Benzimidazole- and benzoxazole-based inhibitors of Rho kinase

Inhibitors of Rho kinase have been developed based on two distinct scaffolds, benzimidazoles, and benzoxazoles. SAR studies and efforts to optimize the initial lead compounds are described. Novel selective inhibitors of ROCK-II with excellent potency in both enzyme and cell-based assays were obtained. These inhibitors possess good microsomal stability, low cytochrome P-450 inhibitions and good oral bioavailability.     

Cleavage of the Phosphodiester Bond of Uridylyl-(3′,5′)-8-Carboxymethylaminoadenosine by Hydronium,Hydroxide and Zinc(Ii) Ions: A Model Study Aimed at Elucidating the Potential of a Carboxylate Function as an Intramolecular Catalyst

Abstract

Uridylyl-(3′,5′)-8-carboxymethylaminoadenosine has been synthesised, and its transesterification to uridine 2′,3′-cyclic phosphate in the presence and absence of Zn²⁺ ion has been studied. The results show that a carboxylate function in the vicinity of the phosphodiester bond accelerates the metal ion promoted cleavage but not the metal ion independent reaction. Under acidic conditions, the predominant reaction is the cleavage of the side chain, giving the 8-amino derivative.     

Evaluating Purifying Selection in the Mitochondrial DNA of Various Mammalian Species

Mitochondrial DNA (mtDNA), the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this selection is fundamental to evolutionary studies and to the identification of deleterious mutations.     

Sperm adaptation in relation to salinity in three goby species

In externally fertilizing species, the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. In this study, the authors compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, Pomatoschistus flavescens, and the sand goby, Pomatoschistus minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity show that the two-spotted goby experiences the least sperm competition, whereas in the sand goby sperm competition is ubiquitous. The authors took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. They then used computer-assisted sperm analysis to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 h after sperm activation. The authors found that sperm performance depended on salinity, but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm, but there was no significant decrease in sperm motility during 20 h. The sand goby was the only species with motile sperm after 72 h.     

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.     

Behavioural fever boosts the inflammatory response in rainbow trout Oncorhynchus mykiss

Behavioural fever, manifested as an increased preferred temperature, was shown in rainbow trout Oncorhynchus mykiss following an injection of bacterial lipopolysaccharide. Simulated behavioural fever, through a 2·5° C water temperature rise following bacterial lipopolysaccharide injection, enhanced the expression of the cytokine interleukin-1β, in comparison with an untreated group held at the initial temperature. The present findings show that an important mediator in the immune response can be boosted through behavioural fever in fishes.     

Opsin gene sequence variation across phylogenetic and population histories in Mysis (Crustacea: Mysida) does not match current light environments or visual-pigment absorbance spectra

The hypothesis that selection on the opsin gene is efficient in tuning vision to the ambient light environment of an organism was assessed in 49 populations of 12 Mysis crustacean species, inhabiting arctic marine waters, coastal littoral habitats, freshwater lakes ('glacial relicts') and the deep Caspian Sea. Extensive sequence variation was found within and among taxa, but its patterns did not match expectations based on light environments, spectral sensitivity of the visual pigment measured by microspectrophotometry or the history of species and populations. The main split in the opsin gene tree was between lineages I and II, differing in six amino acids. Lineage I was present in marine and Caspian Sea species and in the North American freshwater Mysis diluviana, whereas lineage II was found in the European and circumarctic fresh- and brackish-water Mysis relicta, Mysis salemaai and Mysis segerstralei. Both lineages were present in some populations of M. salemaai and M. segerstralei. Absorbance spectra of the visual pigment in nine populations of the latter three species showed a dichotomy between lake (λ(max) =554-562 nm) and brackish-water (Baltic Sea) populations (λ(max) = 521-535 nm). Judged by the shape of spectra, this difference was not because of different chromophores (A2 vs. A1), but neither did it coincide with the split in the opsin tree (lineages I/II), species identity or current light environments. In all, adaptive evolution of the opsin gene in Mysis could not be demonstrated, but its sequence variation did not conform to a neutral expectation either, suggesting evolutionary constraints and/or unidentified mechanisms of spectral tuning.     

Excess copper predisposes photosystem II to photoinhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll

Photoinhibition of photosystem II was studied in vivo with bean (Phaseolus vulgaris) plants grown in the presence of 0.3 (control), 4, or 15 microM Cu(2+). Although photoinhibition, measured in the presence of lincomycin to block concurrent recovery, is faster in leaves of Cu(2+)-treated plants than in control leaves, thylakoids isolated from Cu-treated plants did not show high sensitivity to photoinhibition. Direct effects of excess Cu(2+) on chloroplast metabolism are actually unlikely, because the Cu concentration of chloroplasts of Cu-treated plants was lower than that of their leaves. Excess Cu in the growth medium did not cause severe oxidative stress, collapse of antioxidative defenses, or loss of photoprotection. Thus, these hypothetical effects can be eliminated as causes for Cu-enhanced photoinhibition in intact leaves. However, Cu treatment lowered the leaf chlorophyll (Chl) concentration and reduced the thylakoid membrane network. The loss of Chl and sensitivity to photoinhibition could be overcome by adding excess Fe together with excess Cu to the growth medium. The addition of Fe lowered the Cu(2+) concentration of the leaves, suggesting that Cu outcompetes Fe in Fe uptake. We suggest that the reduction of leaf Chl concentration, caused by the Cu-induced iron deficiency, causes the high photosensitivity of photosystem II in Cu(2+)-treated plants. A causal relationship between the susceptibility to photoinhibition and the leaf optical density was established in several plant species. Plant species adapted to high-light habitats apparently benefit from thick leaves because the rate of photoinhibition is directly proportional to light intensity, but photosynthesis becomes saturated by moderate light.