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.     

Date Palm DNA Mini-Preparation without Liquid Nitrogen

We have developed a new mini-procedure for isolation of total cellular DNA from date palm (Phoenix dactylifera L.). The procedure, which does not use liquid nitrogen, has proved useful due to temporary disruptions in supplies of liquid nitrogen that occur in countries where date palm trees are cultivated. DNA suitable for RFLP and PCR analyses is obtained.     

Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents.

Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.     

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.     

Conditionality of phase resetting by inhibitors of 80 S translation inGonyaulax polyedra

Summary Gonyaulax polyedra was subjected to a cold treatment of 18 h at 10 °C leading to arrhythmicity. Subsequently, the circadian rhythm of bioluminescence was investigated at the permissive temperature of 20 °C. 1-h pulses of 10 M cycloheximide or 2 M anisomycin, when given after the temperature step-up, resulted only in a very weak resetting of the circadian oscillator, in marked contrast to the behaviour of cells kept continuously in oscillatory conditions at 20 °C. The extremely reduced sensitivity to 80 S inhibition was characteristic for the first cycle after the temperature step-up, whereas cells treated with cycloheximide in the second cycle after re-initiation of rhythmicity showed a gradual recovery of resettability, though the phase response curve was still atypical; treatment in the 3rd cycle after step-up led to a relatively normal phase response curve. The observed insensitivity in the 1st cycle was neither a consequence of insufficient drug action, nor of a transient non-oscillatory behaviour after temperature step-up. Already in the first hours after transfer to 20 °C, 80 S translation was strongly suppressed by cycloheximide, and the cells were also efficiently reset by changes of the light-dark zeitgeber. Resettability of the circadian oscillator by 80 S inhibitors is, therefore, conditional.Abbreviations Ani anisomycin - Chx cycloheximide - CT circadian time - LD light-dark cycle - LL constant light - TCA trichloroacetic acid This work was supported by the Deutsche Forschungsgemeinschaft     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry     

Ribosomal DNA as a convenient probe to follow segregation and possible divergency from expected homozygosity after haploidization of an androgenetic process

Summary Restriction fragment length polymorphism of the wheat nuclear ribosomal DNA has been studied in several steps of a breeding scheme, including parental genotypes, F1 hybrid, F9 generation, and anther-derived doubled haploid lines obtained from F9. Ribosomal DNA represents a suitable molecular marker in following segregation and possible divergency from expected homozygosity after haploidization of an androgenetic process. It has been shown to undergo variations among the first cycle-doubled haploid lines in the relative amount of two different sizes of ribosomal DNA repeat units. The specificity and peculiar properties of the plant system used allowed us to assign an intrachromosomal location (short arm of the chromosomes 1B, 1R or 6B) to several ribosomal DNA repeat units that differ by the length of their nontranscribed spacer region.     

Scorpion toxins from Centruroides noxius and Tityus serrulatus. Primary structures and sequence comparison by metric analysis.

The complete primary structures of toxin II-14 from the Mexican scorpion Centruroides noxius Hoffmann and toxin gamma from the Brazilian scorpion Tityus serrulatus Lutz and Mello have been determined. Cleavage of toxin gamma after Met-6 with CNBr produced the 55-residue peptide 7-61, which maintained the four disulphide bonds but was not toxic to mice at a dose 3 times the lethal dose of native toxin gamma. Pairwise comparison by metric analysis of segment 1-50 of toxin gamma and the corresponding segments from two other South American scorpion toxins, five North American scorpion toxins, nine North African scorpion toxins and one Central Asian scorpion toxin showed that the three Brazilian toxins are intermediate between the North American and North African toxins. This result is consistent with the hypothesis that the South American and African continents were joined by a land connection in the distant past.