144 research outputs found
Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism
Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure–activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK·SO4, R57A, and HpSK•shikimate-3-phosphate•ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A•162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism
Threshold intensity factors as lower boundaries for crack propagation in ceramics
BACKGROUND: Slow crack growth can be described in a v (crack velocity) versus K(I )(stress intensity factor) diagram. Slow crack growth in ceramics is attributed to corrosion assisted stress at the crack tip or at any pre-existing defect in the ceramic. The combined effect of high stresses at the crack tip and the presence of water or body fluid molecules (reducing surface energy at the crack tip) induces crack propagation, which eventually may result in fatigue. The presence of a threshold in the stress intensity factor, below which no crack propagation occurs, has been the subject of important research in the last years. The higher this threshold, the higher the reliability of the ceramic, and consequently the longer its lifetime. METHODS: We utilize the Irwin K-field displacement relation to deduce crack tip stress intensity factors from the near crack tip profile. Cracks are initiated by indentation impressions. The threshold stress intensity factor is determined as the time limit of the tip stress intensity when the residual stresses have (nearly) disappeared. RESULTS: We determined the threshold stress intensity factors for most of the all ceramic materials presently important for dental restorations in Europe. Of special significance is the finding that alumina ceramic has a threshold limit nearly identical with that of zirconia. CONCLUSION: The intention of the present paper is to stress the point that the threshold stress intensity factor represents a more intrinsic property for a given ceramic material than the widely used toughness (bend strength or fracture toughness), which refers only to fast crack growth. Considering two ceramics with identical threshold limits, although with different critical stress intensity limits, means that both ceramics have identical starting points for slow crack growth. Fast catastrophic crack growth leading to spontaneous fatigue, however, is different. This growth starts later in those ceramic materials that have larger critical stress intensity factors
Impact Factor: outdated artefact or stepping-stone to journal certification?
A review of Garfield's journal impact factor and its specific implementation
as the Thomson Reuters Impact Factor reveals several weaknesses in this
commonly-used indicator of journal standing. Key limitations include the
mismatch between citing and cited documents, the deceptive display of three
decimals that belies the real precision, and the absence of confidence
intervals. These are minor issues that are easily amended and should be
corrected, but more substantive improvements are needed. There are indications
that the scientific community seeks and needs better certification of journal
procedures to improve the quality of published science. Comprehensive
certification of editorial and review procedures could help ensure adequate
procedures to detect duplicate and fraudulent submissions.Comment: 25 pages, 12 figures, 6 table
Bioformulation of microbial biocontrol agents for a sustainable agriculture
The application of microbial based biopesticides has become a sustainable alternative to the use of chemicals to prevent yield losses due to plant pathogens. However, microbial based biopesticides are often unsuccessfully formulated and do not meet the demanding regulatory standards required by the agencies, which hinders their commercialization. Hence, an outline on the approaches to attain more effective formulations might be useful for the development of future more effective products.
With this aim, this chapter reports the current state of biocontrol strategies and describes the principles of microbial biocontrol formulations. Emphasis is placed on techniques and tools available for the development and characterisation of microbial products. To provide glimpses on the possible formulations, the different existing additives, carriers, inoculation techniques and formulation types are exhaustively reviewed. Finally, requirements and principles for efficacy evaluation of plant protection products in the European Union are include
Revisiting the pH-gated conformational switch on the activities of HisKA-family histidine kinases
Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH
Growth arrest-specific transcript 5 associated snoRNA levels are related to p53 expression and DNA damage in colorectal cancer
BACKGROUND
The growth arrest-specific transcript 5 gene (GAS5) encodes a long noncoding RNA (lncRNA) and hosts a number of small nucleolar RNAs (snoRNAs) that have recently been implicated in multiple cellular processes and cancer. Here, we investigate the relationship between DNA damage, p53, and the GAS5 snoRNAs to gain further insight into the potential role of this locus in cell survival and oncogenesis both in vivo and in vitro.
METHODS
We used quantitative techniques to analyse the effect of DNA damage on GAS5 snoRNA expression and to assess the relationship between p53 and the GAS5 snoRNAs in cancer cell lines and in normal, pre-malignant, and malignant human colorectal tissue and used biological techniques to suggest potential roles for these snoRNAs in the DNA damage response.
RESULTS
GAS5-derived snoRNA expression was induced by DNA damage in a p53-dependent manner in colorectal cancer cell lines and their levels were not affected by DICER. Furthermore, p53 levels strongly correlated with GAS5-derived snoRNA expression in colorectal tissue.
CONCLUSIONS
In aggregate, these data suggest that the GAS5-derived snoRNAs are under control of p53 and that they have an important role in mediating the p53 response to DNA damage, which may not relate to their function in the ribosome. We suggest that these snoRNAs are not processed by DICER to form smaller snoRNA-derived RNAs with microRNA (miRNA)-like functions, but their precise role requires further evaluation. Furthermore, since GAS5 host snoRNAs are often used as endogenous controls in qPCR quantifications we show that their use as housekeeping genes in DNA damage experiments can lead to inaccurate results
Fructooligosacharides Reduce Pseudomonas aeruginosa PAO1 Pathogenicity through Distinct Mechanisms
Pseudomonas aeruginosa
is ubiquitously present in the environment and acts as an opportunistic pathogen on humans,
animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this
bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a
significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was
observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the
swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages)
FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-
a
. Western blot
experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the
NF-
k
B pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is
likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also
with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed
that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism
for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory
cocktails is discussed.The authors acknowledge financial support from FEDER funds and Fondo Social Europeo through grants from the Spanish Ministry of Economy and Competitiveness (grants SAF2011-22922, SAF2011-22812) the Andalusian regional government Junta de Andalucía (grant CVI-7335) and the Centre of Networked
Biomedical Research on Hepatic and Digestive Diseases (CIBERehd) which is funded by the Carlos III Health Institute and the Ramón Areces Foundation, Spain
Evolutionary Diversification of Plant Shikimate Kinase Gene Duplicates
Shikimate kinase (SK; EC 2.7.1.71) catalyzes the fifth reaction of the shikimate pathway, which directs carbon from the central metabolism pool to a broad range of secondary metabolites involved in plant development, growth, and stress responses. In this study, we demonstrate the role of plant SK gene duplicate evolution in the diversification of metabolic regulation and the acquisition of novel and physiologically essential function. Phylogenetic analysis of plant SK homologs resolves an orthologous cluster of plant SKs and two functionally distinct orthologous clusters. These previously undescribed genes, shikimate kinase-like 1 (SKL1) and -2 (SKL2), do not encode SK activity, are present in all major plant lineages, and apparently evolved under positive selection following SK gene duplication over 400 MYA. This is supported by functional assays using recombinant SK, SKL1, and SKL2 from Arabidopsis thaliana (At) and evolutionary analyses of the diversification of SK-catalytic and -substrate binding sites based on theoretical structure models. AtSKL1 mutants yield albino and novel variegated phenotypes, which indicate SKL1 is required for chloroplast biogenesis. Extant SKL2 sequences show a strong genetic signature of positive selection, which is enriched in a protein–protein interaction module not found in other SK homologs. We also report the first kinetic characterization of plant SKs and show that gene expression diversification among the AtSK inparalogs is correlated with developmental processes and stress responses. This study examines the functional diversification of ancient and recent plant SK gene duplicates and highlights the utility of SKs as scaffolds for functional innovation
- …