Association of the PTPN22 R620W polymorphism with increased risk for SLE in the genetically homogeneous population of Crete.

Autoimmune diseases affect approximately 5% of the population, but much work remains to define the genetic risk factors and pathogenic mechanisms underlying these conditions. There is accumulating evidence that common genetic factors might predispose to multiple autoimmune disorders. Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are complex autoimmune disorders with multiple susceptibility genes. The functional R620W (C1858T) polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, a member of the PTPs that negatively regulate T-cell activation, has been recently associated with susceptibility to various autoimmune diseases. The aim of this study was to assess whether the C1858T polymorphism of PTPN22 also confers increased risk for SLE and RA in the genetically homogeneous population of Crete. It was found that the minor T allele of the PTPN22 C1858T SNP was more common in SLE patients than in control individuals (odds ratio [OR] = 1.91, 95% confidence interval [CI] = 1.11 to 3.9, p = 0.017). No significant difference was observed in the frequency of this allele when RA patients were compared with controls (OR = 1.14, 95% CI = 0.65 to 1.9, p = 0.64). Although the PTPN22 1858 T allele is found at decreased frequency in Southern Europe, including Crete, an association was found between this allele and SLE in the population studied.

Effect of acetone feeding on alcohol dehydrogenase activity in the olive fruit fly, Bactrocera oleae.

The purpose of this study is to demonstrate a clear connection between the presence of acetone in larval diet and alcohol dehydrogenase (ADH) activity in laboratory raised populations of Bactrocera oleae. ADH activity of B. oleae is depressed in acetone-impregnated diets. At the same time the change of activity is accompanied by a change in the relative proportions of the multiple forms of ADH. The bulk of activity in the most cathodally migrating form is lost, and all the activity becomes localized in the less cathodally migrating forms of the enzyme. Moreover, ADH activity, expressed in vivo, appears to drop after exposure to acetone, as shown by the fact that larvae become less sensitive to pentenol poisoning. Our results show clear selective differences imposed by acetone on three homozygous genotypes involving the ADH alleles F, S and I in B. oleae. The directions of these differences were found to vary with the fitness component under test. Acetone treatment seems to affect developmental time and larva's viability as well as allele frequencies of ADH under artificial rearing. The effect of acetone on the maintenance of ADH polymorphism in artificially reared populations of B. oleae is further discussed.

Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactrocera oleae.

A 2.2-kb full length cDNA containing an ORF encoding a putative acetylcholinesterase (AChE) precursor of 673 amino acid residues was obtained by a combined degenerate PCR and RACE strategy from an organophosphate-susceptible Bactrocera oleae strain. A comparison of cDNA sequences of individual insects from susceptible and resistant strains, coupled with an enzyme inhibition assay with omethoate, indicated a novel glycine-serine substitution (G488S), at an amino acid residue which is highly conserved across species (G396 of Torpedocalifornica AChE), as a likely cause of AChE insensitivity. This mutation was also associated with a 35-40% reduction in AChE catalytic efficiency. The I199V substitution, which confers low levels of resistance in Drosophila, was also present in B. oleae (I214V) and in combination with G488S produced up to a 16-fold decrease in insecticide sensitivity. This is the first agricultural pest where resistance has been associated with an alteration in AChE, which arises from point mutations located within the active site gorge of the enzyme.

Characterization of two alcohol dehydrogenase (Adh) loci from the olive fruit fly, Bactrocera (Dacus) oleae and implications for Adh duplication in dipteran insects.

We report the cloning and structural characterization of two Adh loci of the olive fruit fly, Bactrocera oleae. Each of the two genes, named Adh1 and Adh2, consists of three exons and two introns for a total length of 1981 and 988 nucleotides, respectively. Their deduced amino acid sequences of 257 and 258 residues exhibit a 77% identity and display the characteristics of the insect ADH enzymes, which belong to the short-chain dehydrogenases/reductases family. The Adh genes of B. oleae are compared to the two genes of the Mediterranean fly, Ceratitis capitata, the only other species of the Tephritidae family in which the Adh genes have been studied. On the basis of amino acid divergence the four genes form two clusters each containing one gene from each species, as expected if there was one duplication event before speciation. On the basis of nucleotide sequence the four sequences form two clusters each containing the two sequences from the same species, as expected if there was a separate duplication event in each species. To help decide between the two alternatives, we compared at both the amino acid and DNA level the Adh genes of five Drosophila species that are known to carry two such genes and observed that, with only one exception at the amino acid level, conspecific loci cluster together. We conclude that the information we have at present does not allow a firm choice between the hypothesis of a single duplication event that occurred before the split of Bactrocera and Ceratitis from their common ancestor and the hypothesis of two independent duplication events, one in each of the two genera.

Biochemical differences between products of the ADH locus in olive fruit fly (Bactrocera oleae).

Purified alcohol dehydrogenases from olive fruit flies of genotypes SS, II, and SI were biochemically compared. The enzymes were found to differ in the specific activity, in the influence of pH and temperature on activity, and in the affinity with different substrate-alcohols. The probable relationships of these findings with the dramatic changes in allele frequencies observed when natural populations are introduced in the laboratory are discussed.

Purification of alcohol dehydrogenase from four genotypes of the olive fruit fly Bactrocera (Dacus) oleae.

This is the first report describing the purification of alcohol dehydrogenase (ADH) from four genotypes of the olive fruit fly Bactrocera oleae, the most important pest of olives in the Mediterranean region. The purified enzyme shows a single band after SDS-PAGE analysis, corresponding to subunit mass of 26 kDa. The native ADH shows a molecular mass of 48 kDa, after gel filtration HPLC analysis. The purification method incorporated a preliminary ammonium sulphate precipitation step, followed by an anion-exchange DEAE chromatography step, a dye affinity chromatography step on Cibacron blue 3GA, and an anion-exchange DEAE chromatography step employing the same column of the first step. The present method offers good overall recovery (40%) and high enzyme purity, and it is applicable to different genotypes. Furthermore, the method is rapid and economical, as it employs two cheap, widely used, and commercially available chromatography materials.