Fresh street: the development and feasibility of a place-based, subsidy for fresh fruit and vegetables.

BACKGROUND: Many UK communities experience food insecurity, and consume diets high in energy-dense, nutrient poor, processed foods and low in fruit and vegetables (FV). We explored a novel area-based approach to promote FV consumption and healthy eating in one such community. METHODS: We developed a weekly subsidy scheme for fresh FV with key local stakeholders in an area of socioeconomic deprivation in Northern England. The scheme (Fresh Street) offered five £1 vouchers to every household, regardless of income or household type. Vouchers were redeemable with local suppliers of fresh FV (not supermarkets). The feasibility of the scheme was assessed in four streets using rapid ethnographic assessment and voucher redemption information. RESULTS: Local councillors and public health teams were supportive of the scheme. Most eligible households joined the scheme (n = 80/97, 83%), and 89.3% (17 849/19 982) of vouchers issued were redeemed. Householders reported that the scheme made them think about what they were eating, and prompted them to buy and eat more FV. CONCLUSIONS: This feasibility study reported high levels of acceptance for a place-based, household-level weekly FV subsidy scheme. Further research is required to evaluate the effectiveness of this approach to creating healthy diets, eating behaviours and food systems.

Three-Dimensional Dosimetry of a Beta-Emitting Radionuclide Using PRESAGE Dosimeters.

Three-dimensional dose distributions from liquid brachytherapy were measured using PRESAGE(®) dosimeters. The dosimeters were exposed to Y-90 for 5.75 days and read by optical tomography. The distributions are consistent with estimates from beta dose kernels.

The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

In an effort to prepare Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia for future spectroscopic and mechanistic studies, two methods for the preparation of Co(II)-L1 were tested. Method A involved adding CoCl2 directly to apo-L1 under anaerobic conditions. The resulting enzyme contained 1.9 moles of cobalt and exhibited very little activity, and UV-Vis, 1H NMR, and EPR studies indicated that most of the cobalt in this sample was Co(III). Method B involved reducing the single and unique disulfide bridge in L1 with tris(carboxyethyl)phosphine prior to adding CoCl2. The resulting enzyme was pink, contained 2.5 moles of cobalt per mole of enzyme, and exhibited kcat and Km values of 18+1 s(-1) and 10+/-1 microM, respectively, when using nitrocefin as the substrate. UV-Vis, 1H NMR, and EPR studies revealed that this enzyme sample contained high-spin Co(II). The UV-Vis spectra also provided evidence for Co(II) bound to one or both of the reduced cysteines. Efforts to block this non-specific Co(II) binding site using a chemical modification agent or site-directed mutagenesis were unsuccessful. The data presented here demonstrate the problem of solvent-exposed disulfides when preparing Co(II)-substituted enzymes and offers a convenient method to circumvent the problem.

Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis.

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.

Analysis of three overexpression systems for VanX, the Zinc(II) dipeptidase required for high-level vancomycin resistance in bacteria.

The gene from Enterococcus faecilis encoding the dipeptidase VanX was subcloned into overexpression vectors pET-5b, pET-27b, and IMPACT-T7, and VanX was overexpressed in BL21(DE3) pLysS Escherichia coli. The pET-5b-vanx overexpression plasmid produces VanX at approximately 12 mg/L under optimum conditions. VanX produced from this overexpression system exists primarily as a dimer in solution, binds ca. 1 Zn(II) ion per monomer, and exhibits K(m) and k(cat) values of 500 +/- 40 microM and 0.074 +/- 0.001 s(-1), respectively, when l-alanine-p-nitroanilide is used as substrate. The IMPACT-T7-vanx overexpression plasmid produces a VanX-fusion protein with a chitin-binding domain that allows for purification of the fusion construct with a chitin column. Cleavage of the fusion protein is completed by an on-column chemical cleavage, resulting in approximately 10 mg/L of purified VanX. VanX produced from this system is identical to that produced from the pET-5b system, except the CD spectrum of the IMPACT-T7-produced VanX suggests a small change in secondary structure. This change in secondary structure does not affect any of the kinetic or metal-binding properties of the enzyme. The pET-27b-vanx overexpression plasmid produces and secretes VanX into the growth medium; this system allows for 20 mg of VanX to be isolated per liter of growth medium. The pET-27b-produced VanX is identical to that produced from pET-5b.

Mutational analysis of metallo-beta-lactamase CcrA from Bacteroides fragilis.

In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-beta-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyses, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat), suggesting that Asn193 does not play a large role in catalysis. However, stopped-flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.

Phosphonamidate and phosphothioate dipeptides as potential inhibitors of VanX.

In an effort to prepare novel inhibitors of VanX, N-[(1-aminoethyl)hydroxyphosphinyl]-D-alanine 1 and S-[(aminoethyl)hydroxyphosphinyl]-thiolacetic acid 2 were synthesized and evaluated as inhibitors of VanX. Phosphonamidate 1 was shown to be a partial competitive inhibitor of VanX with a Ki of 36+/-3 microM, and phosphothioate 2 was shown not to inhibit VanX.

Control and statistical analysis of in vitro reporter gene assays.

The use of in vitro gene reporter assays is becoming increasingly widespread in biology and particularly in drug metabolism, where the need for rapid screening of novel compounds is a driving factor. There is, however, little standardization of technique in the control of such assays, nor in the interpretation of results. This leads to confusion in the literature, with the possibility of a single piece of data being interpreted by several different methods, potentially giving vastly differing results. We have developed a reporter gene assay methodology that controls for many biological and experimental variables in the system and allows the application of a mathematical model to determine statistical significance between groups. Use of this methodology, we feel, allows an accurate and reproducible method of analyzing in vitro reporter gene assay data and increases its value as a biological tool.

Bayesian methods for a growth-curve degradation model with repeated measures.

The increasing reliability of some manufactured products has led to fewer observed failures in reliability testing. Thus, useful inference on the distribution of failure times is often not possible using traditional survival analysis methods. Partly as a result of this difficulty, there has been increasing interest in inference from degradation measurements made on products prior to failure. In the degradation literature inference is commonly based on large-sample theory and, if the degradation path model is nonlinear, their implementation can be complicated by the need for approximations. In this paper we review existing methods and then describe a fully Bayesian approach which allows approximation-free inference. We focus on predicting the failure time distribution of both future units and those that are currently under test. The methods are illustrated using fatigue crack growth data.

Inhibition studies on the metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

In an effort to identify a competitive inhibitor that can be used in future spectroscopic and crystallographic studies and to better understand the interaction of a mercaptoacetic acid-thiolester-containing compound with metallo-beta-lactamase L1 from Stenotrophomonas maltophilia, inhibition studies using two thiol-containing compounds were conducted. N-(2'-Mercaptoethyl)-2-phenylacetamide is a competitive inhibitor of L1 with a K(i) of 50 +/- 3 microM, and this compound is not a time-dependent inactivator of L1. N-Benzylacetyl-d-alanylthioacetic acid is a competitive inhibitor of L1 with a K(i) of 1.6 +/- 0.3 microM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric studies revealed that 2 mol of mercaptoacetate covalently bind to L1 upon incubation of the enzyme with N-benzylacetyl-d-alanylthioacetic acid; however, this covalently modified enzyme has the same activity as wild-type L1. Last, inhibition studies were used to demonstrate that 4-morpholinoethanesulfonic acid does not inhibit L1, even at concentrations up to 300 mM. This work identifies two possible competitive inhibitors which can be used in future structural studies and further demonstrates inhibitory heterogeneity among the metallo-beta-lactamases.

Continuous assay for VanX, the D-alanyl-D-alanine dipeptidase required for high-level vancomycin resistance.

The reaction of L-alanine-p-nitroanilide with VanX was studied in an effort to develop a continuous assay for VanX activity for future kinetic and inhibition studies. VanX, containing Zn(II), Co(II), Fe(II), or Ni(II), catalyzes the hydrolysis of L-alanine-p-nitroanilide producing L-alanine and p-nitroaniline as products; the formation of the latter product (epsilon(404nm) = 10, 700 M(-1) cm(-1)) can be continuously monitored using UV-VIS spectrophotometry. Zn(II)-, Co(II)-, Fe(II)-, and Ni(II)-containing VanX exhibit saturation kinetics when L-alanine-p-nitroanilide is used as the substrate with K(m) and k(cat) values ranging from 300 to 700 microM and 0.028 to 0.080 s(-1), respectively. Inhibition studies using O-[(1S)-aminoethylhydroxyphosphinyl]-D-lactic acid as the inhibitor and L-alanine-p-nitroanilide as the substrate yielded a K(i) of 400 +/- 8 microM at pH 7.0. These studies reveal a continuous assay of VanX activity which could be used to further study the kinetic mechanism of VanX and to allow for the development of high-throughput screening for inhibitors of VanX.

Chloride transport in the ferret trachea.

A net secretion of chloride stimulated by carbamylcholine was observed in whole trachea. Luminal anthracene-9-carboxylic acid inhibited the net secretion of chloride and the transepithelial potential difference across the isolated trachea. Submucosal ouabain inhibited the net secretion of chloride and submucosal ouabain, or submucosal bumetanide inhibited the transepithelial potential difference across the isolated trachea. Short-circuited flat sheets of trachea manifested a net secretion of chloride induced by carbamylcholine. Serosal ouabain inhibited the short-circuit current and net chloride flux across isolated flat sheets of trachea.

Kinetic mechanism of metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

The reaction of nitrocefin with metallo-beta-lactamase L1 from Stenotrophomonas maltophilia was studied using rapid-scan and stopped-flow ultraviolet-visible (UV-vis) studies in an effort to discern the kinetic mechanism used by L1 to hydrolyze penicillins and cephalosporins. Rapid-scan and stopped-flow UV-vis studies of nitrocefin hydrolysis by L1 identified three species: (1) the substrate (nitrocefin) displayed an absorbance peak at 390 nm (epsilon = 11 500 M(-1) cm(-1)) that decreased during the reaction with a rate constant of 170 +/- 30 s(-1); (2) the product (hydrolyzed nitrocefin) displayed an absorbance peak at 485 nm (epsilon = 17 420 M(-1) cm(-1)) that increased during the reaction with rate constant of 40 +/- 1 s(-1); and (3) an intermediate displayed an absorbance peak at 665 nm (epsilon = 32 000 M(-1) cm(-1)) that increased initially with a rate constant of 190 +/- 3 s(-1) and then decreased with a rate constant of 38 +/- 2 s(-1). Single-turnover experiments demonstrated that there were no pre-steady-state bursts in the reaction of L1 with nitrocefin; moreover, the progress curves could be fit to a kinetic mechanism that includes the formation of a transient intermediate by using KINSIM and the rate constants given above. Progress curves from experiments conducted at different reaction conditions or with a different substrate could also be fit to the proposed kinetic mechanism. The evidence for the presence of an intermediate along with kinetic simulations supports a hydrolytic mechanism for L1 that involves an intermediate whose breakdown is rate-determining.

Common genetic determinants of halothane and isoflurane potencies in Caenorhabditis elegans.

BACKGROUND: Genetics provides a way to evaluate anesthetic action simultaneously at the molecular and behavioral levels. Results from strains that differ in anesthetic sensitivity have been mixed in their support of unitary theories of anesthesia. Here the authors use the previously demonstrated large variation of halothane sensitivities in Caenorhabditis elegans recombinant inbred strains to assess the similarities of the determinants of halothane action with those of another volatile anesthetic, isoflurane. METHODS: The recombinant inbred strains, constructed from two evolutionarily distinct C. elegans lineages, were phenotyped. A coordination assay on agar quantified the sensitivity to the volatile anesthetics; median effective concentrations (EC50s) were calculated by nonlinear regression of concentration-response data and were correlated between the drugs for those strains tested in common. Genetic loci were identified by statistical association between EC50s and chromosomal markers. RESULTS: The recombinant inbred strains varied dramatically in sensitivity to halothane and isoflurane, with a 10-fold range in EC50s. Heritability estimates for each drug were imprecise but altogether high (49-80%). Halothane and isoflurane EC50s were significantly correlated (r=0.71, P < 10(-9)). Genetic loci controlling sensitivity were found for both volatile anesthetics; the most significant determinant colocalized on chromosome V. A smaller recombinant inbred strain study of ethanol-induced immobility segregated different genetic effects that did not correlate with sensitivity to either halothane or isoflurane. CONCLUSIONS: The genetic determinants driving the large variation in anesthetic sensitivity in these C. elegans recombinant inbred strains are very similar for halothane and isoflurane sensitivity.

Failure inference from a marker process based on a bivariate Wiener model.

Many models have been proposed that relate failure times and stochastic time-varying covariates. In some of these models, failure occurs when a particular observable marker crosses a threshold level. We are interested in the more difficult, and often more realistic, situation where failure is not related deterministically to an observable marker. In this case, joint models for marker evolution and failure tend to lead to complicated calculations for characteristics such as the marginal distribution of failure time or the joint distribution of failure time and marker value at failure. This paper presents a model based on a bivariate Wiener process in which one component represents the marker and the second, which is latent (unobservable), determines the failure time. In particular, failure occurs when the latent component crosses a threshold level. The model yields reasonably simple expressions for the characteristics mentioned above and is easy to fit to commonly occurring data that involve the marker value at the censoring time for surviving cases and the marker value and failure time for failing cases. Parametric and predictive inference are discussed, as well as model checking. An extension of the model permits the construction of a composite marker from several candidate markers that may be available. The methodology is demonstrated by a simulated example and a case application.

Overexpression, purification, and characterization of the cloned metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

The metallo-beta-lactamase L1 from Stenotrophomonas maltophilia was cloned, overexpressed, and characterized by spectrometric and biochemical techniques. Results of metal analyses were consistent with the cloned enzyme having 2 mol of tightly bound Zn(II) per monomer. Gel filtration chromatography demonstrated that the cloned enzyme exists as a tightly held tetramer with a molecular mass of ca. 115 kDa, and matrix-assisted laser desorption ionization and time-of-flight mass spectrometry indicated a monomeric molecular mass of 28.8 kDa. Steady-state kinetic studies with a number of diverse penicillin and cephalosporin antibiotics demonstrated that L1 effectively hydrolyzes all tested compounds, with k(cat)/Km values ranging between 0.002 and 5.5 microM(-1) s(-1). These characteristics of the recombinant enzyme are contrasted to those previously reported for metallo-beta-lactamases isolated directly from S. maltophilia.

A test for independence of competing risks with discrete failure times.

The identifiability problem in Competing Risks is well known. In particular, it implies that independent action or otherwise of the risks cannot be inferred from data alone. However, Crowder (1996) showed that, in the case of purely discrete failure times, an inference can be made. An algebraic criterion was derived which bears essentially on the independence in question. The condition was presented in a theoretical setting but it was pointed out that the quantities involved can be estimated from data and that, therefore, there is the potential to develop practical tests for the hypothesis of independence. It is the purpose of this paper to construct such a test.

Glyoxalase II from A. thaliana requires Zn(II) for catalytic activity.

Cytosolic glyoxalase II from Arabidopsis thaliana, GLX2-2, was overexpressed and purified to homogeneity using Q-sepharose chromatography. MALDI-TOF mass spectrometry studies indicated a molecular weight of 28 767 Da. Using steady-state kinetics studies, the purified enzyme exhibited a Km of 660 +/- 100 microM and a kcat of 484 +/- 92 s(-1) at 37 degrees C. Metal analyses demonstrated that the enzyme binds 2.1 +/- 0.5 moles of Zn(II) per monomer; the binding of Zn(II) is essential for enzyme viability and activity. Sequence comparison of glyoxalase II enzymes from human, A. thaliana, and yeast and the metallo-beta-lactamases reveal that all metal binding ligands of the metallo-beta-lactamases are conserved in glyoxalase II enzymes, suggesting that all glyoxalase II enzymes are Zn(II) metalloenzymes. These results and their implications are discussed in light of previous studies on glyoxalase II, and an active site for the glyoxalase II enzymes is proposed.

Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis.

In an effort to better understand the structure and function of the metallo-beta-lactamase from Bacteroides fragilis, spectroscopic and metal-binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy was used to confirm that the Co(II)-substituted beta-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are highspin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.0, and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (epsilon approximately 30 M-1.cm-1) and an intense feature at 320 nm (epsilon approximately 1570 M-1.cm-1). The latter is assigned to a cysteine sulfur to Co(II) ligand-to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of a Co(II)-substituted C168S mutant. H NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on kcat. These results are discussed in light of the recent crystal structure of the metallo-beta-lactamase from B. cereus.

On assessing independence of competing risks when failure times are discrete.

The traditional approach to modelling for Competing Risks, via a multivariate distribution of latent failure times, is very natural for many applications but suffers from a well-documented problem of identifiability. However, the demonstrations of this problem in the literature apply to essentially continuous latent failure times where any atoms of probability in their distributions are not too intrusive. It is shown in this paper that for discrete failure times the classic results on the identifiability problem concerning the existence of equivalent independent risks are incomplete.