Genetic Relatedness of Clinical and Environmental Vibrio cholerae Isolates Based on Triple Housekeeping Gene Analysis

Sequence analysis of dnaE, hlyA, and asd housekeeping genes were used to determine the genetic relatedness of our collection of Vibrio cholerae isolated from patients and surface waters over a 5-year period in Iran. The results showed 41, 17, and 9 variable sites throughout the sequenced fragments of dnaE (837 bp), hlyA (495 bp), and asd (295 bp), respectively. The results from sequence typing showed that all our clinical isolates were grouped in the same cluster. Eleven genotypes were identified among the environmental isolates. One environmental isolate was found to be in close genetic relatedness with our clinical isolates. One V. cholerae isolate showed a single-locus variant in the dnaE. For each of the studied genetic loci 10, 7, and 7 sequence types were observed for dnaE, hlyA, and asd, respectively. Only asd sequence analysis could make the distinction between the classical and El Tor isolates which emphasizes on selection of housekeeping locus with better discrimination power for analysis of different groups of isolates. Overall, the results indicated that surface waters in Tehran are a pool of non-toxigenic V. cholerae strains which are rarely related to clinical toxigenic isolates. In addition, our results verified that housekeeping gene sequence analysis could be a suitable approach for determination of the relatedness between clinical and environmental V. cholerae isolates.     

The mechanism of antioxidant activity of IRFI005 as a synthetic hydrophilic analogue of vitamin E

Developing a rational strategy to control intracellular reactive oxygen species (ROS) requires understanding the mechanism of antioxidant activity. In this investigation the properties of a novel synthetic analog of vitamin E (IRFI005) with potent antioxidant activity are described. A mechanism is proposed for its efficient radical-scavenging effects. Cellular antioxidant and antitoxicity assays showed IRFI005 to freely permeate across cellular membranes, enabling it to be an effective suppressor of intracellular ROS and to protect cells against toxicity induced by free radical generating compounds. The free radical-scavenging activity of IRFI005 examined by UV–Vis and electron spin resonance (ESR) techniques clearly confirmed a “two electrons and/or H-atom” donation mechanism for each molecule of IRFI005. Reducing power assay as well as semi-empirical calculations revealed that under physiological conditions (pH∼7) almost all IRFI005 molecules are in the anionic state (IRFI005⁻). Data indicated that the electron donating ability of IRFI005⁻ was dominant at physiological pH because of higher stability of quinine-IRFI005⁻ and less barrier energy of IRFI005⁻ than neutral IRFI005. Consequently, the efficient cellular protection of IRFI005 against toxic free radicals can be explained by a two electron-transfer process, because of reduced inter-frontier molecular orbital energy gap barrier at physiological pH. Our findings suggest that hydrophilic vitamin E-like antioxidants are good candidates in designing novel therapeutic strategies for inhibition of oxidative stress associated with different human diseases.     

Sequence and structural parameters enhancing adaptation of proteins to low temperatures

A systematic analysis compared sequence and structural parameters distributions between 13 pairs of psychrophilic and mesophilic proteins for elucidating the cold adaptation parameters. The results of statistical test (t-test) revealed that helical content, tight turn content, disulfide bonds and hydrogen bonds do not show significant difference between psychrophilic and mesophilic proteins. However, it was demonstrated in this study that a larger proportion of open beta-turn in psychrophilic proteins is an effective parameter in specific activity at low temperature. In addition, substitution of amino acids of charged and aliphatic groups with amino acids of tiny and small groups in protein chains, tight turns and alpha-helices in the direction from mesophilic to psychrophilic proteins is one of the mechanisms of low temperature adaptation. Such sequence and structural parameter differences would help to develop a strategy for designing cold-adapted proteins.     

Evaluation of the two polymorphisms rs1801133 in MTHFR and rs10811661 in CDKN2A/B in breast cancer

The 5,10-Methylenetetrahydrofolate reductase (MTHFR) was the rate-limiting enzyme in the methyl cycle, which was encoded by the MTHFR gene. MTHFR played a key role in homocysteine plasma level and was associated with the risk of breast cancer. The cyclin-dependent kinase (CDK) inhibitor (CDKN2A/B) was the tumor suppressor in the cell cycle regulation. The single-nucleotide polymorphism was thought to be associated with the predisposition of breast cancer and in subsequent immune response in different populations. The current study was conducted on a peripheral blood sample of 100 Iranian women with breast carcinoma and 142 cancer-free healthy female volunteers. The TaqMan real-time polymerase chain reaction technique was applied for genotyping of participants. The correlation of both variants and demographic data were investigated with the risk of breast cancer. Our data showed that the MTHFR allele T and TT genotype had the higher prevalence in patients (P < 0.0001) than the control group. The frequency of risk C allele into the CDKN2A/B rs10811661 was 72%. The correlations of menarche and underlying hormonal disorder with the risk of breast cancer were investigated; also our results showed that the menopause status was statistically significant between patients and controls (P = 0.036). Our investigations demonstrated that the MTHFR rs180113 and CDKN2A/B rs10811661 had a significant correlation with the elevated risk of breast cancer and they might be potentially valuable to apply as a prognostic factor for individual health care.     

Proton-coupled electron-transfer mechanism for the radical scavenging activity of cardiovascular drug dipyridamole

Dipyridamole (DIP) is a well-known pharmaceutical drug used as a coronary vasodilator and anti-platelet agent in clinics for treating several cardiovascular diseases. Primarily, the therapeutic effects of the drug are attributed to its antioxidant potency. In this research, we aim to declare the unknown antioxidant mechanism of DIP as well as its potent chain-breaking antioxidant activity in polar aqueous medium inside the cells, using different experimental methods and theoretical quantum calculations. Data demonstrated the higher antioxidant capacity of DIP against ROS and free radicals in polar cell's interior. DIP is capable of generating long living and noninvasive DIP(?) radicals in oxidant condition that leads to an effective "chain-breaking antioxidant" activity. Quantum computational data indicated that DIP antioxidant has more favorable ionization potential than trolox which means DIP has higher antioxidant activity. Also, data showed that the direct hydrogen-transfer is not a favorable process to construct DIP(?) because of high barrier energy, though electron-transfer process can more easily to produce DIP(?+) with the lowest barrier energy. Altogether, the electron donating potency of DIP to reduce ferric ion, having the low anodic oxidation peak potential, producing long lived stable DIP(?) radicals and protecting myoblast cells from oxidation, proposed the excellent "chain-breaking antioxidant" potency via electron-transfer mechanism of this vasodilator DIP drug in polar aqueous medium.     

Co-solvent mediated thermal stabilization of chondroitinase ABC I form Proteus vulgaris

Chondroitinase ABC I (cABC I) from Proteus vulgaris cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The clinical utilization of this enzyme is mainly limited by its thermal instability. This study has been undertaken to determine the effects of glycerol, sorbitol and trehalose on cABC I activity and thermal stability. The results indicated that the enzyme catalytic activity and intrinsic fluorescence intensity increased in the presence of these cosolvents whereas no considerable conformational changes observed in far-UV CD spectra. Thermal CD experiment revealed an increase in T(m) of cABC I in the presence of cosolvents which was significant for trehalose. Our results support the idea that cABC I has stabilized in the presence of glycerol, sorbitol and trehalose. Therefore, the use of these cosolvents seems to be promising for improvement in shelf-life and clinical applications of this drug enzyme.