JHH modulation while suppressing JXH with broadband heteronuclear decoupling. The resultant spectra show both enhanced resolution in F2 and enhanced signal-to-noise ratio.A method for acquiring pure shift heteronuclear single quantum correlation (HSQC) NMR spectra in real time is described. A windowed acquisition scheme consisting of trains of bilinear rotation decoupling (BIRD)J) coupling provides valuable information for molecular structure elucidation, but the multiplet structure it causes is very expensive in terms of spectral resolution. In 1H NMR spectroscopy, multiplets are often many times the width of a single line. It is routine to suppress heteronuclear couplings (JXH) by broadband decoupling,1H) dimension.  dimension.Scalar spin–spin  to 13C and whose signals are recorded in HSQC. The great advantage of the BIRD method here is that, in contrast to Zangger–Sterk type methods,The HSQC experiment is the most widely used NMR method for correlating the chemical shifts of directly-bonded 1H-13C HSQC spectra,13C, should not itself show homonuclear coupling; thus, for example, the proposed sequence is not suitable for fully 13C-labeled compounds.The BIRD sequence element has already been very effectively used to obtain pure shift n BIRD/180° J-refocusing elements during the acquisition time (at) results in a free induction decay built up of an initial chunk of data of duration at/2n, (n−1) chunks of duration at/n, and a final chunk of at/2n. Provided that n≫(at×JHH), evolution under the homonuclear scalar coupling can be neglected, although care is needed to ensure that chemical shift evolution is accurately refocused during the J-refocusing element. More frequent J-refocusing gives cleaner spectra, but at the expense of some extra line broadening owing to imperfect refocusing and T2 relaxation. The BIRD real-time acquisition scheme differs slightly in timing from that previously proposed,13C, one class of coupling is not refocused, that between geminal protons. Spectra thus show singlet signals for all 1H sites except for nonequivalent methylene protons, for which doublet signals are seen (full details of the sequence are given in the Supporting Information).The pulse sequence used is shown in Figure 1H-13C correlated spectra. The conventional gHSQC spectrum -fucose shows multiplet structure in the 1H frequency (F2) dimension; the structure is collapsed to singlets in the pure shift spectrum  axis show, as expected, that the singlets in the pure shift spectrum are more intense than the corresponding multiplets in the conventional HSQC. Peak heights increase by an average factor of 1.7 for doublets and 2.9 for multiplets. Linewidths in the pure shift spectrum are very similar to those in the conventional spectrum; although signal losses from imperfect pulses, mismatch between τ and 1JCH, and transverse relaxation should, in principle, lead to wider lines in the pure shift spectrum, for this example the degradation is negligible. Similar results were obtained for quinine , will lead to small F2 sidebands at multiples of n/at. In the current work, the level of these sideband artifacts is typically around 1 % -m Figure  obtainedound 1 % .1H-15N correlation, either at natural abundance or in labeled systems where the labels are too far apart for 15N-15N coupling to be significant . Figure 15N-labeled beta-amyloid peptide 1-42 (Aβ). The shaded region in the conventional HSQC spectrum . This homonuclear decoupling produces signals with increased intensity and better resolution, lowering detection limits, speeding up experiments, and improving the ability to distinguish between signals in complex spectra. This method is potentially well-suited to automated spectral analysis, as a single signal is seen for each distinct chemical site or correlation.1H/13C/15N) triple axis gradient probe of maximum z gradient 68.5 G cm−1, using GARPB2/2π=4.2 kHz for 13C, 1.3 kHz for 15N) during data acquisition and BIPM sample of d(+)-fucose in deuterium oxide, containing trimethylsilyl propanoic acid (TSP) as internal reference. The unusually high concentration was used in order to confirm that clean results are obtainable, with artifact signals at around the 1 % level. The following experimental and processing parameters were used: a hard 90° 1H pulse of duration 10.9 μs, a hard 13C 900 pulse of duration 15.2 μs, a BIP composite 180° pulse ; homospoil gradient pulses of 23.0 G cm−1 (G1) and 13.8 G cm−1 (G3) of durations 4.0 ms (δ2) and 2.4 ms (δ4), respectively; and coherence selection (CTP) gradients of 33.4 G cm−1 (G2) and 16.8 G cm−1 (G4) of durations 2.0 ms (δ3) and 1.0 ms (δ5), respectively; 1H spectral width (sw) was 3592.0 Hz; 4 transients were averaged for each of 2×512 free induction decays in which t1 was incremented to provide a 13C spectral width of 11 467.9 Hz (sw1) in the F1 dimension; total number of points (np) stored per FID was 4104, and for Figure n was 27. Data were zero filled to 16 384×8192, and Gaussian weighting was applied before double Fourier transformation. The total experiment times were 4.2 h for Figure All experimental data were obtained using a Varian VNMRS 500 MHz spectrometer equipped with a triple resonance (15N-labeled Aβ in [D6]dimethylsulfoxide containing H2O (5 %). Experimental and processing parameters were: a hard 90° 1H pulse of duration 12.8 μs, a hard 15N 90° pulse of duration 44 μs, a BIP composite 180° pulse ; homospoil gradient pulses of 23.0 G cm−1 (G1) and 13.8 G cm−1 (G3) of durations 4.0 ms (δ2) and 2.4 ms (δ4), respectively; and coherence selection (CTP) gradients of 33.4 G cm−1 (G2) and 16.9 G cm−1 (G4) of durations 2.0 ms (δ3) and 0.4 ms (δ5), respectively; 1H spectral width (sw) was 10.0 kHz; 32 transients were averaged for each of 2×64 free induction decays in which t1 was incremented to provide a 15N spectral width of 3.0 kHz (sw1) in the F1 dimension; number of points (np) sampled per FID was 4096, and for Figure n was 8. Data were zero filled to 16 384×512 and then Fourier transformed without weighting. The total experiment time was approximately 2.7 h in each case.For Figure


Drosophila melanogaster reveals a highly conserved motif that spans the dimeric subunit interface and connects the two active sites.Analysis of a new structure of an Epsilon class glutathione transferase from  Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from  This synthase ; and in synthase .Anopheles gambiae [Anopheles funestus [Musca domestica [In insects, the Delta and Epsilon class GSTs make major contributions to detoxication of insecticides, contributing to insecticide resistance ,8. Curre gambiae ,10, two funestus  and a stomestica . TherefoDrosophila melanogaster. DmGSTE6 has significantly high activity for 4-hydroxynonenal (HNE) [In the present study, we present a novel crystal structure of an Epsilon GST (DmGSTE6) from al (HNE) . HNE wasal (HNE)  and Parkal (HNE) . HNE is al (HNE) . GSTs maal (HNE) . Within al (HNE) . Residueal (HNE) .Escherichia coli BL21 (DE3) cells. Overnight stationary-phase cultures in Luria–Bertani (LB) broth (8 ml) were used to inoculate 800 ml LB broth, both supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. The cells were allowed to grow for 3 h at 310 K and then were cooled to 291 K and induced with 0.1 mM β-D-1-thiogalactopyranoside (IPTG) and incubated overnight for 16 h. The cultures were harvested at 7000 × g for 10 min and the pellets were kept at 253 K until used. The induced culture pellet was suspended with 20 ml of PBS buffer  containing 400 μl of 100 mg/ml lysozyme, 10 mM DTT and 10 μl of 1.4 M β-mercaptoethanol by gentle vortex-mixing. The cell suspension was incubated on ice for 20 min and the crude cell lysate was obtained by sonication. The lysed cells were centrifuged at 10000 × g and 277 K for 30 min. The soluble DmGSTE6 in the supernatant fraction was purified using a GSTrap FF column. The 5 ml column was equilibrated with five column volumes (CV) of PBS buffer. The supernatant was applied to the column with a flow rate of 5 ml/min. The non-specific binding proteins were eluted with 10 CV of PBS buffer. The bound GST was eluted with five CV of elution buffer .The DNA encoding DmGSTE6 was engineered into a pET-21d(+) (Novagen) plasmid and transformed into DmGSTE6 at 15–20 mg/ml was crystallized in the presence of 10 mM GSH under a condition containing 20% PEG3350 and 0.2 M sodium thiocyanate in a 1:1 ratio by sitting drop vapour diffusion at 288 K. Protein crystals appeared within 3 days.A. gambiae (PDB: 2IMI) as the search model. Model building was performed with COOT [X-ray diffraction data were collected on beamline I03 at Diamond Light Source (DLS) with an X-ray wavelength of 0.976 Å (1 Å=0.1 nm), to a resolution of 1.72 Å. The crystal was oscillated in the beam by 0.1° per frame over a range of 360°. Data were processed and scaled using xia2  and AIMLith COOT  and restith COOT  and PHENith COOT . The datith COOT  and PyMohttp://www.psc.edu/biomed/genedoc).Sequence alignment is done by Clustal X, version 2.0.10  and dispIn the present study, we have determined the structure of DmGSTE6 to supplement the currently known Epsilon structures , Table 1101 from one subunit wraps around the His101 from the other subunit to generate the clasp motif also seen in the Delta GST class [69, interacts with clasp His101 from the other subunit , we categorized GSH-binding residues into three different groups namely the GSH glycyl moiety, the GSH glutamyl moiety and the C-terminal domain interactor (53 in DmGSTE6) is highly conserved in both Delta and Epsilon classes of GSTs .on class c and it s of GST , is closD. melanogaster DmGSTE6 has significantly high activity for HNE although not as great as reported for human GSTA4 [9 ; Arg15 (N-terminus of helix 1); Ile107, Met108, Phe111 (helix 4); Tyr212, Val216, Tyr217, Phe220 (helix 8) [12 ; Pro11, Pro13, Pro14 (N-terminus helix 1); Ile36 (coil between beta strand 2 and helix 2); Phe108, Ile112, Arg113, Leu120 (helix 4); Phe174 (C-terminus helix 7); Leu209, Phe213 (helix 9). Although appearing to generate a similar hydrophobic cavity the residues of DmGSTE6 originate from different regions of the protein. This suggests convergent evolutionary pressure occurred in the Epsilon and Alpha GST classes to independently obtain the HNE activity.As stated above, in an GSTA4 , it is gan GSTA4 . Therefoan GSTA4 ) and thehelix 8) . From suSarcoptes scabiei, a parasitic arachnid [Tigriopus japonicus [Eriocheir sinensis [Apis mellifera and Nasonia vitripennis) lack the Epsilon class, which suggests that Epsilon class GSTs appeared after the Hymenoptera divergence, approximately 238.5–307.2 million years ago [Both Delta and Epsilon class GSTs have been shown to be important for contributing to insecticide resistance in mosquito malaria and dengue vectors –11,34. Aarachnid , a copepaponicus  and the sinensis . Genomessinensis . In contears ago ,40. The ears ago –43. As aears ago . Thus thDuring the writing of this manuscript, two newly deposited structures of Drosophila GSTE6 (4PNF) and GSTE7 (4PNG) appeared in the PDB. This DmGSTE6 has structural similarity to the one in the present study (4YH2) with RMSD of 0.24 Å including 2840 atoms.


The X-ray crystal structure of this metallo-receptor bound to arsenate is also reported, which allowed us to establish the binding mode between 3 and this oxyanion. Immobilising 3 onto HypoGel resin yielded a novel adsorbent (Zn–HypoGel) with high affinity for arsenate. Adsorption of arsenate from competitive solutions and natural groundwater was greater than that of the commercially used iron oxide Bayoxide E33. Zn–HypoGel could be efficiently and simply regenerated by washing with sodium acetate solution.A series of copper(II), nickel(II) and zinc(II) dimetallic complexes were prepared and their affinities towards arsenate investigated. Indicator displacement assays (IDAs) were carried out to establish the complexes with best affinities towards arsenate. A di-zinc complex ( Though no structures of this ligand with nickel have previously been reported,, 23 Thre1–6 was investigated by indicator displacement assays (IDAs). It has been reported that binding of pyrocatechol violet (PV) by the di-zinc complex 3 at physiological pH is accompanied by significant colour change, which is reversed upon addition of a guest anion such as phosphate. units bridged by two AsO3(OMe) moieties  and Zn(2), to the left of Figure 1 manner to a metal centre of the other [1LZn2] unit (the one containing Zn(3) and Zn(4), to the right of Figure C2 symmetry about an axis that passes through O(1) and O(41) rather than the iC symmetry that might have been expected. The “unused” coordination sites on Zn(3) and Zn(4) are occupied by water molecules [O(100) and O(101)], and these are involved in intramolecular hydrogen bonds to oxygen atoms of the arsenate groups  complex 2 binding  being slightly shorter [in the range 1.659(2)–1.665(2) Å] than those to the oxygens involved in the monodentate coordination . The methyl groups present in the two AsO3(OMe) moieties were presumably incorporated from the methanol that was used to grow the crystals. Previously, this zinc complex and similar transition metal complexes have been shown to catalyse esterification reactions of various phosphates., where fficient.Qmax was determined to be (10.2±0.6) mg g−1 and the value of b was (4.1±3.4) L mg−1 (average of 3 independent runs). Compared with other reported metal-based ligand-exchange type sorbents,+; found: 531.29; elemental analysis calcd (%) for. C33H34N6O⋅0.5H2O: C 73.44, H 6.54, N 15.57; found C 73.81, H 6.38, N 15.34.This Finkelstein activation was carried out under solvent-free conditions. Di-(2-picolyl)amine , potassium iodide , potassium carbonate  and PEG-400  were ground together with a mortar and pestle for 5 mins to form a yellow paste. 2,6-Bis(chloromethyl)-4-methylphenol  was added and the resulting green paste was ground vigorously for 45 min, yielding a sticky green solid. This material was taken up in CH5 was obtained as large deep-blue crystals suitable for X-ray analysis. ; MS (ESI+) m/z: calcd for C31H35F6N6Ni2O5P⋅MeOH: 659 [M–PF6–OAc–H]+; found: 659; elemental analysis. calcd (%) for C31H35F6N6Ni2O5P⋅MeOH: C 44.38, H 4.54, N 9.70; found C 44.30, H 4.48, N 9.60.1,3-Bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol  was stirred in methanol (5 mL) at 55 °C. Triethylamine  was added and the hot solution was stirred for 10 min. Nickel acetate hydrate  was then added and the temperature was raised to 67 °C. Sodium hexafluorophosphate  was then added and the mixture heated at reflux for 30 min. The solution was then allowed to cool to room temperature and placed in the freezer (−18 °C) for 2 weeks. After this time, pink crystals had formed, which were removed by filtration. The mother liquor was then decanted and concentrated under nitrogen. This methanolic solution was then placed in the fridge, and after slow diffusion of diethyl ether the di-nickel product 31H35N6Ni2O5](PF6)⋅MeOH, M=866.08, monoclinic, P21/c (no. 14), a=10.52058(6), b=14.41429(10), c=24.21878(16) Å, β=96.3228(6)°, V=3650.36(4) Å3, Z=4, ρcald=1.576 g cm−3, μ(CuKα)=2.427 mm−1, T=173 K, blue/pale blue dichroic blocky needles, Oxford Diffraction Xcalibur PX Ultra diffractometer; 7118 independent measured reflections (Rint=0.0214), F2 refinement,, 486 parameters.(BF4)2⋅2meOH, M=1902.43, monoclinic, P21/c (no. 14), a=19.3412(3), b=14.3874(2), c=28.4490(3) Å, β=94.4691(12)°, V=7892.42(18) Å3, Z=4, ρcald=1.601 g cm−3, μ(MoKα)=2.121 mm−1, T=173 K, colourless tablets, Oxford Diffraction Xcalibur 3 diffractometer; 26853 independent measured reflections (Rint=0.0405), F2 refinement,, 1073 parameters.[Cfinement, R1(obs)=http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi(5) and CCDC-995884 http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi(7) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cifCCDC-995883 −1, 0.203 g, 0.18 mmol) was swelled in 8 mL DMF. After 1 hour the solvent was removed by filtration. Compound 11 , EDCI⋅HCl , HOBt  and triethylamine  were dissolved in DMF (8 mL) and this solution was shaken with the swollen resin at room temperature for 48 h. Ligand uptake was monitored by HPLC analysis. After 48 h the reaction solution was removed by filtration. The resin was washed once with 2 mL DMF and then with excess DCM, DMF, MeOH and Et2O, before being dried under vacuum until the weight no longer changed .HypoGel resin  was shaken in 10 mm HEPES buffer at pH 7 (5 mL) for 30 min to allow the resin to swell. After the buffer was removed by filtration, the resin was shaken in a HEPES-buffered solution containing Zn(NO3)2⋅6 H2O  at room temperature for 24 h. After this time the solution was removed by filtration, and the resin was washed 3 times with 3 mL of buffer (30 min each). The resin was then swollen in buffer again, and this loading and washing cycle was repeated twice more. After the final buffer wash, the resin was rinsed with Milli-Q water, methanol and diethyl ether and finally dried under vacuum. The zinc concentration in each of the starting and final solutions, as well as in the 3 buffer washes, was quantified using UV/Vis spectroscopy in conjunction with a colorimetric zinc indicator (PV). These concentration values were used to determine the amount of zinc taken up by the resin. Zn content=0.46 mmol g−1 (91 % sites filled).HypoGel resin loaded with m stock solutions of each receptor were prepared in acetonitrile and stored at −18 °C. A solution of 100 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in Milli-Q water was adjusted to pH 7.5 with 2 m NaOH and used as buffer. A 5 mm stock solution of pyrocatechol violet (PV) indicator was prepared freshly as required using this buffer solution. Working solutions (500 μm) of receptors and indicator were prepared by a twenty-fold dilution in buffer. Working solutions (500 μm) of Na2HAsO4⋅7 H2O, Na2HPO4 and (NH4)HSO4 were also prepared in 100 mm HEPES at pH 7.5 by dilution of 50 mm stocks. The general procedure for carrying out indicator displacement assays was as follows. PV working solution (25 μL) and receptor working solution (25 μL) were added to a series of wells on a 96 well plate. Then, 1 and 10 equivalents of each anion were added to the corresponding wells, and each solution was made to a final volume of 250 μL with HEPES buffer. The absorbance of each solution was then recorded at λ=445 nm.5 m3 were added to 25 μm solutions of PV in HEPES buffer in a 96 well plate. A plot of receptor concentration versus absorbance at 445 nm yields a binding curve, which was fitted in Origin using a 1:1 binding model described by Hargrove et al.[3 and PV (at 50 μm) in HEPES buffer, and then adding increasing amounts of either arsenate or phosphate working solution. A plot of anion concentration versus absorbance at 445 nm yielded a binding curve for each interaction. These data could be fitted in Origin using a displacement assay script reported by Hargrove et al.[The general procedure for determining the binding constant for PV by a metal complex was as follows. Increasing amounts of ve et al. Anion-bive et al.m) were prepared in HEPES buffer (100 mm) at pH 7.5, and stored at −18 °C. Solutions of Na2HAsO4⋅7H2O, Na2H2PO4 and (NH4)HSO4 (3 mm) were also prepared in 100 mm HEPES at pH 7.5 by dilution of 50 mm stocks. All solutions were filtered through a 0.45 μm syringe filter and degassed before use. The basic procedure for the ITC experiments was as follows: a solution of receptor was stirred at constant temperature in the calorimetric cell. A solution of anion was then accurately titrated into the cell, and the heat change upon each addition was measured . This titration data was then integrated using MicroCal Origin software[K, ΔH and the stoichiometric parameter n could all be determined. A control experiment was carried out where the anion was titrated into a cell containing no receptor, to ensure that interaction between the anion and the buffer did not involve a significant heat exchange.Solutions of each receptor 2⋅6H2O (13.5 mm) was prepared in HEPES (10 mm) at pH 7. Aliquots of this zinc standard were then added to a cuvette containing PV (100 μm), and the absorbance spectrum recorded after each addition. This data was used to produce a calibration line of absorbance at 605 nm versus zinc concentration. Aliquots were then taken from the zinc loading solutions and mixed with PV (100 μm). The absorbance at 605 nm was recorded and the zinc concentration in the solutions could be determined.A working solution of Zn(NOm) was prepared in Milli-Q water and adjusted to pH 7 with NaOH (1 m). An arsenic stock (1000 ppm) was prepared by dissolving Na2HAsO4⋅7H2O in 0.1 m HCl, and a 100 ppm solution was prepared by subsequent dilution of the stock. In a typical isotherm study, buffer (5 mL) was added to each of 7 Luer lock syringes, fitted with a frit and cap. Varying volumes of the arsenic stock and working solutions were then added to the syringes, to give an arsenic concentration range from 1 to 25 ppm. Adsorbent (5 mg) was then added to each syringe, and the solution placed on an orbital shaker at 100 rpm for 24 h. After this time, the shaking was stopped and each solution was removed and acidified with HCl (0.1 m). The arsenic concentration was then determined by differential pulse anodic stripping voltammetry .A solution of HEPES (10 mm) was prepared and adjusted to pH 10 with NaOH (1 m). Acetate solution (5 mL) was added to the As-laden sorbent and the syringe returned to the shaker at 480 rpm. The arsenic concentration in the brine was determined after 24 h; this process was repeated until no further arsenic was desorbed. The resin was then washed with copious Milli-Q water and buffer, before the next arsenic solution was added, and the adsorption–desorption cycle was repeated.HEPES buffer  and arsenic stock  were added to a Luer syringe fitted with a frit and lock, followed by HypoGel sorbent (5 mg). After 24 h of shaking at 100 rpm, the solution was removed by filtration and the concentration of arsenic remaining in solution was determined. A solution of NaOAc (1 2SO4 (0.5 m), using a deposition potential of −3 V for 30 s. In a typical ASV determination, the conditioning potential was 0.7 V for 3 s, the deposition potential was −1 V for 10 s. The ASV scan started at −0.2 V and ended at 0.1 V. The background current was determined either by scanning a cell containing only the background electrolyte (0.1 m HCl) or by scanning the sample using a deposition time of only 1 s. Two additions of a Fluka analytical standard were made to the sample and the concentration was determined using the standard addition method. Upon addition to the voltammetric cell, samples were acidified to pH 1 and, where necessary, diluted with Milli-Q water in order that the arsenic concentration was kept within the linear range during the standard addition procedure.Total arsenic concentrations in the sample were determined by DPASV at a gold microwire electrode, similar to methods previously described by Salaün et al., but usin


Synergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated.ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARβ/δ agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements.We present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) β/δ and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (To the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes. The vascular system sits at the center of oxygen delivery in mammals, and its inner layer endothelial cells play an essential role in network formation. In addition to the physiological angiogenesis that occurs in wound healing and during aerobic exercise, hypoxia is involved in various pathological conditions, for example, cardiovascular disease, diabetic complications, inflammatory diseases and cancer. Poor perfusion of vital organs, including the brain, heart, liver and kidney, can result in hypoxia and critical loss of function. In the core of solid tumors, oxygen demand surpasses the capacity of feeding arteries and the cells are exposed to hypoxia, sometimes with deleterious effects on the progress of the disease. In both contexts, the endothelium is the first cell layer that senses hypoxia as well as changes in hemodynamic forces and blood-borne signals, and this evokes the first step in response to hypoxia, namely angiogenesis . RespondIn the gene regulation that takes place under hypoxia, hypoxia-inducible factor (HIF)1 is regarded as one of the master gene regulators  and we pAlthough several key TFs have been shown to be involved in angiogenesis, the detailed underlying hierarchical or mutual interaction of multiple cascades is only partially understood . To dissIn this study, we are focusing on the new molecular mechanism where conformational change could contribute to the co-operative transcriptional regulation of a common target by two different TFs. It was previously reported that synergistic transcription could be achieved by different TFs through enhanceosomes ,19, whicTo confirm the physiological effect of hypoxia and the PPARβ/δ agonists, and to evaluate the physiological crosstalk of these angiogenic stimuli in endothelial cells, we applied PPARβ/δ and hypoxia to HUVECs and studied the effect on cellular migration function by using a monolayer-wound healing assay. Figure 2) stimulation. After normalization and filtering, gene expression change against the normoxia-DMSO sample was calculated. A scatter plot of normalized intensity values of all genes under two conditions is shown in Figure S1A,B in Additional file ANGPTL4), arrestin domain-containing 4 (ARRDC4) and leucine rich adaptor protein 1-like (LURAP1L), exhibiting 35.3-, 2.5-, 2.2-fold induction under PPARβ/δ agonist treatment and hypoxia compared to no stimulation, respectively  and/or hypoxic , with the result showing synergistic activation  using a PPARβ/δ antibody in HUVECs treated with PPARβ/δ agonist stimulation for 24 hours, followed by deep sequencing (ChIP-seq). In total 38,936,258 reads were aligned and 77.6% of the total reads were aligned uniquely to the non-repeating human genomic sequence. Next, we calculated the enrichment of the PPARβ/δ ChIP DNA fragments compared with the input, and determined the significant PPARβ/δ binding sites according to the QuEST algorithm . In totaPDK4), carnitine palmitoyltransferase 1A (CPT1A) and solute carrier family 25, member 20 (SLC25A20), all listed in Table S1 in Additional file VEGFA), solute carrier family 2, member 1 (SLC2A1) and adenylosuccinate synthase like 1 (ADSSL1) are hypoxia induced genes, showing 3.3-, 6.5- and 4.3-fold induction compared with normoxia, respectively  against PPARβ/δ and/or HIF1α. As shown in Figure S8A,B in Additional file ANGPTL4 induction by the agonists was achieved by the siRNA against PPARβ/δ  in ANGPTL4. HUVECs were stimulated with hypoxia (1% O2) and incubated for 24 hours, followed by identification of histone modifications (monomethylated histone 3 lysine 4 (H3K4me1), trimethylated histone 3 lysine 4 H3K4me3, and acetylated histone 3 lysine 27 (H3K27ac)) and HIF1α binding sites by ChIP-seq. As shown in Figure ANGPTL4 gene locus, but five HIF1α binding sites appeared under hypoxia. H3K4me3, known as a promoter marker [ANGPTL4 under hypoxia, suggesting that the ANGPTL4 locus became activated under hypoxia. Three out of the five potential HIF1α binding sites were also accompanied by two markers of enhancer activity, H3K4me1 [Since r marker , was onl H3K4me1  and H3K2 H3K4me1 -25. As t H3K4me1 , to narrAfter luciferase activity was up-regulated in the presence of the promoter region under hypoxia (data not shown), we made a further series of deletion mutant constructs and HRE motif-mutated constructs to identify the hypoxia responsive sites , and both PPARβ/δ agonist and hypoxia (GW501516 + 1% O2) for 24 hours. Unexpectedly, the locations and distribution patterns of the PPARβ/δ binding (shown in red) to ANGPTL4 did not change under the four conditions , PPARβ/δ agonist stimulation (GW501516 + normoxia), hypoxia  activity and increases plasma triglycerides [in vivo needs to be interpreted, taking into consideration communication with other cell types involved in angiogenesis or wound healing.The angiopoietin/angiopoietin-like gene family encodes a glycosylated, secreted protein with a fibrinogen carboxy-terminal domain. In vascular cells, the angiopoietins act as major regulators of angiogenesis and vascular permeability through binding to the Tie-2 receptor . Angiopoal cells ,30. Fullycerides , while tycerides . Since mycerides . In contycerides  and hearycerides . Furtherycerides . Althougycerides . Furtherycerides . TherefoANGPLT4 is HIF1α, and this TF is known to be recruited to target genes immediately after hypoxic stimulation [ANGPTL4 is PPARβ/δ, which belongs to the nuclear receptor superfamily as a subclass of TFs. In general, gene induction by nuclear receptors only happens when a ligand (agonist) is present. PPAR ligands (agonists) change the PPARs’ structures, resulting in modulation of receptor properties [ANGPTL4 locus was not changed under the four conditions we tested β and PPARβ/δ was reported using a human prostatic stromal myofibroblast cell line (WPMY-1), and looping between the TGFβ response element enhancer and PPAR-response element enhancer was in fact postulated previously, although it was not confirmed [To the best of our knowledge, this is the first report of two different TFs cooperating in transcriptional regulation through the conformational change of the target gene. Nuclear receptors have the potential to crosstalk with various other sequence-specific DNA-binding TFs at adjacent sites, resulting in a modification of gene expression. The existence of crosstalk between different TFs has already been reported, and some of the mechanisms have been elucidated . 'Pioneeonfirmed . The devonfirmed  under diANGPTL4 activation comprises DNA looping and histone modification. The mechanism of synergistic ANGPTL4 activation provides an important clue to how different types of stimulation interact with each other.Chromatin conformation capture and ChIP studies clearly identified that the mechanism of synergistic 2 incubator. Primary culture of HUVECs was prepared and maintained in EGM-2 MV  containing 5% FBS. Experiments under hypoxic conditions (1% O2) were performed in a hypoxic cultivation incubator . The cells used in the experiments were from passage 6 or less.Cells were maintained at 37°C in a humidified 5% COet al., PLoS One in press). Recombinant ANGPTL4 (P01) was from Abnova .GW501516  was used as a PPARβ/δ agonist at a concentration of 100 nM. An anti-PPARβ/δ monoclonal antibody was produced in-house . Mouse mTwo million HUVECs were plated on a 15-cm culture plate. HUVECs were maintained in EGM-2 MV containing 5% FBS and the cells were stimulated with hypoxia and/or PPARβ/δ agonist for 24 hours. The cells were crosslinked for 10 minutes using 1% paraformaldehyde at the appropriate time thereafter. After neutralization using 0.2 M glycine, cells were recollected, resuspended in SDS lysis buffer  and fragmented by sonication . Samples were stored at -80°C before use. To perform ChIP, antibodies against PPARβ/δ, H3K27ac and Pol II were used in combination with magnetic beads . Prepared DNA was quantified using Qubit (Life Technologies/Thermo Fisher Scientific) and more than 10 ng of DNA was processed, as described below. The ChIP primer sequences are listed in Table S3C in Additional file All of the protocols for Illumina/Solexa sequence preparation, sequencing and quality control were provided by Illumina. Sequences were aligned using human genome NCBI Builder 36 (UCSC hg18) as the reference genome. Non-immunoprecipitated DNA (input DNA) was used as a negative control to define non-specific binding. All uniquely mapped sequences were analyzed by Quantitative Enrichment of Sequence Tags (QuEST) 2.4 software using the default parameters  . WIG filAll ChIP-seq analyses were performed using a GAII (Illumina). We duplicated ChIP-seq procedures . For PPARβ/δ, in total 36,607,827 and 36,864,223 (normoxia-DMSO), 38,936,258 and 35,654,581 (normoxia-PPARβ/δ), 36,760,328 and 43,029,618 (hypoxia-DMSO), and 35,058,306 and 43,474,951 (hypoxia-PPARβ/δ) reads were aligned and 76.7% and 78.8% (normoxia-DMSO), 77.6% and 53.7% (normoxia-PPARβ/δ), 74.0% and 79.0% (hypoxia-DMSO), and 78.8% and 79.4% (hypoxia-PPARβ/δ) of the total reads were aligned uniquely to the non-repeating human genomic sequence. For H3K27ac, out of a total of 36,530,846 and 41,560,945 (normoxia-DMSO), 37,346,766 and 44,268,585 (normoxia-PPARβ/δ), 38,316,199 and 38,228,969 (hypoxia-DMSO), 36,900,687 and 41,208,985 (hypoxia-PPARβ/δ) sequence reads, 83.0% and 85.7% (normoxia-DMSO), 85.7% and 85.3% (normoxia-PPARβ/δ), 85.8% and 84.2% (hypoxia-DMSO), and 84.6% and 84.8% (hypoxia-PPARβ/δ) were uniquely mapped. For Pol II, 34,732,013 and 41,475,510 (normoxia-DMSO), 28,155,220 and 42,030,082 (normoxia-PPARβ/δ), 31,755,951 and 43,544,244 (hypoxia-DMSO), and 31,015,638 and 41,895,797 (hypoxia-PPARβ/δ) reads were totally aligned and 81.5% and 80.8% (normoxia-DMSO), 83.0% and 81.4% (normoxia-PPARβ/δ), 83.1% and 81.4% (hypoxia-DMSO), and 83.3% and 81.5% (hypoxia-PPARβ/δ) of the total reads were aligned uniquely to the non-repeating human genomic sequence. Detailed information regarding HIF1α, H3K4me1, H3K4me3 was provided previously [de novo motif search was performed using MEME (version 4.6.1) [E-value is an estimate of the expected number of motifs with the given log likelihood ratio for the random sequences.A n 4.6.1) . All then 4.6.1)  was usedq-value. The q-value is the minimum false discovery rate at which the observed similarity would be deemed significant.The enriched sequence was compared to the known consensus TF motifs using TOMTOM (version 4.8.1) with the motif databases JASPAR and UniPROBE . The TomHUVECs maintained with EGM-2 MV containing 5% FBS were stimulated with hypoxia and/or the PPARβ/δ agonist for 24 hours. Total cellular RNA was isolated using an RNeasy Micro kit . Preparation of the cRNA and hybridization of the probe arrays were performed according to the manufacturer’s instructions . Affymetrix Genechip Human Genome U133 plus 2.0 arrays containing over 54,000 probe sets were applied. The expression value for each mRNA was obtained by the robust multi-array analysis (RMA) method. To analyze the expression data at the genetic level, the intensity of the signal values was summarized using Entrez Gene ID . Then the gene set probes were filtered on an expression (20.0 to 100.0) percentile. Genes expressed at lower than the 20 percentile in all of the four arrays were eliminated from the analyses. After excluding the gene set probes that did not have gene symbols, the remaining 21,089 genes were used for further analysis. Hierarchical cluster analysis was performed using average linkage and Pearson correlation as a measure of similarity. All analysis was performed with GeneSpring GX 12.5 . Annotation of the probe numbers and targeted sequences are shown on the Affymetrix web page.Motif estimation for the binding sites of PPARβ/δ was performed using MatInspector, a software for identification of TF binding sites provided by Genomatix Renilla activity. The primers for the site-directed mutagenesis of the HRE and PPRE motifs are shown in Table S3B in Additional file Transfections were performed with Fugene HD . Cells were transfected on 12-well plates at 70 to 80% confluence in Opti-MEM (Life Technologies/Thermo Fisher Scientific) with 0.5 μg of plasmid. Cells were incubated for 24 hours in normal growth medium, then the stimulation was initiated. Luciferase assays were performed 24 hours after stimulation. Luciferase activity was calibrated with the Total cellular RNA was isolated using an RNeasy Micro kit (QIAGEN). The cDNA was synthesized from 100 ng of RNA using oligo (dT) primers and the Superscript III kit (Life Technologies/Thermo Fisher Scientific). qPCR was performed on a CFX96 real-time System  for 45 cycles at an annealing temperature of 60°C. PCRs were carried out using iQ SYBR Green Supermix (BioRad) and a primer concentration of 200 nM, following the manufacturer’s instructions. 18S rRNA was used as the normalizer. The sequences of SYBR Green primers are listed in Table S3A in Additional file Protein samples were separated by 10% SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes . Membranes were blocked with 5% (w/v) nonfat milk in phosphate-buffered saline containing 0.1% Tween for 1 hour, incubated with antibodies for 1 hour, and detected by chemiluminescence using West Dura extended duration substrate  according to the manufacturer’s protocol.Sau3AI at 37°C for 16 hours. After enzyme digestion, the samples were diluted with ligation buffer and treated with DNA ligase at 16°C for 1 hour. Samples were finally reverse crosslinked and purified. All primers and TaqMan probes are shown in Table S3D in Additional file HUVECs were maintained in EGM-2 MV containing 5% FBS, then the cells were stimulated with hypoxia and/or PPARβ/δ agonist for 24 hours. The assay was performed utilizing the TaqMan 3C Chromosome Conformation Kit  according to the manufacturer’s instructions, with certain modifications. Briefly, HUVECs were crosslinked with 1% paraformaldehyde for 10 minutes. The reaction was stopped by the addition of 125 mM glycine for 5 minutes. Nuclei were re-suspended with restriction enzyme buffer. Samples were treated with 400 U of EGM-2 MV used for HUVEC culture was replaced with Opti-MEM culture medium, and the cells were transfected with stealth RNA interference for HIF1α  and PPARβ/δ  at a concentration of 10 nM using Lipofectamine RNAiMax reagent (Life Technologies/Thermo Fisher Scientific). After 6 hours, the culture medium was changed back to EGM-2 MV. After another 24 hours, cells were stimulated with hypoxia and/or the PPARβ/δ agonist. The knockdown efficiencies of HIF1α and PPARβ/δ were validated by qRT-PCR  using the same primers described above  and incubated the cells under usual conditions for 48 hours. After making a scratch in the cell monolayer using a 5 ml serological pipette, EGM-2 MV was replaced with EBM-2 culture medium for Figure P-values were calculated using two-tailed unpaired Student’s t-test. P < 0.05 was considered significant.Data are available from the Gene Expression Omnibus . The ChI3C: chromatin conformation capture; ANGPTL: angiopoietin-like; ChIP: chromatin immunoprecipitation; FBS: fetal bovine serum; HIF: hypoxia-inducible factor; HRE: hypoxia response element; HUVEC: human umbilical vein endothelial cell; PCR: polymerase chain reaction; Pol II: RNA polymerase II; PPAR: peroxisome proliferator-activated receptor; PPRE: PPAR-response element; qRT-PCR: quantitative RT-PCR; siRNA: small interfering RNA; TF: transcription factor; TGF: transforming growth factor; TSS: transcription start site; UTR: untranslated region; VEGF: vascular endothelial growth factor.Hirofumi Aruga, Shoulian Dong and Junko Stevens are employees of Thermo Fisher Scientific, which supplied the materials for Chromosome Conformation Capture (3C) and qPCR assays. Takashi Maejima is an employee of Kowa Company Ltd.TI designed the studies, carried out the molecular genetic studies, and drafted the manuscript. TK, GL, HP, SY, ST, and XR analyzed genome-wide data. TT, YK, TF, HA, MN, YR, and TK designed the studies. IM, MK, AT, TM, JS, AS, KK, HA, SD, and JS prepared the reagents and performed the experiments. YW designed the studies and drafted the manuscript. All authors read and approved the final manuscript.Toshiya Tanaka partial correspondence for PPARβ/δ antibody.Figure S2. Classification of the hierarchical entity tree for the samples. Figure S3. Scatter plot of the genes induced by the PPARβ/δ agonist and/or hypoxia. Figure S4. Real time PCR of ANGPTL4 with primers recognizing intron-exon junctions. Figure S5. Western blotting of ANGPTL4 under the four conditions. Figure S6. Endothelial cell migration is enhanced by ANGPTL4. Figure S7. Genome-wide analysis of PPARβ/δ and/or hypoxia target genes. Figure S8. Efficiency of the siRNA-mediated knockdown of PPARβ/δ and HIF1α. Figure S9. Real time PCR of ANGPTL4 with siPPARβ/δ under PPARβ/δ agonist stimulation, with siHIF1α under hypoxia, or with siHIF1α and siPPARβ/δ under hypoxia and the PPARβ/δ agonist stimulations. Figure S10. Distribution of the H3K27ac binding regions under the four conditions. Figure S11. Sequence of the 3C product. Figure S12. ChIP-PCR of Pol II under the four conditions. Figure S13. ChIP-seq of PPARβ/δ, H3K27ac and Pol II in duplicate.Scatter plot of the genes affected by PPARβ/δ agonist or hypoxia treatment in endothelial cells. Click here for fileTable S2. Hypoxia target genes. Table S3. Primers used.Common and PPARβ/δ agonist target genes. Click here for file


Lactobacillus antri and Lactobacillus sakei 23K, revealing an optimal pH of between 6.0 and 7.0, low stability at basic pHs (>8.0), low optimal temperatures and, especially, low catalytic efficiency compared with their counterparts in group 1 and 2. The mutational analysis carried out showed that a plausible molecular reason for the low activity shown by Lactobacillus antri and Lactobacillus sakei 23k NALs compared with group 1 and 2 NALs could be the relatively small sugar-binding pocket they contain. A functional divergence analysis concluding that group 3 is more closely related to group 2 than to group 1.N-acetyl neuraminate lyases (NALs) catalyze the reversible aldol cleavage of N-acetyl neuraminic acid (Neu5Ac) to pyruvate and N-acetyl-D-mannosamine (ManNAc). Previous phylogenetic studies divided NALs into four different groups. Groups 1 and 2 have been well characterized at both kinetic and molecular levels, but no NAL from group 3 has been studied to date. In this work, a functional characterization of two group 3 members was performed using the recombinant NALs from  The enzyme showed clear biochemical differences from group 1 and 2 enzymes, including an optimal pH close to 7, low stability at basic pH, low temperature stability and low catalytic efficiency. To answer the question whether or not these features were also present in other group 3 NAL members, Lactobacillus sakei 23K NAL (LsNAL) was also cloned and characterized, and similar results were obtained. A detailed functional study through the directed mutagenesis of key residues has allowed us to identity the likely reason for the differences encountered with NALs from groups 1 and 2.To elucidate the underlying biochemical and molecular basis of this NAL classification, this paper describes the cloning, overexpression and detailed characterization of the N-acetylneuraminate lyase gene (L. antri was from the DSMZ collection (#16041) and the genomic DNA from L. sakei 23K was kindly provided by Prof. Monique Zagorec . N-acetyl-D-mannosamine, N-acetylneuraminic acid and other sugars were from Carbosynth . ManNAc dehydrogenase was from Kikkoman . Other reagents were from Sigma-Aldrich .L. antri nanA gene (828 bp) was amplified by PCR using the forward primer 5′CGCGCTAGCATGAAAGATTTTTCAAAGTATCG3′ and reverse primer 5′TATATCTCGAGCTAGTTGAATGCGGCG3′, which introduce NheI-HF and XhoI restriction sites. The corresponding nanA gene from L. sakei 23K (918 bp) was also amplified by PCR, but using the forward primer 5′GCCGCTAGCATGAAGGATTTAACGAAGTATAAAGGTA3′ and reverse primer 5′CGCCTCGAGCTAGCAATATTTTTCAATTGCA3′, which introduce NheI-HF and XhoI sites as above, respectively. The resulting PCR products were purified and digested with NheI-HF and XhoI restriction enzymes, ligated into the same sites of a predigested pET-28a vector  and transformed into electrocompetent E. coli DH5α cells. A selected clone containing the pET28-LaNAL and pET28-LsNAL plasmids was isolated, sequenced, and transformed into E. coli Rosetta 2 competent cells (Merck Biosciences).The cloning and transformation techniques used were essentially those described by Sambrook et al E. coli Rosetta 2 cells harboring the recombinant plasmid pET28-LaNAL and pET28-LsNAL were grown for 4 hours at 37°C in 400 mL Luria-Bertani (LB) medium, containing kanamycin (50 µg mL−1) and chloramphenicol (34 µg mL−1) before being transferred to a 5-L fermenter (Sartorius), containing 4 L Terrific Broth supplemented with the same antibiotics. These cultures were allowed to grow for 3 h at 37°C, and then induced for 12 hours at 30°C with constant stirring and oxygenation by adding 1 mM isopropyl-β-D-thiogalactoside (IPTG) for LaNAL and 1.5 mM in the case of LsNAL. The cultures were diafiltered through a 500-kDa membrane  and cleaned with 50 mM phosphate buffer pH 8.0. Cells were disrupted using a bead homogenizer  and the cell debris was harvested by centrifugation. The recovered supernatant (crude extract) was treated with 3 U/mL DNase I (Sigma) to remove nucleic acids and then centrifuged for 20 min at 6000 g.The 2+-chelating affinity chromatography  into a HiPrep column . The bound enzymes were eluted with a linear imidazole gradient up to 250 mM in 50 mM phosphate buffer pH 8.0. The fractions containing the aldolase activity were pooled, desalted, concentrated and stored at −20°C.The purification in both cases was performed in two steps, starting with tangential ultrafiltration with a 100-kDa cutoff membrane on a QuixStand system . The resulting retentate was purified by NiProtein concentrations were determined using Bradfords reagent (BioRad) T) for Neu5Ac and ManNAc were 10.3 and 4.2 min, respectively. One unit of activity was defined as the amount of enzyme required to cleave 1 µmol of Neu5Ac, releasing 1 µmol of ManNAc in 1 min (HPLC) or consuming 1 µmol of NADH in 1 min at pH 7.0 and 37°C. The synthetic reaction was followed using the above HPLC conditions. The standard reaction medium for LaNAL contained 500 mM ManNAc, 10 mM pyruvate and 2 µg purified LaNAL in 20 mM phosphate buffer pH 7.0 or 500 mM ManNAc, 30 mM pyruvate and 50 µg purified LsNAL in the same buffer in the case of LsNAL. One enzymatic unit was defined as the amount of enzyme required to synthesize 1 µmol of Neu5Ac per minute under the above conditions.Neu5Ac enzymatic cleavage was determined both spectrophotometrically and chromatographically (HPLC). The first method measures the production of pyruvate by LaNAL or LsNAL, as a consequence of Neu5Ac cleavage, through the decrease in absorbance at 340 nm corresponding to the oxidation of NADH by lactate dehydrogenase (LDH) Enzyme inhibition experiments for ManNAc were carried out spectrophotometrically using the above described reaction conditions but with different ManNAc (0–260 mM) and Neu5Ac (0.19–3.3 mM) concentrations. Enzyme inhibition by pyruvate was also measured spectrophotometrically at different pyruvate (0–5 mM) and Neu5Ac (0.19–3.3) concentrations, but using ManNAc dehydrogenase as a coupled enzyme (see below).Substrate specificity tests of LaNAL with different sugars were carried out at 37°C with 0.6 M of each sugar, 1.2 M pyruvate and 1 mg/mL LaNAL in 20 mM sodium phosphate buffer pH 7.0. The samples were measured by HPLC-ELSD-II as described above, except for D-lyxose and 2-deoxy-D-glucose, in which the mobile phase was 70% acetonitrile: 30% 25 mM ammonium acetate.+ when the ManNAc produced by LaNAL is oxidized by the dehydrogenase into its corresponding lactone The pH-stability assay was carried out by incubating the enzyme in the presence of 5 mM sodium pyruvate, or in its absence, at various pHs (6.0–9.0) at 37°C. The buffers used (20 mM) were sodium acetate pH 4.0–5.0, sodium phosphate pH 6.0–7.0, Tris-HCl pH 8.5 and glycine pH 9.0. Aliquots of 100 µL (1 µg LaNAL) were taken at different times, diluted 10-fold in the corresponding buffer to render a reaction medium containing 3 mM NAD+, 10 mM Neu5Ac and 10 U ManNAc dehydrogenase (EC: 1.1.1.233), and measured spectrophotometrically at 340 nm. This method measures the increase in absorbance corresponding to the reduction of NADTemperature melting curves were determined using a commercial solution of SYPRO Orange (Molecular Probes) as previously described E. coli DH5α cells and used as a template for the mutagenesis PCR. After amplification with Pfu Ultra II polymerase (Stratagene), PCR products were digested with DpnI and transformed in E. coli DH5α electrocompetent cells. All mutations were confirmed by automated DNA sequencing.Two single-point mutants of LaNAL (G211S and P192Y) and a double mutant (G210S/Y213G) were constructed using the overlapping extension method N-acetylneuraminate lyase H. influenzae, C. perfringens, L. plantarum and E. coli K-12 . The sequences were aligned using ClustalW http://salilab.org/modweb) using the E. coli NAL structure as template (PDB ID 2WKJ). Superposition of the homology models was performed with the LSQ option of the COOT program http://pymol.org/)Basic Local Alignment Search Tool (BLAST) searches were used to identify homologues of H. influenzae NAL, C. perfringens NAL and L. plantarum NAL; 28% with P. multocida NAL and 26% with S. carnosus NAL), and 33% sequence identity with those of group 2   showed a 28% amino acid sequence identity with functionally characterized group 1 N-acetylneuraminate lyases (29% with eri NAL) . Thus, Leri NAL) .K167NSSMPVQDIQ), a tyrosine (Y139) in block TDF [IV]IY139NIPQLAG and a Neu5Ac binding motif, which included the carboxylate binding block (NGS49S50GECIY) and the carbohydrate binding blocks (FNG192PD194E195Q and IGG211TGYG).Both group 3 NALs (LaNAL and LsNAL) showed the typical conserved residues of the NAL family at the active site (L. antri nanA gene was PCR-cloned into the pET-28a-derived (plasmid pET28-LaNAL), which labels the protein with N-ter His-tag. The expression was carried out in Terrific Broth medium at 30°C and 1 mM IPTG for 12 h with vigorous stirring and oxygenation in a 5-L fermenter. The crude protein was purified to homogeneity , which was purified in three steps (42.3% recovery) C. perfringens NAL, which needed five steps to provide only a 21% recovery −1, which was similar to that reported for the group 1 NALs such as LpNAL (7.65 U/mg) −1 of initial culture in LaNAL was lower than that obtained for group 1 NALs (215–403 mg L−1) L. sakei 23K NAL was cloned, expressed and purified as described for LaNAL, with a 2-fold purification, 51% recovery, specific activity of 1.5 U mg −1 and a similar yield (151 mg L−1). The mass of both isolated proteins was determined by gel filtration (140 kDa), HPLC/MS/ESI  and SDS-PAGE  I for LaNAL and LsNAL were 0.16 mM and 0.4 mM, respectively .In addition, LaNAL and LsNAL showed differences in their optimum temperatures compared with their counterparts from groups 1 and 2. The highest activity for LaNAL was found at 60°C, both in synthetic and hydrolytic reactions . LsNAL sectively . These KTm) were calculated  this value increased to 65°C in LaNAL  was plotted vs pH for LsNAL  and optimal temperature of 30–40°C m values also resembled the pH profiles shown in The low thermal stabilities of LaNAL and LsNAL were also evident when their protein melting temperatures  was used at 37°C in 20 mM phosphate buffer pH 7.0 with a 2∶1 pyruvate:D-arabinose ratio (1.2 M vs 0.6 M), 80% conversion into KDO was obtained in 120 hours (−1).LaNAL preferentially used ManNAc as a substrate in aldol condensation with pyruvate , althoug20 hours . This coM for Neu5Ac cleavage was the lowest of those described (catk/KM) of both group 3 NALs (1.7 mM−1s−1 for LaNAL and 2.8 mM−1s−1 for LsNAL) was slightly lower than those previously reported for other NALs, which ranged from 5.6 to 2 mM−1s−1M values for ManNAc (333 mM for LaNAL and 272 mM for LsNAL) compared with group 1 (149–220 mM) M values for pyruvate were the lowest described (1.1 mM for LaNAL and 12.5 mM for LsNAL), giving rise to the highest catk/KM values reported for pyruvate (5.1 mM−1s−1 for LaNAL and 0.31 mM−1s−1 for LsNAL) (−1s−1 and 0.21 mM−1s−1reported for EcNAL Kinetic parameters were determined for the hydrolytic (Neu5Ac) and synthetic (ManNAc + pyr) reactions. The Kescribed , with a r LsNAL) , compareG[F/Y/P/V]DE) and the other in aldol-cleavage . Th)  was used to identity critical amino acids k >0.75  software  k >0.75 . In fact k >0.75 . This di k >0.75 . This in48SSGE, K167NSS, G192PDE and I209GGT), and were basically located between helices α2–α3 and α4–α6, respectively   after HisTrap column. Each lane contained 15 µg of protein. M: molecular weight standards (New England Biolabs: P7708S).(TIFF)Click here for additional data file.Figure S2Effect of pH on LaNAL and LsNAL stability. A) LaNAL was incubated at 37°C in different buffers of pH 6.0 (□) pH 7.0 (▪), pH 8.0 (○) and pH 9.0 (▴), and samples were taken after the indicated periods for assays of enzyme activity. The activity was measured spectrophotometrycally under the standard reaction conditions at pH 7.0 using Neu5Ac as substrate. Also, the pH-stability of the purified LaNAL incubated at pH 8.0 in the presence of 5 mM sodium pyruvate is shown (•), the activity was measured spectrophotometrically using ManNAc dehydrogenase as described in Material and Methods. B) LsNAL was incubated at 37°C for the indicated periods at pH 6.0 (□) pH 7.0 (▪), pH 8.0 (○) and pH 9.0 (▴), and the activity was measured spectrophotometrically under its corresponding standard conditions.(TIFF)Click here for additional data file.Figure S3Effect of temperature on the LsNAL stability. A) The enzyme was incubated at 40°C (•), 50°C (○), 60°C (▪) and 70°C (□) at pH 7.0, and samples were taken after the indicated periods for enzyme activity assays. The activity was measured spectrophotometrically under the standard reaction conditions at 37°C using Neu5Ac as substrate. B) Thermal inactivation of LsNAL. The enzyme was incubated at the indicated temperatures for 10 min in 20 mM sodium phosphate buffer (pH 7.0) in the absence (•) and presence of 5 mM sodium pyruvate (○). The activity was measured spectrophotometrically using ManNAc dehydrogenase as described in Material and Methods.(TIFF)Click here for additional data file.Figure S4Inhibition of LaNAL and LsNAL by pyruvate. (A) Double-reciprocal plot of LaNAL activity in the presence of different pyruvate concentrations: 0 mM (○), 0.5 mM (▪), 1.5 mM (□) and 5 mM (•). The activity was measured spectrophotometrically using the enzyme-coupled assay with ManNAc dehydrogenase. Inset. Secondary plot of KMapp as a function of pyruvate concentration. The KI value was determined from the abscissa intercept. (B) Double-reciprocal plot of LsNAL activity in the presence of different pyruvate concentrations. The conditions are the same as in (A). Inset. Inset. Secondary plot of KMapp as a function of pyruvate concentration.(TIF)Click here for additional data file.Figure S5Melting curves of LaNAL and LsNAL. (A) LaNAL and (B) LsNAL unfolding was monitored with SYPRO Orange dye with 1 µg of purified enzyme. Curves were obtained in MilliQ water (○) and in the presence of the buffers described in . Effect of pyruvate on melting temperatures of LaNAL and LsNAL at different pHs. Black and light grey bars represent absence or presence of 5 mM sodium pyruvate, respectively. Assays were performed in a real time PCR apparatus with 10X SYPRO Orange.(TIFF)Click here for additional data file.Figure S6Active site comparison of NALs from groups 1, 2 and 3 (LaNAL and LsNAL). The active site of sialic acid alditol for: (A) HiNAL  and (B) EcNAL  are compared with the models of group 3 NALs from (C) LaNAL and (D) LsNAL. The sialic acid alditol was modelled from PDB code 1F73. Residues involved in substrate binding are represented by balls and sticks. Residues subjected to mutational analysis are in yellow. The interactions with the C6 and C7 atoms of sialic acid are shown.(TIF)Click here for additional data file.Figure S7Multiple sequence alignment of N-acetylneuraminate lyases from group 3, indicating position of Cat I and Cat II residues. The background of residues strictly conserved across NAL enzymes is filled. The secondary structure of LsNAL is shown: springs represent helices and arrows represent β-stands. Residues belonging to the active site are indicated by squares. Residues forming Category I in groups 3 and 1 are indicated by triangles up, the residues forming Category II in groups 3 and 1 are indicated by triangles down, the residues forming Category I in groups 3 and 2 are indicated by filled circles, the residues forming Category II in groups 3 and 2 are indicated by open circles, the common Category II residues in groups 3 and 1 and in groups 3 and 2 are indicated by stars.(TIFF)Click here for additional data file.Table S1Oligonucleotide primers used for LaNAL site-directed mutagenesis.(PDF)Click here for additional data file.Table S2Purification of recombinant LaNAL.(PDF)Click here for additional data file.


Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome.The functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy.S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (Km = 0.085 ± 0.04 mM) and low affinity for ornithine (Km = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [3H]-arginine displayed a 16-fold higher efflux of [3H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles.In the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.TcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the  De novo synthesis of amino acids is restricted to those produced via short pathways or those derived from metabolic intermediates of glycolysis, the citric acid cycle, or the pentose phosphate pathway  80–23). This project was approved by the Biophysics Institution Committee of Ethics in Animal Research (IBCCF106) according to resolution 196/96 of the National Health Council of the Brazilian Ministry of Health. The experimental groups consisted of BALB/c mice intraperitoneally infected with 10(BLAST) searches . Candidate amino acid transporters from T. cruzi that shared ≥ 20 % identities with the query sequences and showed ≥ 60 % alignment along the query or hit sequences and an e-value of ≤ 10−5 were selected using the software BioParser [http://www.genedb.org].To clone putative TcCAT transporter genes, basic local alignment search tool searches , 35 wereioParser . The putT. cruzi TcCAT coding sequences were amplified from genomic DNA by polymerase chain reaction (PCR) with Platinum Taq DNA Polymerase High Fidelity (Invitrogen). The forward primer included the EcoRI restriction site (underlined) and Kozak sequence  (5’CGG AAT TCC GCC ACC ATG GAC ACC GAG AGT GGC AAT 3’). The reverse primer contained the XhoI restriction site (underlined) and COOH-terminal region of the ORF (5’ CCG CTC GAG CGG TTA CCG AAC CAC ACC ATA CAG GCT 3’). The resulting PCR-amplified fragment was cloned into a pBAD TOPO TA vector (Invitrogen). The following oligonucleotides were designed for automated sequencing: (1) 5’ GGC TTT CAG ATG AGT GGT GTC 3’; (2) 5’ CAA TCG CGC GGT GAC AAG TGC 3’; (3) 5’ CGA GCG CGA GGC GCA TGA CGC 3’; and (4) 5’ TTG CCT TTT CTG TGG AGT TAT; and the reverse oligonucleotide (5) 5’ GAC ACC ACT CAT CTG AAA GCC 3’(Invitrogen). Automated sequencing was performed with an ABI Prism® 310 Genetic Analyzer (Applied Biosystems) at the Department of Neurobiology, University of Pittsburgh.The corresponding T. cruzi polysomes, purified from Dm28c-WT epimastigotes and other life cycle forms, were used for qPCR and microarray analysis. Polysomal RNA was extracted from replicating epimastigotes, from parasites incubated for 2 h in TAU medium, which introduced nutritional stress and triggered in vitro metacyclogenesis, and from differentiating parasites incubated in TAU3AAG medium for 3 h, 12 h, 24 h, or 5 days to produce metacyclic trypanosomes. Epimastigotes, other life cycle forms, and metacyclic parasites were centrifuged at 2000 g for 20 min at 4 °C and washed three times with NKM buffer, composed of 140 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, and 10 mM Hepes, pH 7.4. Next, parasites were lysed with buffer A, composed of 300 mM KCl, 10 mM MgCl2, 10 mM Tris–HCl, pH 7.4, 10 % Nonidet P-40 and 2 M sucrose, followed by centrifugation at 16,000 g for 5 min at 4 °C. To obtain the post-mitochondrial fraction, the supernatant was again centrifuged at 16,000 g for 30 min at 4 °C and layered onto 15 to 55 % sucrose density gradients prepared in buffer B, composed of 300 mM KCl, 10 mM MgCl2, 10 mM Tris–HCl, pH 7.4, 100 μg/ml cycloheximide, 10 μM E-64, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml heparin, and centrifuged at 200,000 g for 2 h. The pellet containing the polysomal fraction was collected, and RNA was extracted by the hot phenol method and with saturated phenol. Samples containing purified RNA were concentrated by precipitation with one volume of 10 % isopropanol and 3 M sodium acetate, purified with an RNAeasy kit (Qiagen) and stored in liquid nitrogen.T. cruzi life stages. Amplified RNA was generated from 1 μg of polysomal RNA and oligo (dT) primers coupled to the T7 promoter  using a MessageAmp™ aRNA Amplification Kit (Ambion), according to the manufacturer's instructions. Thereafter, cDNA was synthesized from 1 μg of cRNA by incubation with 400 mM of random primers, RT buffer, dNTPs and reverse transcriptase , according to the manufacturer’s recommendations, for 2 h at 42 °C. cDNA was purified and concentrated using a Microcon YM-30 filter (Millipore).Initially, RNA was amplified due to the low yield of RNA obtained from the polysomal fractions of some Real-time PCR was performed with a 7500 Real-Time PCR System (Applied Biosystems), and the results were normalized by expression of L9 ribosomal protein and histone H2B, which were amplified with the following primers: TcL9F (5` CCTTCACTGCCGTTCGTTGGTTTG 3`); TcL9R (5` ATGCGAGAGTGCCGTGTTGAT 3`); and TcH2BF (5` CGGTGGTGCGCGTCAACAAGAAGC 3`); TcH2BR (5` CCAGGTCCGCCGGCAGCACGAG 3`), respectively. PCR was performed in a 20–25 μL reaction mixture containing 10 ng cDNA and the recommended amount of SYBR Green Master Mix (Applied Biosystems). All reactions contained 4 pmol of specific primers (TcCATf 5'-CATCATTGGATGGGATGTGG-3' and TcCATr 5'-ATAAAGAGCCCGAGCAGCAG-3'). The PCR conditions were as follows: 10 min at 95 °C, followed by 45 cycles at 95 °C for 15 s, 60 °C for 30 s and 72 °C for 1 min. For SYBR Green-based assay, melting curve analysis was performed after amplification to ensure that the correct product had been obtained by determining its specific melting temperature.n = 3) was calculated as follows, and was found to exhibit high linearity (r > 0.95): for TcH2B, 2.0 (slope −3.313618); for TcL9, 2.05 (slope −3.210819); and for TcCAT transporter, 1.97 (slope −3.405488). Relative gene expression was determined using the 2 CT-∆∆ method -arginine or [14C]-ornithine substrates (Perkin-Elmer Reagents). The incubation time required for uptake was found to be within the linear range. Apparent Km and Vmax values were calculated by non-linear regression analysis and fitted to the Michaelis-Menten equation . Substrate competition assays were performed with a 100-fold concentration of competitor relative to that of substrate. The optimal pH range for [3H]-arginine uptake was achieved in Krebs buffer without glucose, composed of 146 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 5 mM HEPES and 5 mM MES.Substrate saturation curves were obtained by incubation of induced and repressed yeast cells with increasing concentrations of -arginine at room temperature for uptake assays. For efflux measurements, oocytes preloaded overnight or for 6 h with 1 μM [3H]-arginine were washed twice in ND96 buffer at 4 °C and immediately transferred to ND96 buffer (0.5 ml) to allow for the efflux of radiolabeled compounds. After incubation for the required period of time, an aliquot of incubation buffer was examined in a scintillation counter and the total amount of radiolabel in the buffer was divided by the number of oocytes per well.Trans-stimulation assays were performed using TcCAT1.1-expressing cRNA-injected oocytes and water-injected oocytes preloaded with 1 mM or 10 mM arginine overnight or after 6 h incubation, respectively. Thereafter, the oocytes were washed twice in ND96 buffer at 4 °C and incubated with -arginine uptake was assessed in two clones of Dm28c-EGFP-TcCAT1.1, Dm28c-EGFP, and Dm28c-WT. Epimastigotes were centrifuged at 1500 g for 10 min and washed once with Krebs buffer composed of 146 mM NaCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 5 mM KCl, and 5 mM HEPES, pH 6.8. After centrifugation at 1500 g for 10 min, the excess liquid was drained, and parasite density was adjusted to 108 epimastigotes/ml in Krebs buffer. [3H]-arginine (Perkin Elmer) at a specific activity of 50 Ci/mmol was prepared in Krebs buffer and diluted with cold arginine to achieve the specific activity desired for the various experiments.[7 epimastigotes) to a 100 μl aliquot of two-fold-concentrated [3H]-arginine for a total volume of 200 μM in a 5 ml tube. Following incubation for 15, 30, 60, or 120 min at room temperature and for 30 sec (0.5 min) on ice (binding), uptake was stopped with 3 ml of cold phosphate-buffered saline (PBS), and the suspension was immediately filtered through a nitrocellulose filter with a pore size of 0.45 μm (Millipore). The tube was washed twice, and the filter apparatus was washed once. The radioactivity retained in the nitrocellulose filter was quantified with a liquid scintillation counter (Packard Tricarb) in 2.5 ml of scintillation liquid . Three independent assays were performed in triplicate.Uptake assays were initiated by adding 100 μl of parasite suspension -N,N,N`,N`-tetraacetic acid, 20 mM Hepes, and 60 mM Pipes, pH 7.3, fixed with 4 % paraformaldehyde in PHEM buffer for 15 min on ice, allowed to attach to cover slips that were coated with 0.1 % poly-L-lysine (Sigma), permeabilized in 100 % methanol at −20 °C for 5 min, and blocked with 50 mM NH4Cl and 1 % BSA in PHEM buffer at room temperature for 30 min. Then, further incubation was performed for 1 h with the following primary antibodies diluted in blocking buffer: (1) anti-rabbit PPase (1:200), a vacuolar-type proton-pumping pyrophosphatase; (2) anti-rabbit TcRAB7 (1:20) . TcCLB.506153.10 (TcCAT1.1) and TcCLB.506153.20 (TcCAT1.2) are located in the same contig and share 100 % coding sequence identity; however, TcCAT1.2 is located at the end of the contig, suggesting that the missing 76 amino acid N-terminal region may have been lost due to a gap in the genome sequencing data, or to misassembly -arginine uptake with 100-fold excess of competitor relative to the substrate concentration. Uptake of 10 μM [3H]-arginine was used as a reference to estimate the percentage of [3H]-arginine uptake in the presence of 1 mM competitors after 30 min of incubation at room temperature . The effect observed in the oocytes pre-loaded with arginine suggests that the increased uptake of [3H]-arginine is driven by arginine present on the trans side of the membrane . Arginine uptake driven by trans-stimulation was observed with [3H]-arginine at 100, 250, and 500 μM substrate concentrations (data not shown).Next, a series of experiments were performed using 1.1 Fig. . To invetes Fig. . The traane Fig. . The tra3H]-arginine was investigated in TcCAT1.1-expressing oocytes pre-loaded with 1 μM [3H]-arginine. The time course of [3H]-arginine efflux in oocytes that were pre-loaded for 6 h revealed a rate of 10 ± 2 fmol x min−1 in TcCAT1.1-expressing oocytes and a rate of 0.6 ± 0.08 fmol x min−1 in water-injected oocytes. In oocytes pre-loaded overnight with [3H]-arginine, the efflux rate was approximately 19 ± 3 fmol x min−1 in TcCAT1.1-expressing oocytes and 1 ± 0.2 fmol x min−1 in controls. This rate remained linear for 3 h and was at least 16-fold higher in the TcCAT1.1-expressing oocytes compared with that in the controls, which were water-injected oocytes pre-incubated with [3H]-arginine , with 20 μM or 100 μM nigericin (a proton-potassium antiporter), or with 100 μM ouabain (a sodium-potassium ATPase inhibitor) in ND96 buffer. Uptake assays were performed for 30 min at room temperature with 100 μM [3H]-arginine in ND96 buffer in the presence of each of the three compounds  for 20 μM nigericin and 11.1 ± 7.2 pmol x oocyte−1 (n = 3) for 100 μM nigericin compared with that of the controls, which was 65.0 ± 6.4 pmol x oocytes−1 (n = 5). In the presence of 20 μM CCCP, the level of [3H]-arginine uptake was 17.1 ± 4.1 pmol x oocyte−1 (n = 4), and with 100 μM ouabain, it was 26.7 ± 11.4 pmol x oocyte−1 (n = 4). The water-injected oocytes were subjected to the same treatments, and the background was subtracted.To evaluate the effect of ionophores on -arginine uptake that was observed following expression of EGFP-TcCAT1.1 by whole epimastigotes (Fig. Some intracellular compartments serve as storage sites, and arginine and lysine constitute approximately 90 % of the total amino acids in lcisomes . Howeverlcisomes . This relcisomes . Other tlcisomes , and thelcisomes . Similarmembrane , TcCAT1.tes Fig. .T. cruzi life cycle. The complexity of the expression of the transporters required to obtain arginine and cationic amino acids from the environment is associated with their importance to T. cruzi biology. We have performed a BLAST search, identifying 26 putative amino acid transporters. In addition, we have identified another arginine permease in the T. cruzi genome using a genome database of annotated sequences [http://www.genedb.org/]; thus, it is reasonable to propose that there might be additional cationic amino acid transporters in T. cruzi. An increase in the number of transporters cloned and characterized from T. cruzi will provide new insights into the contributions of various transporters to the metabolism of cationic amino acids throughout the life cycle of this parasite.Substrate affinity, trans-stimulation, and the kinetic parameters of arginine/ornithine transport estimated for TcCAT1.1 can be extended to other TcCAT subfamily members, such as TcCAT1.2, TcCAT1.3, and TcAAP3. Previous characterization of the TcAAP3 transporter has revealed that it is also an arginine transporter that is expressed in the anterior region of epimastigotes. Intracellular expression is an important characteristic of members of the TcCAT subfamily of cationic amino acid transporters, and it could account for their translocation of intracellular pools of cationic amino acids. Members of this transporter subfamily characteristically function as exchangers, consistent with the intracellular localization of the TcCAT1.1 isoform, and its up-regulation under nutritional stress suggests that TcCAT transporters are candidates for regulation of cellular homeostasis during metacyclogenesis and during the


Furthermore, the migration of AZGP1-overexpression cells was decreased. The overexpression of AZGP1 suppressed the activation of the mTOR pathway and endogenous FASN-regulated fatty acid synthesis, mitigating the malignant phenotype of LoVo cells. Herein, we provide evidence that AZGP1 may constitute a novel tumor suppressor for LoVo colorectal cancer cells.AZGP1 is a multifaceted protein associated with lipid mobilization, a process that is regulated by FASN and other metabolic pathways such as mTOR signaling. The active mTOR signaling pathway has been found to be involved in a variety of tumors. However, it remains unclear whether it is involved in the regulation of AZGP1 and FASN. An AZGP1-expressing plasmid was transfected into a human colorectal cancer cell line (LoVo) with a low expression of AZGP1. The expression of AZGP1, FASN, eIF4E, p-mTOR, p-S6,and S6K1 were measured by Western blot analysis, and target genes were detected by RT-PCR. Cell proliferation was studied using the MTT and colony formation assays. The analysis of apoptosis and the cell cycle phase were assessed by flow cytometry. The capacity of cell migration was investigated using the transwell migration assay. We found that the expression of AZGP1 was up-regulated while the expression of FASN, eIF4E, p-mTOR, p-S6, and S6K1 were down-regulated in LoVo cells after AZGP1 was expressed. The proliferation of malignant cells was reduced in AZGP1-overexpression cells, which is consistent with an increased in the G In normal tissue and in tissues with high cellular turnover, fatty acids are supplied by dietary fat and FASN is expressed at undetectable levels; however, various tumors undergo exacerbated endogenous fatty acid biosynthesis irrespective of the levels of extracellular lipids The mammalian target of rapamycin (mTOR) signaling pathway determines the core route from PI3K to AKT and which diverges at mTOR Recent evidences have demonstrated that mTOR signaling and endogenous fatty acid synthesis play a critical role in the development of colorectal cancer; however, it remains to be seen whether AZGP1 is involved in this process. In this study, we investigated the expression of AZGP1, FASN, eIF4E, p-mTOR, p-S6, and S6K1 and characterized the function of AZGP1 in colorectal cancer cells.Four human colorectal cancer cell lines  were used in this study. These cells were obtained from Shanghai Cell Biology, Institute of the Chinese Academy of Sciences . LoVo, HT-29, and HCT116 cells were cultured in RPMI-1640 medium supplemented with 100 units/mL penicillin,100 µg/mL streptomycin, and 10% fetal bovine serum . Caco-2 cells were cultured in high glucose Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum. These cells were incubated in a standard cell culture incubator  at 37°C with 5% CO2.The expression of AZGP1 was detected by RT-PCR and Western blot analysis in order to choose target cells whose expression of AZGP1 was lower than that of other cells. A recombinant pGCMV/EGFP/AZGP1 plasmid with scrambled sequences was synthesized according to the manufacturer’s instructions. The target cell was transiently transfected using the X-treme gene Hp DNA Transfection Reagent . In order to determine transfection efficiency, green fluorescence was detected under a fluorescence microscope , and G418 (600 µg/mL) was used to select for stable clones. In this study, cells were classified into three groups: wild LoVo cells (blank group), LoVo cells were transfected by pGCMV/EGFP/Neo plasmid (control group), and LoVo cells were transfected by pGCMV/EGFP/AZGP1 plasmid .Total RNA from the three groups was isolated using the Trizol Reagent  according to the manufacturer’s protocol. After they were resuspended in 30 µL of RNase free H2O, RNA samples were stored at −80°C. The purity of the isolated RNA was tested by spectrophotometry  at 260/280 nm. The first strand of complementary DNA (cDNA) was synthesized using the Hiscript 1st strand cDNA synthesis kit  according to the manufacturer’s instructions: a 20 µL reaction volume consisting of a 10 mM 2×RT mix (containing dNTP), 2 µL (200 U) HiScipt enzyme mix (containing HiScipt reverse transcriptase and RNase inhibitor), 50 µM oligo (dT) 18 primers, 0.5 µg of extracted RNA, and 20 µl of RNase free ddH2O. These components were added to a 200 µL PCR tube. Amplifications of all transcripts were done using PCR system  under the following conditions: 50°C for 45 min and 85°C for 5 min.Subsequently, 5 µL of complementary DNA (cDNA) was amplified in a reaction mixture containing 25 µL 2×Phanta Master Mix , and 4 µL of primer  in 500 µL RIPA lysis buffer with 5 µL PMSF and protease inhibitor  for cell protein extraction. The amount of cellular protein was measured using a bicinchoninic acid assay. 20 µg of total protein was electrophoresed in an 8% SDS–polyacrylamide mini gel, and then these proteins were transferred to a Polyvinylidene Difluoride (PVDF) membrane . The membrane was incubated for 1 h at 25°C in 5% (w/v) non-fat dry milk. Then, the membrane was washed three times for 5 min at 25°C using blocking buffer . Subsequently, the membrane was incubated overnight at 4°C with monoclonal primary antibodies Table 2 The 3--2,5-diphenyltetrazolium bromide (MTT) assay was employed to examine the proliferation of plasmid transfected LoVo cells and untransfected LoVo cells. All cells were uniformly seeded into 96-well culture plates with a logarithmic growth phase at a concentration of 5 cells/µL in a volume of 100 µL per well. 48 h later, 20 µl of MTT  reagent (5 mg/mL) was added into each well, and the plates were incubated for 4 h at 37°C in 5% CO2. After discarding the mixture, 200 µL dimethylsulfoxide  was added and shaken for 10 min to dissolve the MTT. Then the optical density (OD) value was measured by a spectrophotometric plate reader  at 492 nm. Each group was comprised of eight duplicated wells, and the assay was performed three times independently.An annexin-V-FITC apoptosis detection kit was used  to detect apoptosis. Treated cells were harvested and centrifuged at 1500×g for 5 min and resuspended gently with precooled PBS. Then, resuspended cells (5×105) were taken out and centrifuged at 1500×g for 5 min. After the supernatant was discarded, it was resuspended gently in 500 µL binding buffer, mixed with 3 µL Annexin V-FITC, and 15 µL Propidium iodide . After staining, the cells were protected from the light for 10 min at room temperature . The data was analyzed immediately by flow cytometer  using CXP software.Three groups of cells were concentrated and blended to prepare single-cell suspensions to carry out colony formation assay Cells were placed in a 6-well plate at a density of 1000 cells/mL. Then, 2 mL/well of RPMI-1640 medium with 10% FBS was added. The cells were incubated in a 5% CO2 incubator at 37°C for 2 weeks. The new colonies were rinsed twice with PBS, and the cells were fixed in 1.5 mL of paraformaldehyde for 10 min. The fixed colonies were stained with giemsa  for 20 min and then washed with PBS. The colony counting was performed on clearly visible colonies (colony formation rate (%) = colony number/seeded cell number×100%). Each experiment was repeated three times.Cell invasion is measured by the number of cells that can go through the 8-µm pore of the transwell polycarbonate membrane , which separates the upper and lower chamber. First, the upper chamber was washed three times with PBS. Then, 20 µL of diluted Matrigel  was added uniformly after incubation for 30 min at 37°C to make the artificial base membrane. Secondly, an equal number of cells (5×104) for three groups were seeded in the upper chamber with 200 µL of serum-free RPMI-1640 medium. Next, 500 µL of RPMI-1640 medium containing 10% fetal bovine serum was added to the lower chamber. The fully prepared transwell invasion system was then placed in an incubator for 24 h at 37°C in 5% CO2. Finally, the cells on the surface of the basement membrane were wiped off and were stained with hematoxylin-eosin (HE) and counted using a Leica microscope . Seven randomly selected fields (200 times magnification) were observed for each sample; the experiment was repeated three times.P<0.05 were considered statistically significant.Statistical analysis was performed using SPSS 17.0 software . Statistical comparisons were presented as P<0.05), the result of Western blot test was detected using RT-PCR and Western blot (blot testis similaP<0.05) in experimental group.The pGCMV/EGFP/AZGP1 plasmid and the pGCMV/EGFP/Neo plasmid were constructed and their sequencing were confirmed (P<0.05)  and control group . The colony formation rate of LoVo cells transfected with the overexpression plasmid was 4.38%±0.71%, which is lower than blank group (7.15%±0.82%) and control group (6.88%±0.42%). Furthermore, the size and cell mass of experimental group LoVo cells were smaller compared to the other two groups  when compared with the blank group and the control group (P<0.05), compared to the blank group and control group (P>0.05).Cells in which AZGP1 were overexpressed showed an increased percentage of cells in the GAZPG1 is a well-known lipid mobilizing factor which stimulates lipolysis and leads to cancer cachexia As we know, eIF4E and S6K1 play an important role in promoting cell proliferation FASN expression is also mediated by mTOR signaling, specifically in endogenous fatty acid biogenesis in human cancers Taken together, we found that AZPG1 overexpression blocked eIF4E, S6K1, p-mTOR, p-S6, and FASN expression; simultaneously, the cellular proliferation and migration were also inhibited. These findings suggest that increased expression of AZGP1 can suppress the activity of the mTOR signaling pathway and endogenous fatty acid synthesis. Our research not only demonstrated that AZPG1 over-expression blocked the proliferation and migration of LoVo cells, but also confirmed that AZGP1 regulates biological behaviors of LoVo cells through inhibition of the mTOR signaling pathway and endogenous fatty acid synthesis. These findings might provide better understanding towards the function of the AZPG1 gene in colorectal cancer LoVo cells.Data S1Supporting information file containing figures S1–S6. 1. Figure. S1b: The expression of AZPG1 protein in three colorectal cancer cell lines. 2. Figure. S1d: The expression of AZPG1 gene in three colorectal cancer cell lines. 3. Figure. S1f: The relative expression of the AZPG1 gene in blank group, control group and the experimental group. 4. Figure. S3b: The change in FASN, eIF4E,and S6K1 gene after up-regulation of AZGP1 expression in blank group, control group and the experimental group. 5. Figure. S4b: The expression of AZGP1, FASN, eIF4E, p-mTOR, p-S6, and S6K1 after up-regulation of AZGP1 expression in blank group, control group and the experimental group. 6. Figure. S5a: Up-regulation of AZGP1 expression inhibits the proliferation of LoVo cells. 7. Figure. S5e: Up-regulation of AZGP1 expression inhibits the migration of LoVo cells. 8. Figure. S6a: AZGP1 overexpression induces G2 phase arrest in LoVo cells 9. Figure. S6d: AZGP1 overexpression induces a higher rate of apoptosis in LoVo cells.(XLS)Click here for additional data file.


Numerous evidences suggest that there is a relation between glycemic control and bone fractures in type 2 diabetes individuals. Poorly controlled patients with high blood sugar levels could show higher risk of osteoporosis and/or injuries.This study aims to compare the levels of glycemic control with the serum levels of bone formation and resorpotion markers and the bone mineral density (BMD) in female type 2 diabetes individuals, after menopause.A cross sectional evaluation of 41 female type 2 diabetes individuals was performed. Mean age was 62±5.91 yrs. (mean±SD), time of T2D diagnosis 10.15 ±6.61 yrs., BMI=32.76±6.32. All individuals only used metformin as the anti-diabetic treatment. The glycated hemoglobin (HbA1c) levels were compared to the bone formation markers P1NP and osteocalcin (OC), plus the bone resorption marker CTX as well as lumbar spine, femoral neck and total femoral BMD. Pearson correlation analysis was applied for the total sample, plus comparison between the mean of the groups comprised of quartile 1  of HbA1c and quartile 4 , through t-test.The mean of HbA1c was significantly different between the groups Q1 and Q4 (6.7 ±0.001 vs. 8.1 ±0.003% (p<0.001)). Neither significant correlations nor differences between groups Q1 and Q4 were found related to the HbA1c and P1NP levels , OC , CTX , lumber spine BMD, femoral neck BMD  or total femoral BMD (r=0.10 p=0.575).We found no significant correlation between levels of glycated hemoglobin and bone formation and resorpotion markers and bone mineral density. There was no difference even when the extreme glycemic control groups were compared.