Modelling of β-d-glucopyranose ring distortion in different force fields: a metadynamics study

Modelling of carbohydrate conformations is a challenging task for force field developers. Three carbohydrate force fields, namely GLYCAM06, GROMOS 45a4 and OPLS were evaluated. Free energies of different ring conformations of β-d-glucopyranose were calculated using metadynamics in vacuum as well as in explicitly modelled water. All three force fields model the ⁴C₁ conformation as the most stable by at least 6 kJ/mol, as compared to other conformations. Interconversion from the ⁴C₁ to any other conformation is associated with a barrier of no lower than 26 kJ/mol. The free energy surface calculated in the GLYCAM06 force field is in remarkably good agreement with the recent Car-Parrinello metadynamics study. The effect of a water environment is relatively low and analogous in all tested force fields. Namely, the presence of water stabilizes the upper-left (^3,OB) versus bottom-right (B_3,O) area of Stoddard’s plot, relative to the situation in vacuum. Comparison of free and potential surfaces is also provided for vacuum calculations.     

Phylogeny of the leafy liverwort Ptilidium: cryptic speciation and shared haplotypes between the Northern and Southern Hemispheres

The small, phylogenetically isolated liverwort genus Ptilidium has been regarded as of cool-Gondwanic origin with the bipolar, terrestrial Ptilidium ciliare giving rise to the Northern Hemisphere epiphytes Ptilidium pulcherrimum and Ptilidium californicum. This hypothesis is examined using a dataset including three chloroplast DNA regions from 134 Ptilidium accessions and one accession each of its closest relatives Trichocoleopsis and Neotrichocolea. Maximum likelihood and parsimony analyses point to a close relationship between P. ciliare and P. pulcherrimum, whereas P. californicum is placed sister to the remainder of the genus, separated by a long branch. Haplotype analysis and our phylogeny indicate the presence of Southern Hemisphere haplotypes of P. ciliare in the Northern Hemisphere, and shared haplotypes of P. ciliare and P. pulcherrimum between Europe and North America. Based on our findings, we reject the Gondwana-scenario and propose recent long distance dispersal as an explanation for the bipolar disjunct range. Ptilidium ciliare is resolved as paraphyletic with P. pulcherrimum nested within it. An isolated Ptilidium lineage with the morphology of P. ciliare from the Himalaya region likely represents a hitherto unrecognized cryptic species. Ptilidium pulcherrimum splits into a Japanese clade and a clade with accessions from Europe and North America.     

Bacterial Endophytes from Vitis vinifera L. – Metabolomics Characterization of Plant-Endophyte Crosstalk

Bacterial endophytes are known to protect Vitis vinifera L. against various harmful effects of the environment and support its growth. However, for the most part, biochemical responses of such co-existence have not yet been fully elucidated. In this work, we aimed to characterize the activities of endophytic consortia in a plant-endophyte extract by measuring five indicators of colonization (overall endophyte metabolic activity, microbial ACC deaminase activity, ability to solubilize phosphorus, ability to convert atmospheric nitrogen to ammonia ions, and ability to produce growth promoting indole acetic acid), and find relationships between these activities and metabolome. The V. vinifera canes for the metabolomics fingerprinting were extracted successively with water and methanol, and analysed by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. For data processing, the MS-DIAL – MS-CleanR – MS-FINDER software platform was used, and the data matrix was processed by PCA and PLS-DA multivariate statistical methods. The metabolites that were upregulated with the heavy endophyte colonization were mainly chlorins, phenolics, flavonoid and terpenoid glycosides, tannins, dihydropyranones, sesquiterpene lactones, and long-chain unsaturated fatty acids.     

Kinetic Constant Variability in Bacterial Oxidation of Elemental Sulfur

Wide ranges of growth yields on sulfur (from 2.4 × 10¹⁰ to 8.1 × 10¹¹ cells g⁻¹) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter⁻¹ h⁻¹) of an Acidithiobacillus ferrooxidans strain (CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.     

Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondrial pyruvate dehydrogenase complex?

Monoclonal antibodies against the E1α subunit of pyruvate dehydrogenase (PDH) were used to quantify the mitochondrial pyruvate dehydrogenase complex (mtPDC) by enzyme-linked immunosorbent assay (ELISA). Recombinant Arabidopsis thaliana PDH (E1) was used to calibrate the ELISA. Antibodies against a synthetic phosphopeptide corresponding to phosphorylation site one of E1α were used in an ELISA to quantify phospho-PDC (P-PDC). For calibration of the second ELISA, recombinant E1 was phosphorylated in vitro with recombinant A. thaliana E1-kinase. The two ELISA were used to quantify mitochondrial total- and P-PDC in clarified homogenates from Nicotiana tabacum BY-2 suspension cells. The level of mtPDC remained constant throughout the 7-day growth cycle at 25.1 g⁻¹ FW. During the lag (days 0–2) and stationary (day 7) stages of the growth cycle, the mtPDC was completely phosphorylated (inactive), whereas during the log-growth stage it was completely dephosphorylated (active). Exposure of 3- or 7-day posttransfer suspension cells to osmotic stress significantly decreased proportion of P-PDC. A series of pharmacological studies were undertaken to gain insight into the signal transduction pathways coupling osmotic stress perception with control of mitochondrial respiration. Results from these studies indicate a signal transduction pathway linking stress perception to control of mitochondrial respiration that includes protein kinases and phosphoprotein phosphatases.     

Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm

A vector was constructed for expression of Arabidopsis thaliana mitochondrial pyruvate dehydrogenase (E1) in the cytoplasm of Trichoplusia ni cells. The construct pDDR101 comprises the mature-E1alpha coding sequence under control of the Polh promoter, plus the mature-E1beta coding sequence under control of the p10 promoter. The E1alpha sequence was engineered to include an N-terminal His-tag. When protein samples were subjected to immobilized metal ion affinity chromatography, the alpha- and beta-subunits co-eluted, indicating association. When the recombinant protein sample was analyzed further by gel permeation chromatography, it was demonstrated that a significant amount eluted at a size consistent with assembly into an alpha2beta2 heterotetramer. Recombinant E1 was able to decarboxylate [1-14C]pyruvate and was a substrate for in vitro phosphorylation by E1-kinase.     

Fetal and postnatal development of palmar,plantar, and digital pads,and flexion creases of the rat (Rattus norvegicus)

Fetal development of the hands and feet of rats was investigated to determine the feasibility of using rats as an experimental model for studying the factors influencing early development of the hands and feet, and especially the dermatoglyphics in humans. Eighty rat fetuses of 14–21 days gestational age and 80 newborn rats of 0–7 days of age were used to study the morphological features of the palmar, plantar, and digital areas and to determine the timing of appearance and the location of the volar pads and flexion creases. Comparisons between analogous developmental stages of rat and human fetuses demonstrate striking similarities in overall fetal development. Marked differences, however, were found between rat and human fetuses in the timing of developmental milestones and in some morphological features. The results indicate that rats can serve as a useful experimental model in studies of the utility of the epidermal ridge configurations and flexion creases in medical disorders, provided that the differences in the timing of development are taken into consideration. © 1996 Wiley-Liss, Inc.     

Stress reaction of kidney epithelial cells to inorganic solid-core nanoparticles

A route of accumulation and elimination of therapeutic engineered nanoparticles (NPs) may be the kidney. Therefore, the interactions of different solid-core inorganic NPs (titanium-, silica-, and iron oxide-based NPs) were studied in vitro with the MDCK and LLC-PK epithelial cells as representative cells of the renal epithelia. Following cell exposure to the NPs, observations include cytotoxicity for oleic acid-coated iron oxide NPs, the production of reactive oxygen species for titanium dioxide NPs, and cell depletion of thiols for uncoated iron oxide NPs, whereas for silica NPs an apparent rapid and short-lived increase of thiol levels in both cell lines was observed. Following cell exposure to metallic NPs, the expression of the tranferrin receptor/CD71 was decreased in both cells by iron oxide NPs, but only in MDCK cells by titanium dioxide NPs. The tight association, then subsequent release of NPs by MDCK and LLC-PK kidney epithelial cells, showed that following exposure to the NPs, only MDCK cells could release iron oxide NPs, whereas both cells released titanium dioxide NPs. No transfer of any solid-core NPs across the cell layers was observed.     

Vitamin C for DNA damage prevention

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels>50μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels>50μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels>50μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.