Its structure has cubic symmetry (space group P213) at room temperature. The structure contains a network of corner-sharing ZrO6 octahedra (.3. symmetry) and MO4  tetrahedra (.3. symmetry). Along the main threefold axis of the cubic unit cell, the MO4 tetrahedra are arranged in pairs forming M               2O8 units in which the M1O4 tetrahedra have larger distortions in terms of bond distances and angles than the M2O4 tetrahedra. These units are disordered over two possible orientations, with the M—Oterminal vectors pointing to the [111] or [M               2O8 units results from the concerted flips of these units. The time-averaged proportions of flipped and unflipped M               2O8 units were determined and the fraction of unflipped M               2O8 units is about 0.95. The order degree of the M               2O8 unit orientation is about 0.9. During the reversal process, the M-atom site has a migration about 0.93 Å, one of the O-atom sites has a 0.25 Å migration distance, whereas two other O-atom sites migrate marginally (≃ 0.08 Å). The results prove the constraint strategy to be a reasonable approach based on the ratcheting mechanism.The solid solution in the system Zr–Mo–W–O with composition ZrW Å                           V = 767.79 (1) Å3                                                   Z = 4                           Time-of-flight radiationλ = 0.5–4.4 ÅT = 298 K                           Specimen shape: cylinder30 × 10 × 10 mm                                 GPPD diffractometerSpecimen mounting: standard cylindrical vanadium sample holderScan method: time of flightmin = 53, 2θmax = 145°2θ                                 R                           p = 0.032                           R                           wp = 0.044                           1893 reflections58 parameters                                 et al., 2006GSAS  used to refine structure: GSAS; molecular graphics: VICS-II (Izumi & Dilanian, 2005GSAS.Data collection: IPNS local software (Worlton eele 2000; data re10.1107/S1600536809015281/wm2222sup1.cif            Crystal structure: contains datablocks global, I, DXB-W1.75_p_01, DXB-W1.75_p_02. DOI: 10.1107/S1600536809015281/wm2222Isup2.rtv            Rietveld powder data: contains datablocks I, I, I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Both exist in an octahedral environment composed of four N atoms of the N3               − groups and two N atoms from two monodentate 1-phenyl-3-propan-1-one ligands that are positioned trans to each other. Adjacent CdII centres in the crystal structure are bridged by a pair of N3               − anions in a μ-1,3-fashion, forming an infinite one-dimensional array.In the crystal structure of the title complex, [Cd(N For related literature, see: Guo & Cai 2007.       3)2(C11H11N31)2] = 0.031                           wR(F                           2) = 0.069                           S = 1.09                           8928 reflections667 parametersH-atom parameters constrainedmax = 0.67 e Å−3                        Δρmin = −0.72 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXTL.Data collection: 10.1107/S1600536808012609/bv2096sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808012609/bv2096Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The 2-chloro-6-pyridine molecule acts as a terminal ligand. The thiocyanate ligands function as μ1,3-bridging units connecting adjacent CdII atoms with a separation of 5.7525 (11) Å, forming a one-dimensional chain along the a axis.In the crystal structure of the title complex, [Cd(NCS) DOI: 10.1107/S1600536808030547/is2339Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The 2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinium ring is oriented at a dihedral angle of 44.2 (1)° with respect to the planar phenyl ring systems. The AuIII atom has a square-planar environment defined by four Cl atoms. The crystal structure is stabilized by C—H⋯π and Au⋯π ring–metal (3.551 Å) interactions. In the crystal structure, the molecules stack along the c axis via N—H⋯N hydrogen-bond interactions.Both the cation and anion of the title compound, (C DOI: 10.1107/S1600536809011994/hg2496Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The N-furanoylbenzenamine units are planar (maximum deviations = 0.316 and 0.132 Å). Each electron-deficient acylfuran stacks (centroid–centroid separations of the two pairs of π–π stacked aromatic rings are 3.918 and 3.953 Å) with the electron-rich benzenamine of the other N-furanoylbenzenamine unit, leading to a spiral structure. The conformation is stabilized by two bifurcated intramolecular N—H⋯ interactions.The reaction of 2,2′-dithiobis(benzenamine) with furan-2-carbonyl chloride produced the bis-amide title compound, C DOI: 10.1107/S1600536808038828/ww2132Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The Cu atom is linked by two N atoms from two 3,6-di-4-pyridyl-1,2,4,5-tetrazine ligands and by the N and S atoms from two thiocyanate ligands in a distorted tetrahedral environment. The Cu atom and the thiocyanate ligand occupy a crystallographic mirror plane m, and a crystallographic inversion centre is in the middle of the tetrazine ring, generating the zigzag fashion of the two-dimensional network. The infinite –Cu—SCN—Cu—SCN– chain is due to translational symmetry along the a axis. These chains are further connected through the 3,6-di-4-pyridyl-1,2,4,5-tetrazine ligands that bridge the CuI centers, generating a two-dimensional network. There are π—π stacking interactions between the pyridine and tetrazine rings (perpendicular distances of 3.357 and 3.418 Å), with a centroid–centroid distance of 3.6785 (16) Å.The title compound, [Cu(NCS)(C DOI: 10.1107/S1600536810001431/si2230Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The geometry is distorted towards skew-trapezoidal-bipyramidal owing to the proximity of the double-bonded S atoms. The C2Sn angles range from 129.0 (2) to 136.9 (2)°, the covalent Sn—S lengths from 2.529 (1) to 2.544 (1) Å, and the dative Sn←S lengths from 2.831 (1) to 3.042 (1) Å in the five independent molecules comprising the asymmetric unit. Two of the butyl groups were modelled over two positions of equal occupancy. All butyl groups were refined with distance restraints.The Sn atom in the title compound, [Sn(C DOI: 10.1107/S1600536809015116/tk2429Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The three independent molecules are roughly identical and each MoIII atom is surrounded by three chelating hexafluoroacetonate ligands in a three-bladed propeller-like arrangement, as observed in related compounds with acetylacetonate-type ligands. The structure of the title compound is very similar to the trigonal form of the CrIII analogue. However, the latter crystallizes in a higher-symmetry space group, P               c1. Both crystals are twinned by merohedry with the same twin law (m for the title complex, and m to 6/mmm for the CrIII complex.In the title compound, [Mo(C For a rhite 1979. For thehite 1979.       5HF6O2)3] = 0.030                           wR(F                           2) = 0.068                           S = 0.97                           4635 reflections362 parametersH-atom parameters constrainedmax = 0.78 e Å−3                        Δρmin = −0.44 e Å−3                        Δρ                                 CrysAlis CCD  used to solve structure: SIR97 (Altomare et al., 1999SHELXL97 (Sheldrick, 2008ORTEPIII (Burnett & Johnson, 1996ORTEP-3 for Windows (Farrugia, 1997PLATON (Spek, 2009SHELXL97.Data collection: 10.1107/S1600536810005489/gk2257sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536810005489/gk2257Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Although antagonist G blocks SCLC cell growth (IC50 = 24.5 ± 1.5 and 38.5 ± 1.5 μM for the H69 and H510 cell lines respectively), its exact mechanism of action is unclear. This study shows that antagonist G stimulates apoptosis as assessed by morphology (EC50 = 5.9 ± 0.1 and 15.2 ± 2.7 μM for the H69 and H510 cell lines respectively) and stimulates c-jun-N-terminal kinase (JNK) activity in SCLC cells (EC50 = 3.2 ± 0.1 and 15.2 ± 2.7 μM). This activity is neuropeptide-independent, but dependent on the generation of reactive oxygen species (ROS) and is inhibited by the free radical scavenger n-acetyl cysteine. Furthermore, antagonist G itself induces inflammation (59% increase in oedema volume compared to control) and potentiates (by 35–40%) bradykinin-induced oedema formation in vivo. In view of these results we show that, as well as acting as a ‘broad-spectrum’ neuropeptide antagonist, antagonist G stimulates basal G-protein activity in SCLC cell membranes (81 ± 12% stimulation at 10 μM), thereby displaying a unique ability to stimulate certain signal transduction pathways by activating G-proteins. This novel activity may be instrumental for full anti-cancer activity in SCLC cells and may also account for antagonist G activity in non-neuropeptide-dependent cancers. © 1999 Cancer Research Campaign[Arg


The HgII atom is five-coordinated in a trigonal–bipyramidal configuration by two N atoms from the chelating 4,4′-dimethyl-2,2′-bipyridine ligand, two bridging Br and one terminal Br atom, leading to a centrosymmetric dimeric molecule. There is a π–π contact between the pyridine rings [centroid-to-centroid distance = 3.756 (5) Å].The asymmetric unit of the title compound, [Hg DOI: 10.1107/S1600536808032510/hk2544Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


II—TbIII complex ]diphenolato-1κ4               O               6,O               1,O               1′,O               6′}:2κ4               O               1,N,N′,O               1′-μ-nitrato-1:2κ2               O:O′-dinitrato-1κ4               O,O′-terbium(III)zinc(II)), [TbZn(C18H18N2O4)(NO3)3], with the hexadentate Schiff base compartmental ligand N,N′-bisethylenediamine (H2               L), the Tb and Zn atoms are triply bridged by two phenolate O atoms of the Schiff base ligand and one nitrate ion. The five-coordinate Zn atom is in a square-pyramidal geometry with the donor centers of two imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms. The TbIII center has a ninefold coordination environment of O atoms, involving the phenolate O atoms, two methoxy O atoms, two O atoms from two nitrate ions and one from the bridging nitrate ion. Weak intermolecular C—H⋯O interactions generate a two-dimensional layer structure.In the title heteronuclear Zn DOI: 10.1107/S1600536808006958/at2549Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Each centrosymmetric dicationic unit has a dinuclear structure in which two AgI atoms are bridged by two 1,2-bis(1H-imidazol-1-ylmethyl)benzene ligands in a slightly distorted linear coordination geometry. The 4-amino-2,5-dichlorobenzenesulfonate anion does not coordinate with the AgI center, acting only as a counteranion. In the crystal structure, intermolecular O—H⋯O and N—H⋯O hydrogen bonds form a three-dimensional network.The asymmetric unit of the title compound, [Ag DOI: 10.1107/S1600536808023052/ci2638Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Mucosal-associated invariant T (MAIT) cells display two evolutionarily conserved features: an invariant T cell receptor (TCR)α (iTCRα) chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC) molecule, MHC-related molecule 1 (MR1). MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanVα7.2 antibody and MAIT cell-specific iTCRα and TCRβ transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRα and TCRβ transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT) cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%–4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the periphery in a process dependent both upon B cells and the bacterial flora. Thus, their development follows a unique pattern at the crossroad of NKT and γδ T cells. White blood cells, or lymphocytes, play an important role in defending the body from infection and disease. T lymphocytes come in many varieties with diverse functions. Mucosal-associated invariant T (MAIT) cells constitute a subset of unconventional T lymphocytes, characterized by their invariant T cell receptor (TCR)α chain and their requirement for the nonpolymorphic class Ib (MHC) molecule, MR1. MAIT cells are extremely abundant in human blood and mucosae. Contrary to mainstream T cells, their development requires B cells and commensal microbial flora. To shed light on the little-understood MAIT cells, we used new tools, including an antibody that we recently developed to detect human MAIT cells, and we were able to show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. We show that thymic selection is MR1 dependent but requires neither B cells nor the commensal flora, which are both necessary for the expansion in the periphery. In contrast with the other evolutionarily conserved invariant subset, the natural killer T (NKT) cells, we found that MAIT cells exit the thymus as “naïve” cells before becoming antigen-experienced memory cells and expanding in number to represent a significant 1%–4% of peripheral T cells in human blood. In mice, we found that MAIT cells remain naïve and do not expand substantially. We conclude that MAIT cell development follows a unique scheme, where, unlike NKT cells, MAIT cell selection and expansion are uncoupled events that are mediated by distinct cell types in different compartments. Mucosal-associated invariant T cells, the most abundant invariant T cell subset in humans, arise via a distinct developmental pathway that represents a hybrid of that seen for NKT and γδ T cells, two other unconventional T cell subsets. MR1−/− escribed  by cloniescribed . The TCR+ DN and a proportion of CD8α but few CD4 T cells were selected and cloned. The 3C10 hybridoma was chosen for further characterization because, according to quantitative PCR, the Vα7.2-Jα33 TCRα chain segregated with the 3C10 positive fraction in DN T cells. When transfectants expressing different mouse TCRα and TCRβ chains and a chimeric human iVα7.2-mouse Cα were stained with the 3C10 supernatant, only transfectants harboring the Vα7.2 segment displayed positive staining demonstrating that 3C10 was not an anti-TCRβ chain antibody (unpublished data). Quantitative PCR analysis of the 3C10+ and 3C10− fractions using primers for all Vα segments, demonstrated the absence of significant cross-reactivity between the 3C10 mAb and other Vα segments (unpublished data). The 3C10 antibody was biotinylated and detected with streptavidin PE-Cy7 (BD Pharmingen).The canonical TCRα chain and the associated Vβ13 TCRβ chains were cloned (ET and OL), from an iVα7.2-Jα33 T cell clone . Using this template, L. Teyton (SCRI) generated a recombinant heterodimeric protein, which was used to immunize Balb/c mice. After fusion with SP2/0, hybridoma specifically staining a high proportion of CD3Cell suspensions were prepared from thymus, spleen, peripheral or mesenteric lymph nodes, and gut LPL as previously described .Flow cytometry was performed with directly conjugated antibodies (BD Pharmingen) according to standard techniques with analysis on FACS Aria and LSRII flow cytometers (Becton Dickinson). DAPI and a 405-nm excitation were used to exclude dead cells. The following antibodies, mostly from BD Pharmingen or eBiosciences, were used in mice: anti-CD45.2-Fitc (104), anti-CD3ɛ-PC7 (145–2C11), anti-CD5-APC (Ly-1), anti-Vβ6-PE (RR4–7), anti-CD44-PE or APC (IM-7), anti-CD45.1-PE (A20), anti-βTCR-PC5 (H57–597), anti-CD19-PE, Fitc or APC-Cy7 (1D3), anti-CD8α-APC-Cy7 (Ly-2), and anti-CD4-PE-Texas Red (L3T4).For human stainings, the following were used: anti-CD4-APC-Cy7 (RP4-T4), anti-CD3ɛ-Alexa 700 (UCHT1), anti-TCRγδ-PC5 (IMMU510), anti-CD8β-PE Texas Red (2ST8.5H7), anti-CD45RO-Fitc (UCHL1), anti-CD45RA-PE (HI100), anti-CD27-Fitc (M-T271), anti-CCR7-PE (150503), anti-CD62L-PE (Dreg 56), anti-CXCR6-PE (56811), anti-CD161-APC (DX12), and anti-CD8α-PE Alexa700 (RPA-T8).Fragments of human thymuses were operating residues from children undergoing cardiac surgery. Thymuses were cut into small pieces in cold 0.5% BSA PBS. The resulting cell suspension was centrifuged to exclude aggregates. The supernatant was recovered and the cells were washed again with 0.5% BSA PBS. Thymocytes were then counted and labeled.Blood samples were obtained from healthy donors from the blood bank in accordance with institutional regulations.−/− MR1+ mice were harvested and 107 splenocytes were IV injected into MR1+ or MR1− TCRαβ double Tg Rag−/− mice. 2 wk later, the blood, spleen, and MLN were harvested and analyzed by FACS.Spleens from CD3ɛ−/− MR1−/− or MR1+/+ female mice were caged one night with the respective genotype male mice. Pregnant female mice were sacrificed at day 14. Uterine corns were harvested in CO2-independent medium supplemented with 5% FCS and penicillin/streptomycin (Invitrogen). Fetal thymuses were harvested and cultured in 300 μl of IMDM medium supplemented with 10% FCS, 50 μM beta-mercaptoethanol, 10 mM Hepes, 1 mM Sodium pyruvate, 2 mM L-glutamine, and penicillin/streptomycin in transwell plates from Costar . The medium was changed every 3 d over a period of 6–8 d.iVα19 Tg CαMolecular biology methods: RNA extraction, reverse transcription, TCR primers, PCR methods, and quantitative polyclonal PCR and polyclonal sequencing were carried out as previously described ,22.Figure S1+CD161high or 3C10+CD161lo cells in DN, CD8β, and CD4 subsets. Representative of four independent experiments.Sequence of Vα7.2-Cα amplicons obtained after polyclonal sequencing, with a specific Vα7.2 primer, of αβTCR T cells sorted from either 3C10(866 KB PDF)Click here for additional data file.Figure S2+CD161high) and mainstream T cells (3C10−CD161lo) gated on CD4− αβTCR T cells in PBMCs (upper panel). Representative CD8α/CD8β staining gated on mainstream CD4− T cell and CD4− MAIT cells . CD8α and CD8β expression by CD4− mainstream T cells or CD4− MAIT cells . Percentages of mainstream T and MAIT cells are boxed, upper panel. Representative of three independent experiments.Representative staining of MAIT cells (3C10(98 KB PDF)Click here for additional data file.Figure S3−/− (gray circle) or Cα−/−TAP−/−Ii−/− (black circle) background. Mononuclear cells from thymus (a), spleen (b), peripheral lymph nodes (PLN) (c), MLN (d), and gut LP (e) were harvested as described, counted, and the percentage of TCRβ+ cells was determined by FACS analysis. Each dot represents an individual mouse.(A) Absolute number of mature T cells in lymphoid organs of C57Bl/6 (black square) and iVα19-Jα33 transgenic mice in a Cα−/− or Cα−/−TAP−/−Ii−/− background.(B) Frequency of Vβ6 (left panel) and Vβ8 (right panel) in DN, CD8, and CD4 populations, analyzed by FACS analysis, in αβTCR T cells in the MLN (upper panel) and LP (lower panel) of C57Bl/6 and iVα19-Jα33 transgenic mice in a Cα−/− or Cα−/−MR1−/− (white circle) background. Each dot represents an individual mouse.(C) Vβ6 (left panel) and Vβ8 (right panel) bias in transgenic mice in DN, CD8, and CD4 populations on different backgrounds, analyzed by FACS, in mature thymocytes of C57Bl/6 and iVα19-Jα33 transgenic mice on a Cα+ or MR1−/− C57Bl/6 mice. Each dot represents an individual mouse.(D) Quantitative PCR analysis of iVα19-Jα33 segment expression normalized with respect to Cα gene expression on sorted DN, CD8, or CD4 T cells from thymocytes or MLN lymphocytes of MR1(149 KB PDF)Click here for additional data file.Figure S4+/+ or MR1−/− Cα−/− background either . percentages of CD4, CD8, and DN T cells populations are boxed. Representative of ten independent experiments.Representative CD8/CD4 staining gated on total thymus (upper panels) and on mature thymocytes (TCRβhigh HSAlow) (lower panels) of C57Bl/6 and iVα19-Jα33 transgenic mice on a MR1(71 KB PDF)Click here for additional data file.Figure S5−/− (gray circle) or Cα−/−MR1−/− (white circle) background; upper panels. Frequency of Vβ6 (left panel) and Vβ8 (right panel) in DN, CD8, and CD4 populations, analyzed by FACS, in αβTCR T cells from the MLN of C57Bl/6 (black square) and Cα−/−TAP−/−Ii−/− (black circle) or Cα−/−TAP−/−Ii−/−MR1−/− (white circle) background; lower panels. Each dot represents an individual mouse.Frequency of Vβ6 (left panel) and Vβ8 (right panel) in DN, CD8, and CD4 populations, analyzed by FACS, in αβTCR T cells from the MLN of C57Bl/6 (black square) and iVα19-Jα33 transgenic mice on a Cα(76 KB PDF)Click here for additional data file.Figure S6+CD161hi DN and 3C10+CD161lo CD8 αβTCR T cells sorted from cord blood. Representative of two separate experiments.Sequence of Vα7.2-Jα33 amplicons obtained after polyclonal sequencing, with a specific Vα7.2 primer, of 3C10(229 KB PDF)Click here for additional data file.Figure S7hiHSAlo) from the thymus left part; and from TCRhi T cells from MLN right part, in C57BL/6 and Vβ6, iVα7.2-Jα33/ Cα−/− and in iVα19-Jα33/Vβ6/ Cα−/− transgenic mice. Shaded grey histogram is on an MR1+/+ background while black line is on an MR1−/− background.Representative staining of CD44 expression on DN, CD8, and CD4 gated on mature T cells (TCR(323 KB PDF)Click here for additional data file.Figure S8−/−/TAP−/−/Ii−/− transgenic mice on a MR1+/+ (black diamond) or MR1−/− (white diamond) background.(A) Absolute numbers of mature T cells from the thymus and the MLN of iVα19-Jα33/Vβ6/Cαhi T cells from MLN of C57BL/6 (upper panel) and iVα19-Jα33/Vβ6/ Cα−/−/TAP−/− /Ii−/− transgenic mice on a MR1+/+ (middle panel) or MR1−/− (lower panel) background.(B) Representative CD44 and a pool of endogenVβ staining of DN, CD8, and CD4 gated on TCRβ(188 KB PDF)Click here for additional data file.Text S1(68 KB DOC)Click here for additional data file.


As many as 29 thrombotic patients with PAPS, 10 subjects with idiopathic antiphospholipid antibodies (aPL) without thrombosis, 17 thrombotic patients with inherited thrombophilia and 23 healthy controls were investigated. The following were measured in all participants: β2GPI−oxLDL complexes, IgG anti-β2GPI−oxLig-1, IgG anti-β2GPI−oxLDL antibodies (ELISA), PONa, (para-nitrophenol method), IMT of common carotid (CC) artery, carotid bifurcation (B), internal carotid (IC) by high resolution sonography. β2GPI−oxLDL complex was highest in the control group (p < 0.01), whereas, IgG anti-β2GPI−oxLig1 and IgG anti-β2GPI−oxLDL were highest in PAPS (p < 0.0001). In healthy controls, β2GPI−oxLDL complexes positively correlated to IMT of the IC (p = 0.007) and negatively to PONa after correction for age (p < 0.03). PONa inversely correlated with age (p = 0.008). In PAPS, IgG anti-2GPI−oxLig-1 independently predicted PONa (p = 0.02) and IMT of B (p = 0.003), CC, (p = 0.03) and of IC (p = 0.04). In PAPS, PONa inversely correlated to the IMT of B, CC and IC . IgG anti-2GPI−oxLig-1 may be involved in PAPS related atherogenesis via decreased PON activity.To explore whether antibodies against β


One metal atom (site symmetry 2) adopts a distorted pentagonal-bipyramidal SnC2N2O3 coordination arising from the N,N′,O,O′-tetradentate deprotonated Schiff base, two methyl groups in the axial sites and a μ3-O atom that also bonds to two further Sn atoms. Two symmetry-equivalent Sn atoms adopt very distorted SnC2O4 arrangements that could be described as pentagonal-bipyramidal with one equatorial vertex missing and the C atoms in the axial site. The final Sn atom (site symmetry 2) adopts an SnC2Cl2O trigonal-bipyramidal arrangement, with Cl atoms in the axial sites. As well as the two Sn atoms, one O atom lies on a twofold rotation rotation axis, and another is disordered about the axis. The terminal ethoxy group is disordered over two sets of sites with equal occupancy.In the title tetranuclear tin(IV) complex, [Sn For the al. 2007.       4(CH3)8(C20H22N2O4)Cl2O2] = 0.027                           wR(F                           2) = 0.072                           S = 1.07                           4259 reflections198 parameters10 restraintsH-atom parameters constrainedmax = 0.42 e Å−3                        Δρmin = −0.69 e Å−3                        ΔρAbsolute structure: Flack 1983, 1694 FrFlack parameter: 0.00 (4)                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2009Data collection: 10.1107/S1600536809032255/hb5035sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809032255/hb5035Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


IV atom in the title compound, [Sn(C6H5)2(C10H8NO)(NCS)], is chelated by the 2-methylquinolin-8-olate anion and is five-coordinate in a trigonal-bipramidal geometry [C—Sn—C = 133.47 (13) and 138.77 (12)°]. There are two independent molecules of similar conformation in the asymmetric unit.The Sn DOI: 10.1107/S1600536809055755/bt5150Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


One Pt atom is coordinated by one P atom from the dppmp ligand, and one C atom and two N atoms from a 6-phenyl-2,2′-bipyridine (pbpy) ligand in a square-planar geometry. The other Pt atom is coordinated by one N atom from the dppmp ligand, and one C atom and two N atoms from another pbpy ligand in a square-planar geometry. There are intramolecular π–π interactions between the pbpy ligands, with a centroid–centroid distance of 3.62 (1) Å between two pyridyl rings. The oxygen atoms of both perchlorate anions are disordered, each over two different positions [occupanicies 0.49 (3)/0.51 (3) and 0.48 (2)/0.52 (2)].The title compound, [Pt DOI: 10.1107/S1600536808024525/hy2138Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Adjacent ZnII cations are linked by a 1H-1,2,4-triazole-3,5-dicarboxylate ligand in a μ2,κ3 fashion to form a chain running along the c axis. The crystal packing is stabilized by N—H⋯O, O—H⋯N and O—H⋯O hydrogen bonds.In the title compound, [Zn(C DOI: 10.1107/S1600536808023994/bt2754Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The CoII ion is coordinated by two phenanthroline ligands, a water molecule and a 3-hydroxybenzoate anion, and displays a distorted octahedral geometry. π–π stacking is observed between parallel phenanthroline ligands, the face-to-face separations being 3.454 (19) and 3.435 (7) Å. An extensive hydrogen-bonding network helps to stabilize the crystal structure. The hydroxybenzoate ligand is disordered over two positions, with site occupancy factors 0.6 and 0.4. One solvent water molecule is also disordered over two positions, with site occupancy factors 0.6 and 0.4.The crystal structure of the title compound, [Co(C DOI: 10.1107/S1600536808001815/ng2420Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Two soluble α-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched α-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-α1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 α1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that α1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound α1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of α-mannosidase activity in a kex2Δ null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound α1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic α1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble α1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.Cytosolic  N-Glycosylation is one of the most common post-translational modifications of proteins in eukaryotic cells. The N-glycosylation pathway begins in the endoplasmic reticulum (ER), where the dolichol-bound Glc3Man9GlcNAc2 oligosaccharide is assembled, then transferred to nascent proteins and processed by α-glycosidases which remove the three glucose residues and one mannose residue to generate the Man8GlcNAc2 oligosaccharide  and 47 (EC 3.2.1.113)  to form Man8GlcNAc2 isomer B (M8B)  . Members B (M8B) , b, and 5GlcNAc2 .α-mannosidases II, IIx and III, along with lysosomal, vacuolar acidic and cytosolic/ER neutral α-mannosidases, compose family 38. These enzymes are inhibited by swainsonine and can release α1,2-, α1,3- and α1,6-linked mannose residues  N-oligosaccharide. This study appears to be the first report of an α1,2-mannosidase of family 47 in the cytosolic compartment of a lower eukaryote.Here, we studied the intracellular localization of 9, M8B, M7B, Man5GlcNAc2-Asn, 4-methylumbelliferyl-α-d-mannopyranoside (MUαMan), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), E64, 1,10-phenanthroline, PMSF, pepstatin A, EGTA, lyticase, sucrose, 3,3′-diaminobenzidine, BSA, monensin, monoclonal anti-rat Golgi 58K protein produced in mouse and anti-mouse IgG-horseradish peroxidase antibody generated in goat were obtained from Sigma. A cocktail of protease inhibitors was obtained from Roche. Anti-yeast carboxypeptidase Y (CPY), anti-rat calnexin and anti-yeast hexokinase I polyclonal sera, all generated in rabbit, were from USBiological. Anti-rabbit IgG-horseradish peroxidase antibody generated in donkey was from Amersham Biosciences. Sepharose CL6B and Sephadex G-25 were from Pharmacia LKB. DEAE Bio-Gel A and Bio-Gel P-6 were purchased from Bio-Rad. All other chemicals were of the highest purity commercially available.MCandida albicans ATCC 26555, CAI4 (mns1Δ-CIp10), HMY6 (mns1Δ-CIp10-MNS1)  were used in this study. They were maintained and propagated in YPD medium  as described previously , with or without protease inhibitors and were broken in a Braun MSK cell homogenizer using 0.45 mm diameter glass beads. The homogenate was centrifuged at 1000 g for 10 min and the supernatant was collected and further centrifuged at 105 000 g for 1 h (ultracentrifugation). The high-speed supernatant was collected, freeze-dried and kept at –20 °C until use. In some experiments, the pellet, consisting of a mixed-membrane fraction, was resuspended in buffer A and used to assay α-mannosidase activity.Cell-free homogenates were prepared while maintaining all solutions at 4 °C. α-mannosidase from C. albicans, C. glabrata and S. cerevisiae, the mixed-membrane fraction was incubated for 1 h at 48 °C, followed by incubation at −20 °C for 30 min. This procedure solubilized about 50 % of the non-enzymic protein present in the mixed-membrane fraction, without significantly affecting the total α-mannosidase activity remaining in the pellet. Then, the preparation was ultracentrifuged, and the pellet was recovered, resuspended in 1 ml of buffer A with 3 mM EDTA, incubated 1 h at room temperature and ultracentrifuged as indicated. The pellet was resuspended in 1 ml buffer A containing 0.5 M NaCl, incubated for 1 h at room temperature and ultracentrifuged. Finally, the pellet was resuspended in 1 ml buffer A added with Triton X-100 in a protein : detergent ratio of 1 : 1, and was incubated for 1 h at room temperature and ultracentrifuged. The pellet was then resuspended in 1 ml buffer B  and kept at −20 °C until use.For enzyme E-I purification, the high-speed supernatant obtained in the presence of 1 μM pepstatin A was purified by size-exclusion, anionic-interchange and concanavalin A-Sepharose affinity chromatography as described previously , lyticase (0.25 mg ml−1) and 15 mM β-mercaptoethanol. After incubation at 37 °C for 30 min, nearly 100 % of the cells had been converted into protoplasts. These were washed twice with buffer C and resuspended in 10 mM phosphate buffer, pH 6.0, containing 3.0 mM MgCl2 (buffer D). Samples of protoplasts were lysed with a Potter–Elvehjem and pestle (8–10 strokes) and the lysates were centrifuged at 1000 g for 10 min. The resulting supernatants were collected and 4 ml aliquots were loaded onto the top of a 35 ml, continuous 10–65 % (w/w) sucrose density gradient prepared with buffer D and centrifuged at 232 000 g for 4 h at 4 °C using a VTi 50 rotor (Beckman Coulter). Gradients were fractionated from the top and 1 ml fractions were collected. Alternatively, the protoplasts were incubated with 10 μM monensin for 1 h at 37 °C and lysed as described above.Protoplasts were obtained following the protocol described by C. albicans, C. glabrata or S. cerevisiae, treated or untreated with monensin, were fractionated in a continuous 10–65 % (w/w) sucrose density gradient as indicated above. The soluble fraction  was saved, freeze-dried, resuspended in 150 μl deionized water and applied onto a column (0.3×105 cm) of Bio-Gel P-6. Fractions of 120 μl were collected and those enriched with the oligosaccharides were pooled, and analysed by high-performance anion-exchange chromatography as described previously  was cloned into the bacterial expression vector pET100/D-TOPO (Invitrogen), overexpressed in Escherichia coli and the recombinant protein was purified. Antibodies were raised in a male New Zealand White rabbit after intramuscular injection of 150 μg protein emulsified with complete Freund's adjuvant (day 0). Booster injections were given (150 μg protein emulsified with incomplete Freund's adjuvant) on days 15, 30, 45 and 60 and the animal was bled on day 75.The anti-α1,2-mannosidase, rabbit pre-immune serum, anti-CPY, anti-hexokinase I and anti-Golgi primary antibodies were diluted 1 : 1000, 1 : 3000, 1 : 3000, 1 : 5000, 1 : 5000 and 1 : 5000, respectively, in PBS. The secondary, anti-mouse IgG-horseradish peroxidase and anti-rabbit IgG-horseradish peroxidase antibodies were diluted 1 : 2000 in PBS.Anti-calnexin, anti-α-mannosidase peak, separated after density-gradient fractionation of protoplast homogenates, were subjected to SDS-PAGE (10 %) and electrophoretically transferred to Hybond-C extra nitrocellulose membranes following standard protocols. The membranes were stained with Ponceau S Red to assess the efficiency of transfer. The membranes were blocked with 1 % BSA in PBS for 2 h, washed three times with 0.05 % Tween 20 in PBS for 10 min, and the primary antibody was added and incubated for 2 h at room temperature. Membranes were then washed three times with 0.05 % Tween 20 in PBS for 10 min, incubated for 2 h with the secondary antibody, and washed twice with 0.05 % Tween 20 in PBS for 10 min and once with PBS for 10 min. Bands were revealed with 3,3′-diaminobenzidine (0.5 mg ml−1) and 3 % hydrogen peroxide in PBS. Reactions were stopped with deionized water.Purified protein or samples (10 μg) of each S. cerevisiae and C. glabrata secreted, soluble Kex2 proteins were produced in a commercial Pichia pastoris expression system (Invitrogen). The strain expressing S. cerevisiae Kex2 was a kind gift of G. Boileau .α1,2-mannosidase antibody. For the processing using recombinant Kex2, 20 μg of the partially purified membrane-bound α-mannosidase and 3 μg of recombinant Kex2 from S. cerevisiae, C. albicans or C. glabrata were resuspended in buffer B in a final volume of 20 μl, and incubated for 1 h at 37 °C with gentle shaking. Then, the reactions were applied to 4–12 % NuPAGE Bistris gels (Invitrogen) and analytic zymograms were carried out (see above).Twenty micrograms of protein from peak 2 and peak 3, obtained after treatment with 10 μM monensin (see above), was resuspended in 50 μl buffer D added with 0.5 % Triton X-100, with or without protease inhibitors, and incubated for 1 h at 37 °C with gentle shaking. The reactions were analysed by immunoblotting using the anti-α-mannosidase E-I and the 52 kDa α-mannosidase generated by Kex2 were subjected to double-dimension electrophoresis using isoelectrofocusing ZOOM pH 4–7 strips and 4–12 % NuPAGE Bistris ZOOM gels (Invitrogen). The gels were washed three times with buffer A added with 1 % Triton X-100 for 20 min at room temperature, and α-mannosidase activity was detected after incubation with buffer A added with 40 μM MUαMan for 1 h at 37 °C. The proteins were transferred to a PVDF membrane and N-terminal sequencing was carried out by BioSynthesis.The purified C. albicans were broken in the presence of protease inhibitors and the resulting homogenates were subjected to ultracentrifugation. α-Mannosidase activity was measured in the high-speed supernatant and in the mixed-membrane fraction. The results, summarized in Table 1α-mannosidase was recovered mostly in the supernatant (78–82 %) while only 18–22 % remained associated with the membrane fraction. However, in homogenates prepared in the presence of pepstatin A, total activity was distributed in approximately equal proportions in the soluble (44 %) and membrane (56 %) fractions.Yeast cells of α-mannosidase representing E-I was eluted by 0.1 M NaCl from a DEAE Bio-Gel A column. After eluting with 0.125 M NaCl, a second barely detectable peak corresponding to α-mannosidase E-II was obtained . As was described for α-mannosidases E-I and E-II (5GlcNAc2-Asn oligosaccharide (data not shown) and was more sensitive to 1-DMJ than to swainsonine, with IC50 values of 0.22 mM and 0.54 mM, respectively.The processing of natural substrates such as Mand E-II , the purC. albicans were subjected to centrifugation in a continuous, 10–65 % sucrose density gradient (see Methods). α-Mannosidase activity was distributed in three peaks associated with fractions 1–4 (peak 1), 21–22 (peak 2) and 26–27 (peak 3) with corresponding densities of 1.033±0.002 g cm−3, 1.130±0.007 g cm−3 and 1.183±0.004 g cm−3 and representing 20 %, 27 % and 53 % of total recovered activity, respectively  and 26–27 (peak 3) with corresponding densities of 1.137±0.006 g cm−3 and 1.184±0.006 g cm−3, respectively. Peaks 2 and 3 represented 26 % and 74 % of total recovered activity, respectively α1,2-mannosidase antibody detected a polypeptide of 52 kDa in peaks 1 and 2, which corresponds to the molecular mass of α-mannosidase E-I (see above). The same antibody detected trace amounts of a 65 kDa protein in peak 2 which was enriched in peak 3. Hexokinase I, a cytosolic marker , which is a 65 kDa α1,2-mannosidase that is predicted to be localized in the ER . Therefore, MNS1 encodes both 65 kDa and 52 kDa, α1,2-mannosidase isoforms.An anti-α1,2-mannosidase E-I is associated with the cytosolic and Golgi markers, whereas a 65 kDa α1,2-mannosidase was enriched in the ER and to a lesser extent in Golgi fractions. To determine whether E-I was generated from the 65 kDa protein, the effect of monensin on α1,2-mannosidase localization was analysed. Monensin, a monovalent ion-selective ionophore, blocks the transport of vesicles from the medial to the trans cisternae of the Golgi complex  before their disruption and fractionation in a continuous 10–65 % sucrose density gradient. Under these conditions, the α-mannosidase activity was not affected (data not shown) and was again distributed in three peaks with corresponding densities of 1.032±0.003 g cm−3 for peak 1 (fractions 1–3), 1.139±0.007 g cm−3 for peak 2 (fractions 22–23), and 1.184±0.002 g cm−3 for peak 3 (fractions 27–28). The α-mannosidase activity associated with peaks 1 and 2 was significantly decreased to 3 % and 6 %, respectively, and the rest of the recovered α-mannosidase activity (91 %) was associated with peak 3 . When protoplasts of S. cerevisiae were treated in the same way, the α-mannosidase activity was distributed in two peaks associated with fractions 22–23 (peak 2) and 27–28 (peak 3) with corresponding densities of 1.140±0.003 g cm−3 and 1.186±0.005 g cm−3, respectively. As for the monensin-treated protoplasts of C. albicans, there was a redistribution of the α-mannosidase activity associated with peaks 2 and 3, representing 4 % and 96 % of total recovered activity, respectively .These results suggest that α1,2-mannosidase E-I; however, 5.0 mM PMSF partially inhibited the proteolytic processing of 65 kDa protein . This processing was almost completely inhibited when the concentration of PMSF was increased to 10 mM . High concentrations of pepstatin A (10 μM), trans-epoxysuccinyl-l-leucyl-amido(4-guanidino)butane (E-64) (1 mM) or 1,10-phenanthroline (20 mM) did not affect the conversion of 65 kDa α1,2-mannosidase into α-mannosidase E-I . Addition of 0.5 mM EGTA partially inhibited the proteolytic processing , and almost complete inhibition occurred using 1.0 mM EGTA . The EGTA effect was prevented by the addition of 3 mM CaCl2 . Mock controls containing preimmune serum  or lacking the primary antibodies  run in parallel gave no detectable signals. Therefore a Ca2+-dependent proteolysis is required for the processing of ER α1,2-mannosidase activity into E-I.In order to establish the nature of the proteolytic activity, similar experiments were carried out in the presence of protease inhibitors  min−1 −1], 46 % of the total activity was present in a soluble form as previously demonstrated −1] more than 99 % of the α-mannosidase activity was associated with the mixed-membrane fraction. When wild-type or kex2Δ null mutant cells were broken in the presence of 1 mM EGTA, the α-mannosidase distribution was similar to that of untreated cells. These results suggest that Kex2 participates in the generation of α-mannosidase E-I. To determine whether Kex2 was able to process the 65 kDa α1,2-mannosidase into E-I, incubations of purified 65 kDa α1,2-mannosidase from mixed-membrane fraction and recombinant Kex2 from S. cerevisiae were carried out, and analysed in zymograms using the fluorogenic substrate MUαMan (see Methods). The results, illustrated in Fig. 8α1,2-mannosidase into α-mannosidase E-I (lane 3), and this proteolytic cleavage was sensitive to EGTA (lane 4). The 65 kDa α1,2-mannosidase and E-I were run as controls (lanes 1 and 2). Similar results were obtained when we analysed the products with the anti-α1,2-mannosidase antibodies, or when we performed the reactions using ER membrane-bound α1,2-mannosidase from C. glabrata or recombinant Kex2 from C. albicans or C. glabrata (data not shown). However, recombinant Kex2 failed to hydrolyse the ER membrane-bound α1,2-mannosidase from S. cerevisiae (data not shown).The Golgi protease activity that processed the 65 kDa ase Kex2 . Hence, α-Mannosidase of 52 kDa generated by Kex2 processing  and was analysed by high-performance anion-exchange chromatography (see Methods). M8B and M7B were both present in the cytosol, representing 21 % and 79 % of the total free N-oligosaccharides, respectively  and C. glabrata  (99-Arg100 and Ala101-Arg102 are present. In vitro and in vivo studies have demonstrated that Kex2 can recognize both of these non-canonical sequences, but with lower affinity (α-mannosidase activity was present as E-I. Hence, the results indicate that Kex2 recognizes an atypical cleavage site on the ER membrane-bound α1,2-mannosidase. The mechanism utilized to transport the α1,2-mannosidase E-I to the cytosolic compartment still remains to be determined. Fig. 9α1,2-mannosidase into enzyme E-I.The N-terminal sequence of both the purified affinity . This maα1,2-mannosidase, we determined the levels of free cytosolic N-oligosaccharides in protoplasts treated and not treated with the ionophore monensin, which blocks glycosylation by affecting divalent cation transport into the Golgi. In agreement with previous reports (S. cerevisiae showed only the presence of M8B in the cytosolic preparations. It is believed that this N-oligosaccharide is generated by deglycosylation of glycoproteins during the ERAD (C. albicans, M8B and M7B were detected in the cytosolic compartment, representing 21 % and 79 % of the total oligosaccharides, respectively. Protoplasts depleted of enzyme E-I had reduced amounts of M7B oligosaccharide and increased levels of M8B. These results suggest that enzyme E-I is required for the trimming of M8B into M7B. Similar results were obtained using protoplasts of C. glabrata, indicating that ER membrane-bound α1,2-mannosidase from this yeast can be processed to a cytosolic isoform by Kex2. Similar results have been reported in human cells, where the downregulation of cytosolic α-mannosidase activity directly influences the amount and the structure of the N-oligosaccharides present in the cytosol (To gain further insights into the role of the cytosolic α1,2-mannosidase by Kex2 could represent a regulatory mechanism similar to that recently described for the human ER α1,2-mannosidase (N-glycosylation and the degradation of cytosolic free N-oligosaccharides. To our knowledge this is the first report of a cytoplasmic α1,2-mannosidase in lower eukaryotes belonging to family 47 of glycosyl hydrolases.The limited proteolysis of ER


Symmetry-related CdII ions are linked via bridging 1,3-di-4-pyridylpropane ligands, forming a zig-zag chain-structure parallel to [001]. In the crystal structure, there are weak intrachain π–π stacking interactions between benzene rings, with a centroid–centroid distance of 3.825 (7) Å, and pairs of chains are interdigitated with respect to the 4-methylbenzenethiolate groups.In the title compound, [Cd(C DOI: 10.1107/S1600536809010447/lh2791Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The remaining coordination sites are occupied by two N atoms from one 1,10-phenanthroline (phen) and two O atoms from an L ligand. The six 6-hydroxy-1-naphthoate groups coordinate each EuIII atom in three different ways, namely μ2-η1:η1-bridging, μ1-η1:η1-chelating, and μ2-η1:η2-chelating/bridging modes. Adjacent discrete dinuclear units are linked into a two-dimensional sheet parallel to (011) by intermolecular O—H⋯O hydrogen-bonding interactions. The sheets are cross-linked by water molecules, forming a three-dimensional network. In addition, π–π stacking interactions, with a centroid–centroid separation of 3.547 (2) Å are observed.The title complex, [Eu DOI: 10.1107/S1600536809046091/ci2947Isup2.hklStructure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Toxoplasma gondiistrains . Theastrocytoma cell fine constitutively secreted TNFα and IL-6,but no IL-1α. A positive control was obtained by stimulationwith phorbol esters inducing a significant increase (p < 0.05) in TNFα and IL- 6 secretion but not in IL-1α, whilelipopolysaccharide , interferon gamma,ionophore A 23187 and sera positive to T. gondii didnot induce any increase in cytokine levels. None of the tachyzoites,whatever their virulence, induced a significant increase in cytokineproduction at any time in the study. Tachyzoites did not inhibit thesecretion induced by phorbol esters.The secretion of tumour necrosis factor-α (TNFα),interleukin-1α (IL-α) and interleukin-6 (IL-6) by ahuman astrocytoma cell fine was studied 1 h, 3 h, 6 h and 24 h afterinfection with tachyzoites from three


Since men with chronic kidney disease (CKD) progress faster than women, an accurate assessment of CKD progression rates should be based on gender differences in age-related decline of glomerular filtration rate (GFR) in healthy individuals.A Chinese sample population from a stratified, multistage, and clustered CKD screening study was classified into healthy, at-risk, and CKD groups. The gender differences in estimated GFR (eGFR) and age-related eGFR decline were calculated for each group after controlling for blood pressure, fasting glucose levels, serum lipids levels, education level, and smoking status. After referencing to the healthy group, gender-specific multivariate-adjusted rates of decline in eGFR and differences in the rates of decline were calculated for both CKD and at-risk groups.-1·1.73 m-2·yr-1 vs. 0.74 mL·min-1·1.73 m-2·yr-1 and at-risk group: 0.60 mL·min-1·1.73 m-2·yr-1 vs. 0.73 mL·min-1·1.73 m-2·yr-1). However, in the CKD group, the rates of decline in eGFR in men were similar to those in women (0.96 mL·min-1·1.73 m-2·yr-1 vs. 0.91 mL·min-1·1.73 m-2·yr-1). However, after referencing to the healthy group, the rates of decline in eGFR in men in the at-risk and CKD groups were greater faster than the corresponding rates in women (at-risk group: 0.10 mL·min-1·1.73 m-2·yr-1 vs. -0.03 mL·min-1·1.73 m-2·yr-1 and CKD group: 0.44 mL·min-1·1.73 m-2·yr-1 vs. 0.15 mL·min-1·1.73 m-2·yr-1).The healthy, at-risk, and CKD groups consisted of 4569, 7434, and 1573 people, respectively. In all the 3 groups, the multivariate-adjusted eGFRs in men were lower than the corresponding eGFRs in women. In addition, in the healthy and at-risk groups, the rates of decline in eGFR in men were lower than the corresponding rates of decline in women (healthy group: 0.51 mL·minTo accurately assess gender differences in CKD progression rates, gender differences in age-related decline in GFR should be considered. Men with chronic kidney disease (CKD) progress to end-stage CKD at a faster rate than women,2. This e.g., gender, age, and education), personal and family health history, and lifestyle behaviors . Laboratory measurements also were made by well-trained professionals using uniform standards.We extracted a database of subjects from a stratified CKD cluster sampling study performed in Beijing in 2006. A totalcr) was measured by using a kinetic-rate method described by Jaffe. The glomerular filtration rate was estimated by using a modified Modification of Diet in Renal Disease (MDRD) Study formula[-1·1.73 m-2) = 175 × standard (Scr)-1.234 × age-0.179 × 0.79 .Serum creatinine  and urine creatinine was measured by using Jaffe's kinetic method on a Hitachi 7170 chemistry analyzer . Albuminuria was diagnosed if the urine albumin/creatinine ratio was ≥17 mg·gfor women and protHypertension was defined as measurement of systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥ 90 mmHg, having been diagnosed with hypertension at least twice, or taking an antihypertensive medication at the time of the survey.-1, a previous diagnosis of diabetes, or taking insulin or anti-diabetic medication at the time of the survey.Diabetes was defined as measurement of a fasting glucose level > 6.9 mmol·L-1, triglyceride (TG) ≥2.26 mmol·L-1, high-density lipoprotein (HDL) < 1.04 mmol·L-1, or low-density lipoprotein (LDL) ≥4.14 mmol·L-1; a previous diagnosis of dyslipidemia; or taking lipid-lowering medication at the time of the survey.Dyslipidemia was defined as measurement of total cholesterol (TC) ≥6.22 mmol·L-2 (BMI = weight/(height2)).Obesity was defined as having a body mass index (BMI) ≥28 kg·m-1·1.73 m-2 (n = 33) or < 15 mL·min-1·1.73 m-2 (n = 57) were excluded from this study. Participants were divided into 3 groups according to their health status: those with albuminuria or eGFR < 60 mL·min-1·1.73 m-2 were placed in the CKD group; those without CKD but with hypertension, diabetes, dyslipidemia, or obesity were placed in the at-risk group; those without CKD or other medical conditions were placed in the healthy group. We also used proteinuria as a definition of CKD and performed a separate statistical analysis as described below because currently there is no consensus about defining CKD by microalbuminuria or macroalbuminuria. Some authors only consider microalbuminuria to be a risk factor for CKD such as (pre)hypertension or diabetes and its reproducibility of microalbuminuria tests is limited [Individuals whose eGFR was > 200 mL·min limited .Sociodemographic characteristics , health indicators , and lifestyle behaviors (smoking) of each gender were described by descriptive statistics. The differences in these variables among genders were examined by using chi-square statistics for categorical variables and Student's t-test for continuous values.For each health status group, a linear model was constructed with eGFR as the dependent variable, gender as the independent variable, and confounding factors  as covariates. This model was used to calculate the least squares means of eGFR (adjusted eGFR) and to compare eGFRs between genders within each health status group.A linear model was constructed for each health status group. In these models, the dependent variable was eGFR and the independent variables were age, gender, and interaction of age and gender. These models were used to calculate the gender-specific rates of decline of eGFR and to test gender-specific differences. Subsequently, confounding factors were added to these models as covariates to calculate the adjusted gender-specific rates of decline of eGFR.A linear regression model was constructed for each gender. In these models, the variables were (1) eGFR (dependent variable); (2) age, health status, and interaction of age and health status (independent variables); and (3) the aforementioned confounding factors (covariates). This model was used to calculate the adjusted gender-specific rates of decline of eGFR  for the CKD and at-risk groups. In addition, a linear regression model was constructed with the following variables: (1) eGFR (dependent variable); (2) age, health status, gender, and interactions of age and health status, age and gender, health status and gender, age and health status and gender (independent variables); and (3) the aforementioned confounding factors (covariates). This model was used to test for statistically significant differences between men and women for the adjusted gender-specific rates of decline of eGFR  for the CKD and at-risk groups.All statistical analyses were performed using Statistical Analysis System software, version 9.0 . Tests for association were considered to be statistically significant when the p-values were less than 0.05.-1·1.73 m-2. In the healthy group, men had higher BMIs, blood pressures, fasting glucose levels, serum lipid levels, and lower education levels than women of similar ages. In the at-risk group, men were younger, had higher diastolic blood pressures, TG levels, and education levels but lower levels of LDL, TC, and HDL than women. In the CKD group, men had higher blood pressures, TG levels, and education levels but lower HDL levels than women. In each health status group, men were more likely to be smokers. Compared to individuals in the healthy group, those in the at-risk and CKD groups were older and fatter and had higher blood pressures and serum lipid levels. For men, the prevalence of smoking was similar in all health status groups and the CKD group had significantly lower education levels than the healthy or at-risk groups. For women, the at-risk and CKD groups had significantly lower education levels and more smokers than the healthy group  with microalbuminuria, 104 (0.75%) with proteinuria, and 261 (1.88%) with eGFR < 60 mL·minThe gender differences of adjusted eGFRs in each health status and age groups are shown in Table -1·1.73 m-2·yr-1 for men and 0.75 mL·min-1·1.73 m-2·yr-1 for women in the healthy group (p < 0.05), 0.53 mL·min-1·1.73 m-2·yr-1 for men and 0.67 mL·min-1·1.73 m-2·yr-1 for women in the at-risk group (p < 0.05), and 0.89 mL·min-1·1.73 m-2·yr-1 for men and 0.88 mL·min-1·1.73 m-2·yr-1 for women in the CKD group (p > 0.05). After adjusting for covariates, the rates of decline of eGFR were 0.51 mL·min-1·1.73 m-2·yr-1 for men and 0.74 mL·min-1·1.73 m-2·yr-1 for women in the healthy group (p < 0.05), 0.60 mL·min-1·1.73 m-2·yr-1 for men and 0.73 mL·min-1·1.73 m-2·yr-1 for women in the at-risk group (p < 0.05), and 0.96 mL·min-1·1.73 m-2·yr-1 for men and 0.91 mL·min-1·1.73 m-2·yr-1 for women in the CKD group (p > 0.05). In addition, we calculated the variation in the rates of decline of eGFR before and after the age of 50. However, there was no significant difference for either gender in each health status group.As shown in Table -1·1.73 m-2·yr-1 (p < 0.05) for men and -0.03 mL·min-1·1.73 m-2·yr-1 (p > 0.05) for women in the at-risk group and 0.44 mL·min-1·1.73 m-2·yr-1 (p < 0.05) for men and 0.15 mL·min-1·1.73 m-2·yr-1 (p < 0.05) for women in the CKD group. The gender difference of the adjusted rate of decline of eGFR  was statistically significant for the CKD group  but not the at-risk group .Table -1·1.73 m-2·yr-1 (p < 0.05) (Table -1·1.73 m-2·yr-1 (p < 0.05) (Table -1·1.73 m-2·yr-1 (p < 0.05) (Table -1·1.73 m-2·yr-1 (p < 0.05) (Table -1·1.73 m-2·yr-1 vs. 0.31 mL·min-1·1.73 m-2·yr-1). Moreover, these changes were quantitative but not qualitative, which was partly due to the significantly smaller sample size in the CKD group when proteinuria was used to define CKD.When we used proteinuria to define CKD, we obtained different results for the CKD group Table . For men5) Table . For wom5) Table . HoweverCurrently information about gender differences in GFRs in healthy individuals is limited and conflicting. For example, our observation that men younger than 60 years old had lower eGFRs than women of similar ages is both consistent and incoSome earlier studies showed an increase in the rates of decline of GFR in healthy individuals older than 40 or 50 years old,19,21 orOur observation that the rate of decline of eGFR in men was slower in the healthy group but faster in the CKD group could be explained by epidemiology studies. In general, men have a higher incidence and prevalence of end-stage renal disease (ESRD) than women ,25; howeThis study had 3 potential limitations. First, the use of estimated GFR to assess renal function in healthy individuals could be prone to measurement bias. HoweverThere are gender differences in the decline in GFR and among different health statuses. To accurately evaluate the gender differences in CKD progression rate, the baseline gender differences in age-related decline in GFR in healthy individuals should be considered.The authors declare that they have no competing interests.RX analyzed data and wrote the paper.LXZ, PHZ, and FW performed research.LZ and HYW designed research.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/11/20/prepub


II(C2H8N2)3]2[MoIV(CN)8]·2H2O, crystallized from a mixture of ethane-1,2-diamine (en), octacyanomolybdate(IV), [Mo(CN)8]4−, and the transition metal ion Ni2+. In the crystal structure, the Mo polyhedron has a square-antiprismatic shape, while the geometry around the Ni atom is distorted octahedral. The complex ions and water molecules are linked by hydrogen bonds.The title complex, [Ni DOI: 10.1107/S1600536808024963/br2077Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


N-heterocycles in the centrosymmetric title compound, [La2(C10H9NO)4(NO3)6], exist in the zwitterionic form. One of these binds to one metal center, whereas the other bridges two metal centers. The La atom is chelated by three nitrate groups and is surrounded by nine O atoms in a coordination environment based on a distorted monocapped square-antiprism. The dinuclear structure is further stabilized by intramolecular N—H⋯O(nitrate) hydrogen bonds.The two independent  N-heterocycle exists in the deprotonated and neutral form in hexakis(μ-2-methylquinolin-8-oxido)bis(2-methylquinolin-8-oxido(2-methyl-8-quinolinol)(nitrato)trilanthanum methanol solvate; see: Katkova et al. 4(NO3)6] = 0.022                           wR(F                           2) = 0.057                           S = 1.05                           5258 reflections344 parameters2 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.64 e Å−3                        Δρmin = −0.36 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2009Data collection: 10.1107/S1600536809019746/tk2454sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809019746/tk2454Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


A 1,2,3-benzothiadiazole-7-carboxylate ligand bridges two CuII centres, with a Cu⋯Cu distance of 9.006 (2) Å. The four O atoms in the equatorial planes around each CuII centre form a distorted square-planar arrangement, while the distorted square-pyramidal coordination is completed by the symmetry-related N atoms of the bridging 1,2,3-benzothiadiazole-7-carboxylate ligands. In the crystal structure, intermolecular O—H⋯O and O—H⋯N hydrogen bonds link the molecules into a three-dimensional supramolecular network.In the polymeric title complex, [Cu(C DOI: 10.1107/S1600536809032036/hk2737Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


I complex, [Ag2(NO3)2(C12H10N4)2], each AgI center is coordinated by one pyridine and one benzotriazole N-donor atom of two inversion-related 2-(3-pyridylmethyl)-2H-benzotriazole (L) ligands, and an O atom of a coordinated NO3               − anion in a distorted T-shaped geometry. This forms a unique box-like cyclic dimer with an intramolecular non-bonding Ag⋯Ag separation of 6.327 (2) Å. Weak intermolecular Ag⋯O(nitrate) interactions [2.728 (4) and 2.646 (3) Å] link the binuclear units, forming a two-dimensional network parallel to (100). Intermolecular C—H⋯O hydrogen-bonding interactions, involving the L ligands and the coordinated NO3               − anions, link the sheets, forming a three-dimensional framework.In the title centrosymmetric binuclear Ag DOI: 10.1107/S160053680803479X/su2070Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


II atom in the polymeric title compound, [Pb(C8H4O4)(C12H6N2O2)]n, is chelated by the N-heterocycle, and adjacent atoms are bridged by rigid terephthalate dianions into a linear chain. The PbII atom is stereochemically active in a ψ-square-pyramidal coordination geometry in which the lone-pair electrons occupy a basal site. When three other weaker Pb⋯O interactions are considered, the geometry is a ψ-dodecahedron.The Pb DOI: 10.1107/S1600536809046455/ci2964Isup2.hklStructure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The CdII ion is coordinated by two N atoms from the 1,10-phenanthroline molecule, three O atoms from two crystallographically independent benzene-1,4-dicarboxylate ligands and the O atom of a coordinated water molecule, forming a heavily distorted octahedron. The 1,10-phenanthroline ligand is approximately planar within 0.073 (4) Å. The two different benzene-1,4-dicarboxylate ligands each coordinate to two CdII ions in bidentate and monodentate modes, forming an infinite zigzag chain. Adjacent chains are packed tightly by strong π–π interactions [centroid–centroid distances = 3.851 (2) and 3.859 (2) Å] between the aromatic rings of the benzene-1,4-dicarboxylate ligand and the 1,10-phenanthroline of a neighboring chain, forming a sheet parallel to (011). Different sheets are linked together via O—H⋯O hydrogen bonds between the coordinated water molecules and the O atoms of the carboxylate groups, forming a three-dimensional network.The title compound, [Cd(C DOI: 10.1107/S1600536808041913/ez2152Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Nitrogen-containing bisphosphonates indirectly activate Vγ9Vδ2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vγ9Vδ2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid-pulsed monocytes triggered activation of γδ T cells in a cell contact-dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vγ9Vδ2 T cell activation. Nitrogen-containing bisphosphonate drugs (N-BPs) are effective anti-resorptive agents commonly used for the treatment of postmenopausal osteoporosis and tumour-induced osteolysis. Intravenous administration of N-BPs is commonly associated with a flu-like acute-phase reaction involving the activation of Vγ9Vδ2 T cells . N-BPs, Zoledronic acid (ZOL) and CGP-58318  were isolated using Lymphoprep  density gradient centrifugation and cultured in α-minimal essential medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l glutamine, 10% fetal bovine serum, and 10 U/ml recombinant human interleukin 2, except for detection of drug uptake where PBMCs were isolated using PharmLyse . Murine J774·2 macrophage-like cells were cultured as previously described (6 cells/ml in 75 cm2 flasks) or J774·2 macrophages (5 × 105 cells/well in 6-well plates) were treated as indicated in the figure legends, and T cells or monocytes were purified from human PBMCs by negative selection using MACS beads  according to manufacturer’s instructions. IPP/DMAPP and ApppI were quantified by high performance liquid chromatography negative ion electrospray ionization mass spectrometry (HPLC-ESI-MS) in acetonitrile extracts, as previously described (Human PBMCs (2 × 106 cells/well in 24-well plates) were treated as indicated in the legend of facsdiva software (BD Biosciences), or cytospun onto slides and counterstained with SYTOX Orange  prior to analysis on a Zeiss LSM510 META system  using a 63× objective lens, and aim software (Carl Zeiss Ltd) for image acquisition and analysis.Human PBMCs  were then co-cultured with the monocyte-depleted PBMCs (5 × 105 cells/well) or with γδ T cells (1 × 104 cells/well) isolated from the monocyte-depleted fraction using a negative γδ T cell isolation kit (Miltenyi Biotec GmbH), in a final volume of 500 μl medium in 24-well plates. To assess the role of cell–cell contact, transwell inserts (pore-size 0·4 μm) were used to separate monocyte-depleted PBMCs or γδ T cells from monocytes.Monocytes were isolated from human PBMCs using anti-CD14 magnetic bead separation (Miltenyi Biotec GmbH) according to the manufacturer’s instructions. Purity of the monocyte fraction was ≥93%, as assessed by CD14-labelling and flow cytometric analysis. Isolated monocytes were plated out at 5 × 10After 72 h, conditioned medium was collected and the concentration of interferon (IFN)-γ was determined using an IFN-γ Quantikine enzyme-linked immunosorbent assay kit  according to the manufacturer’s instructions.+ T cells in the PBMC co-cultures was determined by immunostaining with anti-CD3 and anti-Vδ2-TCR antibodies and flow cytometric analysis, as previously described  increase in IFN-γ release when co-cultured with monocyte-depleted PBMCs, as compared to co-culture of monocyte-depleted PBMCs with vehicle-treated monocytes (n = 3) increase (P < 0·05) in Vδ2+ T cell number after a 7 d culture , we investigated whether purified peripheral blood monocytes pretreated with the same concentration and duration of ZOL activated Vγ9Vδ2 T cells in co-cultures with untreated (monocyte-depleted) PBMCs. ZOL-treated monocytes induced a 2·5 ± 1·2-fold (onocytes , althoug culture , and an P < 0·05; Finally, we investigated whether ZOL-pretreated monocytes activated Vγ9Vδ2 T cells in enriched γδ T cell cultures. After 72 h, IFN-γ release was significantly increased in co-cultures of γδ T cells with ZOL-treated monocytes, compared to co-cultures with vehicle-treated monocytes (This study demonstrated that ZOL induced IPP/DMAPP accumulation selectively in monocytes following treatment with a clinically-relevant dose and duration of ZOL, most probably a result of efficient intracellular drug uptake by this highly endocytic cell type. Furthermore, peripheral blood monocytes pretreated with ZOL induced activation and proliferation of Vγ9Vδ2 T cells, both in mixed PBMC cultures, and in co-cultures with enriched γδ T cells. These findings provide a novel explanation for why monocytes are essential for Vγ9Vδ2 T cell activation by N-BPs, and suggest that monocytes, following exposure to ZOL, can directly activate Vγ9Vδ2 T cells in a cell contact-dependent manner.


For example, Gq/phospholipase (PL)Cβ signaling by TPα isdirectly inhibited by the vasodilators prostacyclin and nitric oxide (NO)whereas that signaling by TPβ is unaffected. Herein, we investigated whether TPαand/or TPβ regulate G12/Rho activation and whether thatsignaling might be differentially regulated by prostacyclin and/or NO. Both TPαand TPβ independently regulated RhoA activation and signaling in clonal cellsover-expressing TPα or TPβ and in primary human aortic smooth muscle cells (1°AoSMCs). While RhoA-signaling by TPα was directly impaired by prostacyclin andNO through protein kinase (PK)A- and PKG-dependent phosphorylation,respectively, signaling by TPβ was not directly affected by either agent.Collectively, while TPα and TPβ contribute to RhoA activation, our findingssupport the hypothesis that TPα is involved in the dynamic regulation ofhaemostasis and vascular tone, such as in response to prostacyclin and NO.Conversely, the role of TPβ in such processes remains unsolved. Data hereinprovide essential new insights into the physiologic roles of TPα and TPβ and,through studies in AoSMCs, reveal an additional mode of regulation of VSMcontractile responses by TXA2.In humans, thromboxane (TX) A Hence, the aim of the current study was to investigatewhether TPα and/or TPβ independently regulate G12/Rhoactivation and signaling and to establish whether that signaling isdifferentially regulated by the inhibitory prostacyclin/cAMP/PKA and NO/cGMP/PKGsystems. These studies provide essential new insights into the physiologic rolesof TPα and TPβ and, through studies in primary human aortic smooth muscle cells(1° AoSMCs), reveal an additional mode of regulation of VSM contractileresponses by the potent autocoid TXA2.While both TPα and TPβ are predominantly coupled to Gq/PLCβ activation22.1anti-HA, 3F10anti-HA-horseradish peroxidase (HRP)-conjugatedantibody and chemiluminescence detection kit from Roche;anti-RhoA 26C4 (Sc-418),anti-phospho-RhoASer188(Sc-32954-R), anti-Gα12/13 H-300(Sc-28588), anti-Gαq C15 (SC-392), HRP-conjugatedgoat anti-mouse (Sc-2005), HRP-conjugated mouseanti-goat (Sc-2354) and HRP-conjugated goatanti-rabbit (Sc-2004) antibodies from Santa Cruz;Glutathione-Sepharose 4B  and FITC conjugated goatanti-rabbit antibody from Sigma;anti-cofilin (# 3312) andanti-phospho-cofilin were from Cell Signaling; AlexaFluor® 488 phalloidin  from Molecular Probes;anti-HDJ2 antibody from Neomarkers; Opti-MEM® andOligofectamine® were from Invitrogen. All oligonucleotides were synthesisedby Genosys Biotechnologies; small interfering (si) RNAs by Qiagen. Cicaprostwas a gift from Schering AG .pcDNA3.1(+):hGαqQ209l,D277N,pCis:Gα12G228A andpCis:Gα13G225A were from the UMR cDNAResource Center (Gαq) or from Dr S. Offermanns, University of Heidelberg,Germany.U46619, SQ29,548, BW245C, FK409, FURA2/AM were purchased from CaymanChemical Company; SIN-1 and Y-27632 from Calbiochem; 3F102.2S329A HEK.TPαS331A andHEK.TPαS329,331A cell lines stably over-expressinghemagglutinin (HA) -tagged forms of TPα, TPβ,TPαS329A,TPαS331A andTPαS329,331A respectively, have been previouslydescribed 6 cells/10 cm dish in 8 ml media and co-transfectedwith 10 μg of pADVA and 25 μg of pCMV-based mammalian expression vectorusing the calcium phosphate/DNA co-precipitation procedure Human embryonic kidney (HEK) 293 cells were grown in minimal essentialmedium (MEM), 10% foetal bovine serum (FBS). HEK.TPα, HEK.TPβ,HEK.TPαPrimary (1°) human aortic smooth muscle cells (1° h.AoSMCs) werepurchased from Cascade Biologics (C-007-5C) and routinely grown in SmoothMuscle Cell Growth Medium 2  supplemented with0.5 ng/ml epidermal growth factor, 2 ng/ml basic Fibroblast growth factor,5 μg/ml insulin, 5% FBS.2.32+]i)mobilization were carried out in FURA2/AM preloaded HEK 293 cell linestransiently co-transfected with pCMV:Gαq and pADVA some 48 h previously, asdescribed 2+ mobilized(Δ[Ca2+]i(nM)) as a function of time  following ligandstimulation.Measurements of intracellular calcium in growth media containing 0.1% FBS beforestimulation for 0–30 min with 0–10 μM U46619, as indicated in specificfigure legends. To assess the effect of prostacyclin, nitric oxide (NO) orPGD2 on TP-mediated Rho signaling, cells werepre-incubated for 10 min with either 0.01–10 μM Cicaprost; 0.05–50 μM SIN-1;10 μM FK409 or 1 μM BW245C before stimulation with U46619 . As controls, cells were incubated with an equivalent volume ofthe drug vehicle, agonist or inhibitor in 0.01% ethanol in HBS  for equivalent incubation times.Activated cellular Rho was determined by interaction with a purifiedglutathione-S-transferase: rhotekin Rho-binding domain (GST-RBD) fusionprotein immobilized on Glutathione-Sepharose 4B resin 2, and 10 μg/ml each of leupeptin and aprotinin;anti-RhoA monoclonal antibody(Sc-418), followed by chemiluminescence detection anti-RhoA antibody and/or with the anti-HDJ2antibody. Similarly, to assess U46619-mediated cofilin phosphorylation andactivation, aliquots of whole cells lysates  were first immunoblotted withanti-phosphoSer3 cofilinantibody; thereafter, phospho-cofilin blots were stripped and rescreenedversus anti-cofilin antibody to normalise for totalcofilin protein expression and/or with the anti-HDJ2 antibody to confirmuniform protein loading in each of the assays. All images of RhoAexpression/pulldown or cofilin phosphorylation and/or expression werecaptured using Adobe Photoshop (V6), where band width and intensity wasquantified and represented as fold increases relative to basal levels. Toaccount for biological variations in basal activation levels, experimentswere normalised to within a comparable range based on measurements from morethen 20 individual experiments for each cell type.Thereafter, cells were lysed in 800 μl Lysis Buffer . To assess the role of prostacyclin or NO, cellswere pre-incubated for 10 min with either 0.01–10 μM Cicaprost or 0.05–50 μMSIN-1 before stimulation with U46619 . F-actinpolymerization was stained by the addition of Alexa Fluor® 488 phalloidinessentially as described by the supplier (Molecular Probes) and slides wereimaged using an Axioplan 2 Imaging Universal Microscope.2.6interference(RNAi) experiments, HEK 293cell lines  or 1° h.AoSMCswere plated at ~ 2.5 × 105 cells /35-mm plate and were allowed to attach for24 h, achieving ~ 30 % confluency. Thereafter, cells werewashed twice with serum-free Opti-MEM® and transfected for 4 h at 37 °C with0.2 μM TPα siRNA  or 0.2 μM TPβ siRNA or0.2 μM Lamin A/C siRNA (5'-CUGGACUUCCAGAAGAACAtt) using Oligofectamine®(3 μl/well) in Opti-MEM® (1 ml /well). Thereafter, 1 ml pre-warmed completemedia supplemented with 30% FCS was added per well and cells were harvestedfollowing incubation at 0, 24, 48, and 72 h. As additional controls, HEK.TPαcells were treated according to the latter conditions but using TPβ siRNAsand vice versa.For RNAanti-HA(3F10) antibody and, following stripping, were rescreened versusanti-HDJ antibody to confirm uniform proteinloading. Similarly, 1° h.AoSMCs were screened, under permabilisingconditions, by indirect immunofluorescence microscopy for TPα and TPβexpression using affinity purified isoform specific rabbitanti-TPα (3 μg/ml) andanti-TPβ (3 μg/ml) antibodies To confirm the efficacy of the siRNAs to disrupt TPα or TPβ expression,HEK 293 cell lines were harvested and subject to SDS-PAGE (25–50 μg/lane on12.5% gels) followed by electroblotting onto PVDF membranes (Roche).Membranes were initially screened versus the i disruption of TPα orTPβ expression in respective HEK 293 lines and in 1° h.AoSMCs, experimentswas scaled up 8.2-fold (2 × 106 cells on 10-cm dishes) and functional disruptionwas assessed through Rho pulldown assays or cofilin phosphorylation, aspreviously outlined herein.Thereafter, having optimised the conditions for effectiveRNA2.7d andBmax values. Statisticalanalyses were carried out using the unpaired Student'st test using the Statworks Analysis Package.p-values of less than or equal to 0.05 wereconsidered to indicate a statistically significant difference. Throughoutthe figures, ⁎ < 0.05, ⁎⁎< 0.01,⁎⁎⁎< 0.001.Radioligand binding data was analyzed using GraphPad Prism V3.0 todetermine the K33.12 receptor (TP)expressed in human tissues, to our knowledge, no such study has investigatedthe propensities or relative abilities of the individual TPα or TPβ isoformsto activate and/or regulate Rho-mediated signaling. Hence, herein weinvestigated TPα and TPβ-mediated Rho signaling in response to theTXA2 mimetic U46619 in clonal HEK 293 cell lines thatstably over-express either TPα (HEK.TPα cells) or TPβ (HEK.TPβ cells).Throughout these studies, TPα/TPβ-mediated Gq/PLCβ-dependent[Ca2+]imobilization was monitored as a comparative reference.Whilst a range of studies have investigated Gq/PLCβ-mediated signalingby both the TPα and TPβ isoforms of the TXA2+]i inresponse to U46619 stimulation, with maximal responses generated using 1 μMU46619 (12 (Gα12/Gα13)members but may also occur through Gq coupling, in certain settings at least12(Gα12G228A), but not of Gαq, significantly impairedU46619-mediated RhoA activation through both TPα(p = 0.0011and p = 0.8011respectively) and TPβ , while the dominantnegative form of Gαq  had no significanteffect , it significantly impairedU46619-induced RhoA activation by TPα expressed in HEK.TPα cells in aconcentration-dependent manner and SIN-1 (500 nM) impaired F-actin polymerization in HEK.TPα cells butneither agent affected such responses in HEK.TPβ cells  orthe NO-donor SIN-1 (5 μM) on U46619-mediatedimobilization, Rho activation, F-actin polymerization and cofilinphosphorylation by TPα and its specific site directed variantsTPαS329A, TPαS331A,TPαS329,331A defective in the Cicaprost-sensitive PKA(at Ser329), NO-sensitive PKG (at Ser331)or both  phosphorylation sites, aspreviously described by us 2+]imobilization in HEK.TPα and HEK.TPαS329A cells while havingno affect on such signaling in HEK.TPαS331A andHEK.TPαS329,331A cells  agonist BW245C also impaired RhoA activation by TPα  agonist BW245C alsosignificantly impaired RhoA activation .We next investigated TP-mediated Rho activation and cytoskeletalsignaling in a physiologically relevant, vaso-responsive model byinvestigating U46619-induced signaling in 1° h.AoSMCs, cells that expressboth TPα and TPβ h.AoSMCs A. Consistivation C and cofh.AoSMCs B and D.2 signaling through theprostacyclin (IP) and PGD2 (DP) receptors, respectively,can impair U46619-mediated Rho activation and cytoskeletal signaling in 1°h.AoSMCs. Moreover, our data generated in the HEK.TPα and HEK.TPβ cell linesover-expressing the individual TPα and TPβ isoforms, respectively, suggestthat such inhibitory responses of prostacyclin and NO are mediated, at leastin part, at the interface of the stimulatory GPCR (i.e. the TP). Morespecifically, by directly targeting TPα, prostacyclin and NO may impair itsRhoA-signaling both at the level of TPα itself in addition to at the levelof the well documented targets of cAMP/PKA and cGMP/PKG Hence, taken together, both NO-donors and prostanoid vasodilatoryagents, such as prostacyclin and PGDTPα and siRNATPβ, respectively-targeting agents to selectively disrupt or knock-down expression ofTPα and TPβ in 1° h.AoSMCs. To begin with, the siRNA agents were validatedby examining their ability to affect TPα and TPβ expression and RhoAsignaling in HEK.TPα and HEK.TPβ cells. Under optimized experimentalconditions, we observed effective isoform-specific knock-down of both TPαand TPβ expression following 72 h incubation of HEK.TPα and HEK.TPβ cellswith siRNAectivelyA, with ~Pα cells A therebyell line A. MoreovPβ cells B. ConverPαcells B.RNAi dtivation B. ConsisTPα impaired expression of TPα but not of TPβwhile siRNATPβ reduced expression of TPβ but not of TPαcells; mitogenic and hypertrophic growth of VSM cells; inhibition ofangiogenesis/vascularization 2,its synthase or its receptor have been implicated in various cardiovasculardisorders including thrombosis, myocardial infarction, unstable angina,atherosclerosis, systemic- and pregnancy-induced hypertension and ischemic heartdisease, processes in which RhoA dysfunction is widely implicated 2 signalsthrough 2 distinct isoforms referred to as TPα and TPβ 2 in humans isunknown there is substantial evidence that they may have distinctphysiologic/pathophysiologic roles The prostanoid TXA2+-independent pathways to both normal anddisease-processes within the vasculature Bearing this in mind and the growing appreciation of the critical role ofthe RhoA-mediated Ca2 mimetic U46619.Moreover, we also investigated the ability of TPα and TPβ to regulate eventsdownstream of RhoA: effector coupling by monitoring U46619-induced F-actinpolymerization and cofilin phosphorylation. The ubiquitously expressedactin-depolymerising factor cofilin readily undergoes Rho/Rho kinase-dependentphosphorylation at Ser3 either by the LIM kinase 1/212, mainly Gα13,-dependent mechanism but in certain settings, particularly at higher agonistconcentrations, may also occur through a Gq-mechanism through the specificinvolvement of the LARG (Leukemia-associated Rho guanine-nucleotide exchangefactor), but not the p115- or PDZ-, member of the RGS-containing RhoGEF family12 andGq on TP-mediated RhoA signaling and found that dominant negative forms ofGα12 (Gα12G228A), not ofGαq , significantly impaired U46619-mediatedRhoA activation and cofilin phosphorylation. Collectively, these data confirmedthat both TPα and TPβ can independently couple to Gq/PLCβ activation andGα12/Gα13/RhoGEF-RhoA activation and arein agreement with a host of studies in mouse platelets whereby the single TP inthat species couples to Gq/PLCβ and to G12/RhoA activation toindependently regulate platelet activation (aggregation and secretion) andplatelet shape change responses, respectively To this end, we investigated the ability of TPα and TPβ to mediate RhoAsignaling in established clonal HEK 293 cell lines that over-express theindividual TP isoforms 2+]imobilization by TPα, but not by TPβ, was desensitized in response to bothCicaprost and NO stimulation. In keeping with this, TPα-mediated RhoAactivation, F-actin polymerization and cofilin phosphorylation was alsospecifically impaired by Cicaprost and SIN-1 while neither agent affectedRho-mediated signaling by TPβ. As stated, while both prostacyclin and NOdesensitize TPα-mediated Gq/PLCβ signaling, they do so by entirely independentmechanisms involving direct PKA- and PKG- mediated phosphorylation of TPα atSer329 and Ser331, respectively, withinits unique C-tail domain S329A,TPαS331A, TPαS329,331A, variants of TPαdefective in the prostacyclin-sensitive PKA (at Ser329),NO-sensitive PKG (at Ser331) or both  phospho-target sites 2+]imobilization by TPα and TPαS329A, they had no affect onsignaling by TPαS331A and TPαS329,331A.Conversely, both Cicaprost and the PGD2 receptor agonist BW245Cimpaired RhoA activation, cofilin phosphorylation, F-actin polymerization and[Ca2+]imobilization by TPα and TPαS331A but had no affect on signalingby TPαS329A and TPαS329,331A. Collectively,these data suggest that both Gq/PLCβ-mediated[Ca2+]imobilization and G12/RhoGEF -dependent RhoA activation of itseffector rhotekin and cofilin phosphorylation by TPα, but not by TPβ, arespecifically impaired by the potent vasodilators SIN-1 and Cicaprost in thiscellular context, at least. Of course the inhibitory effects of bothprostacyclin and NO, and other agents that signal through cAMP and cGMP, on RhoAsignaling are widely documented and form an essential component of thehomeostatic regulatory mechanism that determines the balance between activationand inhibition, particularly within the vasculature S331A and TPαS329,331A isunaffected by SIN-1 while that signaling by TPαS329A andTPαS329,331A is unaffected by Cicaprost clearly suggestthat the observed effects of SIN-1 and Cicaprost on TPα, in the HEK 293over-expression system at least, are due to direct effects on TPα itself, namelythrough site specific prostacyclin-induced PKA (at Ser329) andNO-induced PKG (at Ser331) phosphorylation rather than at someother intermediary in the RhoA signaling cascade. Moreover, in keeping with thathypothesis, the finding that agonist-induced G12/RhoA signalingby TPβ is not affected by either prostacyclin or NO again suggests that theeffects of both vasodilators are due to direct effects on TPα itself and isentirely consistent with previous findings involving both prostacyclin- andNO-mediated desensitization of TPα and TPβ signaling through the Gq/PLCβeffector system Thereafter, we investigated the effect of the selective prostacyclinanalogue Cicaprost and the NO donor SIN-1 on TP-mediated RhoA activation andsignaling. Consistent with our previous reports 2 readily desensitize TP-mediated Rho activation andcytoskeletal signaling in 1° h.AoSMCs, findings entirely predicted from and inkeeping with outcomes from other systems TPα andsiRNATPβ reagents were validated whereby we observedeffective isoform-specific knock-down of both TPα and TPβ expression andRhoA-mediated signaling in their respective HEK 293 cell lines. Moreover, theeffective delivery and utility of the latter siRNAs in 1° h.AoSMCs was confirmedwhereby the siRNATPα reduced expression of TPα but not of TPβ,while siRNATPβ reduced expression of TPβ but not of TPα. It wasnotable that the level of siRNA-mediated impairment of TPα and TPβ expression inthe 1° h.AoSMCs was significantly higher than observed in HEK.TPα or HEK.TPβcells. The reason for this apparent discrepancy is simply owing to the fact thatthe stably transfected HEK 293 cell lines express TPα and TPβ in abundance(~ 2 pmol/mg protein) relative to that expressed in 1°h.AoSMCs (20–50 fmol/mg protein) and hence, the inability of the siRNA tocompletely konckdown TPα or TPβ expression in HEK.TPα or HEK.TPβ cells was notsurprising. Consistent with their reduced expression in the 1° h.AoSMCs, therewas a significant reduction in U46619-mediated Rho activation and cofilinphosphorylation in the presence of RNAidirected to either TPα or TPβ but not to Lamin A/C confirming that both TPα andTPβ contribute to the RhoA activation in h.AoSMCs. While SIN-1 and Cicaprostsignificantly impaired U46619-mediated RhoA activation in the presence of thesiRNA directed to Lamin A/C to levels similar to that in vehicle-treated cells,the inhibitory action of both agents on RhoA activation and cofilinphosphorylation in 1° h.AoSMCs exposed to the siRNATPα wassubstantially impaired. On the other hand, in the presence ofsiRNATPβ both SIN-1 and Cicaprost reduced U46619-mediatedRhoA, F-actin polymerization (data not shown) and cofilin phosphorylation tolevels not significantly different to those observed in vehicle-treated cells.Hence, both the NO and prostacyclin impair TP-mediated cytoskeletal changesinvolving RhoA activation, F-actin polymerization and cofilin phosphorylation in1° h.AoSMCs and they do so, at least in part, by specifically and directlytargeting TPα impairing its downstream signaling. On the other hand, neithervasodilatory agent directly target TPβ.Therefore, we extended our studies by investigating TP-mediated Rhoactivation and cytoskeletal signaling in the more physiologically relevantprimary human aortic smooth muscle cells. As expected, stimulation of cultured1° h.AoSMCs with U46619 led to a concentration-dependent RhoA activation,F-actin polymerization and cofilin phosphorylation. Moreover, Cicaprost (IPagonist) and BW245C (DP agonist) and the NO donors SIN-1 and FK409 eachsignificantly impaired such U46619-induced RhoA activation, F-actinpolymerization and cofilin phosphorylation in 1° h.AoSMCs. Hence, collectively,both NO-donors and the vasodilatory prostanoids prostacyclin andPGD12/Rho-mediated shape change while cGMP/PKG signaling inhibitsthe former Gq/Ca2+ dependent mechanism but does not affect thelatter RhoA/Ca2+ independent mechanism 188 within its hypervariable region188 does not apparently alter its association witheither RhoGEFs or RhoGAPs (GTPase activating proteins), it significantlyincreases its interaction with RhoGDI (GDP dissociation inhibitor) therebyreducing the level of membrane bound RhoA and impairing its ability to activateits key effectors including Rho Kinases in vitro and in vivo evidenceto suggest that Ser188 phosphorylation of RhoA impairsactivation of Rho kinase (ROCK 1/2), but does not affect its ability to activateother Rho effectors including rhotekin, mDia-1 and PKN 188 phosphorylation of RhoA may act as a ‘secondarymolecular switch’ capable of overriding GTP-elicited activation of certain RhoAeffectors, such as ROCK, but directing it to signal with (an)other subset of Rhoeffectors, perhaps in a cell specific manner. Returning to studies herein onTPα- and TPβ-mediated RhoA signalling, both NO and prostacyclin directly targetRhoA phosphorylation at Ser188 through their regulation of PKGand PKA signaling, respectively (data not shown). Hence, RhoA-mediated signalingby TPα is subject to regulation by both direct prostacyclin/PKA andNO/PKG-inhibition mediated through their respective phosphorylation ofSer329 and Ser331 within the unique C-taildomain of TPα in addition to the more general type of regulation throughSer188 phosphorylation of RhoA. On the other hand, TPβ isnot a direct target for either PKA or PKG phosphorylation or inhibition, but itsRhoA-mediated signaling would be sensitive to RhoA phosphorylation by eithersecond messenger kinase. Whilst it has not as yet been established whether the“molecular switch mechanism” resulting from RhoAS188phosphorylation proposed by Nusser et al. As stated, it is widely held that agents that signal through either cAMP-or cGMP-second messenger systems play a critical counter-balancing/inhibitoryaffect on RhoA-mediated signaling cascades Ser188 switch mechanism’exists within TXA2-responsive VSM or indeed in platelets, RhoAsignaling through TPβ may be directed away from one effector system, such asROCK signaling, in the direction of another effector(s), such as rhotekin,mDia-1 and PKN (Hence, as presented in our model , we prop and PKN  as propo2+-dependent andG12/RhoA/Ca2+-independent signaling byprostacyclin and NO within the vasculature. On the other hand, as TPβ remainsunaffected by either agent, at the interface of the receptor at least, thefunctional role of TPβ remains to be further clearly defined. The data hereinhighlight further critical differences between the TPα and TPβ receptor isoformsin terms of their regulation of Rho signaling that are likely to bephysiologically relevant in human tissues such as SM and suggest that selectivetargeting and impairment of TPα-mediated signaling may offer a usefultherapeutic approach in the treatment of certain vascular diseases such assystemic- and pulmonary-hypertension in which both TXA2 andRhoA dysfunction has been implicated Hence, in summary TPα- and TPβ-mediated RhoA signaling functionally divergeat the point at which prostacyclin and NO affect the RhoA signaling cascade.These data further support the hypothesis that TPα is the major regulatory TPisoform involved in vascular hemostasis being a direct target for inhibition ofboth its Gq/PLCβ/Ca


I complex, [Ag2(C12H10N4)2](ClO4)2, each AgI center is two-coordinated by one pyridine and one benzotriazole N-donor atom of two inversion-related 1-(2-pyridylmethyl)-1H-benzotriazole (L) ligands. This forms a unique box-like cyclic dimer with an intramolecular Ag⋯Ag separation of 4.479 (2) Å. Intermolecular C—H⋯O hydrogen-bonding interactions, involving uncoordinated ClO4               − ions, link the binuclear units, forming a two-dimensional network parallel to (10In the title centrosymmetric binuclear Ag DOI: 10.1107/S1600536809043669/zq2014Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


One of the two independent Mn atoms is seven-coordinate, binding to five carboxylate O atoms from different chdc ligands and two phenanthrene N atoms from a dipyridophenazine (L) ligand, while the second Mn atom is six-coordinate, binding to six carboxylate O atoms from different chdc ligands. The cis-chdc ligands bridge the trinuclear MnII clusters, forming chains, which are further linked into a three-dimensional network.In the title compound, [Mn For related structures, see: De 2007; Li 200.       3(C8H10O4)3(C18H10N4)2] = 0.060                           wR(F                           2) = 0.176                           S = 1.05                           5830 reflections376 parametersH-atom parameters constrainedmax = 0.64 e Å−3                        Δρmin = −0.74 e Å−3                        Δρ                                 PROCESS-AUTO  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL-Plus (Sheldrick, 2008SHELXL97.Data collection: 10.1107/S1600536808012737/bt2704sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808012737/bt2704Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The second O atoms of the acetate groups provide elongated Cu—O axial contacts, so that the acetates appear to be coordinated in a pseudo-chelate fashion. The pyrazole ligands are situated in cis positions with respect to each other. In the crystal structure, molecules are linked through intermolecular N—H⋯O hydrogen bonds, forming a one-dimensional chain.In the title compound, [Cu(C H-pyrazole and its 3,5-substituted derivatives, see: Fritsky et al. 2(C5H8N2)2] = 0.032                           wR(F                           2) = 0.071                           S = 1.02                           3713 reflections222 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.29 e Å−3                        Δρmin = −0.66 e Å−3                        Δρ                                 X-AREA (Stoe & Cie, 2002X-AREA; data reduction: X-RED (Stoe & Cie, 2002SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008DIAMOND (Brandenburg, 1999SHELXL97.Data collection: 10.1107/S1600536809019400/hy2196sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809019400/hy2196Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: