypic heart slices for cell transplantation and physiological studies

Recent studies have significantly improved our ability to investigate cell transplantation and study the physiology of transplanted cells in cardiac tissue. Several previous studies have shown that fully-immersed heart slices can be used for electrophysiological investigations. Additionally, ischemic heart slices induced by glucose and oxygen deprivation offer a useful tool to investigate mechanical integration and to measure forces of contraction of engrafted cells, at least for short term analysis. A recent and novel model of heart slices, prepared from rat and human tissues, can be maintained in culture for up to two months. This new heart slice model can be used for long term in vitro cell transplantation studies and for pharmacological evaluation. This review will focus on describing these models and demonstrating the use of organotypic heart slices as a novel tool for drugs for studying electrophysiology and developing cellular therapeutic approaches to alleviate cardiac tissue damage.Key words: heart, organotypic, culture, stem cells, transplantation, electrophysiology, pharmacology     

Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease

Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.     

N-3 long chain polyunsaturated fatty acids: a nutritional tool to prevent insulin resistance associated to type 2 diabetes and obesity?

n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), mainly eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), are present in mammal tissues both from endogenous synthesis from desaturation and elongation of 18:3 n-3 and/or from dietary origin (marine products and fish oils). In rodents in vivo, n-3 LC-PUFA have a protective effect against high fat diet induced insulin resistance. Such an effect is explained at the molecular level by the prevention of many alterations of insulin signaling induced by a high fat diet. Indeed, the protective effect of n-3 LC-PUFA results from the following: (a) the prevention of the decrease of phosphatidyl inositol 3' kinase (PI3 kinase) activity and of the depletion of the glucose transporter protein GLUT4 in the muscle; (b) the prevention of the decreased expression of GLUT4 in adipose tissue. In addition, n-3 LC-PUFA inhibit both the activity and expression of liver glucose-6-phosphatase which could explain the protective effect with respect to the excessive hepatic glucose output induced by a high fat diet. n-3 LC-PUFA also decrease muscle intramyofibrillar triglycerides and liver steatosis. This last effect results on the one hand, from a decreased expression of lipogenesis enzymes and of delta 9 desaturase (via a depleting effect on sterol response element binding protein 1c (SREBP-1c). On the other hand, n-3 LC-PUFA stimulate fatty acid oxidation in the liver (via the activation of peroxisome proliferator activated receptor alpha (PPAR-alpha)). In patients with type 2 diabetes, fish oil dietary supplementation fails to reverse insulin resistance for unclear reasons, but systematically decreases plasma triglycerides. Conversely, in healthy humans, fish oil has many physiological effects. Indeed, fish oil reduces insulin response to oral glucose without altering the glycaemic response, abolishes extraggression at times of mental stress, decreases the activation of sympathetic activity during mental stress and also decreases plasma triglycerides. These effects are encouraging in the perspective of prevention of insulin resistance but further clinical and basic studies must be designed to confirm and complete our knowledge in this field.     

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.     

Synthesis and evaluation of 4-triazolylflavans as new aromatase inhibitors

Aromatase is a target of pharmacological interest for the treatment of estrogen-dependent cancers. Azole derivatives such as letrozole or anastrozole have been developed for aromatase inhibition and are used for the treatment of breast tumors. In this paper, four 4-triazolylflavans were synthesized and were found to exhibit moderate to high inhibitory activity against aromatase.