Heterocyclic 1,7-disubstituted indole sulfonamides are potent and selective human EP3 receptor antagonists

We have developed a pharmacophore model for the EP₃ receptor antagonists based on its endogenous ligand PGE₂. This ligand-based design yielded a series of novel peri-substituted [4.3.0] bicyclic aromatics featuring 1-alklyaryl 7-heterocyclic sulfonamide substituents. The synthesized molecules are potent antagonists of human EP₃ receptor in vitro and show inhibition of rat platelets aggregation. Optimized derivatives display high selectivity over IP, FP, and other EP receptor panels.     

Identification of differentially expressed proteins in spontaneous thymic lymphomas from knockout mice with deletion of p53

Background

Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensional gel electrophoresis (2D-PAGE).

Results

Protein spots excised from 2D-PAGE gels, were subjected to in-gel tryptic digestion and identified by liquid chromatography – tandem mass spectrometry. A total of 47 protein spots were identified. Immunological verification was performed for several of the differentially regulated proteins where suitable antibodies could be obtained. Functional annotation clustering revealed similarities as well as differences between the tumours. Twelve proteins that changed similarly in both tumours included up-regulation of rho GDP-dissociation inhibitor 2, proteasome subunit α type 3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid binding protein and down-regulation of adenylosuccinate synthetase, tubulin β-3 chain, a 25 kDa actin fragment, proteasome subunit β type 9, cofilin-1 and glia maturation factor γ.

Conclusion

Some of the commonly differentially expressed proteins are also differentially expressed in other tumours and may be putative diagnostic and/or prognostic markers for lymphomas.     

Determination of active concentrations and association and dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification

Accurate determination of kinetic rate constants for interacting biomolecules requires knowledge of the active concentrations of the participating molecules. Also, in other biomedical and clinical applications, sensitive, precise and accurate methods are needed to determine the concentration of biologically active molecules, which frequently constitute only a fraction of the total molecular pool. Here we report a novel development of the approach to determining active concentrations based on surface plasmon resonance (SPR) technology. The method relies on changes in binding rates with varying flow rates under conditions of partial mass transport, and does not require standards of known concentrations, given that the molecular mass of the molecule of interest is known. We introduce an analytical solution to the differential equations describing the formation of a 1:1 bimolecular complex, taking into account both the association and dissociation reactions, under partial mass transport limitations. This solution can be used in global fitting to binding curves obtained at different flow rates. The accuracy, precision, and sensitivity of this approach were determined in experiments involving binding of tyrosine-phosphorylated recombinant proteins to anti-phosphotyrosine antibodies, where the active concentration could be determined independently by in vitro phosphorylation with (33)P. There was an excellent agreement between the active concentrations determined by the analytical SPR-based method and by determination of the level of radioactivity of the phosphorylated protein. The SPR-based method allows determination of protein concentrations at picomolar levels. A procedure for accurate determinations of association and dissociation rate constants, based on the analytical solution of the mass transport and binding theory, is outlined.     

Changes in protein expression in p53 deleted spontaneous thymic lymphomas

By the use of high-resolution two-dimensional gel electrophoresis and computerized image analysis we investigated and compared the expression of cellular proteins from p53 positive (+/+) mouse thymocytes, p53-/- thymocytes before neoplastic transformation, and from cell lines derived from two spontaneous p53-/- thymic lymphomas, SM5 and SM7. A total of around 1500 proteins were detected on individual gels. Only changes in protein expression by a factor of 2 or more were considered. In the thymic lymphoma cells 3-5% of the proteins were found to be differentially regulated when compared with the protein expression in p53+/+ and p53-/- thymocytes. Only a minority (13 proteins) of the quantitatively changed proteins were common for the two thymic lymphoma cell lines, suggesting that the p53 deficiency mainly results in genetic dysfunctions which are individual for a given tumor. Two of the detected proteins increased their expression levels by more than 10 times from the p53+/+ to the p53-/- thymocytes and these high expression levels were also found in thymic lymphomas. The two proteins were identified by mass spectrometry as acidic ribosomal phosphoprotein P0 and a 33-kDa protein with a primary structure containing motifs of the glyoxalase-bleomycin resistance protein family (MDR) as deduced from the cDNA.     

Simultaneous mutations in CA and Vif of Maedi-Visna virus cause attenuated replication in macrophages and reduced infectivity in vivo

Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.     

Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4-.

In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release.     

Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I.

An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2-oxidation, as a function of temperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures.     

Bacterial composition and succession during storage of North-Atlantic cod (Gadus morhua) at superchilled temperatures

Background  

The bacteriology during storage of the North-Atlantic cod has been investigated for the past decades using conventional cultivation strategies which have generated large amount of information. This paper presents a study where both conventional cultivation and cultivation independent approaches were used to investigate the bacterial succession during storage of cod loins at chilled and superchilled temperatures.     

Isolation and characterization of lambda transducing phages for the E. coli genes ksgA and pdxA

Summary Lambda phages carrying the Escherichia coli genes ksgA and pdxA were isolated from secondary site lysogens in araB. 1) The phage genomes were characterized by genetic complementation tests, restriction endonuclease digestion and electron microscopy. 2) A 6.3 kilobasepair (kb) EcoRI restriction fragment carrying both ksgA and pdxA was cloned in a lambda vector; this fragment has proven useful in further characterization of the ksgA gene (Andrésson and Davies, 1980a, b). The ksgA and pdxA genes are about 14 and 12–13 kb, respectively, counterclockwise of the arabinose operon and 1.5 and 2.5–3.5 kb clockwise of folA.     

Antioxidant Responses of Lentil to Cold and Drought Stress

The effects of cold and drought stress on antioxidant responses and growth parameters in shoots and roots of lentil (Lens culinaris M cv Sultan 1) seedlings were investigated. Ten-day-old hydroponically grown seedlings were subjected to drought and cold (4°C) stress for 5 days. The length and fresh weight of shoots decreased significantly under both stress conditions, contrary to the increase in these growth parameters for roots under the same conditions. The oxidative damage as generation of malondialdehyde and hydrogen peroxide, was markedly higher in shoots under cold. Both stress conditions caused a significant increase in malondialdehyde levels in root tissues. The increase in proline levels was more pronounced under cold stress in shoots and roots. The tested stress conditions had no significant effect on chlorophyll contents. Superoxide dismutase activity was differentially altered in shoot and root tissues under drought and cold stress. The catalase activity was higher in roots under drought stress. On the other hand, ascorbate peroxidase activity increased in root tissues under cold stress. The results indicate that improved tolerance to cold and drought stress in root and shoot tissues of lentil might be correlated to the increased capacity of antioxidative defense system.