Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater

The glycosyltransferase SnogD from Streptomyces?nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6?? resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in?vivo and in?vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction. Database The atomic coordinates and structure factors have been deposited with the RCSB Protein Data Bank under accession numbers 4AMB, 4AMG and 4AN4 Structured digital abstract ? snogD?and?snogD?bind?by?x-ray crystallography?(View Interaction:?1,?2).     

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Abeta and AICD generation

The 37-43 amino acid Abeta peptide is the principal component of beta-amyloid deposits in Alzheimer's disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase. gamma-Secretase also cleaves APP at Val50 in the Abeta numbering (epsilon cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Abeta and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Abeta40 and Abeta42 while increasing Abeta38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Abeta generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N- and C-terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of gamma-secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Abeta and AICD production.     

Membrane filtering properties of the bumblebee (Bombus terrestris) photoreceptors across three spectral classes

Filtering properties of the membrane form an integral part of the mechanisms producing the light-induced electrical signal in insect photoreceptors. Insect photoreceptors vary in response speed between different species, but recently it has also been shown that different spectral photoreceptor classes within a species possess diverse response characteristics. However, it has not been quantified what roles phototransduction and membrane properties play in such diversity. Here, we use electrophysiological methods in combination with system analysis to study whether the membrane properties could create the variation of the response speed found in the bumblebee (Bombus terrestris) photoreceptors. We recorded intracellular responses from each photoreceptor class to white noise-modulated current stimuli and defined their input resistance and linear filtering properties. We found that green sensitive cells exhibit smaller input resistance and membrane impedance than other cell classes. Since green sensitive cells are the fastest photoreceptor class in the bumblebee retina, our results suggest that the membrane filtering properties are correlated with the speed of light responses across the spectral classes. In general, our results provide a compelling example of filtering at the sensory cell level where the biophysical properties of the membrane are matched to the performance requirements set by visual ecology.     

Increased expression of bradykinin type-1 receptors in endothelium of intramyocardial coronary vessels in human failing hearts

In experimental animals, bradykinin type-1 receptors (BK-1Rs) are induced during inflammation and ischemia, and, by exerting either cardioprotective or cardiotoxic effects, they may contribute to the pathogenesis of heart failure. Nothing is known about the expression of BK-1Rs in human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13), due to idiopathic dilated cardiomyopathy (IDC; n = 7) or to coronary heart disease (CHD; n = 6), and from normal hearts (n = 6). The expression of BK-1Rs was analyzed by means of competitive RT-PCR, Western blot analysis, and immunohistochemistry. Expression of BK-1R mRNA was increased in both IDC (2.8-fold) and CHD (2.1-fold) hearts compared with normal hearts. The observed changes were verified at the protein level. Expression of BK-1Rs in failing hearts localized to the endothelium of intramyocardial coronary vessels and correlated with an increased expression of TNF-alpha in the vessel wall. Treatment of human coronary artery endothelial cells with TNF-alpha increases their BK-1R expression. These novel results show that BK-1Rs are induced in the endothelium of intramyocardial coronary vessels in failing human hearts and so may participate in the pathogenesis of heart failure.     

Comparative linkage mapping of the Grey coat colour gene in horses

Grey horses are born coloured, turn progressively grey and often develop melanomas late in life. Grey shows an autosomal dominant inheritance and the locus has previously been mapped to horse chromosome 25 (ECA25), around the TXN gene. We have now developed eight new single nucleotide polymorphisms (SNPs) associated with genes on ECA25 using information on the linear order of genes on human chromosome 9q, as well as the human and mouse coding sequences. These SNPs were mapped in relation to the Grey locus using more than 300 progeny from matings between two Swedish Warmblood grey stallions and non-grey mares. Grey was firmly assigned to an interval with flanking markers NANS and ABCA1. This corresponds to a region of approximately 6.9 Mb on human chromosome 9q. Furthermore, no recombination was observed between Grey, TGFBR1 and TMEFF1, the last two being 1.4 Mb apart in human. There are no obvious candidate genes in this region and none of the genes has been associated with pigmentation disorders or melanoma development, suggesting that the grey phenotype is caused by a mutation in a novel gene.     

Alpha-methylated derivatives of 2-arachidonoyl glycerol: synthesis, CB1 receptor activity, and enzymatic stability

Alpha-methylated analogues of the endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), were synthesized aiming to the improved enzymatic stability of 2-AG. In addition, the CB1 activity properties of fluoro derivatives of 2-AG were studied. The CB1 receptor activity was determined by the [35S]GTPgammaS binding assay, and the enzymatic stability of alpha-methylated analogues was determined in rat cerebellar membranes. The results indicate that even if the alpha-methylated 2-AG derivatives are slightly weaker CB1 receptor agonists than 2-AG, they are clearly more stable than 2-AG. In addition, the results showed that the replacement of the hydroxyl group(s) of 2-AG by fluorine does not improve the CB1 activity of 2-AG.     

Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers

A single accidental event such as the fallout released from the Chernobyl reactor in 1986 can expose millions of people to non-natural environmental radiation. Ionizing radiation increases the frequency of germline mutations in experimental studies, but the genetic effects of radiation in humans remain largely undefined. To evaluate the hereditary effects of low radiation doses, we compared the minisatellite mutation rates of 155 children born to Estonian Chernobyl cleanup workers after the accident with those of their siblings born prior to it. All together, 94 de novo paternal minisatellite mutations were found at eight tested loci (52 and 42 mutants among children born after and before the accident, respectively). The minisatellite mutation rate was nonsignificantly increased among children born after the accident (0.042 compared to 0.036, OR 1.33, 95% CI 0.80-2.20). Furthermore, there was some indication of an increased mutation rate among offspring born after the accident to workers who had received doses of 20 cSv or above compared with their siblings born before the accident (OR 3.0, 95% CI 0.97-9.30). The mutation rate was not associated with the father's age (OR 1.04, 95% CI 0.94-1.15) or the sex of the child (OR 0.95, 95% CI 0.50-1.79). Our results are consistent with both no effect of radiation on minisatellite mutations and a slight increase at dose levels exceeding 20 cSv.