In the complex, the MnII ion is six-coordinated in a considerably distorted cis-N4IO octahedral environment defined by four N atoms of the two chelating di-2-pyridylamine (dpa) ligands, one I− anion and one O atom of a water ligand. As a result of the different trans effects of the I, N and O atoms, the Mn—N bond trans to the I atom is slightly longer than the Mn—N bond trans to the N or O atoms. The dpa ligands are not planar, with dihedral angles between the two pyridine rings of 26.2 (4) and 26.5 (4)°. The complex cations are stacked in columns along the a axis and are linked to the anions by intermolecular O—H⋯I and N—H⋯I hydrogen bonds.The asymmetric unit of the title compound, [MnI(C II complexes with dpa, see: Bose et al. 2(H2O)]I = 0.050                           wR(F                           2) = 0.134                           S = 1.06                           4509 reflections271 parametersH-atom parameters constrainedmax = 1.01 e Å−3                        Δρmin = −1.13 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97  global, I. DOI: 10.1107/S1600536811047349/wm2558Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In one ligand, the dihedral angles between the central pyridine ring, the other bonded pyridine ring, the terminal pyridine ring and the benzene ring are 14.3 (15), 18.3 (18) and 43.9 (16)°, respectively. The equivalent angles in the second ligand are 5.8 (18), 6.3 (18), and 47.0 (17)°, respectively. In the crystal, the complex molecules and lattice water molecules are linked by O—H⋯O and O—H⋯N hydrogen bonds, generating a three-dimensional network.In the title complex, [Mn(C DOI: Click here for additional data file.10.1107/S1600536813006004/hb7038Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Zinc is essential for the activities of pancreatic β-cells, especially insulin storage and secretion. Insulin secretion leads to co-release of zinc which contributes to the paracrine communication in the pancreatic islets. Zinc-transporting proteins  and metal-buffering proteins  tightly regulate intracellular zinc homeostasis. The present study investigated how modulation of cellular zinc availability affects β-cell function using INS-1E cells.N,N,N’,N-tektrakis(2-pyridylmethyl)-ethylenediamine. The changes in intracellular insulin content following exposure to various concentrations of zinc were reflected by changes in the expression patterns of MT-1A, ZnT-8, ZnT-5, and ZnT-3. Furthermore, high zinc concentrations induced cell necrosis while zinc chelation induced apoptosis. Finally, cell proliferation was sensitive to changes in zinc the concentration.Using INS-1E cells, we found that zinc supplementation and zinc chelation had significant effects on insulin content and insulin secretion. Supplemental zinc within the physiological concentration range induced insulin secretion. Insulin content was reduced by zinc chelation with These results indicate that the β-cell-like function and survival of INS-1E cells are dependent on the surrounding zinc concentrations. Our results suggest that regulation of zinc homeostasis could represent a pharmacological target. The toxicity ,15. Paratoxicity -20.2+ traffic by changing the gene expression levels of specific ZnT genes also affects β-cell insulin content and secretion [Modifying intracellular Znecretion ,21-23. Tecretion -20,24,25ecretion -29. Furtecretion . Overexpecretion -32. Finaecretion ,34.Despite intensive research, the full consequence of altered zinc bioavailability on β-cell function remains unclear. Therefore, the present study investigated how cell survival, insulin content/secretion, and the expression of specific β-cell-relevant ZnTs and MTs respond to changes in the zinc environment following supplementation or chelation of zinc. We found that zinc-specific interventions had significant effects on the β-cell-like activity of INS-E1 cells, demonstrating the pharmacological potential of zinc supplementation or chelation.in vitro study. The INS-1E cell line is an established glucose-sensitive cell line with β-cell-like activity [2 atmosphere in complete RPMI 1640 supplemented with 11 mM glucose, 10% (v/v) heat-inactivated fetal bovine serum, 50 μM β-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, as previously described [Rat INS-1E cells were used in this activity ,36. INS-escribed . The zin2)  or 2.5–50 μM of the Zn2+ chelator N,N,N’,N-Tektrakis(2-pyridylmethyl)-ethylenediamine (TPEN) . The basal glucose concentration was kept permanently at 11 mM because we experienced greater insulin response and cell replication of the INS-1E, and continuous growth at this concentration (unpublished data). We used 3–6 replicates for the analyses of mRNA expression, viability, DNA fragmentation assessment, and insulin measurements.For stimulation assays, cells were plated into six-well plates (NUNC) in complete RPMI 1640 supplemented with 11 mM glucose, 10% (v/v) heat-inactivated fetal bovine serum, 50 μM β-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin with the addition of either 5 μM to 1 mM zinc chloride  , a DNA-specific dye, for 15 min at 37°C followed by fluorescence analysis on a NucleoCounter NC-3000 system (Chemometec). Viability was determined by analyzing cell samples on Via1-Cassettes (Chemometec) coated with two different dyes to stain the entire cell population (acridine orange) and nonviable cells (DAPI).INS-1E cells were treated with 50 μM to 1 mM ZnCl2 or 2.5–50 μM TPEN in complete RPMI medium for 24 h. The cells were then incubated for 2 h in serum-free Krebs–Ringer bicarbonate HEPES buffer at pH 7.4 containing 115 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.6 mM CaCl2, 1.2 mM KH2PO4, 20 mM HEPES, 5 mM NaHCO3, 0.1% (v/v) human serum albumin (Sigma), with or without 50 μM to 1 mM ZnCl2 or 2.5–50 μM TPEN and 11 mM glucose. The incubation medium was collected to measure insulin secretion. The cells were collected in Earle’s basal medium  by scraping followed by centrifugation. Half of the intact cells from each sample were re-suspended in a buffer comprising 0.75% (v/v) glycine and 0.25% (v/v) bovine serum albumin at pH 8.8 to measure the insulin concentration, or in 0.1% M NaOH to measure the protein concentration. The total protein concentration was measured using a BCA Protein Assay Reagent Kit from Pierce, USA . The insulin concentration was determined using an ultrasensitive rat insulin enzyme-linked immunosorbent assay kit from DRG Diagnostics .INS-1E cells were treated with 5 μM to 1 mM ZnCl2 or 2.5–50 μM TPEN in complete RPMI medium for 24 h. It was not possible to collect RNA material from cells treated with 50 μM TPEN most likely due to severe toxicity of TPEN at this concentration level. RNA was extracted using the RNeasy Mini Kit Qiagen (VWR) and treated with DNase (VWR). cDNA was synthesized from total RNA using an ImProm-II™ Reverse transcription system .INS-1E cells were treated with 5 μM to 1 mM ZnClTM5 Optical System Software, Version 2.1. Starting quantities were calculated from a standard curve. For each experiment, the most stable housekeeping genes were found using the method described by Vandesompele et al. [Quantitative real-time PCR was performed in duplicate using IQ Sybr Green supermix  in a MyiQ Two-Color Real-time PCR detection system (Bio-Rad). A melting curve was prepared for all reactions. The results were analyzed with iQe et al. . Expresse et al. ,38.The following (forward and reverse) primers were used: UBC-7, 5′-CAG CTG GCA GAA CTC AAC AA-3′ and 5′-TTT GGG TGC CAA ATC TCT GT-3′ ; Cltc, 5′-AAG GAG GCG AAA CTC ACA GA-3′ and 5′-GAG CAG TCA ACA TCC AGC AA-3′ ; HSP, 5′-GAT TGA CAT CAT CCC CAA CC-3′ and 5′-CTG CTC ATC ATC GTT GTG CT-3′ ; CycA, 5′-AGG TCC TGG CAT CTT GTC CA-3′ and 5′-CTT GCT GGT CTT GCC ATT CC-3′ ; β-actin, 5′-CTA CAA TGA GCT GCG TGT GGC 3′ and 5′-ATC CAG ACG CAG GAT GGC ATG-3′ ; Bax, 5′-GTG AGC GGC TGC TTG TCT-3′ and 5′-GTG GGG GTC CCG AAG TAG-3′ ; Bcl-2, 5′-CGA CTT TGC AGA GAT GTC CA-3′ and 5′-ATG CCG GTC AGG TAC TCA G-3′ ; insulin, 5′-CGC TTC CTG CCC CTG CTG GC-3′ and 5′-CGG GCC TCC ACC CAG CTG CTC CA-3′ ; ZnT-3, 5′-TCC TCT TCT CTA TCT GCG CCC-3′ and 5′-TGT GCG GAG GCA ACG TGG TAA-3′ ; ZnT-5, 5′-TCC ACA TGC TCT TTG ACT GC-3′ and 5′-GTC AAG TTC CGG AGG ATC AA-3′ ; ZnT-8, 5′-GGT GGA CAT GTT GCT GGG AG-3′ and 5′-CAC CAG TCA CCA CCC AGA TG-3′ ; MT-1A, 5′-TCC CGA CTT CAG CAG CCC GA-3′ and 5′-GCC CTG GGC ACA TTT GGA GC-3′ ; and MT-3, 5′-TGG TTC CTG CAC CTG CTC GG-3′ and 5′-CAC CAG GGA CAC GCA GCA CT-3′ .P < 0.05 were considered to indicate a significant difference between the experimental and control conditions.Data are presented as mean values with the standard error of the mean (SEM). One-way analysis of variance with Dunnett’s multiple comparison test was used to determine statistical significance among groups. Values of 2 concentration reached 0.4 mM. The percentage of viable cells was decreased by 16.9% at 0.4 mM ZnCl2 and only 47.1% of the cells were viable at the highest ZnCl2 concentration, 1.0 mM  increased the proportion of cells in the G2/M phase while higher ZnCl2 concentrations (0.7–1.0 mM) reduced the number of cells in the G2/M phase. The fraction of cells in the S phase was also affected by the ZnCl2 concentration. The effect was particularly evident at 0.4 mM ZnCl2, where a two-fold increase in the number of cells was detected compared with the control cells  markedly reduced the transcription of ZnT-8  [The pivotal role of Zn (DEDTC) .2+ binding by TPEN than DEDTC, and by the fact that DEDTC chelates other metals, including copper [2+ because TPEN has a higher binding affinity for Zn2+ (dissociation constant 1–2.6 × 10-16 M) than for other metals [ZnT-8 gene expression was reported to be downregulated by the chelator DEDTC ,38, althg copper . In addir metals .2+ chelation also reduced the viability of INS-1E cells. Administered in vitro, TPEN can chelate extracellular zinc ions, cytoplasmic-free Zn2+, zinc located within intracellular compartments, and strip Zn2+ from proteins [2+ in cell division. It seems likely that Zn2+ is required for the passage of cells through the cell cycle. Certainly, several DNA-synthesizing enzymes seem to depend on Zn2+, suggesting that zinc depletion suppresses DNA synthesis [TPEN-based Znproteins . Thus, eproteins ,25,59,60ynthesis ,62.2+ concentrations because it is localized in the Golgi apparatus, secretory vesicles, and in the cell membrane [2+ efflux and influx. A study of ZnT-5–knockout mice revealed a reduction in islet zinc content in these animals [2+ homeostasis may be tissue-specific and might be related to the role of free Zn2+ in individual cell types.Unlike other zinc transporters, ZnT-5 holds a unique position in regulating intracellular Znmembrane ,64. ZnT- animals . Althoug animals , little  animals  and was  animals . Thus, t2+ concentration, adding to the accumulating evidence that links abnormal zinc homeostasis to the development of diabetes. Manipulation of the cellular zinc environment was found to have significant effects on cell survival, cell proliferation, and insulin processing and release. Understanding the finely tuned system involved in zinc transport and zinc buffering might open a new field of research into pharmacological intervention aimed at prolonging and improving pancreatic β-cell function.Using INS-1E cells, the present results indicate that β-cell function is directly related to the surrounding ZnThe authors declare that they have no competing interests.SBN, AL, JR, and KS conceived and designed the experiments, and wrote the manuscript. SBN and AK carried out the experiments. SBN, AL, AK, JR, and KS analyzed the data and approved the final version to be published. All authors read and approved the final manuscript.


MRI is the accepted gold standard for assessment of left ventricular systolic function; however, no standards are available to assess diastolic function at MRI. E/A and E/e’ ratios are currently used in echocardiography to evaluate left ventricular diastolic function. Measuring these ratios with phase contrast MRI may provide a complementary approach to assessing left ventricular function.To validate E/A and E/e’ ratios acquired with phase contrast MRI relative to established values using echocardiography.17 self-reported healthy volunteers were recruited under an IRB approved protocol. Ultra-fast phase contrast data was acquired on a 1.5T Siemens Aera using both breath-hold (30 frames per cardiac cycle) and free breathing (50 frames per cardiac cycle) paradigms. To measure e’ velocities, phase contrast data (Venc 25cm/s) was acquired in the short axis orientation at a slice position where the myocardium on the apical side of the valve ring was within the slice throughout the cardiac cycle. To measure E and A velocities, phase contrast data (Venc 80 cm/s) was acquired in a single slice parallel to the mitral valve annulus, positioned such that the slice stayed below the valve throughout the entire cardiac cycle. E and A velocities as well as septal and lateral e’ velocities were calculated using standard flow post-processing. 2 subjects were excluded from both analyses due to improper gating, and 4 additional subjects were excluded from the breath hold analysis due to too much noise to identify e’ velocities.With free breathing, E/A was measured at 1.7 ± 0.5 (range 0.8 - 2.6), septum E/e’ was measured at 5.4 ± 1.5 (range 2.5 - 7.4), and lateral E/e’ was measured at 5.1 ± 1.7 (range 2.6 - 8.4). With breath hold, E/A was measured at 1.5 ± 0.6 (range 0.7 - 2.7), septum E/e’ was measured at 6.1 ± 2.2 (range 3.6 - 10.2), and lateral E/e’ was measured at 5.4 ± 2.1 (range 3.5 - 10.9). Subjects with E/e’ above 8.0 had normal left atrial size.Measured E/A and E/e’ values are within normal limits using cutoff values that have been published with echocardiography , suggest


The PtII atom has a square-planar coordination environment, with Pt—CCN distances of 1.992 (4) and 2.000 (4) Å. In the cation, there is an N—H⋯N hydrogen bond linking adjacent pyridinium and pyridine rings in positions 4 and 5. Despite this, the organic component is non-planar, as shown by the dihedral angles of 10.3 (2), 6.60 (19) and 15.66 (18)° between the planes of the central imidazole ring and the pyridine/pyridinium substituents in the 2-, 4- and 5-positions. In the crystal, cations and anions are linked via O—H⋯O, O—H⋯N and N—H⋯O hydrogen bonds, forming a three-dimensional network. Additional π–π, C—H⋯O and C—H⋯N contacts provide stabilization to the crystal lattice.The asymmetric unit of the title hydrated complex salt, (C DOI: Click here for additional data file.10.1107/S1600536813011665/su2593Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Background: A bacterial β-etherase pathway uses glutathione-dependent enzymes for catabolism of β-ether-linked substructures found in lignin.Results: Racemic β-ether-linked substructures are stereoselectively converted to thioether-linked monoaromatic compounds by β-etherase pathway enzymes.Conclusion: Multiple enzymes with complementary stereochemical features are needed to catabolize racemic lignin substructures.Significance: Understanding the β-etherase pathway metabolism of lignin enhances our potential to use lignin for agricultural, industrial, and biotechnological purposes. S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave β-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of β-S-thioether linkages. Products of recombinant GSTs  are β-S-glutathionyl-α-keto-thioethers that are degraded by a β-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (β-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (β-guaiacyl-α-veratrylethanone). However, when β-etherase assays were conducted with a racemic model substrate, β-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (β-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the β-position (i.e. its β-epimer)) was produced only in the LigF-catalyzed reaction. Thus, β-etherase catalysis causes stereochemical inversion of the chiral center, converting a β(R)-substrate to a β(S)-product (LigE and LigP), and a β(S)-substrate to a β(R)-product (LigF). Further, LigG catalyzed glutathione-dependent β-S-thioether cleavage with β-S-glutathionyl-α-veratrylethanone and with β(R)-configured β-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced β-S-thioether-cleaving activity with the β(S)-epimer, demonstrating that LigG is a stereospecific β-thioetherase. We therefore propose that multiple Lig enzymes are needed in this β-aryl etherase pathway in order to cleave the racemic β-ether linkages that are present in the backbone of the lignin polymer.Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione  S-transferase (GST) family, the GSH thiol initiates a nucleophilic attack on the substrate during catalysis. Here we report new and novel aspects of GSTs that are implicated in lignin degradation.Glutathione (GSH) and GSH-dependent enzymes play crucial roles in cellular stress responses, protection against cell damage, or detoxification of xenobiotic compounds –4. In we–O-4′-Aryl ether (henceforth termed β-ether) bonds comprise the majority of the linkages between monomer-derived aromatic units in lignin  that allows growth on complex aromatic substrates - and β(S)-enantiomers (A and B) of β-guaiacyl-α-veratrylglycerone.In the best studied β-ether degradation pathway from the α-proteobacterium bstrates , 16, 17 bstrates . Based obstrates , 24, 25.ntiomers , A and Bin vitro assays with racemic substrates, cell extracts containing LigE and LigP separately degrade the β(R)GVG enantiomer while lacking detectable activity with the β(S)GVG substrate under identical assay conditions (A) (S)GVG enantiomer but has no detectable activity with β(R)GVG (B). The LigE, LigP, and LigF reaction products include guaiacol and a previously unidentified compound. The properties of the uncharacterized compound obtained using partially purified LigF were consistent with its being a GSH-conjugated aromatic monomer. However, from available data .In the next step of this pathway, three GSH-dependent GST enzymes  contribute to degradation of substrates containing β-aryl ether linkages like β-guaiacyl-α-veratrylglycerone (βGVG)nditions A 18). I. Iin vit β(R)GVG B. The Lia) cleavage of β(S)GVG and concomitant production of β-deoxy-α-veratrylglycerone (B) in coupled assays containing racemic βGVG, GSH, LigF, and LigG (a subsequent pathway enzyme believed to have β-thioetherase activity) and (b) the finding that a coupled assay containing LigE, LigG, GSH, and racemic βGVG resulted in β(R)GVG degradation but did not produce β-deoxy-α-veratrylglycerone (A)  and β-deoxy-α-veratrylglycerone (B) (Evidence supporting the existence of a GSH conjugate in this pathway includes (lycerone B in couplycerone A 18). B. Ba) clelycerone B 18, 19a cleavagrone (B) . NeverthR)GVG (A) or β(S)GVG (B). We further show that each GST β-etherase is stereoselective, yielding guaiacol and one of two potential β-epimers of GS-βVG. We also demonstrate that LigG is a β-S-thioetherase catalyzing the GSH-dependent cleavage of GS-βVG to β-deoxy-α-veratrylglycerone GVG A or β(S) β(S)GVG B. We furlycerone . To ratiEscherichia coli transformant cells were carried out according to standard methods  were obtained commercially from GeneArt® (Invitrogen) and were codon-optimized for expression in E. coli.Manipulation of DNA and preparation of  methods . All DNA8) affinity tag and tobacco etch virus (Tev) protease cleavage site upstream of the translation start codon.Preparation of expression vector pVP102K required the creation of intermediate plasmids pVP100K and pVP101K as follows. Plasmid pVP100K was first constructed by excising a 1,110-bp fragment from pMK1157550 with restriction enzymes BseRI and HindIII and then ligating into the compatible ends at the AsiSI and HindIII sites of pVP80K (obtained from the DNASU Plasmid Repository). Intermediate plasmid pVP101K was then constructed by excising a 852-bp fragment from the 6,123-bp plasmid pVP100K using AsiSI and ligating the free ends together to form 5,271-bp plasmid pVP101K. Expression vector pVP102K was then constructed by amplifying a 4,169-bp fragment from plasmid pVP101K, restricting the resulting amplicon with SacI, and ligating the free ends together, resulting in removal of the maltose-binding protein-encoding region of pVP101K and formation of the 4,153-bp expression vector pVP102K, which was T5 promoter-inducible and contained an in-frame N-terminal octahistidine  and was cloned into vector pVP202K by the PCR overlap method –33, affo–A 822-bp fragment containing the LigF-encoding ORF was amplified from plasmid pMKT1025979, the amplicon was restricted with AsiSI and SacII, and an 809-bp fragment was inserted into the AsiI-SacII region of pVP102K, plasmid pVP102KSSLigF, which was used for the expression of LigF.A 849-bp fragment containing the LigG-encoding ORF from plasmid pMK1118986 was amplified, both the amplicon and vector pVP102K were restricted with AsiSI and SacII, and the 836-bp and 4,067-bp fragments were ligated together, forming the 4,093-bp plasmid pVP102KSSLigG, which was used for the expression of LigG.E. coli strain B834 were grown aerobically with shaking at 37 °C in Luria-Bertani (LB) medium  of Buffer A -phosphine hydrochloride (TCEP), pH 8.0) supplemented with 1 mg ml−1 lysozyme. Cells were incubated with lysozyme for 30 min at 4 °C, after which time the NaCl concentration was brought up to 300 mm using a 5 m NaCl solution, and the slurry was augmented with 10 μg ml−1 RNase A and 5 μg ml−1 DNase I and allowed to sit for 15 min. Cells were then subjected twice to compression through a Spectronic Instruments French pressure cell at 137 megapascals, and cell debris was removed by centrifugation at 30,000 × g. Histidine-tagged proteins were then purified from cell lysates through a series of chromatographic and size exclusion chromatographic separations using a GE Healthcare ÄKTA Prime Plus FPLC system. First, cell lysates were loaded at 2 ml min−1 onto a GE Healthcare XK-16 column packed with 25 ml of Qiagen nickel-nitrilotriacetic acid (Ni-NTA) resin. The column was then washed with 100 ml of Buffer B , and His-tagged proteins were then eluted with a 30-ml linear gradient to 100% Buffer C . Collected fractions were then dialyzed for ∼12 h at 4 °C using a 10,000 molecular weight cut-off Thermo Scientific Slide-a-Lyzer dialysis cassette into Buffer D . Enzyme-specific methods (see below) were then carried out for the removal of recombinant affinity tags. After proteolysis, affinity tags were removed from the preparation by passage over a second GE Healthcare XK-16 column packed with 30 ml of Qiagen Ni-NTA resin. The preparation was then subjected to size exclusion chromatography in Buffer E  at a flow rate of 1 ml min−1 using a GE Healthcare HiLoad 16/600 SuperdexTM 200 pg column. Finally, each enzyme was concentrated in Buffer E to ≥10 mg ml−1 by centrifugation at 3,500 × g in a 10,000 molecular weight cut-off Pierce concentrator before drop freezing in liquid N2. Protein concentrations were determined by the Bradford method (Tranformant cultures of ) medium  overnigh) medium  supplemeTev protease was expressed as a 70.8-kDa fusion to maltose-binding protein . To ensu8 tag were then cleaved through 100 μm inositol hexaphosphate induction of RtxA (–8 domain. SDS-12% PAGE of denatured 32.1-kDa LigE and 31.0-kDa LigP enzymes showed that each preparation was homogeneous (data not shown).LigE and LigP were expressed as either a 56.1-kDa or 55.0-kDa fusion to RtxA and were dialyzed into Buffer D after Ni-NTA affinity chromatography. The RtxA and CHis of RtxA –42. The 8-fused recombinant enzymes LigF (33.4 kDa) and LigG (34.1 kDa) were expressed with the amino acid motif for the consensus cut site of Tev protease ENLYFQ (where cleavage occurs after Q) encoded immediately after the N-terminal His tag and prior to the encoding sequence of the respective enzyme . Subsequently, the samples were subjected to a second round of Ni-NTA affinity as well as size exclusion chromatography. SDS-12% PAGE of denatured 30.0-kDa LigF and 30.6-kDa LigG preparations showed that each was homogeneous (data not shown).NHise enzyme –46. Afte1H and 13C NMR spectra were recorded on a Bruker Biospin  AVANCE 700-MHz spectrometer fitted with a cryogenically cooled 5-mm TXI gradient probe with inverse geometry (proton coils closest to the sample). All compounds were characterized and assigned using the usual array of homonuclear and heteronuclear two-dimensional correlation  experiments.N2 displacement of the bromide by the phenolate ion of guaiacol, affording crystalline βGVE (The synthesis of βGVE  began wiine βGVE .R)GVG and β(S)GVG were isolated by preparative chiral chromatography. Additional synthetic details can be found in the supplemental material.βGVE was condensed with formaldehyde to yield racemic βGVG from which enantiopure β commenced with the condensation of triethyl phosphonoacetate with 3,4-dimethoxybenzaldehyde, yielding ethyl 3,4-dimethoxycinnamate. Reduction of the ester yielded 3,4-dimethoxycinnamyl alcohol. Oxidation of the ring-conjugated alcohol afforded 3,4-dimethoxycinnamaldehyde. Protection of the carbonyl group then yielded 3,4-dimethoxycinnamaldehyde dimethyl acetal.Synthesis of the dimethyl acetal A commencS)- and β(R)-isomers of γ,γ-dimethoxy-β-S-(methyl N-acetyl cysteinyl)-α-veratrylpropanone (CS-β(S)VP and CS-β(R)VP) were carried out in parallel (B and C), and each thioether was derived from 3,4-dimethoxycinnamaldehyde dimethyl acetal. Then, in separate parallel reactions, the alkene was stereoselectively oxidized to diols γ,γ-dimethoxy-α-veratryl-α(R),β(S)-propanediol and γ,γ-dimethoxy-α-veratryl-α(S),β(R)-propanediol. Benzylic oxidation of the diols then yielded ketones γ,γ-dimethoxy-β(R)-hydroxy-α-veratrylpropanone and γ,γ-dimethoxy-β(S)-hydroxy-α-veratrylpropanone. Triflation of the β-hydroxyls afforded triflate esters γ,γ-di-methoxy-β(R)-O-triflyl-α-veratrylpropanone and γ,γ-di-methoxy-β(S)-O-triflyl-α-veratrylpropanone. Last, SN2 displacement of the triflate groups by the thiolate ion of methyl N-acetyl cysteinate yielded β(S)- and β(R)-isomers of γ,γ-di-methoxy-β-S-(methyl N-acetyl cysteinyl)-α-veratrylpropanone (CS-β(S)VP and CS-β(R)VP) with high diastereometric purity. Because SN1 displacement of the β-O-triflate synthetic intermediates would have yielded a mixture of both CS-βVP β-epimers in the synthesis schemes of both CS-β(S)VP (B) and CS-β(R)VP (C), we conclude that formation of the β-S-thioether linkages in CS-β(S)VP and CS-β(R)VP was governed by SN2 inversion of the chiral center at β, affording highly diastereometrically pure products.Syntheses of βVP B and CS-S-β(R)VP C, we conAll chromatographic separations were carried out using a Beckman 125NM solvent delivery module equipped with a Beckman 168 UV detector.18 (100 g) reversed phase column was used for the purification of all enzymatically synthesized β-S-thioether compounds (GS-βVG and GS-βVE). A mixture of water and methanol was used for the mobile phase at a flow rate of 10 ml min−1. The proportions of the total flow made up by each buffer were adjusted over a gradient: 0–15 min, 0% methanol; 15–20 min, gradient from 0 to 100% methanol; 20–35 min, 100% methanol; 35–40 min, gradient from 100 to 0% methanol; 40–50 min, 0% methanol. Fractions with UV absorption at 280 nm were collected, pooled, dried under a stream of nitrogen gas, and analyzed by 1H, 13C, COSY, HSQC, and HMBC NMR spectroscopy.A prepacked Biotage KP-C−1) with glutathione (2.0 mm) and βGVE (1.5 mm) as cosubstrates. Prior to the addition of enzyme, 0.3 ml of the reaction mixture containing βGVE was collected for analysis by C18-reversed phase chromatography. After 1 h of incubation with one of the β-etherases, 0.3 ml of the reaction mixture was also collected for HPLC analysis. The 0.3-ml samples from 0 h and 1 h reaction times were then injected into an Ultra Aqueous  C18-reversed stationary phase column (4 × 120 mm) for separation of GS-βVE, guaiacol, and βGVE (A). A mixture of water and methanol was used as the mobile phase at a flow rate of 1.0 ml min−1. The methanol fraction of the total flow (with water as the remainder) was adjusted over a gradient as follows: 0–15 min, 0% methanol; 15–40 min, gradient from 0 to 100% methanol; 40–55 min, 100% methanol; 55–60 min, gradient from 100 to 0% methanol; 60–70 min, 0% methanol. GS-βVE, guaiacol, and substrate βGVE eluted from the C18 column after retention times (Rt) = 30.5, 39.6, and 46.3 min, respectively.Parallel β-etherase reactions were carried out using either LigE, LigP, or LigF (1.0 mg mland βGVE A. A mixtR)GVG and β(S)GVG, crystalline racemic βGVG  was dissolved in ethanol (5 ml) and injected into a CHIRALPAK AY-H column (10 × 250 mm). A mixture of ethanol and hexane was used as the mobile phase at a flow rate of 2.0 ml min−1. The ethanol fraction of the total flow (with hexane as the remainder) was adjusted over a gradient as follows: 0–12 min, 50% ethanol; 12–20 min, gradient from 50 to 100% ethanol; 20–90 min, 100% ethanol; 90–95 min, gradient from 100 to 50% ethanol; 95–110 min, 0% ethanol. Fractions containing βGVG enantiomers were collected and pooled, and solvents were dried in vacuo. β(S)GVG or β(R)GVG eluted from the column with Rt = 21.8 and 80.6 min. The aforementioned procedure was repeated four additional times in order to collect ∼5 mg of each enantiomer, to be used for enzymatic syntheses and isolation of GS-βVE, GS-β(S)VG, and GS-β(R)VG.To separate chiral enantiomers β(−1) with glutathione (2.0 mm) and racemic βGVG (1.5 mm) as cosubstrates. Aliquots (0.5 ml) were collected prior to the addition of enzyme (0-h sample) and again after 1 h of incubation with either LigE, LigP, or LigF. The 0.5-ml samples from 0 and 1 h were then each extracted six times with ethyl acetate, and the solvent was subsequently dried in vacuo. Residues from the 0- and 1-h samples were then dissolved in 0.1 ml of ethanol and injected into a Diacel Chemical Industries CHIRALPAK AY-H column (10 × 250 mm) for separation of guaiacol and chiral enantiomers β(R)GVG and β(S)GVG (B). A mixture of ethanol and hexane was used as the mobile phase at a flow rate of 2.0 ml min−1. The ethanol fraction of the total flow (the remainder was hexane) was adjusted over a gradient as follows: 0–12 min, 50% ethanol; 12–20 min, gradient from 50 to 100% ethanol; 20–90 min, 100% ethanol; 90–95 min, gradient from 100 to 50% ethanol; 95–110 min, 0% ethanol. Guaiacol, β(S)GVG, and β(R)GVG eluted from the chiral column with Rt = 8.0, 21.8, and 80.6 min, respectively.Parallel β-etherase reactions were carried out with either LigE, LigP, or LigF (1.0 mg ml β(S)GVG B. A mixtIn vitro reaction assays for enzymatic synthesis of β-S-thioethers were conducted in an aqueous assay buffer . GSH was added just prior to each assay to avoid disulfide formation, and NaOH was used to readjust the buffer to pH 7.5. All aqueous phase reaction products were desalted and purified by preparative C18-reversed phase HPLC, and fractions containing the reaction products were collected and dried over a stream of nitrogen gas. The 1H, 13C, COSY, HSQC, and HMBC NMR spectra were analyzed for each of the isolated reaction products.R)GVG  was added, and the buffer was separated into two 5-ml aliquots. To one 5-ml aliquot, LigE  was added. LigP  was added to the second 5-ml aliquot. Both reactions were incubated at room temperature for a period of 1 h. Guaiacol and trace amounts of β(R)GVG were removed from the reaction mixture by six successive ethyl acetate extractions. The LigE and LigP reaction products eluted from the Biotage KP-C18 column with Rt = 30.9 and Rt = 31.0 min, respectively.To 10 ml of the enzyme assay buffer, β(S)GVG as the LigF substrate, reaction conditions identical to those used for LigE and LigP were applied for LigF-catalyzed synthesis of GS-β(R)VG from β(S)GVG. GS-β(R)VG eluted from the Biotage KP-C18 column with Rt = 31.1 min.Aside from the use of β(m) was added, and the buffer was separated into three 5-ml aliquots. To each, either LigE , LigP , or LigF  was added. The three reactions were incubated at room temperature for 1 h. After ethyl acetate extraction, the aqueous reaction products of LigE, LigP, and LigF eluted from the Biotage KP-C18 column after Rt = 31.0, 31.1, and 31.0 min, respectively.To 15 ml of the enzyme assay buffer, βGVE  and either chemically synthesized GS-βVG  or GS-βVE  were added, and the buffer was readjusted to pH 7.5 using NaOH. Reactions were incubated at room temperature for 1 h. Six ethyl acetate extractions were carried out, and the organic fractions were pooled. Solvent was then removed by evaporation in vacuo, and products (either β-deoxy-α-veratrylglycerone or α-veratrylethanone) were analyzed by NMR spectroscopy VG in the aqueous fraction was purified further by preparative C18-reversed phase HPLC, where GS-β(S)VG eluted from the Biotage KP-C18 column with Rt = 31.0 min.To separate 5-ml aliquots of enzyme assay buffer, LigG -umbelliferyl-α-guaiacyl-ethanone) (C) in assays with the three proposed β-etherases from this strain. We also synthesized  , 17. Thithanone) C in assathesized  a β-S-glA) was accompanied by production of guaiacol and an unidentified compound (later identified as GS-βVE). Thus, we conclude that LigE, LigP, and LigF were each active on the achiral βGVE, as predicted (C).When we incubated βGVE with pure recombinant LigE, LigF, or LigP, we found that, as expected from members of this GST superfamily, the substrate's degradation required the presence of GSH . In the Fig. 5redicted C.supplemental material). The 1H NMR spectrum of the product isolated from either LigE, LigP, or LigF reactions using βGVE and GSH (B–D) was identical to that of chemically synthesized GS-βVE (A). In addition, long range two-dimensional 1H-13C NMR (HMBC) correlations (a) between the β-protons and the 3′-carbon as well as (b) between carbon-β and each 3′Ha and 3′Hb proton in synthetic GS-βVE (A) confirm that the GSH and aromatic moieties are linked via a β-S-3′-thioether. The task of identifying carbon-β and the β-protons was complicated by the β-protons' exchange with solvent (D2O) deuterons. By analyzing the spectra of chemically synthesized GS-βVE in 90/10% H2O/D2O as the NMR solvent , we were able to unambiguously assign the β-carbon in the 13C spectrum and the β-protons in the 1H spectrum . The HMBC spectrum in this solvent also revealed the above correlations between the β and 3′ nuclei, further establishing the existence of the β-S-3′-thioether linkage. Furthermore, both methoxyl moieties (3-OMe and 4-OMe) in GS-βVE could be unambiguously assigned (C) links the β-carbon of the aryl moiety to GSH.The unidentified product of βGVE degradation from each Lig enzyme was purified for analysis by NMR spectroscopy . We therefore conclude that LigG is a GSH-dependent β-S-thioetherase that cleaves the GSH conjugate GS-βVE, the intermediate derived from β-etherase activity with achiral substrate βGVE (C).The identification of GS-βVE as the product of β-etherase activity by the GST family members LigE, LigF, and LigP allowed us to test if this compound is a substrate for the putative GSH-dependent β-ethanone  (see supate βGVE C.C). In addition, we provide direct experimental evidence that LigG catalyzes thioether cleavage at the β-S-glutathionyl linkage in the presence of GSH.The use of the achiral substrate βGVE documents the combined activities of the GSH-dependent enzymes LigE, LigF, LigP, and LigG in a β-etherase pathway that releases two aromatic monomers from a dimeric model substrate C. In addR)GVG (A) and β(S)GVG (B). To aid these studies, we also synthesized (S-glutathionyl-α-veratrylglycerone (GS-βVG), which is the predicted product of β-etherase activity with βGVG. The existence of two β-epimers (GS-β(S)VG and GS-β(R)VG) in chemically synthesized GS-βVG was demonstrated by 1H-1H COSY, HSQC, and 1H NMR spectral analyses (A); the HMBC spectrum confirmed the existence of the β-S-3′-thioether linkage (B).Given the racemic nature of β-ether units in the lignin backbone , 22, we R)GVG A and β(S β(S)GVG B. To aidthesized  the GSH analyses A; the HM linkage B.in vacuo, residual organics were dissolved in ethanol and analyzed by chiral chromatography to determine if the enzymes cleaved one or both of the substrate enantiomers (B). We found that LigF cleaved only the β(S)GVG enantiomer, whereas LigE and LigP each cleaved only the β(R)GVG isomer. This confirmed that each of these enzymes is stereospecific for a single enantiomer GVG or β(S)GVG  was incubated with GSH and either LigE, LigP, or LigF. We found that LigE converted β(R)GVG to GS-β(S)VG with a 13:1 molar excess over the other diastereomer (GS-β(R)VG), as calculated from integration of the 1H NMR spectral peaks (B). LigP exhibited similar stereoselectivity because the product of β(R)GVG cleavage was a ∼4:1:1 mixture of GS-β(S)VG, GS-β(R)VG, and an impurity likely to be a β,γ-unsaturated alkene (C). In contrast, the product of LigF-catalyzed cleavage of β(S)GVG was a 1:7:2 mixture of the two β-epimers of GS-βVG (i.e. primarily GS-β(R)VG) and the above mentioned alkene impurity (D). We found that the abundance of the impurities and the level of the minor isomers in the β-etherase reaction products increased with time after isolation from enzymatic reactions, suggesting that non-enzymatic enolization and dehydration of GS-βVG are responsible for epimerization and side product formation. Thus, we conclude that LigE and LigP each stereoselectively produces GS-β(S)VG from β(R)GVG, whereas LigF stereoselectively yields GS-β(R)VG from β(S)GVG.To further characterize the products of β-etherase activity, we analyzed their activities when either enantiopure βVG and GS-β(R)VG). We found that the β-S-thioether cleavage activity of LigG was insufficient for complete degradation of the synthetic GS-βVG. After chromatographic separation of the LigG assay substrates and reaction products, the 1H NMR spectra of the residual GS-βVG showed that LigG had catalyzed β-S-thioether cleavage with high stereospecificity toward GS-β(R)VG compared with GS-β(S)VG. Integration of the 1H NMR spectral peaks in the residual GS-βVG (E) revealed that GS-β(S)VG was at a 5:1 molar excess over the other diastereomer, GS-β(R)VG. We also found that LigG-catalyzed degradation of the substrate resulted in the production of β-deoxy-α-veratrylglycerone , the expected monoaromatic product of GS-β(R)VG degradation (B). These observations confirm that LigG is a stereospecific β-thioetherase that catalyzes GSH-dependent cleavage of GS-β(R)VG, which arises as an intermediate in the β-etherase pathway.We also tested for the ability of LigG to catalyze GSH-dependent β-l GS-βVG E revealeradation B. These S)VG and GS-β(R)VG are intermediates in the β-etherase pathway (A–E), the absolute configurations of the two GS-βVG β-epimers remained unknown. Thus, to assign absolute orientation of the chiral centers at position β in GS-β(S)VG and GS-β(R)VG, two closely related models with known configurations at position β were synthesized (S)-configured (B) and β(R)-configured (C) γ,γ-dimethoxy-β-S-(methyl N-acetyl cysteinyl)-α-veratrylpropanone (CS-β(S)VP and CS-β(R)VP) syntheses yielded high purity products with only trace amounts of the undesired β-epimer in each case, as indicated by the 1H NMR spectra of CS-β(S)VP (F) and CS-β(R)VP (G). Analysis of the 1H NMR spectra also revealed that the chemical shifts of protons at β, 2′, and 3′ were affected by the chiral configuration at carbon β, with a pronounced effect observed for the splitting of the two protons at carbon 3′.Although our data indicated that both GS-βVP F and CS-S-β(R)VP G. Analysa and 3′Hb regions in CS-β(S)VP (F) and CS-β(R)VP (G) each aligned exclusively with one of the two β-epimers of GS-βVG (A–E). Thus, we propose that the alignment of the CS-β(S)VP (F) and GS-β(S)VG  3′Ha and 3′Hb spectral regions (and likewise of the CS-β(R)VP (G) and GS-β(R)VG (D) alignment) is attributable to the chiral configuration at carbon β. From these results, we conclude that β-etherase catalysis by LigE and LigP causes stereochemical inversion of chiral carbon β from a β(R)-substrate to product GS-β(S)VG (A), whereas LigF carries out inversion of the β(S)-substrate chirality in forming product GS-β(R)VG (B).The features of these synthetic compounds allowed us to compare them with the products of Lig etherase activity. The shifts of the 3′HS-β(S)VP F and CS-S-β(R)VP G each alf GS-βVG , A–E. ThS-β(S)VP F and GS-S-β(R)VP G and GS-S-β(R)VG D alignmeS-β(S)VG A, whereaS-β(R)VG B.R- and S-configured centers in the racemic natural plant lignins  and a second thiol-linked GSH-conjugated monoaromatic product.R)-enantiomer, and LigF cleaves only the β(S)-stereoisomer. We also found that LigE- and LigP-catalyzed β-etherase reactions exhibit stereoselectivity for the β(S)-configured GSH conjugate, whereas LigF yields the β(R)-diastereomer. Although the β-etherase catalytic reaction mechanism remains unknown, our findings reveal that β-etherase catalysis causes SN2-like inversion of the chiral configuration at carbon β, where β-ether cleavage and β-thioether formation are carried out on opposite faces of the molecule. In sum, our data indicate that these GST family members are both substrate-stereospecific and product-selective.Although LigE, LigF, and LigP are each active with β-ether-linked substrates, they are somewhat unusual GST family members because they are stereospecific for the configuration at the β-position of the substrate. We showed that with racemic β-aryl ether-linked model substrates, LigE and LigP each cleave only the β prior to reductive cleavage and release of the second aromatic (by LigG). Overall, this strategy is reminiscent of the chemical “derivatization followed by reductive cleavage” (DFRC) method used to release aromatic monomers from lignin -configuration at carbon β, and concomitant β-S-thioether bond formation. We have also unambiguously identified GSH conjugates as the previously uncharacterized glutathionyl-S-thioether-linked intermediates of the β-etherase pathway in Sphingobium sp. strain SYK-6. Further, given that (a) LigE/LigP and LigF stereoselectively produce GS-β(S)VG and GS-β(R)VG (respectively), (b) LigG converts the LigF β-etherase product GS-β(R)VG to β-deoxy-α-veratrylglycerone, and (c) LigG has little or no activity as a β-thioetherase with the LigE/LigP-produced GS-β(S)VG, we conclude that LigG is a stereospecific β-S-thioetherase that plays a role in the derivation of monoaromatic compounds in the β-etherase pathway. The fate of the LigE/LigP-produced GSH-conjugated β-etherase pathway intermediate, GS-β(S)VG, currently remains unknown. Assumedly, cells either use racemase-like enzymes for the conversion of the inactive β-epimer to the GSH conjugate that is cleaved by LigG, possess a second β-S-thioetherase with the required stereospecificity for GSH-dependent cleavage of the β(S)-isomer, or employ other metabolic activities that enable the bacterium to utilize GS-β(S)VG as a growth substrate.Our data show that β-etherase catalysis by the GST family members LigE, LigP, and LigF is achieved through stereospecific β-ether bond cleavage, inversion of the chiral (R)- and β(S)-configurations in native plant lignins necessitates both β(R)- and β(S)-stereospecific β-etherases and β-thioetherases. Although these enzymes could cleave other structurally related compounds, the stereochemical features of the enzymes described support a hypothesis that they normally function in a pathway that processes racemic substrates similar to those found in nature. These defined properties of the GSH-dependent enzymes could help in producing valuable chiral products from individual stereoisomers of β-aryl substrates that might be derived from lignin degradation.In sum, we have provided new insights into the enzymes, substrates, and products of a novel catabolic β-etherase pathway –20 that –


Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of  An elongation mix (EM) containing EF-Tu (10 µM), EF-Ts (5 µM), phenyl alanine (200 µM), tRNAPhe (5 µM), tRNAPhe-synthetase (0.2 units µl) and EF-G (5 µM), without or with FA (150 µM) and FusB (1 µM) was also incubated at 37°C for 15 min. Equal volumes of IM and EM were mixed by hand and the reaction was quenched after 10 s by adding formic acid . The ribosome-containing pellet was dissolved in potassium hydroxide, and the amount of MF di- and MFF tripeptide was analysed with reversed-phase high-performance liquid chromatography by comparing the respective peaks with the peak of [3H]fMet. All experiments were performed in triplicates.The initiation complexes (IM) were formed by incubating 70S ribosomes (1 µM), [5.11.Phe (3 µM) at 37°C for 20 min. To this complex, a mix containing RRF (10 µM), EF-G (5 µM) and IF3 (2 µM) without or with FA (10 µM) and FusB (1 µM) preincubated at 37°C was rapidly added in a stopped flow apparatus. The kinetics of splitting of 70S ribosomes into subunits was monitored by measuring Rayleigh light scattering as described [A post-termination complex was formed by incubating 70S ribosomes (0.5 µM), XR7 MFF mRNA (2 µM) and tRNAescribed .


The macrocyclic 24-membered ring core is planar with a mean deviation of 0.0311 (15) Å and the four-coordinate AgII cation fits into its center, at 2.0814 (19) and 2.0872 (19) Å, from the surrounding pyrrole-N atoms, in agreement with what is found in related compounds. The p-heptyloxyphenyl groups are rotated 75.51 (5) and 84.45 (8)° with respect to the porphyrin mean plane, due to steric hindrance with the pyrrole-H atoms of the macrocycle.The title compound, [Ag(C DOI: 10.1107/S160053681103385X/bg2416Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the hexanuclear unit, the six octahedrally coordinated NiII atoms are linked by four μ1,1,1-azide and four μ1,1-azide bridges, forming a face-sharing Ni6N8 tetracubane-like unit with four missing corners. The NiII atoms are further bridged by four μ1,2-carboxalate groups. Neighbouring hexanuclear units are connected via N—H⋯O hydrogen-bonding interactions into a three-dimensional structure. Although the H atoms of the methanol OH groups could not be located, O⋯N/O contacts between 2.65 and 2.86 Å suggest that these molecules participate in hydrogen bonding.The crystal structure of the title compound, [Ni DOI: 10.1107/S1600536811003473/wm2438Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The dihedral angles between the 1,2-dicyanoethene-1,2-dithiolato ligands and between the two pyridine rings in the cation are 37.49 (3) and 29.18 (10)°, respectively. Weak C—H⋯N and C—H⋯S hydrogen bonds link the cations and anions into a three-dimensional network.In the title ion-pair complex, (C DOI: Click here for additional data file.10.1107/S1600536813012439/hy2624Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The phenol O atom is a hydrogen-bond donor to the pyridine N atom, resulting in the formation of an eight-membered ring in the molecule. The amino N atom is a hydrogen-bond donor to the phenol O atom of an adjacent molecule; this hydrogen bond leads to the formation of a helical chain that runs along the a axis.The planes of the aromatic rings of the title compound, C For 2- = 0.043wR(F2) = 0.131S = 1.041391 reflections144 parameters2 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.13 e Å−3Δρmin = −0.18 e Å−3ΔρRAPID-AUTO  global, I. DOI: 10.1107/S1600536812031340/xu5581Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536812031340/xu5581Isup3.cmlSupplementary material file. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.We found the cross talk of Ca  Melanoma causes the majority of skin cancer related death, and is prevalent worldwide. The median life span of patients with advanced stage melanoma is less than a year because no therapies are effective once the tumor has spread to vital organs . The tum2+ homeostasis via regulation of membrane voltage-dependent Ca 2+ channels in excitable cells -1,2,3-thiadi azole-5-carboxanilide (YM58483), which is known as an antagonist for Ca 2+-activated Ca 2+ channels (CRAC) -1,2,3-thiadiazole-5-carboxanilide; CRAC: Ca 2+ release-activated Ca 2+ channels; TRP: transient receptor potential;Epac1: exchange protein activated by cAMP; ER: endoplasmic reticulum; IP3: inositol 1,4,5-trisphosphate; Gβγ G-protein βγ subunits; GDPβS: guanosine 5'-O-(2-thiodiphosphate); Gp(CHThe authors declare that they have no competing interests.EB designed the study, performed experiments and wrote the manuscript. MU and L-H X performed calcium experiments and participated in the design of the intracellular calcium experiments. MN participated in the design of the study. MSD, MI, SC and JSG participated in writing the manuscript. KI designed the study, performed experiments and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/256/prepub


The CoIII ion is coordinated by two NH3 ligands and a chelating carbonato ligand in the equatorial sites and by two NH3 groups in the axial sites, forming a distorted octahedral geometry. In the crystal, N—H⋯O hydrogen bonds connect the anions and cations, forming a three-dimensional network.In the title complex, [Co(CO For the al. 2008; Xu et a al. 2009. For the al. 2007; Palania al. 2001; Jothive al. 2013. For rel al. 1998; Massoud al. 2000; Sharma 3)(NH3)4]ClO4 = 0.042wR(F2) = 0.153S = 1.151947 reflections91 parametersH atoms treated by a mixture of independent and constrained refinementmax = 1.06 e Å−3Δρmin = −0.75 e Å−3ΔρAPEX2  used to solve structure: SHELXS97  global, I. DOI: 10.1107/S1600536813018187/lh5627Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.Human immunodeficiency virus-1 (HIV-1) replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR), to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP) cation-based vectors that efficiently deliver nucleotide analogs (PNAs) into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR  The transcriptional transactivation of the HIV-1 genome requires a specific interaction between the highly conserved TAR RNA hairpin fragment with the viral Tat protein and cellular factors (PTEFb-cyclin T1-CDK9 kinase complex). Both the six-nucleotide loop and the three-nucleotide bulge of TAR RNA  are invo24 or R0618, in vitro selection .An NMP solution containing TEA (10 eq.), NaNtR  = 21.6 min; MS :  m/z = 1473.4 (M+H)2+/2 982.4 (M+2H)3+/3, 736.9 (M+3H)4+/4, 589.7 (M+4H)5+/5.HPLC :  tR  = 23.8 min; MS :  m/z = 988.0 (M+2H)3+/3, 741.3 (M+3H)4+/4.HPLC :  tR  = 22.4 min; MS :  m/z = 992.6 (M+2H)3+/3, 744.7 (M+3H)4+/4, 596.1 (M+4H)5+/5.HPLC :  tR  = 13.2 min; MS :  m/z = 1505.4 (M+Na)2+/2.HPLC :  tR  = 12.4 min; MS :  m/z = 1209.23 (M+Na)2+/2.HPLC :  tR  = 12.7 min; MS : , m/z = 1939.5 (M+H)2+/2, 1293.6 (M+2H)3+/3, 970.9 (M+3H)4+/4.HPLC :  tR  = 12.8 min; MS :  m/z = 1654.3 (M+2H)3+/3, 1241.5 (M+3H)4+/4, 994.0 (M+4H)5+/5.HPLC :  tR  = 12.8 min; MS :  m/z = 1656.1 (M+H)2+/2, 1105.2 (M+2H)3+/3.HPLC :  μL (4 μM concentration) of R buffer solution at pH 7.3, that buffer containing cacodylate (20 mM), NaCl (20 mM), KCl (140 mM), and MgCl2 (0.3 mM). The solution was heated at 90°C for 2 min, immediately cooled at 4°C, and maintained at this temperature for 10 min, then kept at 20°C. For preparing hairpin PNAs 4–6, a solution of each compound in R buffer (4 μM) was heated for 3 min at 95°C, then cooled to 20°C with a rate of 0.5°C/min  (0.5 μM–5 μM). After 48 h, the cells were harvested, washed with PBS, and lysed in 1 × passive lysis buffer (Promega) with gentle shaking on a rocker for 15 min at room temperature. The lysed cells were centrifuged at 15,000 rpm for 15 min and an aliquot of the clear lysate was added to 100 μL luciferase assay reporter (Promega) in 96-well plate Fluotrac 200 . The luciferase activity was measured on a Packard Top Count Luminometer. The total light unit was normalized by total protein content in the cell lysate. Total protein was quantified using the DC protein assay kit . Median dose effects (IC50) for individual TPP- were determined using CalcuSyn software  . Similar experiments were carried out with unconjugated PNAs 1a–c and 4–6. The TPP-conjugate of a 16-mer antisense [PNA]TAR targeting the apical stem-loop of TAR was taken as a reference compound  are summarized in In order to evaluate the ter gene , with vacompound  . In addition, it appears that the antiviral activity of TPP-[PNA 1a–c] conjugates is specific. Indeed, TPP-[PNA 2] and TPP-[PNA 3], which, respectively, derive from the mismatched cyclic PNA 2 and from the GA-deleted cyclic PNA 3, have no effect on viral replication. These results, together with the fact that no interaction between mini-TAR and these two cyclic PNAs was detected, tend to further demonstrate that the inhibition of HIV replication by TPP-[PNAs 1a–c] is related to the formation of cyclic PNAs 1a–c/TAR complexes and thus, to the inhibition of the Tat/TAR/cellular factors complex formation.In all cases, except TPP-conjugates of PNAs  3.70 μM . The mic[PNA]TAR . As prev4–6) conjugates, it can be noted that the antiviral activity increases with the stability of the TAR/hairpin PNAs complexes [.] However, the complexes stability is not the only factor that explains biological activity, since cyclic PNAs 1a–c conjugates  are more effective for the inhibition of HIV replication in cells (IC50 1.24–1.94 μM) than the corresponding hairpin PNAs 4-5 conjugates.Concerning TPP-hairpin PNAs  HPLC and ESI MS spectra of PNA 3-6 and TPP- conjugates (p. S2-S13). B) UV-monitored thermal denaturation: First-derivative melting curves of PAA 4-6 and PAA 4-6/TAR complexes (p. S14).Click here for additional data file.


TNF-α produced by CD103−CD11b+ dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα Helicobacter typhlonius triggered intestinal pathology in TRUC mice ► T-bet regulated IL-7R transcription, a key checkpoint in intestinal ILC homeostasis► Chronic colitis in TRUC mice was mediated by IL-17-producing innate lymphoid cells ► TNF-α synergized with IL-23 to induce innate IL-17 production ►  Interactions between the innate immune system and the intestinal microbiota play an important role in the maintenance of mucosal homeostasis. Genetic variation in innate immune components, such as pattern recognition receptors, is associated with Crohn’s disease  and alteTbx21−/−Rag2−/−ulcerative colitis) disease. IBD in this model resolves following treatment with antibiotics , and disease can be ameliorated by neutralizing TNF-α antibodies. However, beyond 12 weeks of age, TNF-α antibodies are ineffective . The innRecently, the repertoire of cells that produce key cytokines implicated in IBD pathogenesis, such as interferon-γ (IFN-γ) and interleukin-17A (IL-17A), has been extended to include a population of cells termed innate lymphoid cells (ILCs) . We hypo+ immune cells. However, few IFN-γ- or IL-4-expressing cells were seen  from TRUC mice induced many IL-17A-expressing CD45ere seen A. In TRU IL-7Rα+ B, consis IL-7Rα+ . Intestifrequent C.+ cells and the number of IL-17A-producing cells in TRUC mice  was administered to TRUC mice. In contrast to effects on isotype-control-treated mice, anti-CD90 substantially reduced the number of CD90RUC mice E, reduceRUC mice B. In conRUC mice E.Tbx21−/−Rag2−/−Il2rg−/− triply deficient mice that additionally lack the common γ chain cytokine receptor. Il2rg−/− mice have >90% reduction in the number of ILCs in secondary lymphoid organs, such as the spleen  in the colon  mice. To identify potentially causative bacterial species, we compared the intestinal bacterial communities of TRUC and TRnUC mice by sequencing bacterial 16S ribosomal RNA (rRNA) genes. Individual samples shared broad similarities in community structure with other members of the same colony but were distinct from mice in the other colony . These data are consistent with an important role for H. typhlonius in triggering disease in TRUC mice.The composition of the intestinal microbiota is now recognized to profoundly impact intestinal immunity. Therefore, in order to better understand the immune pathways activated by the colitogenic microbiota colonizing TRUC mice, we sought to define which components of the microbiota were responsible for driving disease. Prior attempts to define causative bacteria have been hampered by a lack of appropriate control animals. However, we rederived a novel colony of  colitis A and 3B.r colony C. As expbacteres A. At theRUC mice B. Howevephlonius . SpecifiRUC mice D and S3CTRUC IBD E. H. typTbx21+/+ and Tbx21−/− intestinal ILCs in H. typhlonius-associated colitis in TRnUC and Rag2−/− mice. Ex vivo explant cultures from TRnUC mice gavaged with H. typhlonius produced less IFN-γ and more IL-17A in comparison to Rag2−/− mice  cells from TRUC mice, it markedly potentiated IL-23-induced IL-17A production  cells, which results in the activation of numerous key genes involved in the Th1 cell response , Rag2−/− (Jackson Labs), Rag2−/−Il2rg−/− (Taconic), and Ifng−/− (Jackson Labs) mice were sourced commercially. A colony of colitis-free TRnUC mice was generated and remotely maintained at geographically distinct isolators from the TRUC colony that was descendant from the originally described TRUC mice , supplemented with 10% fetal calf serum , 10 mM HEPES (Fisher Scientific), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) and EDTA (5 mM). Tissue was then mechanically disrupted using GentleMACS (Miltenyi Biotec), followed by collagenase digestion through incubation of the crude cell mixture in RPMI-1640 medium (PAA Laboratories) supplemented with 25 μg/ml collagenase D (Roche), 10 μg/ml DNase I (Roche), and 1.5mg/ml dispase (Roche) for 45 min in a shaking water bath (37°C). Cells were resuspended in 40% Percoll  and layered on 80% Percoll prior to centrifugation, and the cLPL-enriched population was harvested.Colons were cut longitudinally, fecal material was removed, and colons were washed with PBS. Colons were then cut in to 5 mm segments, and the epithelium was removed through incubation with Hank’s balanced salt solution (HBSS) without MgOne centimeter segments of colon were fixed in 10% paraformaldehyde and embedded in paraffin blocks. Five micrometer sections were stained with hematoxylin and eosin. For immunofluorescence, colon tissue was removed and snap frozen in Jung tissue-freezing medium (Leica Microsystems). Seven micrometer cryostat sections were fixed in acetone, blocked , and incubated with fluorescein-isothiocyanate-conjugated anti-CD90.2 (eBioscience). Nuclei were counterstained with 1 μg/ml DAPI (Invitrogen). Colitis scores comprising epithelial hyperplasia (0–3), epithelial injury (0–3), polymorphonuclear infiltrate (0–3), and mononuclear infiltrate (0–3) were reported in a blinded fashion (T.T.M.) as described previously .Intracellular cytokine expression was measured after cells were stimulated with IL-23 (20 ng/ml) or with PMA  and ionomycin  for 4 hr, with monensin (2 μM) added for the final 2 hr at 37°C. In order to measure spontaneous cytokine production by DCs, cells were incubated for 4 hr at 37°C in the presence of monensin without additional stimuli. Surface-staining antibodies were added together with LIVE/DEAD stain (Invitrogen) for 20 min at room temperature. Cells were fixed in 1% paraformaldehyde and permeabilized (eBioscience permeabilization buffer), and intracellular staining was performed for 30 min at 4°C. Antibodies were from eBioscience unless otherwise stated: α-CD45 (30-F11), α-CD4 (RM4.5), α-CD11b (M1/70), α-CD11c (N418), α-CD103 (2E7), α-F4/80 (BMP), α-CD90.2 (30H12), α-NKp46 (29A1.4), α-Gr-1 (RB6-8C5), α-RORγt (AFKJS-9), α-CCR6 , α-Sca-1 , α-T-bet (eBio4B10), α-IFN-γ (XMG1.2), α-IL-17A (eBio17B7), α-IL-22 (IH8PWSR), α-TNF-α (MP6-XT22), α-IL-4 , and α-CD127 (A7R34).−ΔCt method.RNA was extracted from colon segments or purified cells with Trizol reagent (Invitrogen) and complementary DNA (cDNA) generated with the cDNA synthesis kit (Bioline). Quantitative PCR was used to quantify mRNA transcripts using TaqMan gene-expression assays . Gene expression was normalized to the expression of β-actin for generation of ΔCt values, and relative abundance was quantified with the 2Antibody treatments comprised: 500 μg anti-TNF-α , 150 μg anti-IL23p19 , 450 μg IL-17A mAb , 1 mg CD90.2 mAb , and 1 mg anti-IL-7R . Control-isotype clones used were MOPC-21 (mouse immunoglobulin G1 [IgG1]), HRPN (rat IgG1), 2A3 (rat IgG2a), and LTF-2 (rat IgG2b). In the anti-CD40 model, 150 μg of mAb (clone FGK4.5) or control isotype was administered to 6- to 8-week-old mice. Monoclonal antibodies were purchased from Bio X Cell  unless otherwise stated and were administered intraperitoneally.DNA was extracted from frozen fecal pellets with the FastDNA SPIN Kit. 16S-rRNA-gene PCR amplicons were generated for Lib-L 454 Titanium sequencing with the use of barcoded primers targeting the V3–V5 regions of the 16S rRNA gene. The primers and barcode sequences used are given in Raw sequences were passed through the PyroTagger pipeline , which fH. typhlonius-specific PCR was performed with the use of primer pairs that have been shown to amplify a 122 bp sequence of the 16S rRNA gene in H. typhlonius, but not other Helicobacter species (Helicobacter-genus-generic PCR was performed with the primer pair (5′-TATGACGGGTATCCGGC-3 and 5′-ATTCCACCTACCTCTCCCA-3′) .  species  (5′-AGGGCCCA-3′) . The 374H. typhlonius CCUG 48335 T was obtained from the culture collection of the University of Goteborg, Sweden . A total of 1–5 × 107 organisms were gavaged into selected mice as described in the text., Sweden  and was 6/ml), mLN (1 × 105/ml), and cLP cells (1 × 105/ml) were cultured in RPMI-1640 medium (PAA Laboratories), supplemented with 10% FCS (PAA Laboratories), 50 μM 2-mercaptoethanol (Invitrogen), 2mM L-glutamine (Sigma-Aldrich), 1 mM sodium pyruvate (Invitrogen), 10 mM HEPES (Fisher Scientific), nonessential amino acids (Sigma-Aldrich), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Cells were cultured in medium alone or in the presence of 20ng/ml recombinant IL-23 and/or TNF-α (R&D Systems). Cytokine concentrations in culture supernatants were measured via ELISA (R&D Systems).Unfractionated splenocytes (2 × 10Three-millimeter punch biopsies (Miltex) were used to acquire full thickness colonic biopsies. Three biopsies were cultured in 300 μl of RPMI supplemented with complete medium (as above) for 24 hr. Cytokine concentrations in culture supernatants were measured by ELISA (R&D Systems).+ T cells from Tbx21−/−Ifng−/− mice were cultured in Th0 conditions with IL-2 only  and transduced with either retrovirus encoding T-bet and green fluorescent protein (GFP) (RV-GFP-T-bet) or control retrovirus encoding GFP only (RV-GFP) as described previously (CD4eviously . Cells wNonparametric data were analyzed with the Mann-Whitney U test using GraphPad Prism. Elsewhere, mean data are expressed with error bars denoting SEM.


Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase  enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds . These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-The online version of this article (doi:10.1007/s00253-012-3943-1) contains supplementary material, which is available to authorized users. Three-dimensional structures have been reported for the N-terminally truncated GTF-180 enzyme of L. reuteri 180  , 135 GH70 sequences are reported in the CAZy database  was amplified from the genomic DNA of L. reuteri DSM 20016 using the primers For (NcoI) 5′-GAT GCA TCC ATG GGC ATA GAT GGT AAG AAC TAC CAC TTC GC-3′ and Rev (BamHI) 5′-ATA TCG ATG GAT CCT ATT AGT GAT GGT GAT GGT GAT GAA TAT TTT CTT GGT TTG CAT AGT AAT CTG C-3′ and high fidelity DNA polymerase (Fermentas), and cloned in pET15b (Novagen), yielding pET15b_4,6-αGT-W. This N-terminally truncated version of 4,6-αGT-W (amino acids 458–1363) carries a fused (His)6-tag at its C-terminal. The removal of the N-terminal variable region is based on the cloning and expression of other GTFs, as this improves protein expression without affecting the catalytic properties  has been identified in L. reuteri ML1, located upstream of a glucansucrase gene (gtfML1). The 5′-end of the gtfML4 gene was obtained by inverse PCR. Briefly, genomic DNA of L. reuteri ML1 was digested by BcuI or NsiI, and the products were ligated at a concentration of ~10 ng/μl using T4 DNA ligase to obtain circular fragments. The ligation mixtures served as PCR template in an inverse PCR with the oligonucleotides HL115, 5′-TGA TCG TCC AGA TGT AGC-3′ and HL116, 5′-CCA GTT ACT TTC ATA GAG G-3′. This yielded PCR fragments of 2 kbp (BcuI) and 8 kbp (NsiI), which were cloned in the pCR-XL-TOPO vector (Invitrogen). The obtained plasmids were used for DNA sequencing, yielding the 5′-end of the gtfML4 gene.Previously, the 3′-end fragment of the 4,6-αGT-ML4 encoding NcoI), 5′-TTT TCC ATG GGG AAC CGC GTT GAT TAC TGG-3′ and HL118 , 5′-AAA AGA TCT TAG TGA TGG TGA TGG TGA TGG TTG TTA AAA TTT AAT GAA ATT GC-3′ were designed and used to amplify the gene from chromosomal DNA. The obtained fragment was cloned in pET15b (Novagen), yielding pET15b_4,6-αGT-ML4. This vector encodes an N-terminally truncated version of 4,6-αGT-ML4 (amino acids 714–1620) and carries a fused (His)6-tag at its C terminus. Removal of the N-terminal variable region previously was shown to improve GTF protein expression without affecting catalytic properties /pET15b_4,6-αGT-W cultivated in Luria broth containing 100 mg/l ampicillin. Protein expression was induced at an OD600 of 0.4–0.5 by adding isopropyl β-d-1-thiogalactopyranoside to 0.1 mM, and cultivation was continued for 4 h at 30°C. Cells were harvested by centrifugation, washed in phosphate buffer  and resuspended in phosphate buffer , containing NaCl (250 mM), imidazole (10 mM), and β-mercaptoethanol (5 mM). Cell-free extracts were made by sonication followed by centrifugation . The 4,6-αGT-W protein was purified by nickel affinity chromatography using an 1-ml Hitrap IMAC HP column  and a gradient of 10–200 mM imidazole in phosphate buffer , containing NaCl (250 mM). Fractions containing 4,6-αGT-W were pooled. The imidazole and NaCl were removed using a 5-ml Hitrap desalting column  run with sodium phosphate buffer , and the protein was loaded on a 1-ml Resource-Q column . Protein was eluted with a gradient of 0–1 M NaCl in phosphate buffer . Fractions containing 4,6-αGT-W were transferred to sodium acetate buffer  using a 5-ml Hitrap desalting column. Protein purity was assessed on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentration measured with Bradford reagent (BioRad) and bovine serum albumin as standard.The 4,6-αGT-W protein was produced in E. coli BL21(DE3)/pET15b_4,6-αGT-ML4 cultivated in Luria broth containing 100 mg/l ampicillin. Protein expression was induced at an OD600 of 0.4–0.5 by adding isopropyl β-d-1-thiogalactopyranoside to 0.1 mM, and cultivation was continued for 4 h at 30°C or 24 h at 18°C. Cells were harvested by centrifugation, washed in Tris/HCl buffer , and resuspended in 10 ml BPer lysis reagent (Pierce), followed by centrifugation . The 4,6-αGT-ML4 protein was purified from inclusion bodies by washing the pellet once with 5 ml BPer plus 2 mg lysozyme for 10 min at room temperature, followed by four washes with 50 ml 10 times diluted BPer. Finally, the inclusion bodies were resuspended in 10 ml 8 M urea, 20 mM Tris/HCl (pH 8.0), 1 mM CaCl2, and 1 mM DTT to obtain unfolded protein. Refolding was done by dialysis against 20 mM Tris/HCl (pH 8.0), 1 mM CaCl2, and 1 mM dithiothreitol. The 4,6-αGT-ML4 protein was then further purified by nickel affinity chromatography using a 1-ml Hitrap IMAC HP column . Following washing with 10 mM imidazole in 20 mM Tris/HCl (pH 8.0) with 1 mM CaCl2, the protein was eluted with 200 mM imidazole in the same buffer. The imidazole was then removed using a 5-ml Hitrap desalting column  run with 20 mM Tris/HCl (pH 8.0) and 1 mM CaCl2. Protein purity was assessed on 8% SDS-PAGE and protein concentration measured with Bradford reagent (BioRad) and bovine serum albumin as standard.The 4,6-αGT-ML4 protein was produced in 2 (1 mM) at 37°C, unless stated otherwise. The substrate spectra of the enzymes were investigated by incubating the various oligosaccharides  with 25 μg/ml enzyme. The progress of the reactions was followed by high-pH anion-exchange chromatography (HPAEC) (described below) and/or thin-layer chromatography (TLC) using silica gel 60F254 plates (Merck) run with butanol/ethanol/water . TLC plates were developed with 10% H2SO4 in methanol/water , followed by heating at 100°C.All reactions were performed in sodium acetate buffer  with CaClFor 4,6-αGT-W activity, the optimal pH and temperature were determined over the pH range of 3.5–6.5 (in 25 mM sodium acetate buffer) and the temperature range of 20–60°C, using 100 mM maltose as substrate by following the release of glucose in time using the GOPOP kit (Megazyme)  at temperatures from 20°C to 55°C in a water bath for 10 min. Samples were then cooled in an ice bath, and residual activity was measured by following the release of glucose using maltose as substrate. The T50 is defined as the temperature at which 50% of the initial enzyme activity is retained after 10-min incubation.The thermal inactivation rate of 4,6-αGT-W was determined by incubating the enzyme at a concentration of 0.26 mg/ml in sodium acetate buffer  with CaClKinetic properties of 4,6-αGT-W were determined with maltose (0–500 mM) as substrate and by following the release of glucose. Reactions were initiated by the addition of 4,6-αGT-W enzyme at a concentration of 64 nM. The rates were fitted to the Michaelis–Menten equation.v/v) as eluent, followed by lyophilization. The desalted fractions were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and 1H nuclear magnetic resonance (NMR) spectroscopy. Fractions containing multiple oligosaccharides were separated again by HPAEC using a different elution gradient.Twenty milligrams of maltose was incubated with 16 μg 4,6-αGT-W in 1.17 ml sodium acetate buffer  at 37°C for 4 days. The reaction mixture was freeze-dried, dissolved in 200 μl Milli-Q water, and 20-μl aliquots were loaded on a 9 × 250 mm CarboPac PA-1 column (Dionex), which was run with a gradient of 0–600 mM sodium acetate in 100 mM NaOH (3 ml/min). Eluting peaks were collected manually and neutralized immediately with 4 M acetic acid. The collected fractions were desalted on 150 mg CarboGraph SPE columns (DiscoverySciences) using acetonitrile/water = 1:3 , or pullulanase M1 . α-Amylase cleaves α1→4 glycosidic bonds in longer α1→4 fragments, amyloglucosidase is an exo-acting enzyme that cleaves terminal α1→4 and α1→6 glycosidic bonds at the non-reducing end to form glucose, and pullulanase M1 cleaves α1→6 glycosidic bonds at the reducing end of an α1→4 glycosidic bond [in (-)α-d-Glcp-(1→4)-α-d-Glcp-(1→6)- sequences] (Domań-Pytka and Bardowski Reaction products (250 μg) of the enzyme incubations with maltose up to maltoheptaose were dissolved in 10 μl sodium acetate buffer (50 mM) and incubated for 1 day at 37°C with α-amylase , amyloglucosidase , run with a gradient of 30–600 mM sodium acetate in 100 mM NaOH (1 ml/min), and detected with an ED40 pulsed amperometric detector. Calibration was done by running samples with known concentrations of glucose and maltotetraose.v/v). The compounds fly as sodium ion adduct.MALDI-TOF-MS experiments were performed on an Axima™ mass spectrometer (Shimadzu), equipped with a nitrogen laser . Positive-ion mode spectra were recorded using the reflector mode at a resolution of 5000 FWHM and delayed extraction (450 ns). The accelerating voltage was 19 kV with a grid voltage of 75.2%; the mirror voltage ratio was 1.12, and the acquisition mass range was 200–3,000 Da. Samples (1 μl) were mixed in 1:1 ratio with 10 mg/ml 2,5-dihydroxybenzoic acid in acetonitrile/water = 1:1 . Conventional work-up, comprising neutralization (by adding 4 M acetic acid) and removal of boric acid by co-evaporation with methanol, followed by acetylation with pyridine/acetic anhydride  , yielded mixtures of partially methylated alditol acetates, which were analyzed by gas-liquid chromatography electron impact mass spectrometry.Samples (~2 mg) were permethylated using CH1H NMR spectra were recorded in D2O on a Varian Inova Spectrometer  or a Bruker DRX-500 spectrometer  at probe temperatures of 300 K. Prior to analysis, samples were exchanged twice in D2O  with intermediate lyophilization and then dissolved in 0.6 ml D2O. Suppression of the HOD signal (on the Bruker DRX-500 spectrometer only) was achieved by applying a WEFT pulse sequence. Chemical shifts (δ) are expressed in parts per million by reference to internal acetone (δ 2.225 for 1H).Resolution-enhanced 1D 500-MHz http://www.ebi.ac.uk) , which encodes a 154-kDa protein, designated 4,6-αGT-W. Sequence comparison indicates that, in analogy with other GH70 enzymes, 4,6-αGT-W possesses a variable N-terminal region (amino acids 1-427), a catalytic domain  as substrate, forming a range of shorter and longer products -α-cts Fig. , but notcts Fig. . Besidescts Fig. . Note thcts Fig. .Fig. 24,kcat of 37 ± 4 s−1 and a KM of 150 ± 40 mM. The enzyme also processes maltopentaose, yielding maltotetraose + glucose (hydrolysis) or maltotetraose + a DP6 compound (transglycosylation). With 43 mM of maltopentaose as substrate, the initial rate of glucose formation was 56% of that of maltotetraose formation, showing that the enzyme is rather hydrolytic at the start of the reaction. However, when reaction products start to accumulate, transglycosylation becomes more efficient, as the reaction products are better acceptor substrates than maltopentaose.The initial rate of 4,6-αGT-W with maltose was determined by measuring the release of glucose  using glucose oxidase, displaying a d-glucooligosaccharides] of DP2 to DP7 -α-1H NMR analysis of the various product mixtures obtained with 4,6-αGT-W and 4,6-αGT-ML4 showed the presence of α1→4 linkages  and newly formed α1→6 linkages . As typical examples, Fig. 1H NMR spectra for the product mixtures generated from maltohexaose, showing a linkage ratio α1→4:α1→6 = 50:50 for 4,6-αGT-W -d-Glcp; Rα H-1, δ 5.225; Rβ H-1, δ 4.652)]. The spectra also revealed the presence of free glucose . Furthermore, small amounts of six-substituted reducing-end glucose residues  -α-d-Glcp-(1→4)-d-Glcp], four compounds could be identified, i.e., α-d-Glcp-(1→4)-α-d-Glcp-(1→4)-d-Glcp , α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-d-Glcp, α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-D-Glcp-(1→6)-α-d-Glcp-(1→4)-d-Glcp, and α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-d-Glcp -α-d-Glcp-(1→6)- elements, the product mixtures obtained from maltose up to maltoheptaose were treated with pullulanase type M1. This enzyme hydrolyzes α1→6 glycosidic linkages in (-)α-d-Glcp-(1→4)-α-d-Glcp-(1→6)- sequences only. Comparison of the HPAEC elution profiles before and after pullulanase treatment showed only a few differences in the larger oligosaccharide and polymeric fractions  that uses (1→4)-α-glucans, but not sucrose, as substrate -α-d-glucooligosaccharides] into linear α-glucans  with about 50% α1→6 glycosidic bonds. It thus appears that 4,6-αGT activity is a common activity within the GH70 family.It was generally accepted that all GH70 enzymes synthesize α-glucans from sucrose, until we recently demonstrated that The 4,6-αGT products are synthesized by cleaving off the non-reducing glucose moiety of a (1→4)-α-glucan and transferring the glucose moiety to the HO-6 function of the non-reducing end of an acceptor α-glucan chain [a (1→4)-α-glucan or a (1→4)-α-glucan already elongated with a number of (α1→6)-linked glucose residues]  is eight residues longer in 4,6-αGTs compared to GTFs Fig.  show thaFurthermore, enzymes such as 4-α-glucanotransferase, cyclodextrin glucanotransferase, branching enzyme, and dextrin dextrinases disproportionate (1→4)-α-glucans /kcat(hydrolysis) ratio of the GH13 Neisseria polysaccharea amylosucrase is 20 -α-glucan substrates into linear α-glucan products with a high percentage of α1→6 glycosidic linkages . These enzymes are potentially very useful for the synthesis of carbohydrate displaying compounds. Indeed, GH13 and GH70 enzymes are being used for the glycosylation of a variety of compounds Below is the link to the electronic supplementary material.


CCAAT enhancer-binding protein (C/EBP)β regulates gene expression in multiple organ systems and cell types, including astrocytes in the central nervous system (CNS). Inflammatory stimuli, interleukin (IL)-1β, tumor necrosis factor-α, human immunodeficiency virus (HIV)-1 and lipopolysaccharide induce astrocyte C/EBPβ expression. C/EBPβ is detectable in brains of Alzheimer’s disease (AD), Parkinson’s disease (PD) and HIV-1-associated dementia (HAD) patients, yet little is known about how C/EBPβ contributes to astrocyte gene regulation during neuroinflammation.The expression of 92 human inflammation genes was compared between IL-1β-treated primary human astrocytes and astrocytes transfected with C/EBPβ-specific small interfering (si)RNA prior to IL-1β treatment for 12 h. Transcripts altered by > two-fold compared to control were subjected to one-way analysis of variance and Newman-Keuls post-test for multiple comparisons. Expression of two genes, cyclooxygenase-2 (COX-2) and bradykinin receptor B2 (BDKRB2) was further confirmed in additional human astrocyte donors. Astrocytes were treated with mitogen-activated protein kinase-selective inhibitors, then with IL-1β for 12 or 24 h followed by COX-2 and BDKRB2, expression analyses.IL-1β altered expression of 29 of 92 human inflammation genes by at least two-fold in primary human astrocytes in 12 h. C/EBPβ knockdown affected expression of 17 out of 29 IL-1β-regulated genes by > 25%. Two genes relevant to neuroinflammation, COX-2 and BDKRB2, were robustly decreased and increased, respectively, in response to C/EBPβ knockdown, and expression was confirmed in two additional donors. COX-2 and BDKRB2 mRNA remained altered in siRNA-transfected astrocytes at 12, 24 or 72 h. Inhibiting p38 kinase (p38K) activation blocked IL-1β-induced astrocyte COX-2 mRNA and protein expression, but not IL-1β-induced astrocyte BDKRB2 expression. Inhibiting extracellular-regulated kinase (ERK)1/2 activation blocked IL-1β-induced BDKRB2 mRNA expression while increasing COX-2 expression.These data support an essential role for IL-1β in the CNS and identify new C/EBPβ functions in astrocytes. Additionally, this work suggests p38K and ERK1/2 pathways may regulate gene expression in a complementary manner to fine tune the IL-1β-mediated astrocyte inflammatory response. Delineating a role for C/EBPβ and other involved transcription factors in human astrocyte inflammatory response may lead to effective therapies for AD, PD, HAD and other neurological disorders. Neuroinflammation is a contributing factor of many central nervous system (CNS) pathologies; yet the details of onset and progression remain enigmatic. Astrocytes, the most numerous cells of the CNS, contribute to homeostasis in the CNS, regulate neural signaling and maintain the blood–brain barrier (BBB) -3. AccorC/EBPβ is evolutionarily conserved among species and is expressed in multiple organ systems -11. The It is well established that IL-1β mediates neuroinflammation through activation of glial cells and subsequent changes in gene expression ,17. In tHuman astrocytes were isolated from first- and early second-trimester  aborted specimens obtained from the Birth Defects Laboratory, University of Washington, Seattle, in full compliance with the ethical guidelines of the NIH, the Universities of Washington and North Texas Health Science Center. The Birth Defects Laboratory is an NIH-funded institution with the mission of disseminating tissues for the advancement of biomedical research. Astrocytes were isolated from specimens as originally described earlier . Activat2 flasks at a density of 8 × 106 cells per flask or six-well plates at a density of 1.6 × 106 cells per well for 24 h. The following day, the medium was exchanged with or without IL-1β (20 ng/ml) for 24 h. Alternatively, astrocytes were cultured in six-well plates at a density of 1.6 × 106 cells per well for 24 h, and then the medium was exchanged with or without MAPK-selective inhibitors  for 1 h and then with inhibitor plus IL-1β (20 ng/ml) for 12 h. Astrocyte RNA was extracted  and reverse-transcribed into cDNA as per the manufacturer’s instructions . The TaqMan® Human Inflammation Array  was used to assay for expression of 92 inflammation genes using total RNA from two independent human astrocyte donors  followed by Newman-Keuls post-test. Selected targets were confirmed individually by RT2PCR TaqMan® gene expression assays performed using the StepOnePlus sequence-detection system (Life Techologies), using primers specific to C/EBPβ (C/N: Hs00270923_m1), COX-2 (C/N: Hs00153133_m1), BDKRB2 (C/N: Hs00176121_m1) and glyceraldehyde phosphate dehydrogenase  or C/EBPβ-specific (siC/EBPβ) siRNA by nucleofection and then cultured as adherent monolayers in 75 cm2 flasks at a density of 8 × 106 cells per flask. The following day, medium was exchanged with or without IL-1β (20 ng/ml) for 24 h. Alternatively, astrocytes were cultured in 75 cm2 flasks at a density of 8 × 106 cells per flask for 24 h, and then the medium was exchanged with or without MAPK-selective inhibitors  for 1 h and then with inhibitor plus IL-1β (20 ng/ml) for 24 h. Cells were lysed; total cell extracts were isolated using mammalian protein extraction reagent . Equal amounts of proteins (15 μg/lane) were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinyl difluoride (PVDF) membrane using i-Blot (Life Techologies). The membrane was incubated in anti-C/EBPβ  or anti-COX-2  at a dilution of 1:200. Blots were then incubated in secondary antibody . β-actin  was used as a loading control. The Western blot was visualized with supersignal chemiluminescent substrate (Thermo Fisher Scientific), and band intensities were quantified by densitometry analysis .Astrocytes were transfected with nonspecific control (siCON) or C/EBPβ-specific (siC/EBPβ) siRNA by nucleofection and then cultured as adherent monolayers in 75 cmMonolayers of astrocytes were treated with 2× the final dose of the MAPK-selective inhibitors, SB203580 (p38K-20 μM), SB202190 (p38K-20 μM), U0126 (ERK1/2-20 μM) and PD184352 (ERK1/2-20 μM) for 1 h before adding equal volume of 2× the final dose of IL-1β treatment.6 cells per well for 24 h, and then with or without MAPK-selective inhibitors  for 1 h and then inhibitor plus IL-1β (20 ng/ml) for 24 h. Experimental cells were fixed with cold acetone:methanol (1:1) for 30 min at −20 °C. Cells were then blocked in phosphate-buffered saline (PBS) with 2% bovine serum albumin (BSA) and 0.1% triton X-100 for 1 h at room temperature. Cells were incubated in PBS with 2% BSA and 0.1% triton X-100 plus anti-COX-2 antibody at 1:500 and anti-glial fibrillary acidic protein (GFAP) antibody at 1:600 for 8 h at 4 °C. Cells were washed with PBS and then incubated in secondary antibody (1:800) for 1 h at room temperature. Micrographs were taken on a Nikon Eclipse Ti. .Astrocytes were cultured as adherent monolayers in a 48-well plate at a density of 0.1 × 10p < 0.05, and data represent mean values ± SEM. Data presented are representative of a minimum of three independent experiments with two or more independent donors, unless noted, in which case, n represents cumulative data from a specific number of independent human donors (TaqMan® Human Inflammation Array and western blots).Statistical analyses were carried out using GraphPad Prism 5.0 software , with one-way ANOVA and Newman-Keuls post-test for multiple comparisons. Data generated from each assay from the TaqMan® Human Inflammation Array  were analyzed independently. Significance was set at ® Human Inflammation Array to evaluate human astrocyte C/EBPβ’s contribution to expression of 92 inflammatory genes in response to IL-1β. Figure As previously reported, IL-1β induces astrocyte C/EBPβ expression and localization to nuclei, where the transcription factor regulates gene expression ,17. Astrp < 0.001). C/EBPβ-specific siRNA transfection achieved a 65% knockdown through 72 h in IL-1β-treated astrocytes . We have previously reported that C/EBPβ-specific siRNA alone reduces basal levels of C/EBBβ mRNA by 65% [p < 0.001). C/EBPβ-deficient astrocytes expressed BDKRB2 mRNA levels at 83 (12 h), 65 (24 h) and 60 fold (72 h) that of control siRNA (siCON)-transfected astrocytes . IL-1β induced 700- (12 h), 533- (24 h) and 400-fold (72 h) increases in astrocyte COX-2 mRNA expression, while C/EBPβ knockdown downregulated this robust induction by 26% (12 h), 39% (24 h) and 31% (72 h) . These data confirm the mRNA expression results from the TaqMan® Human Inflammation Array plate.Differences in genetic background among human astrocyte donors account for variation in readouts; therefore, we confirmed our results for COX-2 and BDKRB2 mRNA in two additional astrocyte donors. Consistent with our previously published work , a singlp < 0.001). IL-1β induced astrocyte COX-2 protein expression at 24 h post-treatment ; however, siC/EBPβ-transfected cells expressed reduced COX-2 compared to siCON-transfected cells . These data suggest that C/EBPβ regulates mRNA and protein expression of multiple human astrocyte inflammation genes.Next, we investigated the changes in COX-2 expression by immunoblot analyses in the context of C/EBPβ-specific siRNA transfection followed by IL-1β activation. To confirm siC/EBPβ effectively blocked IL-1β-induced astrocyte C/EBPβ, we performed immunoblot analysis of 24 h protein lysates; results from 72-h lysates were previously reported [t Figure C. IL-1β-s Figure Dp < 0.05p < 0.001); pretreating the cells with SB203580 had no significant effect on BDKRB2 mRNA levels compared to IL-1β alone. However, pretreating astrocytes with U0126 significantly reduced the IL-1β-mediated increase in BDKRB2 mRNA by 75% . IL-1β induced a 600-fold increase in COX-2 mRNA compared to untreated cells (p < 0.001); SB203580 pretreatment reduced this response by 93% . IL-1β induced a 1,400-fold increase in COX-2 mRNA in U0126-pretreated astrocytes; this was a significant increase compared to astrocytes treated with IL-1β alone . Similar results were obtained using the p38K- and ERK1/2-selective inhibitors SB202190 and PD184352, respectively  was undetectable in total protein lysates from untreated astrocytes; however, a 70-kDa isoform was detected in lysates from IL-1β-treated astrocytes  with COX-2 ; several of our data are corroborated therein. As in this report, multiple arrays have shown IL-1β induces CD40, NOS-2, vascular cell adhesion molecule-1, ICAM-1, TNF and COX-2 ,30. OtheC/EBPβ expression is detectable in immune-activated rodent glia, and it is now clear that this factor is involved in inflammatory processes in tissues throughout the body ,31. In tAs previously reported, NOS-2 and ICAM-1 expression levels were negatively and positively affected by C/EBPβ knockdown, respectively ,19. Of tC/EBPβ knockdown increased IL-1β-induced astrocyte BDKRB1 and BDKRB2 expression as well as that of 11 other genes  Astrocytes were cultured for 24 h, pretreated with pathway selective inhibitors (20 μM) and then treated with IL-1β for 12 h. Total RNA was isolated, reverse-transcribed and then assayed by RT2PCR for (A) BDKRB2 or (B) COX-2 relative transcript levels. GAPDH was used as the normalizing control; p-values are compared to control unless denoted by lines to specific comparisons.IL-1β signals through ERK1/2 and p38K to increase astrocyte BDKRB2 and COX-2 mRNA, respectively. (Click here for file


L) has been found to act as a bidentate ligand in the monomeric title complex, [Cu(CF3O3S)(C12H8N4S)2(H2O)](CF3O3S). The complex shows a distorted octahedrally coordinated copper(II) cation which is linked to two thiadiazole ligands, one water molecule and one trifluoromethanesulfonate anion. The second trifluoromethanesulfonate anion does not coordinate the copper(II) cation. Each thiadiazole ligand uses one pyridyl and one thiadiazole N atom for the coordination of copper. The N atom of the second non-coordinating pyridyl substituent is found on the same side of the 1,3,4-thiadiazole ring as the S atom. The trifluoromethanesulfonate ions are involved in a three-dimensional network of O—H⋯O hydrogen bonds. C—H⋯N interactions also occur.2,5-Bis(pyridin-2-yl)-1,3,4-thiadiazole (denoted  DOI: 10.1107/S1600536812008732/im2358Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The tert-butyl group of the 4-tert-butylpyridine ligand is disordered over two sets of sites in a 0.25:0.75 ratio and was refined using a split model. The CdII cation is coordinated by six N atoms of four tert-butylpyridine ligands and two N-bonded thiocyanate anions within a slightly distorted octahedral coordination environment.The asymmetric unit of the title compound, [Cd(NCS) N-donor ligands, see: Boeckmann & Näther .For the synthesis and properties of coordination polymers based on transition metal thiocyanates and ther 2010, 2011 ▶.2(C9H13N)4] = 0.051wR(F2) = 0.127S = 1.024709 reflections226 parameters3 restraintsH-atom parameters constrainedmax = 1.69 e Å−3Δρmin = −1.58 e Å−3ΔρX-AREA  used to solve structure: SHELXS97  I, global. DOI: Click here for additional data file.10.1107/S1600536812040081/bt6836Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The CoII cation is in a distorted octahedral coordination environment defined by the two N atoms of the 2,9-dimethyl-1,10-phenanthroline ligand and four O atoms. Two of these are from a chelating formate anion, one from a monodentate formate and the last from an aqua ligand. In the crystal, molecules are connected by O—H⋯O hydrogen bonds, forming double chains along [100] with the 2,9-dimethyl-1,10-phenanthroline ligands pointing outwards from each chain. These chains are further linked into layers parallel to (011) by inter-chain π–π stacking interactions with centroid–centroid distances of 3.61 (1) Å.The asymmetric unit of the title compound, [Co(HCO DOI: 10.1107/S1600536810050051/sj5068Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: