Mutational spectrum of dihydropyrimidine dehydrogenase gene (DPYD) in the Tunisian population

Dihydropyrimidine dehydrogenase enzyme (DPD) deficiency is a pharmacogenetic syndrome leading to severe side-effects in patients receiving therapies containing the anticancer drug 5-fluorouracil (5-FU). The aim of this population study is to evaluate gene variations in the coding region of the dihydropyrimidine dehydrogenase gene (DPYD) in the Tunisian population. One hundred and six unrelated healthy Tunisian volunteers were genotyped by denaturing HPLC (DHPLC). Twelve variants in the coding region of the DPYD were detected. Allele frequencies of DPYD*5 (A1627G), DPYD*6 (G2194A), DPYD*9A (T85C), A496G, and G1218A were 12.7%, 7.1%, 13.7%, 5.7%, and 0.5%, respectively. The DPYD alleles DPYD*2A (IVS 14+1g>1), DPYD*3 (1897 del C) and DPYD*4 (G1601A) associated with DPD deficiency were absent from the examined subjects. We describe for the first time a new intronic polymorphism IVS 6-29 g>t, found in an allelic frequency of 4.7% in the Tunisian population. Comparing our data with that obtained in Caucasian, Egyptian, Japanese and African-American populations, we found that the Tunisian population resembles Egyptian and Caucasian populations with regard to their allelic frequencies of DPYD polymorphisms. This study describes for the first time the spectrum of DPYD sequence variations in the Tunisian population.     

Association Between Haptoglobin 2-2 Genotype and Coronary Artery Disease and Its Severity in a Tunisian Population

Haptoglobin (Hp) polymorphism generates three common human genotypes (Hp1-1, Hp2-1, and Hp2-2), having functional differences, related to the risk of development of cardiovascular diseases. These functions are a consequence of hemoglobin binding that leads to the synthesis of an antioxidant like ferritin. We explored the association of Hp polymorphism with significant coronary stenosis (SCS) and its severity within 400 Tunisian patients, using genotyping, biochemical parameters, and the Gensini score. After adjustments for age and gender, Hp2-2 was associated with the highest ferritin but the lowest Hp concentrations. After adjustments for confounding parameters, the OR of SCS associated with Hp2-2 was 1.74 (95% CI 1.18–2.58; p = 0.005). This effect was enhanced within diabetics (OR 1.90, 95% CI 1.11–3.24; p = 0.018), obese subjects (OR 1.98, 95% CI 1.10–4.86; p = 0.034), and smokers (OR 4.17, 95% CI 1.54–1.29; p = 0.005). The Hp2-2 genotype is associated with an increase in SCS especially in diabetics, the obese, and smokers.     

The evolution of seminal ribonuclease: pseudogene reactivation or multiple gene inactivation events?

Two approaches, one novel, are applied to analyze the divergent evolution of ruminant seminal ribonucleases (RNases), paralogs of the well-known pancreatic RNases of mammals. Here, the goal was to identify periods of divergence of seminal RNase under functional constraints, periods of divergence as a pseudogene, and periods of divergence driven by positive selection pressures. The classical approach involves the analysis of nonsynonymous to synonymous replacements ratios (omega) for the branches of the seminal RNase evolutionary tree. The novel approach coupled these analyses with the mapping of substitutions on the folded structure of the protein. These analyses suggest that seminal RNase diverged during much of its history after divergence from pancreatic RNase as a functioning protein, followed by homoplastic inactivations to create pseudogenes in multiple ruminant lineages. Further, they are consistent with adaptive evolution only in the most recent episode leading to the gene in modern oxen. These conclusions contrast sharply with the view, cited widely in the literature, that seminal RNase decayed after its formation by gene duplication into an inactive pseudogene, whose lesions were repaired in a reactivation event. Further, the 2 approaches, omega estimation and mapping of replacements on the protein structure, were compared by examining their utility for establishing the functional status of the seminal RNase genes in 2 deer species. Hog and roe deer share common lesions, which strongly suggests that the gene was inactive in their last common ancestor. In this specific example, the crystallographic approach made the correct implication more strongly than the omega approach. Studies of this type should contribute to an integrated framework of tools to assign functional and nonfunctional episodes to recently created gene duplicates and to understand more broadly how gene duplication leads to the emergence of proteins with novel functions.     

Characterization of a novel cry2Aa-type gene from Bacillus thuringiensis subsp. kurstaki

A 4 kb BamHI-HindIII fragment, corresponding to the cry2A operon of Bacillus thuringiensis subsp. kurstaki strain BNS3, was cloned. The sequencing of the corresponding cry2Aa-type gene, termed crybns3-4, revealed an open reading frame of 1902 bp, encoding a protein of 633 amino-acid residues. Both nucleotide and amino-acid sequences similarity analysis revealed that crybns3-4 is a new cry2Aa-type gene which has several differences from the reported cry2Aa-type genes. The transfer of the cloned operon to an acrystalliferous mutant of BNS3, revealed an expression of the new cry2Aa-type gene and a production of parasporal crystal inclusions in the transformants.     

Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The K(m) value for compound 2 obtained with alpha-2,6-sialyltransferase from rat liver (EC 2.4.99.1) was 160 +/- 20 microM. After incubation of compound 2 with sialyltransferase the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also alpha-2, 6-sialyltransferase from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 microU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification.     

O-GlcNAc protein modification in plants: Evolution and function

The role in plants of posttranslational modification of proteins with O-linked N-acetylglucosamine and the evolution and function of O-GlcNAc transferases responsible for this modification are reviewed. Phylogenetic analysis of eukaryotic O-GlcNAc transferases (OGTs) leads us to propose that plants have two distinct OGTs, SEC- and SPY-like, that originated in prokaryotes. Animals and some fungi have a SEC-like enzyme while plants have both. Green algae and some members of the Apicomplexa and amoebozoa have the SPY-like enzyme. Interestingly the progenitor of the Apicomplexa lineage likely had a photosynthetic plastid that persists in a degenerated form in some species, raising the possibility that plant SPY-like OGTs are derived from a photosynthetic endosymbiont. OGTs have multiple tetratricopeptide repeats (TPRs) that within the SEC- and SPY-like classes exhibit evidence of strong selective pressure on specific repeats, suggesting that the function of these repeats is conserved. SPY-like and SEC-like OGTs have both unique and overlapping roles in the plant. The phenotypes of sec and spy single and double mutants indicate that O-GlcNAc modification is essential and that it affects diverse plant processes including response to hormones and environmental signals, circadian rhythms, development, intercellular transport and virus infection. The mechanistic details of how O-GlcNAc modification affects these processes are largely unknown. A major impediment to understanding this is the lack of knowledge of the identities of the modified proteins.     

A surface acoustic wave sensor functionalized with a polypyrrole molecularly imprinted polymer for selective dopamine detection

A surface acoustic wave sensor operating at 104 MHz and functionalized with a polypyrrole molecularly imprinted polymer has been designed for selective detection of dopamine (DA). Optimization of pyrrole/DA ratio, polymerization and immersion times permitted to obtain a highly selective sensor, which has a sensitivity of 0.55°/mM (≈550 Hz/mM) and a detection limit of ≈ 10 nM. Morphology and related roughness parameters of molecularly imprinted polymer surfaces, before and after extraction of DA, as well as that of the non imprinted polymer were characterized by atomic force microscopy. The developed chemosensor selectively recognized dopamine over the structurally similar compound 4‐hydroxyphenethylamine (referred as tyramine), or ascorbic acid,which co‐exists with DA in body fluids at a much higher concentration. Selectivity tests were also carried out with dihydroxybenzene, for which an unexpected phase variation of order of 75% of the DA one was observed. Quantum chemical calculations, based on the density functional theory, were carried out to determine the nature of interactions between each analyte and the PPy matrix and the DA imprinted PPy polypyrrole sensing layer in order to account for the important phase variation observed during dihydroxybenzene injection. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin

BUPM95 is a Bacillusthuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22 kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62 kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65 kDa compared to the 55 and 100 kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.