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.     

Determination of ochratoxin A in dry-cured meat products by a HPLC-FLD quantitative method

A fast and sensitive method for the quantification of the mycotoxin ochratoxin A (OTA) in dry-cured meat products has been developed, which does not require a clean-up step, by HPLC with an alkaline mobile phase (pH 9.8). Validation procedures for specificity, trueness, ruggedness, stability, recovery and repeatability were performed. The decision limit (CC alpha) and the decision capability (CC beta) were calculated at 1.10 and 1.23 microg/kg, respectively. The procedure was applied to representative dehydration levels of dry-cured meat samples.     

Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.     

Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.     

Identifying ecological drivers of interspecific variation in song complexity in songbirds (Passeriformes,Passeri)

A diversity of selective pressures and stochastic processes have likely created substantial variation in song structure, creating difficulties in quantifying the influence of specific ecological factors. This problem is further compounded by differences in study taxa and methods of data analysis between studies. Large comparative studies offer the potential to mitigate some of these methodological difficulties by maximizing the power of statistical analyses and minimizing the probability of misidentifying the magnitude and direction of relationships between independent and dependent variables. In this study, we quantified song complexity for 367 species of globally distributed songbirds (Passeriformes, Passeri). We quantified eight individual acoustic variables that have previously been linked to audio complexity which we analyzed independently, and after applying multivariate statistics to the variables. We used Bayesian linear mixed effect models to test multiple hypotheses regarding song complexity: that it should be greater in open habitats, in migratory species, for sexually monomorphic species, at higher latitudes and altitudes, and that it should co‐vary with clutch size characteristics. Our results challenge perceptions of the effect of habitat structure on song complexity; for instance, counter to expectation, we found songs in closed environments to have reduced syllable diversity. Additionally, our results suggest song complexity may not be ubiquitously a means of communicating male quality, with no significant difference between recordings from monomorphic and dimorphic species. By estimating song complexity in multiple ways, and quantifying these over large taxonomic and spatial scales, we are able to gain a more nuanced understanding of how song complexity is potentially affected by a range of biotic and abiotic factors. Our results also suggest that caution is required when making generalized statements about the relative influence of different factors on song complexity; more densely‐sampled, group‐specific studies are necessary complements to this taxonomically broad analysis.     

A comparison of the ability of forest and agricultural soils to mineralize chlorinated aromatic compounds

Soils were sampled from two agricultural fields, two relatively pristine forests, and one suburban forest in Ontario, Canada. The ability of these soils to mineralize 2,4-dichlorophenoxyacetate, 3-chlorobenzoate, 4-chlorophenol, 2,4-dichlorophenol, pentachlorophenol, and atrazine was determined using ¹⁴C-labeled substrates. Direct preexposure was necessary before atrazine mineralization could be detected; however, it was not necessary for degradation of any of the other chemicals. 2,4-dichlorophenoxyacetate and pentachlorophenol mineralization was much higher in the agricultural soils relative to the pristine forest soils, but 3-chlorobenzoate and 2,4-dichlorophenol mineralization rates showed the opposite trend. Mineralization of 4-chlorophenol was about equivalent in all soils. Suburban forests soils were indistinguishable from agricultural soils with respect to their degradation of 2,4-dichlorophenoxyacetate and chlorobenzoate. Additionally, they were better able than any of the soils to withstand the toxic effects of pentachlorophenol. Pentachlorophenol mineralization was highly variable in the pristine forest soils, ranging from about 6 to 50%. Abiotic factors such as pH, soil type, and organic and moisture content did not account for these significant site differences. The selective forces responsible for these differences, and the possible differences in microbial populations are discussed.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

Interaction of Pig Kidney and Lentil Seedling Copper-Containing Amine Oxidases with Guanidinium Compounds

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (K(i) =1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a K(i) value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.