Physical and social environmental changes to promote walking among Dutch older adults in deprived neighbourhoods: the NEW.ROADS study.

BACKGROUND: Physical activity is important for healthy ageing, and daily walking is seen as a feasible way to be active at older ages. Yet, many older persons, particularly in lower socioeconomic groups and residing in deprived neighbourhoods, are insufficiently active. Creating a physical and social neighbourhood environment that is more supportive for walking has the potential to improve walking behaviour. Current evidence of the impact of changes to the physical and/or social environmental on walking behaviour is scarce. The aim of the NEW.ROADS study is to design, implement and evaluate changes to the physical and social environment for the purpose of increasing walking behaviour among older residents of deprived neighbourhoods. METHODS: Physical and social environmental interventions were developed by matching scientific evidence on environmental determinants of walking, with input from the target population and stakeholders, and ongoing neighbourhood activities. Specifically, a neighbourhood walking route was designed and marked, and neighbourhood walking groups were organised. These environmental interventions were evaluated in a four-armed experimental study. In addition, the design of the study to evaluate the effect of these environmental changes on walking behaviour is described. DISCUSSION: Designing and implementing environmental interventions is a complex endeavour, challenged by limited available theory and evidence. Input from the target population and professional stakeholders is essential, but may also put constraints on the evaluation. TRIAL REGISTRATION: NTR3800 (registered 9/1/2013).

A hand hygiene intervention to reduce infections in child daycare: a randomized controlled trial.

Infections are common in children attending daycare centres (DCCs). We evaluated the effect of a hand hygiene (HH) intervention for caregivers on the incidence of gastrointestinal and respiratory infections in children. The intervention was evaluated in a two-arm cluster randomized controlled trial. Thirty-six DCCs received the intervention including HH products, training sessions, and posters/stickers. Thirty-five control DCCs continued usual practice. Incidence of episodes of diarrhoea and the common cold in children was monitored by parents during 6 months. Using multilevel Poisson regression, incidence rate ratios (IRRs) with 95% confidence intervals (CIs) were obtained. Diarrhoeal incidence was monitored in 545 children for 91 937 days. During follow-up, the incidence was 3·0 episodes per child-year in intervention DCCs vs. 3·4 in control DCCs (IRR 0·90, 95% CI 0·73-1·11). Incidence of the common cold was monitored in 541 children for 91 373 days. During follow-up, the incidence was 8·2 episodes per child-year in intervention DCCs vs. 7·4 in control DCCs (IRR 1·07, 95% CI 0·97-1·19). In this study, no evidence for an effect of the intervention was demonstrated on the incidence of episodes of diarrhoea and the common cold.

The effect of mutations in EF-Tu on its affinity for tRNA as measured by two novel and independent methods of general applicability.

Elongation factor Tu is essential for binding and a correct delivery of aminoacyl-tRNA during protein biosynthesis. For a good characterization of its interaction with tRNA in terms of structure-function relationship, determinations of kinetic equilibrium parameters are of great value. We describe two novel methods for that purpose. One method is based on EF-Tu protection of the tRNA 3' acceptor end against RNase A cleavage and yields the Kd value together with the corresponding dissociation and association rate constants from one single set of experiments. The other is a rapid method for screening relative affinities of mutant EF-Tus for tRNA. It is based on competition between EF-Tu species with and without a (His)6 extension for the same aminoacyl-tRNA and yields a relative Kd value. The method can be of general importance for the measuring of ligand affinities of all sorts of His-tagged proteins. Both methods are illustrated by their application in the analysis of mutant EF-Tus with changed interactions with tRNA and antibiotics. Raising the assay temperature from 4 to 37 degrees C causes a 30-fold increase of Kd for EF-Tu x GTP x Phe-tRNA complexes. The mutation K237E leads to rapid inactivation at the latter temperature. A parallel is found between the order of increasing Kd values for EF-Tus with mutation G316D, A375T and Q124K, respectively, and their order of increasing resistance to kirromycin.

Synergism between the GTPase activities of EF-Tu.GTP and EF-G.GTP on empty ribosomes. Elongation factors as stimulators of the ribosomal oscillation between two conformations.

A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperativity implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 microM to 0.5 microM for EF-Tu.GTP by the addition of 0.25 microM EF-G and from 0.7 microM to 0.5 microM for EF-G.GTP by the addition of 3 microM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu.GTP and EF-G.GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.

Elongation factors in protein synthesis.

Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

Fluoroaluminates do not affect the guanine-nucleotide binding centre of the peptide chain elongation factor EF-Tu.

EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the 'consensus' amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the gamma-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed AlF4- behaves as a gamma-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences.

The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0.

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.

Molecular properties of two mutant species of the elongation factor Tu.

The molecular properties of two mutant species of the elongation factor Tu (EF-Tu), derived from either tuf A or tuf B, have been studied. One, designated EF-TuAR, is the product of a kirromycin-resistant tufA gene. The other designated EF-TuBO is a tuf B product and is present in a kirromycin-resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF-TuAR species. EF-TuAR has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tuf B gene has been inactivated by an insertion of the bacteriophage Mu. EF-TuBO has been isolated from LBE 2012 together with EF-TuAR in a 1:1 mixture. Fractionation of this mixture of DEAE-Sephadex A-50 resulted in an enrichment of EF-TuBO of about 80%. The properties of EF-TuAR and EF-TuBO have been compared to those of a kirromycin-sensitive species designated EF-TuAS, which was isolated from LBE 2045 by transduction of wild-type tuf A. We show here that all three EF-Tu species are fully competent to sustain polypeptide synthesis. All also appear to interact normally with guanine nucleotides and EF-Ts. Only in the presence of the antibiotic do the following differences appear. (a) Kirromycin causes EF-TuAS (wild-type tuf A gene product) to be retained on, and thus block, the ribosome. (b) EF-TuAR fails to bind the antibiotic and thus is capable of protein synthesis in its presence. (c) EF-TuBO fails to sustain polypeptide synthesis upon binding of kirromycin. It does not, however, block the ribosome, so the strain harbouring both this protein and EF-TuAR (LBE 2012) is kirromycin resistant.

A mutant elongation factor Tu which does not immobilize the ribosome upon binding of kirromycin.

In the accompanying paper we have shown that polypeptide synthesis sustained by the mutant elongation factor EF-TuBO is inhibited by kirromycin. Here we have searched for the primary site of inhibition in the elongation cycle. It is demonstrated that in the presence of the antibiotic EF-TuBO can form a complex with aminoacyl-tRNA and GTP and that the complex is able to bind to ribosomes programmed with poly(U). Like its wild-type counterpart, EF-TuBO . GDP can form a quaternary complex with aminoacyl-tRNA and kirromycin but, unlike the wild-type quaternary complex, the mutant complex fails to associate with the ribosome. This explains the recessive nature of the tuf B mutation in cells producing kirromycin-resistant EF-TuA and EF-TuBO. It also suggests a mechanism for the inhibition by kirromycin of EF-TuBO-dependent polypeptide synthesis.

The primary structure of the coat protein of alfalfa mosaic virus strain VRU. A hypothesis on the occurrence of two conformations in the assembly of the protein shell.

The complete primary structure of the coat protein of strain VRU of alfalfa mosaic virus (AMV) is reported. The strain is morphologically different from all other AMV strains as it contains large amounts of unusually long virus particles. This is caused by structural differences in the coat protein chain. The amino acid sequence has mainly been established by the characterization of peptides obtained after cleavage with cyanogen bromide and digestion with trypsin, chymotrypsin, thermolysin or Staphylococcus aureus protease. The major sequencing technique used was the dansyl-Edman procedure. The VRU coat protein consists of 219 amino acid residues corresponding to a molecular weight of 24056. Compared to the coat protein of strain 425 [Van Beynum et al. (1977) Eur. J. Biochem. 72, 63-78], 15 amino acid substitutions were localized. Most of them have a conservative character and may be explained by single-point mutations. A correction is given for the AMV 425 coat protein: Asn-216 was shown to be Asp-216. The prediction of the secondary structure for the two viral coat proteins was not significantly influenced by the various amino acid substitutions except for the region containing residues 65-100. This led us to the hypothesis that the AMV coat protein may occur in two different conformations favouring its incorporation into either a pentagonal or hexagonal quasi-equivalent position in the lattice of the protein shell. The substitutions in the above-mentioned region of the VRU coat protein may have caused a strong preference for the hexagonal lattice conformation. The model is supported by preliminary sequence data of the same coat protein region in AMV 15/64, a strain morphologically intermediate between 425 and VRU.

Structural studies on the coat protein of alfalfa mosaic virus. The complete primary structure.

The complete amino acid sequence of the coat protein of alfalfa mosaic virus (strain 425) is reported. Sequence determinations were mainly performed on peptides obtained from fragmentation by cyanogen bromide and trypsin. Both manual and automatic sequence methods were used. Some refinements of the solid-phase Edman degradation were introduced. The final alignment of the peptides was established by means of alternative cleavage methods, such as limited tryptic digestion of intact virus particles, tryptic digestion after blockage of lysine residues and chymotryptic digestion. The coat protein consists of 220 amino acid residues corresponding to a molecular weight of 24252. A remarkable clustering of basic residues occurs in the N-terminal part of the protein chain. Several internal hydrophobic clusters and a strongly acidic site at the C-terminus can be observed. Two regions of sequence homology (12 residues) were found. Some features of the secondary structure are predicted.

Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.