X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALMANTH domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALM's role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimer's disease.SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P 2 simultaneously ► Helical N-terminal halves of VAMP SNARE motifs displace the CALM ANTH final helix ► VAMP endocytosis is blocked by mutation of residues in the CALM:SNARE interface► Binding to CALM selects VAMPs 8, 3, and 2 for incorporation into endocytic CCVs ► The CALM ANTH domain binds VAMPs and PtdIns4,5P CALM recognizes the SNARE motif of small R-SNARE proteins as a sorting signal to direct R-SNARE endocytosis and trafficking to the appropriate intracellular compartment while simultaneously shielding the SNARE motif from inappropriate interactions. This unique role for CALM, distinct from other clathrin adaptors, may explain the genetic association of the CALM/PICALM gene with neurological disorders. SNAREs (Soluble NSF Attachment Protein REceptors) are small membrane-anchored proteins that lie at the heart of the vesicle:organelle and organelle:organelle membrane fusion machinery, providing much of the energy and specificity required for membrane fusion . As withabc domain or a longin domain  domain of the endocytic clathrin adaptor CALM.D. melanogaster  was used to quantify the strength of binding. The interaction between GST-VAMP8 and CALMANTH is the tightest with a KD of ∼18 μM , were weaker than the binding to VAMP8 , we carf ∼18 μM B, which f ∼18 μM . Bindingto VAMP8 B. These to VAMP8 B. No int2 similarly to CALMANTH by ITC  A and S2BH by ITC C and lipH by ITC  and thatH by ITC .ANTH and the R-SNARE to which it bound most tightly, VAMP8.Taken together with the in vivo studies published by others and presented here, our biochemical data point to the direct binding of the VAMPs 2, 3, and 8 by CALM being directly responsible for the endocytosis of these small R-SNAREs. Hence, we set out to investigate the molecular basis for this interaction using a combination of structure determination and in vitro and in vivo assays using wild-type and structure-directed mutant versions of both proteins. Because their sequence similarities A and simANTH, a number of deletion constructs were made (D ∼20 μM) to that of wild-type VAMP8 (KD ∼18 μM)  D, indica ∼18 μM) . In agre ∼18 μM) E and S3.ANTH with a peptide corresponding to residues 10–41 of VAMP8, both with the two components free in solution and with residues 10–41 of VAMP8 appended on the C terminus of CALMANTH. While these cocrystallizations did not yield the structure of a complex, they did yield a 1.8Å resolution structure , unexpectedly showed no binding to VAMP8 by GST pull-down experiments and ITC (2 containing liposomes by SPR (data not shown). Multiangle light scattering (MALS) indicated that in solution CALMANTH(1-264) is in fact a tight dimer (ANTH(1-264) at 1.7Å resolution , these data strongly indicated that the hydrophobic trough in which helix 11 (when formed) sits was indeed the VAMP8 binding site on CALMANTH.Attempts were made to crystallize a complex of CALMup 1HF8; , while iup 1HF8;  and S4A.tructure  suggestef a VAMP B. If thi and ITC C and 4D ht dimer B. This win total ) was thein total E and 4F.ANTH and VAMP8, a construct was designed in which residues 11–41 of VAMP8 were fused through an artificial linker of six residues to residues 1–264 of CALMANTH. The resulting chimeric protein (CALMANTH(1-264):VAMP8(11-41)) crystallized and its 2.0 Å resolution structure . There was a small movement of helices 9 and 10 with respect to each other that caused a slight widening of the groove as compared with the unliganded domain structure  does not  over the average of the KDs for the individual ligands when both ligands are present in the same liposome membrane. Thus CALM must be able to bind simultaneously to PtdIns4,5P2 and VAMP8.Since the binding sites on CALMANTH can bind PtdIns4,5P2 and a small R-SNARE VAMP simultaneously and based on the structural identification of the binding sites for a VAMP and PtdIns4,5P2, the following deductions can be made. To allow simultaneous binding to a membrane-embedded VAMP and PtdIns4,5P2 to occur, the amino-terminal half of the VAMP SNARE motif must be able to reach its binding site, which is at a distance of around 50Å from the membrane surface. Thus, the 38 residues between the end of the CALM binding sequence and the start of the transmembrane helix of a VAMP8 molecule must be able to stretch at least this far. The published NMR and CD studies and our own CD measurements (data not shown) indicate that in fact the entire cytoplasmic domain of short VAMPs has no secondary structure in solution . AlternANTH. However, since binding to CALM and participation in SNARE complexes are mutually exclusive processes, as both binding events utilize the same face of the VAMP, it would be more correct to state that VAMPs 8, 3, and 2 are endocytosed in only a potentially active form, as their conserved SNARE motifs are shielded by being bound to CALMANTH. Corecognition of PtdIns4,5P2 markedly increases the strength of binding between CALM and a membrane containing both a small R-SNARE VAMP and PtdIns4,5P2. In order for a small R-SNARE VAMP to bind to CALMANTH, helix 11 of the ANTH domain must be displaced. This dissociation will be facilitated by the fact that helix11 is only poorly associated with the rest of the ANTH domain (helices 1–10)   and may es 1–10) . It shou2 hydrolysis and clathrin cage disassembly. Once PtdIns4,5P2 is hydrolyzed, the avidity effect generated by a single CALM molecule simultaneously binding PtdIns4,5P2 and a small R-SNARE VAMP will vanish leaving only the weak, transient R-SNARE:CALM interaction. The two proteins will therefore quickly dissociate. In order to minimise the energetically unfavorable situation of the hydrophobic face of an amphipathic helix being exposed to the aqueous environment, the N-terminal half of the SNARE motif of the short R-SNAREs, while still remaining as a helix, switches to lying on the surface of the vesicle membrane (Following scission of an endocytic clathrin-coated vesicle (CCV), the vesicle will uncoat by processes that include PtdIns4,5Pmembrane . In the membrane G.ANTH domain. The CALM/PICALM gene has also been directly implicated in alterations in cognitive function with increasing age  or that of its processing proteases   . Furtherretases) . This prIn summary, we have provided insight into the molecular mechanism by which the post-Golgi small R-SNAREs bind specifically to the ubiquitously and highly expressed endocytic clathrin adaptor CALM, recently confirmed in For a list of the constructs and full materials and methods used in this study, please see the HeLaM cells were used for all experiments. For most experiments, the cells were stably transfected with HA-tagged wild-type or mutant VAMPs , and cell lines were selected that expressed the construct at low levels so as not to saturate sorting machinery. For some experiments, the cells were additionally transfected with myc-tagged, siRNA-resistant wild-type or mutant CALM, and cell lines were selected that expressed the tagged CALM at similar levels to endogenous CALM. The steady state localization of the constructs and other proteins, both under control conditions and after siRNA-mediated knockdowns, was observed by immunofluorescence microscopy. Endocytosis kinetics were measured using either a flow cytometry-based antibody uptake assay for the tagged VAMPs, or a radioiodinated ligand uptake assay for other surface receptors. Details of transfection and knockdown conditions, immunolabelling, and endocytosis assays are all described in E. coli for 16 hr at 22°C after induction with 0.2 mM IPTG at 37°C and purified by standard procedures on glutathione sepharose and/or Ni2+-NTA agarose as appropriate. GST-tagged proteins were eluted with free glutathione or by thrombin cleavage of the GST-tag while fusion proteins were bound to the beads. His6-tagged proteins were eluted with buffer containing 300mM imidazole. All proteins were subsequently purified by S200 gel filtration.All recombinant proteins were expressed in BL21(DE3) pLysS 6myc-tagged prey proteins. SNARE complexes were made with a 3-fold excess of SNAP23 and syntaxin3 to GSTVAMP8 and purified by GST sepharose and gel filtration. SNARE complex:adaptor interactions were tested using 2 nmoles of 1:1:1 GST-tagged complex incubated with His6myc-CALMANTH. All binding experiments were carried out in 1 ml of buffer supplemented with 30 μl glutathione beads and incubated with constant agitation at 4°C. The supernatant removed and the beads washed with 1 ml of buffer, three times. Bound proteins were analyzed by SDS-PAGE and western blots probed with anti-myc antibody (9E10 Santa Cruz Biotechnologies).SNARE:adaptor interactions were tested using varying concentrations of GST-tagged SNAREs and relevant His6MycCALMANTH or GST cleaved Epsin1ENTH proteins into 0.15 mM SNARE proteins. Titration curves were fitted using ORIGIN software. Figures show a representative example of each experiment with the KD and associated confidence of the fit (SEM) shown; n = 0.9-1.1. KDs quoted in the text are the average of all runs.Due to yield and solubility issues, GST-tagged SNAREs were used. A VPITC machine  was used to titrate 37 injections of 4–8 μl 2 mM HisANTH and Epsin1ENTH adaptor proteins as analytes and PC/PE liposomes supplemented with 5% PtdIns4,5P2 and/or His12-tagged VAMP8 attached via 5% DGS-NTA(Ni). The binding was monitored during one minute injections at 50 μl/min at concentrations ranging from 10 nM to 50 μM. The kinetic parameters were calculated after background (PC/PE binding) subtraction.Liposome-based SPR was carried out using a Biacore 3000  with GST cleaved CALMANTH (1-289), CALMANTH(1-264), and CALMANTH(1-264): VAMP8(11-41) were grown in sitting drops at 16°C using GST fusion proteins form which the GST had been cleaved. CALMANTH(1-289) was equilibrated against reservoirs containing 100 mM Bis Tris propane, 200 mM sodium malonate, 20% (w/v) PEG 3350; CALMANTH(1-264) was equilibrated against reservoirs containing 15% v/v ethanol, 100 mM imidazole (pH 8.0), 200 mM MgCl2; CALMANTH:VAMP8 was equilibrated against reservoirs containing 100 mM phosphate-citrate (pH 4.2), 200 mM NaCl, 50% (v/v) PEG200. All structures were solved by molecular replacement using previously published ANTH domain of CALM as a starting model (1HF8). Detailed descriptions of structure determination, refinement, and structural analysis can be found in ANTH), 3ZYL (CALMANTH(1-264)), and 3ZYM (CALMANTH(1-264):VAMP8(11-41)).Crystals of CALMExtended Experimental Procedures1-76 (mouse) and mutants thereof, pGEX 4T2 VAMP8(1-31), pGEX 4T2 VAMP8(1-41), pGEX 4T2 VAMP8(16-41), pGEX 4T2 VAMP8(10-41), pGEX 4T2 VAMP3(1-81) (human), pGEX 4T2 VAMP2(1-96) (human), pGEX 4T1 SNAP23 (human), pGEX 4T1syntaxin3(195-253) (human), pGEX 4T2 CALMANTH(1-289) (rat), pMWHis6Myc CALMANTH(1-289), pGEX 4T2 CALMANTH(1-264), pMWHis6Myc AP180ANTH(1-304) (mouse), pGEX 4T1 EpsinENTH(1-163) (human), pGEX 4T2 CALMANTH(1-289):VAMP8(1-76), pGEX CALMANTH(1-264):VAMP8(11-41), pGEX 4T2 VAMP8(1-76)His12, pLXIN VAMP8 and mutants thereof, pLXIN VAMP3 and mutants thereof, pLXIN VAMP2 and mutants thereof.pGEX 4T2 VAMP8pBMN-CALM (made siRNA resistant by the introduction of four silent point mutations in the siRNA oligo binding site) wild-type and mutants Leu219Ser or Met244Lys were derived from IMAGE clone 5537605 into which a myc tag had been inserted in a nonconserved region (position 1308 bp).For expression of HA-tagged VAMP constructs, HeLa M cells were infected with retrovirus using the Phoenix system, and cell lines were selected with 0.5 mg/ml G418, as previously described . EndogenPrimary antibodies used for immunofluorescence and western blotting included a commercial goat polyclonal antibody against CALM (Santa Cruz), commercial mouse monoclonal antibodies against the HA epitope (Covance) and the myc eptiope (Millipore), in-house rabbit polyclonal antibodies against VAMP8 and VAMP3, and (for loading controls) mouse monoclonal antibodies against syntaxin4 and the AP-2 α subunit (both from BD Biosciences). For labeling HA-tagged VAMP8 on the plasma membrane, cells were fixed with 4% paraformaldehyde and labeled with mouse anti-HA, then permeabilized with 0.1% Triton X-100 and labeled with goat anti-CALM (Santa Cruz). For visualizing antibody uptake by immunofluorescence, cells were allowed to endocytose anti-HA antibody for 40 min, then fixed and permeabilized for immunofluorescence. Secondary antibodies used for immunofluorescence were purchased from Invitrogen. Images were acquired using a Zeiss Axioplan fluorescence microscope (Zeiss) equipped with a 63 × oil objective, a Hamamatsu Orca-R2 C10600 camera (Hamamatsu Photonics) and a SEDAT quad pass filter set (Chroma). SDS-PAGE and western blotting were performed as described by To measure the rate of VAMP internalisation, cells expressing HA-tagged VAMPs were incubated with mouse anti-HA (Covance) on ice for 30 min. The cells were washed to remove unbound antibody and warmed to 37°C for various times. Antibody uptake was halted by transferring the cells to ice. The cells were then incubated with Cy5-conjugated antibody against mouse IgG (Jackson ImmunoResearch) for 30 min. The cells were washed and the amount of bound antibody determined using an FACSCalibur flow cytometer (BD Biosciences). Live cells were separated from dead cells by 7-aminoactinomycin D exclusion. Aproximately 10,000 cells were analyzed for each sample and the mean fluorescence was calculated using the software FLOWJO (TreeStar). The percentage of uptake of bound antibody was determined by comparing cells that had been incubated at 37°C with cells that had been kept continuously on ice.125I-labeled transferrin and EGF was carried out as described in  pLysS cells for 16 hr at 22°C after induction with 0.2 mM IPTG. Following lysis, GST fusion proteins were purified on Glutathione Sepharose 4B in 20 mM HEPES pH 7.4, 150 mM NaCl, 4 mM DTT and eluted with buffer containing 30 mM glutathione. His1-76His12 used for SPR was purified using 20 mM HEPES pH 7.4, 120 mM NaCl, 1 mM β-mercaptoethanol. The N-terminal GST tag was cleaved with thrombin for 3hrs at room temperature while the protein was bound to glutathione sepharose beads. The cleaved protein was then eluted and the thrombin cleavage was halted by the addition of AEBSF. The protein was further purified on Ni2+-NTA agarose in 20 mM HEPES (pH 7.4), 150 mM NaCl, 30 mM imidazole, 1 mM β-mercaptoethanol and eluted in the same buffer but now containing 300 mM imidazole. Finally, it was dialysed extensively into buffer lacking both reducing agents and imidazole.GST-VAMP8ANTH(1-289), GST-CALMANTH(1-264) and GST-CALMANTH:VAMP8 fusion constructs destined for crystallization trials were purified using 20 mM HEPES (pH 7.4), 120 mM NaCl, 4 mM DTT. The N-terminal GST-tag was cleaved with thrombin overnight at room temperature while the protein was bound to glutathione sepharose beads. The cleaved proteins were then eluted and the thrombin cleavage was halted by the addition of AEBSF. The proteins were then further purified by S200 gel filtration and concentrated to ∼15mg/ml for crystallization trials.GST-CALM6Myc tagged prey proteins in 20 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM DTT, 4 mM β-mercaptoethanol, 0.1% NP-40, 0.05% BSA. Binding experiments were carried out at 4°C overnight with constant agitation. The supernatants were removed and the beads were washed three times with 1 ml of buffer. Bound proteins were analyzed by SDS-PAGE and western blotting with anti-Myc mouse monoclonal antibody 9E10 (Santa Cruz Biotechnology) used at 1:5000, followed by Rabbit anti Mouse HRP used at 1:8000. For testing the binding to SNARE complexes, the complexes were first formed and purified in isolation before being used in binding assays. SNAP23 and syntaxin3 were themselves made by cleaving the proteins from their N-terminal GST-fusion proteins with thrombin while they were bound to glutathione sepharose beads at 25°C for 2 hr. The cleaved proteins were eluted from beads and the thrombin inactivated by the addition of ABESF. SNARE complexes containing GST-VAMP8, were made by incubating GST-VAMP8 with a three fold molar excess of SNAP23 and syntaxin3 at 4°C for 48 hr with constant agitation in 20 mM HEPES (pH 7.4), 500 mM NaCl, 4 mM DTT, 0.5% NP-40. The complex was purified on glutathione-sepharose beads and eluted with buffer containing 30 mM glutathione. Complexes were further purified on S200 gel filtration in 20 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM DTT. Only fractions containing 1:1:1 complex were pooled.Interactions were tested using appropriate GST tagged bait proteins and His6MycCALMANTH (WT or mutant) or GST cleaved EpsinENTH at 2 mM were titrated in 37 injections of 4–8 μL. The heat of dilution of CALMANTH or EpsinENTH into buffer was subtracted from the relevant data and the titration curves were fitted using ORIGIN software, yielding values for the stoichiometry n, equilibrium association constant KA (= KD-1) and enthalpy of binding. All experiments were carried out at least three times and KDs quoted in the text are the average of all runs. Figures show a representative example of each experiment with the KD and associated confidence of the fit (SEM) shown; n values were always within the range of 0.9 to 1.1.ITC experiments were carried out on a VPiTC machine . All proteins were purified by S200 gel filtration into 100 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM DTT. SNAREs were used while still attached to GST to improve solubility. 1.4 ml of GST-SNARE (WT or mutant) was placed into the cell at 0.15 mM. His2 and VAMP8 was measured with liposomes immobilized on a L1 sensor surface of BIAcore 3000 SPR biosensor. The details of liposome generation, their immobilization on the L1 chip and the subsequent binding are described elsewhere (2 and 18:1 DGS-NTA(Ni) lipid were used at 5% at the expense of PC. Liposomes were captured on the L1 surface during 5 min injections at a flow rate of 10 μl/min. Typically the four flow cells (FCs) of the L1 chip were derivatized with following set of liposomes: PC/PE on FC1, PC/PE+ 5% PtdIns4,5P2 on FC2, PC/PE+5% DGS-NTA(Ni) on FC3 and PC/PE+ 5% PtdIns4,5P2+5% DGS-NTA(Ni) on FC4. To verify that liposomes on flow cells 2 and 4 contained equal amounts of PtdIns4,5P2, the levels of Epsin1ENTH domain binding were monitored. The differences between the flow cells  were less than 4%. After liposome capture, the surface was stabilized by two pulse injections with 50 mM NaOH for 10 s at 30 μl/min. The flow rate was set to 5 μl/min followed by injection of His-tagged VAMP8 (10 μM) for 6 min, which resulted in 600RU of captured protein on the two flow cells with the DGS-NTA(Ni) lipid. Subsequently, we monitored the binding of CALM, using one minute injections at 50 μl/min and concentrations between 10 nM and 50 μM. The kinetic parameters were calculated after background subtraction (binding to PC/PE liposomes) exactly as described  than those obtained for the same ligands in solution by ITC. This likely reflects the fact that in liposome-based SPR the ligands are effectively concentrated on a two dimensional surface rather than in solution, a situation that is more representative of the physiological situation. In this work we do not compare measurements obtained using ITC and liposome-based SPR but only compare KD values obtained using a single technique.CALM binding to PtdIns4,5Plsewhere . In brieescribed . The absMultiangle light scattering (MALS) experiments were performed immediately following size-exclusion chromatography by inline measurement of static light-scattering , differential refractive index  and ultraviolet absorbance . Samples (100 μL) were injected onto an analytical S200 10/300 gel filtration column  equilibrated in 20 mM HEPES (pH 7.4), 120 mM NaCl, 4 mM DTT at a flow rate of 0.5 mL/min and data were analyzed using ASTRA V (Wyatt Technology).ANTH(1-289) was equilibrated against reservoirs containing 100 mM Bis Tris propane, 200 mM sodium malonate, 20% (w/v) PEG 3350; CALMANTH(1-264) was equilibrated against reservoirs containing 15% v/v ethanol, 100 mM imidazole pH 8.0, 200 mM MgCl2; CALMANTH(1-264):VAMP8(11-41) was equilibrated against reservoirs containing 100 mM phosphate-citrate (pH 4.2), 200 mM NaCl, 50% (v/v) PEG-200. Crystals were cryoprotected by brief immersion (5–60 s) in reservoir solution supplemented with 25% glycerol and were rapidly cryocooled in liquid N2 or a 100 K stream of N2 gas.Crystals were grown in sitting drops containing equal amounts of protein and reservoir solution (200–400 nL) and equilibrated against 80μl of reservoir solution at 16°C as follows: CALMDiffraction data were processed using MOSFLM  and SCAL


SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body.Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 ( Drosophila melanogaster, Bombyx mori, Triboloum castaneum, and Anopheles gambiae (Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in vertebrates and invertebrates that can suppress neurotransmission, hyperpolarize postsynaptic membrane, decrease ion inflow, and slow down cellular metabolism, a process that results in the neuron of postsynaptic membrane being brought into a protective inhibitory condition . The GAB gambiae .Anopheles funestes, Drosophila simulons, and Sogatella furcifera can significantly enhanced their resistance to the traditional pesticides  (Leidoptera: Noctuidae), is a prevalent agricultural pest in China, causing enormous economic losses due to its wide range, rapid reproduction, and resistance to pesticides  of a laboratory susceptible strain was amplified via RT-PCR, and developmentand tissue-specific expression patterns were analyzed using quantitative real-time PCR (qRT-PCR).The common cutworm, sticides . Chemicahe world . However serious . AnalysiS. litura of a laboratory susceptible strain were reared with artificial diet under conditions of 26 ± 1° C, 70–80% RH, and 16:8 L:D in a rearing room. The artificial diet contained 100 g of soybeans meal, 80 g of wheat bran, 8 g of casein, 8 g of vitamin C, 2 g of sorbic acid, 0.2 g of choline chloride, 0.2 g of inositol, and 0.2 g of cholesterol in 1 L volume.S. litura were ground in mortar with liquid nitrogen. Total RNA was extracted utilizing the Trizol Reagent  according to the protocol recommended by the manufacturer. 2 µ L total RNA was used as template to synthesize first-strand cDNA by PrimerScript Reverse Transcriptase and oligod(T)18 .Fifth instar larvae of B. mori (NM001099824.1), S. exigua (EF535530.1), P. xylostella (FJ665610.1), and Heliothis virescens (AF006189.1) as reported on NCBI (www.ncbi.nlm.nih.gov). A partial sequence of SlGABARα2 was amplified from firststrand cDNA of 5th instar larvae by SlGARDF and SlGARDR. Thermal cycling was done using the following protocol for touchdown PCR: 94° C for 3 min; 10 cycles at 94° C for 30 sec, annealing temperature decrease from 55 to 50° C for 30 sec, 2 cycles at each annealing temperature, 72° C for 1 min; another 20 cycles at 94° C for 30 sec, 50° C for 30 sec, 72° C for 1 min, and an additional polymerization step at 72° for 10 min. Amplified fragments were purified by 1.5% agarose gels, ligated into pMD 20-T vector (TaKaRa), transformed into Escherichia coli of DH5α to clone, and then positive clones were sequenced .Degenerate primes SlGARDF and SlGARDR  were desSlGABARα2, cloned as below to amplify the cDNA ends of SlGABARα2 using Nested PCR. For 3′ end amplification, according to the instructions of 3′-Full RACE Core Set Version 2.0 (TaKaRa), the first round PCR used primers SlGAR3EF1 and Outer Primer with the following protocol: 94° C for 3 min; 30 cycles at 94° C for 30 sec, 55° C for 30 sec, 72° C for 2 min; and an additional polymerization step at 72° C for 10 min. The second round of PCR used primers of SlGAR3EF2 and Inner Primer, with the first round PCR product as the template. The thermal cycling protocol was as follows: 94° C for 3 min; 30 cycles at 94° C for 30 sec, 60° C for 30 sec, 72° C for 2 min; and an additional polymerization step at 72° C for 10 min. For 5′ end amplification, the 5′ RACE cDNA of S. litura was synthetized using primer SlGAR5ER according to the manufacturer's instructions of 5′ RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Invitrogen). Subsequently, the first round PCR used primers SlGAR5ER1 and Abriged Anchor Primer with the following protocol: 94° C for 2 min; 30 cycles at 94° C for 30 sec, 55° C for 30 sec, 72° C for 1 min; and an additional polymerization step at 72° C for 10 min. The second round PCR used primers SlGAR5ER2 and UAUP Primer, with the first round PCR product as the template. The thermal cycling protocol was as follows: 94° C for 2 min; 30 cycles at 94° C for 30 sec, 60° C for 30 sec, 72° C for 1 min; and an additional polymerization step at 72° C for 7 min. The products of 3′ RACE and 5′ RACE were purified from 2.0% agarose gels and ligated into the T-vector (TaKaRa), then transformed into Escherichia coli of DH5α to clone, and positive clones were sequenced (Invitrogen).Specific primers SlGAR3EF1, SlGAR3EF2, SlGAR5ER, SlGAR5ER1, and SlGAR5ER2 were desS. litura according to the instruction of E.Z.N.A. Insect DNA Kit (OMEGA). Specific primers of genomic DNA SlGADNAF and SlGADNAR  and transformed into E. coli of DH5α to clone; then, positive clones were sequenced.Total genomic DNA was isolated from the adult SlGADNAR  were deswww.dnastar.com). Signal peptide was analyzed by online software SignalP 3.0 Server (http://www.cbs.dtu.dk/services/TMHMM/).Sequence similarity and analysis for conserved domains were performed using BLAST programs on NCBI. Amino acid sequences were derived and analyzed using EditSeq of DNASTAR 7.1  . Transmewww.cbs.dtu.dk) respectively (http://npsapbil.ibcp.fr/cgi-bin/secpred_phd.pl). Multiple sequence alignments were performed by Multalin version 5.4.1. A phylogenetic tree was constructed using MEGA 5.05 (www.megasoftware.net) with the neighbor joining method and reconstructed with 1000 replicate bootstrap analysis.N-glycosylation, O-glycosylation, and phosphorylation sites was analyzed by NetNGlyc 1.0 Server, DictyOGlyc 1.1 Server, and NetPhos 2.0 Server . The template model that was applied for comparative modeling was searched in the 3D structure of Protein Data Bank . Ten models were predicted, and the model that attained the highest score was chosen as the final template model of SlGABARα2. The amino acid rationality of the constructed model was analyzed by Profile-3D and Ramachandran plots.The comparative modeling of the tertiary structure of SlGABARα2 was performed using the module of Build Homology Models of Discovery Studio 2.0  following the manufacturer's recommendations. The final volume of the 25 µL reaction system contained 2 µL cDNA (less then 100ng), 12.5 µL SYBR Premix Ex TaqTm (TaKaRa), 8.5 µL ddH2O, 1 µ L of forward primer (10 µM), and 1 µ L of reverse primer (10 µM). qRT-PCR primers for SlGABARα2  to denature the double-stranded DNA. All samples of real-time PCR were replicated 3 times, the dates were recorded, and data were analyzed by Bio-Rad CFX Manager. Quantification of the transcript level of SlGABARα2 was conducted according to the 2-ΔΔCt method  and 3′ end (1399 bp) of SlGABARα2 was amplified using RACE technique and the specific primers SlGAR3RF1, SlGAR3RF2, SlGAR5RR1, and SlGAR5RR2, respectively, which was designed based on the partial sequence of SlGABARα2. The complete sequence of SlGABARα2 was spliced according to the partial 5′ end and 3′ end sequence by EditSeq of DNASTAR 7.1 and searched on the NCBI(www.ncbi.nlm.nih.gov) at amino acid level. The search result demonstrated that it shared 99.2% identity with GABA receptor a2 subunit gene from S. exigua . It was named as SlGABARα2, and the GenBank accession number is JN792582.A 370 bp fragment was amplified using degenerate primers SlGARDF and SlGARDR from the cDNA of 5th instar SlGABARα2 had 1966 bp that consisted of a 76 bp 5′ end untranslated region, a 389 bp 3′ end untranslated region, and a 1500 bp open reading frame  (The amino acid sequence of SlGABARα2 was blasted in the protein database. The search results indicated that the amino acid sequence of SlGABARα2 shared the highest homology (42%) with glutamate-gated chloride channel in D: 3RHW) . The 3D D: 3RHW) . The entD: 3RHW)  on one sD: 3RHW) , which iS. exigua, 97.6% to H. virescen, 95.4% to Chilo suppressalis, 92.8% to P. xylostella, and 87.2% to B. mori. It also shares remarkable homology among Diptera, with 86.0% to Culex quinquefasciatus, 84.4% to A. gambiae, 84.0% to Anopheles funestus, 83.65% to Musca domestica, 82.0% to Lucilia cuprina, 82.0% to Drosophila simulansv, 81.7% to Aedes aegypti, and 81.6% to D. melanogaster. It has a relatively low identity to Coleoptera and Homoptera, with 83.6% to T. castaneum and 78.2% to L. striatellus, respectively. After comparing homological amino acid sequences of GABA receptors among different insects, it can be concluded that the highly conserved positions were located at 2 N-glycosylation sites (N42 and N237), 1 Oglycosylation site (456S), 2 phosphrylation sites (404S and 461Y), 1 dicysteine-loop (Cys-loop), and 4 transmembrane domains  in SlGABARα2. The phylogenetic tree showed that the sequences of amino acid of GABA receptors had the nearest genetic distance in the same order  with 7 introns in it  within the subunit gene Rdl of the GABA receptor  that bind ions in other conserved domains (β-sheets in the tertiary structure of SlGABARα2) to transport them through the membrane. The DNA sequence information of SlGABARα2 may helpto understand the feedback and regulation mechanism in some physiological process of SlGABARα2 in S. litura.Studies of the GABA receptor subunits are still very limited, except for the RDL subunit. In the present study, the complete sequence of the GABA receptor α2 subunit in SlGABAα2 showed a distinct developmental- and tissue-specific pattern in S. litura. The expression level of SlGABAα2 was the lowest in the eggs, which was probably due to the incomplete development of the nervous system in the embryo. It increased rapidly with larval growth, which is correlated with the rapid growth and complete development of tissues and the nervous system in the 4th, 5th, and 6th instar larvae. Perhaps the low expression level of SlGABARα2 may be the reason why 1st, 2nd, and 3rd instar larvae are more sensitive to abamectin than 4th, 5th, and 6th instar larvae of S. litura. The relative expression level was reduced slightly in the pupal stage compared with 5th and 6th instar larvae and adults, which may be a result of the metamorphic development in the pupae when the nervous system was in the transformative process and the transportation of signals from the external was retarded. The tissue-specific pattern of SlGABARα2 indicated that the GABA receptor was mainly expressed in the brain of the larvae, and was extremely low in the Malpighian tube, midgut, epicuticle, and fat body compared to the brain. These results are in accordance with the ditribution of the nervous system being mainly concentrated in the brain, while just a few neurocytes are distributed in other tissues, especially in the fat body. The characters of expression of the GABA receptor in S. litura suggest that the best period for controlling S. litura using pesticides targeting the GABA receptor is in the early instars. These results may be beneficial to develop some novel, efficient, and safe insecticides targeting the GABA receptor and to create new methods to restrain the GABA receptor in pests, such as RNA interference and pestresistant transgenic plants.The expression of


The cryptic metabolite represents the first non-azinomycin related compound to be isolated and characterized from the soil bacterium, S. sahachiroi. The results from this study suggest that abolishing production of otherwise predominant natural products through genetic knockout may constitute a means to “activate” the production of novel secondary metabolites that would otherwise lay dormant within microbial genome sequences.At least 65% of all small molecule drugs on the market today are natural products, however, re-isolation of previously identified and characterized compounds has become a serious impediment to the discovery of new bioactive natural products. Here, genetic knockout of an unusual non-ribosomal peptide synthetase (NRPS) C-PCP-C module,  Aspergillus nidulans genome was mined for cryptic orphan gene clusters from which a single, unexpressed PKS-NRPS hybrid was identified. The expression of the gene cluster was activated by the ectopic expression of a specific regulatory gene, apdR, resulting in the production of the aspyridones + and GC–MS. The GC–MS fragmentation of the compound matched that provided in the Ultra GC–DSQ  GC–MS database. The structure of the compound was determined by NMR analysis in CDCl3  and C-2”  and double bonds in the aromatic ring at C-3  and C5  were observed by 1H NMR, DEPT, HMQC and HMBC. C-1” (δc 158.53), C-2 (δc 145.44), C-5 (δc 150.14) and C-1’ (δc 162.34) signals were detected by 13C NMR  and XbaI/EcoRI (azia2-D) and cloned into the corresponding sites of pKC1139, resulting in pKCA2UD. pKCA2UD was digested with XbaI and ligated with the thiostrepton-resistance gene (1056 bp), which was amplified with TsrF/TsrR primers using the same amplification condition as detailed for aziA2DF/aziA2DR, to give the final disruption recombinant plasmid pKC-AziA2, where aziA2 is replaced by tsrr in-frame.Disruption of E. coli S-17, a donor host and subsequently transferred into wild type, S. sahachiroi by conjugal transformation, as described by Kieser et al. (with some modification) [E. coli donor strain, 100 µL was transferred into 100 mL fresh LB medium and cultured for 4 h. The cells at OD600 0.6 were harvested, washed twice and solubilized in 500 µL of 2xYT medium. Spores of S. sahachiroi were prepared in MS agar plates and collected after one week. Spores were washed, heat shocked at 70 °C for 10 min and incubated at 37 °C for 4 h. Following incubation, they were washed and resuspended in 500 µL of 2xYT medium. These recipient cells were mixed with an equal volume of E. coli donor cells and 200 µL of the mixture were plated on MS-agar plates. The plates were incubated at 30 °C for 18 h, and overlayed with 1 mL water containing nalidixic acid (500 µg), to inhibit the growth of E. coli, and apramycin (25 µg) to select S. sahachiroi exconjugants. Incubation at 30 °C was continued for 6–8 days to allow growth of the exconjugants. Apramycin exconjugants were screened for several generations until the exconjugants were found to be apramycin sensitive and thiostrepton resistant to detect the double crossover allelic exchange. For further confirmation of deletion of aziA2 from S. sahachiroi, genomic DNA was isolated from both S. sahachiroi wild-type and aziA2 knockout strains (S. sahachiroi/ΔaziA2) and PCR performed. The AziA2UF/TsrR and TsrF/AziA2DR primer set was utilized: initial denaturation at 98 °C for 30 s; 30 total cycles of denaturation for 10 s; annealing at 64.5 °C for 40 s; polymerization at 72 °C for 60 s and finally gap filing at 72 °C for 10 min. Southern hybridization was carried out following standard procedures [32P-ATP labeled probe. The probe was generated through PCR amplification using the aziA2 probe-F/aziA2 Probe R set of primers and following the same PCR condition as detailed above.pKC-AziA2 was transformed into ication)  for deleocedures  with a rm/z.Fermentation was carried out in 10 L following established procedures . After 73 and contained in a 3 mm wilmad 335 pp NMR tube. Proton, carbon, Dept 90, Dept 135, HMQC and HMBC spectra were recorded. A 30° observe pulse was used to acquire the 1H NMR spectrum, while a 45o pulse was used to obtain the 13C NMR spectrum.NMR spectra were acquired on a Bruker Avance III 500 MHz instrument at 25 °C, equipped with a 5 mm H–C–N cryoprobe. The sample was dissolved in 125 μL CDClFile 1Details on genetic knockout, Southern blot analysis, LC–MS analysis and NMR data.


T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.rodent/27human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation. It is likely that skeletal muscle atrophy (muscle wasting) and hypertrophy (muscle growth) are caused by alterations in the balance between protein synthesis and degradation. It has been suggested that one pathway which plays a role in this balance is the p38 mitogen-activated protein kinase  pathway . p38 hasIn vertebrates four isoforms of p38 have been identified: p38α  and SAPK2a), p38β , p38γ  and p38δ,  . Activatrodent/27human) [One substrate of p38 is mitogen-activated protein kinase-activated protein kinase 2  with proposed roles in actin remodeling, cell migration, development and regulation of the cell cycle . In the 27human) , suggest27human) . Phospho27human) ,30. Over27human) .Hsp70, another heat shock protein that has been shown to prevent skeletal muscle atrophy when overexpressed ,33, was Denervation of skeletal muscles generally causes atrophy of the muscles but the hemidiaphragm becomes transiently hypertrophic following denervation -48. We hAll results reported are based on data from 4 sets of 8 animals generating 8 denervated anterior tibial muscles with 8 contralateral innervated controls, 8 denervated pooled gastrocnemius and soleus muscles with 8 contralateral innervated controls, 8 denervated hemidiaphragm muscles and 8 innervated control hemidiaphragms from separate animals.Six days after denervation hemidiaphragm muscles were hypertrophic with a wet weight of 40.3 ± 1.5 mg compared to 29.6 ± 0.6 mg for innervated controls . Six days after denervation anterior tibial muscles were atrophic with a wet weight of 44.7 ± 1.8 mg compared to 60.8 ± 1.9 mg for innervated controls . Six days after denervation pooled gastrocnemius and soleus muscles were atrophic with a wet weight of 149.1 ± 4.4 mg, compared to innervated controls with a wet weight of 198.3 ± 5.7 mg .Successful separation of nuclear and cytosolic fractions was confirmed using Western blots for Lamin A/C and superoxide dismutase 1 (SOD-1), respectively. Very little nuclear protein (Lamin A/C) was present in cytosolic fractions and only a small amount of cytosolic protein (SOD-1) was present in the nuclear fraction  and Hsp25 were increased about 0.6-fold, 1.3-fold and 3.2 fold, respectively, in cytosolic fractions  and Hsp25 were increased about 0.2-fold, 1.4-fold and 30-fold, respectively, whereas the expression of phosphorylated MK2 (T317) decreased about 0.7-fold in cytosolic fractions following denervation  become atrophic following denervation, unfractionated homogenates of pooled gastrocnemius and soleus muscles were examined. In atrophic 6-days denervated pooled gastrocnemius and soleus muscles changes in the expression of total and phosphorylated p38, MK2 and Hsp25 were similar to those seen in denervated atrophic anterior tibial muscle. Expression of total p38 was unchanged whereas MK2 protein expression decreased about 0.5-fold and Hsp25 protein expression increased about 0.6-fold. Phosphorylated p38, MK2 (T205) and Hsp25 increased about 1.2-fold, 1.3-fold and 12-fold, respectively, whereas phosphorylated MK2 (T317) decreased about 0.3-fold following denervation Figure .The mean expression level of total p38 protein Figure a was 86.The mean expression level of total MK2 protein Figure c was 51.The mean expression level of total Hsp25 protein Figure f was 157Hsp70 expression was studied since it has recently been reported to play a role in the phosphorylation of MK2 by p38 (see Background). Unlike the other proteins studied Hsp70 was more abundant in nuclear than in cytosolic fractions of innervated as well as 6-days denervated muscles. In atrophic 6-days denervated anterior tibial muscles the expression of Hsp70 was slightly increased (about 0.3-fold) in the nuclear fraction compared to innervated controls. In 6-days denervated hypertrophic hemidiaphragm the expression of Hsp70 was increased about 5-fold and 2.9-fold in cytosolic and nuclear fractions, respectively  was not reflected in p38 phosphorylation. Thus, in denervated atrophic pooled gastrocnemius and soleus muscles p38 phosphorylation was increased Figure b whereasThe discrepancies between p38 phosphorylation and MK2 T317 phosphorylation may also be due to different isoforms of p38 responding differently to denervation. The antibodies used in the present study do not differentiate between the different p38 isoforms and while all p38 isoforms appear to be expressed to some extent in skeletal muscle -59 p38γ Following denervation the level of Hsp25 was increased in all muscles studied, as was the level of phosphorylated Hsp25. Similar to the discordance between p38 phosphorylation and MK2 T317 phosphorylation, the level of MK2 T317 phosphorylation was not reflected in Hsp25 phosphorylation in atrophic muscle. In atrophic anterior tibial muscles as well as in atrophic pooled gastrocnemius and soleus muscles Hsp25 phosphorylation was increased Figures g despiteThe present study confirms a differential response of MK2 T317 phosphorylation to denervation in hemidiaphragm  and hind-limb muscle  and suggests that factors other than p38 may be important for this differential response, e.g. Hsp70. The study further shows that, despite the differential response of MK2 T317 phosphorylation to denervation in the different muscles, Hsp25 phosphorylation was increased in all denervated muscles studied indicating a role for other kinases and/or phosphatases in the regulation of Hsp25 phosphorylation in denervated muscle.All experiments were performed on adult male NMRI mice . Before surgery the animals were anaesthetized by inhalation of isoflurane. Denervation of either the left hind-limb or the left hemidiaphragm was performed by sectioning and removing a few mm of either the sciatic nerve or the phrenic nerve as described previously . While a2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), pH 7.9 with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail from Thermo Scientific, Rockford, IL). The homogenized tissue was vortexed for 15 s, put on ice for 10 min, vortexed again for 15 s and centrifuged at 16,000 g for 15 s. The supernatant cytosolic extract was immediately frozen (-80°C) for subsequent analyses. The nuclear pellet was resuspended on ice in a high salt nuclear extraction buffer  at a ratio of 4 μl of nuclear extraction buffer per mg muscle wet weight. Preparations were incubated on ice for 30 min and vortexed for 10 s every 5 min before being centrifuged at 16,000 g for 6 min. The supernatant nuclear extract was then removed and frozen (-80°C) for subsequent analyses. Protein concentrations for each fraction were obtained using the Bradford assay [Mouse hemidiaphragm and anterior tibial muscles were used for nuclear and cytosolic protein extraction. The method used was slightly modified from . The musrd assay .Pooled gastrocnemius and soleus muscles were used for preparing protein extracts of unfractionated muscle homogenates. The muscles were homogenized in 2 ml of a buffer containing 100 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail from Thermo Scientific . The homogenate was then centrifuged at 16,000 g for 10 min. The supernatant was recovered and the pellet was resuspended in 2 ml of buffer and re-centrifuged. The supernatants were combined and the protein concentration was determined using the Bradford assay .Western blots were prepared essentially as described in . Ten to The expression levels of total and phosphorylated proteins were studied semi-quantitatively using data from Western blots. Measured levels of total and phosphorylated proteins were expressed without normalization to any specific protein. No loading controls were used and any difference in protein quantifications, pipetting steps, protein transfers etc. are included in the variations of the data sets.http://rsb.info.nih.gov/ij/, 1997-2007). Results were obtained in uncalibrated units.Image analysis was performed using the gel plotting macro of the program ImageJ . Student’s t-test was used for statistical comparisons of normally distributed data . Statistical significance for data that was not normally distributed was determined using the Wilcoxon matched pairs test  and Mann-Whitney test (hemidiaphragm muscles).The authors declare no competing interests.The work presented here was carried out in collaboration between all authors. KE designed the study and carried out the studies on cytosolic and nuclear fractions, most of the protein expression studies, statistical analyses and drafted the manuscript. AKF carried out the studies on total and phosphorylated p38, MK2 and Hsp25 in 6-days denervated atrophic pooled gastrocnemius and soleus muscles. MN and ST conceived of the study, participated in the design, statistical analyses and drafting of the manuscript. All authors have read and approved the final manuscript.


Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant,  Meiosis, the formation of haploid gametes from diploid precursor cells, involves complex changes to chromosomes and their DNA. During meiosis, recombination between homologous chromosomes (homologs) forms a physical connection that provides tension between homologs on proper attachment to microtubules. This ensures proper segregation of recombined homologs to opposite poles at the first meiotic division. Without recombination and crossovers, in most species, chromosomes often missegregrate, and the resulting aneuploid gametes and subsequent progeny are inviable or debilitated . MeioticSchizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have been indispensable in such studies. Particularly useful is the S. pombe temperature-sensitive pat1-114 mutant, which can be synchronously induced by raising the temperature of the culture from 25 to 34°C  formation at 36°C and recombination at 35°C are less frequent than at 25°C  that is inactivated in the presence of an ATP analog, using a procedure successful with many protein kinases (S. pombe and indirectly represses the hundreds of genes whose products are required for meiosis (+/h−h) are starved for nitrogen, Pat1 is inactivated and is unable to phosphorylate and inactivate Mei2, an RNA binding protein that functions in the destruction of meiotic mRNAs during vegetative growth. Nitrogen starvation also activates Ste11, a transcriptional activator of mei2 and many other meiosis-specific genes (S. pombe cells starved for nitrogen arrest at the G1 phase of the cell cycle (pat1-114 at 34°C (5–6 h in pat1-as2 at 25°C), and the first meiotic division (MI) between 5 and 6 h at 34°C (9–10 h at 25°C)   (S. cerevisiae occurs by the invasion of one end of the broken DNA into a homologous duplex to form a displacement (D) loop, also called a single-end invasion (SEI); annealing of the other broken end to the displaced strand forms a double Holliday junction  ,18  . As a Spregions) . DSB repjunction ,21. Clearevisiae . ResolutS. pombe pyrazolopyrimidine; Toronto Research Chemicals] was added to 20 μM for pat1-as2 cultures or the temperature was raised to 34°C without addition of inhibitor for either pat1-as2 or pat1-114 cultures. Cells were harvested, DNA extracted and DSBs and HJs assayed . Whole genome data are in Supplementary Figures S1 and S2, and raw data are at http://www.ncbi.nlm.nih.gov/geo/. To allow comparisons between strains in the same experiment, the log10 value of each data point  was multiplied by a value ranging from 1.1 to 1.3, to make coincident the levels of six prominent DSB hotspots, shown by Southern blots to be indistinguishable, with the values of the array with the highest values at those sites. Data analyses used R (http://www.r-project.org/) and Bioconductor (http://www.bioconductor.org/).The distribution of DSBs was assayed genome-wide by measuring the positions of Rec12-FLAG covalently self-linked to DNA, i.e. without artificial cross-linking . Rec12-Fpat1-as2 cells at 25°C by adding 20 μM 1-NM-PP1 and in pat1-as2 and pat1-114 cells by raising the temperature to 34°C (without addition of inhibitor), extracted DNA and digested it with NotI restriction enzyme   d, althoueratures c.pat1-as2 DSBs assayed at 34°C appeared slightly later than pat1-114 DSBs , suggesting the slower meiosis at 25°C likely reflects the effects both of lower temperature and of the ATP-analog inhibitor. As expected, in each case, DSBs were repaired before MI begins, as measured previously  . To aid comparison of results from two or more strains, we multiplied the normalized log10 values by an appropriate value (ranging from 1.1 to 1.3) so that the signals at six strong DSB hotspots, as determined by Southern blot hybridizations, were nearly equal . Two independent inductions of the pat1-as2 strain at 25°C gave highly reproducible results, as did side-by-side inductions of pat1-as2 rad50S and pat1-114 rad50S strains at 34°C .To analyze the meiotic distribution and relative frequency of DSBs across the entire nalysis) . Chromatbundance a and c . Eleven hotspots, however, reproducibly had significantly more DSBs  in +rad50 strains at 25°C than at 34°C .The data  show tha strains g; thus, reduced at 25°C , some of which are marked with red arrows in ade6-3049, a single bp mutation that creates a binding site for the transcription factor Atf1-Pcr1  and between rad50S strains , in which accumulated DSBs give a more reliable estimate of total DSB frequency , and the six hotspots were within the range of variation, demonstrating that these reductions are not due to timing differences in DSB repair. Furthermore, comparison of the DSBs in pat1-as2 and pat1-114 strains at 34°C also showed high correlation of total hotspots with the six hotspots within the range of variation . Thus, DSB formation, not their repair, differs at 25 and 34°C, and these differences are independent of the pat1 allele used. DSBs at other sites in pat1-as2 meiosis were also modestly reduced. These small reductions are within the 95% prediction interval and hence cannot be differentiated from random variation, although they may be meaningful. We discuss later the implications of these observations.More interestingly, six intense DSB hotspots at 34°C were reproducibly strongly tf1-Pcr1 ; DSBs attf1-Pcr1 d. DSBs apat1-as2 cells, we assayed the formation and resolution of HJs  at the mbs1 hotspot in pat1-as2 cells at 25°C and that in pat1-114 cells at 34°C  at 25°C in pat1-as2 meiosis than at 34°C in pat1-114 meiosis . T. Tmbs1 hpat1-114 b  at 34°C d, the ISpat1-as2 meiosis is that it proceeds slower and DNA intermediates of recombination persist longer, allowing a more sensitive measure of the low-level HJs formed in a +mus81 strain. For example, 1.4% of the total DNA was observed as HJs at mbs1 in pat1-as2 at 25°C but 0.9% in pat1-114 at 34°C . Th. Thpat1-slinking ,25. Shorand 10 h a. These mat-Pc cassette in a homozygous −h diploid restored spore viability and chromosome segregation to wild-type (+pat1) levels. We also observed that pat1-as2 diploid strains, either at 25 or 34°C, had slightly reduced meiotic recombination  and by ∼50% between ade6 and arg1   . Neither repeats .Table 2mbs1 hotspot or the ade6-3049 hotspot. This allowed us to measure the total amount of crossover DNA, rather than just the frequency of recombinants among viable spores ,25mbs1 he spores a and b b at 34°C b, in acceratures b and 4c. at 34°C d and 4d,pat1-as2 mutant studied here provides for the first time a convenient way to prepare large cultures of meiotic S. pombe cells synchronously induced at 25°C, a temperature at which many studies of meiosis have been done. Synchronous cultures are essential for biochemical analyses of transient intermediates, and it is important to have similar conditions for both genetic and biochemical assays. The pat1-as2 mutant affords experimental flexibility and allows comparisons to be made at lower temperature than the pat1-114 temperature-sensitive mutant previously available. Using this allele, we have found that many features of DNA intermediates of recombination are the same as those in the pat1-114 mutant at 34°C, the condition previously used to study DNA recombination intermediates. These results confirm many of the conclusions previously drawn, e.g.  as accession number GSE49961.Raw data in Supplementary Data are available at NAR Online, including .United States of America National Institutes of Health grants [GM031693 and GM032194 to G.R.S.] and Austrian Science Fund (FWF) grants [P23609 and P21437 to J.G.]. The European Community’s Seventh Framework Programme (FP7/2007-2013) grant agreement number [PERG07-GA-2010-268167 to L.C.] and grant agreement number [PCIG11-GA-2012-322300 to J.G.].Funding for open access charge: Conflict of interest statement. None declared.


Inconsistent expression and regulation of drug-metabolizing enzymes (DMEs) are common causes of adverse drug effects in some drugs with a narrow therapeutic index (TI). An important cytochrome, cytochrome P450 3A4 (CYP3A4), is predominantly regulated by a nuclear receptor, pregnane X receptor (PXR). Sesamin, a major lignan constituent in sesame seeds and oil, exhibits a variety of biological functions; however, the effect of sesamin on the modulation of CYP3A4 is not well understood. In this study, the effects of sesamin on the PXR-CYP3A4 pathway were characterized, as well as the underlying mechanisms of those effects. Sesamin potently attenuated CYP3A4 induction in a dose-dependent manner by blocking the activation of PXR. The PXR inducer-mediated inhibition of CYP3A4 was further evidenced by the ability of sesamin to attenuate the effects of several PXR ligands in the CYP3A4 reporter assay. Further mechanistic studies showed that sesamin inhibited PXR by interrupting the interacting with coregulators. These results may lead to the development of new therapeutic and dietary approaches to reduce the frequency of inducer-drug interaction. Sesamin was established as a novel inhibitor of PXR and may be useful for modulating DMEs expression and drug efficacies. Modification of CYP3A4 expression and activity by consumption of sesamin may have important implications for drug safety. Sesamum indicum) and their oil have been used in human diets for thousands of years and are believed to provide health benefits. A major constituent of sesame is a lignan called sesamin. Sesamin has a variety of biological functions including reduction of serum and hepatic cholesterol levels thiazole-5-carbaldehyde O-oxime (CITCO), SR12813, and paclitaxel were purchased from Sigma-Aldrich  and dissolved in DMSO at concentrations appropriate for the specific studies in which they were used. HepG2 and LS174T cells were purchased from the Food Industry Research and Development Institute  and maintained in a minimum essential medium (MEM) α medium supplemented with 10% fetal bovine serum without antibiotics, in a 5% CO2 atmosphere at 37°C. Rifampin (RIF), sesamin (SSM), 5-pregnen-3α, pBIND-PXR, and pACT-HNF4α were prepared as described previously , containing full-length human PXR and CYP3A4 promoter constructs, respectively, have been described previously . Full-leeviously . A full-p-nitrophenyl phosphate (PNPP) disodium salt as a substrate. The cell culture media were aspirated, and the cells were washed with phosphate-buffered saline (PBS). Following the wash, 100 μL of the ACP reagent  were added. After 1 h of incubation at 37°C, the enzyme activity was stopped by adding 10 μL of 1 N NaOH, and the extent of activity was determined photometrically at a wavelength of 405 nm . Therefore, sesamin had significant effects on the basal and induced levels of CYP3A4 mRNA expression  significantly decreased the basal level of CYP3A4 protein expression  as compared to the DMSO-treated group . Coincubation of the cells with 20 and 40 μM sesamin with 20 μM rifampin significantly decreased rifampin-induced CYP3A4 protein expression by 57.7% (P < 0.05) and 66.5% (P < 0.05), respectively, as compared to rifampin-induced cells. The effects were concentration dependent  strongly attenuated the transactivation of the CYP3A4 promoter induced by rifampin in a concentration-dependent manner when cotransfected with human PXR. As shown in P < 0.05), 40.6% (P < 0.01), and 64.8% (P < 0.001), respectively, as compared to the rifampin-induced groups. Similar experiments were also performed using a colon adenocarcinoma cell line (LS174T) to exclude the possibility of a cell-type-specific effect. As shown in μM sesamin producing the greatest inhibitory effect .PXR is a main ligand-activated transcription factor of CYP3A4. PXR transactivation reporter assays were conducted to assess the inhibitory effect of sesamin alone or with rifampin-induced PXR transactivation activity, on the CYP3A4 promoter. Cells were cotransfected with a μM sesamin significantly decreased rifampin-induced CYP3A4 promoter activity in HepG2 cells  was found to markedly attenuate CITCO-induced CYP3A4 promoter activation through CAR  or 40 μM sesamin , as compared to the PCN-treated cells. The results indicate that sesamin activity is not highly species specific.CYP3A4 inhibition is predominantly modulated by the effect of PXR on CYP3A4 expression and partly modulated by CAR. Previous reports have shown that the differences in the ligand-binding domain of human and rat PXR may lead to ligand-induced species-specific effects . Therefo in vitro, the impact of sesamin on PXR protein expression was investigated. PXR binds to DNA as a heterodimer with RXRα. The heterodimer can bind to and induce transcription from response elements located in the CYP450 genes [α on the CYP3A4 promoter transcription complex [α, and HNF4α protein levels in sesamin-treated cells. HepG2 cells were treated with 20 and 40 μM sesamin alone or in combination with 20 μM rifampin and lysed after 48 h of exposure. As shown in α  decreased the promoter activity to 24.9% (P < 0.001) of the level observed in rifampin-treated cells. Transfection with PXR and SRC-1 expression plasmids resulted in a CYP3A4 promoter activity enhancement of 243.6 folds, in the presence of rifampin. Again, sesamin (20 and 40 μM) strongly disrupted the enhancement of the promoter activity; activity was decreased to 64.2% (P < 0.001) or 18.5% (P < 0.001), respectively, of the level observed in rifampin-treated cells. Earlier studies showed that cotransfection of SRC-1 and HNF4activity , 54, 55.rifampin . Sesaminα and PXR was also evaluated. When cells were cotransfected with the vector control and full-length HNF4α prior to rifampin exposure, CYP3A4 activity was enhanced 23.2 folds. This effect was disrupted by sesamin, with the greatest inhibition at a sesamin concentration of 40 μM . The coregulation strength was not as great as that observed in the SRC-1/PXR cells; the presence of HNF4α and PXR enhanced CYP3A4 promoter activity by 53.6 folds. As μM sesamin significantly reduced CYP3A4 promoter activity in these cells when exposed to rifampin . These results indicated that sesamin disrupts the coregulation effects of SRC-1/PXR and HNF4α/PXR on CYP3A4 promoter activity.The coregulation of HNF4α and PXR [α/PXR interaction, a GAL4/VP16 mammalian two-hybrid reporter system was used. The RID of SRC-1 and the full-length PXR gene were fused with the GAL4 DNA-binding domain to become pBIND-SRC-1 and pBIND-PXR, respectively. Similarly, the full-length PXR and HNF4α were fused with the VP-16 vector to become pACT-PXR and pACT-HNF4α, respectively.Ketoconazole has previously been shown to inhibit the expression of CYP3A4 by disrupting the interaction of SRC-1 and PXR as well as that of HNF4 and PXR . To deteμM sesamin decreased the interaction between PXR and SRC-1 by 27% and 49.1% (P < 0.05 and P < 0.001), as compared to control groups in LS174T cells. In contrast, rifampin strongly promoted the specific interaction between PXR and SRC-1 by 8.1 folds; sesamin attenuated the interaction between PXR and SRC-1 concentration-dependent manner, with a 25.8% (P < 0.05) and 67.9% (P < 0.001) inhibition at 20 and 40 μM sesamin, respectively.As shown in α and PXR was also disrupted by sesamin. As shown in α, without rifampin induction, decreased by 45.2% (P < 0.001) compared to DMSO-treated cells. Rifampin enhanced the protein-protein interaction of PXR and HNF4α by 2.2 folds. As expected, sesamin disrupted this interaction in a concentration-dependent manner. A decrease of 31.3% was seen in cells exposed to 20 μM sesamin (P < 0.01), and a decrease of 57.6% was seen in cells exposed to 40 μM sesamin (P < 0.001), as compared to rifampin-treated groups which were not exposed to sesamin. These data suggest that sesamin inhibits SRC-1/PXR and HNF4α/PXR interactions, thus transactivation in the regulation of CYP3A4 gene expression occurs through the transcriptional control.The interaction between HNF4μM paclitaxel enhanced CYP3A4 promoter activity in LS174T cells up to 3 folds as compared to the controls. When the cells were cotreated with paclitaxel and 20 or 40 μM sesamin, CYP3A4 promoter activity reduced by 39.1% (P < 0.05) and 60.5% (P < 0.001), respectively, when compared with paclitaxel-induced controls. These results indicated that sesamin may rescue PXR agonists from autoinduced metabolism. Because many antineoplastic drugs have a narrow therapeutic index, the ability of drugs  to activate PXR may be limited because of autoinduced metabolism . This imμM SR12813, CYP3A4 promoter activity in LS174T cells was enhanced by 46.1 folds, as compared to DMSO-treated cells (μM) resulted in 62.8% (P < 0.001) or 78.4% (P < 0.001) inhibition, respectively, of transcriptional activity induced by SR12813. The results showed that inhibition of CYP3A4 by sesamin was not a rifampin-specific effect.SR12813, the most efficient PXR agonist discovered to date , was useed cells . The preμM) increased UGT1A1 mRNA expression in HepG2 cells in a concentration-dependent manner, resulting in a 3.1-fold (P < 0.01) or 4-fold (P < 0.001) increase at sesamin concentrations of 30 and 40 μM, respectively. The expression of UGT1A1 mRNA also increased 11.7 folds in the presence of 20 μM rifampin, consistent with the results of previous studies [μM sesamin, in combination with 20 μM rifampin, expression of UGT1A1 mRNA increased with increasing concentrations of sesamin. This increase was particularly notable at sesamin concentrations of 30 μM, where mRNA levels increased by 65.6% (P < 0.05), and 40 μM, where mRNA levels increased by 98% (P < 0.01). Presumably, sesamin induced UGT1A1 gene expression through a PXR-independent pathway and may be a nuclear factor E2-related factor 2- (Nrf2-) dependent pathway [P < 0.05) in the presence of 20 μM sesamin, and 2-fold (P < 0.001) at a 30 μM sesamin concentration. The net inductive effect of sesamin on UGT1A1 gene expression is interesting because UGT1A1 is also regulated, in part, by PXR.The discovery that sesamin inhibits CYP3A4 expression led to an investigation of sesamin's effect on the expression of UGT1A1 mRNA in HepG2 cells by RT-PCR. UGT1A1 is an enzyme involved in hepatic phase II metabolism that is also regulated by PXR . As show studies . With th pathway . Sesaminα by disrupting their recruitment and interaction with PXR. These changes were not due to reduced protein levels of those nuclear receptors or coregulators. The inductive effects of SR12813 and paclitaxel could be attenuated by coadministration with sesamin. These results suggest a potential mechanism for preventing inducer-drug interactions between PXR ligands and minimizing the potential for P450 inducer-drug interactions. Although numerous human PXR activators have been identified, to date there are few reports of potent inhibitors of PXR. Sesamin is a novel functional inhibitor of PXR and may be a useful chemical tool for modulating PXR-regulated gene expression in vitro or in vivo.In this study, sesamin was demonstrated to antagonize the induction of CYP3A4 in HepG2 and LS174T cells. It was also determined to be an inhibitor of rat and human PXR in nuclear receptor transactivation assays. Additionally, sesamin was demonstrated to suppress human CAR activation. Furthermore, the underlying mechanisms of human PXR inhibition were explored. Sesamin was found to inhibit coregulation between SRC-1 and HNF4CYP3A4, a cytochrome that is variably expressed in the liver and small intestine cells [Inappropriate PXR activation leads to important and undesirable pathophysiologic consequences . One of ne cells . Drug-inne cells . In this Extensive cross-talk occurs between members of the nuclear receptor superfamily. Both PXR and CAR are closely related nuclear receptors belonging to the nuclear receptor subfamily (NR1) that are involved in CYP3A4 regulation and are endogenously expressed in HepG2 cells . Their lThe full activity of NRs depends on a large number of cellular factors that called coactivator, that do not bind DNA directly, but are selectively recruited to the promoter by the NRs through protein-protein interactions . This suα also plays an essential role in the PXR-dependent induction of human CYP3A4 [α element adjacent to the PXR- and CAR-binding sites in the human CYP3A4 promoter is an obligatory component of a xenobiotic-responsive distal enhancer [α [α and the coactivator, SRC-1.HNF4n CYP3A4 , 62. Thuenhancer . Previouancer [α , 63. Anaα and HNF4α were not affected by sesamin.Differential activation of PXR orthologs between species has been well reported . MoreoveNrf2, which binds to AREs present within the promoter [Nrf2 target gene, NQO1, as well [Nrf2-dependent pathway.Literature reports indicate that UGT1A1 is transcriptionally regulated by both PXR and CAR through DR-3 and NR-1 elements in the 5′-promoter region , 65. Respromoter . The res as well . SesaminAside from its important role in xenobiotic defense, evidence suggests that during carcinogenesis, PXR promotes tumor cell growth . Cancer μg/mL, or approximately 6, 23.1, and 22.6 μM, respectively. Our results indicate that 20 μM sesamin is sufficient to decrease CYP3A4 activity and mRNA and protein expression levels significantly. Therefore, a plasma concentration of 20 μM sesamin, which is sufficient to modulate CYP3A4 according to our in vitro data, may well be achievable by oral ingestion, at least in animal studies. Differences in genetics, geographical origin, growing conditions, seed size, and capsule position may contribute to the variable amounts of sesamin in seeds. Rangkadilok et al. [A genotoxicity report from Hori et al.  includedk et al.  showed rk et al. . Here, win vivo approaches. Moreover, these cells are known to constitutively express CYP1A1/2, 2A6, 2B6, 2C9/19, 2D6, 2E1, and 3A4/5 [CYP3A4 mRNA expression and enzymatic activity levels similar to those in cultured primary human hepatocytes [HepaRG cells, which were derived from a female hepatocarcinoma patient, are capable of differentiating into biliary epithelial cells and hepatocytes . They rend 3A4/5 . Researcatocytes . Therefoα without affecting expression of NR-related modulators. These findings suggest a complementary mechanism by which ingestion of this naturally occurring phytochemical may reduce the frequency of adverse drug reactions secondary to PXR-mediated induction of drug clearance via CYP3A4. This may improve the therapeutic efficacy of certain drugs and may also play a role in reducing the formation of CYP3A4-mediated reactive metabolites.In conclusion, we have identified a naturally occurring lignan, sesamin, that is a potent inhibitor of human and rat PXR. The study provides evidence that sesamin attenuates the induction of CYP3A4 by inhibiting agonist-activated PXR and CAR. Sesamin disrupts the interaction of PXR with SRC-1 and HNF4


VGF level was lower in the spinal cords of sporadic ALS patients than in the control patients. Furthermore, SUN N8075 slowed disease progression and prolonged survival in mutant SOD1 transgenic mouse and rat models of ALS, preventing the decrease of VGF expression in the spinal cords of ALS mice. These data suggest that VGF plays a critical role in motor neuron survival and may be a potential new therapeutic target for ALS, and SUN N8075 may become a potential therapeutic candidate for treatment of ALS.Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease, and recent evidence has suggested that endoplasmic reticulum (ER) stress signaling is involved in the pathogenesis of ALS. Here we identified a small molecule, SUN N8075, which has a marked protective effect on ER stress-induced cell death, in an  In chronic neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), abnormally unfolded proteins are known to aggregate and accumulate in neurons, and these proteins are thought to be closely related to the initiation and development of these neurodegenerative diseases (2S)-1--3-{4-[4-(4-fluorobenzyl) phenyl]-1-piperazinyl}-2-propanol dimethanesulfonate (SUN N8075) was synthesized at Asubio Pharma Co. Ltd. .2 at 37°C. The cells were passaged by trypsinization every 5 to 7 days, as described in a previous report Cultures of human neuroblastoma (SH-SY5Y) cells were maintained in Dulbecco's modified Eagles's medium  containing 10% FBS , 100 U/ml penicillin , and 100 µg/ml streptomycin (Meiji Seika Kaisha Ltd.) in a humidified atmosphere of 95% air and 5% CO4 cells per well into 96-well plates, and then incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 2 days. Tunicamycin  or thapsigargin was added to the cells at a final concentration of 2 µg/ml and 2 µM, respectively. SUN N8075 was added 1 h before the tunicamycin treatment, at which time the cell-culture medium was replaced with D-MEM containing 1% FBS. Assessment of cell viability was performed using two methods, each at 24 h after the addition of tunicamycin. The first method was a single-cell digital imaging-based method employing fluorescent staining of nuclei. Cell death was assessed on the basis of combination staining with fluorescent dyes , observations being made using an OLYMPUS IX70 inverted epifluorescence microscope . Hoechst 33342 freely enters living cells and therefore stains the nuclei of viable cells, as well as those that have suffered apoptosis or necrosis. Apoptotic cells can be distinguished from viable and necrotic cells on the basis of nuclear condensation and fragmentation. YO-PRO-1 is a membrane-impermeant dye that is generally excluded from viable cells, whereas early-stage apoptotic and necrotic cells are YO-PRO-1-positive. At the end of the culture period, Hoechst 33342 and YO-PRO-1 dyes were added to the culture medium  for 30 min. Images were collected using a digital camera . In a blind manner, a total of at least 400 cells per condition were counted using image-processing software . As the second method for measuring cell viability, cell metabolic activity was quantitatively assessed. Cell viability was assessed following immersion in 10% WST-8 solution  with an electron carrier, 1-Methoxy PMS   for 3 h at 37°C, and absorbance was recorded at 450 nm To examine the effects of the SUN N8075 on tunicamycin-induced cell death, cells were seeded at a density of 1×10SH-SY5Y cells or mouse spinal cords were lysed using a cell-lysis buffer . Lysates were solubilized in SDS-sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred to PVDF membrane . Transfers were blocked for 1 h at room temperature with 5% Blocking One-P  in 10 mM Tris-buffered saline with 0.05% Tween 20 (TBS-T), then incubated overnight at 4°C with the primary antibody. The transfers were then rinsed with TBS-T and incubated for 1 h at room temperature in horseradish peroxidase goat anti-rabbit or goat anti-mouse  diluted 1∶2000. The immunoblots were developed using chemiluminescence , and visualized with the aid of a digital imaging system . The primary antibodies used were as follows: mouse anti-cleaved caspase-3 , rabbit anti-VGF  and rabbit anti-β-actin .4 cells per well. After the cells had been incubating for 48 h, they were exposed to tunicamycin at 2 µg/ml with or without SUN N8075 at 3 µM in 1% FBS DMEM for 2, 6, or 12 h. Quantitative real-time PCR was performed using a Thermal Cecler Real Time System  with a TaqMan® Gene Expression Cells-to-CT™ Kit (Applied Biosystems) according to the manufacturer's protocol. mRNA expression was measured by real-time PCR using TaqMan probe (TaqMan Gene Expression Assay): VGF (Assay ID Details: Hs00705044_s1). The thermal cycler conditions were as follows: 2 min at 50°C and then 10 min at 95°C, followed by two-step PCR for 50 cycles consisting of 95°C for 15 sec followed by 60°C for 1 min. For each PCR, we obtained the slope value, R2 value, and linear range of a standard curve of serial dilutions. The results are expressed relative to the GAPDH  internal control.To examine the effect of SUN N8075 on VGF mRNA expression, SH-SY5Y cells were seeded in 96-well plates at a density of 1.0×10For VGF knockdown, the following siRNA sequences specific to human VGF were used: 5′-AUUCACUCGGGUCAGCGUGUGCGUG-3′ (sense), 5′-CACGCACACGCUGACCCGAGUGAAU-3′ (antisense). Stealth™ RNAi Negative Control Medium GC Duplex #2 was used as a control. All siRNAs were obtained from Invitrogen . The SH-SY5Y cells were transfected with 50 pg of siRNA using Lipofectamine™ RNAi MAX Reagent (Invitrogen) according to the manufacturer's protocol.2.For overexpression of VGF, pCMV6-ENTRY containing myc-tagged VGF and same empty vector (control) were obtained from ORIGENE . SH-SY5Y cells were transiently transfected with each plasmid using Lipofectamine™2000 (Invitrogen) according to the manufacturer's protocol. The transfected cells were subjected to immunoblot analysis and cell death assay. The cells transiently transfected with plasmids or siRNAs were incubated for 24 h at 37°C in a humidified atmosphere of 95% air and 5% COWe used two types of transgenic animals overexpressing mutated human SOD1 genes: transgenic mice overexpressing mutated  human SOD1 (G93A) SUN N8075 was dissolved in 6% captisol solution, and subcutaneously administered at doses of 30 mg/kg and 10 mg/kg, respectively, once daily from 10-weeks-old (for SOD1G93A mice) or 15-weeks-old (for SOD1H46R rat) to lifetime. In control group, vehicle (6% captisol solution) was subcutaneously administered at 10 ml/kg.A rotarod test was performed with a rotation at 5 rpm once every 7 days, as previously described by Chiba et al. Symptomatic disease onset and post-symptomatic disease progression were assessed using a 5-point motor score system as described in previous reports Mortality was scored at the day when the mouse was unable to right itself within 30 s after being placed on its back.Total RNA was extracted from SH-SY5Y using the RNeasy Mini kit  including a DNase digestion step, according to the manufacturer's instructions. Total RNA was amplified and labeled with Cyanine 3 for the test sample using Agilent's Low RNA Input Linear Amplification Kit  following the detailed protocol described in the kit manual (version 5.7). Briefly, 500 ng of total RNA was reversed transcribed to double-strand cDNA using a poly dT-T7 promoter primer. Primer, template RNA and quality-control transcripts of known concentration and quality were first denatured at 65°C for 10 min and incubated for 2 hours at 40°C with 5× first strand Buffer, 0.1 M DTT, 10 mM dNTP, MMLV RT, and RNase-out. The MMLV-RT enzyme was inactivated at 70°C for 15 min. cDNA products were then used as templates for in vitro transcription to generate fluorescent cRNA. cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and incubated at 40°C for 2 h. Labeled cRNAs were purified using Qiagen's RNeasy mini spin columns and eluted in 30 µL of nuclease-free water. After amplification and labeling, cRNA quantity and cyanine incorporation were determined using a NanoDrop ND. Microarray expression experiments were performed on 4×44 K Agilent Human and Mouse expression arrays (Agilent technologies) by hybridizing SH-SY5Y cells according to the manufacturer's instructions. Images of the arrays were acquired using a microarray scanner G2565BA (Agilent technologies) and image analysis was performed using Feature Extraction software version 9.5 (Agilent Technologies).Raw data was imported into GeneSpring GX 7.3.1 (Agilent Technologies) and normalized by setting all measurements <0.01 to 0.01, normalizing each chip to the 75th percentile of all measurements taken for that chip, and normalizing each gene to the median measurement for that gene across all chips. To focus on genes with reliable measurements, the normalized data were filtered for data that had present call in ≥33% of all samples (based on flag information of Agilent Feature Extraction Software v.9.5.3) and the genes chosen by parametric test  with a Bejamini and Hochberg false discovery rate (FDR) <0.01 and twofold restriction filters were utilized. Hierarchical clustering was performed on the log transformed normalized data of all the samples. Pearson's correlation was used for the similarity metric and the calculation result was visualized using “Gene Tree” algorism. A red/blue color scheme was used in the heat maps of the gene expressions.SOD1G93A and WT mice at 14 weeks of age (decreasing motor function) were anesthetized with sodium pentobarbital   and perfused with 2% (w/v) paraformaldehyde solution in 0.01 M phosphate-buffered saline (PBS) at pH 7.4. Spinal cord tissues were removed after a 15-min perfusion at 4°C and immersed in the same fixative solution for 24 h. Each spinal cord included L1, L2, and L3 levels, were soaked in 25% (w/v) sucrose at 4°C for 1 day, and then frozen in embedding compound . Embedded tissues were immediately frozen with liquid nitrogen and stored at −80°C. Serial transverse sections were cut on a cryostat to a thickness of 20 µm at 2-mm intervals  and used for cresyl violet staining or immunohistochemistry.Spinal cord segments were taken at autopsy from ten patients with features typical of sporadic ALS. Spinal cord tissue from 6 individuals without history/evidence of neurological or psychiatric disease was taken for the control samples . Three p2O2 in methanol for 30 min at room temperature and blocking with mouse-on-mouse blocking reagent for 1 h at room temperature. Anti-VGF antibody was applied to the sections for overnight at 4°C. After washing the sections with 0.01 M PBS, sections were incubated with biotinylated anti-mouse IgG for 2 h followed by washing, and were incubated with the avidin-biotin-peroxidase complex for 30 min at room temperature. The sections were finally visualized using diamino benzidine/H2O2 substrate for peroxidase .The sections were stained with the following antibodies: (i) mouse anti-VGF monoclonal antibody ; (ii) goat anti-NeuN polyclonal antibody ; (iii) mouse anti-GFAP monoclonal antibody ; and (iv) mouse anti-CD11b monoclonal antibody  within Can Get Signal immunostain solution A . Sections were treated with 0.3% H2O2 substrate. To assess intensity of VGF immunoreactivity in the Rexed laminae IX anterior horn (AH) in the human spinal cord, sections immunostained with VGF were used for the intensity measurement: C7, T8, and L4 in the spinal cords of the control patients and patients with sporadic ALS, respectively. The volume used for the intensity measurement was 438×330×4 µm (section thickness) of each side in the spinal cord. The density measurements were carried out under objective ×40 with bright-field microscope in a masked fashion by a single observer (Y.I.). Anti-VGF antibody revealed the expression pattern of VGF in spinal cord. The density on each section was measured using MetaMorph software (Molecular Devices). Data from each section was averaged for each spinal cord, and the values obtained were used to evaluate the density of VGF immunoreactivity.For paraffin embedding human spinal cord sections, conventional antigen retrieval method for each antigen was performed at its optimal condition with 0.01 M sodium citrate buffer, pH 7.4. Next, coronal sections of spinal cord were washed with 0.01 M PBS, and then treated with 0.3% hydrogen peroxidase in 0.01 M PBS for 30 min at room temperature. Furthermore, the sections were preincubated with 10% normal horse serum (Vector) in 0.01 M PBS for 30 min and then incubated for 1 h at room temperature with specific goat anti-VGF polyclonal antibody  in the following solution: 10% normal horse serum in 0.01 M PBS containing 0.3% (v/v) Triton X-100. They were washed with 0.01 M PBS and then incubated with biotinylated anti-goat IgG before being incubated with the avidin-biotin-peroxidase complex for 30 min at room temperature, and finally visualized using diamino benzidine/HWhen double-immunostaining for VGF/GFAP, VGF/NeuN, or VGF/CD11b were performed, these antibodies were applied to the sections for overnight at 4°C after blocking with 0.01 M PBS containing 10% normal goat serum (Vector) or mouse-on-mouse blocking reagent  for 1 h. After washing the sections with 0.01 M PBS, immunoreactivity was visualized by incubating them for 2 h at room temperature with secondary antibodies conjugated with Alexa 488 rabbit anti-mouse, Alexa 546 rabbit anti-goat, Alexa 488 goat anti-rabbit, or Alexa 546 goat anti-mouse . At the end of immunostaining, Hoechst 33342 (1∶1000) was added to the samples for 30 min to visualize nucleus.Total images of the spinal cord were taken using a microscope  fitted with ×20 and ×40 microscope objective lenses. The images visualized by diamino benzidine were taken using a charge-coupled device camera  and immunofluorescence images were taken using a cooled charge-coupled device camera  at 1360×1024 pixels via Metamorph .t-test using a SPSS 16.0 . Statistical analysis of the cumulative probability of survival was performed with the Kaplan–Meier life test. Statistical analysis of the mean age of onset and survival was performed with log-rank test. A value of P<0.05 was considered to indicate a statistical significance.Data are presented as means ± S.E.M. Statistical comparisons were made by Dunnet's test, Tukey test, or Student's Further n = 8), while in the control group, the percentage (supplemented with 1% FBS) was 10.7±1.3% (n = 8). Treatment with SUN N8075 at 3 µM significantly reduced the increase in YO-PRO-1-positive cells induced by tunicamycin. Exposure to tunicamycin elevated the level of caspase-3 activity, indicative of an early stage of ER stress-induced apoptosis N-acetyl-cysteine (NAC) at 1 mM or edaravone at 1 to 10 µM, did not show any effects on the reduction of cell viability after tunicamycin -sulfoximine (BSO) plus glutamate, suggesting that SUN N8075 protected cell damage against other stresses depending on the antioxidative effect  that counteracts the effects of the original stress. BiP acts as an ER-resident molecular chaperone that is induced by ER stress, and this protein refolds the unfolded proteins, thereby tending to maintain homeostasis in the ER VGF gene as a candidate gene. The induction of VGF was also confirmed by real-time RT-PCR , and neuropeptide Y (NPY) was not affected by SUN N8075 alone or after tunicamycin with or without SUN N8075 (c-fos and early growth response gene 1 (EGR1) but not cAMP response element-binding protein 1 (CREB1) or JUN  inhibitor or Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor. These findings suggest that SUN N8075 may induce VGF by regulating mitogen-activated protein kinase (MAPK) or CaMKII activity. On the other hand, almost all the 15 ER-related genes listed in To generate gene expression profiles after treatment with SUN N8075 or tunicamycin, we performed microarray analyses on 4×44 K Agilent Human expression arrays by hybridizing RNA from SH-SY5Y. At least more than 26,000 genes out of 41,000 gene probes on the array were detected in each sample, and a representative scatter plot comparison in gene expression with DNA microarray between SUN N8075 and vehicle control treatments at 7 h is shown in e RT-PCR . SUN N80 control . Furtherfor 12 h . The tun at 3 µM . VGF is UN N8075 . These r) or JUN . IEGs arVGF siRNA completely abolished the protective effect of SUN N8075 against tunicamycin-induced cell death. Furthermore, VGF siRNA aggravated tunicamycin-induced cell death compared with the control siRNA -dependent phosphorylation of the nuclear transcription factor cAMP-response element binding protein 1 (CREB1) in primary cultures of hippocampal neurons To confirm the effects of SUN N8075 on motor dysfunction and the survival of the familiar ALS model, we used two types of transgenic animals overexpressing mutated human SOD1 genes: transgenic mice overexpressing mutated  human SOD1 (G93A) p = 0.024) and survival (p = 0.0351) in the G93A mice (n = 12) and 136.2±3.4 (n = 12), respectively (p = 0.0178) in the G93A mice compared with that in the vehicle-treated G93A mice. On the other hand, there was no significant difference in the course of body weight between the SUN N8075-treated and the vehicle-treated G93A mice  and survival (p = 0.011) in H46 rats (n = 9) and 214.7±2.0 (n = 10), respectively (p = 0.012) in the H46R rats compared with that in the vehicle-treated H46R rats. On the other hand, there was no significant difference in the course of body weight between the SUN N8075-treated and the vehicle-treated H46R rats . In the present study, SUN N8075 at concentrations of 1 µM or higher inhibited ER stress-induced cell death n = 6)  littermates . Treatmen = 6) . These dn = 4) compared with the WT mice (n = 4) is shown in n = 4) compared with WT mice (n = 4) is shown in To generate gene expression profiles on the spinal cords of WT or G93A mice aged 14 weeks after SUN N8075 treatment, we performed microarray analyses on 4×44 K Agilent Mouse expression arrays. At least more than 27,000 genes out of 41,000 gene probes on the array were detected in each sample, and the Volcano plot of the probe data set averages of gene expression in the spinal cords of the G93A mice Click here for additional data file.Figure S1A) Edaravone at 1 to 10 µM or (B) N‐acetyl‐cystein (NAC) at 1 mM did not show any effects on the reduction of cell viability at 24 h after tunicamycin treatment. (C) Edaravone (Eda) at 10 µM, NAC at 1 mM or trolox (Tro) at 100 µM did not inhibit the reduction of cell viability at 24 h after thapsigargin treatment.Anti‐oxidative agents do not reveal protective effects on ER stress‐induced SH‐SY5Y cell death. ((TIF)Click here for additional data file.Figure S2A) SUN N8075 (SUN) at 0.1 to 1 µM, edaravone (Eda) at 10 µM, and trolox at 100 µM show protective effect on the reduction of cell viability at 48 h after serum deprivation. Each column represents the mean ± S.E.M. (n=6). ##p<0.01 versus control. **p<0.01 versus serum deprivation alone. (B) SUN N8075 (SUN) at 0.1 to 1 µM and trolox at 100 µM show protective effect on the reduction of cell viability at 24 h after l‐buthionine‐‐sulfoximine  plus glutamate (10 mM). Each column represents the mean ± S.E.M. (n=6). ##p<0.01 versus control. **p<0.01 versus BSO plus glutamate alone.SUN N8075 and anti‐oxidative agents protect cell death induced by serum devrivation, or oxidative stress in RGC‐5 cells. ((TIF)Click here for additional data file.Figure S3BiP or CHOP on SH‐SY5Y cells after tunicamycin. BiP and CHOP mRNA levels were measured using a quantitative real‐time PCR.SUN N8075 does not affect gene expression level of (TIF)Click here for additional data file.Figure S4A) SUN N8075 at 0.1 to 3 µM shows protective effect on the reduction of cell viability at 24 h after tunicamycin at 2 µg/ml. Each column represents the mean ± S.E.M. (n=8). ##p<0.01 versus control. **p<0.01 versus tunicamycin alone. (B) ATF4, BiP and CHOP protein levels were measured using Western blotting.SUN N8075 protect cell death induced by tunicamycin, but did not affect protein expression of ATF4, BiP or CHOP in RGC‐5 cells. ((TIF)Click here for additional data file.Figure S5A) Time‐course of changes in phosphorylated‐Akt level after SUN N8075 treatment. (B) Concentration‐dependent changes in phosphorylated‐Akt level at 24 h after SUN N8075 treatment.  Tunicamycin reduced phosphorylated‐Akt (C) and phosphorylated‐ERK1/2 (D) levels, and their reductions were ameliorated by SUN N8075 treatment.  The protective effect of SUN N8075 on tunicamycin‐induced reduction of cell viability was eliminated by LY294002, a PI3kinase inhibitor, at 20 µM (E) or U0126, a MEK1/2 inhibitor, at 5 µM (F).SUN N8075 enhances survival signals via Akt and ERK1/2 activations. ((TIF)Click here for additional data file.Figure S6A) Effect of SUN N8075 at 30 mg/kg/day, s.c. on body weight change in G93A mutant SOD1 mice. (B) Effect of SUN N8075 at 10 mg/kg/day, s.c. on body weight change in H46R mutant SOD1 rats.Effects of SUN N8075 on body weight changes in two type of familial ALS model animals. ((TIF)Click here for additional data file.Figure S7A) Volcano plot of probe data set averages of gene expressions in the spinal cords of G93A (n=4) compared with WT (n=4). The x‐axis and y‐axis correspond to the average fold‐change (FC), and the negative log10‐transformed p value between G93A and WT mice, respectively. Green  and red  squares represent significantly differentially expressed probe sets, and gray squares represent probe sets with no significant difference between G93A and WT mice. The changes in gene expression were considered statistically significant based on the criteria of FC ≥2, p≤0.01 with Benjamini‐Hochberg correction. (B) Heat map representation of hierarchical clustering of 712 different gene expressions in the spinal cords of G93A compared with WT mice. The columns and rows represent the tissue samples  and individual gene expressions, respectively. Shades of red indicate elevated expression while shades of blue indicate decreased expression relative to the median . (C) Principal components analysis (PCA) in the spinal cord of WT, G93A and G93A + SUN N8075 treated mice. Differently expressed 712 genes in the two groups (WT vs. G93A mice) were analyzed using a principal component analysis, and the contribution rates of genes to PCA component 1, 2, and 3 were 81.23%, 6.99%, and 3.90%, respectively. The results are expressed as a one‐dimensional function with PCA component 1 that defines the direction of greatest variation in the probe/gene transcriptomic feature space. ##p<0.01, *p<0.05, Student's t‐test. (D) Gene ontology classification for each category from the 712 different genes.((TIF)Click here for additional data file.Table S1Clinical information about the spinal cord tissues from patients with sporadic ALS (a) and for control (b). M, male; F, female; PMI, post mortem interval; y, years; m, months; h, hours.(PDF)Click here for additional data file.Table S2Gene expression profiles after SUN N8075 treatment in SH‐SY5Y cells.(PDF)Click here for additional data file.Table S3Gene expression profiles for ER‐stress responsible genes after SUN N8075 alone, or tunicamycin with or without SUN N8075 in SH‐SY5Y cells. ↑: two‐fold or more increase, →: no changes.(PDF)Click here for additional data file.Table S4SUN N8075 prolongs disease onset and lifspan in a rat model of familial ALS. Data are the means ± S.E. (n=9 or 10), * P<0.05 vs vehicle‐treated ALS mouse group. a Observable functional deficits (motor score of 4), b Righting reflex failure (motor score of 1), c Motor score of 0.(PDF)Click here for additional data file.


An error was introduced in the preparation of this article for publication. The error is in the penultimate sentence of the "One-step EMP Cloning" section of the Materials and Methods. The correct sentence is: A typical reaction contains 0.02 μM primer R1, 0,5 μM primer F1 and 0.5 μM primer R2 in addition to 1x HF Phusion buffer, 200 μM of each dNTP, 25 ng template DNA for the insert, 25 ng template DNA for the target plasmid, and 0.02 U/μL Phusion DNA Polymerase (NEB) in a 50 μL reaction.