Pathogen-specific TLR2 Protein Activation Programs Macrophages to Induce Wnt-beta-Catenin Signaling

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus non-pathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-beta-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-beta-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-beta-catenin signaling as iNOS(-/-) mice demonstrated compromised ability to trigger activation of Wnt-beta-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-beta-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T(Reg) cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-beta-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

Crystallization and preliminary X-ray diffraction studies of sortase A from Streptococcus pneumoniae

Sortases are cell-membrane-anchored cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. Thus, they play critical roles in virulence, infection and colonization by pathogens. Sortases have been classified into four types based on their primary sequence and the target-protein motifs that they recognize. All Gram-positive bacteria express a class A housekeeping sortase (SrtA). Sortase A from Streptococcus pneumoniae (NP_358691) has been crystallized in two crystal forms. Diamond-shaped crystals of Delta N(59)SrtA diffracted to 4.0 angstrom resolution and belonged to a tetragonal system with unit-cell parameters a = b = 122.8, c = 86.5 angstrom, alpha = beta = gamma = 90 degrees, while rod-shaped crystals of Delta N(81)SrtA diffracted to 2.91 angstrom resolution and belonged to the monoclinic space group P2(1) with unit-cell parameters a = 66.8, b = 103.47, c = 74.79 angstrom, alpha = gamma = 90, beta = 115.65 degrees. The Matthews coefficient (V(M) = 2.77 angstrom(3) Da(-1)) with similar to 56% solvent content suggested the presence of four molecules in the asymmetric unit for Delta N(81)SrtA. Also, a multi-copy search using a monomer as a probe in the molecular-replacement method resulted in the successful location of four sortase molecules in the asymmetric unit, with statistics R = 41.61, R(free) = 46.44, correlation coefficient (CC) = 64.31, CC(free) = 57.67.

X-ray structure of a galactose-specific lectin from Spatholobous parviflorous

A galactose-specific seed lectin from Spatholobous parviflorus (SPL) has been purified, crystallized and its X-ray structure solved. It is the first lectin purified and crystallized from the genus Spatholobus (family: Fabaceae). The crystals belong to the space group P1, with a = 60.792 angstrom, b = 60.998 angstrom, c = 78.179 angstrom, alpha = 78.68 degrees, beta = 88.62 degrees, gamma = 104.32 degrees. The data were collected at 2.04 angstrom resolution under cryocondition, on a MAR image-plate detector system, mounted on a rotating anode X-ray generator. The coordinates of Dolichos biflorus lectin (1lu1) were successfully used for the structure solution by molecular replacement method. The primary structure of the SPL was not known earlier and it was unambiguously visible in the electron density. S. parviflorus lectin is a hetero-dimeric-tetramer with two alpha and two beta chains of 251 and 239 residues respectively. SPL has two metal ions, Ca(2+) and Mn(2+), bound to a loop region of each chain. The SPL monomers are in jelly roll form. (C) 2011 Elsevier B.V. All rights reserved.

Crystal structure of a beta-prism II lectin from Remusatia vivipara

The crystal structure of a beta-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4 A. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the ``monocot mannose-binding'' lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.

Quaternary association in beta-prism I fold plant lectins: Insights from X-ray crystallography, modelling and molecular dynamics

Dimeric banana lectin and calsepa, tetrameric artocarpin and octameric heltuba are mannose-specific beta-prism I fold lectins of nearly the same tertiary structure. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions that undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Energy minimization and simulations were also carried out on models using all possible oligomers by employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also led to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding.

Variation of the gas and radiation content in the sub-Keplerian accretion disk around black holes and its impact to the solutions

We investigate the variation of the gas and the radiation pressure in accretion disks during the infall of matter to the black hole and its effect to the flow. While the flow far away from the black hole might be non-relativistic, in the vicinity of the black hole it is expected to be relativistic behaving more like radiation. Therefore, the ratio of gas pressure to total pressure (beta) and the underlying polytropic index (gamma) should not be constant throughout the flow. We obtain that accretion flows exhibit significant variation of beta and then gamma, which affects solutions described in the standard literature based on constant beta. Certain solutions for a particular set of initial parameters with a constant beta do not exist when the variation of beta is incorporated appropriately. We model the viscous sub-Keplerian accretion disk with a nonzero component of advection and pressure gradient around black holes by preserving the conservations of mass, momentum, energy, supplemented by the evolution of beta. By solving the set of five coupled differential equations, we obtain the thermo-hydrodynamical properties of the flow. We show that during infall, beta of the flow could vary up to similar to 300%, while gamma up to similar to 20%. This might have a significant impact to the disk solutions in explaining observed data, e.g. super-luminal jets from disks, luminosity, and then extracting fundamental properties from them. Hence any conclusion based on constant gamma and beta should be taken with caution and corrected. (C) 2011 Elsevier B.V. All rights reserved.

A new pentameric structure of rotavirus NSP4 revealed by molecular replacement

The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 angstrom. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which unravelled a case of subtle variation between different oligomeric states unknown at the time of solving the structure, are presented in this paper.

The beta-Glucoside (bgl) Operon of Escherichia coli Is Involved in the Regulation of oppA, Encoding an Oligopeptide Transporter

We report that the bgl operon of Escherichia coli, encoding the functions necessary for the uptake and metabolism of aryl-beta-glucosides, is involved in the regulation of oligopeptide transport during stationary phase. Global analysis of intracellular proteins from Bgl-positive (Bgl(+)) and Bgl-negative (Bgl(-)) strains revealed that the operon exerts regulation on at least 12 downstream target genes. Of these, oppA, which encodes an oligopeptide transporter, was confirmed to be upregulated in the Bgl(+) strain. Loss of oppA function results in a partial loss of the growth advantage in stationary-phase (GASP) phenotype of Bgl(+) cells. The regulatory effect of the bgl operon on oppA expression is indirect and is mediated via gcvA, the activator of the glycine cleavage system, and gcvB, which regulates oppA at the posttranscriptional level. We show that BglG destabilizes the gcvA mRNA in vivo, leading to reduced expression of gcvA in the stationary phase. Deletion of gcvA results in the downregulation of gcvB and upregulation of oppA and can partially rescue the loss of the GASP phenotype seen in Delta bglG strains. A possible mechanism by which oppA confers a competitive advantage to Bgl(+) cells relative to Bgl(-) cells is discussed.

Role of Nonplanar Peptide Unit in Regular Polypeptide Helices - New Model for Pply-Beta-Benzyl-L-Aspartate

The effect of non-planarity of the peptide unit on helical structures stabilized by intrachain hydrogen bonds is discussed. While the present calculations generally agree with those already reported in the literature for right-handed helical structures, it is found that the most stable left-handed structure is a novel helix, called the delta-helix. Its helical parameters are close to these reported for poly-beta-benzyl-L -aspartate. Conformational energy calculations show that poly-beta-benzyl-L -aspartate with the delta-helical structure is considerably more stable than the structure it is generally believed to take up (the omega-helix) by about 15 kcal/mol-residue.

The electrical switching and thermal behavior of bulk Ge(15)Te(85-x)Sn(x) and Ge(17)Te(83-x)Sn(x) glasses

Bulk Ge(15)Te(85 - x)Sn(x) and Ge(17)Te(83 - x)Sn(x) glasses, are found to exhibit memory type electrical switching. The switching voltages (V(t)) and thermal stability of Ge(15)Te(85 - x)Sn(x) and Ge(17)Te(83 - x)Sn(x) glasses are found to decrease with Sn content. The composition dependence of v, has been understood on the basis of the decrease in the OFF state resistance and thermal stability of these glasses with tin addition. X-ray diffraction studies reveal that no elemental Sn or Sn compounds with Te or Ge are present in thermally crystallized Ge-Te-Sn samples. This indicates that Sn atoms do not interact with the host matrix and form a phase separated network of its own, which remains in the parent glass matrix as an inclusion. Consequently, there is no enhancement of network connectivity and rigidity. The thickness dependence of switching voltages of Ge(15)Te(85 - x)Sn(x) and Ge(17)Te(83 - x)Sn(x) glasses is found to be linear, in agreement with the memory switching behavior shown by these glasses. (C) 2011 Elsevier B.V. All rights reserved.

C-H functionalization of tertiary amines by cross dehydrogenative coupling reactions: solvent-free synthesis of alpha-aminonitriles and beta-nitroamines under aerobic condition

A solvent-free synthesis of alpha-aminonitriles and beta-nitroamines by oxidative cross-dehydrogenative coupling under aerobic condition is reported. A catalytic amount of molybdenum(VI) acetylacetonoate was found to catalyze cyanation of tertiary amines to form alpha-aminonitriles, whereas vanadium pentoxide was found to promote aza-Henry reaction to furnish beta-nitroamines. Both of these environmentally benign reactions are performed in the absence of solvents using molecular oxygen as an oxidant.