Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux: AN ELECTRON PARAMAGNETIC RESONANCE STUDY*

Transmembrane stretch M_2C from the bacterial K⁺-translocating protein KtrB is unusually long. In its middle part, termed M_2C2, it contains several small and polar amino acids. This region is flanked by the two α-helices M_2C1 and M_2C3 and may form a flexible gate at the cytoplasmic side of the membrane controlling K⁺ translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M_2C2 were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K⁺ ions, M_2C2 residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M_2B residue Asp-222R1 and M_2C3 residue Val-331R1 but not with respect to M_2C1 residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K⁺ ions, structural models of the open and closed conformations were developed.     

Leveraging Distant Relatedness to Quantify Human Mutation and Gene-Conversion Rates

The rate at which human genomes mutate is a central biological parameter that has many implications for our ability to understand demographic and evolutionary phenomena. We present a method for inferring mutation and gene-conversion rates by using the number of sequence differences observed in identical-by-descent (IBD) segments together with a reconstructed model of recent population-size history. This approach is robust to, and can quantify, the presence of substantial genotyping error, as validated in coalescent simulations. We applied the method to 498 trio-phased sequenced Dutch individuals and inferred a point mutation rate of 1.66 × 10⁻⁸ per base per generation and a rate of 1.26 × 10⁻⁹ for <20 bp indels. By quantifying how estimates varied as a function of allele frequency, we inferred the probability that a site is involved in non-crossover gene conversion as 5.99 × 10⁻⁶. We found that recombination does not have observable mutagenic effects after gene conversion is accounted for and that local gene-conversion rates reflect recombination rates. We detected a strong enrichment of recent deleterious variation among mismatching variants found within IBD regions and observed summary statistics of local sharing of IBD segments to closely match previously proposed metrics of background selection; however, we found no significant effects of selection on our mutation-rate estimates. We detected no evidence of strong variation of mutation rates in a number of genomic annotations obtained from several recent studies. Our analysis suggests that a mutation-rate estimate higher than that reported by recent pedigree-based studies should be adopted in the context of DNA-based demographic reconstruction.     

Establishment of the neotropical predator Amblyseius idaeus (Acari: Phytoseiidae) in Benin,West Africa

Populations of the phytoseiid predator Amblyseius(=Neoseiulus) idaeus (Denmark & Muma) from northeastern Brazil, have been successfully introduced into Benin, West Africa, as part of a classical biological control campaign to control the exotic cassava green mite Mononychellus tanajoa (Bondar). Monthly follow‐up surveys revealed the presence of A. idaeus in most release sites. Some populations have persisted for at least 18 months, including two cycles of potentially limiting wet and dry season conditions. In some sites A. idaeus has been the numerically dominant phytoseiid predator on cassava Manihot esculenta, where it is associated with the tetranychids M. tanajoa and Oligonychus gossypii Zacher. During periods of low M. tanajoa densities A. idaeus disappeared from cassava, but were found on weeds with O. gossypii until prey densities on cassava increased.     

Health-Related Quality of Life,Self-Efficacy and Enjoyment Keep the Socially Vulnerable Physically Active in Community-Based Physical Activity Programs: A Sequential Cohort Study

Physical inactivity is most commonly found in socially vulnerable groups. Dutch policies target these groups through community-based health-enhancing physical activity (CBHEPA) programs. As robust evidence on the effectiveness of this approach is limited, this study investigated whether CBHEPA programs contribute to an increase in and the maintenance of physical activity in socially vulnerable groups. In four successive cohorts, starting at a six-month interval, 268 participants from 19 groups were monitored for twelve months in seven CBHEPA programs. Data collection was based on repeated questionnaires. Socio-economic indicators, program participation and coping ability were measured at baseline. Physical activity, health-related quality of life and on-going program participation were measured three times. Self-efficacy and enjoyment were measured at baseline and at twelve months. Statistical analyses were based on a quasi-RCT design (independent t-tests), a comparison of participants and dropouts (Mann-Whitney test), and multilevel modelling to assess change in individual physical activity, including group level characteristics. Participants of CBHEPA programs are socially vulnerable in terms of low education (48.6%), low income (52.4%), non-Dutch origin (64.6%) and health-related quality of life outcomes. Physical activity levels were not below the Dutch average. No increase in physical activity levels over time was observed. The multilevel models showed significant positive associations between health-related quality of life, self-efficacy and enjoyment, and leisure-time physical activity over time. Short CBHEPA programs (10–13 weeks) with multiple trainers and gender-homogeneous groups were associated with lower physical activity levels over time. At twelve months, dropouts'' leisure-time physical activity levels were significantly lower compared to continuing participants, as were health-related quality of life, self-efficacy and enjoyment outcomes. BMI and care consumption scored significantly higher among dropouts. In conclusion, Dutch CBHEPA programs reach socially vulnerable, but not necessarily inactive, groups in terms of socio-economic and health-related quality of life outcomes. Our findings suggest that CBHEPA programs particularly contribute to physical activity maintenance in socially vulnerable groups, rather than to an increase in physical activity behaviour over time.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Supramaximal cycle tests do not detect seasonal progression in performance in groups of elite speed skaters.

Seven female and eight male elite junior skaters performed cycle ergometer tests at four different times during the 1987/1988 season. The tests consisted of a Wingate-type 30-s sprint test and a 2.5-min supramaximal test. The subjects were tested in February, May and September 1987 and in January 1988. Maximal oxygen consumption was measured during the 2.5-min test. With the exception of the maximal oxygen consumption of the women in May which was about 6% lower than in the other three tests, no seasonal changes in the test results could be observed--this, in spite of a distinct increase in training volume (from 10 to more than 20 h.week-1) and training intensity in the course of the season. When the test data were compared to those of elite senior skaters, it appeared that the junior skaters showed the same values for mean power output during the sprint test [14.2 (SD 0.4) W.kg-1 for the men and 12.6 (SD 0.5) W.kg-1 for the women] and maximal oxygen consumption [63.1 (SD 2.8) ml.kg-1.min-1 for the men and 55.3 (SD 3.5) ml.kg-1.min-1 for the women, respectively] as found for senior skaters. It seemed, therefore, that the effects of training in these skaters had already levelled off in the period before they participated in this investigation. In contrast to previous studies, no relationship could be shown between the test results and skating performance. This was most likely due to the homogenous character of the groups (mean standard deviations in power and oxygen consumption were only 5%).(ABSTRACT TRUNCATED AT 250 WORDS)