NMR studies of lantibiotics. The structure of nisin in aqueous solution.

Nisin is a posttranslationally modified protein of 34 amino acids, and is a member of the class of bacteriocidal polypeptides known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of NMR data, i.e. interproton distance constraints derived from nuclear Overhauser enhancement spectroscopy and torsion angle constraints derived from double-quantum-filtered correlated spectroscopy. Translation of the NMR constraints into a three-dimensional structure was carried out with the distance-geometry program DISMAN, followed by restrained energy minimization using CHARMm. The internal mobility of the peptide chain prohibited the determination of a precise overall folding of the molecule, but parts of the structure could be obtained, albeit sometimes with low resolution. The structure of nisin can best be defined as follows. The outermost N-terminal and C-terminal regions of nisin appear quite flexible, the remainder of the molecule consists of an amphiphilic N-terminal fragment (residues 3-19), joined by a flexible 'hinge' region to a rigid double-ring fragment formed by residues 23-28. The latter fragment has the appearance of a somewhat overwound alpha-helix. It is suggested, by assuming the presence of a (transient) alpha-helical structure in this part of prenisin, that the coupling between residues 23 and 26, as well as between 25 and 28, by thioether bridges, and the inversion of the C alpha chiralities at positions 23 and 25, can be rationalized.     

The three-dimensional structure of a DNA hairpin in solution two-dimensional NMR studies and structural analysis of d(ATCCTATTTATAGGAT)

The hairpin formed by d(ATCCTATTTATAGGAT) was studied by means of two-dimensional NMR spectroscopy and conformational analysis. Almost all 1H resonances of the stem region could be assigned, while the 1H and 31P spectra of the loop region were interpreted completely; this includes the stereospecific assignment of the H5' and H5" resonances. The derivation of the detailed loop structure was carried out in a stepwise fashion including some improved and new methods for structure determination from NMR data. In the first step, the mononucleotide structures were examined. The conformational space available to the mononucleotide was scanned systematically by varying the glycosidic torsion angle and pseudorotational parameters. Each generated conformer was tested against the experimental J coupling constants and NOE parameters. In the following stage, the structures of dinucleotides and longer fragments were derived. Inter-residue distances between protons were calculated by means of a procedure in which the simulated NOEs, obtained via a relaxation-matrix approach, were fitted to the experimental NOEs without the introduction of a molecular model. In addition, the backbone torsion angles beta, gamma and epsilon were deduced from homocoupling and heterocoupling constants. These data served as constraints in the next step, in which the loop sequence was subjected to a multi-conformer generation procedure. The resulting structures were tested against the mentioned constraints and disregarded if these constraints were violated. This yielded a family of structures for the loop region, confined to a relatively narrow conformational space. A representative conformation was subsequently docked on a B-type stem which fulfilled the structural constraints (derived from the NMR experiments for the stem region) to yield the hairpin structure. Results obtained from subsequent restrained-molecular-mechanics as well as free-molecular-mechanics calculations are in accordance with those obtained by means of the analysis described above. The structure of the hairpin loop is a compactly folded conformation and the first base of the central TTTA region forms a Hoogsteen T-A pair with the fourth base. This Hoogsteen base pair is stacked upon the sixth base pair of the B-type double-helical stem. The second base of the loop is folded into the minor groove, whereas the third base of the loop is partly stacked on the first and fourth bases. The phosphate backbone exhibits a sharp turn between the third and fourth nucleotides of the loop. The peculiar structure of this hairpin loop is discussed in relation to loop folding in DNA and RNA hairpins and in relation to a general model for loop folding.     

Long-range interactions in mammalian platelet aggregation. II. The role of platelet pseudopod number and length.

In part 1, we reported that human (H) platelets, activated with high concentrations (10 microM) of adenosine diphosphate, aggregate under Brownian diffusion (nonstirred, platelet-rich plasma) with an apparent efficiency of collision (alpha B) approximately 4 times and 8 times larger than observed, respectively, for canine (C) and rabbit (R) platelets. Further evaluations of parallel inhibition of alpha B and shape change suggested a central role for platelet pseudopods in mediating the long-range interactions associated with the elevated alpha B values. We found that greater than 90% of all platelet contacts in the doublets and triplets formed were via at least one pseudopod. We therefore compared pseudopod number and length per platelet generated by approximately 30 s post ADP activation in nonstirred PRP from human, canine, and rabbit donors, using phase-contrast, video-enhanced microscopy of fixed platelets. Theoretical calculations assessing the effects of pseudopod length and number on the collision frequency enhanced by an increased radius of a collision sphere supported the experimental observations that approximately 3 or 4 pseudopods per human or canine platelet, and approximately 5 or 6 pseudopods per rabbit platelet yield optimal alpha B values, with the average pseudopod length: approximately 3:2:1 for H/C/R, paralleling the alpha B differences. After correcting for effects of pseudopods and platelet size on platelet diffusion and sedimentation, it still appeared that the small number of long pseudopods formed on human platelets could largely explain the unusually large alpha B values. The quantitative discrepancies between theory and experiment do not appear related to time-dependent refractoriness within the less than 60 s of observation, but may be related to biochemical differences in dynamics and surface density of adhesive (sticky sites) present on the pseudopod surface.     

Culture of Candida in vinasse and molasses: Effect of acid and salt addition on biomass and raw protein production

Species of the genera Candida grown in vinasse and molasses were studied under the following conditions: agitation of containers, pH 4.6, culture time of 24 hours at 30°C. The greatest biomass production of C. krusei grown in vinasse was obtained with the addition of 0.1% H₃PO₄, and of C. guilliermondii and C. utilis with the addition 0.02% urea plus 0.03% H₃PO₄. Protein levels near 50% were found in C. utilis in vinasse supplemented either with molasses, with 0.05% MgSO₄, or with 0.02% urea plus 0.03% H₃PO₄.     

A strain of <Emphasis Type="Italic">Meyerozyma guilliermondii</Emphasis> isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.     

Semantic Similarity in Biomedical Ontologies

In recent years, ontologies have become a mainstream topic in biomedical research. When biological entities are described using a common schema, such as an ontology, they can be compared by means of their annotations. This type of comparison is called semantic similarity, since it assesses the degree of relatedness between two entities by the similarity in meaning of their annotations. The application of semantic similarity to biomedical ontologies is recent; nevertheless, several studies have been published in the last few years describing and evaluating diverse approaches. Semantic similarity has become a valuable tool for validating the results drawn from biomedical studies such as gene clustering, gene expression data analysis, prediction and validation of molecular interactions, and disease gene prioritization.     

Groundwater promotes emergence of asporogenic mutants of emetic Bacillus cereus

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.     

HLA-DR alpha 2 mediates negative signalling via binding to Tirc7 leading to anti-inflammatory and apoptotic effects in lymphocytes in vitro and in vivo

Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRalpha2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-zeta chain & ZAP70, and inhibition of IFN-gamma and FasL expression. HLA-DRalpha2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRalpha2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway.