Development and Validation of a New Method to Measure Walking Speed in Free-Living Environments Using the Actibelt? Platform

Walking speed is a fundamental indicator for human well-being. In a clinical setting, walking speed is typically measured by means of walking tests using different protocols. However, walking speed obtained in this way is unlikely to be representative of the conditions in a free-living environment. Recently, mobile accelerometry has opened up the possibility to extract walking speed from long-time observations in free-living individuals, but the validity of these measurements needs to be determined. In this investigation, we have developed algorithms for walking speed prediction based on 3D accelerometry data (actibelt®) and created a framework using a standardized data set with gold standard annotations to facilitate the validation and comparison of these algorithms. For this purpose 17 healthy subjects operated a newly developed mobile gold standard while walking/running on an indoor track. Subsequently, the validity of 12 candidate algorithms for walking speed prediction ranging from well-known simple approaches like combining step length with frequency to more sophisticated algorithms such as linear and non-linear models was assessed using statistical measures. As a result, a novel algorithm employing support vector regression was found to perform best with a concordance correlation coefficient of 0.93 (95%CI 0.92–0.94) and a coverage probability CP1 of 0.46 (95%CI 0.12–0.70) for a deviation of 0.1 m/s (CP2 0.78, CP3 0.94) when compared to the mobile gold standard while walking indoors. A smaller outdoor experiment confirmed those results with even better coverage probability. We conclude that walking speed thus obtained has the potential to help establish walking speed in free-living environments as a patient-oriented outcome measure.     

The discrimination between Rb+ and K+ by Escherichia coli is changed after bacteriophage T7 infection

Rb+ and K+ have similar chemical properties. They share the uptake systems in Escherichia coli and can replace each other inside the cell. These common features led to experiments in which the radioactive isotope 86Rb was used to trace intracellular K+ fluxes. However, the E. coli pumps discriminate between these two ions and one should thus be cautious using 86Rb+ as a tracer for K+. We now report that T7 infection alters the degree of discrimination in such a way that changes of intracellular Rb+ do not reflect changes of K+. It has been observed that shortly after infection the 86Rb+ level was strongly reduced (Ponta, H., Altendorf, K.-H. and Schweiger, M. (1976) Mol. Gen. Genet. 149, 145-150). In contrast, determination of the K+ content showed no change directly after infection (Kuhn, A., Jütte, H. and Kellenberger, E. (1983) J. Virol. 47, 540-552). The efflux of 86Rb was only evident when Rb+ was used in trace amounts. In media conditions under which intracellular K+ was mainly replaced by Rb+, 86Rb+ efflux was not observed.     

The Enucleation of Cells on Plastic Leighton Coverslips

A simple enucleation technique that facilitates autoradiographic and electron microscopic examination of cytoplasms is described. Cells were grown on commercially available plastic Leighton coverslips and these were centrifuged in the presence of cytochalasin B. The centrifugation requires no special holders and only a high speed centrifuge. Enucleation frequencies of greater than 90% were obtained for Chinese hamster fibroblasts and mouse B-82 cells.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

Subcellular localization of acetyl-CoA synthetase in leaf protoplasts of Spinacia oleracea

The localization of acetyl-CoA synthetase in the spinach leaf cell was examined. When the different compartments of lysed spinach protoplasts were assayed for marker enzymes and acetyl-CoA synthetase, it was determined that the synthetase was totally localized in the chloroplast compartment. Analysis of spinach leaf for free acetate revealed that this acid was present at a 1 mm level in the leaf cell. It is suggested that free acetate probably derived from a number of sources in the cell diffuses into the chloroplast stroma compartment where it is converted to acetyl-CoA and thence employed for biosynthetic reactions. Thus, free acetate is metabolically inert in the leaf cell until it is transported to the only compartment that contains acetyl-CoA synthetase, namely the chloroplast.     

MECHANICAL PROPERTIES WITHIN THE GROWTH ZONE OF CORN ROOTS INVESTIGATED BY BENDING EXPERIMENTS II. DISTRIBUTIONS OF MODULUS AND COMPLIANCE IN BENDING

A bending technique was used to infer the spatial distributions of rheological properties within the growth zone of the root of corn, Zea mays. “Bending modulus” (ratio of stress to strain, calculated from engineering theory of bending) falls from 20 MPa near the root tip (3 mm from the tip) to 6 MPa at the location 6 mm from the tip and then remains uniform through the basal region of the growth zone. Where growth stops, at 11–12 mm, there is a sharp rise in bending modulus. The profile of bending moduli is not changed by root incubation temperature during the growth period prior to bending, but it is shifted to the left in roots growing more slowly than the average at either of two temperatures (19 and 29 C). The spatial distribution of “compliance” (reciprocal of bending modulus and a measure of tissue extensibility) resembles the distribution of swelling in response to osmotic perturbation. The distribution of compliance does not parallel that of growth rate. Attempts to explain the discrepancy between compliance and growth rate lead us to examine the theoretical basis for the calculations and to suggest that the dependence of compliance on rate of stretching is physiologically important.