Changes in antioxidant enzymes activity and oxidative stress by abscisic acid and salicylic acid in wheat genotypes

Abscisic acid (ABA) and salicylic acid (SA) were sprayed on leaves of wheat genotypes C 306 and Hira at 25 and 40 d after sowing under moderate water stress (−0.8 MPa) imposed by adding PEG-6000 in nutrient solution. ABA and SA increased the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in comparison to unsprayed control plants. Both ABA and SA treatments decreased the contents of hydrogen peroxide and thiobarbituric acid reactive substances, a measure of lipid peroxidation, compared to unsprayed plants. The beneficial effect of increase in antioxidant enzymes activity and decrease in oxidative stress was reflected in increase in chlorophyll and carotenoid contents, relative water content, membrane stability index, leaf area and total biomass over control plants. The lower concentrations of ABA (0.5 mM) and SA (1.0 mM) were generally more effective than higher concentrations.     

Nonparametric estimation of ROC curves in the absence of a gold standard

In the evaluation of diagnostic accuracy of tests, a gold standard on the disease status is required. However, in many complex diseases, it is impossible or unethical to obtain such a gold standard. If an imperfect standard is used, the estimated accuracy of the tests would be biased. This type of bias is called imperfect gold standard bias. In this article we develop a nonparametric maximum likelihood method for estimating ROC curves and their areas of ordinal-scale tests in the absence of a gold standard. Our simulation study shows that the proposed estimators for the ROC curve areas have good finite-sample properties in terms of bias and mean squared error. Further simulation studies show that our nonparametric approach is comparable to the binormal parametric method, and is easier to implement. Finally, we illustrate the application of the proposed method in a real clinical study on assessing the accuracy of seven specific pathologists in detecting carcinoma in situ of the uterine cervix.     

On criteria for evaluating models of absolute risk

Absolute risk is the probability that an individual who is free of a given disease at an initial age, a, will develop that disease in the subsequent interval (a, t]. Absolute risk is reduced by mortality from competing risks. Models of absolute risk that depend on covariates have been used to design intervention studies, to counsel patients regarding their risks of disease and to inform clinical decisions, such as whether or not to take tamoxifen to prevent breast cancer. Several general criteria have been used to evaluate models of absolute risk, including how well the model predicts the observed numbers of events in subsets of the population ("calibration"), and "discriminatory power," measured by the concordance statistic. In this paper we review some general criteria and develop specific loss function-based criteria for two applications, namely whether or not to screen a population to select subjects for further evaluation or treatment and whether or not to use a preventive intervention that has both beneficial and adverse effects. We find that high discriminatory power is much more crucial in the screening application than in the preventive intervention application. These examples indicate that the usefulness of a general criterion such as concordance depends on the application, and that using specific loss functions can lead to more appropriate assessments.     

Vitamin E prevents cell death induced by mild oxidative stress in chicken skeletal muscle cells

Apoptosis and necrosis are two forms of cell death that can occur in response to various agents and oxidative damage. In addition to necrosis, apoptosis contributes to muscle fiber loss in various muscular dystrophies as well participates in the exudative diathesis in chicken, pathology caused by dietary deficiency of vitamin E and selenium, which affects muscle tissue. We have used chicken skeletal muscle cells and bovine fibroblasts to study molecular events involved in the cell death induced by oxidative stress and apoptotic agents. The effect of vitamin E on cell death induced by oxidants was also investigated. Treatment of cells with anti-Fas antibody (50 to 400 ng/mL), staurosporine (0.1 to 100 microM) and TNF-alpha (10 and 50 ng/mL) resulted in a little loss of Trypan blue exclusion ability. Those stimuli conducted cells to apoptosis detected by an enhancement in caspase activity upon fluorogenic substrates but this activity was not fully blocked by the caspase inhibitor Z-VAD-fmk. Oxidative stress induced by menadione (10, 100 and 250 muM) promoted a significant reduction in cell viability (10%, 20% and 35% for fibroblasts; 20%, 30% and 75% for muscle cells, respectively) and caused an increase in caspase activity and DNA fragmentation. H2O2 also promoted apoptosis verified by caspase activation and DNA fragmentation, but in higher doses induced necrosis. Vitamin E protected cells from death induced by low doses of oxidants. Although it was ineffective in reducing caspase activity in fibroblasts, this vitamin diminished the enzyme activity in muscle cells. These data suggested that oxidative stress could activate apoptotic mechanisms; however the mode of cell death will depend on the intensity and duration of the stimulus, and on the antioxidant status of the cells.     

Analysis of thyroglobulin iodination by tandem mass spectrometry using immonium ions of monoiodo- and diiodo-tyrosine

Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion.     

Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.     

Receptor fragment approach to the binding between CCK8 peptide and cholecystokinin receptors: a fluorescence study on type B receptor fragment CCK(B)-R (352-379)

Fluorescence titrations in a membrane mimetic solvent system allowed us to estimate that the dissociation constant of the bimolecular complex between CCK8 peptide and cholecystokinin type B receptor fragment CCK(B)-R (352-379) is in the micromolar range. When considered in the context of the full receptor/ligand model, these experiments demonstrate that the receptor fragment chosen on the basis of previous structural studies represents a reliable model system to monitor the ability of CCK8 or CCK8 analogs to bind the cholecystokinin receptor. Together with previous studies, this confirms that the receptor fragment approach adopted to define the binding mode of the CCK8 fragment of cholecystokinin with its two receptors, CCK(A) and CCK(B,) can be used to characterize the binding from the equilibrium standpoint. In this context, fluorescence spectroscopy proves to be the favored technique to measure dissociation constants in the nanomolar to micromolar range.     

Does the anesthetic 2,2,2‐trifluoroethanol interact with bovine serum albumin by direct binding or by solvent‐mediated effects? A calorimetric and spectroscopic investigation

Thermal unfolding of bovine serum albumin (BSA) has been studied in the presence of 2,2,2-trifluoroethanol (TFE) using high-sensitivity microdifferential scanning calorimetry. Quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. TFE is observed to be a stabilizer or a destabilizer of the folded state of BSA depending on the pH. CD spectroscopy revealed an increase in the alpha-helical content of BSA and a decrease in the tertiary structure in the presence of increasing molalities of TFE. Isothermal titration calorimetric results do not indicate appreciable binding of the TFE molecules to BSA. TFE quenches the steady-state tryptophan fluorescence of BSA only at higher molalities and there is no effect on the tryptophan fluorescence at lower molalities. The calorimetric and spectroscopic results obtained in this work suggest that solvent-mediated effects dominate the interaction of TFE with BSA and the binding component may be very weak. Since the binding component is very weak, one of the possibilities of anesthetic action of TFE molecules on the actual targets may be through perturbation of the structural and dynamic properties of the lipid bilayer so that the function of crucial but unspecified membrane proteins is affected.     

Production of fuculose-1-phosphate aldolase using operator-repressor titration for plasmid maintenance in high cell density Escherichia coli fermentations

We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.