High-throughput sequencing reveals key genes and immune homeostatic pathways activated in myeloid dendritic cells by Porphyromonas gingivalis 381 and its fimbrial mutants.

The human microbiome consists of highly diverse microbial communities that colonize our skin and mucosal surfaces, aiding in maintenance of immune homeostasis. The keystone pathogen Porphyromonas gingivalis induces a dysbiosis and disrupts immune homeostasis through as yet unclear mechanisms. The fimbrial adhesins of P. gingivalis facilitate biofilm formation, invasion of and dissemination by blood dendritic cells; hence, fimbriae may be key factors in disruption of immune homeostasis. In this study we employed RNA-sequencing transcriptome profiling to identify differentially expressed genes (DEGs) in human monocyte-derived dendritic cells (MoDCs) in response to in vitro infection/exposure by Pg381 or its isogenic mutant strains that solely express minor-Mfa1 fimbriae (DPG3), major-FimA fimbriae (MFI) or are deficient in both fimbriae (MFB) relative to uninfected control. Our results yielded a total of 479 DEGs that were at least two-fold upregulated and downregulated in MoDCs significantly (P ≤ 0.05) by all four strains and certain DEGs that were strain-specific. Interestingly, the gene ontology biological and functional analysis shows that the upregulated genes in DPG3-induced MoDCs were more significant than other strains and associated with inflammation, immune response, anti-apoptosis, cell proliferation, and other homeostatic functions. Both transcriptome and quantitative polymerase chain reaction results show that DPG3, which solely expresses Mfa1, increased ZNF366, CD209, LOX1, IDO1, IL-10, CCL2, SOCS3, STAT3 and FOXO1 gene expression. In conclusion, we have identified key DC-mediated immune homeostatic pathways that could contribute to dysbiosis in periodontal infection with P. gingivalis.

Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition.

Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.

Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures.

The crystal structures of pyruvate decarboxylase from the yeast Saccharomyces uvarum and Saccharomyces cerevisiae have been determined at 2.4 and 2.3 A resolution, respectively. These structures provide details about the protein fold and domain assembly within subunits, about subunit assembly to form dimers and about dimer assembly to form tetramers. They also provide a clear picture of the active site centered on the thiamin diphosphate cofactor, and have allowed amino acids critical for catalysis and involved in stabilization of the unusual cofactor conformation to be identified. The structural information has enabled identification of the site of allosteric activation to be centered on Cys-221, and suggests that a six residue segment leading from the regulatory site to the catalytic site may be involved in transmission of a binding signal. The importance of several amino acids within this segment in the regulatory process, as well as some involved in stabilizing and activating the cofactor has been confirmed by analyzing the behavior of recombinant enzymes with single point mutations introduced at these sites. Additional structures have been determined for pyruvate decarboxylase in multiple crystal forms, some of which were obtained from crystals grown with known allosteric activators present in the media. Currently four distinct types of tetramers have been observed, with each showing a different mode of association of dimers to form the tetramers. In some of the cases involving the presence of allosteric activators drastic changes in the mode of dimer assembly to form tetramers is seen.

Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 A resolution.

The crystal structure of pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate-dependent enzyme isolated from Saccharomyces cerevisiae, has been determined and refined to a resolution of 2.3 A. Pyruvate decarboxylase is a homotetrameric enzyme which crystallizes with two subunits in an asymmetric unit. The structure has been refined by a combination of simulated annealing and restrained least squares to an R factor of 0.165 for 46,787 reflections. As in the corresponding enzyme from Saccharomyces uvarum, the homotetrameric holoenzyme assembly has approximate 222 symmetry. In addition to providing more accurate atomic parameters and certainty in the sequence assignments, the high resolution and extensive refinement resulted in the identification of several tightly bound water molecules in key structural positions. These water molecules have low temperature factors and make several hydrogen bonds with protein residues. There are six such water molecules in each cofactor binding site, and one of them is involved in coordination with the required magnesium ion. Another may be involved in the catalytic reaction mechanism. The refined model includes 1074 amino acid residues (two subunits), two thiamin diphosphate cofactors, two magnesium ions associated with cofactor binding and 440 water molecules. From the refined model we conclude that the resting state of the enzyme-cofactor complex is such that the cofactor is already deprotonated at the N4' position of the pyrimidine ring, and is poised to accept a proton from the C2 position of the thiazolium ring.

Technique for the isolation of secondary phases in processed magnesite.

The study describes a new wet chemical technique, termed ANM, for the isolation of secondary phases in magnesite processed at a temperature of 1650 degrees C. The major MgO (periclase) phase in processed magnesite was preferentially extracted as ammonium nitrate magnesium double salt by ammonium nitrate in ethanol medium. The residues containing enriched secondary phases were identified by XRD.

Structural studies on DNA-binding drugs: crystal structure and molecular dynamics studies of triazoloacridinones.

Structural and conformational studies on two 8-substituted triazoloacridinone antitumor agents, C1295 and C1303, have been carried out to compare the conformation of the (aminoalkyl)amino side chain and the effect of C-8 substitution. Crystal data for 5-[2-(diethylamino)ethylamino]-8-methyl-6H-[1,2,3]-triazolo[4,5,1- de]acridin-6-one (C1295), C20H23N5O, M(r) = 349.4, triclinic, P1, a = 12.200 (1), b = 14.890 (1), c = 5.185 (1) A, alpha = 93.54 (1), beta = 102.21 (1), gamma = 80.61 (1)degree, V = 907.9 (1) A3, Z = 2, Dx = 1.278 g cm-3, lambda(Cu K alpha) = 1.54178 A, mu = 6.2 cm-1, F(000) = 372, T = 293 K, R = 0.061 for 1631 observed reflections; for 5-[2-(diethylamino)ethylamino]-8- hydroxy-6H-[1,2,3]triazolo[4,5,1-de]acridin-6-one (C1303), C17H17N5O2, M(r) = 323.4, monoclinic, P2(1)/n, a = 15.823 (2), b = 5.790 (1), c = 16.856 (2) A, beta = 98.59 (1)degree, V = 1526.9 (2) A3, Z = 4, Dx = 1.404 g cm-3, lambda(Cu K alpha) = 1.54178 A, mu = 7.5 cm-1, F(000) = 680, T = 293 K, R = 0.054 for 1303 observed reflections. There is a difference in the orientation of the (aminoalkyl)amino side chain in the two compounds in the solid state, but molecular dynamics simulations indicate that in the gas phase the orientation is very similar. This difference could result from the different crystal packing in the case of C1303, due to the presence of an intermolecular hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Naphthyridinomycin-DNA adducts: a molecular modeling study.

Monocovalent groove binding complexes of antitumor antibiotic naphthyridinomycin and its analogs with DNA sequence d(ATGCAT)2 have been studied by molecular mechanics to understand which enantiomer of the drug and what chirality at C(7) of the drug are preferred for forming better drug-DNA adducts. The effect of hydroquinone intermediate and the substitution at C(11) on drug-DNA interactions have also been investigated. The results indicate that the enantiomer that forms the best adduct is different from the one reported earlier in the literature. The drug with an R configuration at C(7) is preferred for binding. The hydroquinone models do not necessarily provide a given analog of the drug with additional favorable DNA interactions. The substitution at C(11) by OH provides the best binding model. This finding agrees well with the results from previous biochemical studies. The sequence specific studies indicate that the sequence d(ATGCAT)2 is slightly preferred over others.

Structural, conformational, and theoretical binding studies of antitumor antibiotic porfiromycin (N-methylmitomycin C), a covalent binder of DNA, by X-ray, NMR, and molecular mechanics.

X-ray, NMR, and molecular mechanics studies on antitumor antibiotic porfiromycin (C16H20N4O5), a covalent binder of DNA, have been carried out to study the structure, conformation, and theoretical interactions with DNA. The crystal structure was solved by direct methods and refined to an R value of 0.052. The configurations at C(9), C(9a), C(1), and C(2) are S, R, S, and S, except for the orientation of the aziridine ring and (carbamoyloxy)methyl side chain. The five-membered ring attached to the aziridine ring adopts an envelope conformation. The solution conformation is similar to that observed in the solid state except for the (carbamoyloxy)methyl side chain. Monovalent and cross-linked models of the drug bound to DNA have been energetically refined by using molecular mechanics. The results indicate that, in the case of monocovalent binding, the drug clearly prefers a d(CpG) sequence rather than a d(GpC) sequence. In the case of the cross-linked model there is no clear-cut preference of d(CpG) over d(GpC), indicating that the binding preference of the drug may be kinetic rather than thermodynamic.

Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase.

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.