Characterizing the complexity of enzymes on the basis of their mechanisms and structures with a bio-computational analysis

Enzymes are basically composed of 20 naturally occurring amino acids, yet they catalyse a dizzying array of chemical reactions, with regiospecificity and stereospecificity and under physiological conditions. In this review, we attempt to gain some understanding of these complex proteins, from the chemical versatility of the catalytic toolkit, including the use of cofactors (both metal ions and organic molecules), to the complex mapping of reactions to proteins (which is rarely one-to-one), and finally the structural complexity of enzymes and their active sites, often involving multidomain or multisubunit assemblies. This work highlights how the enzymes that we see today reflect millions of years of evolution, involving de novo design followed by exquisite regulation and modulation to create optimal fitness for life.     

The Activity of Neutral α-Glucosidase and Selected Biochemical Parameters in the Annual Cycle of Breeding Carp (Cyprinus carpio L.)

The aim of the study was to demonstrate seasonal changes in the hydrolytic and transferase activity of neutral α-glucosidase, the level of glucose, cholesterol, triglycerides and total protein in the annual breeding cycle of the carp. The study was conducted on fish from a fish farm in Lower Silesia (Poland). Blood serum was collected from the heart in: June, September and December of two consecutive years. The results of the study show that the hydrolytic and transferase activity of neutral α-glucosidase, as well as the results of basic biochemical parameters are highest in summer, when the fish seek and intake food intensively. The lowest values were observed in spring, when carp have the lowest metabolism after the wintering period.     

Macro-Climatic Distribution Limits Show Both Niche Expansion and Niche Specialization among C4 Panicoids

Grasses are ancestrally tropical understory species whose current dominance in warm open habitats is linked to the evolution of C₄ photosynthesis. C₄ grasses maintain high rates of photosynthesis in warm and water stressed environments, and the syndrome is considered to induce niche shifts into these habitats while adaptation to cold ones may be compromised. Global biogeographic analyses of C₄ grasses have, however, concentrated on diversity patterns, while paying little attention to distributional limits. Using phylogenetic contrast analyses, we compared macro-climatic distribution limits among ~1300 grasses from the subfamily Panicoideae, which includes 4/5 of the known photosynthetic transitions in grasses. We explored whether evolution of C₄ photosynthesis correlates with niche expansions, niche changes, or stasis at subfamily level and within the two tribes Paniceae and Paspaleae. We compared the climatic extremes of growing season temperatures, aridity, and mean temperatures of the coldest months. We found support for all the known biogeographic distribution patterns of C₄ species, these patterns were, however, formed both by niche expansion and niche changes. The only ubiquitous response to a change in the photosynthetic pathway within Panicoideae was a niche expansion of the C₄ species into regions with higher growing season temperatures, but without a withdrawal from the inherited climate niche. Other patterns varied among the tribes, as macro-climatic niche evolution in the American tribe Paspaleae differed from the pattern supported in the globally distributed tribe Paniceae and at family level.     

The alternative aerobic ribonucleotide reductase of Escherichia coli, NrdEF, is a manganese-dependent enzyme that enables cell replication during periods of iron starvation

The genome of Escherichia coli encodes two class I ribonucleotide reductases. The first, NrdAB, is a well-studied iron-dependent enzyme that is essential for aerobic growth. The second, NrdEF, is not functional under routine conditions, and its role is obscure. Recent studies demonstrated that NrdEF can be activated in vitro by manganese as well as iron. Since iron enzymes are potential targets for hydrogen peroxide, and since the nrdHIEF operon is induced during H(2) O(2) stress, we hypothesized that H(2) O(2) might inactivate NrdAB and that NrdEF might be induced to compensate. This idea was tested using E. coli mutants that are chronically stressed by H(2) O(2) . Contrary to expectation, NrdAB remained active. Its resistance to H(2) O(2) depended upon YfaE, which helps to activate NrdB. The induction of NrdEF during H(2) O(2) stress was mediated by the inactivation of Fur, an iron-dependent repressor. This regulatory arrangement implied that NrdEF has a physiological role during periods of iron starvation. Indeed, NrdEF supported cell replication in iron-depleted cells. Iron bound to NrdF when it was expressed in iron-rich cells, but NrdEF was functional only in cells that were both iron-depleted and manganese-rich. Thus NrdEF supports DNA replication when iron is unavailable to activate the housekeeping NrdAB enzyme.     

Rainfall manipulation experiments as simulated by terrestrial biosphere models: Where do we stand?

Changes in rainfall amounts and patterns have been observed and are expected to continue in the near future with potentially significant ecological and societal consequences. Modelling vegetation responses to changes in rainfall is thus crucial to project water and carbon cycles in the future. In this study, we present the results of a new model‐data intercomparison project, where we tested the ability of 10 terrestrial biosphere models to reproduce the observed sensitivity of ecosystem productivity to rainfall changes at 10 sites across the globe, in nine of which, rainfall exclusion and/or irrigation experiments had been performed. The key results are as follows: (a) Inter‐model variation is generally large and model agreement varies with timescales. In severely water‐limited sites, models only agree on the interannual variability of evapotranspiration and to a smaller extent on gross primary productivity. In more mesic sites, model agreement for both water and carbon fluxes is typically higher on fine (daily–monthly) timescales and reduces on longer (seasonal–annual) scales. (b) Models on average overestimate the relationship between ecosystem productivity and mean rainfall amounts across sites (in space) and have a low capacity in reproducing the temporal (interannual) sensitivity of vegetation productivity to annual rainfall at a given site, even though observation uncertainty is comparable to inter‐model variability. (c) Most models reproduced the sign of the observed patterns in productivity changes in rainfall manipulation experiments but had a low capacity in reproducing the observed magnitude of productivity changes. Models better reproduced the observed productivity responses due to rainfall exclusion than addition. (d) All models attribute ecosystem productivity changes to the intensity of vegetation stress and peak leaf area, whereas the impact of the change in growing season length is negligible. The relative contribution of the peak leaf area and vegetation stress intensity was highly variable among models.     

Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARalpha and PPARdelta (also known as PPARbeta) (but not PPARgamma). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARalpha (but not PPARdelta or PPARgamma) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4beta-PMA and PGF(2alpha), and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4beta-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4beta-PMA in the absence of a PPAR ligand was decreased by the NF-kappaB (nuclear factor kappaB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-kappaB in addition to PPAR phosphorylation. Use of NF-kappaB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARalpha to increase PTGS2 levels in bovine endometrial stromal cells.     

Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells.

BACKGROUND: Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution. METHODS: Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison. RESULTS: Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution. CONCLUSIONS: This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.     

Estrella lausannensis, a new star in the Chlamydiales order

Originally, the Chlamydiales order was represented by a single family, the Chlamydiaceae, composed of several pathogens, such as Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia abortus. Recently, 6 new families of Chlamydia-related bacteria have been added to the Chlamydiales order. Most of these obligate intracellular bacteria are able to replicate in free-living amoebae. Amoebal co-culture may be used to selectively isolate amoeba-resisting bacteria. This method allowed in a previous work to discover strain CRIB 30, from an environmental water sample. Based on its 16S rRNA gene sequence similarity with Criblamydia sequanensis, strain CRIB 30 was considered as a new member of the Criblamydiaceae family. In the present work, phylogenetic analyses of the genes gyrA, gyrB, rpoA, rpoB, secY, topA and 23S rRNA as well as MALDI-TOF MS confirmed the taxonomic classification of strain CRIB 30. Morphological examination revealed peculiar star-shaped elementary bodies (EBs) similar to those of C. sequanensis. Therefore, this new strain was called "Estrella lausannensis". Finally, E. lausannensis showed a large amoebal host range and a very efficient replication rate in Acanthamoeba species. Furthermore, E. lausannensis is the first member of the Chlamydiales order to grow successfully in the genetically tractable Dictyostelium discoideum, which opens new perspectives in the study of chlamydial biology.     

Helicobacter felis�CAssociated Gastric Disease in Microbiota-Restricted Mice

Human Helicobacter pylori infection leads to multiple pathological consequences, including gastritis and adenocarcinoma. Although this association has led to the classification of H. pylori as a type 1 carcinogen, it is not clear if additional nonhelicobacter gastric microbiota play a role in these diseases. In this study, we utilized either specific pathogen-free C57BL/6 mice (B6.SPF) or mice colonized with altered Schaedler flora (B6.ASF) to evaluate the role of nonhelicobacter gastric microbiota in disease development after Helicobacter felis infection. Despite similar histological changes, H. felis persisted in B6.ASF stomachs, while H. felis could no longer be detected in the majority of B6.SPF mice. The B6.SPF mice also acquired multiple Lactobacillus spp. in their stomachs after H. felis infection. Our data indicate that potential mechanisms responsible for the ineffective H. felis clearance in the B6.ASF model include the absence of new gastric microbiota to compete for the gastric niche, the lack of expression of new gastric mucins, and a reduced ratio of H. felis–specific IgG2c:IgG1 serum antibodies. These data suggest that although H. felis is sufficient to initiate gastric inflammation and atrophy, bacterial eradication and the systemic immune response to infection are significantly influenced by pre-existing and acquired gastric microbiota.