Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.     

Oxidative stress in the testis of hyperglycemic rabbits treated with repaglinide

In the present study, the induction of oxidative stress was examined in the testis of alloxan-induced diabetic rabbits. In addition, the protective effect of repaglinide, an oral anti-diabetic, at a dose of 1 mg daily was studied after four and eight weeks of the treatment. For these purposes, the levels of superoxide dismutase (Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione (GSH), ascorbic acid (AA), lipid peroxidation products (LPO) and protein carbonyl groups (PCG) were quantified. Hyperglycemia resulted in significant increases in the antioxidative enzymes, Cu, Zn-SOD, CAT, GSH-Px, and GSSG-R after four and eight weeks, respectively. There was also an increase in GSH level, and a decrease in the level of AA. These effects were accompanied by an elevation in testicular LPO levels and PCG levels. Repaglinide was found to normalize the activity of GSSG-R and levels of GSH and AA, and blunted the increased lipid peroxidation, however no decrease in PCG levels were observed. In conclusion, some oxidative changes provoked in the testis of rabbits by hyperglycemia, were found to be reduced with repaglinide treatment at therapeutic dose.     

Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis

Bacterial lipoproteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N-terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten-fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N-terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration-dependent interference with essential functions of chloroplastic membranes required for primary metabolism.     

Effect of feeding dehydrated and ensiled tanniferous sainfoin (Onobrychis viciifolia) on nitrogen and mineral digestion and metabolism of lambs

The effects of tanniferous sainfoin on digestion and metabolism have been investigated in 12 lambs in an incomplete cross-over design (n = 6). Effects of condensed tannins (CT) were evaluated by comparing dehydrated and ensiled sainfoin treated with and without polyethylene glycol (PEG). Dehydrated and ensiled grass-clover mixtures served as controls. The lambs were fed the treatment diets, including a mineral supplement, for 21 d. During the last 7 d excreta, rumen fluid and blood were sampled. The CT of sainfoin decreased rumen fluid ammonia concentration (p < 0.001) and increased the plasma concentration mainly of essential amino acids (p < 0.001). Body retention of phosphorus, calcium and magnesium was lower with sainfoin compared to PEG-treated sainfoin (p < 0.05). Sainfoin without PEG resulted in lower digestibilities of organic matter and neutral detergent fibre than sainfoin with PEG and the grass-clover mixture (p < 0.001). Ensiling of sainfoin led to the lowest N-retention. In conclusion, the reduction in ruminal ammonia and urine-N losses by sainfoin CT did not improve N-retention.     

Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.     

Inhibition of cobalamin-dependent methionine synthase by substituted benzo-fused heterocycles

The cobalamin-dependent cytosolic enzyme, methionine synthase (EC.2.1.1.13), catalyzes the remethylation of homocysteine to methionine using 5-methyltetrahydrofolate as the methyl donor. The products of this remethylation--methionine and tetrahydrofolate--participate in the active methionine and folate pathways. Impaired methionine synthase activity has been implicated in the pathogenesis of anaemias, cancer and neurological disorders. Although the need for potent and specific inhibitors of methionine synthase has been recognized, there is a lack of such agents. In this study, we designed, synthesized and evaluated the inhibitory activity of a series of substituted benzimidazoles and small benzothiadiazoles. Kinetic analysis revealed that the benzimidazoles act as competitive inhibitors of the rat liver methionine synthase, whilst the most active benzothiadiazole (IC(50) = 80 microm) exhibited characteristics of uncompetitive inhibition. A model of the methyltetrahydrofolate-binding site of the rat liver methionine synthase was constructed; docking experiments were designed to elucidate, in greater detail, the binding mode and reveal structural requirements for the design of inhibitors of methionine synthase. Our results indicate that the potency of the tested compounds is related to a planar region of the inhibitor that can be positioned in the centre of the active site, the presence of a nitro functional group and two or three probable hydrogen-bonding interactions.     

Early Cambrian record of failed durophagy and shell repair in an epibenthic mollusc

Predation is arguably one of the main driving forces of early metazoan evolution, yet the fossil record of predation during the Ediacaran-Early Cambrian transition is relatively poor. Here, we present direct evidence of failed durophagous (shell-breaking) predation and subsequent shell repair in the Early Cambrian (Botoman) epibenthic mollusc Marocella from the Mernmerna Formation and Oraparinna Shale in the Flinders Ranges, South Australia. This record pushes back the first appearance of durophagy on molluscs by approximately 40Myr.