Two continuous coupled assays for ornithine-δ-aminotransferase

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ¹-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.     

Movement of the S4 segment in the hERG potassium channel during membrane depolarization

Abstract

The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison of effects of static,proprioceptive neuromuscular facilitation and Mulligan stretching on hip flexion range of motion: a randomized controlled trial

The aim of this study was to compare the effects of static stretching, proprioceptive neuromuscular facilitation (PNF) stretching and Mulligan technique on hip flexion range of motion (ROM) in subjects with bilateral hamstring tightness. A total of 40 students (mean age: 21.5±1.3 years, mean body height: 172.8±8.2 cm, mean body mass index: 21.9±3.0 kg · m^-2) with bilateral hamstring tightness were enrolled in this randomized trial, of whom 26 completed the study. Subjects were divided into 4 groups performing (I) typical static stretching, (II) PNF stretching, (III) Mulligan traction straight leg raise (TSLR) technique, (IV) no intervention. Hip flexion ROM was measured using a digital goniometer with the passive straight leg raise test before and after 4 weeks by two physiotherapists blinded to the groups. 52 extremities of 26 subjects were analyzed. Hip flexion ROM increased in all three intervention groups (p<0.05) but not in the no-intervention group after 4 weeks. A statistically significant change in initial–final assessment differences of hip flexion ROM was found between groups (p<0.001) in favour of PNF stretching and Mulligan TSLR technique in comparison to typical static stretching (p=0.016 and p=0.02, respectively). No significant difference was found between Mulligan TSLR technique and PNF stretching (p=0.920). The initial–final assessment difference of hip flexion ROM was similar in typical static stretching and no intervention (p=0.491). A 4-week stretching intervention is beneficial for increasing hip flexion ROM in bilateral hamstring tightness. However, PNF stretching and Mulligan TSLR technique are superior to typical static stretching. These two interventions can be alternatively used for stretching in hamstring tightness.     

Estimation of hurdle clearance parameters using a monocular human motion tracking method

This paper presents a method of monocular human motion tracking for estimation of hurdle clearance kinematic parameters. The analysis involved 10 image sequences of five hurdlers at various training levels. Recording of the sequences was carried out under simulated starting conditions of a 110 m hurdle race. The parameters were estimated using the particle swarm optimization algorithm and they are based on analysis of the images recorded with a 100 Hz camera. The proposed method does not involve using any special clothes, markers, inertial sensors, etc. As the quality criteria, the mean absolute error and mean relative error were used. The level of computed errors justifies the use of this method to estimate hurdle clearance parameters.     

Anatomical glenohumeral internal rotation deficit and symmetric rotational strength in male and female young beach volleyball players

Beach volleyball is a sport with a high demand of shoulder structures that may lead to adaptations in range of motion (ROM) and strength like in other overhead sports. Despite of these possible alterations, no study evaluated the shoulder adaptations in young beach volleyball athletes. The aim of this study was to compare the bilateral ROM and rotation strength in the shoulders of young beach volleyball players. Goniometric passive shoulder ROM of motion and isometric rotational strength were evaluated in 19 male and 14 female asymptomatic athletes. External and internal ROM, total rotation motion, glenohumeral internal rotation deficit (GIRD), external rotation and internal rotation strength, bilateral deficits and external rotation to internal rotation ratio were measured. The statistical analysis included paired Student’s t-test and analysis of variance with repeated measures. Significantly lower dominant GIRD was found in both groups (p < 0.05), but only 6 athletes presented pathological GIRD. For strength variables, no significant differences for external or internal rotation were evident. Young beach volleyball athletes present symmetric rotational strength and shoulder ROM rotational adaptations that can be considered as anatomical. These results indicate that young practitioners of beach volleyball are subject to moderate adaptations compared to those reported for other overhead sports.