The reaction mechanism of Na, K-ATPase

To help characterize the Na,K-ATPase active site with enzyme incorporated into phospholipid vesicles, the activities with alternative substrates were compared, 22Na/Na-transport was equivalent with ATP, CTP, carbamylphosphate and acetylphosphate, but slower with CTP, 3-O-methylfluoresceinphosphate (3-O-MFP), nitrophenylphosphate and umbelliferonephosphate. It indicates a slower rate of formation of phosphorylating enzyme complex in conformation position of E1 (E1P) when the second group of substrates is bound with enzyme active center. 22Na/K-transport was half as effective with CTP as with ATP and was far slower with the other substrates. It indicates a more stringent selectivity at the low-affinity site of enzyme in conformation E2 that accelerates the slow step of this transport mode. Although enzyme modification with fluoresceinisothiocyanate blocks the high-affinity site to ATP, the K-phosphatase reaction catalyzed by E2 is retained, even with a substrate, 3-O-MFP, that binds to the adenine pocket. Dimethylsulfoxide inhibits hydrolysis of the nucleotides and of the carboxylic phosphate substrates of the K-phosphatase reaction, but stimulates hydrolysis of the phenolic phosphate substrates (nitrophenylphosphate and umbelliferone phosphate) which normally are hydrolyzed more slowly than the other substrates. On the basis of these data the authors propose the model of Na,K-ATPase active center.     

Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor.

The P1 plasmid partition system is responsible for segregation of daughter plasmids during division of the Escherichia coli host cell. The P1-encoded elements consist of two essential proteins, ParA and ParB, and the cis-acting incB region. The incB region determines partition-mediated incompatibility and contains the centromere-like site parS. We have isolated and purified the two proteins. ParB binds specifically to the incB region in vitro. DNase I footprinting assays place a strong binding site over the 35-bp parS sequence previously shown to be sufficient for partition when the Par proteins are supplied in trans. A weaker site lies within the incB region in sequences that are important for specifying incompatibility, but are not essential for partition. Gel band retardation assays show that a host factor binds specifically to the incB sequence. The factor strongly stimulates binding of ParB. Cutting the region at a site between the two ParB binding sites yields two fragments that can bind ParB but not host factor. Thus, information for host-factor binding lies in the region determining the specificity of plasmid incompatibility. The roles of parB and the host factor in partition and the specificity of plasmid incompatibility are discussed.     

Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae.

Two dimensional gel electrophoretic techniques were used to locate all functional DNA replication origins in a 22.5 kb stretch of yeast chromosome III. Only one origin was detected, and that origin is located within several hundred bp of an ARS element.     

Modified inoculum for radiometric susceptibility testing ofMycobacterium tuberculosis

Two-hundred and fifteen isolates ofMycobacterium tuberculosis were evaluated with the BACTEC 460 radiometric method for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin (SM); a revised protocol for inoculum preparation was used. Fresh clinical isolates were subcultured into 7H9 broth and then photometrically adjusted to the equivalent of a 0.5 McFarland standard, one-half the recommended inoculum density. This method produced an overall 98.3% correlation with a conventional agar method. The sensitivity of this procedure was good for all drugs tested except for the lowest concentration of SM (2 g/ml). Specificity was excellent for all drugs tested. After repeat testing, only four discrepancies were found, yielding a 99.8% correlation between the two systems. The time required for susceptibility tests averaged 4.6 days. This method for inoculum preparation effectively minimized the number of susceptibility tests exceeding the threshold value before the fourth day of incubation. This allowed for definite trends of the growth index values to become established before interpretation of results.     

General practitioners' use of written materials during consultations

Russell et al have shown that the compliance of patients can be enhanced by general practitioners'' providing them with written materials.'' We examined what educational materials for patients were used by general practitioners and where the materials came from.     

Differential survival of chaparral seedlings during the first summer drought after wildfire

Summary Big Pod Ceanothus (Ceanothus megacarpus) is an obligate seeder after fire; Laurel Sumac (Rhus laurina) is primarily a resprouter after fire. Both species commonly occur together in mixed stands and are dominant members of the coastal chaparral of southern California. We compared the mean survival of post-fire seedlings of each species during the first summer drought after fire and found C. megacarpus to have a mean survival of 54% while R. laurina had a mean survival of only 0.1%. Rooting dephs were similar between species but predawn water potentials and leaf temperatures were higher for R. laurina seedlings. Leaf temperatures for R. laurina reached a mean value of 46.8° C on hot, summer days, about 5° C higher than seedlings of C. megacarpus. By the end of the first growing season, 92% of all C. megacarpus seedlings had suffered herbivory compared to only 17% of all R. laurina seedlings. Herbivory did not appear to be the immediate cause of seedling mortality. Transect data indicated that full recovery of prefire species composition and density at our study site was likely but the mode of recovery was different for the species examined. R. laurina recovered primarily by sprouting, C. megacarpus totally by seedling establishment and a third species, Adenostoma fasciculatum (chamise), by a combination of sprouting and seedling establishment. We attribute the higher mortality of R. laurina seedlings to the greater sensitivity of its tissue to water stress. It may be that differential survival of shrub seedlings and differential modes of reestablishment after fire play an important role in maintaining species diversity in the chaparral communities of coastal, southern California.