An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Large-scale evaluation of candidate genes identifies associations between VEGF polymorphisms and bladder cancer risk

Common genetic variation could alter the risk for developing bladder cancer. We conducted a large-scale evaluation of single nucleotide polymorphisms (SNPs) in candidate genes for cancer to identify common variants that influence bladder cancer risk. An Illumina GoldenGate assay was used to genotype 1,433 SNPs within or near 386 genes in 1,086 cases and 1,033 controls in Spain. The most significant finding was in the 5′ UTR of VEGF (rs25648, p for likelihood ratio test, 2 degrees of freedom = 1 × 10⁻⁵). To further investigate the region, we analyzed 29 additional SNPs in VEGF, selected to saturate the promoter and 5′ UTR and to tag common genetic variation in this gene. Three additional SNPs in the promoter region (rs833052, rs1109324, and rs1547651) were associated with increased risk for bladder cancer: odds ratio (95% confidence interval): 2.52 (1.06–5.97), 2.74 (1.26–5.98), and 3.02 (1.36–6.63), respectively; and a polymorphism in intron 2 (rs3024994) was associated with reduced risk: 0.65 (0.46–0.91). Two of the promoter SNPs and the intron 2 SNP showed linkage disequilibrium with rs25648. Haplotype analyses revealed three blocks of linkage disequilibrium with significant associations for two blocks including the promoter and 5′ UTR (global p = 0.02 and 0.009, respectively). These findings are biologically plausible since VEGF is critical in angiogenesis, which is important for tumor growth, its elevated expression in bladder tumors correlates with tumor progression, and specific 5′ UTR haplotypes have been shown to influence promoter activity. Associations between bladder cancer risk and other genes in this report were not robust based on false discovery rate calculations. In conclusion, this large-scale evaluation of candidate cancer genes has identified common genetic variants in the regulatory regions of VEGF that could be associated with bladder cancer risk.     

The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress

The 29-kDa surface antigen (thiol-dependent peroxidase; Eh29) of Entamoeba histolytica exhibits peroxidative and protective antioxidant activities. During tissue invasion, the trophozoites are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS). In this investigation, attempts have been made to understand the role of the 29-kDa peroxidase gene in parasite survival and pathogenesis. Inhibition of eh29 gene expression by antisense RNA technology has shown approximately 55% inhibition in eh29 expression, maximum ROS accumulation, and significantly lower viability in 29-kDa downregulated trophozoites during oxidative stress. The cytopathic and cytotoxic activities were also found to decrease effectively in the 29-kDa downregulated trophozoites. Size of liver abscesses was substantially lower in hamsters inoculated with 29-kDa downregulated trophozoites compared to the normal HM1:IMSS. These findings clearly suggest that the 29-kDa protein of E. histolytica has a role in both survival of trophozoites in the presence of ROS and pathogenesis of amoebiasis.     

Stress fiber growth and remodeling determines cellular morphomechanics under uniaxial cyclic stretch

Biomechanics and Modeling in Mechanobiology - Stress fibers in the cytoskeleton are essential in maintaining cellular shape and influence cellular adhesion and migration. Cyclic uniaxial stretching...     

Ultrashort nanosecond electric pulses evoke heterogeneous patterns of Ca2+ release from the endoplasmic reticulum of adrenal chromaffin cells

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High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum

AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4′-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C₁₈ analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50–2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml.