Increased xylose affinity of Hxt2 through gene shuffling of hexose transporters in Saccharomyces cerevisiae.

AIMS: Optimizing D-xylose transport in Saccharomyces cerevisiae is essential for efficient bioethanol production from cellulosic materials. We have used a gene shuffling approach of hexose (Hxt) transporters in order to increase the affinity for D-xylose. METHODS AND RESULTS: Various libraries were transformed to a hexose transporter deletion strain, and shuffled genes were selected via growth on low concentrations of D-xylose. This screening yielded two homologous fusion proteins (fusions 9,4 and 9,6), both consisting of the major central part of Hxt2 and various smaller parts of other Hxt proteins. Both chimeric proteins showed the same increase in D-xylose affinity (8·1 ± 3·0 mmol l-1 ) compared with Hxt2 (23·7 ± 2·1 mmol l-1 ). The increased D-xylose affinity could be related to the C terminus, more specifically to a cysteine to proline mutation at position 505 in Hxt2. CONCLUSIONS: The Hxt2C505P mutation increased the affinity for D-xylose for Hxt2, thus providing a way to increase D-xylose transport flux at low D-xylose concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The gene shuffling protocol using the highly homologues hexose transporters family provides a powerful tool to enhance the D-xylose affinity of Hxt transporters in S. cerevisiae, thus providing a means to increase the D-xylose uptake flux at low D-xylose concentrations.

Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption.

AIMS: Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. METHODS AND RESULTS: Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. CONCLUSIONS: Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.

Functional characterization and expression analysis of the androgen receptor in zebrafish (Danio rerio) testis.

The biological activity of androgens, important for male sexual differentiation and development, is mediated by the androgen receptor (AR) that binds to specific DNA recognition sites regulating the transcription of androgen target genes. We investigated androgen production by adult zebrafish testis tissue, and identified 11beta-hydroxyandrostenedione, 11-ketoandrostenedione (OA), and 11-ketotestosterone (11-KT) as main products, and hence potential ligands, for the zebrafish Ar. These androgens were then included in the pharmacological characterization of the zebrafish Ar. The zebrafish Ar responded well in terms of binding and transactivation to synthetic androgens as well as to testosterone and 11-KT, and reasonably well to OA and androstenedione. In situ hybridization analysis of zebrafish testis revealed that ar mRNA expression was detected in the subpopulation of Sertoli cells contacting early spermatogonia.

No evidence of the usefulness of eye blinking as a marker for central dopaminergic activity.

This study aimed to evaluate eye blinking as a marker for central dopaminergic activity by investigating the effects of sulpiride (D2-antagonist) and lisuride (D2-agonist) on spontaneous eye blinks. Twelve healthy subjects were included in a randomized, double-blind, placebo-controlled, three-period crossover trial. They received sulpiride 400 mg, lisuride 0.2 mg and placebo on different occasions. Eye blinks, prolactin, finger tapping, eye movements and visual analogue scales were measured at baseline and regularly for 12 h after administration. No effect of sulpiride or lisuride was observed on the number of eye blinks. Sulpiride caused an increase in prolactin (643 U/ml) [confidence interval (CI) 549-737). Lisuride caused a decrease in smooth pursuit eye movements (-4.1%) (CI -7.3 to -0.9) and visual analogue scales for mood (-2.1 mm) (CI -3.7 to -0.4). Spontaneous eye blink rate was not affected by sulpiride and lisuride, which makes eye blinking not suitable as a marker for central D2 activity.

Biomarkers for the effects of benzodiazepines in healthy volunteers.

Studies of novel centrally acting drugs in healthy volunteers are traditionally concerned with kinetics and tolerability, but useful information may also be obtained from biomarkers of clinical endpoints. A useful biomarker should meet the following requirements: a consistent response across studies and drugs; a clear response of the biomarker to a therapeutic dose; a dose-response relationship; a plausible relationship between biomarker, pharmacology and pathogenesis. In the current review, all individual tests found in studies of benzodiazepine agonists registered for anxiety in healthy volunteers since 1966 were progressively evaluated for compliance with these requirements. A MedLine search yielded 56 different studies, investigating the effects of 16 different benzodiazepines on 73 different (variants of ) neuropsychological tests, which could be clustered into seven neuropsychological domains. Subjective and objective measures of alertness were most sensitive to benzodiazepines. The most consistent effects were observed on saccadic peak velocity (SPV) and visual analogue scores ( VAS) of alertness, where 100% and 79% of all studies respectively showed statistically significant effects. A dose-response relationship could be constructed for temazepam and SPV, which was used to determine dose equivalencies relative to temazepam, for seven different benzodiazepines. These dose equivalencies correlated with the lowest recommended daily maintenance dose (r2 = 0.737, P < 0.05). This relationship between SPV reduction and clinical efficacy could reflect the clinical practice of aiming for maximum tolerated levels, or it could represent a common basis behind SPV reduction and anxiolytic activity for benzodiazepines (probably sedation). The number of tests used in human psychopharmacology appears to be excessive and their sensitivity and reproducibility low.