Functional analysis of TatA and TatB in Streptomyces lividans

Recently, genes encoding TatA, TatB, and TatC homologues were identified in Streptomyces lividans and the functionality of the twin-arginine translocation (Tat) pathway was demonstrated. Previously, we have shown that TatC is indispensable for Tat-dependent secretion in S. lividans. In the present work, we demonstrate that as TatB, S. lividans TatA is important but not essential for efficient secretion of xylanase C and tyrosinase. The results presented here indicate that in the presence of TatC, still partially functional translocation systems composed of TatAC or TatBC can be formed, suggesting that TatA and TatB have at least partially overlapping activities. However, the dissimilar effect caused by a tatA deletion or a tatB deletion on Tat-dependent secretion together with the fact that TatA cannot fully functionally substitute TatB and vice versa indicates that in S. lividans TatA and TatB are not functionally equivalent. Interestingly, soluble GST-tagged TatA and TatB were able to specifically bind Tat-dependent preproteins. The ability to bind Tat-dependent preproteins together with their cytoplasmic localization in S. lividans strongly suggests that both TatA and TatB, independently or associated, serve to recruit Tat-dependent preproteins to the translocase.     

Phenotypic and molecular typing of Salmonella strains reveals different contamination sources in two commercial pig slaughterhouses

This study aimed to define the origin of Salmonella contamination on swine carcasses and the distribution of Salmonella serotypes in two commercial slaughterhouses during normal activity. Salmonellae were isolated from carcasses, from colons and mesenteric lymph nodes of individual pigs, and from the slaughterhouse environment. All strains were serotyped; Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Derby isolates were additionally typed beyond the serotype level by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiling (ARP); and a subset of 31 serotype Typhimurium strains were additionally phage typed. PFGE and ARP had the same discriminative possibility. Phage typing in combination with PFGE could give extra information for some strains. In one slaughterhouse, 21% of the carcasses were contaminated, reflecting a correlation with the delivery of infected pigs. Carcass contamination did not result only from infection of the corresponding pig; only 25% of the positive carcasses were contaminated with the same serotype or genotype found in the corresponding feces or mesenteric lymph nodes. In the other slaughterhouse, 70% of the carcasses were contaminated, and only in 4% was the same genotype or serotype detected as in the feces of the corresponding pigs. The other positive carcasses in both slaughterhouses were contaminated by genotypes present in the feces or lymph nodes of pigs slaughtered earlier that day or from dispersed sources in the environment. In slaughterhouses, complex contamination cycles may be present, resulting in the isolation of many different genotypes circulating in the environment due to the supply of positive animals and in the contamination of carcasses, probably through aerosols.     

From individual leaf elongation to whole shoot leaf area expansion: a comparison of three Aegilops and two Triticum species

BACKGROUND AND AIMS: Rapid leaf area expansion is a desirable trait in the early growth stages of cereal crops grown in low-rainfall areas. In this study, the traits associated with inherent variation in early leaf area expansion rates have been investigated in two wheat species (Triticum aestivum and T. durum) and three of its wild relatives (Aegilops umbellulata, A. caudata and A. tauschii) to find out whether the Aegilops species have a faster leaf area expansion in their early developmental stage than some of the current wheat species. METHODS: Growth of individual leaves, biomass allocation, and gas exchange were measured on hydroponically grown plants for 4 weeks. KEY RESULTS: Leaf elongation rate (LER) was strongly and positively correlated with leaf width but not with leaf elongation duration (LED). The species with more rapidly elongating leaves showed a faster increase with leaf position in LER, leaf width and leaf area, higher relative leaf area expansion rates, and more biomass allocation to leaf sheaths and less to roots. No differences in leaf appearance rate were found amongst the species. CONCLUSIONS: Aegilops tauschii was the only wild species with rapid leaf expansion rates similar to those of wheat, and it achieved the highest photosynthetic rates, making it an interesting species for further study.     

Substitution of glycine-661 by serine in the α1(I) and α2(1) chains of type I collagen results in different clinical and biochemical phenotypes

We have characterised a point mutation causing the substitution of serine for glycine at position 661 of the 1(I) chain of type I collagen in a child with a severe form of osteogenesis imperfecta. An identical glycine substitution in the 2(I) chain was previously detected in a woman with post-menopausal osteoporosis. Two of her sons were heterozygous for the mutation and the third son was homozygous as a result of uniparental isodisomy. Biochemical profiles of the type I collagen heterotrimers were studied in each of the patients and compared with a control. Medium and cell-layer collagens were overmodified in all patients. Overmodification was obvious in the patient with the 1(I) mutation but mild in the patients with the 2(I) mutation, being slightly less evident in the heterozygote than in the homozygote. Investigation of the melting curves of the mutant collagen trimers in all three patients showed the same slight decrease in thermal stability and, hence, a lack of correlation with phenotypic severity. In contrast, the degree of overmodification of the collagen alpha chains was correlated with the phenotypic severity. The clinical observations in these patients illustrate the possibly predominant role of mutations in the collagen 1(I) chains over the same mutations in the 2(I) chains in determining the clinical outcome.     

Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-dihydropyrimidinones

A series of 4-substituted 3,4-dihydropyrimidine-2-ones (DHPM) was synthesized, characterized by IR, ¹H NMR, ¹³C NMR and HRMS spectra. The compounds were evaluated in vitro for their antiviral activity against a broad range of DNA and RNA viruses, along with assessment for potential cytotoxicity in diverse mammalian cell lines. Compound 4m, which possesses a long lipophilic side chain, was found to be a potent and selective inhibitor of Punta Toro virus, a member of the Bunyaviridae. For Rift Valley fever virus, which is another Bunyavirus, the activity of 4m was negligible. DHPMs with a C-4 aryl moiety bearing halogen substitution (4b, 4c and 4d) were found to be cytotoxic in MT4 cells.     

Effects of an Enhanced Human Interaction Program on shelter dogs’ behaviour analysed using a novel nonparametric test

The aim of this study was to investigate the effects of introduction and interruption of an Enhanced Human Interaction Program (EHIP) on shelter dogs’ behaviour and welfare and to apply a novel statistical method to analyse the behavioural data. Twenty-two dogs, which were never subjected to similar programs, were studied. The EHIP consisted of walking and petting the dogs for 15 min once a week. Dogs were divided into two groups: dogs participating in the program (EHIP, n = 9) and control dogs receiving no such program but exposed to human visual stimuli (n = 13). EHIP dogs started being walked on day 8 of the study and continued until day 42 when the walking schedule was stopped. Behavioural observations were performed by instantaneous scan sampling every 3 min for 3 h on days 1, 3, 8, 10, 15, 36, 38, 42, 57, 59 and 64. Hence three periods were identified: the first in which all the dogs were not walked, the second in which EHIP dogs were walked (subdivided into early interaction and late interaction phase), the third in which all the dogs were not walked. Data were analysed using the inferential multivariate nonparametric methodology called NPC (Nonparametric Combination) Test. EHIP increased the time the dogs were visible from the front of the pen and the time they were tail wagging (p < 0.05). These changes may make the animals more likely to be re-homed. No behavioural sign of stress was recorded when the program was discontinued. These findings lead to conclude that EHIPs may be useful enrichment programs for shelter dogs. Furthermore, the NPC Test proved to be a suitable and powerful statistical tool since it does not need a modelling for dependence among variables and it is not affected by the problem of loss of degrees of freedom when the number of variables is large compared to sample size as was the case in the present study.     

Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.