Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae

Hydrogenase expression in Chlamydomonas reinhardtii can be artificially induced by anaerobic adaptation or is naturally established under sulphur deprivation. In comparison to anaerobic adaptation, sulphur-deprived algal cultures show considerably higher expression rates of the [FeFe]-hydrogenase (HydA1) and develop a 25-fold higher in vitro hydrogenase activity. Based on this efficient induction principle we have established a novel purification protocol for the isolation of HydA1 that can also be used for other green algae. From an eight liter C. reinhardtii culture 0.52 mg HydA1 with a specific activity of 741 μmol H₂ min^− 1 mg^− 1 was isolated. Similar amounts were also purified from Chlorococcum submarinum and Chlamydomonas moewusii. The extraordinarily large yields of protein allowed a spectroscopic characterization of the active site of these smallest [FeFe]-hydrogenases for the first time. An initial analysis by EPR spectroscopy shows characteristic axial EPR signals of the CO inhibited forms that are typical for the H_ox-CO state of the active site from [FeFe]-hydrogenases. However, deviations in the g-tensor components have been observed that indicate distinct differences in the electronic structure between the various hydrogenases. At cryogenic temperatures, light-induced changes in the EPR spectra were observed and are interpreted as a photodissociation of the inhibiting CO ligand.     

Habitat Association in Two Genetic Groups of the Insect-Pathogenic Fungus Metarhizium anisopliae: Uncovering Cryptic Species?

Strains of insect-pathogenic fungi with high virulence toward certain pest insects have great potential for commercial biological control applications. Identifying such strains has been a central theme in using fungi for biological control. This theme is supported by a persistent paradigm in insect pathology which suggests that the host insect is the predominant influence on the population genetics of insect-pathogenic fungi. In this study, a population genetics analysis of the insect-pathogenic fungus Metarhizium anisopliae from forested and agricultural habitats in Ontario, Canada, showed a nonrandom association of alleles between two distinct, reproductively isolated groups (index of multilocus association = 1.2). Analyses of the mitochondrial DNA showed no differences between the groups. The two groups were associated with different habitat types, and associations with insect hosts were not found. The group from forested areas showed an ability for cold-active growth (i.e., 8°C), while the group from the agricultural area showed an ability for growth at high temperatures (i.e., 37°C) and resilience to UV exposure. These results represent a significant paradigm shift; habitat selection, not host insect selection, drives the population structure of these insect-pathogenic deuteromycetous fungi. With each group we observed recombining population structures as well as clonally reproducing lineages. We discuss whether these groups may represent cryptic species. Worldwide, M. anisopliae may be an assembly of cryptic species, each adapted to certain environmental conditions. The association of fungal genotypes with habitat but not with host insects has implications on the criteria for utility of this, and perhaps other, fungal biocontrol agents.     

Predictive Models to Determine Imagery Strategies Employed by Children to Judge Hand Laterality

A commonly used paradigm to study motor imagery is the hand laterality judgment task. The present study aimed to determine which strategies young children employ to successfully perform this task. Children of 5 to 8 years old (N = 92) judged laterality of back and palm view hand pictures in different rotation angles. Response accuracy and response duration were registered. Response durations of the trials with a correct judgment were fitted to a-priori defined predictive sinusoid models, representing different strategies to successfully perform the hand laterality judgment task. The first model predicted systematic changes in response duration as a function of rotation angle of the displayed hand. The second model predicted that response durations are affected by biomechanical constraints of hand rotation. If observed data could be best described by the first model, this would argue for a mental imagery strategy that does not involve motor processes to solve the task. The second model reflects a motor imagery strategy to solve the task. In line with previous research, we showed an age-related increase in response accuracy and decrease in response duration in children. Observed data for both back and palm view showed that motor imagery strategies were used to perform hand laterality judgments, but that not all the children use these strategies (appropriately) at all times. A direct comparison of response duration patterns across age sheds new light on age-related differences in the strategies employed to solve the task. Importantly, the employment of the motor imagery strategy for successful task performance did not change with age.     

Penetration of 2,4,6-trinitrobenzenesulfonate into human erythrocytes: Consequences for studies on phospholipid asymmetry

The glutathione content of human erythrocytes rapidly diminishes when cells are exposed to 2,4,6-trinitrobenzenesulfonate (20 μmol/l cells) at 37°C. Even at 0°C a slow decrease in glutathione content is observed. The uptake of trinitrobenzenesulfonate by the cells is retarded by inhibitors of the inorganic anion exchange system, indicating that trinitrobenzenesulfonate enters the cells by this pathway.The disappearance of glutathione most probably results from the reaction: 2 GSH + trinitrobenzenesulfonate → GSSG + aminodinitrobenzenesulfonate The reaction of trinitrobenzenesulfonate with glutathione occurs prior to its covalent binding to amino groups of hemoglobin which makes this reaction a more sensitive method of detection of penetration of trinitrobenzenesulfonate into erythrocytes. Results of studies on the asymmetric distribution of phospholipids using trinitrobenzenesulfonate as the only probe should be reconsidered in the light of these new data.     

Stay Focused! The Effects of Internal and External Focus of Attention on Movement Automaticity in Patients with Stroke

Dual-task performance is often impaired after stroke. This may be resolved by enhancing patients’ automaticity of movement. This study sets out to test the constrained action hypothesis, which holds that automaticity of movement is enhanced by triggering an external focus (on movement effects), rather than an internal focus (on movement execution). Thirty-nine individuals with chronic, unilateral stroke performed a one-leg-stepping task with both legs in single- and dual-task conditions. Attentional focus was manipulated with instructions. Motor performance (movement speed), movement automaticity (fluency of movement), and dual-task performance (dual-task costs) were assessed. The effects of focus on movement speed, single- and dual-task movement fluency, and dual-task costs were analysed with generalized estimating equations. Results showed that, overall, single-task performance was unaffected by focus (p = .341). Regarding movement fluency, no main effects of focus were found in single- or dual-task conditions (p’s ≥ .13). However, focus by leg interactions suggested that an external focus reduced movement fluency of the paretic leg compared to an internal focus (single-task conditions: p = .068; dual-task conditions: p = .084). An external focus also tended to result in inferior dual-task performance (β = -2.38, p = .065). Finally, a near-significant interaction (β = 2.36, p = .055) suggested that dual-task performance was more constrained by patients’ attentional capacity in external focus conditions. We conclude that, compared to an internal focus, an external focus did not result in more automated movements in chronic stroke patients. Contrary to expectations, trends were found for enhanced automaticity with an internal focus. These findings might be due to patients’ strong preference to use an internal focus in daily life. Future work needs to establish the more permanent effects of learning with different attentional foci on re-automating motor control after stroke.     

An in vivo 31P-NMR study of the possible regulation of glycogen phosphorylase a by phosphagen via phosphate in the abdominal muscle of the shrimp Crangon crangon

31P-NMR spectroscopy has been used to determine the concentrations of Pi and phosphagen in the abdominal muscle of the shrimp Crangon crangon at rest and to follow the changes during their recovery after exhaustive work in vivo. Additionally, the effect of physiological Pi concentrations to the phosphorylase a activity has been examined. At rest, the cytoplasmic Pi and phosphagen concentrations were 1 mM and 38 mM, respectively. After exhaustive work the concentrations of both metabolites were in the same range (20 mM). During recovery, the time course of phosphagen regeneration correlates with the decrease of Pi, as the sum of the concentrations of these metabolites is constant. The ATP level was stable throughout the experiment. The phosphorylase a, which was already present in the resting muscle, was virtually inactive at the resting Pi level (1 mM). During work, however, Pi, which is probably generated by phosphagen-ATP splitting, might ensure a sufficient activation of phosphorylase a.     

Transfection of Escherichia coli by Mu DNA

Summary Infectivity of Mu DNA was demonstrated in Ca⁺⁺-treated Escherichia coli cells that lacked the nucleases Exo V and Endo I. The efficiency of transfection is about 10^-7 per phage equivalent. Infectivity is destroyed by denaturation of Mu DNA, and cannot be restored by renaturation.