Association of BANK1 and cytokine gene polymorphisms with type 1 diabetes in Tunisia

Type 1 diabetes (T1D) is an autoimmune disease (AID) with both genetic and environmental components. We aimed to investigate the genetic association of polymorphisms in genes previously linked with other AIDs, namely BANK1, IL15 and IL2/IL21 region.     

Toxicological Study and Oxidative Stress Evaluation for Safety Assessment of Xylanase Preparations in Wistar Rats

Acute and 90‐day subchronic oral toxicity studies were conducted to establish the safety evaluation of xylanases preparations. A potential oxidative stress evaluation was also performed through testing the generation of oxidative radicals, depletion of antioxidants via oxidative modification of lipids, proteins and DNA of organ cells. During the subchronic oral toxicity study, no mortality was observed, obvious treatment‐related clinical signs and urinalysis parameters were in normal range. Differences in some hematological parameters, biochemistry, relative organ weight, and histopathology examinations between the treated group and the control group were not judged to be adverse. Our results indicated that the no‐observed‐adverse‐effect level for xylanases was 1,500 TXU/kg/day and the plasma antioxidant assays showed that these xylanases did not produce free‐radicals nor oxidative injuries. On the basis of the bacterial reverse mutation assay data, it is concluded that the expressed xylanase in Pichia pastoris do not present any mutagenic potential when tested in relevant genotoxicological assays.     

Antifungal activities of an endophytic <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens</Emphasis> strain Pf1TZ harbouring genes from pyoluteorin and phenazine clusters

Pseudomonas fluorescens Pf1TZ was inhibitory in vitro to a number of phytopathogenic fungi and could protect vine plantlets against Botrytis cinerea. Total protection was reached after 3 weeks of bacterial inoculation. The endophytism of Pf1TZ was confirmed by confocal microscopy using its inherent fluorescence. The molecular characterization of Pf1TZ indicated the presence of genes from clusters encoding pyoluteorin and phenazine. The chromatographic purification and LC-MSⁿ analysis revealed that the most active one has a molecular mass of 504 Da.     

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.     

The Calmodulin Regulator Protein,PEP-19, Sensitizes ATP-induced Ca2+ Release

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca²⁺ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca²⁺ binding activity, which may modulate Ca²⁺ binding to CaM by stabilizing an initial Ca²⁺-CaM complex or by electrostatically steering Ca²⁺ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca²⁺ dynamics, we tested the hypothesis that it influences ligand-dependent Ca²⁺ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca²⁺ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca²⁺ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca²⁺ binding to CaM greatly inhibit its effect on ATP-induced Ca²⁺ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca²⁺ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.     

Changes in the catalytic properties and substrate specificity of Bacillus sp. US149 maltogenic amylase by mutagenesis of residue 46

Maltogenic amylase from Bacillus sp. US149 (MAUS149) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of residue Asp46 in the specificity and catalytic properties of MAUS149 by using site-directed mutagenesis. Three mutated enzymes (D46V, D46G and D46N) were constructed and studied. The three mutants were found to be similar to the wild-type MAUS149 regarding thermoactivity, thermostability and pH profile. Nevertheless, the kinetic parameters for all the substrates of the mutant enzymes D46V and D46G were altered enormously as compared with those of the wild type. Indeed, the K _m values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward β-CD were increased fivefold as compared with those of MAUS149. Molecular modelling suggests that residue D46 forms a salt bridge with residue K282. This bond would maintain the arrangement of side chains of residues Y45 and W47 in a particular orientation that promotes access to the catalytic site and maintains the substrate therein. Hence, any replacement with uncharged amino acids influenced the flexibility of the gate wall at the substrate binding cleft resulting in changes in substrate selectivity.     

Chromosomal Aberrations (CA), Sister Chromatid Exchanges (SCE), and High-Frequency Cells (HFC) Frequencies in Peripheral Blood Lymphocytes of Tunisian Gasoline Truck Loaders

Gasoline constitutes a mixture of chemicals that contain well-known genotoxicants. Thus, chronic occupational exposure to gasoline may be considered to possess genotoxic risk. In this study, the frequencies of total chromosomal aberrations (TCA), aberrant cells (Ab.c.), sister chromatid exchanges (SCE), high-frequency cells (HFC), and high-frequency cell individual (HFI) were investigated in peripheral blood lymphocytes from 17 gasoline-exposed workers (10 smokers and 7 non-smokers) and 22 unexposed reference subjects (12 smokers and 10 non-smokers). The exposed subjects were gasoline truck loaders at a gasoline company from Tunis City, north of Tunisia. The results indicate multiple CA, such as dicentrics (DIC), chromatid breaks (SB), and chromosome breaks (DB). A significant difference was observed in TCA and Ab.c. frequencies between exposed and unexposed groups (p < 0.01). A significant difference was found in frequencies of SCE (p < 0.01) and HFI (p < 0.05) between exposed and unexposed groups. SCE and TCA frequencies of smokers were found to be significantly higher than those of non-smokers in both groups. There was an interaction between gasoline exposure and smoking habit for TCA (p = 0.020), but not for SCE. Our findings indicate that gasoline truck loaders were under risk of significant cytogenetic damage that was enhanced by their smoking habit.