Uncultured archaea dominate in the thermal groundwater of Uzon Caldera,Kamchatka

The thermoacidophilic microbial community inhabiting the groundwater with pH 4.0 and temperature 50°C at the East Thermal Field of Uzon Caldera, Kamchatka, was examined using pyrosequencing of the V3 region of the 16S rRNA gene. Bacteria comprise about 30% of microorganisms and are represented primarily by aerobic lithoautotrophs using the energy sources of volcanic origin—thermoacidophilic methanotrophs of the phylum Verrucomicrobia and Acidithiobacillus spp. oxidising metals and reduced sulfur compounds. More than 70% of microbial population in this habitat were represented by archaea, in majority affiliated with “uncultured” lineages. The most numerous group (39% of all archaea) represented a novel division in the phylum Euryarchaeota related to the order Thermoplasmatales. Another abundant group (33% of all archaea) was related to MCG1 lineage of the phylum Crenarchaeota, originally detected in the Yellowstone hot spring as the environmental clone pJP89. The organisms belonging to these two groups are widely spread in hydrothermal environments worldwide. These data indicate an important environmental role of these two archaeal groups and should stimulate the investigation of their metabolism by cultivation or metagenomic approaches.     

Dependence of Deformability of Geometries and Characteristics of Intramolecular Hydrogen Bonds in Canonical 2′-Deoxyribonucleotides on DNA Conformations

Abstract

The molecular structure and deformability (with respect to average geometry) of methyl ethers of canonical 2′-deoxyribonucleotides thymidine-5′-phosphate (mTMP), 2-deoxycytidine-5′- phosphate (mCMP), 2-deoxyadenosine-5′-phosphate (mAMP) and 2′-deoxyguanosine-5′- phosphate (mGMP) in different types of DNA have been calculated using B3LYP/cc-pvdz method. Comparison of energy at equilibrium conformations of nucleotides and conformations with torsion angles of backbone fixed to average values for different types of DNA reveals that incorporation of nucleotides to A-DNA macromolecules requires the minimum amount of deformation energy. Therefore, this type of DNA should be the least strained from viewpoint of intramolecular deformations of monomers. Modeling of environmental effects within the PCM approach reveals that the immersion of nucleotides in polar medium results in significant decrease of energy differences between anti conformers of all DNTs and syn conformers of mGMP This also leads to reduction by almost a half nucleotides' deformation energy facilitating formation of DNA macromolecule. Change of DNTs conformation causes switch between different types of intramolecular H bonds. Every type of DNA possesses unique set of intramolar hydrogen bonds in nucleotides.     

Adamantyl thioureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing one or two thiourea groups has been developed. Inhibition potency of the described compounds ranges from 50?μM to 7.2?nM. 1,7-(Heptamethylene)bis[(adamant-1-yl)thiourea] (6f) was found to be the most potent sEH inhibitor, among the thioureas tested. The inhibitory activity of the thioureas against the human sEH is closer to the value of activity against rat sEH rather than murine sEH. While being less active, thioureas are up to 7-fold more soluble than ureas, which makes them more bioavailable and thus promising as sEH inhibitors.     

Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha)

Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data.     

Improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV).

An improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV) is proposed. The method is based on the use of IgG purified from immune rabbit serum conjugated with biotin. Optimized and validated materials for the test can be stored for a long time in the form of ready-to-use kits. Optimization included selection of anti-poliovirus rabbit antibody batches with the best specificity to D-antigen as well as finding the most efficient parameters for all steps of ELISA protocol. The assay is based on direct ("sandwich") ELISA scheme, in which antigens are captured on ELISA plates coated with purified rabbit polyclonal D-antigen specific IgG raised against wild polioviruses of three serotypes. D-antigen specificity of the IgG was at least 10 times higher than to H-antigen (heat-inactivated virus). The presence of antigen was detected using biotin-conjugated IgG from the same source. Eight-point dose-response curves were obtained for each sample and the reference vaccine. The protocol ensured low background (less than 0.2 OD), linear response over the entire range of optical density measurements (up to 3.0 OD), and high precision of data (assay variability was about 3%). The quantitative results and the validity of the test were determined by two numerical approaches, linear regression and a new analysis procedure called the local interpolation method. For the first approach we also proposed a new method for testing of parallelism of regression lines. The ELISA protocol for all three types of poliovirus is based on standard off-the-shelf reagents, and is highly reproducible and reliable. An in-house Reference Reagent was formulated and calibrated against the International Reference for IPV.     

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

Low molecular weight chitosan is an efficient inhibitor of ribonucleases

RNase inhibitors are commonly used to block the RNase activity in manipulations with RNA-containing preparations. Recently RNase inhibitors, either synthetic or natural, have been intensively sought because they appeared to be promising for therapy of cancer and allergy. However, there is only a limited number of efficient RNase inhibitors. We have shown that a low molecular weight chitosan (M(r) approximately 6 kDa) inhibits activity of pancreatic RNase A and some bacterial RNases with inhibition constants in the range of 30-220 nM at pH 7.0 and ionic strength 0.14 M. The preferential contribution to the chitosan complex formation with RNases is due to establishment of 5-6 ion pairs. The results of this work show that polycations may efficiently inhibit ribonuclease activities.     

Exploring sequence-structure relationships in the tyrosine kinome space: functional classification of the binding specificity mechanisms for cancer therapeutics

MOTIVATION: Evolutionary and structural conservation patterns shared by more than 500 of identified protein kinases have led to complex sequence-structure relationships of cross-reactivity for kinase inhibitors. Understanding the molecular basis of binding specificity for protein kinases family, which is the central problem in discovery of cancer therapeutics, remains challenging as the inhibitor selectivity is not readily interpreted from chemical proteomics studies, neither it is easily discernable directly from sequence or structure information. We present an integrated view of sequence-structure-binding relationships in the tyrosine kinome space in which evolutionary analysis of the kinases binding sites is combined with computational proteomics profiling of the inhibitor-protein interactions. This approach provides a functional classification of the binding specificity mechanisms for cancer agents targeting protein tyrosine kinases. RESULTS: The proposed functional classification of the kinase binding specificities explores mechanisms in which structural plasticity of the tyrosine kinases and sequence variation of the binding-site residues are linked with conformational preferences of the inhibitors in achieving effective drug binding. The molecular basis of binding specificity for tyrosine kinases may be largely driven by conformational adaptability of the inhibitors to an ensemble of structurally different conformational states of the enzyme, rather than being determined by their phylogenetic proximity in the kinome space or differences in the interactions with the variable binding-site residues. This approach provides a fruitful functional linkage between structural bioinformatics analysis and disease by unraveling the molecular basis of kinase selectivity for the prominent kinase drugs (Imatinib, Dasatinib and Erlotinib) which is consistent with structural and proteomics experiments.