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Chemical modification by phenylglyoxal, an arginine-specific reagent, of both native and recombinant rat brain inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the substrate ATP but not Ins(1,4,5)P3. The modification reaction obeyed pseudo-first-order rate kinetics. Complete inhibition of activity corresponded to incorporation of 1.2 mol of phenylglyoxal per mol of protein. A single [14C]phenylglyoxal-modified peptide was isolated following alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P3 3-kinase and reverse-phase HPLC. ATP prevented the incorporation of radioactivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corresponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivative showed the modified amino acid to be Arg-317. The data directly identify a reactive arginine residue as part of the ATP-binding site. Arg-317 is located within a sequence segment which is conserved among the catalytic domain of Ins(1,4,5)P3 3-kinase isoenzymes A and B in human and rat species., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Chinese hamster ovary cells have been engineered to secrete an anchorless form of the varicella-zoster virus gpII protein. Purification of the recombinant product was achieved by a combination of hydrophobic and gel filtration chromatography giving rise to a protein more than 85% pure. Recombinant gpII was composed of several polypeptides which, on the basis of amino-terminal sequence analysis, corresponded to a 93-kDa precursor and to the N- and C-terminal subunits of the molecule (64 and 39/36 kDa, respectively). All polypeptides carried N-linked high-mannose and complex glycosylations, whereas O-glycosylations were carried by the precursor and the N-terminal subunits only. Surprisingly, purified recombinant gpII spontaneously formed large oligomeric structures of variable size. These complexes contained noncovalently linked lipids. Mice inoculated with the recombinant gpII absorbed onto the weak adjuvant, aluminium hydroxide, produced virus neutralizing antibodies. The recombinant gpII may thus constitute a good candidate for the development of a subunit vaccine against varicella-zoster virus., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Monocytes stimulated with bacterial lipopolysaccharide (LPS) generate a procoagulant activity (PCA) related to the induction of tissue factor (TF) expression at their surface. Since interleukin-10 (IL-10) was recently shown to inhibit LPS-induced cytokine production and is currently considered as a potential therapeutic agent in septic shock, we were interested to determine its effects on LPS-induced monocyte PCA. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with 1 microgram/ml LPS in the presence of serial dilutions of recombinant human IL-10 and PCA was determined after 6 h in a one-stage clotting assay. IL-10 inhibited in a dose-dependent manner LPS-induced TF-dependent PCA: a significant effect was already observed with 30 pg/ml IL-10 while 64-97% inhibition was achieved with 120 pg/ml IL-10. In parallel flow cytometry experiments, IL-10 was shown to block LPS-induced TF expression at the surface of monocytes. In order to inhibit LPS-induced PCA, IL-10 had to be added to PBMC at least 6 h before LPS challenge. This inhibitory effect of IL-10 was already apparent at the TF mRNA level and was prevented by co-incubation with cycloheximide (20 micrograms/ml). These data suggest that IL-10 acts via the induction of protein(s) which might interfere with TF gene transcription or mRNA stability. We conclude that the protective effects of IL-10 in endotoxinemia might be related not only to cytokine synthesis blockade but also to inhibition of LPS-induced PCA., In Vitro, Journal Article, Research Support, Non-U.S. Gov't, FLWNA, info:eu-repo/semantics/published

Transfected CV1 cells were used to compare the in vivo effects of various domains of villin and gelsolin. These two homologous actin modulating proteins both contain a duplicated severin-like sequence. Villin has in addition a carboxy-terminal domain, the headpiece, which accounts for its bundling activity. The effects of the villin-deleted mutants were compared with those of native villin. Our results show that essential domains of villin required to induce the growth of microvilli and F-actin redistribution are present in the first half of the core and in the headpiece. We also show that the second half of the villin core cannot be exchanged by its homolog in gelsolin. When expressed at high levels of CV1 cells, full length gelsolin completely disrupted stress fibers without change of the cell shape. Addition of the villin headpiece to gelsolin had no effect on the phenotype induced by gelsolin alone. Expression of the first half of gelsolin induced similar modifications as capping proteins and rapid cell mortality; this deleterious effect on the cell structure was also observed when the headpiece was linked to the first half of gelsolin. In cells expressing the second half of gelsolin, a dotted F-actin staining was often seen. Moreover elongated dorsal F-actin structures were observed when the headpiece was linked to the second gelsolin domain. These studies illustrate the patent in vivo severing activity of gelsolin as well as the distinct functional properties of villin core in contrast to gelsolin.

The extensive amino acid sequence conservation among G protein-coupled receptors has been exploited to clone new members of this large family by homology screening or by PCR. Out of four such receptor cDNAs we cloned recently, RDC7 corresponds to a relatively abundant transcript in the brain cortex, the thyroid follicular cell and the testis. We have now identified RDC7 as an A1 adenosine receptor. The A1 agonist CPA [N6-cyclopentyladenosine] decreased by 80% cAMP accumulation in forskolin-stimulated CHO cells stably transfected with RDC7. Specific binding of another A1 adenosine agonist, [3H]CHA [N6-cyclohexyladenosine], was demonstrated on membranes from Cos cells transfected with a pSVL construct harbouring the RDC7 cDNA insert. The binding characteristics were similar to those of the natural brain A1 receptor. The recombinant and the natural receptors behaved also in the same way in displacement experiments involving a series of A1 adenosine agonists. The binding characteristics of RDC7 were compared to those of RDC8, another orphan receptor recently identified as an A2 adenosine receptor. The two molecular species RDC7 and RDC8 correspond clearly to the A1 and A2 receptor entities defined hitherto on a purely pharmacological basis., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., Review, info:eu-repo/semantics/published

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another., info:eu-repo/semantics/published