Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.     

Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity

Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.     

Anchor profiles of HLA-specific peptides: analysis by a novel affinity scoring method and experimental validation

The study of intermolecular interactions is a fundamental research subject in biology. Here we report on the development of a quantitative structure-based affinity scoring method for peptide-protein complexes, named PepScope. The method operates on the basis of a highly specific force field function (CHARMM) that is applied to all-atom structural representations of peptide-receptor complexes. Peptide side-chain contributions to total affinity are scored after detailed rotameric sampling followed by controlled energy refinement. A de novo approach to estimate dehydration energies was developed, based on the simulation of individual amino acids in a solvent box filled with explicit water molecules. Transferability of the method was demonstrated by its application to the hydrophobic HLA-A2 and -A24 receptors, the polar HLA-A1, and the sterically ruled HLA-B7 receptor. A combined theoretical and experimental study on 39 anchor substitutions in FxSKQYMTx/HLA-A2 and -A24 complexes indicated a prediction accuracy of about two thirds of a log-unit in Kd. Analysis of free energy contributions identified a great role of desolvation and conformational strain effects in establishing a given specificity profile. Interestingly, the method rightly predicted that most anchor profiles are less specific than so far assumed. This suggests that many potential T-cell epitopes could be missed with current prediction methods. The results presented in this work may therefore significantly affect T-cell epitope discovery programs applied in the field of peptide vaccine development.     

Hypothyroidism induces expression of the peptide transporter PEPT2

The kidney is a target organ for thyroid hormone action and a variety of renal transport processes are altered in response to impaired thyroid functions. To investigate the effect of thyroid hormone on the expression of the renal proximal tubular high-affinity-type H(+)-peptide cotransporter (PEPT2) in rats, hypothyroidism was induced in animals by administration of methimazole (0.05%) via drinking water. After 7 weeks of treatment, hypothyroidism was confirmed by determining serum free T(3) and free T(4) concentrations. Northern blotting was used to examine the expression of PEPT2 mRNA in kidney tissues from hypothyroid rats compared to control rats. Hypothyroidism resulted in an increased level of total renal PEPT2 mRNA (121.1+/-3.3% vs. control 100+/-2.8%; p=0.008). The mRNA results were confirmed by immuno-blotting, which demonstrated significantly increased protein levels (162% vs. control 100%; p<0.01). Immunohistochemistry also revealed increased PEPT2 protein levels in the proximal tubules of treated compared to non-treated rats. In summary, PEPT2 is the first proximal tubule transporter protein that shows increased expression in states of hypothyreosis. As PEPT2 reabsorbs filtered di- and tripeptides and peptide-like drugs, the present findings may have important implications in nutritional amino acid homeostasis and for drug dynamics in states of altered thyroid function.     

Asymmetric binding of membrane proteins to GroEL

The interaction of GroEL with non-native soluble proteins has been studied intensively and structure-function relationships have been established in considerable detail. Recently, we found that GroEL is also able to bind membrane proteins in the absence of detergents and deliver them to liposomes in a biologically active state. Here, we report that three well-studied membrane proteins (bacteriorhodopsin, LacY, and the bacteriophage lambda holin) bind asymmetrically to tetradecameric GroEL. Each of the membrane proteins was visualized in one of the center cavities of GroEL using single particle analysis.     

Vesicle mobility studied in cultured astrocytes

Astrocytes release many neuroactive substances, which are stored in membrane bound vesicles and may play a role in synapse modulation and in the coupling between neuronal activity and the local blood flow. However, the mobility of these vesicles in astrocytes has not been studied yet. We here used a fluorescently tagged proatrial natriuretic peptide to label single vesicles and dynamic microscopy to monitor their mobility. To track and analyze labeled vesicles, we employed a computer software. We found two modes of vesicle mobility, directional and non-directional. The mobility of non-directional vesicles is likely determined mainly by free diffusion. Only directional vesicles displayed a straight-line motion. The relationship of mean square displacement with time in directional vesicles resembled a quadratic function, indicating that in addition to free diffusion other mechanisms may contribute to vesicle movements in astrocytes, the biophysical properties of which are similar to those of neurons.     

ATPase activity of non-ribosomal peptide synthetases

Adenylation domains of non-ribosomal peptide synthetases (NRPS) catalyse the formation of aminoacyl adenylates, and in addition synthesize mono- and dinucleoside polyphosphates. Here, we show that NRPS systems furthermore contain an ATPase activity in the range of up to 2 P(i)/min. The hydrolysis rate by apo-tyrocidine synthetase 1 (apo-TY1) is enhanced in the presence of non-cognate amino acid substrates, correlating well with their structural features and the diminishing adenylation efficiency. A comparative analysis of the functional relevance of an analogous sequence motif in P-type ATPases and adenylate kinases (AK) allowed a putative assignment of the invariant aspartate residue from the TGDLA(V)R(K) core sequence in NRPS as the Mg(2+) binding site. Less pronounced variations in ATPase activity are observed in domains with relaxed amino acid specificity of gramicidin S synthetase 2 (GS2) and delta-(L-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), known to produce a set of substitutional variants of the respective peptide product. These results disclose new perspectives about the mode of substrate selection by NRPS.     

Microbiological external quality assurance program (MEQAP)--evaluation of results obtained in sanitary epidemiological laboratories in 2002

The aim of this study was to evaluate reliability of identification and determination of sensitivity to antibiotics and chemotherapeutics of some Gram positive cocci strains in 34 sanitary-epidemiological stations. All laboratories engaged in this study received 3 strains: S. aureus (S. aureus SC+ CF+, resistant to methicillin., or S. aureus SC - CF+, sensitive to methicillin), coagulase-negative staphylococci (S. epidermidis or S. haemolyticus or S. saprophyticus) and Enterococcus sp. (E. faecalis HLAR-positive or E. faecalis HLAR-negative or E. faecium or E. gallinarum). All these strains previously were identified in Department of Bacteriology of National Institute of Hygiene. Of the 68 staphylococci strains tested, 66 isolates were correctly identified. Among the 34 enterococcal strains studied the greatest difficulty in identification was caused by E. gallinarum strain--(8 out of 13 strains were incorrectly recognised). The determination of the sensitivity of the control strains to antibiotics and chemotherapeutic agents, generally was performed correctly in accordance with to the NCCLS and national recommendations. Some incorrect results of the antibiograms were caused by an erroneous interpretation of the zones of inhibition.