EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media

Free radicals and/or hydrogen peroxide produced by exposure of cells to ultrasound are potentially cytotoxic and mutagenic. The formation and type of free radical species can be substantially modulated by the chemical composition of the media in which the ultrasound exposures of cells are carried out. In the current study, we examined the free radical intermediates formed during ultrasound exposure of a typical cell culture medium (RPMI-1640); the dominant free radicals that were identified by spin trapping were derived from the hydrophobic amino acids Trp, Leu, and Phe, and were formed by hydrogen abstraction from these amino acids. Compared to exposures in phosphate-buffered saline, the yield of *OH radicals and H2O2 was significantly reduced in the cell culture medium, glucose (the main organic component in the medium), and the hydrophobic amino acids (Trp, Phe, Tyr, Leu, Val, Met) being chiefly responsible for this effect. In contrast, other nonhydrophobic amino acids did not contribute significantly to the *OH or H2O2 decrease. These findings are consistent with the accumulation of hydrophobic solutes at the liquid-gas interface of the collapsing cavitation bubbles resulting in increased efficiency of radical scavenging.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

El carcinoma diferenciado incidental de tiroides es menos prevalente en la enfermedad de Graves que en el bocio multinodular

ObjectiveRisk factors for differentiated thyroid carcinoma (DTC) are poorly understood, but serum TSH levels, thyroid nodularity, and presence of autoimmunity are well-recognized factors that modulate DTC prevalence. TSH stimulates proliferation of both normal and neoplastic follicular cells. Consequently, thyroid-stimulating immunoglobulins (TSI), because of its TSH-like action, should induce DTC progression in patients with Graves’ disease (GD). The study objective was to compare the prevalence of incidental DTC in patients undergoing thyroidectomy for benign thyroid disease.MethodsThe pathology reports of 372 patients with preoperative diagnosis of euthyroid multinodular goiter (EMG) or hyperthyroidism were reviewed. Scintigraphy results and serum TSI levels were used to diagnosed either GD or hyperactive MG (HMG) to hyperthyroid subjects. Prevalence of DTC in each category was calculated using a Chi-square test.ResultsEMG, GD, and HMG were diagnosed in 221, 125, and 26 patients. There were 58 DTCs, distributed as follows [n (%)]: EMG, 49 (22.2%); GD, 8 (6.4%), and HMG, 1 (3.8%). Difference in prevalence of incidental DTC between the groups was statistically significant (p < 0.001). After adjustment for age, patients with EMG had a greater DTC prevalence than GD patients, with an OR of 4.17 (p < 0.001). Tumor size (mm, mean ± SD) was 6.92 ± 11.26, 1.97 ± 1.85, and 9.0 for EMG, GD and HMG respectively (p = 0.017).ConclusionsIncidental DTC was less prevalent in GD as compared to EMG irrespective of age. This finding may suggest a predisposition to develop DTC in patients with thyroid nodular disease and/or a potential effect of autoimmunity to protect against development of neoplastic disease.     

The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures

Protein aggregation is a major bottleneck during the bacterial production of recombinant proteins. In general, the induction of gene expression at sub-optimal growth temperatures improves the solubility of aggregation-prone polypeptides and minimizes inclusion body (IB) formation. However, the effect of low temperatures on the quality of the recombinant protein, especially within the insoluble cell fraction, has been hardly ever explored. In this work, we have examined the conformational status of a recombinant GFP protein when produced in Escherichia coli below 37 degrees C. As expected, the fraction of aggregated protein largely decreased at lower temperatures, while the conformational quality of both soluble and aggregated GFP, as reflected by its specific fluorescence emission, progressively improved. This observation indicates that physicochemical conditions governing protein folding affect concurrently the quality of the soluble and the aggregated forms of a misfolding-prone protein, and that protein misfolding and aggregation are clearly not coincident events.     

Anti-Osteoporotic Drug Release from Ordered Mesoporous Bioceramics: Experiments and Modeling

The release of a potent bone-resorption inhibitor such as zoledronate from a versatile drug delivery system such as SBA 15 has been modeled. The initial and boundary conditions have been defined, together with the system parameters, including the determination of equilibrium and transport parameters. Additionally, the experimental model of the same system has been observed to validate the prediction here developed. This approach represents a powerful tool for the designing of mesoporous implantable drug delivery systems because their release kinetics can be predicted in advance, and this leads to a considerable time and resources saving.     

Potential of the luminol reaction in the sensitive detection of pesticide residues by flow injection analysis.

This study presents the first analytical application of the luminol chemiluminescence (CL) reaction for the sensitive detection of carbamate residues. Some experiments have been carried out to check the influence of the presence of traces of a N-methylcarbamate (carbaryl) on the CL emission produced from the oxidation of luminol using different oxidants, showing a significant enhancing effect on the CL emission when the oxidation of luminol is produced by potassium permanganate in alkaline medium, this enhancement being proportional to the carbaryl concentration. This fact has permitted the establishment of a sensitive chemiluminescence flow-injection (CL-FIA) method for the direct determination of carbaryl. The optimization of instrumental and chemical variables influencing the CL response has been carried out by applying experimental designs. Under the optimal conditions, the CL intensity was linear for a carbaryl concentration over the range 5-100 ng/mL with a detection limit of 4.9 ng/mL. This luminol-KMnO4-based FIA-CL system in basic medium shows an easy, fast and cheap alternative detection mode for the analysis of carbaryl residues in environmental water samples.     

Oxidative refolding of lysozyme assisted by DsbA, DsbC and the GroEL apical domain immobilized in cellulose

Expression of recombinant proteins in Escherichia coli often leads to formation of inclusion bodies (IB). If a recombinant protein contains one or more disulfide bonds, protein refolding and thiol oxidation reactions are required to recover its biological activity. Previous studies have demonstrated that molecular chaperones and foldases assist with the in vitro protein refolding. However, their use has been limited by the stoichiometric amount required for the refolding reaction. In search of alternatives to facilitate the use of these folding biocatalysts in this study, DsbA, DsbC, and the apical domain of GroEL (AD) were fused to the carbohydrate-binding module CBD_Cex of Cellulomonas fimi. The recombinant proteins were purified and immobilized in cellulose and used to assist the oxidative refolding of denatured and reduced lysozyme. The assisted refolding yields obtained with immobilized folding biocatalysts were at least twice of those obtained in the spontaneous refolding, suggesting that the AD, DsbA, and DsbC immobilized in cellulose might be useful for the oxidative refolding of recombinant proteins that are expressed as inclusion bodies. In addition, the spontaneous or assisted refolding kinetics data fitted well (r² > 0.9) to a previously reported lysozyme refolding model. The estimated refolding (k _N) and aggregation (k _A) constants were consistent with the hypothesis that foldases assisted the oxidative refolding of lysozyme by decreasing protein aggregation rather than increasing the refolding rate.     

Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.     

The role of fecundity and sexual selection in the evolution of size and sexual size dimorphism in New World and Old World voles (Rodentia: Arvicolinae)

Evolutionary ecologists dating back to Darwin (1871) have sought to understand why males are larger than females in some species, and why females are the larger sex in others. Although the former is widespread in mammals, rodents and other small mammals usually exhibit low levels of sexual size dimorphism (SSD). Here, we investigate patterns of sexual dimorphism in 34 vole species belonging to the subfamily Arvicolinae in a phylogenetic comparative framework. We address the potential role of sexual selection and fecundity selection in creating sex differences in body size. No support was found for hyperallometric scaling of male body size to female body size. We observed a marginally significant relationship between SSD and the ratio of male to female home range size, with the latter being positively related to the level of intrasexual competition for mates. This suggests that sexual selection favours larger males. Interestingly, we also found that habitat type, but not mating system, constitutes a strong predictor of SSD. Species inhabiting open habitats – where males have extensive home ranges in order to gain access to as many females as possible – exhibit a higher mean dimorphism than species inhabiting closed habitats, where females show strong territoriality and an uniform distribution preventing males to adopt a territorial strategy for gaining copulations. Nonetheless, variation in the strength of sexual selection is not the only selective force shaping SSD in voles; we also found a positive association between female size and litter size across lineages. Assuming this relationship also exists within lineages (i.e. fecundity selection on female size), this suggests an additional role for variation in the strength of fecundity selection shaping interspecific differences in female size, and indirectly in SSD. Therefore our results suggest that different selective processes act on the sizes of males and females, but because larger size is favoured in both sexes, SSD is on average relatively small.