In the title hydrated salt, which was obtained from the hydrothermal reaction between between potassium 1,1,3,3-tetracyano-2-ethoxypropenide and 4,4′-bipyridine in the presence of iron(II) sulfate heptahydrate, the ionic components are linked into a three-dimensional network by C—H⋯N hydrogen bonds. 30H26N62+·2C9H5N4O−·3H2O, was obtained as an unexpected product from the hydrothermal reaction between potassium 1,1,3,3-tetracyano-2-ethoxypropenide, 4,4′-bipyridine and iron(II) sulfate heptahydrate. The cation lies across a twofold rotation axis in the space group I2/a with the other components all in general positions. In the cation, the H atom linking the pyridine units is disordered over two adjacent sites having occupancies of 0.66 (4) and 0.36 (4), i.e. as N—H⋯N and N⋯H—N. The water molecules of crystallization are each disordered over two sets of atomic sites, having occupancies of 0.522 (6) and 0.478 (6) for one, and 0.34 (3) and 0.16 (3) for the other, and it was not possible to reliably locate the H atoms associated with these partial-occupancy sites. In the crystal, four independent C—H⋯N hydrogen bonds link the ionic components into a three-dimensional network.The title hydrated salt, C The cation lies across a twofold rotation axis, selected for the reference cation as that along , while the other components all lie in general positions. Within the cation, the H atom linking the 4,4′-bipy units is disordered over two adjacent sites having occupancies of 0.66 (4) and 0.36 (4), and the two independent water molecules are also disordered, both over two atomic sites, with one having occupancies of 0.522 (6) and 0.478 (6) and the other having occupancies of 0.34 (3) and 0.16 (3).The structure of compound (I)cf. Fig. 12 units containing the atoms C41 and C43 are very similar. In addition, the C—C distances in the C(CN)2 fragments are all short for their type +·X−, (II)   2Cu]2+·2X−, (V)   XCu]2(μ-X)2, (VIII) ClCu]n, (IX) , (III) and (IV) are most closely related to compound (I)et al., 1958v/v, 20 ml). This mixture was sealed in a Teflon-lined autoclave and held at 423 K for 2 d, and then cooled to ambient temperature at a rate of 10 K h−1 (yield 25%). Pale-yellow blocks of the title compound suitable for single-crystal X-ray diffraction were selected directly from the synthesized product.The salt K(tcnoet) was prepared according to a published method (Middleton 3) or 0.99 Å (CH2), and with Uiso(H) = kUeq(C) where k = 1.5 for the methyl group, which was permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms. The unique H atom bonded to N was disordered over two atomic sites, labeled H11 and H21, adjacent to atoms N11 and N21, respectively, and having unequal occupancies; for these two sites, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N), leading to the N—H distances shown in Table 1A and O5B, which are separated by 0.963 (4) Å, while O6 is disordered over two sites, labelled O6A and O6B, which are separated by 0.627 (9) Å. Free refinement of the site occupancies of O5A and O5B gave values of 0.579 (7) and 0.512 (7), respectively; these values are not physically possible and both are over-estimates because of the lack of H atoms in the modelling of the water sites. Accordingly, the occupancies of O5A and O5B were constrained to sum to unity, giving values of 0.522 (6) and 0.478 (6). Free refinement of the site occupancies for O6A and O6B gave values of 0.36 (3) and 0.16 (3), and these values were subsequently restrained to sum to 0.500 (2), giving final values of 0.34 (3) and 0.16 (3). The final analysis of variance showed a large value, 4.522, of K = [mean(Fo2)]/[mean(Fc2)] for the group of 541 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.014.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016012160/hb7603sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016012160/hb7603Isup2.hklStructure factors: contains datablock(s) I. DOI: 1496221CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Although the delicate mechanism in which IL-7Rα downregulation allows the homeostasis of T cell with limited IL-7 has been well known, the exact mechanism behind the interaction between IL-7Rα and γc in the absence or presence of IL-7 remains unclear. Additionally, we are still uncertain as to how only IL-7Rα is separately downregulated by the binding of IL-7 from the IL-7Rα/γc complex. We demonstrate here that 4G3, TUGm2, and 3E12 epitope masking of γc protein are induced in the presence of IL-7, indicating that the epitope alteration is induced by IL-7 binding to the preassembled receptor core. Moreover, the epitope masking of γc protein is inversely correlated with the expression of IL-7Rα upon IL-7 binding, implying that the structural alteration of γc might be involved in the regulation of IL-7Rα expression. The conformational change in γc upon IL-7 binding may contribute not only to forming the functional IL-7 signaling complex but also to optimally regulating the expression of IL-7Rα. IL-7 signaling via IL-7R They have a common feature of sharing γc as the signaling subunit of their receptors [α-chain (IL-7Rα)/γc, forming functional heterodimeric receptor complex [The common eceptors . Among teceptors . IL-7 bi complex –10. Thusγc and IL-7Rα. One is a stepwise cytokine-induced heterodimerization mechanism, in which IL-7 interacts initially with IL-7Rα, followed by recruitment of γc to form functional signaling complex [γc cytokine-independent preassembled IL-2Rβ, IL-4Rα, and IL-9Rα homodimer or heterodimer [α/γc heterodimer and IL-7Rα homodimer on the cell membrane have been reported [α/γc heterodimer is converted into the signaling form by a conformational change of the heterodimer in presence of IL-7, denoting that a conformational change in the preassembled IL-7Rα/γc receptor complex is required for normal signal transduction [α and γc heterotypic receptor has not been fully mapped.There are two proposed models of interaction mechanism between IL-7 and functional receptor complex, which is composed of  complex . Moreove complex , 13. Theerodimer –17. The reported , 18. Strsduction , 13, 19.γc protein in rapid and energy-independent manner. Anti-γc polyclonal antibody (pAb) detects surface γc protein, while anti-γc monoclonal antibody (mAb) fails to detect the surface γc protein upon IL-7 stimulation. Flow cytometry studies indicate that at least 3 epitopes in the extracellular domain of γc—recognized by 4G3, TUGm2, and 3E12 antibodies—are concealed in the presence of IL-7. We also report that the masking of γc epitopes rapidly arises, and it is IL-7 specific and energy independent. These results reveal that conformational changing sites in γc protein may be located at the 4G3, TUGm2, and 3E12 epitope regions and that it is important to form a functional IL-7R core. Finally, we suggest that these data might support IL-7 signaling model through the IL-7 independent preassembled IL-7Rα/γc receptor complex.Herein, we show that IL-7 induces epitope masking of γc−/− mice were bred in our own colony. The γc-transgenic construct was made by ligating the γc cDNA into the human CD2 (hCD2) enhancer-promoter-based vector and was injected into the fertilized B6 oocytes to generate γcTg mice [γc−/− mice were bred with γcTg mice to generate sγc deficient mice. Animal experiments were approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-2014-0620), and all mice were cared for in accordance with the guidelines set forth by the Pusan National University School of Medicine.C57BL/6 (B6) mice were obtained from the Jackson Laboratory, and cTg mice . The γc−α (53-6-7), TCRβ (H57-597), γc , IL-4Rα (M1), phosphorylated STAT5 , B220 , γc , and IL-7Rα . Anti-human CD3ε  was used as the isotype control. Anti-mouse CD16/32 antibody  was incubated to block Fcγ receptors. All antibodies were incubated at 4°C for 30 min. For FACS analysis of pSTAT5, cells were fixed with 2% paraformaldehyde solution and were subsequently permeabilized with acetone/methanol solution. y-axis of all histograms is log scale with maximum value 104. x-axis of all histograms is linear scale and maximum values are indicated in each figure legend.The cells were harvested, stained, and analyzed on a FACS Aria or FACS Canto (BD). Dead cells were excluded by forward light scatter gating and propidium iodide staining. Data were analyzed using FlowJo version 10 (TreeStar). Antibodies with the following specificities were used for staining: CD4 (GK1.5 and RM4.5), CD8https://doi.org/10.1155/2017/9096829). In case of sodium azide condition, around 20% of cells survived in 16-hour incubation regardless of IL-7 treatment, while cell viability was maintained by 6 hours . Dead cells were excluded by forward light scatter gating and propidium iodide staining. All FACS analysis was performed only with live cells.A single cell suspension of all LNs from mouse was prepared by applying light pressure with two glass slides to crush the nodes and suspended with chilled full RPMI medium and then stored in ice before using. A single cell of thymus was prepared by tearing into little pieces and suspended with chilled full RPMI medium and then stored in ice before using. The prepared cells were simultaneously stimulated with conditional medium prewarmed at 37°C for the indicated time. The cell viability was comparably maintained during 16-hour incubation regardless of IL-7 treatment . They were stimulated with recombinant human IL-2 (10 ng/ml), murine IL-4 (20 ng/ml), IL-7 (10 ng/ml), IL-9 (100 ng/ml), IL-15 (100 ng/ml), IL-21 (100 ng/ml), or IL-6  or stimulated in presence of sodium azide (Sigma-Aldrich) in a 37°C or 4°C humidified incubator.Thymocytes and LN cells were cultured in RPMI 1640 medium (Welgene) containing 10% FBS (Gemini), L-glutamine, 100,000 U/ml penicillin plus 100 mg/ml streptomycin (GIBCO), nonessential amino acid (Sigma-Aldrich), sodium pyruvate (Sigma-Aldrich), and γc pAb (R&D system), followed by horseradish peroxidase- (HRP-) conjugated streptavidin (BioLegend).Cell lysates were obtained from IL-7 stimulated T cells and were resolved by SDS-PAGE on 12% acrylamide (Invitrogen) under reducing conditions, which were then transferred to the PVDF membranes (Amersham Biosciences). Blots were incubated with biotin-conjugated anti-γc , IL-7Rα , and RPL13 . Gene expression values were normalized to those of Rpl13 in the same sample.Total RNA was immediately isolated with the Ribospin (GeneAll). RNA was reverse transcribed into the cDNA by oligo(dT) priming with the Reverse Transcription Kit (GeneAll). Quantitative RT-PCR (qRT-PCR) was performed with SYBR Green Master Mix (Bio-Rad) on the LightCycler 96 Real-Time PCR System (Roche) with the following primers: γc-specific monoclonal antibodies (TUGm2: BD) and γc-specific polyclonal antibodies (R&D Systems) at a previously optimized dilution. The Streptavidin Alexa Fluor® 594 was used as a secondary agent. The slides with stained cells were inverted over a drop of vectashield/4,6-diamidino-2-phenylindole (DAPI) and sealed with varnish. Slides were analyzed by confocal microscopy. All images were acquired on an Olympus FV 1000 (Olympus Corp.) using a PLAPON60x/1.35 oil-immersion objective. DAPI fluorescence was detected with a violet 405 nm and Streptavidin Alexa Fluor 594 fluorescence was detected using a violet 543 nm. All confocal images were acquired with frame size of 1024 × 1024 pixels.The lymph node (LN) T cells were harvested in RPMI 1640 and stimulated with recombinant human IL-7 (10 ng/ml) at 37°C for the indicated time. After stimulation, cells were washed and fixed with 4% PFA incubation for 10 min. The fixed cells were stained with biotin-conjugated t-test. p values of less than 0.05 were considered significant. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. All statistical analyses were performed using GraphPad Prism.Statistical differences were analyzed by unpaired two-tailed Student's α with transcriptional mechanism [γc is essentially required for normal IL-7 signaling pathway, we wondered how the γc expression is altered in the presence of IL-7 compared with IL-7Rα. To test this, LN cells were stimulated with IL-7 and analyzed in alteration of γc and IL-7Rα expression at different time points. Consistent with a previous study [α expression is time-dependently downregulated by IL-7. Surprisingly, surface γc level of CD4+ and CD8+ LNT cells was rapidly reduced compared with the medium controls, and later their expression was time-dependently rescued, as detected by FACS   , which iNT cells . In ordepression . These dγc cytokines, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, share γc as the functional subunit of their signaling. We thus ask whether other γc cytokines change γc surface expression. To address this question, CD8+ T cells were stimulated with other γc cytokines and IL-6 as non-γc cytokine control. Upon short-term stimulation (10 minutes), there was a notable and specific decrease in the surface γc protein only for IL-7, whereas no early reduction of surface γc protein was detected for other γc cytokines and IL-6 stimulation  thymocytes, lacking IL-7Rα, were stimulated with IL-7, there was also no notable reduction of surface γc protein for IL-7 compared with mature CD8+ (CD8SP) thymocytes   from when IL-7Rα expression started to be significantly downregulated, as shown in Figures α expression was downregulated at early time (to 30 minutes), IL-7Rα was still expressed at around 80% compared to medium group. However, γc expression was not detected (around 10%) at the same time points upon IL-7 stimulation. In order to further confirm whether the alteration of γc epitopes is reversely correlated to IL-7Rα expression, we analyzed pattern of γc and IL-7Rα expression on different conditions, such as medium, sodium azide, and low temperature   left. Onmulation , right,  Figures . We founmulation . Collectγc surface expression was observed only under IL-7 stimulation through a decrease of immunofluorescence analysis by FACS, we assumed that the epitope recognized by 4G3 may be masked on IL-7 binding but may not actually be regulated. To verify this, we first checked the γc protein and mRNA levels of T cells upon either medium or IL-7 stimulation. Both γc protein and mRNA levels were not significantly changed by IL-7  and a membrane-proximal portion, respectively, to stain T cells under IL-7 stimulation. Interestingly, FACS analysis of different mAb against TUGm2 and 3E12 epitopes has shown similar reduction of γc expression in comparison with 4G3  patients are less than 30 Å, which spontaneously activate the IL-7 signaling pathway to malignant proliferation of lymphocyte in absence of IL-7 [α homodimer detected in both T and B cells in ALL patients [α has preassembled mechanism but not sequential dimerization with IL-7 [γc cytokines. Compared with other γc cytokines acting on their unique receptor subunit in high affinity, IL-7 interacts with IL-7Rα in lower binding affinity because its interface has relatively narrow contact area and less polarized and uncharged contact surface and and lacks knobs-into-holes shape complementarity [γc cytokine specific receptor subunits that scarcely interact with γc, IL-7Rα has low binding affinity with γc [γc epitope with IL-7 was immediately induced just within 1 minute and even at 4°C at which IL-7Rα was not downregulated. In keeping with previous reports, these results lead us to consider that recruitment of γc to IL-7/IL-7Rα which occurs in sequential dimerization model would be impossible in 4°C and sodium azide medium, suggesting that the epitope masking may result from conformational changes of surface γc protein with preassembled heterodimer model [γc epitopes could be directly concealed by IL-7/IL-7Rα heterodimer in which 4G3 and TUGm2 epitopes were concealed by IL-7 binding and 3E12 epitope was masked by IL-7Rα. Along with previous reports, our data may support the preassemble heterodimer model of IL-7Rα/γc, as shown through the rapid and energy-independent nondetection of surface γc protein.Questions still remained regarding the cytokine-induced receptor heterodimerization. For instance, the distances between the C-terminal of mutated IL-7R of IL-7 , 19. Thupatients , 30. Morith IL-7 , 13, 19.er model , 13, 19.α and β subunits located in the extracellular domains [γc cytokine binding motif in γc, and identified that these epitopes are critical for conformational change. Olosz and Malek have reported that 4G3 epitope, which is located near Val-121 and Asn-128 of mγc, is a potential residue of IL-7 binding, although it rarely affects IL-2 and IL-15 binding. The TUGm2 epitope has been predicted to be centered near Lys-158 of mγc, and the antagonistic effect of 4G3 and TUGm2 in the γc cytokine signaling pathway has been demonstrated. Thus, previous studies regarding the cytokine cross-linking assay with mutant molecules, the predicted location of these epitopes, and the inhibitory properties of the mAbs suggest that they are the pivotal sites in direct ligand-receptor interactions [γc surface protein on LNT cells was not detected by immunostaining with 4G3 and TUGm2 mAbs upon IL-7 stimulation, although the expression of γc was intact upon IL-7 stimulation as proved by the immunostaining with anti-γc pAbs, indicating that 4G3 and TUGm2 are essential sites of conformational change as well as ligand binding. Although 3E12 epitope is located in a membrane-proximal portion of mγc, near Arg-197, 3E12 mAb is able to interfere with γc cytokine signaling. Both our research based on the results of flow cytometry assay and previous reports of 3E12 epitope support that 3E12 is a critical site of receptor-γc interaction or conformational change to form the functional receptor core.Conventionally, it has been thought that the different capacity of antibodies in detecting the specific epitopes reflects conformational change . The res domains , 33. In  domains . Here, wractions . In addiγc cytokine-independent preassembled receptor core mechanism in the IL-7 signaling pathway by flow cytometry evaluation. Walsh et al. assert that this mechanism might be applied to other γc cytokines receptors that form nonfunctioning homodimers and heterodimers [Here we report evidence that may support rodimers . Relatedrodimers , 36. We α and γc can spontaneously associate by their extracellular domains on the surface of T cells and form a receptor core, the primary functional target of IL-7. IL-7 binding may induce the conformational change of γc, which migrates to complete the functional receptor core and reconstitutes active protein kinases, such as JAK1-JAK3 pair by clustering their protein components together. Then, the associated JAKs transphosphorylate themselves. The IL-7 signaling pathway induces the internalization and degradation of the surface IL-7Rα. At this point, epitopes masked by IL-7/IL-7Rα are reexposed in free γc and are detected with relevant mAbs. Our results thus suggest that the conformational change of γc not only leads the IL-7R core to be functional but also induces the split of IL-7Rα from γc to be internalized. Controlling the internalization of IL-7Rα could be one of the alternative strategies to regulate γc responsiveness on IL-7. Further studies are required to further elucidate how γc epitopes are reexposed as IL-7Rα expression is reduced.In conclusion, our data suggest that IL-7RSurvival kinetics of WT LN T cells in presence or absent of IL-7 with or without Na-azide. Cell survival was determined by gating on propidium iodide negative cells. Data are summary of three independent experiments (mean and SEM).


The solid-state structure of a 4-bromobenzoxazine has been determined. The whole molecule of the title compound is generated by inversion symmetry. This is a potential benzoxazine monomer for the preparation of phenolic materials. 18H18Br2N2O2, was prepared by a Mannich-type reaction of 4-bromophenol with ethane-1,2-diamine and formaldehyde. The title compound crystallizes in the monoclinic space group C2/c with a centre of inversion located at the mid-point of the C—C bond of the central CH2CH2 spacer. The oxazinic ring adopts a half-chair conformation. The structure is compared to those of other functionalized benzoxazines synthesized in our laboratory. In the crystal, weak C—H⋯Br and C—H⋯O hydrogen bonds stack the molecules along the b-axis direction.The title benzoxazine molecule, C The six-membered oxazine heterocyclic ring adopts a half-chair conformation, with puckering parameters Q = 0.512 (2) Å, θ =129.6 (2)°, φ = 283.6 (3)°. This ring is analysed with respect to the plane formed by O1/C3/C4/C5, with deviations of the C2 and N1 atoms from this plane of 0.300 (6) and −0.320 (4) Å, respectively.The asymmetric unit of the title compound Fig. 1, C18H18Bet al., 2012aet al., 2011p-chloro derivative where C—O = 1.421 (2) Å  and 1.529 (2) Å, respectively; Rivera et al., 2010p-substituent on the aromatic ring  Å] is comparable with other previously reported C—O bond lengths for related structures [1.370 (10) and 1.388 (9) Å  ,  monohydrate  bis was added dropwise to a mixture of ethane-1,2-diamine  and 4-bromophenol  dissolved in dioxane (10 ml). The reaction mixture was stirred for 4 h at room temperature. Single crystals were obtained from this solution by slow evaporation of the solvent.Uiso(H) set to 1.2Ueq of the parent atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016016509/sj5510sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016016509/sj5510Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016016509/sj5510Isup3.cmlSupporting information file. DOI: 1510085CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Z)-2-[2-(morpholin-4-yl)-2-oxoethylidene]thiazolidin-4-one and (Z)-N-(4-methoxyphenyl)-2-(4-oxothiazolidin-2-ylidene)acetamide are described and compared with a related structure.The crystal structures of two (oxothiazolidin-2-ylidene)acetamides, namely ( Z)-2-[2-(morpholin-4-yl)-2-oxoethylidene]thiazolidin-4-one, C9H12N2O3S, (I), and (Z)-N-(4-methoxyphenyl)-2-(4-oxothiazolidin-2-ylidene)acetamide, C12H12N2O3S, (II), are described and compared with a related structure. The Z conformation was observed for both the compounds. In (I), the morpholin-4-yl ring has a chair conformation and its mean plane is inclined to the thiazolidine ring mean plane by 37.12 (12)°. In (II), the benzene ring is inclined to the mean plane of the thiazolidine ring by 20.34 (14)°. In the crystal of (I), molecules are linked by N—H⋯O hydrogen bonds, forming C(6) chains along the b-axis direction. The edge-to-edge arrangement of the molecules results in short C—H⋯O and C—H⋯S interactions, which consolidate the chain into a ribbon-like structure. In the crystal of (II), two N—H⋯O hydrogen bonds result in the formation of C(8) chains along the b-axis direction and C(6) chains along the c-axis direction. The combination of these interactions leads to the formation of layers parallel to the bc plane, enclosing R44(28) rings involving four molecules.The crystal structures of two (oxothiazolidin-2-ylidene)acetamides, namely ( The dihi Table 1 hydrogenC(8) and C(6) chains, running parallel to the b- and c-axes, respectively °. The combination of these chain motifs generates a two-dimensional network lying parallel to the bc plane. Each molecule acts as both a double donor and a double acceptor of N—H⋯O hydrogen bonds. The molecules of (II)In crystal of (II)et al., 2016N-phenylacetamide (III) . The precipitates formed of the 1,3-thiazolidinones, were filtered off. The crude products were additionally purified by refluxing a suspension of the thiazolidine in MeCN, followed by hot filtration. Colourless crystals of compounds (I)Thiazolidinones (I)(Z)-2-[2-(Morpholin-4-yl)-2-oxoethylidene]-1,3-thiazolidin-4-one (I). Yield 1.54 g (45%), white powder, m.p. 503–505 K. 1H NMR spectrum, δ, p.p.m. : 3.42 ; 3.54 ; 3.57 ; 5.83 ; 11.25 .(Z)-N-(4-Methoxyphenyl)-2-acetamide (II). Yield 2.85 g (72%), white powder, m.p. 534–536 K.  = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other C-bound H atoms. The NH H atoms were located in difference-Fourier maps and freely refined.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017016061/su5403sup1.cifCrystal structure: contains datablock(s) Global, II, I. DOI: Click here for additional data file.10.1107/S2056989017016061/su5403Isup2.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017016061/su5403IIsup3.cmlSupporting information file. DOI: 1582018, 1582019CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The single-crystal X-ray diffraction investigation of a substituted quinoline derivative, which may serve as a basis for the development of a family of cytotoxic agents, confirms the anticipated covalent structure with an unusual twisted conformation and reveals a densely packed molecular lattice. 17H14N2O5, was prepared as a lead compound in a project dedicated to the development of therapeutic agents binding to the colchicine binding site on tubulin, thereby interfering with the cell division in cancer cells. It holds many of the main structural characteristics for colchicine binding and has the potential for further modification and functionalization. In the title molecule, the benzene ring is inclined to the quinoline ring by 76.10 (8)°. In the crystal, molecules are linked by two pairs of C—H⋯O hydrogen bonds, forming tubular-like arrangements, propagating along the direction of the diagonals of the ab plane, and enclosing R22(26) and R22(16) ring motifs.The protein tubulin is central for maintaining normal cellular processes, and molecules interfering with the tubulin dynamics have potential in the treatment of cancerous diseases. The title compound, C It can be seen that only one previous compound, 4-[6-methoxy-4-oxoquinolin-1(4H)-yl]benzonitrile . Even though (I)The frequencies of molecules in the Cambridge Structural Database  Å] is clearly visible. While the bicyclic ring systems of DAHWOE and PEBDIL  are perfectly coplanar with the C6 and C7 substituents as well as the C1′-atom attached to N1, this is not the case for (I)ca 167.7°; see Fig. 2a and 2b. The more extended search fragment in Fig. 1c found 157 such torsion angles in 62 CSD entries, and in only nine compounds does this torsion angle deviate by more than ca 13.3° from planarity.The molecular structure of (I)IL Fig. 1a are peb and 2c, where close contacts to two neighbouring molecules are apparent; these force the methoxy group and the nitrophenyl group out of the quinolinone mean plane. In the crystal, molecules are linked by two pairs of C—H⋯O hydrogen bonds, forming tubular-like arrangements propagating along the direction of the diagonals of the ab plane, and enclosing Cg1⋯Cg1 = 3.659 (1) Å . The crystal density is comparatively high at 1.415 g cm−3, and no voids were calculated by Mercury s Table 1. Within 2CO3  and 6,7-dimethoxyquinolin-4-ol  were weighed out in a round-bottom flask, to which was added 3 ml DMF and 1 ml MeCN. The mixture was then stirred for 15 min. 1-Fluoro-4-nitrobenzene  in 2 ml 1:1 DMF:MeCN was then added, and the reaction mixture was stirred for 20 h at 328 K. The crude product was washed with water (4 × 10 ml) and brine (10 ml), and then purified by column chromatography [Hep:EtOAc (4:1) → Hep:EtOAc:MeOH (10:10:1)]. The title compound (I)1H NMR : δ 8.48 , 7.79 , 7.67 , 7.48 , 6.35 , 6.32 , 3.98 , 3.72 . 13C NMR : δ 176.98, 153.56, 147.96, 147.71, 146.91, 140.54, 136.08, 128.64, 125.92, 120.99, 110.68, 106.17, 98.10, 56.46, 56.21. HRMS (ESI+) m/z calculated for C17H15N2O5 [M+H]+: 327.0975, found 327.0976. Yellow crystals of compound (I)CsUiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017002948/su5354sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017002948/su5354Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017002948/su5354Isup3.cmlSupporting information file. DOI: 1533984CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine , in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine  and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown.Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity.The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities. Alpha-N-acetylgalactosaminidase  EC 3.2.1.49] is a lysosomal hydrolase that catalyzes the removal of terminal α-N-acteylgalactosamine  and, to a lesser extent, galactose monosaccharide from polysaccharides, glycolipids and glycoproteins .49 is a . α-GalNABecause of similar substrate specificity and physical properties including subunit molecular mass and homodimeric structure, α-GalNAcase was initially referred to as α-galactosidase B. However, subsequent studies of kinetics, immunoreactivity, peptide and gene mapping demonstrated that α-GalNAcase and α-galactosidase are two distinct enzymes derived from two different genes . Upon crα-GalNAcase has been purified from various animal tissues including human, bovine, pig, chicken, earthworm, snail and gastropod , 9–12. TTodarodes pacificus), a marine invertebrate [Asterina amurensis) and purified two different α-GalNAcases, α-GalNAcase I and II. The two enzymes appeared to be different with respect to enzymatic characteristics such as molecular mass, optimum pH, pI, pH stability, enzyme kinetics (KM and Vmax), and substrate specificities. α-GalNAcase II was monomer and heat stable, while α-GalNAcase I was multimer and heat labile. Both α-GalNAcase I and II removed terminal α-GalNAc of natural compounds investigated: Forsman hapten glycolipid, blood group A type trisaccharide, GalNAc-α1-O-serine. Contrary, oligosaccharide and glycolipid containing α-galactosyl terminal were hydrolyzed by α-GalNAcase I only, but not by α-GalNAcase II [KM and Vmax values of α-GalNAcase II for pNP-α-GalNAc (3.97 mM and 126 μmole/min/mg) were much higher than that of α-GalNAcase I (1.04 mM and 49.5 μmole/min/mg). α-GalNAcase II activity was significantly inhibited by Ag+ and Hg+2 salts, while α-GalNAcase I activity was not much affected. However, the interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their enzymological differences remain to be known.Marine animals are well known as potential sources of novel enzymes and provide useful information in understanding the structure and function of mammalian enzymes. We previously reported the occurrence of an unusual form, α-GalNAcase II, which was free from α-galactosidase activity, in addition to a typical form, α-GalNAcase I in squid  , also called N-acetyl recognition loop, in α-GalNAcase are replaced with glutamate and leucine, respectively in human α-galactosidase A, to accommodate smaller hydroxyl group of α-galactosyl ligand but not the larger N-acetyl group [172APAY176) in α-GalNAcase II and thus generate a larger space for the pocket for accommodation of the N-acetyl group as compared to typical α-GalNAcases. Similarly, the small hydroxyl group of galactose moiety loosely fits in the loop cavity and hence showed no or too little α-galactosidase activity in absence of a bulkier indole ring of tryptophan , pNP-α-galactoside and PNGase F were purchased from Sigma Aldrich Chemical Co . Di-isopropyl-fluorophosphate (DFP), carbodiimide cyanamide HCl (CNM-HCl), N-bromosuccinimide (NBS), bovine serum albumin (BSA) and lysylendopeptidase were procured from Wako Pure Chemical Industries . Peroxidase conjugated sheep anti-rabbit IgG was obtained from Organon Teknika Corporation . Unless otherwise directed, all other chemicals and reagents were of analytical grade and obtained from commercial sources.The purification and enzymatic characterization of α-GalNAcase I and II from digestive organ of starfish were done by the procedures as reported . Enzyme The purified α-GalNAcase I and II (10 μg) were denatured in 50 μl sodium phosphate (50 mM) buffer, pH 7.0 by heating at 100 °C for 10 min in presence of 0.02% SDS and 10 mM β-mercaptoethanol. The denatured protein was treated with Triton X-100 (Conc. 1.5%) and incubated at 37 °C with 1 unit of PNGase F (Sigma) for 24 h. The PNGase F treated enzyme were separated on SDS-PAGE and stained with CBB R-250 solution.Purified enzyme (5 μg) was separated on SDS-PAGE and transferred to polyvinyledine fluoride (PVDF) membrane  by electro-blotting in a Biorad mini-trans blot cell using Tris-glycine buffer. Nonspecific binding sites of membrane were saturated by incubation with 3% BSA in 50 mM Tris–HCl containing 150 mM sodium chloride and 0.1% sodium azide (buffer A) for 1 h at room temperature. The membrane was then incubated with primary antibody at an appropriate dilution for 30 min. After rinsing, the membrane was incubated for 1 h with peroxidase conjugated sheep anti-rabbit IgG in buffer A. The protein which crossreacted with the antibody was visualized using 3, 3-diaminobenzidine (DAB) intensified with 0.001% hydrogen peroxide.−1 followed by elution with a 90 min linear gradient of acetonitrile (5-50%) in 0.1% trifluroacetic acid (TFA). The peptide peaks were detected at 215 nm.Purified enzyme (100 μg) was separated on SDS-PAGE and visualized by CBB staining. The expected protein band was excised with a sharp blade and washed with water. The gel containing protein band was cut into pieces and destained with 40% acetonitrile in 100 mM ammonium carbonate for 24 h. Then protein in gel was reduced with DTT (10 mM) at 56 °C and alkylated with iodoacetamide (50 mM) in 100 mM ammonium carbonate for 1 h in every treatment. The polyacrylamide gel was dehydrated with acetonitrile after completion of each treatment. The reduced and alkylated proteins were digested with lysylendopeptidase at protein to peptidase ratio 50:1 in 50 mM Tris–HCl buffer, pH 8.5 for 24 h. After digestion, the peptides were extracted from polyacrylamide gel once with 1.0% formic acid, once with 9% acetonitrile in 20 mM ammonium carbonate and twice with 60% acetonitrile in 0.1% formic acid. The extracted peptides were lyophilized by freeze drying, reconstituted in water and subjected to RP-HPLC separation on a column TSK gel ODS-120 T  at a flow rate 1 ml minThe individual peptide fractions obtained from RP-HPLC separation were adsorbed on PVDF membrane by repeated pipetting of the sample solution. The resulting bound samples were desalted with 10 μl 0.1% TFA and spotted on a MALDI target plate that was double layered with 2, 5-dihydroxybenzoic acid (10 mg/ml in acetone). The spots were air dried and MS/MS fragmentation was performed using a MALDI-TOF-MS Autoflex  under a 20 kV accelerating voltage in the positive ion mode. The Bruker Data Analysis software (Version 4.0) was employed for data acquisition, processing and analyses. The mass tolerance in all experiments was ≤ ±0.4 Da.Purified enzyme α-GalNAcase I and II (10 μg each) was resolved on SDS-PAGE and transferred to PVDF membrane by electro-blotting. The protein was stained with 0.1% Ponceau S (Sigma) in 5% acetic acid. After staining, expected protein band was excised and washed with methanol and subjected to sequence the N-terminal amino acids , 16, wit−1cm−1 [Chemical modifications were performed by incubating the enzyme with DFP in 20 mM Tris–HCl, pH 4.0, and CNM-HCl and NBS in 50 mM citrate-phosphate buffer, pH 4.0 (CPB). Purified enzyme (45 μg per ml) was incubated at 25 °C in various concentrations of modifying agent for 5 min [−1cm−1 . ModificThe deduced peptide sequences of starfish α-GalNAcase I and II were substituted in chicken α-GalNAcase (PDB code: 1Ktb) aligned sequence and constructed a fold fit structure by DeepView/Swiss-PdbViewer based on the chicken α-GalNAcase structure as a template . From th


Taxodium ascendens (B.).The title compound, 7β-hydroxyroyleanone, an abietane-type diterpene, was isolated from  20H28O4 , is an abietane-type diterpene, which was isolated from Taxodium ascendens (B.). The compound crystallizes in the chiral space group P21, but it was not possible to determine the absolute structure of the molecule in the crystal by resonant scattering. The molecular structure is stabilized by two intramolecular O—H⋯O hydrogen bonds, enclosing S(5) and S(6) ring motifs. In the crystal, molecules are linked by O—H⋯O and C—H⋯O hydrogen bonds, forming chains along the [010] direction. The crystal structure of the 10R stereoisomer of the title compound, isolated from the roots of Premna obtusifolia (Verbenaceae), has been reported. It crystallized in the chiral space group P212121, and the absolute structure was determined as , by resonant scattering using Cu Kα radiation .The title compound, C al. 2010. Acta Cr Taxodium ascendens Brongn belongs to the Taxodiaceae species, which is native to the south-east of North America and has spread widely over southern China  have described many diterpenes, such as 6,7-dehydroroyleanone, salvinolone and xanthoperol  has a chair conformation , while ring B (C1/C2/C10–C13) has an envelope conformation, with atom C2 as the flap . Benzoquinone ring C (C12–C17) has a screw-boat conformation . The mean planes of the various rings are inclined to one another in the following manner: A/B = 22.97 (10)°, A/C = 34.52 (10)° and B/C = 12.84 (9)°.The molecular structure of the title compound is shown in Fig. 1R stereoisomer of the title compound, isolated from the roots of Premna obtusifolia (Verbenaceae), has been reported twice (see §4Database survey). It crystallized in the chiral space group P212121, and the absolute structure was determined as  by resonant scattering using Cu Kα radiation .The crystal structure of the 10A⋯O3i and O4—H4⋯O1ii, both approximately running along the b axis, are formed via the hydroxy group and the carbonyl groups . The air-dried pollen (1.8 kg) was extracted with 95% ethanol and then partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) to give a PE extract (80 g), an EtOAc extract (120 g) and a n-BuOH extract (100 g). The PE extract (80 g) was subjected to normal-phase silica-gel column chromatography (300–400 mesh) with a gradient solvent system of petroleum ether–acetone  to give eight major fractions, denoted F1–F8. Fraction F4 (6 g) was sequentially subjected to normal-phase silica-gel column chromatography (300–400 mesh) with an isocratic elution  to give three major fractions, denoted F4.1, F4.2 and F4.3. Fraction F4.3 was purified by semipreparative HPLC , to give an orange solid, which was recrystallized from the mixed solvents of CH2Cl2–MeOH (5:2 v/v), affording orange block-like crystals suitable for X-ray diffraction analysis. The 1H and 13C NMR data of 7β-hydroxyroyleanone have been reported elsewhere  = 1.5Ueq for hydroxy and methyl groups, and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017011987/su5382sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989017011987/su5382Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017011987/su5382Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017011987/su5382Isup4.cmlSupporting information file. DOI: 1551129CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Plasmodium falciparum malaria develops after several episodes of infection. Fc gamma receptors (FcγRs) bind to immunoglobulin G (IgG) antibodies and mediate phagocytosis of opsonized microbes, thereby, linking humoral and cellular immunity. FcγR polymorphisms influence binding affinity to IgGs and consequently, can influence clinical malaria outcomes. Specifically, variations in FcγRIIA -131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 modulate immune responses through altered binding preferences to IgGs and immune complexes. Differential binding, in turn, changes ability of immune cells to respond to infection through production of inflammatory mediators during P. falciparum infection.Naturally-acquired immunity to n = 274; aged 6–36 months) presenting for their first hospital visit with P. falciparum malaria in a holoendemic transmission region of western Kenya. FcγRIIA-131Arg/His and FcγRIIIA-176F/V genotypes were determined using TaqMan® SNP genotyping, while FcγRIIIBNA1/NA2 genotypes were determined using restriction fragment length polymorphism. Hematological and parasitological indices were measured in all study participants.We determined the association between haplotypes of FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 variants and severe malarial anemia  in children (P = 0.036), while the FcγRIIA-131His/ FcγRIIIA-176F/ FcγRIIIB NA1 haplotype was marginally associated with enhanced susceptibility to SMA  and higher levels of parasitemia (P = 0.009). Individual genotypes of FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 were not associated with susceptibility to SMA.Carriage of FcγRIIA-131Arg/FcγRIIIA-176F/FcγRIIIBNA2 haplotype was associated with susceptibility to SMA  contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria holoendemic transmission regions, such as western Kenya, malaria manifests with a milieu of life-threatening conditions including severe malarial anemia (SMA), metabolic acidosis, high-density parasitemia , respiratory distress, hypoglycaemia and other infrequent complications such as hypotension  of both sexes were recruited at SCRH during their initial hospitalization for treatment of malaria. Recruitment followed a two-phase tier of screening and enrolment. The parent/legal guardian of the child received detailed explanation of the study. Enrollment decisions were made after initial HIV-1 screening of the child and a signed informed consent, which included authority to publish the findings. Questionnaires and written informed consent were administered in the language of choice . Children with acute malaria were stratified into two categories: non-severe malarial anemia (non-SMA) group defined as a positive smear for asexual P. falciparum parasitemia (of any density) and Hb ≥ 6.0 g/dL; and SMA group defined by a positive smear for asexual P. falciparum parasitemia (of any density) and Hb < 6.0 g/dL  than those in the non-SMA group , P < 0.001. Parasitemia values (log10 of parasites/μL) was comparable between the study groups, SMA  and non-SMA , P = 0.088). The proportion of participants with high-density parasitemia (HDP) was also comparable between the clinical groups . Similarly, there was no difference in body temperature (°C) between the study groups, SMA  and non-SMA , respectively, P = 0.430. Further analysis revealed that children with SMA had higher respiration rate (breaths/min),  than non-SMA, , P < 0.001. Analysis of hematological parameters revealed that red blood cells counts (RCBs × 1012/μL) were higher in children with non-SMA  than those with SMA, , P < 0.001. The SMA group were also characterized by elevated levels of white blood cells counts (WBC × 103/uL)  relative to the non-SMA group , P < 0.001. The platelet counts (platelets ×103/μL) were lower in children with SMA,  as compared to the non-SMA, , P = 0.025. The distribution of G6PD in SMA and non-SMA were comparable . Similarly, the distribution of those with sickle cell trait in SMA and non-SMA were comparable . These results are presented on Table We conducted a cross-sectional analysis of children (2) analysis showed that the distributions of the FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 genotypes were not significantly different between the clinical groups  . FcγRIIA-131Arg/His genotypes distribution in non-SMA were 39 (24.3%) Arg/Arg, 71 (44.4.0%) Arg/Hist and 50 (31.1%) His/His. Frequencies of the genotypes in non-SMA showed deviation from HWE . The overall genotype distribution for the FcγRIIA-131Arg/His did not deviate from HWE  with an overall variant allele frequency of the FcγRIIA-131Arg/His at 0.49 (Arg). The genotypic distribution of the FcγRIIIA-176 F/V in SMA group was 61 (53.5%) FF, 45 (39.5%) FV and 8 (7.0%) VV. The distribution of these genotypes in SMA showed consistency with HWE . Within the non-SMA group, the distributions was 77 (48.1%) FF, 60 (37.5%) FV and 23 (14.4%) for VV and the genotypes showed consistency with HWE . The distribution of these genotypes in overall population showed consistency with HWE  and had an overall mutant allele frequency of 0.30 (V). FcγRIIIB-NA1/NA2 genotypes distribution in the SMA group were 6 (5.3%) NA1, 73 (64.0%) NA1/NA2 and 35 (30.7%) NA2, while in non-SMA there was 8 (5.0%) NA1, 94 (58.8%) NA1/NA2 and 58 (36.2%) NA2. The distributions of the genotypes in both SMA and non-SMA revealed deviation from HWE normality . In addition, HWE deviation was revealed by the FcγRIIIB-NA1/NA2 genotypes’ distribution considering the whole study group  with variant allele frequency of 0.36 (NA1), Table Chi square χ analysisP = 0.077). Further analysis did not reveal association between SMA using the additive  or the recessive model . The dominant  and the additive  model of the FcγRIIIA-176 F/V dimorphism did not show associations with SMA. However, the recessive model of FcγRIIIA-176 F/V showed a trend towards protection against SMA, albeit with marginal significance . Analysis of all the genetic models of FcγRIIIB-NA1/NA2 variation did not reveal any association with SMA; dominant , additive  and recessive  relative to non-carriage , P = 0.009), was associated with higher parasitemia. Additional analysis showed that the level of parasitemia was comparable between the carriage and non-carriage of FcγRIIA-131Arg/FcγRIIIA-176F/FcγRIIIBNA2 haplotype  versus non-carriage , P = 0.973) and FcγRIIA-131His/FcγRIIIA-176F/FcγRIIIBNA2  versus non-carriage . Further analysis also showed that the level of parasitemia was also comparable between those with FcγRIIA-131His/FcγRIIIA-176 V/FcγRIIIBNA1 haplotype  versus those without the haplotype , P = 0.587), Fig. Since the FcγRs are important determinants in phagocytosis of parasites, we determined if carriage of FcγRs haplotypes was associated with parasitemia levels. Results revealed that carriage of FcγRIIA-131His/FcγRIIIA-176F/FcγRIIIBNA1 haplotype [mean (SEM); 4.37 (± 0.079), P. falciparum infections. The current study demonstrated that the FcγRIIA-131Arg/ FcγRIIIA-176F/FcγRIIIBNA2 haplotype was associated with an increased susceptibility to SMA, while the FcγRIIA-131Arg/ FcγRIIIA-176F/ FcγRIIIBNA1 haplotype was associated with increased levels of circulating parasites during infection. However, there was no association between the individual genotypes and SMA in this pediatric population from western Kenya.Based on the observations that Fc gamma receptors (FcγRs) are important contributory factors for infectious disease immuno-pathogenesis, the association between the FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 polymorphisms and pediatric severe malaria anemia  was determined. We further assessed whether the carriage of different haplotypes of FcγRs were associated with parasite levels in 2 as opposed to FcγRIIA-131Arg/Arg. In addition, the IgG2 and IgG3 antibodies have been shown to confer resistance to malaria by some studies [The FcγRs constitute a crucial arm of host immune defense against extracellular challenges by infectious agents through engagement of IgGs to enable innate immune effectors cells carry out phagocytosis and other downstream processes leading to immunity , 31. Som studies , 35. In  studies  in Ghana studies , 37. TheThe FcγRIIIA-176F/V gene displays a functional allelic polymorphism that generates allotypes exhibiting different receptor properties . Our stuIt is important to note that FcγRs function synergistically, especially via crosslinking, resulting in phagocytosis of immunoglobulin-opsonized immune complexes or through stimulation of neutrophil granulation leading to production of reactive oxygen species (ROS) , 42. MorThe finding that the FcγRIIA-131His/FcγRIIIA-176F/FcγRIIIBNA1 haplotype was associated with higher parasitemia levels is fascinating given the fact that the FcγRIIA-131His/His and FcγRIIIB-NA1 allotypes in this haplotype construct are associated with effective binding to cytophillic IgGs , leadingP. falciparum  that does not affect the neighboring FcγRIIA and FcγRIIIA genes. This, however, remains to be determined most preferably by whole genome sequencing so as to develop a conclusive explanation.Differences in the allelic frequencies of the FcγRs SNPs observed in the current study likely suggest their indirect influence on malaria susceptibility and pathogenesis in the current population. The deviation from HWE of FcγRIIIB NA1/NA2 genotypes in the current study remains consistent with the results of FcγRIIIB genetic polymorphisms performed in our previous reporting in which we included 528 children . It is lIn summary, the current study demonstrates that FcγRs haplotypes, but not individual genotypes are associated with malarial disease severity, demonstrating the combinatorial effects of FcγRs on influencing clinical malaria outcomes. Future studies aimed at longitudinally measuring immune complexes over time will help to delineate the important role of FcγR haplotypes on susceptibility to severe malaria in pediatric populations.


Hypoxia is a typical character of locally advanced solid tumours. The transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator under the hypoxic environment. HIF-1α regulates various genes to enhance tumour progression, angiogenesis, and metastasis. Sphingosine kinase 1 (SPHK-1) is a modulator of HIF-1α.To investigate the molecular mechanisms of pristimerin in association with SPHK-1 pathways in hypoxic PC-3 cancer cells. Vascular endothelial growth factor (VEGF) production, cell cycles, and SPHK-1 activity were measured, and western blotting, an MTT assay, and an RNA interference assay were performed.Pristimerin inhibited HIF-1α accumulation in a concentration- and-time-dependent manner in hypoxic PC-3 cells. Pristimerin suppressed the expression of HIF-1α by inhibiting SPHK-1. Moreover, inhibiting SPHK-1 with a sphingosine kinase inhibitor enhanced the suppression of HIF-1α, phosphorylation AKT, and glycogen synthase kinase-3β (GSK-3β) by pristimerin under hypoxia. Furthermore, a reactive oxygen species (ROS) scavenger enhanced the inhibition of HIF-1α and SPHK-1 by pristimerin.Taken together, these findings suggest that pristimerin can exert an anti-cancer activity by inhibiting HIF-1α through the SPHK-1 pathway.The online version of this article (doi:10.1186/s12885-016-2730-2) contains supplementary material, which is available to authorized users. Hypoxia is a common characteristic of locally advanced solid tumours  and up tThe hypoxia inducible factor-1 (HIF-1) is a transcription factor and also a key factor that maintains oxygen homeostasis in mammalian cells . HIF-1 iPristimerin is a naturally occurring triterpenoid quinone methide , 25. SevHere, we demonstrate that pristimerin inhibits HIF-1α via the SPHK-1 signaling pathway in a prostate cancer cell lines. The results we have yielded provide the mechanism for inhibitory action of HIF-1α and angiogenesis by pristimerin in hypoxic prostate cancer cell lines.Pristimerin (purity: ≥98 % as determined by HPLC) was purchased from Sigma Aldrich .2 incubator at 37 °C. The cells cultured under hypoxia were grown in a hypoxic chamber  containing 1 % oxygen, 5 % carbon dioxide, and 94 % nitrogen at 37 °C.The human castration-resistant prostate cancer cell lines PC-3 and DU145 cells were preserved in RPMI1640 , supplemented with 10 % FBS and 1 % antibiotics . The human androgen responsive prostate cancer cell line LNCaP was maintained in RPMI1640, supplemented with 25 % HEPES , 10 % FBS and 1 % antibiotics . Normoxically conditioned cells were cultured in a 5 % CO4 per well) were seeded in 96-well plates  and treated with various concentrations  of pristimerin. After 24 h, 50 μL of MTT reagent (1 mg/mL) was added. After incubation for 1 h, optical density was measured by an ELISA-Reader  at a wavelength of 570 nm.A colorimetric 3--2,5-diphenyltetrazolium bromide (MTT) assay  was used to assess cell viability. Cells . The protein extract were separated on SDS–polyacrylamide gels and were electrotransferred to a nitrocellulose membrane . The membranes were blocked in 5 % non-fat dry milk and probed with primary antibodies for SPHK-1 , HIF-1α , AKT , p-AKT , GSK-3β , p-GSK-3β , VEGF , PCNA , PI3K , and β-actin  overnight at 4 °C and HRP-conjugated secondary antibodies. Detection of specific proteins was carried out with an enhanced chemiluminescence (ECL) assay .To measure sphingosine kinase activity, sphingosine kinase activity assay kit  was used. The Sphingosine kinase activity assay method was previously described in our other study , 38. ProVEGF ELISA kit  was used to assess VEGF levels in pristimerin and/or SKI exposed PC-3 cells. The VEGF production level measurement methods was previously described in our other study . The culThe cell cycle was determined according to the protocol described previously . Cells wThe siRNA transfection method was previously described in our other study , 38. A pP <0.05 was considered to indicate statistical significance.The data showed as means ± S.D. (standard deviation) of three replications each experiment in this study. Analysis of variance (ANOVA) was used to assess the significance of differences between groups. To measure whether pristimerin affects cell viability under hypoxic and normoxic conditions, cells were treated with various concentrations of pristimerin in PC-3 cells under hypoxia or normoxia for 24 h. Pristimerin significantly decreased cell viability under hypoxia than it did under normoxia Fig. . As showTo examine whether pristimerin inhibits hypoxia-induced HIF-1α, pristimerin was treated into PC-3 cells under hypoxia for 4 h. As shown in Fig. To investigate whether pristimerin affects SPHK-1 in PC-3 cells, the cells were incubated under hypoxia for 4 h with 0.5 or 1 μM of pristimerin. Pristimerin at 1 μM reduced SPHK-1 to 55 % under hypoxia compared with the control Fig. . As SPHKTo confirm the involvement of SPHK-1 in the pristimerin-mediated inhibition of HIF-1α during hypoxia, the effects of pristimerin was evaluated by using SPHK-1 siRNA and an SPHK-1 inhibitor, on SPHK-1 activity and the phosphorylation of AKT and GSK-3β. This is because the SPHK-1 dependent stabilization of HIF-1α is known to be mediated by AKT/GSK-3β, downstream of SPHK-1. The phosphorylation of AKT and GSK-3β was induced under hypoxia Fig. . PristimTo examine whether ROS mediate pristimerin-induced inhibition of HIF-1α and SPHK-1, PC-3 cells were treated with pristimerin or/and NAC. The treatment with either pristimerin or NAC reduced hypoxia-mediated HIF-1α, and SPHK-1 expressions and activity Fig. .As shown in Fig. As shown in Fig. PC-3 cells were treated with SKI and pristimerin for 48 h under hypoxic conditions. Treatment with pristimerin and SKI significantly increased G1-arrest and decreased the expression of G1 regulatory proteins, such as cylinD1 and CDK4, in hypoxic PC-3 cells Fig. . PCNA isMost solid tumours are more aggressive and resistant to chemotherapy or radiation under hypoxic conditions , 41. HypN-acetylcysteine (NAC), an ROS scavenger, suppresses HIF-1α by blocking SPHK-1 under hypoxia.Hypoxia leads to an increase in mitochondrial production of ROS,  and ROS To confirm whether pristimerin suppresses hypoxia-induced HIF-1α accumulation via the inhibition of SPHK-1 and ROS generation in prostate cancer cells, we evaluated the effect of NAC on HIF-1α and SPHK-1 abundance in hypoxic PC-3 cells, treated with pristimerin. The co-treatment of pristimerin with NAC affected HIF-1α and SPHK-1 abundance.PI3K is necessary for cell growth and survival, and PI3K can be activated by growth factors binding to cell surface receptor and hypoxia. PI3K induces the accumulation, activation, and stabilization of HIF-1α proteins during hypoxia in cancer cells . To confThere is evidence that HIF-1α can regulate VEGF secretion in cancer cells , 58. In Our study shows that pristimerin inhibits HIF-1α, SPHK-1 expression or activity, and phospho-AKT/GSK-3β and decreases VEGF production in hypoxic PC-3 cells. These results suggest that pristimerin may inhibit HIF-1α accumulation by inactivation of SPHK-1 including the free radical scavenging effect in PC-3 cells under hypoxia.


In this study, a successive treatment with dilute acid (0.25–1.0 % aqueous H2SO4 concentration (0.5 %) for a reaction time of 2.0 h. During the alkali post-treatment, 14.2 g lignin with a higher purity of 99.2 % and 30.3 g glucose with a higher conversion rate by enzymatic hydrolysis were obtained from the successively treated substrates with 100 g RS as starting material. As the pretreatment temperature, H2SO4 concentration, or time increased, more β-O-4 linkages in lignins were cleaved, which resulted in an increase of phenolic OH groups in lignin macromolecules. The signal intensities of G2 and G6 in HSQC spectra gradually reduced and vanished, indicating that a condensation reaction probably occurred at C-2 and C-6 of guaiacyl with the side chains of other lignin.During the dilute acid pretreatment, the maximum production of XOS (12.8 g XOS/100 g RS) with a relatively low level of byproducts was achieved at a relatively low temperature (130 °C) and a low HThe present study demonstrated that the successive treatments with dilute acid and alkali had a synergetic effect on the fractionation of the three main constituents in RS. It is believed that the results obtained will enhance the availability of the combined techniques in the lignocellulosic biorefinery for the application of the main components, cellulose, hemicelluloses, and lignin as biochemical and biofuels.The online version of this article (doi:10.1186/s13068-016-0632-9) contains supplementary material, which is available to authorized users. A significant type of biomass burning is a common way to eliminate waste after harvesting in China, such as in Shandong and Hebei Provinces, which is an important source of greenhouse gases and particulate pollutants in the atmosphere and has a remarkable effect on global atmospheric chemistry and global warming , 2. To aThe ultimate goal of rice straw separation according to the biorefinery is to obtain the fractionation of the three primary constituents via an effective and economical process, in which the three fractions can be converted into multiple biobased products, respectively. To date, a series of biorefinery processes for biomass including dilute acid, alkali, and steam explosion treatments have been investigated –12. AmonAs far as we know, although both acid and alkali pretreatments for the enzymatic hydrolysis of biomass have been intensively studied –20, very2SO4, 100–150 °C, 0.5–3.0 h) and alkali  treatments was proposed to enhance availability for the application of three major components  as biochemical and biofuels . The composition of XOS was determined by a high-performance anion-exchange chromatography (HPAEC). The yield, purity, molecular weight, and structural transformation of alkaline lignin isolated have been thoroughly investigated. Meanwhile, the cellulose-rich substrates were characterized by chemical constituent, scanning electron microscopy (SEM), Fourier transform infrared (FT-IR), and X-ray diffraction (XRD), and the yield of glucose was also determined. These results will provide useful information in the utilization of rice straw for the value-added biochemicals or bioethanol in biorefinery industry.In this study, a combination of successive dilute acid , the characteristic bands of acetyl ester units of hemicelluloses at 1728 (C=O conjugates) and 1246 cm−1 (C–O) were observed by FT-IR spectra. As elevating severity, the intensities of the two bands were gradually reduced due to the deacetylation of hemicelluloses during the pretreatment [−1 in the post-treated substrates revealed that alkali treatment completely cleaved the ester bands of hemicelluloses, such as acetyl and uronic ester groups [A biorefinery with successive dilute acid and alkali treatments was applied to enhance the value-added application of RS in this study. The scheme of the biorefinery process is shown in Fig. reatment . In ordereatment  demonstrreatment  also poiThe morphological changes of the pretreated and post-treated substrates were measured by SEM images , which was probably related to the partial degradation of crystalline cellulose. As expected, the further alkali treatment resulted in higher CrI values in the post-treated substrates, which was ascribed to the removal of lignins during the alkali post-treatment. Bi et al. [Crystallinity is an essential factor that reflects the hydrolysis properties of substrates. The crystalline region hinders cellulases accessing to the cell walls of fibers, resulting in the reduction of cellulose hydrolysis . The cry2SO4, the yield of XOS increased. However, further prolonging the reaction time to 3.0 h, the yield of XOS rapidly reduced. Interestingly, the effect of dilute acid concentration on yield of XOS showed a similar tendency. It was found that a maximum yield of XOS was achieved (12.8 g XOS/100 g RS) at 130 °C with 0.5 % H2SO4 for 2.0 h in this study. Recently, a relatively high XOS production from oil palm empty fruit bunch by hot compressed water pretreatment was studied [The XOS production was influenced by the pretreatment conditions Fig. . As the  studied . Results2SO4 concentration, or time. It was found that the maximum concentration of all the degraded byproducts was observed at 150 °C with 0.5 % H2SO4 for 2.0 h. This indicated that the pretreatment temperature was more effective than the H2SO4 concentration and reaction time on the degraded byproducts from the polysaccharides. Due to the existence of the degraded byproducts from polysaccharides during the dilute acid pretreatment, the liquor phase was not a very pure source of XOS. In industry, to purify and then produce commercial XOS, the liquors have to be refined by removing non-XOS compounds [Pinus pinaster wood, and forest waste [When XOS was produced by the dilute acid pretreatment, a variety of other compounds, such as formic acid, acetic acid, levulinic acid, lactic acid, hydroxymethylfurfural (HMF), and furfural was produced and appeared in the reaction media. The concentrations of the degraded byproducts of polysaccharides in the liquor phase are listed in Additional file ompounds . In the ompounds –36. Amonompounds . In thisst waste –39.2SO4 concentration, or time. The increasing trend was related to the dilute acid pretreatment conditions, which cleaved the chemical bonds between lignin and hemicelluloses to different degrees [Mw) of control lignin was 2910 g mol−1, and its polydispersity (Mw/Mn) was 1.87. During the pretreatment, it was observed that the Mw (2320–2870 g mol−1) of the lignins reduced slightly. This suggested that the lignins underwent a slight depolymerization under the conditions given [The yields of alkali lignins, which were calculated based on the lignins in respective substrate, are shown in Additional file  degrees . Thus, tns given . In addiβ-ether  and phenylcoumaran  are identified due to the presence of cross-peaks at δC/δH 71.7/4.83 (Aα), 59.7/3.54 (Aγ), and 62.7/3.55 (Bγ), respectively. The cross-peaks of methoxy groups in the lignins  were clearly observed. The aromatic lignin units syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) units showed prominent correlations at δC/δH 103.8/6.70 , 110.7/6.95 (G2), 115.3/6.76 (G5), 118.9/6.76 (G6), and 127.9/7.20 , respectively. Minor amounts of oxidized S units (S′) were detected due to the presence of a correlation at δC/δH 106.0/7.30 . Moreover, the lignins obtained appeared to contain small amounts of p-hydroxycinnamates (p-coumaric and ferulic acids). The p-coumaric acid (PCA) was characterized by some relatively intense correlations at δC/δH 129.7/7.50  and 143.9/7.48 (PCA7), while ferulic acid (FA) was found at δC/δH 110.9/7.27 (FA2), 116.6.0/6.39 (FA8), and 143.9/7.48 (FA7). As can be seen, as the pretreatment temperature, H2SO4 concentration, or time increased, some changes appeared in the HSQC spectra of lignins. For example, the correlated signals of β-O-4 were diminished, and the correlated signals of PCA and FA were reduced simultaneously. Interestingly, the signal intensities of C2–H2 and C6–H6 in G unit were decreased gradually and disappeared under the harsh conditions, which is attributable to the fact that the condensation reaction probably occurred at C-2 and C-6 of G unit in lignin with the side chains of other lignin [To further reveal the structural transformations of the lignins isolated during the various pretreatment conditions, the samples were investigated by 2D-HSQC NMR spectra Figs. , 4 and tr lignin .Fig. 32D31P NMR based on the integration area of individual peaks . Figure 2SO4 for 2.0 h. Enhancement of the pretreatment time, temperature, or H2SO4 concentration reduced the yield of XOS and increased the concentrations of the byproducts. Based on the above process condition, during the further alkali post-treatment, a relatively high yield of lignin (45.58 %) and cellulose-rich substrate (82.29 % cellulose) were collected. For the cellulose-rich substrate, the glucose yield reached to 88.4 % after enzymatic hydrolysis for 72 h. Process yield was normalized to a common basis of 100 g of dried RS as the starting material  was obtained with a relatively low level of byproducts under the conditions of 130 °C with 0.5 % Hial Fig. . By the 2SO4 for 2.0 h. On further alkali treatment, 14.2 g lignin with a higher purity of 99.2 % and 30.3 g glucose by enzymatic hydrolysis were also obtained from the successively treated substrates per 100 g starting material. The cleavage of β-O-4 linkages of lignin resulted in an increase of the content of phenolic OH groups with the elevating severity. During the treatment processes, the C-2 and C-6 of guaiacyl were condensed with the side chains of other lignin. The successive treatments of rice straw for production of XOS, lignin, and glucose can be further extended to the value-added application of various lignocellulosic biomasses.Successive treatments with dilute acid and alkali have been performed for the production of XOS, lignin, and cellulose-rich substrates for bioethanol production from rice straw. During the dilute acid pretreatment, a maximum production of XOS (12.8 g XOS/100 g RS) with a relatively low level of byproducts was obtained at 130 °C with 0.5 % HRS was obtained from Shandong Province of China. RS was firstly dried in an oven at 60 °C for 24 h and then ground in a mill to obtain a 40–60 mesh powder. Then, the powder was extracted with toluene–ethanol  in a Soxhlet apparatus for 6 h to remove extractives and then dried in an oven at 60 °C for 24 h to serve as control substrate (control-S). The chemical composition of control-S  was determined to be cellulose 38.29 %, hemicelluloses 27.46 %, and lignin 20.50 %, according to National Renewable Energy Laboratory’s (NREL) standard analytical procedure. All chemicals were analytical grade and purchased from Sigma–Aldrich and Megazyme.2SO4 for 1.0 h; 130 °C with 0.5 % H2SO4 for 0.5, 1.0, 1.5, 2.5, and 3.0 h; and 130 °C with 0.25, 0.6, 0.7, 0.8, and 1.0 % H2SO4 for 2.0 h, respectively) were performed in a batch reactor  at a solid-to-liquor ratio of 1:15 (g/mL). The reactor was made of Hastelloy C-276 to mitigate the effects of acid corrosion at high temperatures. The substrates pretreated with dilute acid were filtered off with a Buchner funnel, washed thoroughly with distilled water, and further dried in a cabinet oven with air circulation at 60 °C for 16 h and then labeled as S(100-0.5-2), S(110-0.5-2), S(120-0.5-2), S(130-0.5-2), S(140-0.5-2), S(150-0.5-2), S(130-0.5-0.5), S(130-0.5-1), S(130-0.5-1.5), S(130-0.5-2.5), S(130-0.5-3), S(130-0.25-2), S(130-0.6-2), S(130-0.7-2), S(130-0.8-2), and S(130-1-2), respectively, corresponding to temperature, H2SO4 concentration, and time of pretreatment.Control-S was successively treated by dilute acid and alkali. The dilute acid pretreatments , L(110-0.5-2), L(120-0.5-2), L(130-0.5-2), L(140-0.5-2), L(150-0.5-2), L(130-0.5-0.5), L(130-0.5-1), L(130-0.5-1.5), L(130-0.5-2.5), L(130-0.5-3), L(130-0.25-2), L(130-0.6-2), L(130-0.7-2), L(130-0.8-2), and L(130-1-2) according to the pretreatment conditions of the temperature, the H2SO4 concentration, and the time. Control lignin  was also isolated from the control-S under the same alkali treatment condition. Meanwhile, the alkaline post-treated substrates obtained from the corresponding pretreated substrates were filtered off with a Buchner funnel, washed thoroughly with distilled water, and further dried in a cabinet oven with air circulation at 60 °C for 16 h and then labeled as R(100-0.5-2), R(110-0.5-2), R(120-0.5-2), R(130-0.5-2), R(140-0.5-2), R(150-0.5-2), R(130-0.5-0.5), R(130-0.5-1), R(130-0.5-1.5), R(130-0.5-2.5), R(130-0.5-3), R(130-0.25-2), R(130-0.6-2), R(130-0.7-2), R(130-0.8-2), and R(130-1-2), respectively. As a control substrate, control-R was also obtained from control-S under the same alkali treatment condition without dilute acid pretreatment. All of the prepared substrates  were used to produce glucose by enzymatic hydrolysis in the present study.A further treatment was performed with 1.5 % aqueous NaOH at 80 °C for 3 h under a solid-to-liquid ratio of 1:10 (g/mL) to isolate lignin from the pretreated substrates and obtain post-treated substrates (cellulose-rich substrates). The collected liquid fractions were concentrated to 40 mL and poured into 200 mL of acidic water  to precipitate the alkali-soluble lignins. The precipitations were centrifuged, freeze-dried, and then named as L®CTec2, 17 FPU/g substrate) was provided by Novozymes North America, Inc.  and employed for all the enzymatic hydrolyses. The hydrolyzates were sampled periodically and analyzed by a HPAEC  system equipped with an integral amperometric detector and CarboPac PA 100  analytical column. The detailed procedures were previously described in a publication [Enzymatic hydrolysis was performed on the substrate (2 % w/v) in 10 mL 50 mM sodium acetate buffer (pH 4.8) with a 25 mL Erlenmeyer flask at 50 °C in a double-layer shaking incubators (ZWYR-2102C)  at 150 rpm for 72 h. Commercial enzyme  with Ni-filtered Cu Kα radiation (χ = 1.54 Å) generated at 40 kV and 30 mA. The scattering angle (2θ) was from 10 to 36° at a scanning speed of 2°/min. The CrIs of the substrates were calculated from the ratio of the crystalline peak area to the total area of crystalline and amorphous peaks.The chemical compositions  of all the substrates were determined by NREL standard analytical procedure. The content of cellulose and hemicelluloses in the substrates was determined by HPAEC system on a CarboPac PA20  analytical column. SEM images were performed with a S-3400 N II  instrument at 10 kV and 81 mA. FT-IR spectroscopic measurements were conducted on a Nicolet iN10 spectrophotometer in the range of 1760–860 cm2SO4 at 121 °C for 1 h to determine the total concentration of XOS. The increased concentrations of monosaccharides after post-hydrolysis were related to the concentration of XOS [2SO4 solution with a volumetric flow rate of 0.5 mL/min. Column temperature was set at 50 °C. The temperature of RI detector was 40 °C and injection volume was 10 μL.The liquor obtained from dilute acid pretreatment was stored and filtered to determine XOS and byproducts . Liquor sample of 3 mL was post-hydrolyzed with 4 % Hn of XOS . Accordid6. Functional groups  of the lignins were determined by 31P NMR spectra according to a previous publication [The associated polysaccharides in the lignins were calculated using HPAEC as reported previously . Molecullication .


S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) was identified as an endogenous ligand for leukotriene B4 receptor 2 (BLT2), which is important in maintenance of epithelial homeostasis. We hypothesized that NSAID-dependent corneal damage is caused by reduced production of 12-HHT. Diclofenac eye drops decreased the abundance of downstream products of COX and delayed corneal wound healing in BALB/c mice. Expression of BLT2 was observed in murine ocular tissues including cornea, and in human corneal epithelial cell line and human primary corneal epithelial cells. In BLT2-knockout mice, corneal wound healing was delayed, but the diclofenac-dependent delay in corneal wound healing disappeared. 12-HHT accelerated wound closure both in BLT2-transfected corneal cell line and human primary corneal epithelial cells. Thus, our results reveal that NSAIDs delay corneal wound healing by inhibiting 12-HHT production, and suggest that stimulation of the 12-HHT/BLT2 axis represents a novel therapeutic approach to corneal wound healing.Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce inflammation by suppressing cyclooxygenases (COXs). NSAID eye drops are frequently prescribed after ocular surgery to reduce inflammation and pain, but this treatment has clinically significant side effects, including corneal ulcer and perforation. The molecular mechanisms underlying these side effects remain unknown. Recently, the COX product 12( In mice, BLT2 is expressed in small intestine, colon, and skin, whereas it is expressed ubiquitously in human2. We recently generated BLT2-deficient mice and used them to analyze the in vivo function of BLT2. Using BLT2-knockout (KO) mice, we identified the crucial roles of BLT2 in barrier function in intestine and skin4. Subsequently, we reported that BLT2 potentiates epithelial cell adhesion by up-regulating tight junctional proteins, including claudin 45. Although LTB4 was assumed to be the ligand for BLT2, we recently identified 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), a compound without known biological functions, as the endogenous ligand for BLT26. 12-HHT is synthesized from arachidonic acid by cyclooxygenase (COXs) and/or thromboxane A2 synthase (TXA2S), and its production is suppressed by non-steroidal anti-inflammatory drugs (NSAIDs)7. Previous reports suggested that the 12-HHT/BLT2 axis plays important roles, especially in tissues exposed to the external environment, such as intestine and skin. Currently, however, the function of the 12-HHT/BLT2 axis in the eye, which also faces the external environment, remains unknown.Leukotriene B10. However, the molecular mechanisms by which NSAIDs delay corneal wound healing are unclear. Based on the observation of BLT2 expression in murine and human corneal epithelium, we hypothesized that delayed corneal wound healing by NSAIDs might be due to reduced production of 12-HHT and subsequent inhibition of BLT2 signaling. We investigated this possibility in this study.NSAID-containing eye drops are used clinically to suppress inflammation after intraocular surgery. Despite its importance and utility, topical eye application of NSAIDs can cause corneal epithelial injuries, ulceration, and perforation, and can also delay corneal epithelialization9, this phenomenon has not been explored experimentally in a detailed manner. To address this issue, we generated a murine model of corneal wound healing and used it to examine the effects of NSAIDs. In BALB/c mice, corneal wound area decreased in a time-dependent fashion, with complete wound closure occurring after 32 hr  . All eicosanoids detected in murine eye disappeared after treatment with diclofenac , thromboxane Alow Fig. , suggestI2) Fig. , but leunac Fig. , and shu2, PGE2, 6-keto-PGF1α, and PGF2α in whole murine eye . Because BLT2 was the only receptor we detected in murine cornea   Fig. . In addior) Fig. . Furtheror) Fig. . Collectin vitro and in vivo  and neuroprotectin D1 (NPD1), but not pro-inflammatory lipid mediators such as LTB4 and 12R-hydroxyeicosatrienoic acid, play important roles in corneal wound healing by recruiting neutrophils21. Lipid mediators such as LXA4 and NPD1 are produced by 12/15-lipoxygenase–dependent pathways, which are not inhibited by NSAIDs. Corneal wound healing is a multi-step process that includes cell proliferation, migration, differentiation, stratification, and desquamation/apoptosis23. Therefore, to efficiently cure corneal wounds, multiple lipid mediators are required. Accordingly, in future work we plan to investigate the role of other lipid mediators, including lysophospholipids, sphingosine 1-phosphate (S1P), and pro-resolving lipid mediators27.In addition, our results revealed the critical role of the COX-dependent lipid mediator 12-HHT in corneal epithelial wound healing. Previous reports showed that Lipoxin Ain vitro and in vivo. These findings resolve a longstanding problem in ophthalmology, and suggest a novel therapeutic target for corneal damage.In summary, our results provide evidence that the 12-HHT/BLT2 axis promotes corneal epithelial cell migration, and that inhibition of 12-HHT production by NSAIDs delays corneal wound healing 4. Sex matched BLT2 KO and BLT2 WT mice, 11–13 weeks old, were used for the experiments. For the diclofenac treatment experiments, 10–12 weeks old female BALB/c mice were purchased from Charles River Inc . All animals were reared under specific pathogen–free conditions and provided with food and water ad libitum.BLT2 KO mice were generated as previously described, and backcrossed with BALB/c mice for more than 12 generationsAll animal experiments were approved by the Ethical Committee for Animal Experiments in Juntendo University. All methods in this manuscript were carried out in accordance with the approved guidelines and regulations.i.e., two eyes/sample), and total RNA was extracted using the RNeasy Fibrous Tissue Kit (Qiagen) according to the manufacturer’s instructions. Total RNA (1 µg) was subjected to reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen) with random and oligo-dT mixed primers. For the human corneal epithelial cell line and primary human corneal epithelial cells, total RNA was extracted from 1 × 106 cells using the Trizol reagent (Life Technologies). Total RNA (1 µg) was subjected to reverse transcription using the same kit. Target genes were detected by real-time PCR using Fast SYBR Green Master Mix (Applied Biosystems) on an ABI7500 Real-Time PCR system (Life Technologies). Semi-quantitative PCR was performed by using KOD -Plus- (TOYOBO). Condition of PCR was as follows: mouse BLT2 and mouse β-actin; 92 °C 2 min, 35cycles of 94 °C 48 sec, 60 °C 42 sec, 68 °C 30 sec and 68 °C 10 min, mouse DP; 94 °C 2 min, 35cycles of 94 °C 48 sec, 62 °C 42 sec, 68 °C 48 sec and 68 °C 10 min, mouse IP and mouse FP; 94 C° 2 min, 35cycles of 94 °C 48 sec, 62 °C 42 sec, 68 °C 59 sec and 68 °C 10 min, mouse EP1; 94 °C 2 min, 35cycles of 94 C° 48 sec, 65 °C 42 sec, 68 °C 48 sec and 68 °C 10 min, mouse EP2; 94 °C 2 min, 35cycles of 94 °C 48 sec, 65 °C 42 sec, 68 °C 48 sec and 68 °C 10 min, mouse EP4; 94 °C 2 min, 35cycles of 94 °C 48 sec, 64 °C 42 sec, 68 °C 25 sec and 68 °C 10 min, mouse TP; 94 °C 2 min, 35cycles of 94 °C 45 sec, 64 °C 42 sec, 68 °C 90 sec and 65 °C 10 min.Tissue samples, including skin, intestine, eye, cornea, conjunctiva, lens, and retina, were collected from five mice  Fw, 5′-TACCACGCAGTCAACCTTCTG-3′; human BLT2 Rv, 5′-GCGGTGAAGACGTAGAGCAC-3′; human GAPDH Fw, 5′-TGCCCTCAACGACCACTTTG-3′; human GAPDH Rv, 5′-CTCTTCCTCTTGTGCTCTTGCTG-3′; mouse cadherin 1 (Cdh1) Fw, 5′-CAGGTCTCCTCATGGCTTTGC-3′, mouse cadherin 1 (Cdh1) Rv, 5′-CTTCCGAAAAGAAGGCTGTCC-3′; mouse cadherin 2 (Cdh2) Fw, 5′-ATAGCCCGGTTTCACTTGAGA-3′, mouse cadherin 2 (Cdh2) Rv, 5′-CAGGCTTTGATCCCTCTGGA-3′; mouse vimentin (Vim) Fw, 5′-CGGCTGCGAGAGAAATTGC-3′, mouse vimentin (Vim) Rv, 5′-CCACTTTCCGTTCAAGGTCAAG-3′; mouse BLT2 (Ltb4r2) Fw, 5′- ACAGCCTTGGCTTTCTTCAG-3′; mouse BLT2 Rv, 5′-TGCCCCATTACTTTCAGCTT-3′; human BLT2 (LTB4R2) Fw, 5′-TACCACGCAGTCAACCTTCTG-3′; human BLT2 Rv, 5′-GCGGTGAAGACGTAGAGCAC-3′; mouse β-actin (Actb) Fw, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′; mouse β-actin (Actb) Rv, 5′-ATGGAGCCACCGATCCACA-3′; mouse EP1 (Ptger1) Fw, 5′-CGTCGCTCTCGACGATTCCGAAAGACCGCA-3′, mouse EP1 (Ptger1) RV, 5′-CGATGGCCAACACCACCAACACCAGCAGGG-3′; mouse EP2 (Ptger2) Fw, TTCATATTCAAGAAACCAGACCCTGGTGGC-3′, mouse EP2 (Ptger2) Rv, 5′-AGGGAAGAGGTTTCATCCATGTAGGCAAAG-3′; mouse EP4 (Ptger4) Fw, 5′-TTCCGCTCGTGGTGCGAGTGTTC-3′, mouse EP4 (Ptger4) Rv, 5′-GAGGTGGTGTCTGCTTGGGTACG-3′; mouse TP (TbxA2r) Fw, 5′-TGCCTTGTTGGACTGGCGAGCCACTGACCC-3′, mouse TP (TbxA2r) Rv, 5′-CAGGTAGATGAGCAGCTGGTGCTCTGTGGC-3′; mouse IP (Ptgir) Fw, 5′-CCTGCAGTGTTTGTGGCCTATGCTCGAAAC-3′, mouse IP (Ptgir) Rv, 5′-CTGCTGTCTGGGGCGATGGCCTGAGTGAAG-3′; mouse DP (Ptgdr) Fw, 5′-AAAGGAACTGCTGCCTGCCTCAGGCAATCA-3′ mouse DP (Ptgdr) Rv, 5′-GTTCTCAAGTTTAAAGGCTCCATAGTACGC-3′; mouse FP (Ptgfr) Fw, 5′-GCATAGCTGTCTTTGTATATGCTTGTGATA-3′, mouse FP (Ptgfr) Rv, 5′-GTGTCGTTTCACAGGTCACTGGGGAATTAT-3′.Primer sequences were as follows: human BLT2 . The column was subsequently washed with petroleum ether and water containing 0.1% formic acid. The samples were eluted with 200 µl of methanol containing 0.1% formic acid. Eicosanoids in each sample were quantified by LC-MS/MS as described previously7. LC-MS/MS analyses were performed by using a Shimadzu liquid chromatography system and tandem-connected a TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with an electrospray ionization system (Thermo Fisher Scientific). Each sample was injected into the trap column, an Opti-Guard Mini C18 and concentrated sample was analyzed with an analytical column, a Capcell Pak C18 MGS3 . Separation of lipids was achieved by a step gradient with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The LC column eluent was introduced directly into a TSQ Quantum Ultra. All compounds were analyzed in a negative ion polarity mode. 12-HHT and eicosanoids were quantified by selective reaction monitoring (SRM); m/z 279  → 179 (daughter ion MS) for 12-HHT. A mixture of deuterium-labeled eicosanoids was used as internal standards. Because deuterium-labeled 12-HHT is not commercially available, 12-HHT was calibrated with deuterium-labeled LTB4. Automated peak detection, calibration, and calculation were carried out by using the XCalibur 1.2 software package.Eyes were collected after perfusion of mice with 50 ml of saline containing 4 U/ml heparin  11 generated in our laboratory. Secondary antibody was biotin-conjugated anti-rabbit IgG, and streptavidin-conjugated horseradish peroxidase was used for the third reaction. Signals were visualized with 3,3′-diaminobenzidine (DAB). For electron microscopy, mice were euthanized, and the eyes were enucleated, fixed by immersion in 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer, postfixed with 1% OsO4, dehydrated with a graded series of ethanol, and embedded in epoxy resin. Ultrathin sections were cut with an ultramicrotome , stained with uranyl acetate and lead citrate, and observed with a Hitachi HT7700 electron microscope. To quantify the number of desmosomes, electron micrographs of superficial and basal layers of the cornea were randomly taken with a primary magnification of 5,000x. Data are expressed as the average number of desmosomes in three electron micrographs.Eyes were fixed in 10% formalin, embedded in paraffin, sectioned (10 µm thickness), and stained with hematoxylin and eosin (HE). For immunohistochemical analysis, antigen retrieval was performed by boiling the sample for 10 min in citrate buffer (pH 6.0). Primary antibody was a rabbit anti-mouse BLT2 polyclonal28. Briefly, the day before the wound was made, 1% atropine eye drops  were applied to avoid damage to the iris. After local anesthesia with oxybuprocaine hydrochloride , a trephine was used to introduce a 2 mm diameter wound in the cornea of the right eye. Epithelial defects were created using a spatula (INAMI Inc.). Each corneal wound was visualized with 1% fluorescein and monitored after surgery every 8 hr until the wound was completely healed. After wounding, levofloxacin eye drops  were used to prevent corneal infection. The area of the epithelial defect was measured using the ImageJ software. For diclofenac treatment, diclofenac was dissolved into PBS at a concentration of 0.1% (w/v). Diclofenac administration (4 times/day) started 2 days before the wound was made.The mouse model of corneal wound injury was generated as described previously® BM supplemented with growth factors (KURABO).The human corneal epithelial cell line HCET was maintained in DMEM/F12 , 10% FBS (Gibco), and 1% P/S . Primary human corneal epithelial cells were purchased from KURABO LTD  and maintained in OcuLife29 using FuGENE® HD. Forty-eight hours after transfection, medium was replaced with selection medium , and the cells were cultured for an additional 4 weeks. Cells were collected and resuspended in FACS buffer (PBS + 2% FBS), incubated with 1 µg/ml anti-FLAG antibody 2H830 at 4 °C for 60 min, and then stained with 1 µg/ml Alexa Fluor 488–conjugated goat anti-mouse IgG (Life Technologies) at 4 °C for 30 min. To isolate BLT2-expressing cells, the cells were sorted three times on a MoFlo (Beckman Coulter).HCET cells were plated on 10 cm dishes 1 day before transfection. Cells at 60% confluence were transfected with pCXN2.1-FLAG-human BLT2 (hBLT2) plasmid or pCXN2.1 (Mock)4 cells/well) and HCET cells overexpressing FLAG-hBLT2 (1 × 104 cells/well) were plated onto collagen-coated 96-well ImageLock tissue culture plates (Essen Bioscience) and cultured for 48 hr. Wounds were made with a 96-well WoundMaker (Essen Bioscience). The wounded wells were washed twice with medium and treated with 100 µl of medium containing 12-HHT. Images were acquired automatically every 3 hr until the wound was completely closed (48 hr). In wound healing assays, cells were starved for 3 hr in 0.1% BSA containing medium before treatment with 12-HHT.Primary human corneal epithelial cells (1 × 104 cells/well) were seeded in glass-bottom 96-well plates and incubated for 24 hr. Cells were loaded with 5 µM Fluo-8 AM  in HBSS (pH 7.4) buffer containing 2.5 mM probenecid and 20 mM HEPES (pH 7.4) at 37 °C for 30 min, and further incubated at room temperature for 30 min. After two washes with buffer, the cells were stimulated with HBSS containing various concentrations of ligand. Agonist-induced intracellular calcium mobilization was determined using a FLEX Station .HCET-Mock and HCET-BLT2 cells .All data are expressed as mean ± SEM. Statistical significance was evaluated by Student’s


N,N,N′,N′-tetrakis[(6-methylpyridin-2-yl)methyl]ethane-1,2-diamine-κ6N}copper(II) bis(tetrafluoridoborate) shows a distorted octahedral environment around the CuII cation. The presence of the 6-methyl substituent hinders the approach of the pyridine group to the CuII core. The bond lengths about CuII are significantly longer than those of analogues without the 6-methyl substituents.The mononuclear copper complex { N,N,N′,N′-tetrakis[(6-methylpyridin-2-yl)methyl]ethane-1,2-diamine-κ6N}copper(II) bis(tetrafluoridoborate), [Cu(C30H36N6)](BF4)2, is conveniently prepared from the reaction of Cu(BF4)2·6H2O with N,N,N′,N′-tetrakis[(6-methylpyridin-2-yl)methyl]ethane-1,2-diamine (tmpen) in acetonitrile at room temperature in air. The complex shows a distorted octahedral environment around the CuII cation (site symmetry 2) and adopts the centrosymmetric space group C2/c. The presence of the 6-methyl substituent hinders the approach of the pyridine group to the CuII core. The bond lengths about the CuII atom are significantly longer than those of analogues without the 6-methyl substituents.The mononuclear copper title complex { N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine] (TPEN) is reported as a versatile and highly active catalyst for acrylic, methacrylic and styrenic monomers ethylenediamine (bztpen) displays high catalytic activity for electrochemical proton reduction in acidic aqueous solutions, with a calculated hydrogen-generation rate constant (kobs) of over 10000 s−1 2(NO3)2](PF6)2 [m-xpt = m-xylylenebis(pyridyltriazole)] can selectively capture CO2 from air and reduce it to oxalate, in the form of an oxalate-bridged complex  Å] are significantly longer than those for the other four nitrogen atoms . The long Cu—N3 distance indicates a weak connection between copper and pyridine, which is apt to dissociate under reductive conditions , assigned to CuII/I ethylenediamine (TPEN) ligand is most similar to title complex in configuration. In the presence of ascorbic acid as a reducing agent, Cu2+(TPEN) displays high activity in atom-transfer radical addition (ATRA) reactions , the title complex exhibits greater steric hindrance, which results in an evident Jahn–Teller effect on the configuration. In the title complex, the axial Cu—N bonds to pyridyl nitrogen atoms [2.5742 (13) Å)] are significantly longer than in Cu2+(TPEN) [2.377 (3) and 2.308 (2) Å] while the differences in the equatorial Cu—N distances are negligible methyl]ethane-1,2-diamine (tmpen) was prepared according to literature procedures : δ 7.44 , 7.31 , 6.94 , 3.74 , 2.75 , 2.48 . ESI–MS: calculated for [M + H]+: m/z 481.65.19; found: 481.31.The tetrapyridinediamine ligand 4)2 (1), Cu(BF4)2·H2O  was added to an acetonitrile solution (5 ml) of tmpen . The mixture was stirred at room temperature for 6 h. The blue solution was then transferred to tubes, which were placed in a flask containing ether. Block-shaped crystals were obtained in a yield of 85% (0.25 g). Analysis calculated for C30H36B2CuF8N6 (%): C, 50.52; H, 5.09; N, 11.78; found: 50.51; H, 5.08; N, 11.75; MS (TOF–ES): m/z =272.6641 {[M − 2(BF4)−]/2}+, 579.3025 [M − 2(BF4)−+Cl−]+.For the preparation of [Cu(tmpen)](BF4 group were split into two groups and their ccupancies determined via a free variable refinement. All hydrogen atoms were refined in riding mode with C—H= 0.93–0.97 and Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017004492/pj2042sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017004492/pj2042Isup2.hklStructure factors: contains datablock(s) I. DOI: 1440025CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures have been elucidated by means of NMR and HRESIMS spectroscopic data. The new alkaloids were evaluated for their cytotoxicity against glioma initiating cell lines (GITC-3# and GITC-18#), glioblastoma cell lines (U-87MG and T98G), and lung cancer cell line A-549, but none of them was active at 20 μg/mL concentration.Three new pyridine type alkaloids, (−)-vinmajpyridines A–C (The online version of this article (doi:10.1007/s13659-017-0137-7) contains supplementary material, which is available to authorized users. Vinca (Apocynaceae), distributed through Europe, Northwest Africa, and South-west Asia, represents a group of species which are rich in indole alkaloids of diverse structural patterns, many of which are of considerable therapeutic value +  in the positive HRESIMS. Its IR spectrum revealed a characteristic absorption band at 1724 (C=O). The 1H and 13C NMR spectral data of 1  and 8.53 (s)] assigned to be pyridine aromatic protons + .(−)-vincapyridine A : C13H17N2): C12H15NO4. white amorphous powder; c 0.04, MeOH); UV (MeOH) λmax (log ε): 204 (4.44), 219 (4.13), 269 (3.57); IR (KBr) νmax 3419, 2926, 1724, 1606, 1383, 1042 cm−1; 1H and 13C NMR data, see Table m/z 238.1072 [M+H]+ .(−)-vincapyridine B (3): C11H15NO2. white amorphous powder; c 0.02, MeOH); UV (MeOH) λmax (log ε): 204 (3.35), 279 (2.64); IR (KBr) νmax 3425, 2926, 1720, 1623, 1383, 1035 cm−1; 1H and 13C NMR data, see Table m/z 194.1172 [M+H]+ .(−)-vincapyridine C (# and GITC-18# (glioma initiating cell lines) were established previously in Kunming Institute of Zoology from three different human glioblastoma multiform samples. These cell lines were cultured in serum-free medium supplemented with 1XB27 (Life 12587-010), 50 ng/mL EGF (PeproTech AF-100-15), and bFGF (PeproTech AF-100-18B). GITCs were cultured in laminin (Gibco 1725712) pre-mdish. The cells could adhere and normally grow without differentiation. Culture dishes were pre-coated with laminin for 4-6 h at 10 mg/mL concentration. The T98G, U-87MG, and A549 cell lines were purchased from the American Type Culture Collection (ATCC), the cells were cultured in DMEM basic medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37 °C and an atmosphere of 5% CO2. Cells were digested with TryplE express (Gibco 12604-021) for 3–5 min at 37 °C in cell incubator and centrifuged at 1000 rpm/min for 3 min.GITC-3#G3581] assay  assay , 21. The1–3 are available as Supplementary Information.1D and 2D NMR spectra, HRESIMS, and UV spectra of compounds Supplementary material 1 (PDF 1903 kb)Below is the link to the electronic supplementary material.