Modelling of Aniline-Vermiculite and Tetramethylammonium-Vermiculite; Test of Force Fields

Molecular mechanics simulations in Cerius² have been used for modelling vermiculite intercalated with tetramethylammonium and aniline cations. The published structure data obtained for these intercalated structures from X-ray single crystal diffraction have been used to test the force fields and modelling strategy for organo-clays. The strategy of modelling was based on the nonbond host-guest interactions and on rigid silicate layers and rigid guest species. The rigidity of silicate layers requires that the cell parameters a, b and % are kept fixed during the energy minimisation. The energy term was set up using the nonbond interaction terms only and the Crystal Packer module in Cerius² has been used for the energy minimisation. In Crystal Packer the rigid units, i.e. the silicate layers and guest species can be translated and rotated during energy minimisation and the cell parameters c, !, and # have been varied. Three sets of Van derWaals (VDW) parameters available in Crystal Packer: Tripos, Universal and Dreiding have been used in present molecular simulations. Ab initio MP2 calculations were performed to justify the application of the force field. The best agreement of molecular mechanics simulations with both: experimental and ab initio data was obtained with the Tripos VDW parameters for both intercalates. The results of modelling are in good agreement with the experimental data as to the cell parameters and the interlayer packing. The cell parameters reported by Vahedi-Faridi and Guggenheim (1997) for tetramethylammonium-vermiculite are: c = 13.616 Å, ! = 90°, # = 97.68° ; from the present modelling we obtained: c = 13.609 Å, ! = 90.19°, # = 97.56°. Tetramethylammonium-cations are arranged in one layer in the interlayer space. One C-C edge of NC₄ tetrahedra is perpendicular to the silicate layers. The deep immersion of the methyl groups into the ditrigonal cavities suggested by Vahedi-Faridi and Guggenheim was not confirmed by modelling. Slade and Stone (1984) presented the measured cell parameters for aniline vermiculite: c = 14.89 Å, ! = 90°, # = 97°; present result is: c = 14.81 Å, ! = 90.72°, # = 96.70° for partially exchanged vermiculite and c = 14.84 Å, ! = 90.53°, # = 97.17° for fully exchanged vermiculite. The aniline cations are positioned over the ditrigonal cavities alternating in their anchoring to lower and upper silicate layer. The C-N bonds are perpendicular to layers.     

Critical evaluation of the JDO API for the persistence and portability requirements of complex biological databases

Background  

Complex biological database systems have become key computational tools used daily by scientists and researchers. Many of these systems must be capable of executing on multiple different hardware and software configurations and are also often made available to users via the Internet. We have used the Java Data Object (JDO) persistence technology to develop the database layer of such a system known as the SigPath information management system. SigPath is an example of a complex biological database that needs to store various types of information connected by many relationships.     

Alternative splicing and protein function

Background  

Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.     

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

A novel heterobinuclear mixed valence complex [Fe^IIICu^II(BPBPMP)(OAc)₂]ClO₄, 1, with the unsymmetrical N₅O₂ donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H₂BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe^III–()OH–Cu^IIOH₂]⁺ species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe^III-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe^IIIM^II, where M^II is Zn^II, Mn^II, Ni^II, or Fe^II). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 M), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7×10⁷ over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.Electronic Supplementary Material Supplementary material is available for this article at .     

Macrophages expressing triggering receptor expressed on myeloid cells-1 are underrepresented in the human intestine

Triggering receptor expressed on myeloid cells (TREM)-1 is a cell surface molecule on neutrophils and monocytes/macrophages implicated in the amplification of inflammatory responses by enhancing degranulation and secretion of proinflammatory mediators. Macrophages play an important role in the intestinal mucosal immune system, because they are preferentially localized in the subepithelial region. Despite the presence of enormous numbers of bacteria in the colonic mucosa and the close proximity between mucosal macrophages and luminal bacteria, the intestinal mucosa normally displays minimal signs of inflammation. In this study, we show that the resident macrophage population in normal human small and large intestine contains only few TREM-1-expressing macrophages (<10%), whereas the overwhelming majority of monocytes (>90%) and macrophages from lymph nodes or tonsils (>80%) express TREM-1 on the cell surface. These findings were confirmed by FACS analysis and immunostainings of frozen tissue sections. The differential expression of TREM-1 greatly affects the functional capacities of monocytes and tissue macrophages. Although monocytes and macrophages from spleen, lymph nodes, or tonsils show a substantial increase in oxidative burst after TREM-1 cross-linking, no effect is seen in intestinal macrophages. Intriguingly, in contrast to monocytes, intestinal macrophages fail to up-regulate TREM-1 in response to TNF. This refractory state may be induced in intestinal macrophages by the local presence of IL-10 and TGF-beta, because these two immunoregulatory cytokines synergistically down-regulate TREM-1 expression on monocytes in vitro. The absence of TREM-1 expression on lamina propria macrophages is likely to prevent excessive inflammatory reactions, and thus, excessive tissue damage in the intestine.     

Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia

ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X(7)R antagonists. Isolation of shed vesicles, followed by IL-1beta evaluation by a specific ELISA revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1beta efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pretreatment with the P2X(7) antagonist oxidized ATP, thus indicating a crucial involvement of the pore-forming P2X(7)R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1beta release from these cells.     

Membrane protein reconstitution and crystallization by controlled dilution

Efficient reconstitution of membrane proteins for functional analyses can be achieved by dilution of a ternary mixture containing proteins, lipids and detergents. Once the dilution reaches the point where the free detergent concentration would become lower than the critical micellar concentration, detergent is recruited from the bound detergent pool, and association of proteins and lipids is initiated. Here we show that dilution is also suitable for the assembly of two-dimensional crystals. A device has been designed that allows controlled dilution of a protein-lipid-detergent mixture to induce formation of densely packed or crystalline proteoliposomes. Turbidity is used to monitor the progress of reconstitution on-line, while dilution is achieved by computer-controlled addition of buffer solution in sub-microliter steps. This system has mainly been tested with porin OmpF, a typical beta-barrel protein, and aquaporin-1, a typical alpha-helical protein. The results demonstrate that large, highly ordered two-dimensional crystals can be produced by the dilution method.     

Cross-presentation of Listeria monocytogenes-derived CD4 T cell epitopes

Listeriolysin O (LLO) mediates the evasion of Listeria monocytogenes from the phagolysosome into the cytoplasm of the host cell. The recognition of infected cells by CD4 T cells is thought to be limited by the evasion of bacteria from the phagolysosome and also by the direct LLO-mediated inhibition of CD4 T cell activation. To analyze the influence of these immunoevasive mechanisms on the antilisterial CD4 T cell response, the expansion of L. monocytogenes-specific CD4 and CD8 T cells was monitored in infected mice. It was found that expansion of L. monocytogenes-specific CD4 T cells occurred synchronously with CD8 T cell expansion. The analysis of Ag presentation by macrophages and dendritic cells isolated from spleens of infected mice revealed efficient presentation of L. monocytogenes-derived CD4 T cell epitopes that was not dependent on the actA-mediated intercellular spread of bacteria. The further in vitro Ag presentation analysis revealed that although L. monocytogenes-infected macrophages and dendritic cells were poor presenters of CD4 T cell epitopes, more efficient presentation occurred after cocultivation of noninfected dendritic cells or macrophages with infected cells. These data indicate that the suppressive effect of LLO on the antilisterial CD4 T cell response is maintained only in infected APC and support the hypothesis that cross-priming plays a role in the induction of the strong CD4 T cell response in Listeria-infected mice.     

Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation

Proteolytic cleavage in an exposed loop of human tartrate-resistant acid phosphatase (TRAcP) with trypsin leads to a significant increase in activity. At each pH value between 3.25 and 8.0 the cleaved enzyme is more active. Substrate specificity is also influenced by proteolysis. Only the cleaved form is able to hydrolyze unactivated substrates efficiently, and at pH >6 cleaved TRAcP acquires a marked preference for ATP. The cleaved enzyme also has altered sensitivity to inhibitors. Interestingly, the magnitude and mode of inhibition by fluoride depends not only on the proteolytic state but also pH. The combined kinetic data imply a role of the loop residue D158 in catalysis in the cleaved enzyme. Notably, at low pH this residue may act as a proton donor for the leaving group. In this respect the mechanism of cleaved TRAcP resembles that of sweet potato purple acid phosphatase.