Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.     

Maximal strength testing in healthy children

Strength training has become an accepted method of conditioning in children. However, there is concern among some observers that maximal strength testing may be inappropriate or potentially injurious to children. The purpose of this study was to evaluate the safety and efficacy of 1 repetition maximum (1RM) strength testing in healthy children. Thirty-two girls and 64 boys between 6.2 and 12.3 years of age (mean age 9.3 +/- 1.6 years) volunteered to participate in this study. All subjects were screened for medical conditions that could worsen during maximal strength testing. Under close supervision by qualified professionals, each subject performed a 1RM test on 1 upper-body (standing chest press or seated chest press) and 1 lower-body (leg press or leg extension) exercise using child-size weight training machines. No injuries occurred during the study period, and the testing protocol was well tolerated by the subjects. No gender differences were found for any upper- or lower-body strength test. These findings demonstrate that healthy children can safely perform 1RM strength tests, provided that appropriate procedures are followed.     

Controls on methane production in a tidal freshwater estuary and a peatland: methane production via acetate fermentation and CO2 reduction

Rates of total methane production, acetate fermentation andCO₂ reduction were compared for two different wetland sites. On aper-liter basis, sediments from the White Oak River estuary, a tidal freshwatersite in eastern North Carolina, had an annual methane production rate (53.3mMyr^–1) an order of magnitude higher thanthat ofBuck Hollow Bog (5.5 mMyr^–1), a peatlandinMichigan. Methane was produced in the White Oak River site on an annual basisbyboth acetate fermentation (72%) and CO₂ reduction (28%) in a ratiotypical of freshwater methanogenic sites. Competition for acetate bynon-methanogenic microorganisms in Buck Hollow peat limited methane productionfrom acetate to only a few months a year, severely impacting annual methaneproduction rates. However, when acetate was available to the methanogens in thepeat during early spring, the percentage of methane production from acetatefermentation (84%) and CO₂ reduction (16%) and rates of totalmethaneproduction were similar to those of the White Oak River sediments at the sametemperature. Rates of CO₂ reduction and acetate fermentationconducted at both sites at various temperatures showed that Buck Hollow peatmethane production was also limited by a colder temperature regime as well asdifferences in the response of the CO₂ reducing and aceticlasticmethanogens to temperature variations.     

Differential regulation of NFAT and SRF by the B cell receptor via a PLCgamma-Ca(2+)-dependent pathway

NFAT and SRF are important in the regulation of proliferation and cytokine production in lymphocytes. NFAT activation by the B cell receptor (BCR) occurs via the PLCgamma-Ca(2+)-calcineurin pathway, however how the BCR activates SRF is unclear. We show here that like NFAT, BCR regulation of SRF occurs via an Src-Syk-Tec-PLCgamma-Ca(2+) (Lyn-Syk-Btk-PLCgamma-Ca(2+)) pathway. However, SRF responds to lower Ca(2+) and is less dependent on IP(3)R expression than NFAT. Ca(2+)-regulated calcineurin plays a partial role in SRF activation, in combination with diacylglycerol (DAG), while is fully required for NFAT activation. Signals from the DAG effectors protein kinase C, Ras and Rap1, and the downstream MEK-ERK pathway are required for both SRF and NFAT; however, NFAT but not SRF is dependent on JNK signals. Both SRF and NFAT were also dependent on Rac, Rho, CDC42 and actin. Finally, we show that Ca(2+) is not required for ERK activation, but instead for its association with nuclear areas of the cell. These data suggest that combinatorial assembly of signaling pathways emanating from the BCR differentially regulate NFAT and SRF, to activate gene expression.     

Cell cycle- and age-dependent activation of Sod1p drives the formation of stress resistant cell subpopulations within clonal yeast cultures

Phenotypic heterogeneity describes non-genetic variation that exists between individual cells within isogenic populations. The basis for such heterogeneity is not well understood, but it is evident in a wide range of cellular functions and phenotypes and may be fundamental to the fitness of microorganisms. Here we use a suite of novel assays applied to yeast, to provide an explanation for the classic example of heterogeneous resistance to stress (copper). Cell cycle stage and replicative cell age, but not mitochondrial content, were found to be principal parameters underpinning differential Cu resistance: cell cycle-synchronized cells had relatively uniform Cu resistances, and replicative cell-age profiles differed markedly in sorted Cu-resistant and Cu-sensitive subpopulations. From a range of potential Cu-sensitive mutants, cup1Delta cells lacking Cu-metallothionein, and particularly sod1Delta cells lacking Cu, Zn-superoxide dismutase, exhibited diminished heterogeneity. Furthermore, age-dependent Cu resistance was largely abolished in cup1Delta and sod1Delta cells, whereas cell cycle-dependent Cu resistance was suppressed in sod1Delta cells. Sod1p activity oscillated approximately fivefold during the cell cycle, with peak activity coinciding with peak Cu-resistance. Thus, phenotypic heterogeneity in copper resistance is not stochastic but is driven by the progression of individual cells through the cell cycle and ageing, and is primarily dependent on only Sod1p, out of several gene products that can influence the averaged phenotype. We propose that such heterogeneity provides an important insurance mechanism for organisms; creating subpopulations that are pre-equipped for varied activities as needs may arise (e.g. when faced with stress), but without the permanent metabolic costs of constitutive expression.     

Characterization of the selectivity filter of the epithelial sodium channel

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed alpha, beta, and gamma. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the alpha-subunit to cysteine and co-expressing these mutants with wild type beta- and gamma-subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (alpha-Val(569)-alpha-Gly(579)) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (alpha-Ser(580)-alpha-Ser(592)) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing alphaG587C or alphaS589C were permeable to K(+), and mutation of a GSS tract (positions alpha587-alpha589) to GYG resulted in a moderately K(+)-selective channel. Our results suggest that the C-terminal portion of the pore region within the alpha-subunit contributes to the selectivity filter of ENaC.     

Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10(5) to 10(6)/ml) at 22 degrees C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.     

Chromosome aberrations in in vitro-produced bovine embryos at days 2-5 post-insemination

Availability of embryos of high quality is required to obtain satisfactory embryonic developmental rates and normal calves following transfer of in vitro-produced (IVP) bovine embryos. One relevant quality parameter is the frequency of chromosome aberrations, which can be evaluated using multicolor fluorescent in situ hybridization (FISH) with chromosome 6- and chromosome 7-specific probes in cattle. In this study, interphase nuclei (n = 3805) were analyzed from 426 bovine IVP embryos. We found that 73%, 72%, 81%, and 58% of the embryos from Days 2, 3, 4, and 5 post-insemination (pi), respectively, displayed a normal diploid chromosome number in all cells. When looking at the types of chromosome aberrations, the percentages of mixoploidy at Days 2, 3, 4, and 5 pi were 22%, 15%, 16%, and 42%, respectively, whereas the percentages of polyploidy (i.e., all nuclei in an embryo were analyzed and were polyploid) were 5%, 13%, 3%, and 0%, respectively. In conclusion, numerical chromosome aberrations were detected as early as Day 2 pi. The development of polyploid embryos is slow and is apparently arrested during the third cell cycle, whereas the mixoploid embryos seem to continue development.     

Effects of temperature gradients on in vitro maturation of bovine oocytes

The effect of temperature gradients on in vitro maturation of bovine oocytes was examined in this study. Six treatment groups were made by combining 3 different maturation periods (0 to 10 h, 10 to 18 h and 18 to 24 h) with 2 different culture temperatures (37.0 degrees C and 38.5 degrees C). The frequency of oocytes matured to the metaphase II stage was apparently gradually increased as the culture temperature was increased from 37.0 degrees C to 38.5 degrees C at 0, 10 and 18 h after the onset of culture (75.2 vs 80.5, 82.3 and 84.3%, respectively), but this difference was not significant. Neither was the minor decrease in the proportion of oocytes reaching metaphase II when the temperature was decreased from 38.5 degrees C to 37.0 degrees C at 10 and at 18 h after the onset of maturation (84.3 vs 82.4 and 78.0%, respectively). However, more oocytes cleaved (79.2%; P = 0.0653) and developed to morulae (43.6%; P = 0.0019) and blastocysts (27.4%; P = 0.1568) when they were in vitro matured at 38.5 degrees C between 0 and 10 h, and then at 37.0 degrees C from 10 to 24 h. Although only the morula group was statistically different, cleavage- (79.2 vs 69.8, 72.5, 74.2, 76.3, 74.3%, respectively) and blastocyst formation (27.4 vs 23.2, 24.6, 25.2, 19.6, 21.9%, respectively) from this group was the highest among the 6 treatments.