Crystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with β-lactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 dd-peptidase structures reveal the presence of a specific binding site for the d-α-aminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential high-molecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 dd-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.     

Patterns of Growth Associated With the Timing of Adiposity Rebound

This study was undertaken to evaluate the effects of age of adiposity rebound (AR) on measures of fat mass between ages 7 and 11 years, maturity, and adiposity in 458 children from a birth cohort studied to age 26 years. Patterns of growth between ages 3 and 26 years and changes in fat mass index between 7 and 11 years in groups with early (<5.5 years for boys and <5 years for girls), average (between 5.5 and 7.5 years for boys and between 5 and 7 years for girls), and late AR (≥7.5 years for boys and ≥7 years for girls) are described. The mean z‐scores for BMI, height, and weight increased between age 3 years and adolescence in the early‐rebound group and decreased in the late‐rebound group. The differences were maintained until adulthood for BMI and weight. Disproportionately high increases in fat mass index during growth (7–11 years), more advanced bone age in boys at age 7 years, and earlier menarche in girls were evident in the early‐rebound group. The relative risks at 26 years of being overweight (BMI 25–29.9 kg/m²) and obese (BMI ≥30 kg/m²) were 2.70 (95% confidence interval (CI): 1.55, 4.66) and 5.91 (95% CI: 3.03, 11.55) respectively, using the average group as the reference. The corresponding relative risks for adult waist girths exceeding international cut points were 2.12 (95% CI: 1.09, 4.13) and 3.32 (95% CI: 1.46, 7.54). Thus, early rebound is associated with increased depositions of fat in middle childhood, and risks associated with early rebound persist at least until early adulthood.     

Evaluating morphometric and metabolic markers of body condition in a small cetacean,the harbor porpoise (Phocoena phocoena)

Mammalian body condition is an important individual fitness metric as it affects both survival and reproductive success. The ability to accurately measure condition has key implications for predicting individual and population health, and therefore monitoring the population‐level effects of changing environments. No consensus currently exists on the best measure to quantitatively estimate body condition in many species, including cetaceans. Here, two measures of body condition were investigated in the harbor porpoise (Phocoena phocoena). First, the most informative morphometric body condition index was identified. The mass/length² ratio was the most appropriate morphometric index of 10 indices tested, explaining 50% of the variation in condition in stranded, male porpoises with different causes of death and across age classes (n = 291). Mass/length² was then used to evaluate a second measure, blubber cortisol concentration, as a metabolic condition marker. Cortisol is the main glucocorticoid hormone involved in the regulation of lipolysis and overall energy balance in mammals, and concentrations could provide information on physiological state. Blubber cortisol concentrations did not significantly vary around the girth (n = 20), but there was significant vertical stratification through the blubber depth with highest concentrations in the innermost layer. Concentrations in the dorsal, outermost layer were representative of concentrations through the full blubber depth, showed variation by sex and age class, and were negatively correlated with mass/length². Using this species as a model for live cetaceans from which standard morphometric measurements cannot be taken, but from which blubber biopsy samples are routinely collected, cortisol concentrations in the dorsal, outermost blubber layer could potentially be used as a biomarker of condition in free‐ranging animals.     

Utility of Microsatellite Markers and Amplified Fragment Length Polymorphism in the Study of Potentially Ochratoxigenic Black Aspergilli

Microsatellite markers and the results of amplified fragment length polymorphism (AFLP) were compared in the characterization of 68 Aspergillus carbonarius and A. niger aggregate strains of differing ochratoxin-producing ability and from different geographic areas, isolated mainly from grapes and soil. AFLP was applied to both A. carbonarius and A. niger aggregate strains, and it clearly differentiated these species. Microsatellite markers were only applied to A. niger aggregate strains because of the species-specific nature of these markers. Both AFLP and microsatellite marker analyses were able to divide A. niger aggregate strains into the two recognized internal transcribed spacer (ITS)-5.8S rDNA RFLP types, N and T. Clustering of A. niger aggregate strains was similar in both AFLP and microsatellite analyses, yielding an additional separation of N type strains into two groups. Both microsatellite marker and AFLP analyses showed high levels of polymorphism in the A. niger aggregate (index of discriminatory power 0.991 and 1.0, respectively). Of the two techniques, microsatellite marker analysis was quicker and more straightforward to perform. In addition, microsatellite marker analysis is more reproducible, and the results can be expressed as quantitative data, making microsatellite markers a good candidate for use in large-scale studies of genetic diversity in A. niger aggregate species.     

Involvement of sphingosine kinase in plant cell signalling

In mammalian cells sphingosine-1-phosphate (S1P) is a well-established messenger molecule that participates in a wide range of signalling pathways. The objective of the work reported here was to investigate the extent to which phosphorylated long-chain sphingoid bases, such as sphingosine-1-phosphate and phytosphingosine-1-phosphate (phytoS1P) are used in plant cell signalling. To do this, we manipulated Arabidopsis genes capable of metabolizing these messenger molecules. We show that Sphingosine kinase1 (SPHK1) encodes an enzyme that phosphorylates sphingosine, phytosphingosine and other sphingoid long-chain bases. The stomata of SPHK1-KD Arabidopsis plants were less sensitive, whereas the stomata of SPHK1-OE plants were more sensitive, than wild type to ABA. The rate of germination of SPHK1-KD was enhanced, whereas the converse was true for SPHK1-OE seed. Reducing expression of either the putative Arabidopsis S1P phosphatase (SPPASE) or the DPL1 gene, which encodes an enzyme with S1P lyase activity, individually, had no effect on guard-cell ABA signalling; however, stomatal responses to ABA in SPPASEDPL1 RNAi plants were compromised. Reducing the expression of DPL1 had no effect on germination; however, germination of SPPASE RNAi seeds was more sensitive to applied ABA. We also found evidence that expression of SPHK1 and SPPASE were coordinately regulated, and discuss how this might contribute to robustness in guard-cell signalling. In summary, our data establish SPHK1 as a component in two separate plant signalling systems, opening the possibility that phosphorylated long-chain sphingoid bases such as S1P and phytoS1P are ubiquitous messengers in plants.     

A new chromogenic medium for the isolation of Listeria spp

A new medium is described for the isolation of Listeria spp. from foods and environmental samples. It is based on a modified Oxford medium in which 3,4-cyclohexenoesculetin-beta-D-glucoside replaces aesculin. Positive colonies are intensely black with the advantage that the pigment does not diffuse into the medium. The medium, when tested alongside the US Department of Agriculture (spiked samples) and Food and Drug Administration (naturally contaminated samples) isolation procedures, performed significantly better than the current formulations (34% more confirmed positives from naturally contaminated samples) with a reduction of 1 d in the assay time for most samples.     

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium’s nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.     

Plasticity of purine release during cerebral ischemia: clinical implications?

Adenosine is a powerful modulator of neuronal function in the mammalian central nervous system. During a variety of insults to the brain, adenosine is released in large quantities and exerts a neuroprotective influence largely via the A₁ receptor, which inhibits glutamate release and neuronal activity. Using novel enzyme-based adenosine sensors, which allow high spatial and temporal resolution recordings of adenosine release in real time, we have investigated the release of adenosine during hypoxia/ischemia in the in vitro hippocampus. Our data reveal that during the early stages of hypoxia adenosine is likely released per se and not as a precursor such as cAMP or an adenine nucleotide. In addition, repeated hypoxia results in reduced production of extracellular adenosine and this may underlie the increased vulnerability of the mammalian brain to repetitive or secondary hypoxia/ischemia.     

DXA measurements confirm that parental perceptions of elevated adiposity in young children are poor

Objective: To compare parental assessments of child body weight status with BMI measurements and determine whether children who are incorrectly classified differ in body composition from those whose parents correctly rate child weight. Also to ascertain whether children of obese parents differ from those of non‐obese parents in actual or perceived body weight. Research Methods and Procedures: Weights, heights, BMI, and waist girths of New Zealand children ages 3 to 8 years were determined. Fat mass, fat percentage, and lean mass were measured by DXA (n = 96). Parents classified child weight status as underweight, normal‐weight, slightly overweight, or overweight. Centers for Disease Control and Prevention 2000 percentiles of BMI were used. Results: Parents underestimated child weight status. Despite having 83% more fat mass than children with BMI values below the 85th percentile, only 7 of 31 children with BMI values at or above the 85th percentile were rated as slightly overweight or overweight. In the whole sample, participants whose weight status was underestimated by parents (40 of the 96 children) had l9% less fat mass but similar lean mass as children whose weight status was correctly classified. However, children of obese and non‐obese parents did not differ in body composition or anthropometry, and obese parents did not underestimate child weight more than non‐obese parents. Discussion: Because parents underestimate child weight, but BMI values at or above the 85th percentile identify high body fat well, advising parents of the BMI status of their children should improve strategies to prevent excessive fat gain in young children.