N′-[(E)-(3-hydroxy-4-methoxyphenyl)methylidene]furan-2-carbohydrazide (L1) and N′-[(E)-(3-hydroxy-4-methoxyphenyl)methylidene]thiophene-2-carbohydrazide (L2) are reported. Schiff-base ligands L1 and L2 were derived from condensation of 3-hydroxy-4-methoxybenzaldehyde (iso-vanillin) with furan-2-carboxylic acid hydrazide and thiophene-2-carboxylic acid hydrazide, respectively. Complexes of the general formula [M(L)2]Cl2 (where M = Co(II) or Cd(II), L = L1 or L2) have been obtained from the reaction of the corresponding metal chloride with the ligands. The ligands and their metal complexes were characterised by spectroscopic methods , elemental analysis, metal content, magnetic measurement, and conductance. These studies revealed the formation of four-coordinate complexes in which the geometry about metal ion is tetrahedral. Biological activity of the ligands and their metal complexes against gram positive bacterial strain Bacillus (G+) and gram negative bacteria Pseudomonas (G−) revealed that the metal complexes become less resistive to the microbial activities as compared to the free ligands. New monomeric cobalt and cadmium complexes with Schiff-bases, namely,  Schiff-bases and their complexes have a variety of applications in the biological systems and industry –6. FurthAll reagents were commercially available and used without further purification. Solvents were distilled from appropriate drying agents immediately prior to use.−1. Electronic spectra of the prepared compounds were measured in the region 250–1100 nm for 10−3 M solutions in DMSO at 25°C using a Shimadzu 160 spectrophotometer with 1.000 ± 0.001 cm−1 matched quartz cell. 1H and 13C NMR spectra were acquired in DMSO-d6 solution using a Brucker AMX 400 MHz spectrometer with tetramethylsilane (TMS) as an internal standard for 1H NMR. Metals were determined using a Shimadzu (A.A) 680 G atomic absorption spectrophotometer. Chloride was determined using potentiometer titration method on a 686-Titroprocessor, 665 Dosimat Metrohm, Swiss. Conductivity measurements were made with DMSO solutions using a PW 9526 digital conductivity meter and room temperature magnetic moments were measured with a magnetic susceptibility balance .Melting points were obtained on a Buchi SMP-20 capillary melting point apparatus and are uncorrected. FTIR spectra were recorded as KBr disc using a Shimadzu 8400 FTIR spectrophotometer in the range 4000–400 cm−1): 3473 ν (O–H), 3202 ν (N–H), 1664 ν (C=O), 1629 ν (C=N). NMR data (ppm), 1H NMR data showed peaks at δH : 3.84 , 6.92 , 7.214–7.340 , 7.85–7.911 , 8.57 , 8.74 . 13C NMR data showed peaks at δc : 56.51 (O–CH3),  assigned to carbon of furan ring,  assigned to aromatic ring carbon, 149.50 (HC=N), 154.51 (C=O).A solution of 3-hydroxy-4-methoxy-benzaldehyde  in methanol (5 mL) was added to a mixture of furan-2-carboxylic acid hydrazide  in methanol (5 mL), and then (2–4) drops of glacial acetic acid was added. The reaction mixture was refluxed for 4 h, filtered off, and then cooled to RT. Solvent was allowed to slow evaporation and a pale yellow solid was obtained. Yield , m.p = 183°C. IR data  was used in place of furan-2-carboxylic acid hydrazide. The quantities of other reagents used were adjusted accordingly. An identical work-up procedure gave the title compound as an orange-yellow solid. Yield , m.p. = 170°C. IR data (cm−1): 3375 ν (O–H), 3163 ν (N–H), 1669 ν (C=O), 1618 ν (C=N). NMR data (ppm), δH : 3.83 , 6.69 , 6.83–6.95 , 7.06–7.40 , 7.92 , 8.34 (H–C=N–). 13C NMR data δc : 56.30 (O–CH3),  assigned to carbon of furan ring,  assigned to aromatic ring carbon, 161.34 (C=O), 151.86 (H−C=N).The method used to prepare L2·XH2O , was stirred into methanolic solution of the Schiff-base ligand (2 mmoL) in methanol (15 mL). The reaction mixture was then refluxed for 2 h to give a coloured precipitate which was collected by filtration, washed with cold ethanol (5 mL), and dried at room temperature. Elemental analysis data, colours, and yields for the complexes are given in A methanolic solution (10 mL) of the hydrated metal salt (1 mmoL), MClμL) of the test sample 1 mg/mL in DMSO was introduced in the respective wells. The plates were incubated immediately at 37°C for 20 h. Activity was determined by measuring the diameter of zones showing complete inhibition (mm). In order to clarify the role of DMSO in the biological screening, separate studies were carried out with the solutions alone of DMSO and they showed no activity against any bacterial strains. Ligand found to be potentially active against these bacterial strains were compared with its complexes.Bioactivities were investigated using agar-well diffusion method . The wel1 and L2, respectively 2]+2 (where M = CoII and CdII) were obtained , ν (C=N) functional groups, respectively. The bands at 3473, 3203, and 3375, 3163 cm−1 assigned to the ν (O–H) and ν (N–H) stretching group for ligands L1 and L2, respectively, Cl2 (where M = Co(II) and Cd(II); L = L1 and L2) were obtained. Physico-chemical analysis indicated the formation of four coordinate dicationic metal complexes. Biological activities revealed that the ligands have higher antimicrobial activity than their metal complexes.In this paper, we have explored the synthesis and coordination chemistry of cobalt and cadmium complexes derived from the Schiff-base ligands L


One CoII atom is coordinated by two N atoms from one pyridine-2-carbaldehyde oxime ligand, by one terminal and one bridging Cl− ion, by one O atom from an ethanol molecule, and by one O atom from a bridging pyridine-2-carboxylate (picolinate) anion. The second CoII atom is coordinated by two N atoms from another pyridine-2-carbaldehyde oxime ligand, one N and one O atom from the bridging picolinate anion, and by one terminal Cl− and one bridging Cl− anion. The structure displays intramolecular O—H⋯O and O—H⋯Cl hydrogen bonds. Weak C—H⋯Cl hydrogen-bonding interactions connect the molecules into a three-dimensional network.The dinuclear title compound, [Co II complexes with pyridine-2-carbaldehyde oxime ligands, see: Stamatatos et al. (C6H6N2O)2(C2H6O)] = 0.036wR(F2) = 0.165S = 1.184520 reflections319 parameters14 restraintsH-atom parameters constrainedmax = 1.09 e Å−3Δρmin = −1.31 e Å−3ΔρAPEX2  used to solve structure: SHELXS97  I, global. DOI: Click here for additional data file.10.1107/S1600536812041736/wm2687Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


We previously reported that nerve injury-induced neuropathic pain is initiated by newly produced lysophosphatidic acid (LPA).2 (cPLA2) and calcium-independent phospholipase A2 (iPLA2), key enzymes for LPA synthesis, at 1 h, while there was no significant change in phospholipase A1 activity. Pharmacological studies revealed that NMDA and neurokinin 1 receptors, cPLA2, iPLA2 and microglial activation, as well as LPA1 and LPA3 receptors were all involved in the nerve injury-induced LPA production, and underlying cPLA2 and iPLA2 activations. In the cells expressing LPA1 or LPA3 receptor, the receptor-mediated calcium mobilization was most potent with 18:1 LPA, compared with 16:0 or 18:0 LPA. Moreover, the intrathecal injection of 18:1 LPA, but not 16:0 or 18:0 LPA, caused a spinal LPA production and neuropathic pain-like behavior.In this study, we developed a quantitative mass spectrometry for detecting LPA species by using Phos-tag. Following nerve injury, the levels of 18:1, 16:0 and 18:0 LPA in the spinal dorsal horn significantly increased at 3 h and declined at 6 h. Among them, 18:1 LPA level was the most abundant. In the same preparation, there were significant elevations in the activities of cytosolic phospholipase A1 and LPA3 receptors-mediated amplification of LPA production through microglial activation.These results suggest that 18:1 LPA is the predominant ligand responsible for LPA The reactions were stopped by the addition of 5,5’-dithiobis(nitrobenzoic acid) for 5 min. The activity of PLA2 was defined as the percentage of the control activity as follows: injured tissues (absorbance/mg of protein)/normal tissues (absorbance/mg of protein) × 100.The activities of cPLAeviously ,30,53. Bnhibitor , and a s1 activity assay was performed according to the manufacturer’s protocol of EnzChek® phospholipase A1 assay kit . Briefly, isolated ipsilateral spinal dorsal horn was sonicated in PLA1 reaction buffer  and centrifuged at 20000 × g for 20 min at 4°C. The supernatant was collected and its protein concentration was determined by the Lowry method. In the PLA1 assay, the substrate-liposome mix was prepared by mixing 9 μl of lipid mix  and 501 μl of PLA1 reaction buffer. Subsequently, 1 μl of tissue sample, standard PLA1  or negative control (PLA1 reaction buffer) was incubated with 19 μl of substrate-liposome mix in a 384-well microplate  for 30 min. Then the fluorescence was measured using a PHERAstar FS reader  equipped for excitation at 485 nm and fluorescence emission at 520 nm. The activity of PLA1 was defined as the percentage of the control activity as follows: injured tissues (absorbance/mg of protein) / normal tissues (absorbance/mg of protein) × 100.PLAMice were deeply anesthetized with pentobarbital (50 mg/kg) and perfused with potassium-free phosphate buffered saline (pH7.4), followed by 4% paraformaldehyde solution. L4-6 spinal cord segments were then isolated, postfixed in the same fixative for 3 h, and replaced with 25% sucrose overnight. Tissues were fast-frozen in cryo-embedding compound and cut on a cryostat at a thickness of 10 μm.2 antibody . Sections were subsequently incubated with secondary antibody, Alexa Fluor 488-conjugated anti-rabbit IgG , for 2 h at 20°C. Stained sections were washed and cover-slipped with Perma Fluor . Images were collected using an LSM 710 confocal microscope with ZEN software .Immunofluorescence labeling was performed by blocking the sections with 2% BSA in TBST  for 2 h at 20°C, followed by incubation with anti-NeuN antibody  or anti-Iba1 antibody  overnight at 4°C. After washing, sections were incubated with Alexa Fluor 594-conjugated anti-mouse IgG  or Alexa Fluor 546-conjugated anti-goat IgG , respectively, for 2 h at 20°C. Then, sections were blocked with 2% BSA in TBST for 2 h at 20°C and incubated overnight at 4°C with anti-phospho-cPLA1-B103 or LPA3-B103 cells were used for calcium mobilization assay. The cells were harvested by centrifugation and re-suspended with 0.1% BSA supplied-DMEM. The cell suspension was plated 30 μl per well in a 384-well plate with the density of 5.0 × 103 cells/well. Following incubation at 37°C in a 5% CO2 atmosphere overnight, cells were loaded with 10 μl Fluo-8 (8 μM) in 0.1% BSA supplied-DMEM containing 1 mg/ml amaranthl. After 30 min, 20 μl various LPA species at defined concentration was added, and fluorescence was recorded by Functional Drug Screening System/μCell  immediately. The fluorescence intensity was described as signal ratio .LPApost hoc analysis following one-way ANOVA. The criterion of significance was set at p < 0.05. All results are expressed as mean ± SEM.Statistical analysis was evaluated using the Tukey’s multiple comparison Lpar1- or Lpar3-deficient mice; LPC: Lysophosphatidylcholine; MALDI-TOFMS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Glu: Glutamate; SP: Substance P; NK1: Neurokinin 1; cPLA2: Cytosolic phospholipase A2; iPLA2: Calcium-independent phospholipase A2; PC: Phosphatidylcholine; AACOCF3: Arachidonyl trifluoromethyl ketone; BEL: Bromoenol lactone; i.p.: Intraperitoneal; PLA1: Phospholipase A1; RT-PCR: Reverse transcription polymerase chain reaction; NeuN: Neuronal nuclei; Iba1: Ionized calcium-binding adaptor molecule 1; p-cPLA2: Phosphorylated cPLA2; LPA1-B103 or LPA3-B103: B103 cells expressing LPA1 or LPA3 receptor; ED50: Half-maximal effective dose; DMEM: Dulbecco’s modified Eagle’s medium; BSA: bovine serum albumin; TBST: 0.1% Triton X-100 in Tris Buffered Saline.LPA: Lysophosphatidic acid; i.t.: Intrathecal; Lpar1-/- or Lpar3-/-: The authors declare that they have no competing interests.1 activity assay. JC generated Lpar1-/- and Lpar3-/- mice. HU was responsible for experimental design and manuscript revision for publication. All authors read and approved the final manuscript.LM was responsible for experimental design, performance of all the experiments except for calcium mobilization assay, and writing the manuscript. JN conducted MALDI-TOFMS analyses and performed calcium mobilization assay as well as PLA


Calendula officinalis L. (Asteraceae) were found to show an inhibitory effect on amylase. From the crude extract fractions, one new phenolic acid glucoside, 6′-O-vanilloyl-β-D-glucopyranose, was isolated, together with twenty-four known compounds including five phenolic acid glucosides, five phenylpropanoids, five coumarins, and nine flavonoids. Their structures were elucidated based on chemical and spectral data. The main components, isoquercitrin, isorhamnetin-3-O-β-D-glucopyranoside, 3,5-di-O-caffeoylquinic acid, and quercetin-3-O-(6′′-acetyl)-β-D-glucopyranoside, exhibited potent inhibitory effects on amylase.An ethanolic extract and its ethyl acetate-soluble fraction from leaves of  Calendula officinalis L., commonly called marigold, or pot marigold is widely cultivated as an ornamental, culinary and valuable medicinal herb due to its various pharmacological properties: anti-inflammatory, antioxidant, antibacterial, antifungal, and so forth  to give quercetin-3-O-(6′′-acetyl)-β-d-glucopyranoside   to give 1′-O-p-hydroxybenzoyl-β-d-glucopyranose  + . +FAB-MS, m/z: 331 [M + H]+. 1H-NMR  and 13C-NMR data ; see UV C. officinalis were extracted with 60% ethanol (5 mL) in an ultrasonic bath for 40 min. The extracted solutions were filtered through a 0.22 μm PTFE syringe filter before injection into the LC system for analysis.The dried and powdered leaves (200 mg) from the different varieties of ∅ 5 μm; Metrohm AG). Mobile phase A was 0.2 M LiClO4 in 0.006 M HClO4 and mobile phase B was acetonitrile. The injection volume was 1 μL, and elution was at 150 μL min−1 with a gradient program . The detector wavelength was 270 nm. Reference compounds with purity greater than 96% were used for the establishment of calibration curves. This included 3-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, caffeic acid, isoquercitrin, and rutin from Sigma-Aldrich ; Isorhamnetin-3-O-β-d-glucoside was from Extrasynthese ; 4,5-di-O-caffeoylquinic acid was from ChemFaces . Thyphaneoside, quercetin-3-O-(6′′-acetyl)-β-d-glycoside, and isorhamnetin-3-O-(6′′-acetyl)-β-d-glycoside were isolated previously from C. officinalis flowers  × 100, where A510NC is the absorbance of the negative control, A510PC is the absorbance of the positive control, and A510Sample is the absorbance of the sample solution. The IC50 value is the effective concentration at which amylase activity was inhibited by 50%. Values are expressed as mean obtained from 5 independent experiments.Amylase inhibitory activity was measured using a microplate method: 10 C. officinalis leaves was carried out for nine varieties cultivated widely in the territory of Russia. These varieties are characterized by high productivity and simplicity of cultivation. As a result, it was found that flavonoid content in the leaves examined ranged from 8.49 (“Indian Prince”) to 13.08 mg g−1 (“Radio”), while the total content of phenolic compounds varied from 29.21 (“Russian Size”) to 50.24 mg g−1 (“Radio”)  . The maxC. officinalis leaves of “Radio” variety was partitioned with CHCl3, EtOAc, and n-BuOH to yield three fractions. The crude extract was found to exhibit inhibitory activity on an amylase with an inhibitory value (IC50) of 38.02 ± 1.29 μg mL−1 , prep. HPLC, and prep. TLC, yielding one new (6) and twenty-four known compounds. The known compounds, including five phenolic acid glucosides , five phenylpropanoids , five coumarins , and nine flavonoids , were identified by comparing their UV, MS, and NMR data with those reported in the literature , 7.52 , and 6.80 , which are typical for 1,3,4-trisubstituted benzene rings  showed a correlation with the aromatic proton of H-6 at δ 7.63 and the aromatic carbon at δ 147.5 (C-3) correlated with methoxyl protons at δ 3.97  showed correlation with the proton at δ4.05, suggesting that the vanilloyl moiety is connected to C-6′ of the glucose moiety. Moreover, the results of the 13C-NMR analysis showed a downfield shift in the resonance of the glucose C-6′, as well as an upfield shift of the C-5′ resonance comparing with those of free glucose. Assignment of the protons and carbons signals was achieved by a combination of the HMBC and 1H-1H COSY spectral data. Since the anomeric hydroxyl group of the glucose moiety of 6 is unacylated, it may be obtained as anomers (α- and β-forms) mixture. The 13C-NMR data obtained showed a presence of a week signal at δ92.1 that corresponded to α-form of 6. However, the ratio of the peak areas for C-1′ atoms of α- (δ92.1) and β-forms (δ98.3) is about 1 : 92. Thus, we can conclude that the predominant component of mixture is β-forms . These results enabled compound 6 to be identified as 6′-O-vanilloyl-β-d-glucopyranose.Alkaline hydrolysis of 6 has been detected previously in different compounds like glehlinosides from Glehnia littoralis (Euphorbiaceae) [O-vanilloylsucrose from Saccharum officinarum (Poaceae) [O-vanilloyicariside B5 from Baccaurea ramiflora (Euphorbiaceae) [Amburana cearensis (Fabaceae) [Acer saccharum (Aceraceae) [O-galloyl-6′-O-vanilloyl-β-d-glucopyranose from Alchornea trewioides (Euphorbiaceae) [6 has been isolated as a separate compound.The moiety of biaceae) , 6′-O-vaPoaceae) , 6′-O-vabiaceae) , amburosabaceae) , sacchareraceae) , and 1′-biaceae) . This isC. officinalis leaves was performed using microcolumn HPLC-UV method which allowed separating 15 components  was the dominant compound, as well as 3-O-caffeoylquinic acid—6.48 (“Egypt Sun”)—13.25 mg g−1 (“Flame Dance”) —5.34 mg g Dance”) . The totβ-d-glycoside prevailed. Thyphaneoside in “Egypt Sun,” “Flame Dance,” “Indian Prince,” “Radio,” “Russian Size,” isorhamnetin-3-O-β-d-glucoside in “Green Heart Orange” and “Red Black Centered,” and isorhamnetin-3-O-(6′′-acetyl)-β-d-glycoside in “Geisha” and “Touch of Red” are all simple derivatives of isorhamnetin.The content of quercetin derivatives was higher than the content of derivatives of isorhamnetin, ranging from 1.64- (“Green Heart Orange”) to 4.79-fold (“Radio”). It should be noted that isoquercitrin dominated in almost all species except “Geisha” and “Touch of Red” where quercetin-3-O-(6′′-acetyl)-C. officinalis varieties studied can be divided into three groups:varieties with a predominant content of flavonoids —“Touch of Red,” “Green Heart Orange,” “Red Black Centered” and “Russian Size”;varieties with similar contents of phenylpropanoids and flavonoids —“Radio,” “Egypt Sun,” “Geisha” and “Indian Prince”;varieties with a predominance of phenylpropanoids —“Flame Dance.”According to the data on the phenylpropanoids, flavonoid ratio, 7, 13, 17, and 21 showed significantly higher inhibitory activity at concentration ranging 1.02–2.64 μg mL−1 comparing with the reference compound acarbose (9.54 μg mL−1) , 22 , 9 , and 19 (quercetin-3-O-(6′′-acetyl)-β-d-glucopyranoside; 32.70 μg mL−1).All substances isolated were tested for their amylase inhibiting activity by the microplate method. Compounds μg mL−1) . It shouO-rutinoside) and narcissin (isorhamnetin-3-O-rutinoside), have been previously reported as inhibitors of glucosidase [C. officinalis leaves.It is known that the phenolic phytosubstances are the perspective natural compounds which exhibited antidiabetic activity . Flavonocosidase . The resC. officinalis leaves. As a result, twenty-five compounds were isolated, of which sixteen had not previously been reported in this plant source. The presence of undiscovered benzoic acid glucosides early in the Calendula genus, one of which was novel (6′-O-vanilloyl-β-d-glucopyranose), as well as the amylase inhibiting activity of the crude extract and individual compounds, illustrates the need for in-depth research even on very familiar species such as C. officinalis.The present investigation is the first extended study of the phenolic composition from


High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL. Epidemiological studies have shown that high plasma levels of high-density lipoproteins (HDLs) protect against development of atherosclerosis . HDLs upSphingosine 1-phosphate (S1P), a bioactive lysophospholipid, is produced by activated platelets and erythrocytes and enters circulation in HDL- or albumin-bound forms , 9. In p32P]ATP  and [32P]orthophosphate were purchased from PerkinElmer Life Sciences . HUVECs were purchased from the Medical Center for Cells, Central South University . Pertussis toxin (PTX), NS398, and 4′-6-diamidino-2-phenylindole were obtained from Sigma . GF109203X, PD98059, JTE013, and VPC23019 were from Cayman . Competitive enzyme immunoassay kit for 6-keto PGF1α, nuclear and cytoplasmic protein extraction kit, and chromatin immunoprecipitation (ChIP) assay kit were obtained from Beyotime Institute of Biotechnology . Mouse GAPDH was from Boster . Rabbit anti-COX-2 antibodies were from Epitomics . Rabbit polyclonal anti-SphK-2 and anti-SphK1 antibodies were from Abcam . P-CREB, ERK1/2, P-ERK1/2, P-PKCα, and PKCα antibodies were from Cell Signaling . HDL was purchased from Millipore Corporation . Lipofectamine 2000 was from Invitrogen . The small interfering RNA (siRNA) for SphK-2 was obtained from Ruibo Co., Ltd. . RealSuper Mixture (with ROX) was obtained from Cowin Biotech Co., Ltd. .orthophosphate (70 μCi/ml) at 37 °C for 4 h, and subsequently treated with PTX , JTE013 , VPC23019 , PD98059 , GF109203X , or their respective vehicle controls. Cells were then washed once with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium and stimulated with HDL.HUVECs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum, 100 U/ml of penicillin, and 100 mg/L of streptomycin at 37 °C, 5 % COCytoplasm and nuclei were isolated using a Nuclear and Cytoplasmic Protein Extraction Kit  according to the manufacturer’s instructions. Lipids were extracted and sphingolipids were determined by liquid chromatography coupled with electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) using LIPID MAPS™ Incinerator calendar provided by Onyalai Lipids . Internal standards were added in 0.5-fold quantities.32P]ATP at 37 °C for 30 min. Reactions were stopped by the addition of 500 μl of chloroform/methanol/concentrated HCl . After addition of 100 μl chloroform and 100 μl 2 M KCl for phase separation, samples were vortexed and centrifuged at 700×g for 5 min. Aliquots (100 μl) of organic phase containing labeled lipids were separated on TLC using a mobile phase of chloroform/acetone/methanol/acetic acid/water .Cells were lysed by repeated freeze and thaw cycles. SphK-2 activity in cell lysates was determined in the presence of 1 M KCl, under which SphK-2 exhibits optimal activity, while SphK-1 is strongly inhibited . In brie6 cells/well). At 30–50 % confluence, HUVECs were transfected with 50 or 100 nM of SphK-2 siRNA or scrambled control siRNA in OptiMEM  using Lipofectamine 2000 according to manufacturer’s protocol. Target sequences for human SphK-2 siRNA were 5-CAAGGCAGCUCUACACUCA-3 (sense) and 3-GUUCCGUCGAGAUGUGAGU-5 (antisense), and those for scrambled control siRNA were 5-CGGCCUACUUCUGCAUCUA-3 (sense) and 3-GCCGGAUGAAGACGUAGAU-5 (antisense). After 24 h, the medium was replaced with fresh serum-free DMEM. After another 48 h incubation, HUVECs were treated with 120 μg/ml of HDL for 8 h. COX-2 expression and PGI-2 release were determined.HUVECs were plated in six-well plates  for 8 h, washed twice with phosphate buffered saline (PBS), and incubated in PBS buffer containing 0.75 % fatty acid-free BSA and exogenous AA (10 μmol/L) at 37 °C, 5 % CO2 for 30 min in a humidified incubator [Nuclear and cytoplasmic proteins were extracted using cell lysis buffer  according to manufacturer’s instructions.Equal amounts of proteins were separated by 10 % sodium dodecyl sulfate-based polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to polyvinylidenedifluoride (PVDF) membranes. Membranes were incubated with COX-2, SphK-2, P-CREB, P-PKCα, PKCα, P-ERK1/2, ERK1/2, and GAPDH antibodies, respectively, washed, and incubated with anti-rabbit or anti-mouse secondary antibodies conjugated with horseradish peroxidase for detection. Immunopositive bands were visualized by enhanced chemiluminescence and quantified using a densitometer .COX-2 and GAPDH were obtained from Invitrogen  and had sequences as follows: COX-2: 5′-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3 (sense), 5′-AGA TCA TCT CTG CCT GAG TAT CTT-3′ (antisense); GAPDH: 5′-CAA GGT CAT CCA TGA CAA CTT TG-3′ (sense), 5′-GTC CAC CAC CCT GTT GCT GTA G-3′ (antisense). mRNA expression levels of COX-2 and GAPDH were quantified using SYBR Green Master Mix (with ROX) on a real-time PCR detection system . mRNA levels of COX-2 were normalized to GAPDH.Total RNA was isolated using TRIzol reagent  and reverse transcribed by random priming and Moloney Murine leukemia virus reverse transcriptase  according to manufacturer’s instructions. Primers specific for human 2, 1 % NP-40) containing 1X Halt Protease Inhibitor Cocktail  [HUVEC cells cultured for 48 h were harvested by scraping and lysed in 1 ml lysis buffer  . Lysate IL, USA) . PrecipiCOX-2 promoter region using 5′ primer −119TAA GGG GAG AGG AGG GAA AAA T−97 and 3′ primer +6ACA ATT GTC GCT AAC CGA G−14 [ChIP was conducted using a ChIP assay kit from Beyotime  with slight modifications. Immunoprecipitated DNA was purified and amplified across the CGA G−14 .t test or ANOVA analysis. Differences with P < 0.05 were considered statistically significant.Data are presented as mean ± SE. Statistical analysis was performed using Student’s Vascular endothelial cells play an important role in many pathological and physiological processes and are extensively studied in medical research. HUVECs resemble arterial endothelial cells and are widely used in studies of vascular endothelial cells, since they are more easily available than cells from many other vessels . Since sWe postulated that S1P receptors coupled to PTX-sensitive Gαi/Gαo cells with PTX. Our data showed that incubation of HUVECs with PTX, a specific inhibitor of Gαi/Gαo-protein, prior to HDL treatment markedly attenuated HDL-induced COX-2 expression  are expressed in endothelial cells, whereas S1PR1–4 are expressed in myocardial tissues . We founSphK-2 silencing by siRNA transfection. Western blot analysis showed that SphK-2 protein level was much lower in HUVECs transfected with SphK-2 specific siRNA than those transfected with control siRNA  for 20 min and then incubated with anti-SphK-2 antibody or anti-SphK-1 antibody coupled to agarose beads. Results from ChIP showed that HDL treatment significantly increased SphK-2-associated, but not SphK-1-associated COX-2 promoter activity  ligands and cytokines , 34. OurECs Fig. . These en by HDL , 35. We COX-2/PGI-2 activation, JTE013, an inhibitor of S1P2 did not show significant effect (Fig. It has been reported that HDL binds to and activates S1PR1–3, which are Gαi/αo-coupled GPCRs, leading to activation of intracellular signaling pathways and COX-2 expression . In thisect Fig. . These rCOX-2 expression and PGI-2 release in HUVECs (Fig. COX-2 transcriptional [COX-2 transcription in HUVECs via SphK-2/S1P activation in the nucleus. In addition, we found that specific inhibitors of Gαi/αo, ERK1/2, and PKC markedly reduced HDL-induced phosphorylation of SphK-2 (Fig. COX-2 expression and PGI-2 release in HUVECs.In the nucleus, S1P activates target gene expression by inhibiting histone deacetylases HDAC1 and HDAC2 . AlthougECs Fig. . FurtherECs Fig. . CREB, aiptional . HoweverK-2 Fig. , suggestIn summary, our results provide the first evidence that HDL induces COX-2 expression and PGI-2 release by SphK-2 phosphorylation and activation. The significance of SphK-2 in cardioprotective effects of HDL and in lipid metabolism needs to be further investigated.


Each Cu atom is in a distorted square-pyramidal coordination environment, with one O atom of an adipate dianion and three N atoms from the 2,2′:6′,2′′-terpyridine ligand occupying the basal plane, and one chlorine in the apical site. In addition, there is weak Cu—O interaction opposite of the chlorine with a distance of 2.768 (1) Å. The adipate ligand adopts a gauche–anti–gauche conformation. The interstitial water molecules form hydrogen-bonded tertramers that are connected to the complexes via O—H⋯O and O—H⋯Cl hydrogen bonds, thus leading to the formation of tightly hydrogen-bonded layers extending perpendicular to the b-axis direction.In the title compound, [Cu Cl2(C15H11N3)2]·4H2O = 0.025                           wR(F                           2) = 0.080                           S = 1.25                           4046 reflections245 parametersH-atom parameters constrainedmax = 0.48 e Å−3                        Δρmin = −0.51 e Å−3                        Δρ                                 RAPID-AUTO (Rigaku, 1998RAPID-AUTO; data reduction: CrystalStructure (Rigaku/MSC, 2002SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEPII (Johnson, 1976SHELXL97.Data collection: 10.1107/S1600536810027005/zl2287sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536810027005/zl2287Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Carbolines inhibited platelet aggregation induced by five agents with the potency being 3-amino-1,4-dimethyl-5H-pyridoindole > 3-amino-1-methyl-5H-pyridoindole > 1-methyl-9H-pyridoindole. The most potent 3-amino-1,4-dimethyl-5H-pyridoindole showed 50% aggregation-inhibitory concentrations of 6–172 μM. Both γ-carbolines interacted with phosphatidylcholine membranes to lower the lipid phase transition temperature with the potency correlating to the antiplatelet activity, suggesting that the interaction with platelet membranes to increase their fluidity underlies antiplatelet effects. Given their possible concentration and accumulation in platelets, γ- and β-carbolines would provide cigarette smokers and alcohol drinkers with reduced platelet aggregability, and they may be responsible for the occurrence of hemorrhagic diseases associated with heavy smoking and alcoholics.Cigarette smoking and alcohol consumption possibly affect platelet functions. To verify the hypothesis that some  Cigarette smoking has the possibility to affect the functions of platelets. Platelet aggregation stimulated by collagen is diminished in smokers compared with nonsmokers, suggesting that habitual smoking reduces platelet aggregability . SmokersCigarette smoke contains a variety of bioactive compounds . AlcoholCigarette smoking and alcohol drinking have been referred to as risk factors for intracerebral hemorrhage, subarachnoid hemorrhage, and hemorrhagic stroke –16. Thesα-, β-, and γ-carbolines, are contained in cigarette smoke and alcoholic beverages. Their levels in mainstream smoke condensates are 2.01–10.3 ng/cigarette for α-carboline, 0.25–2.53 μg/cigarette for β-carbolines, and 0.29–1.10 ng/cigarette for γ-carbolines indole (AMαC), 1-methyl-9H-pyridoindole (MβC), 3-amino-1,4-dimethyl-5H-pyridoindole (ADγC), and 3-amino-1-methyl-5H-pyridoindole (AMγC) , and AMγC  were purchased from Wako Pure Chemicals . Collagen , PAF , epinephrine , ADP  and thrombin  were used for inducing platelet aggregation. 1,2-Dipalmitoylphosphatidylcholine (DPPC) was obtained from Avanti Polar Lipids . Dimethyl sulfoxide (DMSO) of spectroscopic grade  and water of liquid chromatographic grade (Kishida) were used for preparing sample solutions. All other reagents were of the highest grade available.AMn = 6) of nonsmokers and nonalcoholics without taking any drugs at least for one month according to the previous method indole (γ-carboline) is important for inhibiting platelet aggregation, followed by the structure of 9H-pyridoindole (β-carboline). An additional methyl group in the pyrido moiety provides γ-carbolines with higher activity. Since ADγC and AMγC themselves do not aggregate platelets despite partly resembling serotonin in structure [γ-carbolines are referred to as potent antiplatelet compounds.The comparative studies have revealed that tructure , these γγ-carbolines showed relatively low concentrations to produce 50% inhibition of AUC of aggregation response. Since the antiplatelet effects of aspirin were recently reported by using the same method for platelet aggregation assay from our laboratory [γ-carbolines. Aspirin showed 116.1 ± 6.4 μM for collagen-induced, 71.3 ± 5.5 μM for epinephrine-induced first phase and 19.3 ± 3.6 μM for epinephrine-induced second phase response, although it did not inhibit PAF-, ADP-, and thrombin-induced responses by 50% at 500 μM. In this study, ADγC and AMγC have been found to show 50% AUC-inhibitory concentrations of 84.0 ± 7.1 and 179.5 ± 20.7 μM, 106.1 ± 8.5 and 153.6 ± 8.5 μM, and 21.5 ± 1.9 and 117.2 ± 11.0 μM for collagen-induced, epinephrine-induced first phase and epinephrine-induced second phase response, respectively. ADγC and AMγC are also effective in inhibiting AUC of aggregation responses induced by PAF, ADP and thrombin to show 50% inhibitory concentrations of 6.01 ± 0.58 and 32.0 ± 5.8 μM, 65.2 ± 2.4 and 115.5 ± 8.8 μM, and 105.8 ± 19.5 and 230.9 ± 25.8 μM, respectively. With respect to platelet aggregation inhibition, γ-carbolines, especially ADγC, are comparable to or more potent than aspirin.Antiplatelet boratory , its 50%α-, β-, and γ-carbolines are contained in mainstream cigarette smoke , and higher levels are found in sidestream cigarette smoke (≥3.0 μg/cigarette for MβC) [β-carbolines are also present in various alcoholic beverages at the concentrations of ≥3.2 μM for MβC [βC are higher in smokers (≥48 pM) and increase to ≥165 pM by smoking a cigarette [βC are ≥0.015 pmol/109 platelets for nonsmokers but ≥0.060 pmol/109 platelets for smokers, and MβC shows the increasing platelet concentrations to be ≥0.142 pmol/109 platelets 13 minutes after smoking [β-carbolines increase in blood rapidly following cigarette smoking and alcohol drinking, and their concentrations in platelets are much higher than in plasma, indicating their significant concentration and accumulation in platelets. The accurate γ-carboline concentrations in platelets of smokers and alcoholics have been unknown. In the dosing experiment using rabbits, however, ADγC was present in blood, especially in red blood cells, for a long time after oral dosing [γC, it has been frequently used as an antiplatelet drug. Considering that cigarette smoke- and alcoholic beverage-derived β- and γ-carbolines are concentrated and accumulated in platelets, the possibility for them to affect platelet aggregability is not necessarily excluded.A question arises as to whether carboline components actually affect the aggregability of platelets of cigarette smokers and alcohol drinkers. for MβC) , 25. β-c for MβC . Compare smoking . β-carbol dosing . AlthougγC and AMγC were inhibitory on platelet aggregation induced by all of these aggregants, and MβC by two aggregants. These results indicate that γ- and β-carbolines influence the step common to different aggregation agonists in addition to their specific action at the receptor and enzyme levels. The physicochemical property of biomembranes, such as fluidity, plays a crucial role in signal transduction and affects the activity of platelets. Membrane fluidity modulates platelet aggregability and membrane-fluidizing compounds attenuate collagen- and thrombin-induced platelet aggregation [Although the platelet aggregation mechanisms for collagen, PAF, epinephrine, ADP, and thrombin differ, ADregation . A changregation , 54.γ-carbolines, because they are naturally fluorescent with the maximal excitation and emission wavelengths almost similar to those of typical fluorescent probes. Therefore, turbidimetry was used for them. The changes in turbidity of liposome suspensions reflect the promotion of a gel to liquid crystalline transition of membrane phospholipids, indicating an increase of membrane fluidity [γC and AMγC were found to lower the phase transition temperature of membrane DPPC at 500 μM–1.5 mM with the potency correlating to their relative antiplatelet effects. Antiplatelet γ-carbolines appear to interact with lipid membranes and increase their fluidity at platelet aggregation-inhibitory concentrations.The membrane effects of toxins and drugs have been most frequently studied by measuring fluorescence polarization of liposomal and cellular membranes labeled with fluorescent probes –39, 54. fluidity , 48. Conβ- and γ-carbolines and their relating structures include the inhibition of aggregation-relevant enzymes and receptors, and the suppression of cytosolic calcium mobilization and arachidonic acid liberation. β- and γ-carbolines influence cyclooxygenase activity and arachidonic acid metabolism to reduce the production of prostaglandins and thromboxane [γ-carbolines (ADγC and AMγC) and β-carbolines (MβC) are the potent inhibitors of monoamine oxidase and serotonin uptake of platelets [β-carbolines like MβC inhibit phospholipase Cγ2 and protein tyrosine phosphorylation [The antiplatelet mechanisms previously reported for omboxane . Antiplalatelets , 50. β-crylation . Biologirylation , 39. Theγ- and β-carbolines. However, the influences of cigarette smoking and alcohol consumption on platelet aggregation have been conflicting, with some studies reporting a reduction of aggregability [γC and AMγC, rigid membranes (with decreased fluidity) show the enhanced platelet aggregability in response to epinephrine and thrombin [The present results suggest that platelet hypoaggregability might be induced in smokers and alcoholics by gability –7 but otgability –11. In cthrombin , 41. Thethrombin , 57. Varthrombin . Plateleγ- and β-carboline components in cigarette smoke and/or alcoholic beverages are pharmacotoxicologically relevant to these serious hemorrhagic diseases as well as antiplatelet agents by decreasing platelet aggregation responses to different aggregants.Reduced platelet aggregability is important in the development of intracranial hemorrhage , 28. Antγ- and β-carbolines are in line with several studies that cigarette smoking and alcohol drinking protect against coronary heart disease and also lower the risk of coronary artery disease and myocardial infarction [Another etiological role of cigarette smoking and alcohol consumption has been indicated in cardiovascular events , 63 althfarction . Howeverfarction , 64. Chrfarction , by whicγ- and β-carbolines may be responsible for the occurrence of hemorrhagic diseases associated with heavy smoking and alcoholics by inhibiting platelet aggregation.In conclusion, the reduced platelet aggregability found in cigarette smokers and alcoholic drinkers would be attributable to antiplatelet carboline components in cigarette smoke and alcoholic beverages.


The structure is the first example of β-diketonate in a dianionic κ2               C-coordination complex containing a PdII—PdII bond. Both PdII atoms adopt a pseudo square-planar coordination geometry. The molecular packing involves π-interactions between the phenyl rings of the 1,3-diphenylpropane-1,3-dionato ligands with centroid–centroid distances in the range 3.823 (2)–3.868 (2) Å.The title compound, [Pd DOI: 10.1107/S1600536811017144/ff2010Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: interactive version of Fig. 1            Enhanced figure:


To evaluate the expression and differential significance of c-Jun, p73, Casp-9 and N-ras in thymic epithelial tumors (TETs) with the aim to provide useful information for tumor biology and prospective therapy.In this study, we analyzed the expression of four chromosome 1-related genes, namely c-Jun, p73, Casp-9 and N-ras, in 60 cases of thymic epithelial tumors. The tumors included 52 thymomas and 8 thymic carcinomas which were categorized according to the current WHO classification systems.Compared with the normal thymus tissue, all thymic epithelial tumors demonstrated higher expression of c-Jun and p73. The expression of c-Jun and p73 in type B2, B3 thymoma and thymic carcinomas was similar, and significantly higher than that in all other subtypes of thymomas. Unlike type A thymoma, the expression of Casp-9 was relatively lower in type B thymoma and thymic carcinomas. With respect to the clinical staging systems, c-Jun was more expressed in progressive tumors harboring higher stages. In contrast to c-Jun, p73 and Casp-9, there was no significant aberration with N-ras expression irrespective of either tissue or tumor types.The overexpression of c-Jun, p73 and Casp-9 in thymic epithelial tumors is closely related with the pathogenesis and biological behavior of the neoplasms. These candidate biomarkers provided useful information for prospective personalized therapy in the clinical management.背景:评估c-Jun, p73, Casp-9 和 N-ras在胸腺上皮性肿瘤诊断和鉴别诊断中的运用.方法:根据世界卫生组织最新的诊断标准60例胸腺上皮性肿瘤分类,运用Envision法检测c-Jun,p73,Casp-9 和N-ras在不同亚型肿瘤中的表达情况,并结合临床病理学特征进行分析.结果:c-Jun和p73在肿瘤中的表达明显高于正常胸腺组织;c-Jun和p73在B3,B2型胸腺瘤和胸腺癌的表达类似,且表达明显高于其他类型的胸腺肿瘤;Caspase-9在B型胸腺瘤和胸腺癌中的表达相对低于A型胸腺瘤;c-Jun的表达更常见于高级别的胸腺肿瘤.结论:c-Jun,p73和Casp-9在胸腺肿瘤中的表达很好地反映了肿瘤的生物学特点,为胸腺肿瘤的诊断和鉴别诊断提供了较好的理论基础.http://www.diagnosticpathology.diagnomx.eu/vs/1521774814749726 Thymic epithelial tumors (TETs) are a panel of rare neoplasms, located in anterior mediastinum, accounting for approximately 0.2-1.5% of all human malignancies -5. Basedet al.[c-Jun (1p32-31), p73 (1p36.3), Casp-9 (1p36.21), and N-ras (1p13.2) were such kind of genes, which might involved in the process of origination, proliferation, differentiation, and apoptosis of the malignant cell [et al. indicated that the behavior of primary thymic neuroendocrine carcinomas seems correlated with tumor differentiation [et al. declared that autoimmune related disorders of thymoma contained with a significant population of CD20+ intratumoral B lymphocytes, and strong CD57 expresssion in thymomas may indicated with a concomitant neuromuscular disorder [Zettl et al. declaredet al.,12. Evid N-ras 1p.2 were sant cell . Howeverntiation . Alexievdisorder . BesidesTo our knowledge, the combination of expression of c-Jun, p73, Casp-9, and N-ras was firstly evaluated in different subtypes of thymic epithelium tumors and normal thymus tissue. By trying to investigate the expression characteristics of those antibodies, we aim to build an efficient panel of biomarkers for clinical differential diagnosing between subtypes of TETs.TETs cases included were formaldehyde-fixed, paraffin-embedded (FFPE) archival samples from the Department of Pathology in First Affiliated Hospital of Xinjiang Medical University between January 2001 and February 2010. All of the archival slides were reviewed by two independent senior pathologists (YQM and QXL) according to the latest WHO criteria, and any discrepancy between these two investigators was resolved with a third reviewer (WZ) in order to reach an ultimate decision on all of the items. Finally, 60 cases were recruited based on the following criteria: 1) pathologically confirmed TETs (thymoma and thymic carcinoma), 2) integrated clinicopathological information, 3) without any chemotherapy/radiotherapy performed prior to recruitment. Among those, including 26 male and 34 female (1:1.3) with an average age of 48.5 years (range 25–73 years). In addition, 11 normal biopsy thymic tissues were used as normal control . Informed consent was obtained from all of the case and control subjects. All specimens were handled and approved by the hospital’s ethics committee.http://www.cap.org > cancer protocols > thorax > thymoma and thymic carcinoma). Briefly, information of specimen integrity, histologic subtypes, regional lymph nodes, tumor extension, and procedure treatment were obtained from the surgical document, if any. Pathologic staging for thymomas according to Modified Masaoka Stage system; and staging for thymic carcinomas according to pTNM system [Information of the total TETs was extracted based on the criteria from the CAP website data ; p73 ; Casp-9  and N-ras .Experimental procedures were performed as previously described . BrieflyImmunoreactivity was assessed by two senior pathologist (YQM and XL) who were blinded to clinicopathologic data, and any disagreements were resolved with a third reviewer (WZ) using a multi-headed microscope. Scoring of immunohistochemistry was based on two parameters: intensity of immunoreactivity and the exact location of immunoreaction. The immunostaining intensity was scored using the following semi-quantitative scale: 1) -, no reactivity (no staining or weak staining less than 5% of the target cells), 2) +, cases presented specific staining of more than 5% of the target cells, regardless of staining intensity, were scored as positive for c-Jun, p73, Casp-9 or N-ras.P-values were two-sided, P-values less than 0.05 were considered to be statistically significant, less than 0.01 meant highly significant.Statistical analysis was performed using SPSS version 13.0. Group comparisons of categorical variables were evaluated using the Fisher’s exact or Pearson’s chi-square test. All 2N0M0, four T3N0M0, two T4N0M0 and one T4N1M0 based on the pTNM system; however, in order to obtained a powerful statistic results, we transform pTNM of thymic carcinoma into Mosaoka tumor stages based on comparison of Masaoka tumour stages and corresponding TNM classification [Among the total 60 TETs, most of which involved anterior mediastinum (58/60), only one of these TETs involved superior mediastinum and right mid-lower mediastinum, respectively. The maximum diameters of the samples in our study ranged from 1.8 to 14.0 cm , among those, two type A, 19 type AB, four B1, 14 B2, 11 B3, two metaplastic thymoma, and eight thymic carcinoma were observed. 21 TETs cases employed an uninvolved-margin, as well as 39 margins involved by tumor. Concerning to the staging information, among 52 thymomas, there were 21 stage I, 19 stage II, 11 stage III, and one stage IV based on the Modified Masaoka Stage system; among eight thymic carcinomas, there were one TIn order to evaluate the diagnostic significance of c-Jun, p73, Casp-9, and N-ras expression in the distinction of TETs, we detected these markers with immunohistochemistry. The results of immunohistochemistry were seen in Figure P < 0.05, Table P < 0.05), either. Thymic carcinoma , Type B3 and Type B2 thymoma ranked the first higher expression rate of c-Jun; they were 87.5% (7/8), 45.5% (5/11), and 42.9% (6/14), respectively. However, immunoreactions were not seen in Type A, B1, and metaplastic thymoma (Table P < 0.05), c-Jun expression were definitely higher in high stage (III + IV) when compared with the low stage (I + II) TETs (P < 0.01). However, no statistical discrepancy was observed in stage I vs. II, as well as stage III vs. IV, respectively (P > 0.05)  was observed, as showed in Table P > 0.05, Table Caspase-9 expression: Similar Caspase-9 expression was observed both in thymic epithelium tumors and normal tissue, no statistics difference between them was observed (P < 0.05), as well as compared among subtypes (P < 0.05). The respective expression of p73 in type B3, type B2 and type B1 were 72.2% (8/11), 64.3% (9/14), and 0% (0/4), as showed in Table P < 0.05). Meanwhile, there was no significant difference of p73 expression between type B3, B2 thymoma and thymic carcinoma . p73 positive expression in different clinical stages of thymoma was 38.1% (Stage I), 55.0% (Stage II), 53.3% (Stage III), and 75.0% (Stage IV), respectively. No statically significant among different clinical stages of p73 expression was observed   Table , similarP-values were 0.002, 0.019, and 0.004, respectively. No statistically significant discrepancy was found between the expression of those antibodies and some other clinicopathological characteristics.We also evaluated the expression distribution of those four antibodies between low-grade TETs  and high-grade TETs , and found that c-Jun, Caspase-9, and p73 expression was statistically significant with high-grade TETs, the Thymomas were neoplasms arising from or exhibiting differentiation toward thymic epithelial cells. It has been reported that different subtypes of thymoma have different genetic characteristics, recent studies indicated that chromosomal 1 gain plays an important role in molecular genetic mechanism of thymic epithelium tumors ,23-25.C-Jun (cellular Jun), a member of nucleus transcription factor, is an oncogene locating on chromosome 1. It was indicated that the expression of c-Jun immunohistochemistry can reveal the mRNA level of c-Jun . In thisN-ras is one of the ras gene family members locating on chromosome 1. N-ras function as an important factor in the process of cell proliferation, senescence, immortalization and carcinogenesis. N-ras can also inhibit the cancer cells proliferation by Suv39h1 and H3K9 methylation. Mutational ras protein can affect cell proliferation, cell cycle regulation and anti-apoptotic signal by decreasing the activity of endogenous GTPase, or transcriptional decreasing the expression of Fas receptor and regulating the last time of the p38 activity of Jun N-terminal protein kinase (JNK) by Ral-GEF (Ras related GTPase-guanine exchange factor) pathway  MeanwhilCaspase-9 gene locates on 1p36.3-p36.1. It precipitates in mitochondria induced cell senescence pathway . Severalp73 gene locates on human chromosome 1p36.33. Many isomers of p73 were identified, and the expression and interaction of those different isomers involved the process of regulate transcription and growth inhibition ,36. The In summary, the results indicated that c-Jun and p73 expressed significantly higher in thymic epithelium tumors than in normal control tissues. c-Jun and p73 also had similar positive expression level in high-grade TETs, which is significantly higher than low-grade TETs. In addition, Caspase-9 expression was relative lower in type B thymoma and thymic carcinoma. However, no significant difference of N-ras expression among different tissues of the thymus and different thymic epithelium tumors was observed. What we observed suggested that different genes on chromosome 1 might employ different functions in the generation and development of thymic epithelium tumors. c-Jun and p73 may promote the tumor formation. Previous studies of our group also suggested that chromosome 1 gain was significantly higher in thymic epithelium tumors than normal thymus tissue, and it was also higher in type B3 thymic epithelium tumors than other subtypes of thymoma . It is hTETs: Thymic epithelial tumors; WHO: World health organization; FFPE: Formaldehyde-fixed, paraffin-embedded.The authors declare that they have no competing interests.Ma Y. participated in the design of the study and given final approval of the version to be published. Li Q. participated in its design and coordination and helped to draft the manuscript. Cui W. performed the statistical analysis. Miao N. carried out the immunohistochemistry method. Liu X. was response for the quality of the immunohistochemistry result. Zhang W. was response for the quality of the study. Zhang C. helped to draft the manuscript. Wang J. participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.This study was supported by the grant from “Project of the Foundation and Application of the Comprehensive Pathologic Diagnostic Platform” Recruitment at technology program of Xinjiang Uygur Autonomous Region (201233142).


Each of the four NiII atoms occupies every other corner of a cube, with the alternate corners occupied by μ3-methanolate bridging groups linking to three NiII atoms. Each NiII atom is in an O6 octahedral coordination environment formed by three O atoms from three μ3-methanolate groups, one from methanol, and two others from a bidentate 2-formyl-6-methoxyphenolate ligand. The Ni—O bond distances range from 2.0020 (14) to 2.0938 (14) Å, the cis bond angles range from 81.74 (6) to 97.63°, and the trans bond angles range from 168.76 (5) to 175.22 (6)°. There are bifurcated hydrogen-bonding interactions between the coordinated methanol OH groups and both the phenolic and methoxy O atoms of an adjoining 2-formyl-6-methoxyphenolate moiety. In addition, there are weak intermolecular C—H⋯O interactions involving the methoxy O atoms.The molecule of the title compound, [Ni DOI: 10.1107/S1600536810043497/bt5390Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Background: Endocrine-disrupting chemicals (EDCs) influence the activity of estrogen receptors (ERs) and alter the function of the endocrine system. However, the diversity of EDC effects and mechanisms of action are poorly understood.Objectives: We examined the agonistic activity of EDCs through ERα and ERβ. We also investigated the effects of EDCs on ER-mediated target genes.Methods: HepG2 and HeLa cells were used to determine the agonistic activity of EDCs on ERα and ERβ via the luciferase reporter assay. Ishikawa cells stably expressing ERα were used to determine changes in endogenous ER target gene expression by EDCs.p-hydroxyphenyl)-1,1,1-trichloroethane (group 1) strongly activated ERα estrogen responsive element (ERE)-mediated responses. Daidzein, genistein, kaempferol, and coumestrol (group 2) activated both ERα and ERβ ERE-mediated activities. Endosulfan and kepone (group 3) weakly activated ERα. Only a few EDCs significantly activated the “tethered” mechanism via ERα or ERβ. Results of real-time polymerase chain reaction indicated that bisphenol A and bisphenol AF consistently activated endogenous ER target genes, but the activities of other EDCs on changes of ER target gene expression were compound specific.Results: Twelve EDCs were categorized into three groups on the basis of product class and similarity of chemical structure. As shown by luciferase reporter analysis, the EDCs act as ER agonists in a cell type– and promoter-specific manner. Bisphenol A, bisphenol AF, and 2-2-bis tended to have comparable ERα and ERβ ERE-mediated activities, similar chemical structure did not correlate with previously reported ligand binding affinities of the EDCs. Using ERα-stable cells, we observed that EDCs differentially induced activity of endogenous ER target genes. These effects, inhibited by appropriate antagonists, indicate that steroids and growth factor–induced up-regulation of WISP-2 may be mediated through ERs  was identified as a key target of estrogens in ER-positive breast and ovarian cells (Estrogens regulate a large number of target genes through the ER. et genes . GREB1 (ve genes . Recentlve genes . WISP2 (ough ERs . SDF-1 (an cells . The corp-Hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the pesticide methoxychlor, has estrogenic effects similar to that of BPA (n-Nonylphenol (4n-NP) is a resistant alkylphenol that is degraded from alkylphenol ethoxylates and is generally present in food  have been identified as estrogenic EDCs. Daidzein (Dai) is a soy-derived isoflavone that originates from plants and herbs . GenisteIn vitro studies have shown that Kep has ligand binding affinity to ERα , kepone (Kep), and 1-bromopropane (1-BP). Endo is a fluorinated organic insecticide, and animal studies have indicated that it affects the male reproductive system . Kep, aly to ERα . 1-BP, cy to ERα .In the present study, we used two ER-negative cell lines, HepG2 and HeLa, to analyze the effects of 12 estrogenic EDCs—which were grouped based on chemical structure and product class—on the estrogenic ERE- and AP-1/Sp1–mediated responses of ERα and ERβ. Using Ishikawa cells that stably express ERα, we evaluated changes in endogenous ER target gene expression after EDC treatment.Chemicals. 17β-Estradiol (E2) was purchased from Sigma-Aldrich , and ICI 182,780 (ICI) was obtained from Tocris Bioscience . The 12 EDCs used in this study were provided by the Midwest Research Institute  via a contract with the National Toxicology Program. The chemical names, Chemical Abstracts Services Registry Numbers, and the sources are summarized in Supplemental Material, Table S1 (http://dx.doi.org/10.1289/ehp.1205951).EDC groups. The 12 EDCs were categorized into three groups based on their chemical and product classes , pRL-TK vector plasmid from Invitrogen , and 7×AP-1 Luc from Stratagene . pcDNA/mouse wild-type (WT)-ERα (pcDNA/ERα) and pcDNA/ΔNmERβ310G (former pcDNA/mouse WT-ERβ) have been described previously (http://dx.doi.org/10.1289/ehp.1205951). The luciferase reporters 3×ERE (modified reporter) and pS2ERE (endogenous pS2 gene reporter) have been described previously  were purchased from ATCC . The human endometrial adenocarcinoma stable cell lines Ishikawa/vector (Ishikawa/vec) and Ishikawa/WT ERα (Ishikawa/ERα) have been described previously (l-glutamine (Invitrogen). HeLa cells were maintained in phenol red–free Dulbecco’s modified Eagle medium  supplemented with 10% FBS and 4 mM l-glutamine. The stable cell lines Ishikawa/vec and Ishikawa/ERα were maintained in phenol red–free DMEM:F12 medium (Invitrogen) supplemented with 10% FBS and geneticin . For serum-starved conditions, 10% HyClone charcoal/dextran-stripped FBS  was substituted for FBS in the medium (starve medium).eviously . HepG2 cTransient transfection and luciferase assay. HepG2 and HeLa cells were seeded in 24-well plates with starve medium overnight. A total of 0.5 μg of DNA, including 0.2 μg of expression plasmid, 0.2 μg of reporter plasmid, and 0.1 μg of pRL-TK plasmid, were transfected overnight using Effectene transfection reagent  according to the manufacturer’s protocol. E2, ICI, and EDCs were dissolved in 100% ethanol (EtOH) before being diluted in media. The final EtOH concentration was 0.01%. The cells were changed to fresh starve medium; after 8 hr, cells were treated with EtOH vehicle (control), 10 nM E2, 100 nM ICI, or EDCs for 18 hr. For experiments with pRSV/c-Jun on 7×AP-1 Luc, cells were transfected with a total of 0.7 μg of DNA, including 0.2 μg ERα or ERβ, 0.2 μg pRSV/c-Jun, 0.2 μg 7×AP-1 Luc, and 0.1 μg pRL-TK plasmids. Luciferase assays were performed using the Dual Luciferase Reporter Activity System . Transfection efficiency was normalized by renilla luciferase using pRL-TK plasmid. All experiments were repeated at least three times. Data represent mean fold change  relative to the control.RNA extraction and real-time polymerase chain reaction (PCR). Ishikawa/vec and Ishikawa/ERα cells were cultured in starve medium for 2 days and then treated with 10 nM E2, 100 nM EDCs, or EtOH vehicle (control) for 18 hr. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). First-strand cDNA synthesis was performed using Superscript reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. The mRNA levels of ER target genes were measured using SYBR green assays . The Genbank accession numbers (http://www.ncbi.nlm.nih.gov/genbank/) and sequences of primers used for real-time PCR were as follows: human PR (NM_000926.4): forward 5´-GACGTGGAGGGCGCATAT-3´, reverse 5´-GCAGTCCGCTGTCCTTTTCT-3´; human pS2/TFF1 (NM_003225.2): forward 5´-GCCCTCCCAGTCTGCAAATA-3´, reverse 5´-CTGGAGGGACGTCGATGGTA-3´; human GREB1 (NM_014668): forward 5´-CAAAGAATAACCTGTTGGCCC-3´, reverse 5´-GACATGCCTGCGCTCTCATAC-3´; human SPUVE (NM_007173): forward 5´-ATGCCCGAGCAGATGAAATT-3´, reverse 5´-CCAACCCTTGGGCACATG-3´; human WISP2 (NM_003881): forward 5´-TGAGCGGCACACCGAAGAC-3´, reverse 5´ACAGCCATCCAGCACCAG-3´; human SDF-1 (NM_000609): forward 5´-GTGGTCGTGCTGGTCCTC-3´, reverse 5´-GATGCTTGACGTTGGCTCTG-3´. Cycle threshold (Ct) values were obtained using the ABI PRISM 7900 Sequence Detection System and analysis software . Each sample was normalized to its β-actin transcript content: forward 5´-GACAGGATGCAGAAGGAGATCAC-3´, reverse 5´-GCTTCATACTCCAGCAGG-3´. The experiments were repeated three times, and results are presented as the mean fold change  relative to control (vehicle-treated) Ishikawa/vec cells.Statistical analysis. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test and two-way ANOVA with Bonferroni posttests were performed using GraphPad Prism, version 6.00 .ERE-mediated estrogenic activation of ERα and ERβ by EDCs. To evaluate the ERE-mediated transcriptional activity of ERα and ERβ, we examined promoter activation in two ER-negative cell lines, HepG2 and HeLa. The two luciferase reporters, 3×ERE (modified reporter) and pS2ERE (endogenous pS2 gene reporter) (2 (data not shown). Because we observed estrogenic effects of BPA and BPAF at 100 nM concentrations in cells with WT-ERα (eporter)  were useh WT-ERα , we used2, with up to 50-fold increases in 3×ERE-mediated transactivation  showed weak activity via ERα , left anThe effects of EDCs on expression of ER target genes. To characterize the ER-dependent response of EDCs, we examined their effects on ERα target genes  using real-time PCR in Ishikawa/ERα stable cells  had a mutation of 310 glutamic acid (E) to glycine (G). Using this mutated ERβ plasmid, we found that BPAF (group 1 EDC) and Kaem (group 2 EDC) lost the majority of ERE-mediated activity in HepG2 cells relative to full-length ERβ . Additionally, Endo and Kep (group 3) exhibited weak activation of ERα in a cell type–specific manner (only in HeLa cells), suggesting that cell type–specific factors are involved in regulating ER ERE-mediated activity.Many EDCs adversely affect estrogen signaling by interacting with two ERs: ERα and ERβ. We are interested in defining the roles of ERs in mediating cellular and physiological responses to EDCs based on the similarities in chemical structure. One of the most significant findings of our study is that the structural similarities of the EDCs correlate with their estrogenic activity for ERs. The 3×ERE Luc reporter contains a series of three 13-base-pair inverted repeats , whereas pS2ERE Luc, derived from the human pS2 gene promoter, contains an imperfect ERE sequence (GGTCAnnnTGGCC) and several AP-1 sites . Using t for ERα . The grog to ERβ . These r2’s ability to affect gene expression through the nonclassical “tethered” mechanism, which involves ER modulating the activity of other transcription factors such as AP-1 and Sp1. EDCs activate the nonclassical “tethered” ER mechanism (AP-1/Sp1–mediated ER activation) in a manner not correlative to chemical structure similarity There is growing literature supporting E2 2 hr after treatment  PDFClick here for additional data file.


The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 3 times higher than the rate constant for dehydration from L-β-Asp peptide to L-succinimidyl peptide. The rate constant for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide was about 5 times higher than the rate constant for hydrolysis from L-succinimidyl peptide to L-α-Asp peptide. The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 2 times higher than the rate constant for dehydration from D-α-Asp peptide to D-succinimidyl peptide. The rate constants for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide and for hydrolysis from D-succinimidyl peptide to D-β-Asp peptide were almost equal. Using these rate constants, we calculated the change in the abundance ratios of the 4 Asp isomers during a human lifespan. This result is consistent with the fact that isomerized Asp residues accumulate in proteins during the ageing process.Although proteins consist exclusively of L-amino acids, we have reported that aspartyl (Asp) 58 and Asp 151 residues of αA-crystallin of eye lenses from elderly cataract donors are highly inverted and isomerized to D-β, D-α and L-β-Asp residues through succinimide intermediates. Of these Asp isomers, large amounts of D-β- and L-β-isomers are present but the amount of D-α-isomer is not significant. The difference in abundance of the Asp isomers in the protein may be due to the rate constants for the formation of the isomers. However, the kinetics have not been well defined. Therefore, in this study, we synthesized a peptide corresponding to human αA-crystallin residues 55 to 65 (T Proteins consist exclusively of L-amino acids in living tissues. However, D-aspartyl (Asp) residues have been found in various proteins of lensvia a succinimide intermediate as follows: (i) When the carbonyl group of the side chain of the L-α-Asp residue is attacked by the nitrogen of the amino acid residue following the Asp residue, L-succinimide is formed by intramolecular cyclization; (ii) L-succinimide may be converted to D-succinimide through an intermediate that has the prochiral α-carbon in the plane of the ring; (iii) The D- and L-succinimide are hydrolyzed at either side of their two carbonyl groups, yielding both α- and β-Asp residues, respectively. Thus, 4 isomers, L-α-Asp, L-β-Asp, D-α-Asp and D-β-Asp, are simultaneously formed in the protein  55VLD58SGISEVR65) and its isomers in which L-α-Asp at position 58 was replaced with L-β-, D-β- and D-α-Asp by Fmoc (9-fluorenylmethoxycarbonyl) solid-phase chemistry using an automated solid-phase peptide synthesizer. These peptides were designated T6-Lα, T6-Lβ, T6-Dα and T6-Dβ  peptides were obtained by heating T6-Lα and T6-Dα peptides at 90°C for 6 h in water The purified T6-suc-peptide eluted between T6-Lα and T6-Dα as shown in T6-L-suc and T6-D-suc peptides were incubated at 37°C under physiological conditions. Therefore, the derivative of the fitted curve is%/dt, therefore, d[L-α-Asp]%/dt can be expressed as follows:−1, because the units of t are min.On the other hand, F1′(t) is expressed as F1′(t) = d[L-α-Asp]%, −k1[L-α-Asp]%which was almost equal to 0. The experimental value of [L-succinimide]% was 92.415% at t = 0 min.At t = 0 min, as compared with k2[L-succinimide]−1 (4.27 day−1).Thereforek4, k6 and k8 were determined by the same method using the fitted curves in −1, because the units of t are min.Therefore, the derivative of the fitted curve is% was almost equal to 0%. The experimental value of [L-succinimide]% was 92.415% at t = 0 min. Therefore−1 (0.529 day−1). k10 was determined by the same method ) in −1, because the units of t are day in this section.Therefore, the derivative of the fitted curve is% was almost equal to 0%. [L-α-Asp]% was 100% at t = 0 day. Therefore−1. The rate constants, k3, k5 and k7 at 70°C were determined by the same method.At t = 0 day, [L-succinimide]In addition, the rate constants, k1, k3, k5 and k7 at 50°C–90°C were calculated using the same method. The temperature dependence of these rate constants is shown in an Arrhenius plot . Figures% = 100%, [L-β-Asp]% = 0%, [L-succinimide]% = 0%, [D-α-Asp]% = 0%, [D-β-Asp]% = 0%, [D-succinimide]% = 0%. Using the values of k1–k10 , we calc55VLD58SGISEVR65), and to discuss the stability or instability of the Asp isomers in the protein. The T6 peptide and its isomers in which L-α-Asp was replaced with L-β-, D-β- and D-α-Asp at position 58, that is, T6-Lα, T6-Lβ, T6-Dα and T6-Dβ peptides were synthesized. TheT6-Lα, T6-Lβ, T6-Dα and T6-Dβ peptides were clearly separated into 4 peaks by RP-HPLC chromatography as shown in Our previous studies have indicated that specific Asp residues in αA-crystallin (Asp 58 and Asp 151) The succinimidyl peptides (T6-L-suc and T6-D-suc) are not stable, therefore, the succinimidyl peptides hydrolyze to α-Asp and β-Asp containing peptides rapidly. As shown in The rate constants  for the racemization of the succinimide were calculated by heating of T6-L-suc and T6-D-suc peptides at 37°C . The racIn order to determine the rate constants  for succinimide formation in the peptides, we subjected T6-Lα, -Lβ, -Dα and -Dβ peptides to heating experiments at 50–90°C for 10 days. Subsequently, we estimated the rate constants k1, k3, k5 and k7 for succinimide formation in peptides at 37°C using Arrhenius plots.The rate constants, k1–k10 in in vivo, because we might be underestimating the rate constants for dehydration from β-Asp to succinimide (k3 and k7). Such an underestimation could be due to the rapid rate of hydrolysis from succinimide to β-Asp. Therefore the actual values of k3 and k7 might be near the values of k1 and k5. In fact, Geiger and Clarke −3day−1and k7 = k5 = 1.20×10−3day−1. The lens is a metabolically inactive tissue and therefore significant amounts of abnormal Asp isomers accumulate in lens. Our recent study showed that the ratios of L-α-:L-β: D-β-:D-α-isomers of Asp 58 in αA-crystallin from84-year-old human lens to be about 32%: 23% : 35% :10% by LC-MS analysis The accumulation of abnormal Asp isomers in proteins induces age-related diseases. Our previous study clearly showed thatα-crystallin containing large amounts of abnormal isomers undergoes abnormal aggregation to form massive and heterogeneous aggregates, leading to loss of its chaperone activity Synthesis and purification of the T6 peptide and its isomeric peptides containing the four different Asp isomers .55VLD58SGISEVR65) and its diastereoisomers in which L-α-Asp was replaced with L-β-, D-β- and D-α-Asp at position 58 were synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid-phase chemistry using an automated solid-phase peptide synthesizer . Fmoc-L-Asp(OtBu)-OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-L-Asp-OtBu and Fmoc-D-Asp-OtBu were used as building blocks to synthesize L-α-, D-α-, L-β- and D-β-isomers, respectively. The coupling reaction was carried out using each Fmoc amino acid (10 equiv), PyBOP (10 equiv), 1-hydroxybenzotriazole (HOBt) (10 equiv) and N-methylmorpholine (7.5 equiv) in dimethylformamide (DMF). The N-terminal Fmoc group was de-blocked with 20% piperidine in DMF. Simultaneous cleavage of the peptide from the resin and removal of the protective groups was achieved by treatment with a cocktail containing 90% TFA, 5% 1,2-ethanedithiol and 5% thioanisole.The T6 peptide corresponding to human αA-crystallin sequence from residues 55 to 65  with a linear gradient of 0–50% acetonitrile in the presence of 0.1% TFA, at a flow rate of 3.0 mL/min, with monitoring at 215 nm. The purity of each peptide was confirmed to be >95% by analytical RP-HPLC and TOF-MS analysis.In order to prepare the T6 peptide containing L- or D-succinimide in which L-α-Asp or D-α-Asp were replaced with succinimide at position 58, the T6-Lα- orT6-Dα-peptides were incubated at 90°C for 6 h in water. T6 peptides containing L- and D-succinimides were obtained from the heating.55−Val-Leu- L-suc58-Ser-Gly-Ile-Ser-Glu-Val-Arg65T6-L-suc: Thr55−Val-Leu-D-suc58-Ser-Gly-Ile-Ser-Glu-Val-Arg65T6-D-suc: ThrThe T6-L-suc and T6-D-suc peptides were purified by RP-HPLC using a C18 column  with a linear gradient of 15–35% acetonitrile in the presence of 0.1% TFA, at a flow rate of 3.0 mL/min, with monitoring at 215 nm. The purity of each peptide was confirmed to be >90% by analytical RP-HPLC and TOF-MS analysis.2; Shimadzu, Kyoto, Japan). The MALDI-TOFMS spectrometer operated with a 337 nm nitrogen laser and an ion acceleration voltage of 20 kV. Data were collected in positive ion reflection mode. 1 µL of 20 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) in acetone was applied onto a stainless steel MALDI plate and allow to air dry before addition of 1 µL of the peptide solution.All spectra were obtained using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOFMS)  and were incubated at 50, 60, 70, 80, and 90°C for 0.4–31 days.%, [L-β-Asp]%, [D-α-Asp]%, and [D-β-Asp]% are relative amounts (%) of L-α-Asp, L-β-Asp, D-α-Asp and D-β-Asp, and [L-α-Asp], [L-β-Asp], [D-α-Asp] and [D-β-Asp] represent the concentration of L-α-Asp, L-β-Asp, D-α-Asp and D-β-Asp respectively, which are calculated from the peak area in the RP-HPLC chromatogram. We plotted the relative amounts against incubation time. Subsequently, we drew fitted curves for this plot as follows:f1(t) for L-α-Asp, f2(t) for L-β-Asp, f3(t) for D-α-Asp, f4(t) for D-β-Asp.After the incubation of the 4 isomeric peptides, these peptides were subjected to HPLC. Since the four isomeric T6 peptides are clearly separated by RP-HPLC chromatography, we can measure the relative amounts of the peptides produced by the incubation from the peak areas of the 4 isomeric peptides in the chromatogram. The results are as follows:% and [D-succinimide]% represent the relative amounts of L-succinimide and D-succinimide as a percentage, t is time (day), k1 is the rate constant for the conversion of L-α-Asp to L-succinimide (day−1), k2 is the rate constant for the conversionof L-succinimide to L-α-Asp (day−1), k3 is the rate constant for the conversion of L-β-Asp to L-succinimide (day−1), k4 is the rate constant for the conversion of L-succinimide to L-β-Asp (day−1), k5 is the rate constant for the conversion of D-α-Asp to D-succinimide (day−1), k6 is the rate constant for the conversion of D-succinimide to D-α-Asp (day−1), k7 is the rate constant for the conversion of D-β-Asp to D-succinimide (day−1), k8 is the rate constant for the conversion of D-succinimide to D-β-Asp (day−1), and f1’(t), f2’(t), f3’(t) and f4’(t) are derivatives of f1(t), f2(t), f3(t) and f4(t). At t = 0 day, [L-α-Asp]% and [L-succinimide]% are 100% and 0% respectively, therefore, Eq. (5-1) can be expressed as:The Asp residues invert to isomerize through succinimide intermediates in peptide as shown in −3 kcal/Kmol), A is the frequency constant (day−1), and T is the absolute temperature (K). From Eq. (7), we calculated k1, k3, k5 and k7 at 37°C using k1, k3, k5 and k7 from five temperatures .We determined k1, k3, k5 and k7 at five temperatures  by using Eq. (6-1), (6-2), (6-3), (6-4). The Arrhenius equation shown below gives the activation energy of the degradation reaction.To determine the rate constant for the conversion from the succinimide intermediate to the peptides containing Asp isomers and the racemization rate of succinimide, samples were dissolved in PBS (pH7.4) and incubated at 37°C.After incubation, the T6-L-suc and T6-D-sucpeptideswere subjected to RP-HPLC. We measured the amounts of theT6-Lα, T6-Lβ, T6-Dα and T6-Dβ, T6-L-suc and T6-D-suc peptides from the peaks areas in the RP-HPLC chromatogram. The results are as follows:−1 in this section, F1’(t), F2’(t), F3’(t) and F4’(t) are derivatives of F1(t), F2(t), F3(t) and F4(t). Because the dehydration rate of isomers is 1000–10000 times slower than the hydrolysis rate of succinimide, Eq. (9–1), (9–2), (9–3) and (9–4) can be expressed as follows at t = 0:0 and [D-succinimide]0 are relative amounts (%) of L- succinimide and D-succinimide at t = 0 min. We determined k2, k4, k6 and k8 at 37°C by using Eq. (10–1), (10–2), (10–3) and (10–4). Then the units of k2, k4, k6 and k8 (min−1) was converted to day−1.The change of percentage content of isomers can be expressed as follows:% is relative amounts of D-Asp (%), [L-Asp]% is relative amounts of L-Asp (%), k9 is rate constant for L-succinimide to D-succinimide (min−1), k10 is rate constant for conversion of D-succinimide to L-succinimide (min−1), and G1’(t) and G2’(t) are derivative of G1(t) and G2(t) respectively. Because [D-succinimide]%is probably 0 at t = 0, Eq. (11-1) can be expressed as follows at t = 0 min :Racemization of succinimides in T6 peptide can expressed as follows;Using these rate constants, we calculated the change in relative amounts of the Asp isomers in T6 peptide during a human life span. This calculation was performed using the Runge-Kutta method see .Appendix S1(DOCX)Click here for additional data file.


II atom of the molecular title complex, [CuCl2(C2H3N3)4], is situated on a site with symmetry 2.22. It is six-coordinated in an elongated octahedral geometry, with the equatorial plane defined by four N atoms of four 1,2,4-triazole ligands and the axial positions occupied by two Cl atoms situated on a twofold axis. The molecules are connected via N—H⋯Cl hydrogen bonds and the crystal consists of two interpenetrating three-dimensional hydrogen-bonded frameworks.The central Cu For the al. 2003, Haasnoo al. 2003. For proyama 2001; Morillo al. 2002.2(C2H3N3)4] = 0.025wR(F2) = 0.067S = 1.10952 reflections59 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.40 e Å−3Δρmin = −0.26 e Å−3ΔρCOLLECT  used to solve structure: SIR08  I, global. DOI: 10.1107/S1600536812008872/gk2463Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


It is isotypic with the previously reported CuII analog [Zhong et al.  ligands, two O atoms from two sulfate ions and two aqua O atoms. The second NiII atom is surrounded by two N atoms from 4,4′-bipy ligands and four O atoms, two from bridging sulfate ions and from two ethane-1,2-diol ligands. The sulfate anion acts as a bridging ligand, linking adjacent NiII atoms, leading to the formation of linear ⋯Ni1—Ni2—Ni1—Ni2⋯ chains along the a-axis direction. Adjacent chains are further bridged by 4,4′-bipy ligands, resulting in a two-dimensional layered polymer parallel to (001). In the crystal, the polymeric layers are linked by extensive O—H⋯O hydrogen-bonding interactions involving the O atoms of the water molecules and the ethane-1,2-diol molecules, resulting in a three-dimensional supramolecular network.The title compound, [Ni(SO al. 2011. Acta Cr For an  al. 2011. For bac al. 2005; Robin & al. 2005; Sarma e al. 2009; Zhang e al. 2010.4)(C10H8N2)(C2H6O2)(H2O)] = 0.033wR(F2) = 0.086S = 1.063420 reflections214 parametersH-atom parameters constrainedmax = 0.56 e Å−3Δρmin = −0.41 e Å−3ΔρCrystalClear  used to solve structure: SHELXS97  global, I. DOI: Click here for additional data file.10.1107/S1600536813003772/zq2195Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


We used flow cytometry and ELISA to define the early cytokine profiles of Vγ9Vδ2 T cells stimulated in vitro with isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP) in the absence and presence of IL-2 and IL-15. We show that fresh Vγ9Vδ2 T cells produce interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) within 4 hours of stimulation with phosphoantigen, but neither IL-10, IL-13, nor IL-17 was detectable up to 72 hours under theseconditions. Cytokine production was not influenced by expression or lack, thereof, of CD4 or CD8. Addition of IL-2 or IL-15 caused expansion of IFN-γ-producing Vγ9Vδ2 T cells, but did not enhance IFN-γ secretion after 24–72 hours. Thus, phosphoantigen-stimulated Vγ9Vδ2 T cells have potential as Th1-biasing adjuvants for immunotherapy.Human V The Vγ2Vδ2 TCR recognises small, nonpeptide phosphate antigens (phosphoantigens) independently of major histocompatibility complex (MHC) molecules  by administering phosphoantigens along with T-cell growth factors, most commonly IL-2, or by using aminobisphosphonates which inhibit isoprenoid synthesis resulting in the accumulation of endogenous phosphoantigens [γ9Vδ2 T cell functions presents a double-edged sword, with the potential to treat disease balanced against the possibility of triggering undesirable immune responses, or cell exhaustion. Thus, further characterisation of activated Vγ9Vδ2 T cells is required if such cells are to be used to generate reliable immunological outcomes.The imperative to reliably define Vesenting , 18 and esenting –16 activin vitro , 19–21, in vitro –26. Currantigens . Such stγδ T-cell subsets can produce cytokines that control distinct adaptive immune responses, such as those categorised as T helper type 1 (Th1), Th2, Th17, or regulatory T (Treg) responses [γ9Vδ2 T cells can readily release the Th1 cytokines IFN-γ and TNF-α [γ9Vδ2 T cells taken from the peripheral blood of healthy volunteers and stimulated in vitro with phosphoantigens in the presence and absence of T-cell growth factors IL-2 or IL-15. The antigens used were isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP), intermediates of the mevalonate and nonmevalonate pathways of isoprenoid synthesis, respectively [γδ T cell stimulating antigen described to date [γ9Vδ2 T cells stimulated with this novel phosphoantigen under clinically relevant conditions have similar cytokine profiles to IPP-stimulated Vγ9Vδ2 T cells, with rapid production of cytokines that promote Th1 responses, but little or no cytokines associated with Th2, Th17, or Treg responses.Numerous studies have demonstrated that murine esponses –32. Humand TNF-α , 33–37, nd TNF-α , 38, 39.ectively , 41. HMB to date  but has γδ T-cell enrichments, blood from buffy coat packs from the Irish Blood Transfusion Service Board was used. Peripheral blood mononuclear cells (PBMCs) were prepared by standard density gradient centrifugation over Lymphoprep .Anticoagulated venous blood samples were obtained from healthy donors. For γδ T cells were enriched from PBMC by positive selection using the Anti-TCR γδ Microbead Kit . T cells negative for the γδTCR (hereafter denoted αβ T cells) were prepared by positive selection of CD3+ cells from the γδ TCR-negative fractions using CD3 Microbeads (Miltenyi Biotec). Purity of αβ and γδ T-cell fractions was assessed by flow cytometry.αβ and γδ T cells were plated at 106 cells/mL in complete RPMI medium  and stimulated for 4–72 hours with phorbol myristate acetate  and ionomycin  or various concentrations of the phosphoantigens IPP  or HMB-PP  [PBMC or enriched henberg) , with anμg/mL, Sigma-Aldrich) to promote intracellular accumulation of cytokines. Cells were harvested and stained for cell surface expression of Vγ9, CD3, CD4, and/or CD8 and intracellular expression of IFN-γ, TNF-α, IL-10, IL-13, or IL-17 using fluorochrome-conjugated monoclonal antibodies (mAb) obtained from BD Biosciences, , Immunotools , eBioscience , and R&D Systems  [Total PBMCs were stimulated for 4 hours as described in don, UK) . Cells wαβ and γδ T cells were enriched from PBMC by magnetic bead selection as described in 6 cells per well of a 96-well plate were stimulated in vitro as described in γ, IL-10, IL-13, and IL-17 were quantified by ELISA using antibody pairs purchased from R&D Systems (DuoSet ELISA Development kits).t-test, and P values of <.05 were considered significant.Statistical analysis of data was carried out using Prism GraphPad Version 5.0. Differences between groups were assessed using paired γ9Vδ2 T cells was examined by stimulating freshly isolated PBMC for 4 hours with medium alone, PMA and ionomycin, or various concentrations of IPP or HMB-PP in the presence of brefeldin A (10 μg/mL). Cells were then washed and stained for cell surface expression of CD3 and Vγ9 or Vδ2 and intracellular expression of IFN-γ, IL-10, IL-13, or IL-17. Virtually all Vγ9+ cells expressed the Vδ2 chain and vice versa, with single-positive Vγ9+ or Vδ2+ being very rare [γ9Vδ2 T cells were subsequently identified by a single Vγ9 mAb stain. We found that the numbers of Vγ9+ cells detectable by flow cytometry after stimulation of PBMC with PMA and ionomycin were usually lower than those detectable in unstimulated PBMC from the same donors (not shown) or after stimulation with phosphoantigen , HMB-PP (γ9+ cells that produced IFN-γ did not increase with greater stimulation times (data not shown). Cytokine production by non-Vγ9Vδ2 cells never exceeded background levels (0.4%) in response to phosphoantigens yet was evident in response to PMA and ionomycin treatment, for all cytokines assayed . The percentages of Vγ9Vδ2 T cells producing IFN-γ increased in a dose-dependent manner in response to IPP and HMB-PP, with half-maximal stimulation occurring when 10 μM IPP or 1 nM HMB-PP were used  T cells using ELISA. αβ and γδ T cells were enriched using magnetic beads, and 0.2 × 106 cells of each type were stimulated with various concentrations of HMB-PP or IPP in the absence or presence of IL-2 or IL-15. After 24 hours, IFN-γ was detectable in the supernatants of Vγ9Vδ2 T cells, with half-maximal levels seen when 20 μM IPP or 1 nM HMB-PP were used  of IFN-γ were detectable in the culture supernatants. However, IL-2 or IL-15 only slightly augmented the amounts of IFN-γ released by phosphoantigen-stimulated Vγ9Vδ2 T cells in 3 experiments. Thus, although IL-2 and IL-15 increase the frequencies of pyrophosphate-stimulated Vγ9Vδ2 T cells that express IFN-γ at an early time point  after 72 hours  cells, which also recognise nonpeptide antigens via a conserved TCR and mediate antitumour immunity but are found at ~100-fold lower frequencies in human blood compared to Vγ9Vδ2 T cells [Veceptors , 46, streceptors , and phoeceptors , 48. Theeceptors , 19–21, eceptors –18, and eceptors , 10, 11. T cells .γ9Vδ2 T cell activating agents, such as aminobisphosphonates and phosphoantigens, are ongoing for treatment of cancer [γ9Vδ2 T cells may also prove to be beneficial in numerous other disease settings, due to their potent adjuvant and effector functions in innate and adaptive immunity [γ9Vδ2 T cells respond to different stimuli is required. Clinical trials using various Vf cancer –26. Suchimmunity –16. Howeγ9Vδ2 T cells treated in vitro in a similar manner to current immunotherapeutic protocols, that is, using phosphoantigens and T-cell growth factors. We used two phosphoantigens, the prototype IPP [γ9Vδ2 T cells even at subnanomolar concentrations [γ9Vδ2 T cells in vitro, in the presence and absence of IL-2 and IL-15, and the early production of cytokines that help polarise adaptive immune responses was examined.In this study, we analysed the cytokine production profiles of Vtype IPP  and the trations , 41. Theγ9Vδ2 T cells promptly produce the proinflammatory cytokines IFN-γ and TNF-α, in response to phosphoantigen stimulation, with IFN-γ-producing cells detected as early as 4 hours after stimulation. In contrast, we were unable to detect any upregulation of IL-10, IL-13, or IL-17 production in response to phosphoantigen stimulation. This Th1 cytokine profile was observed exclusively in Vγ9Vδ2 T cells when freshly isolated PBMCs were stimulated with either IPP or HMB-PP, and intracellular cytokine expression was analysed by flow cytometry. The same profile was seen when magnetic bead-enriched γδ T cells were stimulated for up to 72 hours and released cytokine was quantified by ELISA. The frequencies of IFN-γ expressing Vγ9Vδ2 T cells and amounts of cytokine released followed dose-dependent responses to each pyrophosphate, with HMB-PP being ~10,000-fold more potent than IPP, in agreement with previous reports [Our results show that V reports , 42. γ9Vδ2 T cells that produced IFN-γ within 4 hours of stimulation, but these increases only translated into a slight enhancement in IFN-γ levels, when supernatants were analysed after 72 hours. This suggests that IL-2 and IL-15 speed up the kinetics of the Vγ9Vδ2 response, without synergising with phosphoantigens in inducing IFN-γ production, perhaps by stimulating cell division and proliferation of IFN-γ-expressing Vγ9Vδ2 T cells rather than directly stimulating IFN-γ secretion. This result may also be explained by the presence of other accessory cells, such as antigen-presenting cells [γδ T-cell preparations used for ELISA.The addition of IL-2 or IL-15 to pyrophosphate stimulations led to moderate increases in the proportions of Vng cells , within γδ T cells incubated with IL-2 or IL-15 in the absence of phosphoantigen stimulation released moderate amounts of IFN-γ and IL-13, but not IL-10 nor IL-17. This IFN-γ production was augmented by simultaneous stimulation with HMB-PP or IPP, but the IL-13 production was not. This suggests that phosphoantigen stimulation of Vγ9Vδ2 T cells results in secretion of IFN-γ but not IL-13, while treatment with IL-2 or IL-15 can induce secretion of both cytokines. Alternatively, the IFN-γ and IL-13 released in response to IL-2 or IL-15 may emanate from non-Vγ9Vδ2 T cells present in the enriched γδ T-cell preparations, such as Vδ1+ T cells. However, our results clearly show that Vγ9Vδ2 T cells release IFN-γ and not IL-13 in response to phosphoantigen stimulation. Interestingly, γ9Vδ2 T cells produce IFN-γ and TNF-α, we next investigated whether this cytokine profile is restricted to CD4+, CD8+, or double-negative Vγ9Vδ2 T cells. We found that all 3 subtypes of Vγ9Vδ2 T cells were found in all 6 donors tested, with double negative cells predominating. Significant proportions of all 3 Vγ9Vδ2 T cell subsets produced IFN-γ and TNF-α upon stimulation, showing CD4 or CD8 expression does not impact on cytokine production by these cells. Following confirmation that phosphoantigen-stimulated Vγ9Vδ2 T cells release cytokines that promote Th1 responses [+, CD8+, and double-negative Vγ9Vδ2 T cell subsets all exhibit this Th1 cytokine profile. Our data fit with previous observations from us and others that phosphoantigen-stimulated Vγ9Vδ2 T cells possess Th1-promoting adjuvant activity for dendritic cells, inducing them to mature into antigen-presenting cells that release IL-12 but not IL-10 [γ9Vδ2 T cells can induce maturation of monocytes into antigen-presenting cells that stimulated Th1 and Th17 cells. Although Vγ9Vδ2 T cells appear to be Th1 inducers, other studies have shown that subsets of these cells can release other Th cell-polarising cytokines under different conditions. Wesch and co-workers [γ9Vδ2 T cells stimulated with IPP in the presence of IL-12 and anti-IL-4 mAb (Th1-priming conditions) produced IFN-γ and TNF-α, but when stimulated in the presence of IL-4 and anti-12 mAb (Th2 priming) they produced IL-4. Vermijlen and co-workers [γ9Vδ2 T cells stimulated with HMB-PP and IL-2 can also produce IL-5 and IL-13, but in agreement with our results, these authors also found that treatment of Vγ9Vδ2 T cells with IL-2 in the absence of phosphoantigen stimulation was sufficient to induce IL-13 production [γ9Vδ2 T cells characterised by the expression of CXCR5 and CD27 has also been reported to release IL-4 and IL-10 following stimulation with a synthetic phosphoantigen [γ9Vδ2 T cells stimulated with HMB-PP in the presence of IL-1β, TGF-β and IL-6, or IL-23 has also been reported [γ9Vδ2 T cells can be induced to produce various cytokines, but whether they do so under physiological conditions is unclear. Furthermore, such cells appear to account for very small proportions of peripheral blood Vγ9Vδ2 T cells, which under the conditions used in the present study make very small contributions to the overall cytokine profile of phosphoantigen-stimulated Vγ9Vδ2 T cells.The present findings are in agreement with previous studies that have shown that phosphoantigen-stimulated Vesponses , 33–37. esponses , 38. We ot IL-10 , 16, alt-workers  reportedoantigen . IL-17 areported . Thus, iγ9Vδ2 T cells and thus shows promise as an immunotherapeutic agent, worthy of further evaluation. Furthermore, we confirm that Vγ9Vδ2 T cells are also responsive to lymphocyte growth factors. These responses seen with fresh, uncultured Vγ9Vδ2 T cells are similar to those seen with Vγ9Vδ2 T cells that were expanded in vitro with HMB-PP and IL-2 [γ9Vδ2 T cells through other non-TCR stimulatory receptors, such as toll-like receptors [γ9Vδ2 T-cell subsets or functions and thus greatly enhance the gamut of immunotherapeutic tools available.In conclusion, we have shown here that IPP and the novel phosphoantigen HMB-PP rapidly activates a strong proinflammatory response specific to Vand IL-2 . Future eceptors , 46 and eceptors  and inhieceptors , and the