In the complex, the Mn2+ ion is seven-coordinated in an approximately pentagonal–bipyramidal environment by six N atoms from the hexadentate N,N,N′,N′-tetrakis(2-pyridylmethyl)cyclohexane-1,2-diamine (tpdach) ligand and one O atom from a perchlorate anion. The complex displays intermolecular π–π interactions between adjacent pyridine rings (centroid-to-centroid distance 4.133 Å). Moreover, there are weak intra- and intermolecular C—H⋯O hydrogen bonds. The Cl atom and two O atoms of the perchlorate are disordered, with a site-occupancy factor of 0.59 (2) for the major component.The asymmetric unit of the title compound, [Mn(ClO DOI: 10.1107/S1600536808025804/zl2134Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The ZnII atom is coordinated by the N,N-bidentate 6,6′-dimethyl-2,2′-bipyridine ligand and two iodide ions, resulting in a distorted ZnN2I2 tetrahedral geometry for the metal. In the crystal, there are weak π–π contacts between the pyridine rings [centroid–centroid distance = 3.978 (3) Å].The complete molecule of the title compound, [ZnI DOI: 10.1107/S1600536809043049/hb5152Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The complex is square planar and contains HL–  as a tridentate 2N (amino),O (carboxylate) donor. The metal to ligand bond distances are Pt-Cl 2.287(1) Å, Pt-O2.002 (1) Å, Pt-Ntrans Cl 2.014(1) Å and Pt-Ntrans O 2.073 Å. There is extensivehydrogen bonding, each molecule of Pt(HL)Cl being intermolecularly hydrogenbonded to ten others giving a 3-dimensional network. There is also oneintramolecular H-bond.The synthesis and crystal and molecular structures of the platinum(II) complexPt(HL)Cl where H


II atom in the title compound, [Ni(C12H8N5)2(H2O)4]·2H2O, lies on a center of inversion and is coordinated by the N atoms of two 3,5-di-4-pyridine-1,2,4-triazolate ligands and by four water O atoms in a slightly distorted octahedral geometry. The coordinated and uncoordinated water molecules interact with the N-heterocycles through O—H⋯N and O—H⋯O hydrogen bonds, generating a three-dimensional supramolecular architecture.The Ni DOI: 10.1107/S1600536809027688/hg2531Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The two on general positions bind through their four O atoms to five 2-phenyl-1H-1,3,7,8-tetraazacyclopenta[l]phenanthrene-chelated CoII ions, whereas the two on special positions bind to only four. Of the three Co atoms, two are chelated by N-heterocycles; the third is bonded to six O atoms. The bonding mode of the dianion gives rise to a three-dimensional network structure; the network is further consolidated by N—H⋯O hydrogen bonds.In the title polymer, [Co H-1,3,7,8-tetraazacyclopenta[l]phenanthren-2-yl)phenol transition metal dicarboxylate compounds. See, for example: Che 3(C19H12N4)2] = 0.067                           wR(F                           2) = 0.178                           S = 1.02                           10947 reflections712 parametersH-atom parameters constrainedmax = 0.64 e Å−3                        Δρmin = −0.59 e Å−3                        Δρ                                 RAPID-AUTO (Rigaku, 1998RAPID-AUTO; data reduction: CrystalStructure (Rigaku/MSC, 2002SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2008Data collection: 10.1107/S1600536808010738/bh2161sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808010738/bh2161Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The asymmetric unit is compossed of an NiII atom, which sits on an inversion center, a μ-1,5-bridging dicyanamide anion, a water molecule, and a free 4-hydroxypyridine molecule present in the zwitterionic pyridinium-4-olate form. The NiII atom is coordinated in a slightly distorted N4O2 octahedral geometry by four bridging dicyanamide ligands and two trans water molecules. In the crystal, the two-dimensional networks are linked via N—H⋯O and O—H⋯O hydrogen bonds, forming a three-dimensional network.The title compound, {[Ni(C DOI: 10.1107/S1600536809048880/su2153Isup2.hklStructure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Each zigzag chain is constructed from one CoII ion, one O-monodentate 5-nitroisophthalate (ndc) dianion, one N,N′-bidentate 2,2′-bipyridyl ligand and one water molecule. A symmetry-generated O,O′-bidentate ndc dianion completes the cobalt coordination environment, which could be described as very distorted cis-CoN2O4 octahedral. The bridging ndc ligands result in parallel chains running along the a direction, and O—H⋯O hydrogen bonds arising from the water molecules complete the structure.In the crystal structure of the title compound, [Co(C For uses of carboxylic acids in materials science, see: Church & Halvorson 1959; and in 8H3NO6)(C10H8N2)(H2O)] = 0.037                           wR(F                           2) = 0.106                           S = 1.01                           6672 reflections535 parameters6 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.95 e Å−3                        Δρmin = −0.29 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXTL.Data collection: 10.1107/S1600536808038178/hb2844sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808038178/hb2844Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Metal binding to 2-thiooxo-1,2-dihydropyridin-1-olate occurs in a bidentate fashion via O and S, and in a monodentate manner via the N-oxide O atom. π–π Interactions between polymeric chains are evident from centroid-to-centroid distances of pyridinethione fragments of 3.461 (6)–3.607 (6) Å. The N—O and C—S bond lengths are distinctively different from those in hitherto investigated NiII, ZnII and (H3C)2TlIII complexes of 2-thiooxo-1,2-dihydropyridin-1-olate, but correlate with those reported for 1-hydroxy- and 1-alkoxypyridine-2(1H)-thiones in the solid state.The title compound, [Li DOI: 10.1107/S1600536808003619/cv2377Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


MDA-VP cells were more resistant to etoposide than to amsacrine and had more resistance to etoposide than did MDA-parental cells. MDA-VP cells also expressed lower topo IIα RNA and protein levels than parental cells but had comparable topo IIβ levels. After infection with Ad-topo IIα, topo IIα, RNA and protein levels increased significantly, as did the cells' sensitivity to etoposide. In contrast, topo IIβ levels remained constant with little alteration in the cells' sensitivity to amsacrine. Band-depletion immunoblotting assays indicated that topo IIα was depleted in etoposide-treated, Ad-topo IIα-transduced MDA-VP cells but not in amsacrine-treated cells. Topo IIβ was depleted in amsacrine-treated, Ad-topo IIα-MDA-VP cells, with little change in the topo IIα levels. These results suggest that topo IIα gene transfer does not alter topo IIβ expression and that enhanced sensitivity to etoposide is therefore secondary to change in topo IIα levels. These studies support the theory that etoposide preferentially targets topo IIα, while amsacrine targets topo IIβ. © 2001 Cancer Research Campaign


II atom in the title compound, [Zn(C8H7N3O2S)(C12H8N2)]·2C2H6OS·H2O, is N,N′-chelated by the N-heterocycle and N,O,S-chelated by the deprotonated Schiff base in a distorted square-pyramidal enviroment. Hydrogen bonds link the mononuclear molecule, the water and the dimethyl sulfoxide (DMSO) molecules into a linear chain motif. One DMSO molecule is disordered over two positions in respect of the S atom in an approximate 1:1 ratio.The Zn DOI: 10.1107/S160053680804169X/sj2562Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The Mn—O bond lengths are in the range 2.1281 (13)–2.2011 (12) Å and the Mn—N bond lengths are 2.269 (2) and 2.278 (2) Å. Mn(II) atoms are double-bridged along the a axis by two pairs of bi-monodentate carboxyl groups, forming a double-stranded chain, while the bidentate 4,4′-bipyridine ligand bridges the Mn atom along the b axis. This results in a two-dimensional structure constructed of oblong grids with the sides of length 11.634 and 5.075 ÅThe Mn atom in the title compound, [Mn DOI: 10.1107/S1600536810034495/br2146Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


HIV-1 specific HLA-B-restricted CD8+ T cell responses differ from HLA-C-restricted responses in antiviral effectiveness. To investigate possible reasons for these differences, we characterized the frequency and polyfunctionality of immmunodominant HLA-B*57/B5801- and HLA-Cw*07-restricted CD8+ T cells occurring concurrently in nine study subjects assessing IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a by flow cytometry and analyzed sequence variation in targeted epitopes. HLA-B*57/5801 and HLA-Cw*07 restricted CD8+ T cells did not differ significantly in polyfunctionality (p = 0.84). Possession of three or more functions correlated positively with CD4+ T cell counts  and monofunctional CD8+ T cells inversely correlated with CD4 cell counts . There were no differences in polyfunctionality of CD8+ T cells specific to wildtype versus mutated epitopes. These results suggest that loss of polyfunctionality and increase in monofunctional HIV-1-specific CD8+ T cells are associated with disease progression independent of restricting HLA allele. Furthermore, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic HIV-1 infection. HIV-specific CD8+ T cells play a vital role in the control of HIV replication and disease progression . Howeverin vivo, HLA-C is not down-regulated by HIV-1 Nef from the surface of infected cells to the same extent that HLA-A and HLA-B molecules are  RNA copies/ml and the median CD4 count was 271 (range 202–411) cells/μl. The subject gender, age, CD4 count, viral load, HLA type and epitopes examined for each subject are shown in 6 PBMCs.The study subjects were seven females and two males, with a median age of 40 (range 27–58) years, all coexpressing HLA-B*57/5801 and Cw*07. The subjects were selected on the basis of possession of concurrent dominant HLA-B*57/5801 and HLA-Cw*07 restricted HIV-1 specific CD8+ T cell responses ≥500 SFC/106 SFCs for both HLA-B*57/5801 and HLA-Cw*07; these high magnitude responses were examined further in subsequent assays. The immunodominant HLA-B*57/5801-restricted responses were to the following four epitopes: TSTLQEQIAW (TW10), ISPRTLNAW (ISW9), QATQDVKNW (QW10) and KAFSPEVIPMF (KF11). In contrast, only one HLA-C response was targeted by the study subjects: KRQEILDLWVY (KY11) restricted by HLA-Cw*07  (data not shown) with HLA-B*57/5801-restricted responses in terms of breadth of positive IFN-γ epitope-specific ELISPOT responses detected. ISPRTLNAW (ISW9) and KAFSPEVIPMF (KF11) were dominantly presented by HLA-B*5703; whereas the presentation of QW9 and TW10 was variable between B*5702 and B*5801 and were least presented by HLA-B*5703 C. NotablPrevious studies of long term non-progressors have shown that HIV-1-specific CD8+ T cells restricted by protective alleles such as HLA-B*57 and HLA-B*27, may be more polyfunctional than CD8+ T cells restricted by other HLAs . To deteHIV-specific CD8+ T cells polyfunctionality was evaluated using multicolor flow cytometry by simultaneous measurement of five functions: IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a as previously described in other studies . On singThe vast majority of both HLA-B and HLA-C restricted CD8+ T cells (75%) in this study cohort were monofunctional B. The moTo further investigate potential differences in functional profiles upon stimulation with both HLA-B*57/5801- and HLA-Cw*-restricted peptides on all patients, we compared the frequencies of the different HLA-restricted CD8+ T cells expressing different functions detected in these individuals. The fraction of each function was determined as a percentage of the total CD8+ T cell response as previously described . No signPrevious studies have suggested that HLA-B-restricted HIV-1-specific CD8+ T cells are more polyfunctional than HLA-A and HLA-C  and thatGag and nef genes were sequenced in all nine patients as the epitopes studied here were located in these viral proteins. HLA-B*57/5801-restricted HIV-1-specific CD8+ T cell epitopes had more sequence variation (6/9) when compared to HLA-Cw*07-restricted epitopes (1/9), , despite the fact that the HLA-C-restricted response examined is in the highly variable Nef protein. The most frequent sequence changes were noted in the TSTLQEQIAW (TW10) and ISPRTLNAW (ISW9) Gag epitopes restricted by HLA-B*57/5801, and a single sequence variation was noted in the Nef Cw*07-restricted epitope KRQEILDLWVY (KY11) restricted by HLA-Cw*07  ELISPOT, defined as a minimum magnitude of 500 spot forming cells per million (SFC/million) PBMC for each of these responses.Viral loads (VL) were determined from plasma using Roche Amplicor (version 1.5) and CD4+ T cell counts were enumerated from fresh blood by Tru-Count technology using a four-color FacsCalibur flow cytometer (Becton Dickinson) as previously described .DNA for HLA typing was isolated using Puregene DNA isolation kit for blood  according to the manufacturer's instructions. HLA class I typing was done by DNA PCR using sequence-specific primers as described before .Ex vivo measurement of T cells for IFN-γ production was undertaken by the ELISPOT assay as previously reported (A panel of 410 overlapping peptides (18mers with 10–12 amino acid overlap) spanning the entire HIV-1 clade C consensus sequence were synthesized and used in matrix screening assays as previously described . Previoureported . The antEx vivo measurement of CD8+ T cells for expression of IFN-γ, IL-2, MIP-1β, TNF-α and CD107a was assessed by multicolor flow analysis as previously described  and rested for 2 h at 37 °C and 5% CO2. One million cells/ml were stimulated with optimal HLA-B*57/5801 or HLA-Cw*-restricted peptides that represented immunodominant (≥ 500 SFC on ELISPOT) responses in the study subjects (Staphylococcus enterotoxin B (SEB) were included in the assays. Anti-CD107a-PE-Cy5 (BD Biosciences) antibody was added and incubated for 30 min at 37 °C, 5% CO2, followed by addition of Brefeldin A  and Monensin  and incubated at 37 °C, 5% CO2 for total of 6 h. The cells were then washed with PBS (2% FCS), stained to differentiate between live/dead cells  and incubated for 30 min at 4 °C. Cells were then washed and stained with the following surface antibodies: anti-CD3-PE Cy5.5 , CD4-APC, CD8-APC Cy7 (both from BD Biosciences) and incubated for 20 min in the dark at room temperature. Cells were again washed and fixed in 1% paraformaldehyde  for 20 min in the dark at room temperature and were then permeabilized  and stained intracellularly with the following antibodies: anti-IFN-γ PE Cy7, anti-TNF-α Alexa 700, anti-MIP-1β PE, anti-IL-2 FITC  before incubation for 20 min in the dark at room temperature. The cells were washed twice with PBS and resuspended in 200 μl of PBS before acquisition on LSRII flow cytometer (BD Bioscience).escribed . In briesubjects A in the Between 200,000 and 1,000,000 events were collected per sample. Analysis was performed using DIVA and FlowJo 8.3.3 software . Initial gating was on the lymphocytes population, then forward scatter height (FSC-H) versus forward scatter area (FSC-A) to remove doublets . SubsequGag PCR primers were 5′-CTAGCAGTGGCGCCCGAACA-3′ and 5′GCAGTCTTTCATTTGGTGTCCTCC-3′. The same primers were used in the second round PCR reaction using the following conditions: 94 °C for 2 min, followed by 35 cycles of 94 °C for 15 s, 58 °C for 30 s, 72 °C for 1:30 min, with a final extension at 72 °C for 7 min. Nef PCR primers were 5′-TTCAGCTACCACCGATTGAGA-3′ and 5′-TGAGGGTTGGCCACTCC-3′. The PCR conditions were similar to those for gag except for the second round PCR reaction, where the annealing temperature was set at 56 °C for 30 s. Purified (Qiagen) PCR products were sequenced using BigDye v3.1 Terminator sequencing kit  on a XL-3100 automatic DNA sequencer (Applied Biosystems). Nucleotide sequences were analyzed using Sequencer 4.8 software . Alignments of reference and newly generated gag and nef sequences were performed using ClustalX and edited by Bioedit Sequence Alignment editor.HIV-1 RNA was extracted from 500 μl of plasma (VL < 5000 HIV-1 RNA copies/ml) or 140 μl plasma (VL > 5000 RNA copies) using QIAmp viral RNA extraction kit (Qiagen). RNA was reversed transcribed using one-step RT-PCR kit (Invitrogen) and gene specific primers under the following conditions: reverse transcription at 55 °C for 30 min, followed by amplification at 94 °C for 2 min, and 35 cycles of 94 °C for 15 s, 55 °C for 30 s, 68 °C for 2 min; and final extension at 68 °C for 5 min. Mann–Whitney U test was used to compare the median magnitude of HLA-B versus HLA-C responses. The same test was used to compare individual effector functions or polyfunctional responses between HLA-B*57/5801 and HLA-C responses in individuals. The Spearman rank correlation test was used to correlate individual and polyfunctional responses with viral loads and CD4 counts. Fisher's exact test was used to compare proportions of HLA-B and HLA-C epitopes with sequence variation.


The dihedral angles between the five-membered rings of the ligands are 4.1 (2), 9.4 (2) and 10.5 (2)°. In the crystal, molecules are linked by N—H⋯N hydrogen bonds, generating a layered network lying in (11In the title compound, [Co(C DOI: 10.1107/S1600536810011785/hb5377Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Acta Cryst. (2008), E64, m884–m885.Corrigendum to  Acta Cryst. (2008), E64, m884–m885] is corrected.The chemical name in the title of the paper by Suksai, Watchasit, Tuntulani & Pakawatchai [ Acta Cryst. (2008), E64, m884–m885], the chemical name in the title is incorrect. The correct title should be ‘Diaquabis{2,6-bis[N-(2-pyridinylmethyl)carbamoyl]phenolato-κ2            O            1,O            2}zinc(II)’.In the paper by Suksai, Watchasit, Tuntulani & Pakawatchai [


The asymmetric unit contains one CuII ion, one 3,5-dinitro-2-oxidobenzoate ligand and a 3-hydroxypyridine ligand. Coordination by one N atom and three O atoms from two different 3,5-dinitro-2-oxidobenzoate ligands and a 3-hydroxypyridine ligand creates a square-planar CuII center, which is augmented by a less tightly bonded fifth phenol O atom to form a square-pyramidal five-coordinate complex with an essentially planar base. The double-stranded chains are stabilized by intrachain π–π interactions [the centroid-to-centroid distance between adjacent aromatic rings is 3.719 (7) Å], and further linked through O—H⋯O hydrogen bonds, forming a three-dimensional supramolecular network.A new coordination polymer, [Cu(C DOI: 10.1107/S1600536808009392/zl2099Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Two of the O atoms originate from one naphthalene-1,4-dicarboxylate anion, whereas the remaining two O atoms derive from two symmetry-equivalent naphthalene-1,4-dicarboxylate anions. Two Mn atoms are connected via the anions into dimers, which are further linked by the anions and the N-donor ligands into a three-dimensional coordination network.In the crystal structure of the title compound, [Mn(C II, see Boeckmann et al. (C10H8N2)] = 0.063                           wR(F                           2) = 0.119                           S = 1.17                           7902 reflections523 parametersH-atom parameters constrainedmax = 0.31 e Å−3                        Δρmin = −0.36 e Å−3                        Δρ                                 X-AREA  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008DIAMOND (Brandenburg, 2008XP in SHELXTL (Sheldrick, 2008XCIF in SHELXTL.Data collection: 10.1107/S1600536809009301/bt2868sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809009301/bt2868Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


But after a 20 min incubation of α2-macroglobulin at 37°Cwith 2 × 106 human polymorphonuclear leukocytes activated byN-formyl-L-methionyl-L-leucyl-L-phenylalanine(10−7 M) and cytochalasin B (10−8 M), 100% of trypsinactivity was recovered, indicating a total inactivation ofα2-macroglobuHn. Incubation with granulocyte myeloperoxidase alsoinactivates α2-macroglobulin. Hypochlorous acid, a by-product ofmyeloperoxidase activity, at a concentration of 10−7 M alsoinactivates α2-macroglobulin, which indicates that an importantcause of α2-macroglobulin inactivation by activatedpolymorphonuclear leukocytes could be the activity of myeloperoxidase.The proteolytic activity of trypsin releases the dye RemazolBrilliant Blue from its high molecular weight substrate, the skinpowder , with an increase in absorbance at595 nm. Active α


In both 2--1,10-phenanthroline ligands, the 1,2,4-triazolyl ring and the 1,10-phenanthroline ring system are essentially coplanar . The dihedral angle between the mean planes of the ligands is 89.65 (4)°. In the crystal structure, there is a weak π–π stacking interaction between a pyridine ring and a symmetry-related benzene ring with a centroid–centroid distance of 3.772 (3) Å.In the title complex, [Cd(C DOI: 10.1107/S1600536809038768/lh2911Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the crystal structure, one O,S-dibenzyl dithiophosphate counter-anion links with the CuII complex cation through N—H⋯O and N—H⋯S hydrogen bonding. During the synthesis, the structure of the anion re-arranged from O,O′-dibenzyl dithiophosphate in the starting material to O,S-dibenzyl dithiophosphate in the title compound.In the crystal structure of the title compound, [Cu(C II complex, see: Xie et al. ](C14H14O2PS2)2                         = 0.051                           wR(F                           2) = 0.154                           S = 1.04                           4420 reflections275 parametersH-atom parameters constrainedmax = 0.45 e Å−3                        Δρmin = −0.69 e Å−3                        Δρ                                 CAD-4 Software (Enraf–Nonius, 1989CAD-4 Software; data reduction: XCAD4 (Harms & Wocadlo, 1995SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997SHELXL97.Data collection: 10.1107/S1600536809030037/xu2568sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809030037/xu2568Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the monomeric compound, the CuII atom is five-coordinated in a square-pyramidal configuration by one O atom and one N atom from one 1H-1,2,4-triazole-3,5-dicarboxylate (TZDCA2−) ligand and three O atoms from water molecules. In the centrosymmetric binuclear complex, each CuII atom is six-coordinated in an octahedral geometry by one O atom and one N atom from one TZDCA2− ligand and four O atoms from water molecules, two of which bridge the CuII atoms. In the structure, there are intramolecular O—H⋯O and N—H⋯O hydrogen bonds, and in the crystal, intermolecular O—H⋯O, O—H⋯N and N—H⋯O hydrogen bonds link symmetry-related molecules, forming a three-dimensional supramolecular structure.In the title compound, [Cu(C DOI: 10.1107/S1600536809026993/su2127Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Correction to Ramírez-Rodríguez VR, Frías-Treviño G, Aviña-Padilla K, Silva-Rosales L, Martínez-Soria JP: Presence of necrotic strains of Potato virus Y in Mexican potatoes. Virology Journal 2009, 6:48 The published version of this article  included


Consistent with the presence of three cysteines within its unique carboxyl-terminal domain, metabolic labelling and site-directed mutagenesis confirmed that TPβ is palmitoylated at Cys347 and, to a lesser extent, at Cys373,377 whereas TPα is not palmitoylated. Impairment of palmitoylation did not affect TPβ expression or its ligand affinity. Conversely, agonist-induced [Ca2+]i mobilization by TPβC347S and the non-palmitoylated TPβC347,373,377S, but not by TPβC373S or TPβC373,377S, was significantly reduced relative to the wild type TPβ suggesting that palmitoylation at Cys347 is specifically required for efficient Gq/phospholipase Cβ effector coupling. Furthermore, palmitoylation at Cys373,377 is critical for TPβ internalization with TPβC373S, TPβC373,377S and TPβC347,373,377S failing to undergo either agonist-induced or temperature-dependent tonic internalization. On the other hand, whilst TPβC347S underwent reduced agonist-induced internalization, it underwent tonic internalization to a similar extent as TPβ. The deficiency in agonist-induced internalization by TPβC347S, but not by TPβC373,377 nor TPβC347,373,377S, was overcome by over-expression of either β-arrestin1 or β-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPβ at Cys373,377 is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys347 has a role in determining which pathway is followed, be it by the β-arrestin-dependent agonist-induced pathway or by the β-arrestin-independent tonic internalization pathway.Palmitoylation is a prevalent feature amongst G protein-coupled receptors. In this study we sought to establish whether the TPα and TPβ isoforms of the human prostanoid thromboxane (TX) A The presence of Cys residues within the unique C-tail domain of TPβ raises the possibility that they may be palmitoylated and that such modification(s) may contribute to the functional differences between the two TP isoforms. Hence, the aim of the current study was to investigate whether TPβ is subject to palmitoylation and to examine the functional consequences thereof. Our studies confirm that TPβ, but not TPα, is palmitoylated at Cys347 and, to a lesser extent, at Cys373/377, a modification that not only regulates agonist-induced second messenger signalling but also differentially influences agonist- and tonic-induced internalization of TPβ.Another notable difference between the TP isoforms, the significance of which has yet to be investigated, is the presence of multiple cysteine (Cys) residues within the unique C-tail of TPβ at Cys22.13H]SQ29,548 (50.4 Ci/mmol) was obtained from DuPont NEN. Rat monoclonal 3F10 anti-HA-horse radish peroxidase-conjugated antibody (25 μg/ml) was obtained from Roche. Mouse monoclonal anti-hemagglutinin (HA)-101R antibody (1 mg/ml) was obtained from Eurogentec; goat anti-β-arrestin1 (K-16), goat anti-β-arrestin2 (N-16), horse radish peroxidase (HRP)-conjugated goat anti-mouse (400 μg/ml), HRP-conjugated mouse anti-goat (400 μg/ml) and HRP-conjugated goat anti-rabbit (400 μg/ml) secondary antibodies were from Santa Cruz; goat anti-mouse FITC-conjugated antibody, propidium iodide, protein G sepharose 4B Fast Flow (∼ 50% v/v slurry) were obtained from Sigma. AlexaFluor594 goat anti-mouse antibody (2 mg/ml) was from Molecular Probes.  Palmitic acid (60 Ci/mmol) was purchased from TOCRIS.U46619 and SQ29,548 were obtained from Cayman Chemical Company. G418 sulfate and FURA2/AM were from Calbiochem. SQ29,548. In this way, HEK.TPβC347S, HEK.TPβC373S, HEK.TPβC373,377S and HEK.TPβC347,373,377S cell lines stably over-expressing HA-tagged forms of TPβC347S, TPβC373S, TPβC373,377S and TPβC347,373,377S, respectively, were established. HEK.TPα, HEK.TPβ and HEK.hIP cells stably over-expressing HA-tagged form of TPα, TPβ and of the human IP, respectively, have been previously described Routinely, approximately 48 h prior to transfection cells were plated at a density of 2 × 102.43H]SQ29,548 for Scatchard analyses or in the presence of 20 nM [3H]SQ29,548 for saturation radioligand binding experiments as previously described TP radioligand binding assays were carried out at 30 °C for 30 min in the presence of 0–40 nM [2.55 cells/10-cm dish (∼ 80% confluency) on the day of metabolic labelling. Cells were washed once in PBS and then metabolically labelled in serum-free MEM (1.5 ml) containing 1.5 mCi of [3H] palmitic acid (60 Ci/mmol). Following incubation for 2 h at 37 °C, the labelling reaction was stopped by washing the cells in ice-cold PBS. Thereafter, cells were lysed by the addition of 600 μl RIPA buffer  and were disrupted by passing through needles of decreasing bore size. The lysate was centrifuged at 13,000 rpm for 5 min and the supernatant (600–700 μg) was subjected to immunoprecipitation using the anti-HA 101R antibody (1:300 dilution) followed by the addition of 10 μl protein G Sepharose 4B (50% v/v slurry) and further incubation at room temperature for 1 h, as described Palmitoylation was carried out essentially as previously described 2.62+]i) mobilization in response to the TXA2 mimetic U46619 (1 μM) were carried out in FURA2/AM preloaded HEK.TPβ, HEK.TPβC347S, HEK.TPβC373S, HEK.TPβC373,377S and HEK.TPβC347,373,377S cells that had been transiently co-transfected with pCMV:Gαq and pADVA some 48 h previously, essentially as described 2+/Mg2+-free Hank's buffered salt solution, containing 10 mM HEPES, pH 7.67, 0.1% bovine serum albumin) and the agonist in vehicle (20 μl) or, as controls, the vehicle alone (20 μl) were added to the 2 ml cells (0.8 × 106 cells/ml) to achieve the desired working concentration. It was established that the vehicle had no effect on [Ca2+]i mobilization by either TP isoform and had no effect on experimental data. The ratio of the fluorescence at 340 nm to 380 nm is a measure of [Ca2+]iKd of 225 nM Ca2+ for FURA2/AM. The results presented in the figures are representative data from at least three/four independent experiments and are plotted as changes in intracellular Ca2+ mobilized (Δ[Ca2+]i (nM)) as a function of time  following agonist stimulation.Measurements of agonist-induced intracellular calcium , non-specific sites were blocked with Blocking Buffer (5% dried skimmed milk powder in TBS) and then cells were incubated with anti-HA 101R antibody (1:1000 in Blocking Buffer) for 1 h at room temperature. The antibody solution was removed and the cells were washed twice in TBS, prior to incubation with goat anti-mouse HRP (1:1000) for 45 min at room temperature. Thereafter, the cells were washed trice in TBS and HA-tagged receptor expression was detected colorimetrically at 650 nm using the K-Blue substrate Cells were seeded at a density of 5 × 10To assess tonic internalization, 48 h post-seeding, cells were washed in MEM, 10% FBS, prior to incubation in media containing mouse anti-HA 101R (1 in 1000 dilution), at 4 °C for 1 h. Thereafter, the cells were rinsed briefly in MEM to remove unbound antibody prior to incubating the cells at 4 °C and 37 °C for 2 h. Cells were washed twice in ice-cold PBS prior to fixation in 3.7% paraformaldehyde, 1× PBS, pH 7.4, for 15 min at room temperature. After washing the cells twice in 1× TBS , non-specific sites were blocked with Blocking Buffer followed by incubation with the secondary goat anti-mouse HRP (1:1000) for 45 min at room temperature. Detection of the HA-tagged receptors was via colorimetric K-Blue substrate (Neogen Corp), as previously described 2.8l-lysine pre-treated coverslips in 6-well plates to achieve 60–70% confluency following 48 h incubation at 37 °C. Thereafter, the media was changed to MEM, 10% FBS and cells were incubated with anti-HA 101R (1 in 1000 dilution) at 4 °C for 1 h. Thereafter, unbound antibody was removed by washing twice with media. To investigate agonist-induced internalization, cells were washed once with serum-free MEM prior to incubation with 1 μM U46619 for 0, 2 and 4 h at 37 °C. To examine tonic internalization, following labelling with anti-HA 101R (1 in 1000 dilution) at 4 °C for 1 h, cells were washed briefly in MEM to remove unbound antibody prior to incubation at 4 °C and 37 °C for 2 h in MEM, 10% FBS.Cells were seeded onto poly-2O). Excess propidium iodide was washed away with H2O prior to mounting coverslips in Vectashield (Vector Laboratories) mounting medium. Slides were then imaged using Carl Zeiss Lazer Scanning System LSM510 using Zeiss LSM Imaging software.After the appropriate incubation, cells were washed twice in ice-cold PBS prior to fixation in 3.7% paraformaldehyde, 1× PBS, pH 7.4, for 15 min at room temperature and thereafter washed three times with PBS. Cells to be permeabilized were then incubated with 0.2% Triton X-100 in PBS for 10 min on ice followed by washing in 1× TBS. Non-specific sites were blocked by incubating cells with Blocking Buffer. Thereafter, receptors that had been previously labelled with the anti-HA 101R antibody were labelled with the secondary goat anti-mouse FITC-conjugated antibody solution. Cells were washed 3 times in 1× TBS, prior to counterstaining with propidium iodide  pre-coated glass coverslips (22 mm diameter) in 6-well plates and incubated for a further 48 h. Thereafter, cells were incubated with anti-HA 101R  at 4 °C for 1 h. Unbound antibody was removed by washing twice with media prior to incubating cells with 1 μM U46619 (prepared in media) for 30 min at 37 °C. Thereafter, cells were fixed in 3.7% paraformaldehyde, 1× PBS, pH 7.4, for 15 min at room temperature. Following washing (3 times with 1× PBS), cells were permeabilized by incubation on ice for 10 min in 0.2% Triton X-100 in PBS. Following washing twice in 1× TBS  and further incubation for 30 min with Blocking Buffer (5% dried skimmed milk powder in 1× TBS), HA-tagged receptors, previously labelled with the anti-HA 101R antibody, were visualized using the AlexaFluor594 goat anti-mouse IgG (1:1500) as secondary antibody. Cells were washed trice in TBS, prior to counterstaining with DAPI . Excess DAPI was washed away with H2O prior to mounting coverslips in Vectashield mounting medium. A Zeiss fluorescence microscope coupled with AxioVision software (version 4.4) was used for acquiring multichannel, Z-stack images  with filters appropriate for enhanced GFP and AlexaFluor 594 fluorescence. Subsequently, the multichannel, Z-stack images were processed by AxioVision Deconvolution Analysis, with enabled multichannel and Z-stack correction.HEK 293 cells  were transiently co-transfected with pHM6-based plasmid encoding HA-tagged TPβ (365 ng), or its mutated variants, along with either pEGFPN3:β-arrestin1 or pEGFPN1:β-arrestin2, encoding green fluorescent protein (GFP)-tagged forms of the β-arrestin1/2 (365 ng), in the presence of pADVA (270 ng) using the Effectene (Qiagen) transfection reagent, as per manufacturer's instructions. Some 24 h post-transfection, cells were replated onto fibronectin  isoforms are palmitoylated, human embryonic kidney (HEK) 293 cells stably over-expressing hemagglutinin (HA) epitope-tagged forms of TPα and TPβ were metabolically labelled with [3H] palmitic acid. As a control, metabolic labelling was also investigated in HEK.hIP cells stably over-expressing HA-tagged human (h) prostacyclin receptor (hIP), a GPCR previously confirmed to be palmitoylated To investigate whether the human thromboxane (TX) A347, Cys373 and Cys377 to Ser residues, either individually and collectively, to generate TPβC347S, TPβC373S, TPβC373,377S and TPβC347,373,377S. HEK 293 cell stably over-expressing TPβC347S, TPβC373S, TPβC373,377S and TPβC347,373,377S were generated and initially characterised by saturation radioligand binding analysis using the selective TP antagonist SQ29,548. HEK.TPβC347S, HEK.TPβC373S, HEK.TPβC373,377S and HEK.TPβC347,373,377S cells were confirmed to display similar ligand binding characteristics (Kd and Bmax) relative to HEK.TPβ cells and the mutations per se had no affect on the receptor affinity  were significantly reduced relative to TPβ . However, as it was not possible to generate stable cell lines over-expressing TPβC377S, we cannot conclude with certainty whether Cys377 along with Cys373 is palmitoylated.Thus, in summary, it is evident that TPβ is palmitoylated at multiple Cys residues within its C-tail domain and that mutation of Cys3.22+]i) mobilization by TPβC347S, TPβC373S, TPβC373,377S and TPβC347,373,377S in response to the TXA2 mimetic U46619. Consistent with previous reports q, showed efficient [Ca2+]i mobilization in response to U46619 . On the other hand, while TPβC347S did show a substantial signalling response . Moreover, consistent with this, U46619-induced [Ca+]i mobilization by TPβC347,373,377S was also impaired  whereas there was no significant difference in signalling between TPβC347S and TPβC347,373,377S (p = 0.79). Taken together, these data confirm that palmitoylation of TPβ plays a significant role in mediating its efficient coupling to Gq/PLCβ activation and, moreover, established that palmitoylation of Cys347 is specifically required for this regulation.Numerous studies have indicated that the covalent attachment of palmitate has been implicated in the regulation of receptor G protein coupling and subsequent downstream signalling events o U46619 A. Stimulresponse B, the leimpaired E, showin3.3C347S, TPβC373S, TPβC373,377S and TPβC347,373,377S, initially using an ELISA-based assay. Cells were treated with U46619 (1 μM) over a 4 h period, prior to fixation and immunolabelling with anti-HA 101R. Consistent with previous reports 347, Cys373 or Cys377 either singly or in combination, significantly impaired agonist-induced internalization .Agonist-induced internalization of TPβ, TPβcroscopy . Owing tnditions A, 4 h. Inditions B. MoreovC347S, TPβC373S, TPβC373,377S and/or TPβC347,373,377S, the ability of TPβ to undergo agonist-induced internalization is impaired, showing no significant reduction in cell surface expression by either mutated variant.Taken collectively, these data are consistent with previous studies confirming that TPβ is readily internalized in response to exposure to agonist 3.4C347S, TPβC373S, TPβC373,377S and TPβC347,373,377S were pre-labelled at 4 °C with anti-HA 101R prior to incubating the cells for 2 h at 4 °C and 37 °C, to evaluate tonic internalization initially using an ELISA-based assay. Consistent with previous reports C347S also underwent similar temperature-dependent tonic internalization to levels similar to that of the wild type TPβ. Specifically, it was found that cell surface expression of TPβC347S was reduced by approx. 40% at 37 °C and to a level that was not significantly different from that of the wild type TPβ (p = 0.32). On the other hand, the level of tonic internalization of TPβC373S, TPβC373,377S or TPβC347,373,377S was substantially impaired relative to TPβ (C347S (p < 0.0005) showing, on average, only a 15% loss of cell surface receptor expression following 2 h at 37 °C.As stated, TPβ, but not TPα, has also been reported to undergo tonic internalization through a temperature- and dynamin-dependent but GRK- and β-arrestin-independent manner e to TPβ  or TPβ CC347S, TPβC373,377S or TPβC347,373,377S were pre-labelled with anti-HA 101R at 4 °C; thereafter images were captured either directly (0 h), or following incubation for 2 h at 4 °C or 37 °C under non-permeabilizing (data not shown) and permeabilizing conditions .To further investigate the influence of palmitoylation on internalization, we also monitored temperature-dependent tonic internalization of TPβ and its palmitoylation defective variants by confocal microscopy . In brienditions . At time347, Cys373 or Cys377, either singly or in combination, abolishes agonist-induced internalization. However, like TPβ, TPβC347S actually retains the ability to undergo tonic internalization while that of TPβC373S, TPβC373,377S and TPβC347,373,377S is substantially impaired.Hence, in summary, whilst TPβ undergoes agonist-induced and tonic internalization, disruption of palmitoylation by mutating Cys3.5C347S, TPβC373,377S and TPβC347,373,377S with the view to assessing their effect on agonist-mediated internalization. In the case of TPβ, consistent with previous reports C347S does not undergo significant agonist-induced internalization . On the other hand, consistent with previous data . Over-expression of β-arrestin1 and β-arrestin2 was confirmed in HEK.TPβ cells by Western blot analysis using anti-β-arrestin1 and anti-β-arrestin2 , TPβC373,377S (data not shown) and TPβC347,373,377S along with GFP-tagged forms of either β-arrestin1 and β-arrestin2. HA-tagged cell surface receptors were initially immunolabelled prior to stimulation with U46619 for 30 min; thereafter, cells were permeabilized and HA-tagged receptor or GFP-tagged β-arrestin expression and co-localizations were captured by fluorescence microscopy  nor TPβC347,373,377S underwent internalization even in the presence of over-expressed β-arrestins  or TPβC347,373,377S  7, which may potentially be palmitoylated A typical feature of palmitoylation is that it is viewed as a reversible modification, with the propensity to regulate protein:membrane or protein:protein association, protein localization and/or function in a dynamic manner 347 is required for its efficient Gq-mediated PLCβ activation, the primary effector of TP signalling. Specifically in the case in TPβC347S and TPβC347,373,377S, there was a 30% reduction in U46619-mediated [Ca2+]i mobilization compared to that of the wild type TPβ. In contrast, U46619-mediated [Ca2+]i mobilization by TPβC373S and TPβC373,377S was not significantly different from TPβ, indicating that palmitoylation at Cys373 and/or Cys377 does not influence Gq/PLCβ effector signalling. The reduction in [Ca2+]i mobilization by TPβC347S or TPβC347,373,377S was not due differences in their ligand binding properties as each of the palmitoylation deficient mutants displayed similar affinities for the selective TP radioligand [3H]SQ29,548 and their overall level of expression was not significantly different to that of the wild type TPβ. The specific requirement for palmitoylation at Cys347 for TPβ-mediated Gq/PLCβ activation is somewhat similar to that previously reported for the hIP where it was established that palmitoylation at Cys308, but not at Cys311, is specifically required for its Gq/PLCβ coupling Numerous studies have indicated that the consequences of palmitoylation of GPCRs are wide-ranging with many aspects of receptor function being potentially implicated including receptor processing, G protein/effector coupling, phosphorylation and desensitization, sequestration and internalization, trafficking and/or protein turnover per se does not affect the overall level of surface expression and does not appear to affect the maturation or surface expression of TPβ. As TPβ is reported to undergo agonist-dependent and agonist-independent or tonic internalization, albeit through distinct mechanisms, we next sought to determine whether palmitoylation may affect TPβ internalization 347, Cys373 or Cys377, either alone or in combination, significantly impaired agonist-induced TPβ internalization reducing the movement of the respective cell surface receptors  into intracellular compartment(s) relative to the wild type TPβ. On the other hand, while tonic internalization by TPβC373S, TPβC373,377S and TPβC347,373,377S was also significantly impaired, it was striking that the variant TPβC347S retained the ability to undergo tonic internalization. Furthermore, over-expression of either β-arrestin1 or β-arrestin2 was able to overcome the inability of TPβC347S to undergo agonist-induced internalization, an effect not observed with the other palmitoylation deficient mutants including TPβC373S, TPβC373,377S and TPβC347,373,377S. These data indicated a critical role for palmitoylation of Cys373,377 in both agonist-dependent and tonic internalization of TPβ, while palmitoylation of TPβC347 is also required for agonist-induced, but not tonic internalization.A number of studies have demonstrated that impairment of receptor palmitoylation, as exemplified by the CCR5 receptor, can result in the accumulation of GPCRs in intracellular stores and in the lowering of cell surface expression 347 and Cys373,377 separated by some 30 amino acid residues within its unique C-tail domain, to regulate G protein coupling and receptor internalization. One of the structural consequences of GPCR palmitoylation is that, by increasing local hydrophobicity, the palmitoyl moiety(s) is thought to become inserted into the membrane lipid bilayer resulting in the formation of a putative fourth intracellular loop within its C-tail domain, such as originally demonstrated for rhodopsin 4(a) receptor, Ponimaskin et al. 328/Cys329) located proximal to TM7 but is also palmitoylated at Cys386, located one amino acid residue from the C-terminus. They proposed that membrane insertion of the palmitates at the proximal Cys328/Cys329 and distal Cys386 residues resulted in the formation of a double loop structure within the C-tail of the 5-HT4(a) receptor. While palmitoylation deficient mutants of 5-HT4(a) receptor were indistinguishable from the wild type receptor in mediating G protein/effector signalling, mutation of the proximal and distal residues was shown not only to differentially modulate constitutive activation but also to modulate agonist-dependent phosphorylation, desensitization and β-arrestin internalization of the 5-HT4(a) receptor 308/Cys311, and by a distal farnesyl isoprenoid permanently attached to its C-terminus and that collectively these dual lipidations modulate G protein coupling and effector signalling Taken together, our results indicate that TPβ is palmitoylated at 2 independent sites Cys347 and at Cys373,377 also leads to the formation of a double loop structure within its C-tail domain. In our model, palmitoylation at Cys347 and membrane insertion leads to the formation of Loop A which, though not obligatory for Gq coupling, leads to more efficient Gq/PLCβ activation and [Ca2+]i mobilization. On the other hand, palmitoylation at the couplet Cys373,377, leads to the formation of Loop B or C, depending on whether Cys347 is also palmitoylated or not, respectively 2/3 phosphorylation leading to β-arrestin recruitment and TPβ desensitization and internalization 347 and Cys373,377 within the C-tail of TPβ. Hence in our model (347 and Cys373,377, leading to the formation of Loops A and B, would expose the agonist-induced internalization motif and GRK phosphorylation site at Ser357 thereby enabling β-arrestin recruitment. Whilst formation of Loop C by palmitoylation at Cys373,377 alone, as observed in TPβC347S, may also expose the internalization motif and GRK site at Ser357, we propose that the C-tail is in an altered, less favorable conformation for β-arrestin interaction and with reduced affinity for β-arrestin binding. Hence, palmitoylation of Cys373,377 alone, as in the case of TPβC347S, yields a receptor with impaired agonist-induced internalization owing to its reduced affinity for β-arrestin1/2. Consistent with this, through our experimental observations, over-expression of either β-arrestin1/2 overcame this reduced affinity by TPβC347S and facilitated its internalization in response to U46619-stimulation to levels that were indistinguishable from those of the wild type TPβ. Moreover, our immunolocalization data supported the agonist-dependent recruitment of GFP-tagged forms of β-arrestin1 and β-arrestin2 to both the agonist-activated TPβ and TPβC347S but not to TPβC347,373,377S, for example.In order to rationalise the functional consequences of palmitoylation of TPβ we present a model, as outlined in ectively . Whilst ur model  and as sOur proposed model accounting for the role of palmitoylation in the regulation of β-arrestin-dependent agonist-induced internalization of TPβ is consistent with a number of other studies. In the case of the V2 vasopressin receptor, for example, Charest and Bouvier established that palmitoylation enhances the β-arrestin recruitment to the activated V2R, facilitating internalization and other processes requiring the scaffolding action of β-arrestin including activation of the mitogen-activated protein kinase cascades 339X3Φ343, where X is any residue and Φ is a bulky hydrophobic isoleucine, within the C-tail domain of TPβ. Hence, the critical residues required for tonic internalization lie between residues 339 and 343 in close proximity to the Cys347 within the proposed Loop A structure associated with TPβ (347 may modulate tonic internalization of TPβ. Our experimental finding that TPβC347S, in addition to the wild type TPβ, undergoes tonic internalization suggests that the conformation of Loop C is sufficient to present or orientate the latter motif for internalization to occur. However, the fact that TPβC373,377S which contains Loop A, but not Loop B, does not undergo tonic internalization leads us to propose that other unidentified sequence determinants within Loop B, associated with TPβ, may also be required for efficient tonic internalization and that the conformation of Loop C may also orientate those sequences to facilitate that internalization. The identities of those additional sequences required for tonic internalization remain to be established.Whilst TPβ undergoes agonist-induced internalization via a dynamin-, GRK2/3- and β-arrestin-dependent mechanism, requiring an active or dynamic actin cytoskeleton with TPβ . Hence, 347 and Cys373,377, within its unique C-tail domain. Whilst palmitoylation at Cys347 is required for efficient Gq/PLCβ effector coupling, palmitoylation at Cys373,377 is essential for receptor internalization, with palmitoylation at Cys347 acting as a structural determinant of the route by which the receptor is internalized, be it β-arrestin-dependent or independent internalization. Due to the importance of TXA2 in vascular hemostasis, a clearer understanding of how the TP isoforms are differentially regulated is critical. Palmitoylation provides a dynamic and reversible post-translational modification that can regulate TPβ, in terms of its signalling, receptor desensitization and internalization and, as such, acts as a major determinant of TPβ function. Moreover, data presented herein provide a further rational explanation for the clear functional and mechanistic differences between the TPα and TPβ isoforms.In summary, various studies have indicated that many GPCRs are palmitoylated and that the functional consequences are diverse between receptors


The PtIV atom is six-coordinated in an octahedral configuration by two N atoms of the 4,4′-dimethyl-2,2′-bipyridine ligand and four terminal Cl atoms. In the crystal structure, there are weak π–π interactions between pyridine rings, with a centroid–centroid distance of 4.365 (3) Å.The asymmetric unit of the title compound, [PtCl DOI: 10.1107/S1600536808016796/hk2470Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Silencing of HIPK2 led to a 51 ± 0.2% decrease in Ser-269 phosphorylation in MIN6 β-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature β-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in β-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 β-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1  The analysis of (de)phospho-Ser-269-specific mutants suggest that phosphorylation at this site, whilst having no effect on PDX1 protein stability or PDX1 DNA-binding property, is involved in nucleoplasmic (versus nuclear-peripheric) localization in the β-cell in response to glucose.Humphrey and colleagues 2EC Directive 86/609/EEC for animal experimentshttp://europa.eu.int/scadplus/leg/en/s23000.htm; and the Uniform Requirements for manuscripts submitted to Biomedical journalshttp://www.nejm.org/general/text/requirements/1.htm.The work described in this article has been carried out in accordance with the 2.1Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza) containing 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin and 100 IU/ml streptomycin. INS-1(832/13) cells  and MIN6 β (mouse insulinoma pancreatic beta) cells were cultured as in c-myc antibody was from Roche. Rabbit polyclonal anti-PDX1 antibody was as described 262PSGLSVpSPQPSSIAPLRPQEPR284 . HIPK2 was purchased from Upstate .Anti-2.2Islets were isolated from CD1 mice and cultured as previously described 2.3c-myc has been described -c-myc and pcDNA3-PDX1-S269E-c-myc were generated using a QuikChange site-directed mutagenesis kit (Stratagene). Wild-type and mutant PDX1 myc-tagged coding sequences were inserted (HindIII/EcoRV) into the shuttle vector pAdTrack-CMV multiple cloning site c-myc was cloned (NcoI/BamHI) into 6xHis-MBP  plasmid. Mutant plasmid His-MBP-PDX1 (S269A) was generated as above.Plasmid pcDNA3-PDX1-2.4Escherichia coli BL21 with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Proteins were purified on a nickel–nitrilotriacetic acid column according to Qiagen and dialyzed for 16 h at 4 °C in 50 mM Tris pH 7.9, 150 mM NaCl, 5 mM MgCl2, 1 mM β-mercaptoethanol. The MBP moiety was cut with Tobacco Etch Virus (TEV) protease, AcTEVTM protease (Invitrogen). MBP, histidine tag and histidine-tagged Ac-TEV protease were removed respectively with Amylose beads (New England BioLab) and Ni–NTA agarose beads.His-MBP-PDX1 proteins expression was induced in 2.5Recombinant adenoviruses expressing wild-type (WT) and mutant  PDX1 and control adenovirus, expressing green fluorescent protein (Ad-GFP) were prepared using the AdEasy system 2.6Total mRNA and real-time quantitative RT-PCR analysis was as Data were analyzed by ABR PRISM SDS v1.3.1 (Applied Biosystems).2.7MIN6 β-cells infected with wild-type or mutant forms of PDX1 viruses at 50 MOI (multiplicity of infection). Immunocytochemical analysis was performed as described in the figure legends and in 2.83VO4 (sodium orthovanadate); 1 × CompleteTM protease inhibitor cocktail solution (Roche) and 1 × phosphatase inhibitor cocktail 1 & 2 (Sigma)] were rotated on wheel at 4 °C for 30 min. The lysate was clarified by centrifugation at 16,000g for 5 min. c-myc antibody-conjugated beads  were added and tubes rotated at 4 °C overnight. After centrifugation at 1,000g at 4 °C for 30 s, beads were washed five times with lysis buffer.Cells lyzed on ice with 750 μl of IP buffer cysteine/methionine (Perkin–Elmer), after which cells were washed and cultured for 2, 4, 8, and 16 h in 5 ml chase media with 10,000-fold molar excess of cold l-cysteine, l-methionine or harvested as the zero time point. Cells were lyzed in 1 ml RIPA . Lysates were immunoprecipitated with anti-myc antibody (Roche). After five washes with RIPA buffer, proteins were eluted and analyzed by SDS–PAGE. PDX1 proteins were detected by fluorography and quantified in a Cyclone (Perkin–Elmer) phosphoimager storage system.MIN6 β-cells were infected with PDX1 wild-type, S269A and S269E adenoviruses. 24 h after infection, media were replaced with fresh DMEM containing 3 mM glucose and cells were cultured for 16 h. Cells were transferred for 1 h to cysteine/methionine-free medium, pulsed for 3 h with 0.6 mCi  pulse chase labeling experiments which revealed equal stability of phospho/de-phospho-Ser-269 mimetic mutants of PDX1 . The turnover rates of S269A and S269E PDX1 were not significantly different from wild-type PDX1  and accessory components of the transcription machinery  . ImportaIn contrast to earlier studies 5Our data provide evidence that serine 269 in mouse PDX1 is phosphorylated in living pancreatic β-cells, as revealed through the use of mass spectrometry and confirmed by the generation of a phosphoserine-specific antibody. We also show that this event affects the subnuclear localization of PDX1 in β-cells.


Here, we show that the transient transfection of the proline allele in p53 null cancer cells exposed to low oxygen tension or to the hypoxia-mimetic drug Desferoxamine induces a higher amount of cell death than the arginine allele. Accordingly, proline allele transiently transfected cell lines express lower levels of hypoxia pro-survival genes , compared to those transiently transfected with the arginine allele. Further, we report that the exposure of the arginine/proline heterozygote MCF-7 breast cancer cell line to cytotoxic concentration of Desferoxamine for several weeks, gives raise to hypoxia-resistant clones, carrying the arginine, but not the proline allele. These data indicate that the p53 codon 72 proline allele is less permissive for the growth of cancer cells in a hypoxic environment, and suggest that the preferential retention of the arginine allele in the tumour tissues of arginine/proline heterozygous patients may depend upon its lowered capacity to induce cell death in a hypoxic tumour environment.The preferential retention of the arginine allele at the p53 codon 72 locus is commonly observed in tumours from arginine/proline heterozygotes. Considering that cancer cells are harboured in a hypoxic environment  MCF-7 cells were grown in RPMI-1640 medium, supplemented with 10% FBS; PC-3 cells were grown in F-12K medium supplemented with 10% FBS; HEP-3B cells were grown in low glucose DMEM, supplemented with 10% FBS; MDA-MB-157 cells were grown in 50 : 50 mixture of DMEM/F-12K media, supplemented with 5% FBS. All the cultures were kept at 37°C in a 5% CO2-humidified atmosphere.All culture media -1640, F-12K, Low glucose Dulbecco's modified Eagle's medium (DMEM)), fetal bovine serum (FBS), 2) was generated in a humidified incubator supplied with 95% N2/5% CO2 . Desferoxamine (DFX)  was used as hypoxia mimetic. MCF-7 cells were exposed to various concentrations of DFX (100–500 μM), for 4 weeks. After a massive cell death, several (20) clones were isolated in 100 μM administered cultures, and were genotyped for the p53 codon 72 polymorphism  was extensively passaged and cultured for at least 1 year in absence of DFX, without appreciable changes in morphology and gene expression . Transfection efficiency was scored using fluorescent microscopy of the GFP-expressing cells.Plasmids encoding the p53 codon 72 proline or arginine allele were obtained by cloning the entire p53cDNA into a pCMS-GFP expression vector , as described elsewhere , provided by Dr M. Oren . P53D is a C-terminal fragment of p53 that retains the multimerisation, but not the transcriptional transactivation domain, and it forms transcriptionally inactive multimers with endogenous wild-type p53 protein, which in turn accumulate in the cells owing to the lack of MDM2-mediated degradation (μm pore size filter. After supplementation with 4 μg ml−1 polybrene (Sigma), the augmented medium was applied to MCF-7 cells at 50% for 24 h. Puromycin 2 μg ml−1 (Sigma) was used as a selection agent to isolate successfully transduced cells.Retroviral gene transfer was performed as described previously  and a non-specific short interfering RNA (siRNA) control oligonucleotide , . Transfection procedures were performed using Lipofectamine 2000 (Invitrogen). Briefly, 60% confluent MCF-7 cells were incubated with 1 FW, 5′-GAC CCAGGTCCAGATGAAGCT-3′, RV, 5′-ACCGTAGCTGCCCTGGTAGGT-3′. PCR conditions were as follows: initial denaturation at 94°C for 3 min, 29 cycles with denaturation at 94°C for 1 min, annealing at 63.5°C for 1 min and extension at 72°C for 1 min, followed by final extension at 72°C for 7 min. The 156 bp PCR product was digested with restriction enzyme BstUI , which recognises a restriction site on the arginine allele, yielding a 109 bp fragment (and an undetectable band of 47 bp). The entire p53 cDNA was amplified by using the following primers, 5′-GCCATG GAGGAGCCGCAGTC-3′ and 5′-TCAGTCTGAGTCAGGCCCTT-3′. The 1189 bp fragment was sequenced in a CEQ8000 Automatic Sequencer , using the following primers: 5′-GCCATGGAGGAGCCGCAGTC-3′; 5′-GCCCCTCCTCAGCATCTTAT-3′; 5′-TCAGTCTGAGTCAGGCCCTT-3′; 5′-GACCCAGGTCCAGATGAAGCT-3′; 5′-ACCGTAGCTGCCCTGGTAGGT-3′. The sequence analysis revealed the presence of a wild type TP53 in both MCF-7 and HYPO-7 (data not shown). Loss of heterozygosity at p53 locus was assessed by amplifying the 100–120 bp region located in Intron I of the TP53 gene, using the following primers: 5′-ACTCCAGCCTGGGCAATAAGAGCT-3′; 5′-ACAAACATCCCCTACCAAACAGC-3′. Previous reports indicate that the above region is in tight linkage with p53 codon 72 locus , following the manufacturer's protocol. Oligo-(dT)12-18 primers (Invitrogen) were used for the first strand synthesis. The following primers were used to amplify the target genes mRNA: vascular endothelial growth factor (VEGF): FW: 5′-GAGAATTCGGCCTCCGAAACCATGAACTTTCTGCT-3′ and RV: 5′-GAGCATGCCCTCCTGCCCGGCTCACCGC-3′, annealing temperature 65°C, amplicons length: 520, 600 and 675 bp; carbonic anhydrase IX (CA-IX): FW: 5′-CAGGGACAAAGAAGGGGATGAC-3′; RV: 5′-TTGGAAGTAGCGGCTGAAGTCA-3′, annealing temperature 61°C, amplicon length 589 bp; Heme Oxigenase-I (HO-I): FW: 5′-CCCGACAGCATGCCCCAGGAT-3′; RV: 5′-GGAGTTCATGCGGGAGCGGTAGAG-3′, annealing temperature 60°C, amplicon length 549 bp; hepatocyte growth factor receptor (c-MET): FW: 5′-ACAGTGGCATGTCAACATCGCT-3′; RV: 5′-CTTAGACATCTGATGGCTCG-3′, annealing temperature 62°C, amplicon length 656 bp; vascular endothelial growth factor receptor-2 (KDR): FW: 5′-TATAGATGGTGTAACCCGGA-3′; RV: 5′-TTTGTCACTGAGACAGCTTGG-3′, annealing temperature 62 C, amplicon length 656 bp; Hypoxia Induced Factor-1alpha (HIF-1α): FW: 5′-GGTGAATATGTCTGGGTTGAAAC-3′, RV: 5′-TGGGACTATTAGGCTCAGGTGAA-3′, annealing temperature 56°C, amplicon length 615 bp; PUMA: 5′-CAGACTGTGAATCCTGTGCT-3′ RV: 5′-ACAGTATCTTACAGGCTGCC-3′, annealing temperature: 62°C, amplicon length: 285 bp; NOXA: FW: 5′-GTGCCCTTGGAAACGGAAGA-3′, RV: 5′-CCAGCCGCCCAGTCTAATCA-3′, annealing temperature: 64°C, amplicon length: 258 bp; BNIP3L: FW: 5′-CTCAGTCAGAAGAAGAAGTTGTAG A-3′, RV: 5′-CTCAGTCGCTTTCCAATATAGAT-3′, annealing temperature 51°C, amplicon length: 283 bp; β2-microglobulin (β2μ): FW:5′-ACCCCCACTGAAAAAGATGA-3′; RV: 5′-ATCTTCAAACCTCCATGATG-3′, annealing temperature 58°C, amplicon length 114 bp . All PCR protocols were performed as follows: predenaturation step at 95°C for 2 min; 28 cycles of denaturation at 95°C for 1 min, annealing at the appropriate temperature for 1 min, extension at 72°C for 1 min; final extension at 72°C for 7 min. Amplified fragments were resolved onto a 1.8% agarose gel.Total RNA was extracted from cells using TRIzol® Reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription reaction was performed in a 20 M HEPES, 142 mM KCl, 2.5 MgCl2, 1 mM EDTA, 1 mM DTT, 0.2% NP-40) supplemented with a protease and phosphatase inhibitors cocktail (Sigma). Total protein lysate (100 μg) was loaded onto an 8% acrylamide/bis-acrylamide gel, and transferred to Hybond, nitrocellulose membrane . Antibodies for WB analysis were directed against: p53 , CA-IX , HIF-1α and VEGF , β-actin (Sigma). HRP-conjugated secondary antibodies were visualised by the luminol/enhancer chemiluminescent substrate (Amersham Biosciences).Cell lysates were prepared using coimmunoprecipitation buffer t-test or analysis of variance (ANOVA). Bonferroni-corrected post hoc test was used for pairwise comparisons following a significant F-test. Statistical calculations were executed with SPSS 10.1 Software Package . Data in graphs are expressed as mean±s.d.Associations among variables were verified using Student's 2) or to the hypoxia mimetic DFX, at a concentration of 100 μM, and we found that both p53D-transduced cells and p53siRNA-transfected MCF-7 cells exhibited a significantly lower rate of cell death in comparison to the matched controls , which hampers the activity of p53 by accumulating the protein in the cytoplasm , or encoding the p53 codon 72 arginine (p53Arg) or proline (p53Pro) allele. We found that the p53Pro allele induced a higher rate of cell death than the p53Arg allele in all the three cell lines  were exposed to <0.1%. Oll lines . Since tted ones . Neverthted ones . These d2, the p53Pro allele transiently transfected cells exhibited a higher rate of cell death, . In conclusion, we propose that the result here presented may provide a functional explanation for the preferential retention of the p53Arg allele  in tumour tissues of p53 codon 72 heterozygote individuals. Moreover, these data may provide a functional cue for explaining the recently reported reduction in vivo of the amount of spontaneous cell death in the p53Arg-retaining tumours arisen in heterozygote individuals , whose promoters contain the consensus sequence for the hypoxia-induced transcription factor HIF-1


Of the three La atoms in the asymmetric unit, two are eight-coordinate with bicapped trigonal–prismatic configurations; the third is nine-coordinated and has a tricapped trigonal–prismatic coordination geometry. Of the seven Cu atoms, two are two-coordinate with CuBrN and CuN2 ligand sets, three have trigonal configurations, viz. CuBrN2, CuBr2N and CuBr3, while the remaining two adopt distorted tetrahedral CuBr3N geometries. In the crystal structure, adjacent La centers are linked by isonicotinate (IN−) and benzene-1,2-dicarboxylate ligands to form a two-dimensional La–carboxylate layer in the ab plane. These layers are further interconnected with each other by bridging [Cu(IN)2] motifs, leading to an unusual three-dimensional heterometallic Cu–halide–lanthanide–organic framework, with the inorganic [Cu6Br6]n chains located in the resulting channels. Two Cu atoms are disordered over two positions, both with site occupancy factors of 0.80 and 0.20. O—H⋯O hydrogen bonding between water molecules and carboxylate O atoms helps to consolidate the crystal packing.A new lanthanum(III)–copper(I) heterometallic coordination polymer, [Cu For som al. 2003; Prasad  al. 2007; Cheng e al. 2008; Deng et al. 2008; Wang, L al. 2008. For the al. 2008; Wang, D al. 2008.       7La3Br6(C6H4NO2)8(C8H4O4)(H2O)4] = 0.052                           wR(F                           2) = 0.126                           S = 1.18                           13363 reflections955 parametersH-atom parameters constrainedmax = 1.73 e Å−3                        Δρmin = −2.22 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXL97.Data collection: 10.1107/S1600536809014081/sj2614sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809014081/sj2614Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The ZnII ion displays a distorted tetragonal-pyramidal geometry with four N atoms from two chelating 4-amino-3,5-di-2-pyridyl-1,2,4-triazole (2-bpt) ligands in the basal plane and one water molecule occupying the apical site. Another water molecule at the opposite of the apical site has a weak interaction with the ZnII ion [Zn—O = 2.852 (5) Å]. The ZnII ion and the two water molecules lie on a twofold rotation axis. An extensive system of hydrogen bonds involving the NH2 groups of the 2-bpt ligands, water molecules and nitrate anions links all residues into a three-dimensional network.The asymmetric unit of the title compound, [Zn(C For rar al. 1986; Rheingo al. 1993. For hyd al. 1998.       12H10N6)2(H2O)2](NO3)2                         = 0.045                           wR(F                           2) = 0.101                           S = 1.03                           3210 reflections228 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.32 e Å−3                        Δρmin = −0.64 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008DIAMOND (Brandenburg, 1999SHELXTL (Sheldrick, 2008SHELXTL.Data collection: 10.1107/S1600536809032103/hy2215sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809032103/hy2215Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Each PbII centre is surrounded by eight O atoms; four belong to bidentate acetate ions, three to neighbouring bridging acetate groups and one to the 2,5-dimethylbenzene-1,4-diol molecule. The PbII ions are chelated symmetrically and asymmetrically by acetate ligands. The coordination environment of the PbII ion can be described as a hemidirected PbIIO6 core with the empty space around the metal ion filled by the stereochemically active 6s               2 electron pair and two longer Pb—O contacts. The Pb—O distances are in the range of 2.355 (3)–2.994 (3) Å. Additionally, the crystal structure is stabilized by O—H⋯O hydrogen bonds.The title compound, [Pb Van der al. 1996. The syn al. 2006. Hemi- a al. 1998.       2(C2H3O2)4(C8H10O2)] = 0.017                           wR(F                           2) = 0.043                           S = 1.07                           2432 reflections132 parametersH-atom parameters constrainedmax = 1.30 e Å−3                        Δρmin = −1.67 e Å−3                        Δρ                                 CrysAlis CCD  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008Mercury (Macrae et al., 2006SHELXL97.Data collection: 10.1107/S1600536808030535/nc2114sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808030535/nc2114Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Alkylation of PTEN by α,β-enals/enones and interference with its restraint of cellular PKB/Akt signaling may accentuate hyperplastic and neoplastic disorders associated with chronic inflammation, oxidative stress, or aging.PTEN, a phosphoinositide-3-phosphatase, serves dual roles as a tumor suppressor and regulator of cellular anabolic/catabolic metabolism. Adaptation of a redox-sensitive cysteinyl thiol in PTEN for signal transduction by hydrogen peroxide may have superimposed a vulnerability to other mediators of oxidative stress and inflammation, especially reactive carbonyl species, which are commonly occurring by-products of arachidonic acid peroxidation. Using MCF7 and HEK-293 cells, we report that several reactive aldehydes and ketones, e.g. electrophilic α,β-enals  and α,β-enones (prostaglandin A Innate immune mechanisms are prime candidates for investigation.Inflammation and cancer are intricately linked 2, into anti-inflammatory metabolites, Δ12-PGJ22 and Δ12-PGJ2Usually, innate immune inflammation consists of a “wounding” phase to annihilate pathogens, and a “healing” phase to repair and regenerate damaged host tissue PTEN gene is frequently mutated or inactivated in advanced cancers 2) inactivate the PTEN protein – not the gene - by alkylation. Inactivation of PTEN by α, ß–unsaturated carbonyls leads to increased Akt signaling, enhanced nuclear β-catenin signaling, and augmented cellular proliferation. Redox signaling by PTEN may have evolved to enable cells (tissues) to stratify their response to oxidative stress. For example, transient inhibition of PTEN by reactive oxygen or carbonyl species, and the corresponding signaling through Akt/GSK3β/β-catenin/TCF4/Lef1 might benefit the host via increasing proliferation and regeneration of tissue damaged by acute inflammation or oxidative stress. Errant and persistent PTEN inactivation by the same molecular mechanism might favor tumor progression and provide an etiological link between ‘smouldering’ inflammation and certain cancers, especially colorectal cancer, where both the PTEN and the APC tumor suppressors restrict nuclear β-catenin signaling PTEN (phosphatase tensin homolog on chromosome 10) is a phosphoinositide-3-phosphatase with two physiological roles: tumor suppressor and regulator of anabolic/catabolic cell signaling. The 2O2, then selectively tagged any proteins that had oxidized or carbonylated thiols using NEM-biotin . We then sequestered proteins with a biotin epitope onto NeutrAvidin (NA) beads and identified carbonylated PTEN by SDS-PAGE and immunoblotting. MCF-7 cells treated with 10 µM Δ12-PGJ2, 4-HNE, or acrolein contained carbonylated PTEN in amounts comparable to cells treated with 100 µM H2O2 . This method does not distinguish between oxidized and carbonylated thiols on PTEN. However, electrophoresis under non-reducing conditions, followed by western blotting, showed that PTEN migrated as a discrete isoform due to an oxidized disulfide 2O2, but not in cells treated with α, ß–unsaturated carbonyl  or 15-HpETE, a lipid hydroperoxide RT–) is prone to oxidation because it is a strong nucleophile, pKa ∼5. This trait should also facilitate alkylation of PTEN by α, ß–unsaturated carbonyls. We used CyPG-biotin analogs, which have an electrophilic β-carbon capable of nucleophilic addition (Michael reaction), as chemical models to test this hypothesis de novo  was more reactive than PGA1-biotin , agreeing with others who reported ∼20-30 protein targets modified by cyPG-biotin in 3T3 cells or mitochondria de novo biotinylated cellular proteins on NA beads, followed by immunoblot with anti-PTEN antibodies, showed that PTEN formed a covalent adduct ∼10-fold more readily with Δ12-PGJ2-biotin than with PGA1biotin .The cysteinyl thiolate in the PTEN active site  Akt in MCF-7 cells. As little as ∼2 µM Δ12-PGJ2 caused a half-maximal response (308)Akt were detectable at 10 min, maximal at 30 min, and durable for >120 min (2 increased formation of phospho-(T308)Akt without altering formation of phospho-(S241)PDK1 (the kinase that phosphorylates T308 of Akt), and without altering PTEN protein expression Akt by inhibiting PI3-K, at the apex of the PI3-K/→PDK1/2→Akt kinase cascade . Furthermore, 1 µM of various PGA and PGJ isomers, and other α, ß–enones directly inhibited isolated PTEN enzyme , 10% v/v for 1 h. The precipitate was washed twice with acetone to remove traces of TCA, NEM, and IAA. Precipitated proteins were solubilized and oxidized or modified cys residues were reduced in 0.1 ml of O2-free reducing buffer  for 30 min at 50°C. Reduced proteins were subsequently biotinylated with 0.9 ml of a solution containing 50 mM NaHPO4 pH 7.0, 1 mM EDTA, and 1 mM biotin conjugated to polyethylene oxide-maleimide for 30 min at 50°C. Proteins were precipitated in 10% v/v TCA for 1 h. The precipitate was isolated by centrifugation, washed with dry ice-chilled acetone, and solubilized in 0.3 ml of 50 mM Hepes-NaOH, pH 7.7, 1 mM EDTA, and 2% SDS. The sample was then diluted with 0.3 ml of the same solution without SDS. 15 µg protein was assayed by immunoblot for PTEN. A separate sample (200 µg protein) was added to 100 µL immobilized NA beads in 1 ml PBS, 0.4% v/v Tween 20. This suspension was rotated 16 hr at 4°, centrifuged, and beads were washed twice with PBS/0.4% v/v Tween 20. Loading buffer (50 µL) with 5% BME was added directly to beads, boiled for 10 min to release maleimido-biotinylated proteins, and 20 µL was assayed by immunoblot for total and oxidized PTEN.MCF-7 cells (MEM 1% v/v FBS) were treated for 30 min with vehicle, or reactive electrophiles  in the lysates were fractionated by non-reducing SDS-10% PAGE, followed by anti-PTEN immunoblot to distinguish native PTEN from oxidized PTEN occurring as an intra-molecular Cys124-Cys71 disulfide MCF-7 cells (MEM 1% v/v FBS) treated for 30 min with 20 µM Δ12-PGJ1 or Δ12 PGJ2, were lysed, sonicated 10× for 1 s at 4°C, then centrifuged 10,000× g for 10 min. Supernatant with 100 µg of protein was incubated with 100 µl of NA beads in 1 ml PBS with 0.4% Tween 20 for 16 h at 4°C to sequester proteins containing a biotin epitope introduced de novo by reaction with cyPG-biotin analogs. The beads were then centrifuged at 500× g for 5 min to isolate neutravidin-biotin complexes (NA pulldown). The beads were washed 3× with 1 ml of PBS/0.4%Tween 20 then boiled 5 min in Laemmli loading buffer with 5% BME to release bound proteins. These samples were analyzed by immunoblotting for PTEN or proteins with a biotin epitope.MCF-7 cells (MEM 1% v/v FBS) treated 1 hr with 1–10 µM of the aminopentylbiotinamide analogs of PGA2O2, or enzyme inhibitors, MCF-7 or HEK-EBNA cells were lysed in 250 mM sucrose, 50 mM Tris pH 7.4, 5 mM MgCl2, 1 mM EGTA, 1× Complete™ protease inhibitor, 2 mM NaF and 2 mM sodium orthovanadate. Samples were dissolved in 50 µl of Laemmli loading buffer, 0.5% BME and heated at 95°C for 10 min. Samples (15–30 µg protein) were fractionated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% w/v nonfat dry milk in TBS-T, then incubated for 16 h at 4°C with primary antibodies directed against PTEN (1∶1000), biotin , Akt (1∶1000), phospho-(T308)Akt (1∶1000), phospho-(S473)Akt (1∶1000), phospho-(S241)PDK1 (1∶1000), GSK3β (1∶1000), phospho-(S9)-GSK3β (1∶1000), RxRxx-phospho-(S/T) (1∶1000) or β-catenin (1∶1000) followed by HRP-conjugated secondary antibody (1∶5000). Antigen-antibody complexes were detected with Western Lightning™ ECL reagents. The intensity of chemiluminescent protein-antibody complexes was quantified with a Kodak Image Station 440™. Bar graphs depict the mean ± S.E. from densitometric analyses of separate experiments.Following treatment with reactive electrophiles, H6 MCF-7 cells (MEM 1% v/v FBS) were treated with 0–20 µM Δ12-PGJ2 for 30 min at 37°C, or with 20 µM Δ12-PGJ2 for 0–120 min to determine concentration and time dependence. ∼1×106 MCF-7 cells were also treated 30 min at 37°C with 10 µM of various PGs, including PGD2 and its cyPG dehydration products PGJ2, Δ12-PGJ2 and 15-deoxy-Δ12, Δ14-PGJ2; PGE2 and its cyPG dehydration products PGA2, its epimer 8-iso-PGA2, and its isomer PGB2 to determine structure-activity relationships. In certain experiments cells were also treated with 50 µM LY294002, 5 µM troglitazone, or 10 µM Akt inhibitor IV. Lysates from treated cells (15 µg protein) were fractionated on SDS-10% PAGE and proteins were transferred to PVDF membranes for immunoblot analysis of Akt, phospho-(T308)Akt, phospho-(S473)Akt, Akt substrate proteins containing the K/R-x-K/R-x-x-S/T(PO4) epitope, phospho-(S241)PDK1, and PTEN.∼1×102. We measured wnt/β-catenin signaling in STF3A cells by quantifying their Super Topflash® luciferase reporter signal. STF3A cells  were plated in white, clear bottom 96-well plates coated with poly-L-lysine and grown 24 h at 37°C. Medium was removed and replaced with 200 µl fresh medium containing 0–300 ng/ml DKK1; 2–20 µM of α,β-enone containing cyPGs; 2–20 µM of α,β-enal metabolites derived from lipid peroxidation ; 20 µM of primary PGs, PGH2, PGE2, PGF2α, PGD2, PGI2; 20 µM BSO, a glutathione synthesis inhibitor; other inhibitors, or DMSO vehicle. STF3A cells were incubated for 24 h at 37°C, washed with 200 µl PBS at 4°C, and lysed with 20 µl lysis buffer. Luciferin (60 µl/well) was added and luciferase activity in the lysates was quantified by fluorimetry. LDH activity in the lysate, an index of cell count, was quantified by spectroscopy. The ratio of luciferase/LDH activity is proportional to nuclear β-catenin signaling in STF3A cells.STF cells are HEK-293 cells containing a stably integrated SuperTopFlash (STF) transgene with TCF binding sites upstream of a luciferase reporter gene Inhibition of PTEN with 0–30 µM reactive electrophiles was quantified by using a PTEN enzyme assay kit .Statistical significance at p<0.05 was assessed by analysis of variance (ANOVA) with Bonferroni's post-hoc test for comparisons among groups.


IκBζ is a novel member of the IκB family of NFκB regulators, which modulates NFκB activity in the nucleus, rather than controlling its nuclear translocation. IκBζ is specifically induced by IL-1β and several TLR ligands and positively regulates NFκB-mediated transcription of genes such as IL-6 and NGAL as an NFκB binding co-factor. We recently reported that the IL-1 family cytokines, IL-1β and IL-18, strongly synergize with TNFα for IFNγ production in KG-1 cells, whereas the same cytokines alone have minimal effects on IFNγ production. Given the striking similarities between the IL-1R and IL-18R signaling pathways we hypothesized that a common signaling event or gene product downstream of these receptors is responsible for the observed synergy. We investigated IκBζ protein expression in KG-1 cells upon stimulation with IL-1β, IL-18 and TNFα. Our results demonstrated that IL-18, as well as IL-1β, induced moderate IκBζ expression in KG-1 cells. However, TNFα synergized with IL-1β and IL-18, whereas by itself it had a minimal effect on IκBζ expression. NFκB inhibition resulted in decreased IL-1β/IL-18/TNFα-stimulated IFNγ release. Moreover, silencing of IκBζ expression led to a specific decrease in IFNγ production. Overall, our data suggests that IκBζ positively regulates NFκB-mediated IFNγ production in KG-1 cells. We previously showed that the Interleukin-1 (IL-1) family members, IL-1β and IL-18, synergize with tumor necrosis factor-α (TNFα) for interferon-γ (IFNγ) production in the human acute myeloid leukemic KG-1 cell line Although the IL-1β and TNFα receptors (IL-1R and TNFR) belong to different families, their signaling pathways utilize similar adaptor molecules, such as TRAFs, and lead to the activation of NFκB and MAPKs IκBζ expression is immediately induced upon stimulation with TLR ligands and IL-1β We recently showed that both, IL-1β and IL-18, synergize with TNFα for IFNγ production in KG-1 cells Escherichia coli (E. coli) LPS (serotype 0111:B4) was obtained from Axxora  and cell culture tested ATP disodium salt from Sigma-Aldrich . Anti-human IL-8 ELISA capture monoclonal Ab (clone 6217), IL-18 monoclonal Ab (clone 159-12B), IL-18 receptor (IL-18R) monoclonal Ab (clone 70625), and human rIL-1β, rIL-18 and rIL-8 were purchased from R&D Systems . An IκBα polyclonal Ab was obtained from Upstate , actin monoclonal Ab (clone C4) from MP Biomedicals , p50 monoclonal (clone E-10) Ab and p65 polyclonal Ab from Santa Cruz , lamin B1 polyclonal Ab from Abcam , TNFα capture monoclonal Ab (clone 2C8) from Advanced Immunochemical , IL-8 ELISA detection polyclonal Ab from Endogen , rTNFα from Knoll Pharmaceuticals , Interleukin-1 receptor antagonist (IL-1ra) from Amgen , and IFNγ and IL-6 ELISA kits from eBioscience . Rabbit anti-serum against IκBζ and IL-1β and pre-immune serum were developed in our laboratory. The NFκB inhibitor, JSH23, was purchased from Calbiochem . A mixture of three different small interfering RNA (siRNA) oligonucleotides against IκBζ and three different scrambled siRNA oligonucleotides were purchased from Dharmacon RNA Technologies . The cell line nucleofector kit R was purchased from Amaxa .Purified 2. Peripheral blood monocytes were isolated from human blood by density gradient centrifugation using lymphocyte separation media  followed by CD14 positive selection , which yields>95% purity as determined by FACS. Isolated monocytes were cultured at 106/ml or at 12.5×106/ml in RPMI 1640 , supplemented with 5% FBS (<0.0005 EU/ml)  and 1% penicillin/streptomycin , in a 37°C humidified incubator with 5% CO2. Monocytes were stimulated, or not, with E. coli LPS (10 ng/ml) for 4 h and ATP (5 mM) for the last 15 min of LPS stimulation, or with LPS or ATP alone. The supernatants (conditioned media) from monocytes cultured at 106/ml were harvested by centrifugation  and used to stimulate KG-1 cells.KG-1 cells  were maintained in RPMI 1640 , supplemented with 20% FBS  and 1% penicillin/streptomycin in a 37°C humidified incubator with 5% CO6/ml and incubated with test samples  or recombinant proteins (10 ng/ml each). In selected experiments, neutralizing agents for IL-1β , IL-18 , and/or TNFα (TNFα Ab – clone 2C8) were used at the indicated concentrations to neutralize the activities of rIL-18, rIL-1β and/or rTNFα, or the same endogenous cytokines present in the monocyte conditioned media, prior to incubation with KG-1 cells. Alternatively, the neutralizing agents, IL-1ra and IL-18R Ab, were used to neutralize IL-18 or IL-1 receptors prior to addition of rIL-18, rIL-1β, or monocyte conditioned media, to the KG-1 cells. KG-1 supernatants and cells were harvested at various time points for subsequent ELISA and Western blot analysis. In selected experiments, cells were treated with the NFκB inhibitor, JSH-23 (30 µM) (an NFκB nuclear translocation inhibitor), in combination with rIL-1β, rIL-18 and rTNFα for 24 h. KG-1 cells were harvested at 12 h for subsequent nuclear extraction and Western blot analysis. KG-1 supernatants were harvested at 24 h for IL-6 and IFNγ ELISA.KG-1 cells were plated at a final cell density of 10KG-1 cells were lysed in a 60 mM Tris-HCl (pH 6.8), 2% SDS buffer. Total cell extracts were sonicated and spun  to remove cell debris. KG-1 nuclear/cytosolic extracts were also analyzed by Western blotting. Protein concentration in total, nuclear or cytosolic extracts was estimated using the Bio-Rad Dc protein Lowry assay (Bio-Rad). Samples were boiled in Laemmli's buffer for 5 min or heated at 70°C for 10 min in NuPAGE Sample Reducing Agent (Invitrogen). 10–40 µg of total protein were loaded per well on pre-cast 10% Tris-Glycine or 7% Tris-Acetate SDS-PAGE gels and transferred to a PVDF or nitrocellulose membrane. Membranes were blocked with 10% nonfat milk  in 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween for 1 h at R.T. The membranes were probed with the indicated primary Abs, followed by peroxidase-conjugated secondary antibodies. Protein bands were visualized by chemiluminescence .Sandwich ELISAs were used to measure cytokine release in the supernatants of KG-1 cells.6/ml) were stimulated, or not, with the indicated combinations of rIL-1β, rIL-18 and rTNFα (10 ng/ml each) for 24 h. Cells were Fc-blocked by treating with 1 µg of human IgG/105 for 15 min at R.T. Cells (105/25 µl*reaction) were transferred to a 5 ml tube. Phycoerythrin (PE)-conjugated anti-IL-18Rα or fluorescein (FITC)-conjugated anti-IL-1R1 reagent (10 µl of each per reaction) were added to the cells. Cells were incubated for 30 min at 4°C, washed twice with 1×PBS and re-suspended in 1×PBS (105/200 µl) for flow cytometric analysis. As controls, cells were also treated with phycoerythrin-labeled murine IgG1 and fluorescein-labeled goat IgG.KG-1 cells (106/ml) were stimulated, or not, with rIL-1β, rIL-18, rTNFα (10 ng/ml each), or a combination of this cytokines for the indicated time points. Cells were washed twice in 1×PBS and gently re-suspended in cold Buffer A  at 400 µl/0.5−1×106 cells. Cells were allowed to swell for 15 min. 10% Nonidet NP-40 was added to the solution (25 µl per 400 µl). Samples were vortexed for 10 sec and centrifuged for 30 sec . Supernatants containing cytosolic contents were transferred to fresh tubes containing an equal volume of Buffer B  and stored immediately at −20°C. The pellets containing the nuclear contents were re-suspended in cold Buffer C ; 1×Complete Mini protease inhibitor cocktail, Roche) at 50 µl/0.5−1×106 cells. The samples were vigorously shook for 15 min at 4°C on a shaking platform and then centrifuged for 5 min . The supernatants with the nuclear contents were stored at −20°C. Nuclear and cytosolic extracts were subsequently analyzed for protein concentration using the Bio-Rad Dc protein Lowry assay (Bio-Rad). Nuclear and cytosolic extracts were then prepared for Western blot analysis.KG-1 cells (106/ml) were nucleofected following the protocol for KG-1 cell nucleofection provided with the cell line nucleofector kit R from Amaxa  with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IκBζ or 3 different scrambled siRNA oligonucleotides (3 µg per 2×106 cells). After 2 h, cells were stimulated with a combination of rIL-1β, rIL-18 and rTNFα (10 ng/ml each). Cells and supernatants were harvested at 24 h for subsequent RNA and protein analysis .KG-1 cells (2×106/ml) were lysed in TRIzol reagent (Invitrogen Life Technologies) and mRNA was extracted and converted to cDNA using the Thermoscript RT-PCR system (Invitrogen Life Technologies). qPCR was performed using specific primers for IFNγ, IL-6 and IL-8. Values were normalized to two house-keeping genes, CAP-1 and GAPDH.KG-1 cells  of IκBα expression upon rTNFα stimulation . Moreover, IL-6 and IFNγ release were significantly reduced with NFκB inhibition . TherefoIκBζ has been shown to either negatively or positively regulate NFκB activity depending upon the context. Genes, such as E-selectin, are negatively regulated In order to test this hypothesis, KG-1 cells were nucleofected with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IκBζ or 3 different scrambled siRNA oligonucleotides. Cells were then stimulated with a combination of rIL-1β, rIL-18 and rTNFα. Western blot analysis indicated a reduction in IκBζ protein expression with anti-IκBζ siRNA delivery . In ordeRegulation of IFNγ gene transcription involves the action of many different transcripition factors including STATs, AP-1, GATA-3, NFAT, T-bet, Eomesodermin, NFκB, NFAT, T-bet, YY-1, DREAM, ERM and SMADs. In most cases, multiple signals synergize for IFNγ production via induction of different transcription factors that act in concert to induce gene expression In general, IL-12 and IL-18 require each other for IFNγ gene expression. However, at high doses (50 ng/ml), IL-18 alone can induce IFNγ production in the human acute myeloid leukemic KG-1 cell line NFκB has been shown to regulate the expression of many pro-inflammatory genes, IFNγ being no exception. Two putative NFκB binding sites have been identified in the IFNγ promoter region (κBB site and CD28RE) and one in the first intron (C3) We have previously described a novel synergistic role for the members of the IL-1 family, IL-1β and IL-18, in combination with TNFα in IFNγ production in KG-1 cells The novel member of the IκB family of NFκB regulators, IκBζ, is known to be induced by IL-1R/TLR ligands. Moreover, even though the TNFR signaling pathway shares some similarities with the IL-1R/TLR pathway, such as the use of TRAF adaptor molecules, TNF signaling alone does not result in IκBζ protein expression IκBζ has been shown to positively regulate NFκB-mediated transcription of secondary response genes such as IL-6 and NGAL, as a co-factor binding to the p50 NFκB subunit cis-element in the N-terminal half of the IκBζ gene appears to be crucial for this stabilization Interestingly, we observed that TNFα enhanced IL-1β/IL-18-mediated IκBζ protein expression, even though by itself it had not effect on IκBζ protein expression. However, TNFα, IL-1β and LPS have all been shown to induce IκBζ mRNA transcription in NIH3T3 and A549 cells In support of the latter findings, the NGAL promoter has been shown to be specifically induced by IL-1β, not by TNFα Based on this information, we conclude that while IL-1β and IL-18 may provide the signal(s) required for IκBζ transcriptional activation, mRNA stabilization and subsequent IκBζ protein expression, TNFα may enhance IL-1β/IL-18-mediated IκBζ expression by providing strong transcriptional activation of the IκBζ gene. Moreover, TNFα stimulation of KG-1 cells provides robust binding of the p50 and p65 NFκB subunits to EMSA probes containing the NFκB binding sites present in the IFNγ promoter and first intron (data not shown), compared to weaker binding provided by IL-1β and IL-18. Therefore, robust NFκB activation provided by TNFα may also result in increased IFNγ gene expression since IκBζ regulates transcription as a co-factor for NFκB and KG-1 IFNγ production in response to IL-1/TNFα combined stimulation is NFκB dependent . Thus, tIn summary, we have shown a positive role for IκBζ on IFNγ production in response to IL-1β, IL-18 and TNFα combined stimulation in KG-1 cells. This regulation is most likely dependent on the ability of IκBζ to regulate NFκB mediated transcription of the IFNγ gene. This finding represents a new addition to the complex and continuously growing literature on the regulation of IFNγ expression.


In the 2,9-diethoxy-1,10-phenanthroline ligand, the O and C atoms of the ethoxy groups are located almost in the plane defined by the phenanthroline ring system. Two dicyanamide ions bridge two CdII ions, which are located on a twofold axis, forming a one-dimensional zigzag chain along the [001] direction. The 2,9-diethoxy-1,10-phenanthroline molecules act as bidentate terminal ligands. There are π–π interactions between polymeric chains, characterized by a centroid–centroid distance of 3.7624 (2) Å between the phenanthroline rings of two neighbouring chains.In the title polymer, [Cd(C DOI: 10.1107/S1600536808021727/bh2180Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Complexation of 1 with methyl-β-cyclodextrin 2 results in clearly water-soluble structures due to incorporation of the ferrocene moiety into the cyclodextrin cavity. After free radical polymerization of the water-soluble complexed monomer, corresponding to polyvinylferrocene (PVFc), the water-soluble polymer is obtained due to the host guest interactions. Those polymeric complexes are stable in water up to about 90 °C. Above this temperature the polymer precipitates due to decomplexation. The complex was investigated by 1H NMR spectrometry, dynamic light scattering (DLS), differential scanning calorimetry (DSC), and lower critical solution temperature (LCST) measurements.We report the radical initiated homopolymerization of a soluble vinylferrocene cyclodextrin-complex in water. Uncomplexed vinylferrocene  N-Isopropylacrylamide (NiPAAM) was obtained from Aldrich, Germany. Vinylferrocene (VFc) was obtained from Sigma-Aldrich, Germany.Methyl-β-cyclodextrin was obtained from Wacker-Chemie GmbH, Burghausen, Germany, and used after drying overnight with a vacuum oil pump over P−1 at room temperature. 1H NMR spectra were recorded with a Bruker AC 500 at 20 °C. Chemical shifts were referenced to the solvent value δ 2.51 for dimethyl-d6 sulfoxide. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDITOF-MS) was performed on a Bruker Ultraflex TOF mass spectrometer. Cyclic voltammetry measurements were performed on a simplot instrument with a resolution of <100 pA. Dynamic light scattering (DLS) experiments were carried out with a Malvern HPPS-ET at 25 °C. The particle size distribution was derived from a deconvolution of the measured intensity autocorrelation function of the sample by the general purpose mode algorithm included in the DTS software. DLS measurements were performed in water; the concentrations of the substances were 10 mg × mL−1. Each experiment was performed at least five times to obtain statistical information. Differential scanning calorimetry (DSC) measurements were performed on a Mettler DSC-30 instrument in a temperature range of −25 to 200 °C at a heating rate of 15 °C × min−1 as the average of five measurements using the midpoint method.IR spectra were recorded with a Nicolet 5 SXB FTIR (Fourier transform infrared) spectrometer equipped with an ATR unit. The measurements were performed in the range of 4000–300 cm2 in water  was prepared and purged with argon for 5 min. Vinylferrocene 1  was added under an argon atmosphere and the mixture heated to 50 °C. VA-044  was added to the mixture followed by the addition of a similar amount of the initiator after 8 h. After a reaction time of 48 h, the reaction mixture was freeze-dried. No residual monomer was evident by thin layer chromatography. According to differential scanning calorimetry (DSC), the obtained polyvinylferrocene 3 had a glass transition temperature (Tg) of 145 °C.An aqueous solution of methyl-β-CD 3: 2902 cm−1 (–C–H), 1643 cm−1 (–C=C), 1452 cm−1 (CH2), 1153, 1033 cm−1 (–C–O), 1082 cm−1 (C–O–C), 964, 758, 702 cm−1 (=C–H); 1H NMR:  3: δ (ppm) = 1.83 (CH2), 3.10–3.31  3.45–3.65  4.11 , 4.25 , 4.44 , 4.75–5.30  6.47 , 3J = 17.34 Hz (trans)).FT-IR (film) 2  was prepared and purged with argon for 5 min. Vinylferrocene 1  was added under an argon atmosphere. After vinylferrocene 1 had completely dissolved, N-isopropylacrylamide (NiPAAM) 5  was added. The mixture was heated to 50 °C and VA-044  added. After 3 h an orange solid precipitated.An aqueous solution of methyl-β-CD 7: 2970 cm−1 (–C–H), 1637 cm−1 (–C=C), 1533 cm−1 (–N–H), 1458 cm−1 (-CH2), 1385 cm−1 (–CH3), 1365 cm−1 (–C–N); 1H NMR:  7: δ (ppm) = 1.05 , 1.46 , 1.98 , 3.10–3.30 , 3.45–3.65 , 3.86 , 4.25–3.96 , 4.71–5.20 , 6.18 , 7.20 ; MALDI-TOF 7: m/z 1.6 × 104.FT-IR (film)


The metal centre  has a distorted tetrahedral coordination formed by two N atoms from one 2,9-dimethyl-1,10-phenanthroline ligand and two Cl atoms. There is intermolecular π–π stacking between adjacent 2,9-dimethyl-1,10-phenanthroline ligands, with a centroid–centroid distance of 3.733 (2)Å.In the title compound, [CuCl DOI: 10.1107/S1600536809034187/at2860Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: