Cloning, sequencing, expression, and single-step purification of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) from Salmonella typhimurium ATCC 19585

The bi-functional enzyme, adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU), is involved in the biosynthesis of cobalamin in Salmonella typhimurium, and, therefore, can be used for the in vitro synthesis of analogs of B(12). Previously, five different steps were required to purify the recombinant enzyme from Escherichia coli. Here, we describe the cloning, sequencing, and expression of the cobU gene from S. typhimurium ATCC 19585 and, without introducing a purification tag sequence to the N- or C-terminus of the recombinant enzyme, a new single-step purification method based on hydrophobic interaction chromatography.     

Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.     

Anillin binds nonmuscle myosin II and regulates the contractile ring

We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.     

Ablation of PRC1 by small interfering RNA demonstrates that cytokinetic abscission requires a central spindle bundle in mammalian cells, whereas completion of furrowing does not

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.     

Proposed tests for measuring the running velocity at the oxygen consumption and heart rate thresholds for treadmill exercise

The purposes of this study were to (a) determine if the mathematical model used to estimate the physical working capacity at the oxygen consumption threshold (PWC(VO(2))) and physical working capacity at the heart rate threshold (PWC(HRT)) for cycle ergometry could be applied to treadmill running; (b) propose new fatigue thresholds called the running velocity at the oxygen uptake threshold (RV(VO(2))) and running velocity at the heart rate threshold (RV(HRT)) for treadmill exercise; and (c) statistically compare the velocities at the RV(VO(2)), RV(HRT), and ventilatory threshold (VT). Seven aerobically trained adult volunteers (mean +/- SD: age 24.0 +/- 3.9 years, Vo(2) max 56.7 +/- 7.1 ml.kg(-1).min(-1)) performed a maximal treadmill test to determine Vo(2) peak and VT as well as four 8-minute submaximal workbouts for the determination of RV(VO(2)) and RV(HRT). One-way repeated-measures analysis of variance indicated that there were no significant (p > 0.05) mean differences among the running velocities for the RV(VO(2)), RV(HRT), and VT. The results of this study indicated that the mathematical model used to estimate PWC(VO(2)) and PWC(HRT) for cycle ergometry could be applied to treadmill running. Furthermore, the RV(VO(2)) and RV(HRT) test may provide submaximal techniques for estimating the VT.     

Influence of playing position on the site, nature, and cause of rugby league injuries

This study investigated the influence of playing position on the site, nature, and cause of injuries in rugby league. The incidence, site, nature, and cause of playing injuries was prospectively studied in 156 rugby league players over 2 seasons. An injury was defined as any pain or disability suffered by a player during a match that resulted in the player missing a subsequent match. The hooker (101 per 1,000 playing hours) and prop (92 per 1,000 playing hours) positions had the highest incidence of injury. Injuries sustained by the fullback (32 per 1,000 playing hours) and halfback (44 per 1,000 playing hours) positions were less common. Compared with other individual playing positions, props had a significantly higher incidence of overexertion injuries (22 per 1,000 playing hours), thigh and calf injuries (47 per 1,000 playing hours), and hematomas (19 per 1,000 playing hours), whereas the five-eighth position (31 per 1,000 playing hours) and the hookers and halves positional group (17 per 1,000 playing hours) had a significantly higher incidence of falling and stumbling injuries. These results demonstrate that the hooker and prop positions have higher injury rates than other rugby league positions. Furthermore, the site, type, and cause of injuries are different among individual playing positions and playing groups. These findings suggest that individual position training for injury prevention is warranted in rugby league.     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

A novel approach to determine water content in DMSO for a compound collection repository

The quality of a corporate compound collection can be significantly affected by a complex combination of storage and operational processing factors. Water content in DMSO solutions is one factor that is of great interest as it can affect solubility, degradation, and freeze-thaw cycle parameters. To the authors' knowledge, this is the first report of using near-infrared (NIR) spectroscopy to assess water content in DMSO compound stock solutions within the common storage vessel format of polypropylene microtubes. The precision and accuracy of the NIR technique was benchmarked against a Karl Fisher titration method, and a correlation coefficient was determined to be 0.985 over a range of 1% to 10% water in DMSO by weight. The advantages of the NIR technique include accuracy, precision, speed, nondestructiveness, and the capability of assessing compounds under in situ storage conditions within microtubes. In this report, the authors demonstrate the accuracy and precision of using NIR to assess water content in DMSO solutions and present a case study to demonstrate the utility of the technique to aid in assessing a pharmaceutical compound collection.     

Activation of defense responses in Chinese cabbage by a nonhost pathogen, Pseudomonas syringae pv. tomato

Pseudomonas syringae pv. tomato (Pst) causes a bacterial speck disease in tomato and Arabidopsis. In Chinese cabbage, in which host-pathogen interactions are not well understood, Pst does not cause disease but rather elicits a hypersensitive response. Pst induces localized cell death and H2O2 accumulation, a typical hypersensitive response, in infiltrated cabbage leaves. Pre-inoculation with Pst was found to induce resistance to Erwinia carotovora subsp. carotovora, a pathogen that causes soft rot disease in Chinese cabbage. An examination of the expression profiles of 12 previously identified Pst-inducible genes revealed that the majority of these genes were activated by salicylic acid or BTH; however, expressions of the genes encoding PR4 and a class IV chitinase were induced by ethephon, an ethylene-releasing compound, but not by salicylic acid, BTH, or methyl jasmonate. This implies that Pst activates both salicylate-dependent and salicylate-independent defense responses in Chinese cabbage.