Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

A Novel,Single Algorithm Approach to Predict Acenocoumarol Dose Based on CYP2C9 and VKORC1 Allele Variants

The identification of CYP2C9 and VKORC1 genes has strongly stimulated the research on pharmacogenetics of coumarins in the last decade. We assessed the combined influence of CYP2C9 *2 and *3, and VKORC1 c.-1639G>A, 497C>G, and 1173C>T variants, on acenocoumarol dosage using a novel algorithm approach, in 193 outpatients who had achieved stable anticoagulation. We constructed an “acenocoumarol-dose genotype score” (AGS, maximum score = 100) based on the number of alleles associated with higher acenocoumarol dosage carried by each subject for each polymorphism. The mean AGS was higher in the high-dose (>28mg/week) compared with the low-dose (<7mg/week) group (mean(SEM) of 84.1±3.4 vs. 62.2±4.8, P = 0.008). An AGS>70 was associated with an increased odds ratio (OR) of requiring high acenocoumarol dosage (OR: 3.347; 95%CI: 1.112–10.075; P = 0.032). In summary, although more research is necessary in other patient cohorts, and this algorithm should be replicated in an independent sample, our data suggest that the AGS algorithm could be used to help discriminating patients requiring high acenocoumarol doses to achieve stable anti-coagulation.     

Proton leak modulation in testicular mitochondria affects reactive oxygen species production and lipid peroxidation

Mitochondrial proton leak can account for almost 20% of oxygen consumption and it is generally accepted that this process contributes to basal metabolism. In order to clarify the role of basal proton leak in testicular mitochondria, we performed a comparative study with kidney and liver mitochondrial fractions. Proton leak stimulated by linoleic acid and inhibited by guanosine diphosphate (GDP) was detected, in a manner that was correlated with protein levels for uncoupling protein 2 (UCP2) in the three fractions. Modulation of proton leak had an effect on reactive oxygen species production as well as on lipid peroxidation, and this effect was also tissue‐dependent. However, a possible role for the adenine nucleotide transporter (ANT) in testicular mitochondria proton leak could not be excluded. The modulation of proton leak appears as a possible and attractive target to control oxidative stress with implications for male gametogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

Calculation of NMR relaxation, covolume, and scattering-related properties of bead models using the SOLPRO computer program

The hydrodynamic properties of macromolecules and bioparticles, represented by bead models, can be calculated using methods implemented in the computer routine HYDRO. Recently, a new computer routine, SOLPRO, has been presented for the calculation of various SOLution PROperties. These include (1) time-dependent electro-optic and spectroscopic properties related to rotational diffusion, (2) non-dynamic properties like scattering curves, and (3) dimensionless quantities that combine two or more solution properties in a form which depends on the shape of the macromolecule but not on its size. In the present work we describe the inclusion of more of those types of properties in a new version of SOLPRO. Particularly, we describe the calculation of relaxation rates in nuclear magnetic resonance (NMR). For dipolar coupling, given the direction of the dipole the program calculates values of the spectral density, from which the NMR relaxation times can be obtained. We also consider scattering-related properties, namely the distribution of distances for the bead model, which is directly related to the angular dependence of scattered intensity, and the particle's longest distance. We have devised and programmed a procedure to calculate the covolume of the bead model, related to the second virial coefficient and, in general, to the concentration dependence of solution properties. Various shape-dependent dimensionless quantities involving the covolume are calculated. In this paper we also discuss some aspects, namely bead overlapping and hydration, that are not explicitely included in SOLPRO, but should be considered by the user. Received: 25 May 1998 / Revised version: 30 July 1998 / Accepted: 30 July 1998     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

Efficient Transformation of <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> by Electroporation and Conjugation with <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.     

Validation of the Diagnostic Interview for Genetic Studies (DIGS) in Colombia

An interview tool, Diagnostic Interview for Genetic Studies (DIGS 3.0), was translated into Spanish for application in studies of psychiatric disorders in Colombia. Two Spanish translations of the original English version of DIGS were prepared and back-translated into English. A review committee verified the linguistic and cultural equivalence of the translations. The evaluator and test-retest reliability were assessed calculating Cohen's kappa for samples of 65 and 91 patients respectively. DIGS proved valid in both appearance and content. The confidence interval (C.I.) was excellent for schizophrenia (kappa = 0.81, C.I. 95% = 0.68-0.93), bipolar disorder (kappa = 0.87, C.I. 95% = 0.75-0.99), major depressive disorder (kappa = 0.86, C.I. 95% = 0.70-1.00), and for a normal diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89); it was good for other psychiatric diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89) and poor for schizoaffective disorder (kappa = 0.37, C.I. 95% = -0.02-0.76). Test-retest reliability was excellent for all diagnoses (kappa > 0.8), except for "other psychiatric diagnoses" (kappa = 0.64, C.I. 95% = 0.31-0.96). The Spanish translation of the DIGS was comprehensible, with face and content validity, and good test-retest and evaluator reliability. This translation will be a useful tool for genetic studies of psychiatric disorders in Latin America, particularly where schizophrenia and affective disorders are involved.