Ubiquitous soluble Mg(2+)-ATPase complex. A structural study.

We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.     

Root-microbial effects on plant iron uptake from siderophores and phytosiderophores

Collaborative experiments were conducted to determine whether microbial populations associated with plant roots may artifactually affect the rates of Fe uptake and translocation from microbial siderophores and phytosiderophores. Results showed nonaxenic maize to have 2 to 34-fold higher Fe-uptake rates than axenically grown plants when supplied with 1 μM Fe as either the microbial siderophore, ferrioxamine B (FOB), or the barley phytosiderophore, epi-hydroxymugineic acid (HMA). In experiments with nonsterile plants, inoculation of maize or oat seedlings with soil microorganisms and amendment of the hydroponic nutrient solutions with sucrose resulted in an 8-fold increase in FOB-mediated Fe-uptake rates by Fe-stressed maize and a 150-fold increase in FOB iron uptake rates by Fe-stressed oat, but had no effect on iron uptake by Fe-sufficient plants. Conversely, Fe-stressed maize and oat plants supplied with HMA showed decreased uptake and translocation in response to microbial inoculation and sucrose amendment. The ability of root-associated microorganisms to affect Fe-uptake rates from siderophores and phytosiderophores, even in short-term uptake experiments, indicates that microorganisms can be an unpredictable confounding factor in experiments examining mechanisms for utilization of microbial siderophores or phytosiderophores under nonsterile conditions.     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.     

Fixed-bin analysis for statistical evaluation of continuous distributions of allelic data from VNTR loci, for use in forensic comparisons.

The detection of DNA polymorphisms by RFLP analysis is having a major impact on identity testing in forensic science. At present, this approach is the best effort a forensic scientist can make to exclude an individual who has been falsely associated with an evidentiary sample found at a crime scene. When an analysis fails to exclude a suspect as a potential contributor of an evidentiary sample, a means should be provided to assess suitable weight to the putative match. Most important, the statistical analysis should not place undue weight on a genetic profile derived from an unknown sample that is attributed to an accused individual. The method must allow for limitations in conventional agarose-submarine-gel electrophoresis and Southern blotting procedure, limited sample population data, possible subpopulation differences, and potential sampling error. A conservative statistical method was developed based on arbitrarily defined fixed bins. This approach permits classification of continuous allelic data, provides for a simple and portable data-base system, and is unlikely to underestimate the frequency of occurrence of a set of alleles. This will help ensure that undue weight is not placed on a sample attributed to an accused individual.     

Modulation of M-current by intracellular Ca2+

IM is a voltage- and time-dependent K+ current that is suppressed by muscarinic receptor activation. IM augmentation following agonist washout was blocked by heavily buffering [Ca2+]i using BAPTA. Although IM is not primarily Ca2+ dependent, small increases in [Ca2+]i by photolysis of the "caged" Ca2+ chelator nitr-5 or by evoking action potentials augmented, while larger increases inhibited, IM. Raising [Ca2+]i for prolonged periods, by nitr-5 photolysis, reduced its sensitivity to agonist, leaving a poorly reversible response. These results suggest that IM can be regulated by physiologically relevant changes in [Ca2+]i, placing IM in a unique position to modulate cell excitability.     

Effects of varied air velocity on sweating and evaporative rates during exercise.

This study was designed to determine the extent to which changes in the evaporative power of the environment (Emax) affect sweating and evaporative rates. Six male subjects undertook four 60-min bouts of cycle ergometer exercise at 56% maximal O2 uptake (VO2max).Emax was varied by differences in ambient temperature and airflow; two exercise bouts took place at 24 degrees C and two at 35 degrees C, with air velocity at < 0.2 and 3.0 m/s in both. Total sweat production was estimated from body weight loss, whereas whole body evaporative rate was measured continuously from a Potter beam balance. Body core temperature was measured continuously from a thermocouple in the esophagus (T(es)), with mean skin temperature (Tsk) computed each minute from thermocouples at eight sites. Total body sweat loss was significantly greater (P < 0.05) in the 0.2- than in the 3.0-m/s condition at both 24 and 35 degrees C. Tsk was higher (P < 0.05) in the still-air conditions at both temperatures, but final T(es) was significantly higher (P < 0.05) in still air only in the 35 degrees C environment. Thus the reduced Emax in still air caused a greater heat storage, thereby stimulating a greater total sweat loss. However, in part because of reduced skin wettedness, the slope of the sweat rate-to-T(es) relation at 35 degrees C in the 3.0-m/s condition was 118% that at 0.2 m/s (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete moles have paternal chromosomes but maternal mitochondrial DNA

Summary Complete hydatidiform moles contain only paternal chromosomes. To learn more of their origin, we used restriction endonuclease site polymorphisms found in the parental mitochondrial DNAs to demonstrate that moles contain exclusively maternal mitochondrial DNA. Thus, moles must arise from the fusion of one or two sperm with a mature but anucleate ovum.