Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32.

An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.     

Stochastic Models of Soil Denitrification

Soil denitrification is a highly variable process that appears to be lognormally distributed. This variability is manifested by large sample coefficients of variation for replicate estimates of soil core denitrification rates. Deterministic models for soil denitrification have been proposed in the past, but none of these models predicts the approximate lognormality exhibited by natural denitrification rate estimates. In this study, probabilistic (stochastic) models were developed to understand how positively skewed distributions for field denitrification rate estimates result from the combined influences of variables known to affect denitrification. Three stochastic models were developed to describe the distribution of measured soil core denitrification rates. The driving variables used for all the models were denitrification enzyme activity and CO₂ production rates. The three models were distinguished by the functional relationships combining these driving variables. The functional relationships used were (i) a second-order model (model 1), (ii) a second-order model with a threshold (model 2), and (iii) a second-order saturation model (model 3). The parameters of the models were estimated by using 12 separate data sets (24 replicates per set), and their abilities to predict denitrification rate distributions were evaluated by using three additional independent data sets of 180 replicates each. Model 2 was the best because it produced distributions of denitrification rate which were not significantly different (P > 0.1) from distributions of measured denitrification rates. The generality of this model is unknown, but it accurately predicted the mean denitrification rates and accounted for the stochastic nature of this variable at the site studied. The approach used in this study may be applicable to other areas of ecological research in which accounting for the high spatial variability of microbiological processes is of interest.     

A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction

The biosynthesis of S-adenosylmethionine occurs in a unique enzymatic reaction in which the synthesis of the sulfonium center results from displacement of the entire polyphosphate chain from MgATP. The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trapping measurements. Replacement of 12C by 14C at the 5' carbon of ATP yields a primary Vmax/Km isotope effect (12C/14C) of 1.128 +/- 0.003 in the absence of added monovalent cation activator (K+). At saturating K+ concentrations (10 mM) the primary isotope effect diminishes slightly to 1.108 +/- 0.003, indicating that the step in the mechanism involving bond breaking at the 5' carbon of MgATP has a small commitment to catalysis at conditions near Vmax. No alpha-secondary 3H isotope effect from [5'-3H]ATP was detected, (1H/3H) = 1.000 +/- 0.002, even in the absence of KCl. There was no significant primary sulfur isotope effect from [35S]methionine at KCl concentrations from 0 to 10 mM. Substitution of the methyl group of methionine with tritium yielded a beta-secondary isotope effect (CH3/C3H3) = 1.009 +/- 0.008 independent of KCl concentration. The reaction of selenomethionine and [5'-14C]ATP gave a primary isotope effect of 1.097 +/- 0.006, independent of KCl concentration. Substrate trapping experiments demonstrated that the step in the mechanism involving bond making to sulfur of methionine does not have a significant commitment to catalysis at 0.25 mM KCl, therefore intrinsic isotope effects were observed. Substrate trapping experiments indicated that the step involving bond breaking at carbon 5' of MgATP has a 10% commitment to catalysis at 0.25 mM KCl. The isotope effects are interpreted in terms of an Sn2-like transition state structure in which bonding of the C5' is symmetric with respect to the departing tripolyphosphate group and the incoming sulfur of methionine. With selenomethionine as substrate an earlier transition state is implicated.     

A film technique for the biological evaluation of pyrethrum-in-oil insecticides for use against stored product insects in warehouses

There is need for a technique whereby a given insecticidal preparation can be compared with a standard containing 0.8% (w/v) pyrethrin 1 in a heavy white oil and pronounced biologically equal or inferior to the standard within known and reasonable limits. A film method was investigated, Tribolium castaneum beetles being used as the test insects.
Numerous factors which may influence the kill achieved are discussed. The results of preliminary work, planned to help in the standardization of some of these factors, are summarized.
Several materials were tested to ascertain their suitability for reception of the insecticidal film and, as thin greaseproof paper showed promise, its possibilities were explored. Although 0.3 % pyrethrin I could be distinguished from 0.4%, 0.5% could not be distinguished from 0.8%, and the results throughout were very heterogeneous.
Woven Nylon proved more satisfactory, since the test just failed to establish a significant difference between 0.6 and 0.8% pyrethrin 1. The origin of a pink coloration of the substrate, when T. castaneum beetles were confined on Nylon sprayed with pyrethrum in oil, was investigated.
Whatman filter paper no. 544 proved the best substrate, and a comparative test on it will distinguish 0.65% pyrethrin 1 from the standard 0.8%; the limit could probably be raised to 0.7%.
The influence of several factors on the homogeneity of the results was investigated, and the importance was shown of allowing beetles time to recover from the mechanical shock inherent in the method of counting into batches.
Directions are given for carrying out the film technique for comparing preparations of relatively high pyrethrin content. The method does not measure the direct spray or fumigant action of the insecticide.
The technique described is compared with the Peet-Grady method for evaluating liquid household insecticides of low pyrethrin content. A simplified, but less accurate, design for the test is indicated.     

Soccer Player Characteristics in English Lower-League Development Programmes: The Relationships between Relative Age,Maturation, Anthropometry and Physical Fitness

The relative age effect (RAE) and its relationships with maturation, anthropometry, and physical performance characteristics were examined across a representative sample of English youth soccer development programmes. Birth dates of 1,212 players, chronologically age-grouped (i.e., U9’s-U18’s), representing 17 professional clubs (i.e., playing in Leagues 1 & 2) were obtained and categorised into relative age quartiles from the start of the selection year (Q1 = Sep-Nov; Q2 = Dec-Feb; Q3 = Mar-May; Q4 = Jun-Aug). Players were measured for somatic maturation and performed a battery of physical tests to determine aerobic fitness (Multi-Stage Fitness Test [MSFT]), Maximal Vertical Jump (MVJ), sprint (10 & 20m), and agility (T-Test) performance capabilities. Odds ratio’s (OR) revealed Q1 players were 5.3 times (95% confidence intervals [CI]: 4.08–6.83) more likely to be selected than Q4’s, with a particularly strong RAE bias observed in U9 (OR: 5.56) and U13-U16 squads (OR: 5.45–6.13). Multivariate statistical models identified few between quartile differences in anthropometric and fitness characteristics, and confirmed chronological age-group and estimated age at peak height velocity (APHV) as covariates. Assessment of practical significance using magnitude-based inferences demonstrated body size advantages in relatively older players (Q1 vs. Q4) that were very-likely small (Effect Size [ES]: 0.53–0.57), and likely to very-likely moderate (ES: 0.62–0.72) in U12 and U14 squads, respectively. Relatively older U12-U14 players also demonstrated small advantages in 10m (ES: 0.31–0.45) and 20m sprint performance (ES: 0.36–0.46). The data identify a strong RAE bias at the entry-point to English soccer developmental programmes. RAE was also stronger circa-PHV, and relatively older players demonstrated anaerobic performance advantages during the pubescent period. Talent selectors should consider motor function and maturation status assessments to avoid premature and unwarranted drop-out of soccer players within youth development programmes.