Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs). In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC) cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC. Lung cancer is the leading cause of cancer-related mortality worldwide and the number of cases and deaths related to lung cancer is on the rise in many parts of the world. Non-small cell lung carcinoma (NSCLC) accounts for nearly 80% of all cases of lung cancer. Despite aggressive efforts, treatments are unsatisfactory and survival rates remain dismal (<20%) Acetylcholine (ACh) is an important neurotransmitter in the central and peripheral nervous systems and plays key roles in learning, memory, autonomic control, and muscular contraction via activation of acetylcholine receptors (AChRs), including the muscarinic (mAChRs) and nicotinic receptors (nAChRs). Recently, it has been found that ACh is also widely synthesized by a variety of non-neuronal cell types, including airway epithelial cells et al reported that interruption of autocrine muscarinic cholinergic signaling with M3 receptor antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) or darifenacin has potential to inhibit SCLC cell growth both in vitro and in vivoSimilarly, non-neuronal ACh stimulates cell growth through either muscarinic cholinergic or nicotinic cholinergic pathways. It has been reported that the growth of tumor cells was accelerated via activation of mAChRs in colon in vitro and its potential mechanisms are explored. To our knowledge, this is the first study to investigate the effect of M3 antagonist on NSCLC cells.Penehyclidine hydrochloride (PHC) is an anti-cholinergic drug derived from hyoscyamine and developed independently by our institute. As its potential anticholinergic activity, it has been marketed for treatment of acute organophosphorus pesticides poisoning and chronic obstructive pulmonary disease (COPD) D) values for [3H] NMS at human M1, M2, M3, M4 and M5 of 0.29±0.04, 0.81±0.17, 0.53±0.09, 0.19±0.03, and 0.48±0.13 nM, respectively. The relative selectivity for M1–M5 AChR subtypes of R2HBJJ was first determined at the level of receptor binding affinity in human mAChRs proteins  or atropine as a control antagonist, were added cumulatively into the organ bath. The inhibitory concentration response against carbachol-induced contraction was obtained. The results showed that R2HBJJ concentration-dependently inhibited the contractile responses of guinea-pig tracheae to the carbachol (50) was 7.58±1.05 nM, which was statistical significantly less than that of atropine . The data revealed that R2HBJJ exhibited a strong antagonistic activity on mAChRs expressed in guinea-pig trachea.To determine the blocking effect of R2HBJJ on mAChR, an ex vivo model of guinea-pig tracheal contraction was applied. The contraction of tracheal strip isolated from guinea-pig was induced by 1 µM of carbachol, a non-selective muscarinic agonist. And then, increasing concentrations of R2HBJJ , mAChRs and/or nAChRs for ACh to provide autocrine cholinergic stimulation to NSCLC cell growth. To determine whether NSCLC cell lines express AChRs and ChAT, the mRNAs of five subtypes of mAChRs (M1–M5), three subtypes of nAChRs  and ChAT were examined by RT-PCR in human NSCLC cell lines H1299, H157, H460, A549 and human immortalized bronchi epithelial cell BEP2D. As shown in 50) of 8.5, 8.8 and 28.5 µM, respectively. In comparison, A549 and BEP2D cells were resistant to R2HBJJ, with a GI50 of more than 100 µM, the highest concentration tested  for 72 h and cell viability was determined by using the sulforhodamine B (SRB) assay. The results showed that treatment with R2HBJJ markedly suppressed cell growth in a concentration-dependent manner in H1299, H460 and H157 cells, with a 50% growth-inhibitory concentration . Post-hoc tests confirmed that the following pretreatment concentrations of ACh, 10 µM and 100 µM, significantly attenuated the growth inhibition induced by 10 µM or 20 µM of R2HBJJ. When compared with R2HBJJ treatment alone at the level of 10 µM, 10 µM and 100 µM of ACh pretreatment increased the cell viability from 54.13±3.89% to 82.29±5.82% (P<0.01) and 91.1±3.65% (P<0.01), respectively. When compared with R2HBJJ treatment alone at the level of 20 µM, 10 µM and 100 µM of ACh pretreatment increased the cell viability from 23.23±2.14% to 35.57±4.48% (P<0.05) and 58.55±2.83% (P<0.01), respectively  and the inhibition of 55.5% was achieved within a 96 h time frame (P<0.001). For ChAT-siRNA, its anti-proliferative effect was presented at 72 h post transfection (P<0.001), which was slower than M3 siRNA; and the inhibition of 37.9% was achieved 96 h post transfection compared to the negative control siRNA (P<0.001).To further demonstrate that endogenous ACh and M3 receptor signal were involved in cell proliferation of human NSCLC cells, H1299 cells were transfected with M3- or ChAT-specific siRNAs and their proliferation was observed over time. Firstly, western blot analysis was used to identify the levels of these genes. As shown in ol siRNA , P<0.001P<0.05) and 57.4% (P<0.01) after R2HBJJ 8 µM and 16 µM treatment. However, after the cells were treated with M3 siRNA, the cell viability was inhibited to 54.4%; R2HBJJ (8 µM and 16 µM) didn't further influence the cell growth, which suggested that silencing M3 receptor deprived of cellular sensitivity to R2HBJJ and accentuated the role of M3 in the effect of R2HBJJ  was decreased time-dependently. The decrease of Rb phosphorylated at the position of Ser807/811 occurred as early as 3 h after treatment and before apparent cell viability change. In contrast, the expression of basal Rb showed a significant persistent increase and then rapidly declined to an undetectable level at 48 h after treatment when cell growth suppression has been induced (data not shown), which may suggest a potential cellular adaptation to the decrease of phosphorylated Rb after R2HBJJ treatment. Further we evaluated the concentration response of Rb phosphorylation reduced after treatment with R2HBJJ for 48 h (807/811 by R2HBJJ was concentration-dependent and occurred at the lowest concentration tested (1.25 µM). Consistently, the down-regulation of Cyclin D1, CDK4 and CDK6 proteins was observed in the cells treated by increasing concentrations of R2HBJJ. Similarly, the expression of basal Rb showed a significant increase over concentration at first and then rapidly declined to basal level. Together, these results suggested that Rb protein and the kinase composite of cyclin D1/CDK4, 6 were predominantly involved in the cellular G0/G1 arrest induced by R2HBJJ.Cell cycle transition from G1 to S is mainly activated by two different kinase composites: cyclin D1/CDK4, 6, and cyclin E, A/CDK2. Rb is the primary substrate of cyclin D-dependent kinase and plays a key role in the network of cell cycle regulation. To investigate the underlying molecular mechanisms of G0/G1 arrest induced by R2HBJJ, the time-dependent changes of several related cell cycle regulatory molecules were evaluated after treatment with 20 µM R2HBJJ in H1299 cells by using western blot analysis. As shown in for 48 h . The resMany studies have demonstrated that ACh and other components of cholinergic signaling, including ChAT, cholinesterase, M and N cholinergic receptors, are present in a variety of non-neuronal tissues, including most common cancer cells such as NSCLC, SCLC, colon, glial, breast and ovarian carcinomas. In the local environment of tumors, concentrations of ACh at surface receptors may be even higher due to high cell densities in solid masses, increased secretion of ACh proximity to receptor location and decreased levels of cholinesterase in NSCLC in vitro.R2HBJJ, as a derivative of anticholinergic agent R2-PHC, was synthesized by our institute. In the present study, we evaluated the selectivity of R2HBJJ for M1–M5 muscarinic receptors and its anticholinergic activity. We also determined if interruption of endogenous cholinergic signaling with R2HBJJ has potential to inhibit NSCLC growth Our results demonstrated that R2HBJJ had significantly higher affinity for M3 and M1 receptor subtypes than M2 subtype, which suggested fewer M2 receptor-associated cardiovascular side effects. The rank of affinity of R2HBJJ for five different mAChRs was M3>M1>M4>M5>M2. The result is consistent with that obtained in its parental compound PHC before et al reported that SCLC synthesize and secrete acetylcholine, which acts as an autocrine growth factor through both nicotinic and muscarinic cholinergic mechanisms in vitro, we found that only M3 receptor antagonist darifenacin presented a significant inhibition on cell growth. Meantime, our compounds R2HBJJ and R2-PHC also markedly inhibited the cell viability of H1299 cells, with a preferable effect than darifenacin. In the study of Song et al, treatment of SCLC cells with M3 receptor antagonists of 4-DAMP or darifenacin inhibited cell growth both in vitro and in vivo. Our results suggest that M3 receptor antagonists have inhibitory activity on NSCLC as well. The anti-proliferative effect of R2HBJJ on NSCLC may be related to its selective M3 receptor antagonistic activity. Russo P et al reported that inhibition of α7-nAChR with a powerful high affinity antagonist α-CbT induced antitumor activity in NSCLC and pleural mesothelioma by triggering apoptosis Song In the present study, we failed to observe the proliferative effect of exogenous ACh on H1299 cells, which may imply that endogenous ACh has provided enough stimulant effect on cell proliferation. However, exogenous replenish of ACh is needed to antagonize competitively the inhibition of R2HBJJ on H1299 cells. The result stressed that the effect of R2HBJJ on H1299 cell growth was achieved via impacting on endogenous ACh signaling. In the present study, ChAT siRNA was proved to inhibit cell growth, which further supported this hypothesis. Moreover, it is noteworthy that treatment with M3 receptor siRNA resulted in a significant growth inhibition on H1299 cells when compared with negative control siRNA treated cells. The potency of inhibition achieved 50% or more within a 96 h time frame. When M3 receptor was effectively knocked down in H1299 cells, the cells lossed their sensitivity to R2HBJJ. The results further accentuated the role of M3 in the anti-proliferative effect of R2HBJJ.807/811 was inhibited by R2HBJJ time-dependently, which could abolish the interaction between c-Abl and Rb and retard the activation of c-Abl and the cell cycle progression Cell cycle arrest is a major mechanism preventing tumor growth. Our results from flow cytometric analysis showed that R2HBJJ markedly suppressed the cell mitotic progression through arresting cells in G0/G1 phase. Cell cycle transition from G1 to S controls DNA synthesis and requires hyperphosphorylation of a tumor suppressor, Rb In fact, regulatory molecules governing early cell cycle progression such as cyclin D1-CDK4/6-Rb are the frequent targets of genetic alterations in an exceptionally high percentage of lung cancers In conclusion, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, a novel muscarinic antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth. The mechanism for R2HBJJ-induced cell cycle G0/G1 arrest involves down-regulation of cyclin D1-CDK4/6-Rb. The results suggest that M3 receptor antagonists possibly become a new potent chemotherapeutic regimen for NSCLC.3H] N-methylscopolamine ([3H] NMS), human muscarinic receptor  proteins and BetaPlate Scint of membrane were obtained from Perkin Elmer Life and Analytical Sciences. R2-PHC and R2HBJJ , Cyclin D1, Cyclin E, CDK2, CDK4, CDK6. M3-AChR was obtained from Santa Cruz. ChAT, β-actin, goat anti-rabbit and goat anti-mouse immunoglobulin labeled with horseradish peroxidase were purchased from Beijing Zhongshan Jinqiao Biotechnology.Radiolabeled compound [3H] NMS  in the absence and presence of atropine (500 µM) to determine nonspecific binding. [3H] NMS, mAChRs proteins and R2HBJJ were individually diluted with the binding buffer, which consisted of 110 mM NaCl, 5.3 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 25 mM glucose, 58 mM sucrose, 20 mM HEPES (pH 7.4). Binding assays were performed in a final volume of 0.5 ml containing 18 to 42 µg of receptor protein and were incubated for 2 h at 37°C. Competition binding assays were performed using 2.4 nM of [3H] NMS in the absence and presence of a range of antagonist concentrations (10−6 - 10−13 M). Bound radioligand was separated from free by rapid vacuum filtration through GF/C glass fiber filters (Whatman). The filters were rinsed three times with ice cold PBS. After being dried, the fibers were placed in scintillation vials with 0.5 ml of BetaPlate Scint. Radioactivity trapped on the filters was quantified by a BECKMAN Beta counter. The assay was repeated three times with duplicate samples.Saturation binding was performed using a range of concentration of [2 2.52, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2, and glucose 11.0. The tissue bath solution was maintained at 37°C and gassed with 95% O2 plus 5% CO2. Tracheal preparations were equilibrated in the medium for 60 min with four changes of KH solution and maintained under an optimal tension of 1.5 g before specific experiment protocols were initiated. Segments were first pre-contracted with 1 µM carbachol for 10 min till reaching stable plateaus, then washed completely until the segments reached basal tension and maintained in KH solution for another 60 min with four changes. Thereafter, segments were incubated with 1 µM carbachol for 10 min again and the experiments were started with this preparation. R2HBJJ or atropine was then administered cumulatively to produce their concentration-response curves. Female guinea-pigs (350–450 g) were used in this study, which were purchased from Beijing Animal Center. Animal experiments were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals (1996) and the institutional guidelines of Beijing Institute of Pharmacology and Toxicology. The experimental protocol was approved by the Animal Research Advisory Committee of Beijing Institute of Pharmacology and Toxicology. The experiment was performed as described previously Human NSCLC cells H1299, H157, H460 and A549 were commercially obtained from the Cell Resource Center, IBMS, CAMS/PUMC . The human papillomavirus (HPV18) immortalized bronchi epithelial cell BEP2D was developed by the research group of Dr. Curtis C. Harris  15 Primer at 42°C for 60 min, 95°C for 5 min, 4°C for 5 min. PCR was carried out in 25 µl of a reaction mixture with the following conditions: 95°C for 5 min, 35 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min. The primers used for PCR were shown in Total RNA was extracted from cells using TRIzol reagent (Invitrogen). 1 µg aliquot of each RNA sample was reverse-transcribed to synthesize cDNA in a 20 µl reaction volume by using an AMV-RT kit (Promega). Incubate the reaction using Oligo (dT)3 cells/well in 96-well plates. After overnight incubation, the cells were treated with R2HBJJ (1.6–100 µM) or other antagonists (0.3–100 µM) for 72 h. The inhibitory effects of the antagonists on cell growth were determined using SRB assay, as described previously 50) was calculated with Hill equation after curve fitting by GraphPad Prism 5 software.Cells were seeded at a density of 6.0×105′-CCTGGTAATTGTGTCATTT-3′and 5′-GCAAGCCATTGGCAACAAA-3′, respectively. The negative control siRNA consists of a proprietary siRNA sequence not corresponding to any eukaryotic gene.The M3 receptor and ChAT specific small interfering RNA (siRNA), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) positive control siRNA , the negative control siRNA were designed and synthesized by Guangzhou Ruibo Biotechnology . The target sequences for M3 receptor and ChAT are 5/well in six-well plates one day prior to transfection and 1.5 mL of the medium without antibiotics was added to each well. The siRNA were transfected at a concentration 200 nM each in serum-free Opti-MEM I medium with Lipofectamine™ 2000 reagent (Invitrogen) according to the manufacture's instruction. Following 6 h incubation, the transfection medium was replaced with RPMI-1640 containing 10% (v/v) FBS. 72 h after transfection, the cells were harvested for Western blotting analysis. For cell proliferation assay, H1299 cells were seeded at 3.5×103/well in 96-well plates one day prior to transfection. 48 h, 72 h and 96 h after transfection, cell growth viability was respectively determined using SRB assay. An independent experiment was performed to observe the combination effect of M3 siRNA knockdown and R2HBJJ treatment. While the transfection medium was replaced with complete RPMI-1640 medium 6 h after transfection, cells were treated with or without R2HBJJ (8 µM and 16 µM) for 72 h. Each experiment was performed in four replicates and repeated for three times. Cell viability of negative control siRNA was set at 100%.H1299 cells were seeded at 1×105 cells/well in six-well plates and synchronized by plating in RPMI-1640 containing 1% FBS for 24 h. For time-dependent response, cells were incubated in 2 ml fresh RPMI-1640 containing 10% FBS with 20 µM R2HBJJ for 24, 48, 72 h. For concentration-dependent response, cells were incubated for 48 h with R2HBJJ at final concentrations of 2.5, 5, 10, 15, 20 µM in 2 ml fresh RPMI-1640 containing 10% FBS. Fresh media added in the control well simultaneously. After treatment, the cells were harvested with trypsin, washed once with PBS, and then fixed in 70% ethanol overnight at 4°C. Before flow cytometry analysis, cells were stained with 200 µg/ml Ribonuclease A and 50 µg/ml propidium iodide (Sigma) for 30 min at room temperature away from light. 1×104 cells per sample were analyzed using a FACSCalibur flow cytometer (BD Biosciences). Data were evaluated using Modfit software.Alterations in cell cycle were determined by using flow cytometric assay. Cells were seeded at a density of 1×10Cells were harvested and subjected to lysis in the buffer X . Equal amounts of lysates (30 µg) were separated on 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore). Membranes were then blocked with PBS buffer containing 5% low fat milk and 0.1% Tween 20 (PBST) for 1 h at room temperature and then incubated with primary antibodies (1∶1000–2000) overnight at 4°C. After being washed three times with PBST, membranes were incubated with peroxidase-conjugated secondary antibodies (1∶5000) for 1 h at room temperature. The membranes were washed with PBST again and developed with a pro-light HRP chemiluminescence detection kit . β-actin was used as a loading control.50 values were determined by using the non-linear curve-fitting program. The inhibition constants  were calculated by using the Cheng-Prusoff equation. For the experiments of exogenous ACh combined with R2HBJJ, two-way ANOVA was used to compare the interaction of two treatment factors. Post-hoc analyses were performed with Bonferroni's tests for comparisons to controls. For the cell viability assay after siRNA transfection, one-way ANOVA followed by Dunnett's t test was used. A P value of less than 0.05 was considered statistically significant.GraphPad Prism 5 software was used for statistical analysis. For competition binding and Guinea-pig tracheal contraction assay, the IC


NPP1–NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5′-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28°C/23°C (12 h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33°C/28°C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions.Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes ( The nucleotide sequence reported in this paper has been submitted to the DDJB with accession number AB196673.N-glycosylated enzymes that catalyze the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of numerous nucleotides and nucleotide sugars (Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed e sugars . Unlike e sugars  and diphe sugars , plant Ne sugars . NPPs are sugars . Howevere sugars . Moreovee sugars . The ovee sugars , NPP1 ise sugars .npp1 null mutants cultured under different temperature and CO2 concentration conditions strongly indicated that NPP1 exerts a negative effect on plant growth and starch accumulation, especially under high CO2 concentration conditions, which provides the first in vivo evidence about the possible involvement of NPP1 in the control of plant growth and reserve carbohydrate accumulation in rice under high CO2 concentration conditions.Mammalian NPPs have been shown to be involved in a variety of cellular processes such as nucleotide signaling, cell differentiation, nucleotide recycling and control of the levels of nucleotides linked to glycosylation and sulfation reactions . As a fiNPP1–NPP6) were obtained from rice shoot cDNA libraries using an NPP1-encoding cDNA , 8 (NPP1), 9 (NPP6) and 12  in rice (see Supplementary Table S1). A comparison of the deduced amino acid sequences of proteins using ClustalW  showed that (i) the degrees of similarity between NPP1 and other NPPs ranged between 61.1 and 71.6%, and (ii) 53.7% of the amino acids of NPP1, 2 and 6 are identical. NPP amino acid sequences are highly similar to those of metallophosphatases from yellow lupin NPP1–NPP6NPP1, NPP2 and NPP6 and purified the corresponding recombinant proteins as described in the Materials and Methods. Details of the processes for purification of recombinant proteins and quality of the final preparations are shown in Supplementary Table S2 andSupplementary Fig. S1A.Previous studies using recombinantly produced NPP3 revealed that this enzyme hydrolyzes several nucleoside diphosphates, but not nucleotide-sugars such as ADP-ribose, ADP-glucose or UDP-glucose . To carrSupplementary Fig. S2A). SDS–PAGE and subsequent staining of the purified NPPs revealed a single polypeptide band in each preparation, strongly indicating that NPP1, NPP2 and NPP6 form homo-oligomeric structures composed of 70, 71 or 74 kDa polypeptides, respectively . This, and the fact that rice NPPs exhibit no requirement for a metal ion , ADP (kcat/Km of 0.73 × 105 s−1 M−1) and adenosine 5′-phosphosulfate , a characteristic similar to that of NPP3 (kcat/Km of 2.50 × 105 s−1 M−1), ADP-ribose (kcat/Km of 2.48 × 105 s−1 M−1), TDP-glucose (kcat/Km of 2.06 × 105 s−1 M−1) and UDP-glucose (kcat/Km of 1.93 × 105 s−1 M−1) the best, while ADP and ADP-glucose were the best substrates for NPP6 . NPP1, NPP2 and NPP6 did not recognize phosphomonoester bond-containing compounds such as sugar phosphates or nucleoside monophosphates .Substrate specificity and kinetic analyses revealed clearly distinguishable characteristics between NPP1, NPP2 and NPP6 , 2. Thus of NPP3 . NPP1 hyosphates , and disNPP1, NPP2 and NPP6 in rice plant was examined by Northern blot analyses. As shown in NPP1 was widely expressed in shoots, young roots, scutella, mature leaves and roots, but not in calli. NPP6 was expressed in the whole plant and also in calli. In clear contrast, NPP2 was predominantly expressed in roots  and UDP-glucose pyrophosphatase (UGPPase) activities. As illustrated in NPP1 mRNA content was below detection limits in the npp1 mutant shoots. In clear contrast, the expression of NPP6 mRNA in the npp1 mutant shoots was comparable with that of WT plants. It is noteworthy that AGPPase and UGPPase activities in npp1 shoots were approximately 8% and 38% of that in WT shoots, respectively  and two temperature regimes: 28°C/23°C (12 h light/12 h dark) and ‘high temperature’ 33°C/28°C (12 h light/12 h dark) regimes. As shown in npp1 shoots was markedly higher than that of WT shoots when plants were cultured under high CO2 concentration conditions at any temperature regime. No difference in starch content could be found between npp1 and WT shoots when plants were cultured at normal (40 Pa) CO2 under the 28°C/23°C (12 h light/12 h dark) temperature regime  CO2 concentration under the 33°C/28°C (12 h light/12 h dark) ‘high’ temperature regime. No significant differences in starch content could be found between roots of WT and npp1 plants cultured under the various CO2 concentration and temperature conditions employed in this work  in shoots of WT and npp1 plants. These analyses revealed that: (i) maximum AGPase and SuSy activities in rice shoots are comparable with those of AGPPase  and mature leaves from npp1 plants cultured at ambient CO2 concentration under a 28°C/23°C (14 h light/8 h dark) regime. As shown in NPP1 mRNA levels in UNP1 leaves were approximately 330% of that found in WT leaves. Furthermore, the UNP1 leaves exhibited a low starch phenotype, whereas the npp1 leaves showed a high starch phenotype . Suspension-cultured cells of rice actively synthesize and accumulate starch molecules. The cultured cells are frequently used as a model system for studying the biosynthesis and degradation of starch in rice. As shown in NPP1-overexpressing lines and WT cells  when plants were cultured under the 28°C/23°C (12 h light/12 h dark) regime. Shoots and roots of npp1 plants were also larger than those of WT plants at normal atmospheric CO2 concentration (40 Pa) when cultured under the 33°C/28°C (12 h light/12 h dark) ‘high’ temperature regime  this hypothesis.npp1 rice plants impaired in the NPP1 function possess approximately 8% of the WT AGPPase activity and accumulate higher starch content than WT shoots would indicate that NPP1: (i) is a major determinant of AGPPase activity; and (ii) prevents metabolic flux towards starch in rice plants by reducing the plastidic pool of ADP-glucose linked to starch biosynthesis.Two different protein entities are responsible for the ADP-glucose hydrolytic activity in plants: ‘Nudix’ hydrolases  and NPPsnpp1 shoots is higher than in WT shoots at normal (400 p.p.m.) CO2 concentration conditions under the ‘high temperature’ regime, but not under the ‘low temperature’ regime, would indicate that, under normal CO2 concentration and ‘high temperature’ conditions, NPP1 is a major site of regulation of starch synthesis. This is probably because under ‘high temperature’ conditions: (i) ADP-glucose hydrolytic NPP1 activity increases to levels exceeding those of AGPase and/or SS; and/or (ii) AGPase and/or SS activities decrease far below that of ADP-glucose hydrolytic NPP1 activity at normal CO2 concentration and ‘high temperature’ conditions.Maximal in vitro AGPase activity greatly exceeds the minimum required to support the normal rate of starch accumulation in some organs . The flu+, cyclic ADP-ribose and poly(ADP)-ribose. Because of its free aldehydic group, high intracellular ADP-ribose levels can result in non-enzymatic ADP-ribosylation of proteins, a deleterious process that inactivates enzymes and interferes with recognition processes that rely on enzymatic ADP-ribosylation  was obtained from the National Institute of Agrobiological Sciences , and 32P-labeled using the Rediprime II DNA Labeling System . Total DNA from rice leaves was digested with XbaI, fractionated by agarose gel electrophoresis, alkali-transferred to Hybond-N+, hybridized for 16 h at 65°C with the 32P-labeled Tos17 and NPP1 probes, washed under high stringency conditions and exposed to IP (Fuji film). The exposed IP was scanned by BAS-5000 (Fuji film).The rice variety used in this study was −1 2,4-D and 5 mg l−1 thiamine-HCl, placed on a reciprocal shaker operated at 110 strokes min−1 with 70 mm amplitude, at 28°C in darkness. The established suspension-cultured cells were subcultured at 7 d intervals. All these procedures were performed under aseptic conditions.Rice calli derived from the embryo portions of seeds were cultured as described by NPP1 cDNA was cloned from rice shoot cDNA libraries as described by NPP1 cDNA as a radiolabeled probe, we screened the shoot cDNA libraries to isolate other NPP-encoding cDNAs. The complete cDNAs thus obtained, designated as NPP2, NPP3, NPP4, NPP5 and NPP6, were cloned into pBluescript SK(–) (Stratagene) to create pOsNPP2, pOsNPP3, pOsNPP4, pOsNPP5 and pOsNPP6.A complete rice Supplementary Table S4. For overexpression of rice NPP2 and NPP6 in rice plants and cultured cells, BamHI–KpnI PCR fragments were amplified from pOsNPP2 and pOsNPP6, and cloned into the BamHI and KpnI sites of the p2K-1+ plant expression vector . The DNA probe for rice actin 1 was prepared as described earlier (Measurements of mRNA contents were performed by reverse transcription–PCR (RT–PCR) and by Northern blot analyses. For RT–PCR analyses, total RNA was extracted from each mutant or transgenic rice plant using an RNeasy Plant Mini Kit (Qiagen). First-strand cDNA synthesis was carried out via a TaKaRa RNA PCR™ Kit (AMV) Ver.3.0 (TAKARA) with an Oligo dT-Adaptor Primer. PCR was performed with a set of forward and reverse primers as shown in eviously . The DNA earlier .g for 10 min at 4°C. The pellets including starch were re-extracted with 80% ethanol four times and dried using a SpeedVac. The dried pellets were resuspended in dimethylsulfoxide and boiled for 30 min, then centrifuged at 16,000 × g for 10 min at room temperature. An aliquot of the collected supernatant was incubated in 50 mM acetate buffer (pH 5.0) with 44 U ml−1 α-amylase and 28 U ml−1 amyloglucosidase at 37°C overnight. The reaction mixture was further incubated in 260 mM Tris–HCl (pH 7.6) with 4 mM MgCl2, 1.3 mM ATP, 0.78 mM β-NAD+, 3.9 U ml−1 hexokinase and 1.3 U ml−1 glucose-6-phosphate dehydrogenase at 37°C for 30 min. The amount of glucose resulting from starch was determined spectrophotometrically by measuring the absorbance of NADH at 340 nm.Measurements of starch content were carried out as follows. Harvested shoots and roots were immediately frozen and powdered in liquid nitrogen using a pestle and mortar. Samples were homogenized in 5 vols. of 80% (v/v) ethanol, boiled for 1 min and centrifuged at 16,000 × g for 10 min and the collected supernatant was extracted with diethyl ether to remove TCA, and lyophilized. The dried samples were suspended with 0.5 ml of water and passed through a solid-phase extraction cartridge . Contents of ADP-glucose in the flow-through fraction were determined employing reverse-phase ion-pairing liquid chromatography coupled to mass spectrometry described by −1. Solvent A was 97 : 3 water : methanol with 10 mM tributylamine and 15 mM acetic acid; solvent B was methanol. The gradient is: 0 min, 0% B; 2.5 min, 0% B; 5 min, 20% B; 7.5 min, 20% B; 13 min, 55% B; 15.5 min, 95% B; 18.5 min, 95% B; 19 min, 0% B; 25 min, 0% B. Other liquid chromatography parameters are autosampler temperature 4°C, injection volume 10 µl and column temperature 30°C. Various instrumental settings were optimized as follows: sheath gas flow rate 25 (arbitrary units) and sweep gas flow rate 0 (arbitrary units), spray voltage 2.5 kV, capillary temperature 300°C. The instrument was mass calibrated using the polytyrosine-1,3,6 standards every 3 d.Frozen tissues (600 mg) were ground with liquid nitrogen and homogenized with 5 ml of 10% (w/v) trichloroacetic acid (TCA). The homogenates were centrifuged at 10,000 × Harvested tissues were immediately ground to a fine powder in liquid nitrogen with a pestle and mortar. A 1 g aliquot of the frozen powder was suspended with 5 ml of extraction buffer consisting of 100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol at 4°C. The suspension was desalted and assayed for enzymatic activities. We verified that this procedure did not result in loss of enzymatic activity by comparing activity in extracts prepared from the frozen powder with extracts prepared by homogenizing fresh tissue in extraction medium. The methods of measurement of starch phosphorylase , solubleProtein contents were determined by the procedure of Supplementary Table S2. Rice callus cells (200 g) derived from the seed embryos of UNP1, UNP2 and UNP6 lines were homogenized in 5 vols. of 10 mM Tris–HCl (pH 8.8) and filtered through four layers of gauze. The homogenates were centrifuged at 20,000 × g for 10 min. The supernatants were then adjusted to acidic pH 5.4 with acetate buffer and centrifuged at 20,000 × g. After re-adjustment to pH 7.4 with 1.5 M Tris–HCl (pH 8.8) and further centrifugation at 20,000 × g, the resulting supernatants were applied to a Con A–Sepharose 4B column  equilibrated with 40 mM Tris–HCl (pH 7.4), 0.5 M NaCl, 1 mM MnCl2 and 1 mM CaCl2, and eluted with 25 ml of 0.5 M α-methyl-d-mannopyranoside in 10 mM Tris–HCl (pH 7.4). The eluents were applied to a Q Sepharose HP column  equilibrated with 10 mM Tris–HCl (pH 7.4) and eluted with 50 ml of a linear gradient of 0–0.5 M NaCl in 10 mM Tris–HCl (pH 7.4). Finally, the enzyme preparation was desalted by ultrafiltration on a Microcon YM-100 (Amicon).Purification procedures of NPP1, NPP2 and NPP6 expressed in the transgenic rice cells  were essentially identical to those used in the earlier study . They arNPP1 , NPP2 (AB196673), NPP3 (AK101976), NPP4 (AK073512), NPP5 (AK121432) and NPP6 (AK102346).Rice NPP genes whose complete cDNAs were isolated were: Supplementary data are available at PCP online.Ministry of Education, Culture, Sports, Science, and Technology, Japan [Scientific Research on Innovative Areas (22114507) and Grants-in-Aid for Scientific Research (B) (22380186)]; the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) [grant BIO2010-18239]; the Government of Navarra [grant IIM010491.RI1].This research was supported by the


Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7.Der f 7 is the group 7 allergen from the dust mite Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel β-sheets – one 4-stranded and the other 5-stranded – that wrap around a long C-terminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the β1–β2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded β-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH.Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens. D. pteronyssinus cDNA library and named Der p 7 For decades, the house dust mite has been known as a major causative agent of various allergic diseases including asthma, atopic dermatitis (AD) and allergic rhinitis. The group 7 allergen of dust mite was first isolated from a D. farinae, is a 196-residue protein with 86% identity to Der p 7. A Der f 7 fusion protein has been shown to react with IgE antibodies in sera of 19/41 (46%) asthmatic children et al. identified residue Asp159, located in the loop region (based on the model of Der f 7), as an important residue that is responsible for the IgE-mediated cross-reactivity between Der f 7 and Der p 7 The homologous allergen, Der f 7, isolated from As the continuation of our efforts to understand the structure and function of allergens, here we report the crystal structure of Der f 7, its ligand binding and stability when compared with Der p 7. The overall structure of Der f 7 is homologous to Der p 7, except with significant differences in the loop region proximal to the putative IgE epitope residue. Der f 7 binds PB at a similar site as Der p 7 with weak affinity. Der f 7, however, differs significantly in thermal stability as compared to Der p 7 and the stabilities of both proteins are pH dependent. The current study provides the structural basis for studying the allergenicity, function and stability of this group of allergens.free  = 0.28) (We purified a Der f 7 construct (Asp1 to Asn196) and determined its crystal structure by molecular replacement method. In this isoform of Der f 7, residue 130 is a Pro instead of a Ser, as reported in other Der f 7 sequences; the other residues are identical  = 0.28) . The eleGalleria mellonella hemolymph  Epiphyas postvittana Takeout 1  In the crystal structure of Der f 7, the 11 β-strands are arranged to form an elongated, curved anti-parallel β-sheet that wraps around the long C-terminal helix, α2; this is compared with the 10 β-strands in Der p 7. The single β-sheet has loops at the middle of each β-strand, making it appear as if the two β-sheets are joined together. The 4-stranded β-sheet comprised β-strands β4/β5, β6, β9 and β10, whereas the 5-stranded β-sheet comprised β-strands β1, β2/β3, β7, β8 and β11 . The N-tBased on B-factor distribution of Der f 7 , the looThe surface of Der f 7 consists of many charged amino acids, typical of an inhalant allergen . The ExPD. farinae1H-15N HSQC NMR spectrum of Der f 7 following titration of ligands  were Ala15, Ile16, Leu64, Gly80, Ile135 and Glu194  but binds bacterial lipopeptide polymyxin B (PB) with weak affinity in the predicted binding site of Der p 7  ligands . The resd Glu194 . These rm for thermal denaturation values of Der f 7 at pH 7.0 and pH 9.0 were 73.8°C and 65.7°C, respectively; whereas the Tm for thermal denaturation values of Der p 7 at pH 7.0 and pH 9.0 were 88.4°C and 81.6°C, respectively  derived from the β1–β2 loop region of Der f 7 can inhibit mAb binding to Der f 7, but this peptide cannot inhibit IgE binding to Der f 7 Significant structural difference was observed between Der f 7 and Der p 7 in the β1–β2 loop region, which contains the linear epitope residues, Leu48 and Phe50, of the Der f 7 specific mAb HD12 et al. used direct IgE binding method instead of the indirect mAb method. D159A mutant of Der f 7 has a lower IgE reactivity and cannot inhibit IgE binding to Der f 7 to the same degree as wild type Der f 7 D. pteronyssinus infestations over infestation with D. farinae. Further study of the Der f 7 epitopes and cross-reactivity needs to include the examination of sera from subjects predominantly exposed to D. farinae.To locate the exact position of the IgE epitope in Der f 7, Clhou 1H-15N-HSQC NMR spectrum of Der f 7 and determined the binding of PB, JH3 and methoprene to Der f 7, based on chemical shift perturbation. PB is a natural peptide and a potent antibiotic that binds to and neutralizes LPS It has been shown that Der p 7 binds weakly to PB, but not to LPS D. pteronyssinus to the IgE binding of Der f 7 by sera from subjects exposed to D. farinae. Further studies examining the stability and allergenicity of allergens are required to establish a correlation, if any exists.Although Der f 7 and Der p 7 both interact with the same ligand, their stabilities are found to be significantly different and highly depend on the pH of the buffer. The correlation between the allergenicity, function and stability of Der f 7 and Der p 7 is not clear. The recombinant Der f 7 is much less stable than Der p 7 at both pH 7.0 and pH 9.0 showing that it is important to compare the results of the IgE binding activity to Der p 7 by subjects sensitized by In summary, Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and 3D structure. However, there are significant differences in the β1–β2 loop region proximal to the putative IgE epitope residue. Both Der f 7 and Der p 7 weakly bind to PB in a similar binding site, but their thermal stabilities are different and highly depend on pH. The crystal structure of Der f 7 provides a basis for studying the allergenicity, function and stability of proteins in this group of allergens.et al., 2011 Der f 7 was sub-cloned, expressed and purified as a His-tag labeled protein using Ni-NTA affinity column (Qiagen) followed by a Superdex-200 gel filtration column . The details of expression and purification of Der f 7 are given in Tan HKL-2000 program suite Prior to crystallization, purified Se-Met labeled Der f 7 protein was dialyzed into 10 mM HEPES buffer at pH 7.0 and concentrated to 8 mg/ml with an Amicon filter . The diffraction quality crystal was obtained after 1 week from the reservoir solution containing 0.1 M Bis-Tris pH 7.4 and 28% polyethylene glycol monomethyl ether (PEG MME) 2000. The complete diffraction data was collected using the in-house Bruker MICROSTAR X-ray generator equipped with a PLATINUM 135 CCD detector. Subsequent data set processing was done using the m was determined by curve fitting according to the equation by Ruiz-Sanz et al.Far-UV CD experiments were carried out with 15 μM protein (Der f 7 and Der p 7) in 50 mM Tris-HCl at different pH levels (pH 7 and pH 9). The spectra were acquired with a J–810 Spectropolarimeter  using a quartz cuvette with 1 mm path length . Thermal denaturation experiments were carried out for Der f 7 and Der p 7 by recording the molecular ellipticity at 220 nm for which the change was most drastic with temperature increments. Spectra were recorded at the temperature slope of 1°C/min with the resolution of 0.1°C across a range of 55–95°C. T1H-15N HSQC as a reference spectrum. All NMR experiments were carried out on a Bruker AVANCE 800 MHz spectrometer equipped with a cryoprobe. Samples were loaded into a 5 mm NMR tube and all experiments were carried out at 40°C. All data acquired were processed using NMRPipe 1H-15N HSQC spectrum was acquired with 1280 complex points over a spectral width of 11160.7 Hz on the amide proton dimension. In the 15N dimension, 128 points were collected over 1865 Hz. Carrier frequencies for 1H and 15N were at 4.7 ppm and 117.5 ppm, respectively. Backbone assignments were carried out using HNCACB 1H) of the experiment, 1280 points were collected over a spectral width of 11160.7 Hz. In the F2 dimension (15N), 92 points were collected over 1865 Hz of spectral width, while the F3 dimension (13C) contained 116 points covering 12072.4 Hz spectral width. The carrier frequencies for F1, F2 and F3 dimensions were at 4.7 ppm, 118.5 ppm and 41.7 ppm, respectively. The 3D CBCA(CO)NH experiment was carried out under similar conditions, using the same data points and spectral widths as above, except that the F2 dimension (15N) was recorded with 40 points. For ligand binding studies, the highly purified Der f 7 was dialyzed into 50 mM Tris buffer at pH 7.0 and concentrated to 0.5 mM. 2D 1H-15N HSQC spectra were acquired with four scans using similar parameters as described earlier, with and without the addition of PB, insect juvenile hormone III (JH3) or methoprene at 1∶1 molar ratios. Perturbation of peaks by ligand titration was estimated using the formula: √(ΔH)2+(ΔN*0.17)2 and plotted against residue numbers.3D heteronuclear NMR experiments were carried out to assign the backbone chemical shifts using a 2D The crystal structure of Der f 7 has been deposited at the Protein Data Bank with accession ID of 3UV1.Figure S1Thermal stabilities of wild type and P130S mutant of Der f 7 at different pH values. Thermal denaturation of Der f 7 at pH 7.0 (closed circles) or pH 9.0 (closed squares) and P130S mutant of Der f 7 at pH 7.0 (open squares) or pH 9.0 (open circles) were monitored by Far-UV CD signals at 220 nm.(TIF)Click here for additional data file.


Accumulating evidence suggests that Th17 cells play a role in the development of chronic allograft injury in transplantation of various organs. However, the influence of current immunosuppressants on Th17-associated immune responses has not been fully investigated. We prospectively investigated the changes in Th17 cells in peripheral blood mononuclear cells (PBMCs) collected before and 1 and 3 months after KT in 26 patients and we investigated the suppressive effect of tacrolimus on Th17 in vitro. In the early posttransplant period, the percentage of Th17 cells and the proportion of IL-17-producing cells in the effector memory T cells (TEM) were significantly increased at 3 months after transplantation compared with before transplantation (P<0.05), whereas Th1/Th2 cells and TEM cells were significantly decreased. The degree of increase in Th17 during the early posttransplant period was significantly associated with allograft function at 1 year after transplantation . In vitro, tacrolimus suppressed Th1 and Th2 cells in a concentration-dependent manner, but did not suppress Th17 cells even at high concentration. This suggests that current immunosuppression based on tacrolimus is inadequate to suppress Th17 cells in KTRs, and dysregulation of Th17 may be associated with the progression of CAD. After kidney transplantation, alloimmune responses by CD4+ T cell activation mediate most cases of allograft rejection The Tac-based immunosuppressive protocol has been shown to be more effective in preventing the development of acute rejection episodes and improving 1 year allograft survival than the previously used regimen based on azathioprine and steroids Th17 is a third subset of effector T cells and is characterized by the secretion of the proinflammatory cytokine IL-17 In this study, we prospectively and sequentially investigated the changes in immune cell subsets, including Th17, in renal transplant recipients during the early posttransplant period, during which most significant changes in immune reactivity develop, and evaluated the relationship of the change in Th17 cells with 1-year allograft function. Second, we investigated the percentage of Th17 cells in another renal transplant recipient group with long-term follow-up, comparing patients with normal allograft function and those with chronic allograft dysfunction (CAD). Third, we tested the effect of Tac on each effector T cell subset in vitro.The patient population comprised of 26 living-donor renal transplant recipients (RTRs). Second transplants, highly sensitized patients and ABO-incompatible kidney transplants were excluded from this study because they were treated with modified or intensified immune suppressant protocols. For the included patients, the initial immunosuppressant was Tac in combination with MMF and prednisolone. Basiliximab was used as additional induction therapy at 2 hours before transplantation and on day 4 after transplantation. The initial dose of Tac was 0.16 mg/kg per day orally, and target trough levels were 8–12 ng/mL during the first 3 months and 3–8 ng/mL afterward . MethylpWe followed all RTRs for at least 1 year from transplantation. We calculated the differences between the Th17 percentage before transplantation and at 3 months post transplant, calling this ΔTh17. We evaluated whether ΔTh17 had a significant association with the 1-year allograft function to investigate the clinical impact of the changes in Th17 during the early post-transplant period.2) since KT and have showed the change of eGFR within 20% of the value at 1 year from KT. Another 8 patients who have showed gradually deteriorated allograft function since KT  and MDRD eGFR at enrollment was lower than 60 mL/min/1.73 m2. All patients in CAD group took at least 1 time of allograft biopsy during follow up and allograft tissue showed chronic change  ≥2). We compared the proportion of Th1, Th2 and Th17 cells in the CD4+ T cell populations of the two groups to investigate the clinical impact of Th17 on chronic allograft injury. This study was approved by the Institutional Review Board (KC10SISI0235) of the Seoul St. Mary's Hospital, and we obtained written informed consent from all patients and all clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki.We enrolled another patient group who were being treated with Tac (n = 16) with a long-term follow-up (9.5 years (5.1–13.1)) and who showed stable allograft function without significant change (>20% from baseline) and no acute rejection for at least the past 1 year. These patients comprised two groups that were matched for age and post-transplant duration. One group, designated as the no CAD group (n = 8), was comprised of 8 patients who have showed favorable allograft function  was dispensed into 24-well multiwell plates . For cytokine detection at the single-cell level, PBMC were stimulated with 50 ng/mL phorbol myristate acetate (PMA) and 1 µg/mL ionomycin for 4 h.PBMC were prepared from heparinized blood by Ficoll–Hypaque  density-gradient centrifugation. Cell cultures were performed as described previously Flowcytometic study of PBMC was done within a few hours after the sampling of peripheral blood as a fresh state. For analysis of human intracellular cytokine production, PBMC were stimulated with PMA and ionomycin in the presence of GolgiStop  for 4 hours. For intracellular staining, cells were stained with combinations of the following mAbs: CD4–PE/Cy7 ; CD45RA–FITC  and CD25–APC . Staining for chemokine receptors was performed using the following mouse mAbs : anti-CCR4 , anti-CCR6  and anti-CCR7 . Cells were washed, fixed, permeabilized and stained to detect intracellular cytokines with mAbs to IL-17 ; interferon (IFN)-γ ; IL-4 ; IL-17 ; Foxp3  and IFN-γ . Appropriate isotype controls were used for gate setting for cytokine expression. Cells were analyzed on a FACS Calibur flow cytometry system (BD Biosciences).2 and 2 µL of template DNA. All capillaries were sealed, centrifuged at 500×g for 5 s, and then amplified in a LightCycler instrument with activation of polymerase (95°C for 10 min), followed by 45 cycles of 10 s at 95°C, 10 s at 60°C (β-actin) or 57°C , and 10 s at 72°C. The temperature transition rate was 20°C/s for all steps. The double-stranded PCR product was measured during the 72°C extension step by detection of fluorescence associated with the binding of SYBR Green I to the product. Fluorescence curves were analyzed with LightCycler software v. 3.0 (Roche Diagnostics). The LightCycler was used to quantify IL-1beta and HMGB-1 mRNA. The relative expression level of each sample was normalized to the endogenously expressed housekeeping gene (β-actin). Melting curve analysis was performed immediately after the amplification protocol under the following conditions: 0 s (hold time) at 95°C, 15 s at 71°C, and 0 s (hold time) at 95°C. The rate of temperature change was 20°C/s, except for 0.1°C/s in the final step. The crossing point (Cp) was defined as the maximum of the second derivative from the fluorescence curve. Negative controls were also included and contained all the elements of the reaction mixture except template DNA. All samples were processed in duplicate.After incubation of PBMC for 4 h with PMA and ionomycin, mRNA was extracted using RNAzol B  according to the manufacturer's instructions. Reverse transcription of 2 µg of total mRNA was performed at 42°C using the Superscript™ reverse transcription system . Polymerase chain reaction (PCR) was performed in a 20 µL final volume in capillary tubes in a LightCycler instrument . The reaction mixture contained 2 µL of LightCycler Fast Start DNA Master Mix for SYBR® Green I (Roche Diagnostics), 0.5 µM of each primer, 4 mM MgCl5) from healthy people were incubated under appropriate conditions to stimulate Th0 cells for 48 h. Anti-CD3 (1 µg/mL) and anti-CD28 (1 µg/mL) were used to differentiate Th0 cells. Anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), IL-1b (20 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml) were added to stimulate the differentiation of Th17 cells. For analysis of the suppressive effect of Tac, PBMC from healthy people and renal transplant recipients were preincubated for 1 h in the presence of Tacrolimus and stimulated with Th0 or Th17-polarizing condition for 48 h. Neutralizing antibodies to IFN-gamma (2 µg/ml) and IL-4 (2 µg/ml) were added in some experiments (R&D Systems).Isolated PBMC cells . P values<0.05 were considered significant.Statistical analysis was performed using SPSS software . The comparison of values before transplantation and at 1 month and 3 months after transplantation was made using a paired The age at transplantation was 42.8±11.6 years. Eighteen patients were male and eight female. The HLA mismatch number was 3.2±1.8 and all cases were first kidney transplants. Thirteen patients (50.0%) were on hemodialysis and five patients (19.2%) on peritoneal dialysis before transplantation and the duration of dialysis was 13.7±18.8 months. Eight patients (30.8%) took preemptive transplantation. The most common cause of end-stage renal disease was chronic glomerulonephritis  followed by diabetes mellitus , hypertension . Eighteen cases (69.2%) were living-related donor transplants and eight (30.8%) were living-unrelated donor transplants .P>0.05) (+ T cells showed no significant change at 1 month (41.2%±10.0%) and 3 months (43.7±11.6%) after transplantation compared with before transplantation  (As shown in P>0.05) . The per P>0.05) .+ T cells were also IFN-γ+ (Th1). At 1 month after transplantation, this had not changed significantly , but after 3 months, it had significantly decreased to 17.9±9.1% (P = 0.02 vs. before transplant) (+/CD4+T cells (Th2) was 3.6±1.8% before transplantation, and decreased significantly to 2.4±1.2% at 1 month after transplantation (P = 0.007) and 2.6±1.0% at 3 months after transplantation (P = 0.04) (+FoxP3+/CD4+ T cells (Treg) did not change at either 1 month  or 3 months  after transplantation compared with before transplantation (6.5±2.6%) (+/CD4+ T cells (Th17) significantly increased after transplantation. Before transplantation, it was 1.9±0.7% and it increased to 2.2±1.0% (P = 0.04) at 1 month and 2.6±0.8% at 3 months (P = 0.008) after transplantation  . The per = 0.04) . The per.5±2.6%) . The perantation .+CD45RA+/CD4+ T cells (Tnaive/CD4+ T cells) was 34.1±10.3% and this did not change significantly at 1 month  and 3 months  after transplantation (+CD45RA−/CD4+ T cell (TCM/CD4+ T cells) also did not change significantly after transplantation  (−CD45RA−/CD4+ T cells (TEM/CD4+ T cells) were significantly decreased after transplantation   and 3 months  after transplantation compared with before transplantation  (EM was 4.2±3.2% before transplantation and increased to 6.7±3.2% at 1 month (P = 0.021) and 3 months  after transplantation  . In cont= 0.002) . The per.4±0.9%) . The perantation .P = 0.612; at 3 months, P = 0.09)   . The exp = 0.36) .r = 0.45, P = 0.02)  and Th2  cells showed no significant correlation with allograft function 1 year post transplant. The median value of ΔTh17 was 1.08% (−1.06 to 3.26). The patients with ΔTh17>1.08 were designated the ΔTh17-high group and those with ΔTh17<1.08 were the ΔTh17-low group; comparison of the two groups showed that allograft function was significantly poorer in ΔTh17-high patients at 1 year post transplant ( = 0.02) . In contansplant .+ T cells and compared them in patients with and without CAD. The serum creatinine (Scr) in the group without CAD (n = 8) was 0.9±0.1 mg/dL and the MDRD eGFR was 73.3±8.7 mL/min/1.73 m2. In the group with CAD (n = 8), Scr was 1.8±0.3 mg/dL (P<0.01 vs. patients without CAD) and MDRD eGFR was 37.0±14.0 mL/min/1.73 m2 (P<0.01 vs. patients without CAD). The duration of follow-up  and age at sampling  were matched between the two groups (P<0.01) (P = 0.38) and Th2  showed no significant differences between the groups.In another cohort, which was at least 5 years after transplantation, we evaluated the proportion of Th1, Th2 and Th17 cells in the CD4o groups . The per(P<0.01) . In contin vitro Th1, Th2, Th17 and Treg subpopulations of CD4+ T lymphocytes. PBMC were preincubated for 1 h in the presence of Tac and stimulated with 1 µg/ml anti-CD3 and anti-CD28 for 48 h. As shown in P = 0.429 compared with anti-CD3 and anti-CD28), 10.0±0.2% in the anti-CD3, anti-CD28 and Tac 1 ng/ml (P = 0.0001 compared with anti-CD3 and anti-CD28). As shown in P = 0.03 compared with anti-CD3 and anti-CD28), 0.09±0.05% in the anti-CD3, anti-CD28 and Tac 1 ng/ml (P = 0.03 compared with anti-CD3 and anti-CD28), and 0.01±0.004% in the anti-CD3, anti-CD28 and Tac 10 ng/ml (P = 0.009 compared with anti-CD3 and anti-CD28). By contrast, the frequency of Th17 cells did not differ significantly by Tac . The frequency of Treg cells was also significantly lower in the anti-CD3, anti-CD28 cultured cells by Tac. The values were 36.2±5.8 (anti-CD3 and anti-CD28), 31.3±8.4% in the anti-CD3, anti-CD28 and Tac 0.1 ng/ml (P = 0.09 compared with anti-CD3 and anti-CD28), 21.0±5.8% in the anti-CD3, anti-CD28 and Tac 1 ng/ml . We tested the effect of Tac on Th17 in early-post transplant recipients in Th17 condition and Th17 was not suppressed in high doses of Tac  on Th17 responses, only a small number of animal study has been conducted. In heart transplant animal model, CNI could not suppress Th-17 associated immune responses. EM showed a significant decrease after transplantation, the proportion of IL-17-producing cells in TEM cells significantly increased. This suggests that the increase in Th17 cells resulted from an increase in activated T cells, not of quiescent T cells.Usually, the intensity of immune suppression is highest and the immune reaction to allograft most robust during the early posttransplant period. Therefore, we investigated the change in Th17 cells at 1 month and 3 months after transplantation compared with before transplantation. In contrast to other effector T cell subsets, the percentage of Th17 showed a gradual increase after transplantation, although the clinical course of the recipients was stable. In addition, even though the entire cohort of TWe investigated the expression of markers associated with the development of Th17 cells, including IL-1beta and HMGB1. It is well known that IL-1beta plays an important role in the development of Th17 The clinical impact of Th17 cells in kidney transplantation has not been fully investigated. However, many previous reports suggested a role for Th17 in the progression of fibrosis in various organs. Fibrosis is a major component of the progression of chronic allograft injury in transplanted kidney To investigate its clinical impact, we further evaluated the association between the changes in Th17 cells during the early posttransplant period and allograft function at 1 year after transplantation. The size of the change in Th17 was negatively correlated with MDRD eGFR at 1 year after transplantation, which suggests that chronic injury is more significant in patients who showed a greater increase in Th17 during the early posttransplant period. In addition, we investigated the significance of Th17 in another group of kidney transplant recipients with long-term follow-up. The CAD group showed a gradual deterioration in allograft function during follow-up, which suggests that chronic allograft injury may cause the allograft dysfunction. Interestingly, the proportion of Th17 cells was significantly increased in patients with CAD. It is possible that a sustained elevation in Th17 may be associated with the progression of chronic allograft injury through the mechanisms of both fibrosis and alloimmune reactions.Finally, we tested the suppressive effect of Tac on Th1, Th2, Treg and Th17 cells. In kidney transplantation, only one study has shown that Tac can suppress Th17-related alloimmune responses in vitro In conclusion, Tac-based immune suppression could not suppress Th17 cell as well as other effector T cell subsets such as Th1 or Th2. The non-suppression of Th17 may be associated with the progression of chronic rejection even in stable renal allograft recipients. The results of this study might indicate a need to develop therapeutic options to suppress IL-17-producing effector T cells in renal transplant recipients.


Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide  enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated the DADLE-mediated neurite outgrowth in PC12h cells as well as phosphorylation of MAPK and Akt in PC12h and F11 cells, respectively. These data together strongly suggest that delta opioid peptide DADLE acts through the NGF-induced functional G protein-coupled Oprd1 to provide its neuroprotective and differentiating effects at least in part by regulating survival and differentiating MAPK and PI3K/Akt signaling pathways in NGF-responsive rodent neuronal cells.Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene ( Opioids have been used as painkillers for thousands of years. In addition, opioids are also involved in cell proliferation ,2, angiod-Ala2, d-Leu5] enkephalin (DADLE) -Enkephalin acetate salt (DADLE), dibutyryl cAMP sodium salt (db-cAMP), and naltrindole (NTI) from Sigma. LY294002 (LY), PD98059 (PD) and K252a were purchased from CalbioChem. . Effectene® transfection reagent was purchased from Qiagen, Valencia, CA, USA . MTT assay kit was purchased from Promega . Nerve growth factor (2.5S) (NGF) was purchased from Harlan Bioproducts for Science, Inc. .[4.2.2 incubator. F11 cells were differentiated with 0.5 mM db-cAMP in 0.1% serum containing medium.Seed PC12h (a subclone of PC12) cells , were ge4.3.For PC12h cell experiments, PC12h cells (0.8 million cells per 60-mm dish) were plated in 2 mL of medium A. After 24 h of plating, the medium was removed and the cells were treated simultaneously with 100 ng/mL NGF and with or without DADLE in 2 mL of 0.1% serum medium for 72 h. After 48 h, the cells were re-fed with 0.5 mL of 0.1% serum containing medium supplemented with 100 ng/mL NGF and with or without DADLE. For F11 cell experiments, F11 cells (0.3 million cells/60 mm dish) were plated in 2 mL of DMEM containing 10% FBS. After 24 h of plating, the medium was removed and the cells were treated simultaneously with 0.5 mM db-cAMP, with or without 50 ng/mL NGF and with or without DADLE in 2 mL of 0.1% serum medium for 72 h. After 72 h the total RNA was harvested. All treatments were done in duplicate or triplicate dishes.4.4.Total RNA was harvested with the Tri Reagent RT kit according to the manufacturer’s protocol . Purity of RNA was determined by spectrophotometric analysis at 260 nm and 280 nm.4.5.NGF and Oprd1 PCR products are confirmed by sequencing . Total of 2 μg RNA was used for reverse transcription for each sample in a total volume of 20 μL. PCR was carried out using the Platinum Blue PCR Supermix . A total reaction volume of 25 μL was used with 2 μL of cDNA and 0.4 μM each of forward and reverse primers. The primers used for PCR are given in quencing .4.6.For PC12h cell experiments, PC12h cells (0.8 million/60-mm tissue culture dish) were plated in 2 mL of medium A. After 24 h of plating, medium in each dish was replaced by 2 mL of 1:1 F-12/DMEM containing 0.1% serum and 100 ng/mL NGF. The cells were differentiated with 100 ng/mL NGF for 72 h. The cells were re-fed once with 0.5 mL of 0.1% serum medium containing 100 ng/mL NGF, after 48 h of plating. After 72 h of differentiation, medium was removed from each dish and supplemented with medium without NGF for further 24 h. After 24 h of NGF deprivation, cells were treated with DADLE in varied times. For experiments with naltrindole and PD, after 24 h of NGF deprivation (following 72 h of differentiation with 100 ng/mL NGF), the cells were pretreated with or without 10 μM PD and 1 μM naltrindole for 30 min before treating them with or without DADLE for 15 min. For F11 cell experiments, F11 cells (0.3 million/60-mm tissue culture dish) were plated in 2 mL of medium B. After 24 h of plating, medium in each dish was replaced by 2 mL of DMEM containing 0.1% FBS, 0.5 mM db-cAMP, and with or without 50 ng/mL NGF. The cells were administered with 1 μM DADLE, 100 μM naltrindole and 100 nM K252a for different time points. Cells were pretreated for 30 min with K252a and naltrindole before DADLE and NGF were added, respectively. After 72 h of differentiation, the cells were harvested for the lysates and immunoblotting was carried out according to the manufacturer’s protocol  for phosphorylated (p)-Akt (ser473), total Akt, phosphorylated (p) MAPK (p-44/42), total MAPK using beta actin or tubulin as the comparison for sample loading control. Either 10 μL or 10 μg of each sample was loaded.4.7.For PC12h cell experiments, PC12h  were plated in 96 well plates in 100 μL of medium A per well. After 24 h of plating, medium in each well was replaced with 100 μL of 0.1% serum medium supplemented with 100 ng/mL NGF. After 48 h, the cells were re-fed with 50 μL of the same fresh medium. The cells were differentiated for 3 days. After 3 days the serum containing media was removed and washed with serum free media once. The cells were then fed with 100 μL of serum free media (without NGF) with or without 10 nM DADLE, pretreated for 30–40 min with 1 μM naltrindole or 10 μM of LY or PD before DADLE was added for 48 h. For F11 cell experiments, F11 cells  were plated in 96 well plates and differentiated for 72 h in the presence of 0.5 mM db-cAMP with or without 50 ng/mL NGF. Cells were also differentiated in the presence of db-cAMP, NGF and K252a with or without DADLE. Cells were pretreated with 100 nM K252a for 30 min before addition of 50 ng/mL NGF. After 72 h, the medium was removed and cells were washed with serum free medium and incubated with serum free medium with or without 1 μM DADLE for 48 h. As a positive control, cells were differentiated in db-cAMP in the presence or absence to NGF for a total of 5 days. After a total of 5 days in culture the cells were harvested and MTT assay was carried out according to the manufacturer’s protocol .4.8.www.imagescience.org/meijering/software/neuronj) [PC12h cells were plated  in 2 mL of medium A. After 24 h of plating, medium in each dish was replaced by 2 mL of 0.1% serum medium containing 100 ng/mL NGF, with or without (control) 10 nM DADLE. Cells were also treated with 10 μM LY or 10 μM PD or 1 μM naltrindole. Cells were re-fed with NGF and their respective compounds after 48 h of plating. After total 72 h of treatment, random pictures were taken (10 per dish for neuronj) . Each br4.9.Oprd1 gene (PC12 cells) or mouse Oprd1 gene (F11 cells) . There was no visible change in cell morphology after transfection with transfection efficiency of around 100% (data not shown). After transfection, 500 μL of pre-warmed differentiating medium were added to each cuvette for the respective cell lines. Cells (0.5 or 0.8 million) were then aliquoted into 60-mm dishes containing 2 mL of pre-warmed (37 °C) medium supplemented with or without DADLE . An “untransfected” control was also included.Cells (3 million/100 μL nucleofactor reagent) were transfected for 72 h with a cocktail of 3 siRNAs (500 nM each) against the rat 1 cells)  and 3 an4.9.1.Oprd1 siRNA and DADLE; (c) transfected with Oprd1 siRNA without DADLE; (d) transfected with scrambled siRNA and DADLE; and (e) transfected with scrambled siRNA without DADLE. After 72 h cells were harvested for total RNA isolation. Reverse transcription was carried out with 2 μg of total RNA for each sample in a total volume of 20 μL as described above. PCR was performed for rat NGF (36 cycles) rat Oprd1 (36 cycles), mouse Oprd1 (27 cycles) and GAPDH (23 cycles).The experimental conditions were: (a) cells untransfected without DADLE as negative control; (b) transfected with 4.9.2.www.imagescience.org/meijering/software/neuronj) [Before harvesting the cells for total RNA, 5 to 6 random frames were captured from each dish by VistaVision USB camera . Neurite length was measured using the Neuron J free software (neuronj) ) as desc4.9.3.Oprd1 siRNA; and (c) transfected with scrambled siRNA. PC12h cells (0.2 million cells) were plated in each of 60-mm dish for each treatment after transfection. After 48 h of transfection the cells were re-fed with 0.5 mL of 0.1% serum medium containing 100 ng/mL NGF. After 72 h of transfection, the medium was removed and cells were fed with 2 mL of medium without NGF. After 24 h of NGF deprivation, cells were treated with or without (vehicle only) 10 nM DADLE for 15min. After 15 min, cell lysates were obtained for each treatment condition and Western blotting was carried out for p-MAPK with alpha tubulin as the loading control as described previously. For the F11 cell experiment, F11 cells (0.2 million cells) were plated in each of the 60-mm dishes after transfection in 2 mL of 0.1% serum medium containing 0.5 mM db-cAMP and 50 ng/mL NGF for 72 h and treated with or without 1 μM DADLE for 1 h. The cells were harvested and immunoblotting was carried out according to the manufacturer’s protocol , total Akt, p-MAPK, total MAPK using beta actin as the comparison for sample loading control. Each sample (10 μL) was loaded into each well.The conditions for Western blotting analysis were: (a) without any transfection; (b) transfected with 4.9.4.PC12h or F11 cells  were plated in each well of a 96-well plate in 100 μL of pre-warmed medium containing 100 ng/mL of NGF for PC12h cells or 50 ng/mL of NGF with 0.5 mM db-cAMP for F11 cells, respectively. After 72 h, medium was removed from each well and washed once with serum free medium. The cells were re-fed with serum- and NGF-free medium (100 μL/well) with or without 10 nM DADLE in PC12h cells and with or without 1 μM DADLE in F11 cells for 48 h, respectively. MTT assay was performed as described above.4.9.5.Oprd1 gene in F11 cells as described above. cDNA samples from 2 independent experiments were pooled together to run the PCR. PCR for each treatment were performed in 4 replicates using the iQ5 multicolour real time PCR detection system . The protocol used for running mouse Oprd1 and GAPDH using 1QTM SYBR Green Supermix  was Cycle 1: (1X) Step 1: 95.0 °C for 3 min. Cycle 2: (45X), Step 1: 95.0 °C for 10 s. Step 2: 60.0 °C for 30 s. Cycle 3: (1X) Step 1: 95.0 °C for 1 min. Cycle 4: (1X) Step 1: 55.0 °C for 1 min. Cycle 5: (81X) Step 1: 55.0–95.0 °C for 15 s. The protocol for running rat NGF and GAPDH using TaqMan gene expression master mix  was Cycle 1: (1X) Step 1: 50.0 °C for 2 min. Cycle 2: (1X) Step1: 95.0 °C for 10 min. Cycle 3: (55X) Step 1: 95.0 °C for 10 s. Step 2: 60.0 °C for 1 min. cDNA (1 μL) was used in a total reaction volume of 20 μL for each reaction. Primers for rat NGF  and GAPDH used in the TaqMan assay (RN9999916) were obtained from Applied Biosystems . Mouse Oprd1 and rat GAPDH primers for SYBR Green are given in siRNA was used to knockdown the mouse 4.10.PCR products were run on a 2% or 3% agarose gel for 40–45 min at 96 V and stained with ethidium bromide (0.5 μg/mL). Pictures were taken using a KODAK EDAS 290 High Performance UV transilluminator .4.11.Gel bands were quantified with the Quantity One Basic Software  using local background subtraction.4.12.®  for Windows. Differences were considered to be significant (* p < 0.05). The graphs were made using KaleidaGraph, version 4.03 .Statistical differences were determined by ANOVA using StatView5.Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. In this cell model study, our data together strongly suggest that delta opioid peptide DADLE acts through the NGF-induced functional G protein-coupled Oprd1 to provide its neuroprotective and differentiating effects at least in part by regulating survival and differentiating MAPK and PI3K/Akt signaling pathways in NGF-responsive rodent neuronal cells. Our data together with others’ studies also suggest that delta opioid agonists may be a potential lead compound for developing the NGF signaling activators and such NGF activators may be further developed for the prevention and treatment of NGF or other neurotrophic factor-dependent neurological disorders.


Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×105 transformants per µg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.Among the diverse alkaliphilic  Bacillus strains are of industrial importance because they produce various alkaline extracellular enzymes exhibiting great values in industrial application Bacillus strains are promising metabolite producers of carotenoids, cholic acid derivatives, and organic acids Alkaliphiles are defined as microorganisms that grow optimally at pH of over 9 Bacillus strains at the molecular level, the development of genetic transformation systems is indispensable. To date, most investigations on physiological alkaline adaptation mechanisms of alkaliphiles are limited in the two facultatively alkaliphilic strains, Bacillus pseudofirmus OF4 and Bacillus halodurans C-125 Bacillus strains are very rich Bacillus strains may be helpful to broaden the study on the physiological alkaline adaptation mechanisms of alkaliphiles. Besides, it can be useful to metabolically engineer these strains for production of industrial valuable metabolites. Bacillus sp. N16-5 is a recalcitrant facultatively alkaliphilic strain, producing multiple extracellular hydrolases and organic acids. It is phylogenetically related to Bacillus agaradhearensBacillus pseudofirmus and Bacillus haloduransBacillus sp. N16-5.For in-depth study of the metabolic mechanisms and physiological alkaline adaptation mechanisms of the alkaliphilic Escherichia coli (E. coli), the shuttle plasmid would be methylated by the MTase, which was encoded by the MTase gene from the methylation plasmid.It is well known that the restriction and modification (RM) systems act as “immune” systems in prokaryotes for defending the hosts against the invasion of exogenous DNA Bacillus sp. N16-5 with a developed hard agar regeneration medium. Additionally, an in vivo methylation strategy was exploited to methylate plasmid DNA for improving the transformation efficiency. Prior to the successful protoplast transformation of Bacillus sp. N16-5, the method of electroporation transformation In this study, an efficient protoplast transformation procedure was introduced for Bacillus sp. N16-5 was a very easy process. Over 99% of the bacillary cells formed a spherical state at the concentration of 0.1 mg/ml of lysozyme after a 30 min treatment at 37°C under the observation of the optical microscopy. But the cells were treated with lysozyme of 0.2 mg/ml at 37°C for 60 min for complete protoplastization, which was necessary for efficient protoplast transformation The protoplast formation of Bacillus sp. N16-5. After incubation of the protoplasts plated on the medium at 37°C for 5–7 days, only small, round and transparent colonies with a diameter of about 0.5 mm grew up , a soft agar regeneration medium modified from DM3 medium  grew up . However–10 days . Besides3 transformants per µg plasmid DNA. Also plasmid pMK4 and pHP13 could be transformed into Bacillus sp. N16-5. All of the four plasmids could be maintained well in Bacillus sp. N16-5 at both neutral complex medium (pH 7.7) Protoplast transformation was firstly accomplished by plasmid pHCMC04 and pBK15 with the transformation efficiency of about 10Bacillus sp. N16-5 was achieved with SG25 medium. However, the protoplast regeneration took about 8 days and therefore became a time-consuming step for the protoplast transformation as a result of the low incubation temperature of the medium (25°C). To shorten the time of protoplast regeneration, we tried to find out a regeneration medium that could be incubated at higher temperature. Previous report found that the regeneration frequency was strongly affected by the agar concentration in the regeneration medium Bacillus subtilis with the regeneration medium containing 0.9% agar produced a combination of L colonies and bacillary colonies, whereas the medium with 2.5% agar could provide a hard substratum to induce the complete protoplast reversion Bacillus sp. N16-5 on the adjusted medium at 37°C for 2–3 days, both bacillary colonies and L colonies appeared. However, over 99% of the total colonies were L colonies. The predominance of L colonies suggested that the regeneration medium with 2.5% agar probably could not provide a physical substratum strong enough for the complete protoplast regeneration of Bacillus sp. N16-5. So we tried to further increase the concentration of agar in SA1.2 medium to promote the physical strength of the medium and test its effect on the regeneration frequency. As shown in −3, which was over 10 times higher than that with SG25 medium. What's more, the protoplast regeneration with SA5 medium only needed 2–3 days, compared with about 8 days with SG25 medium.Protoplast transformation of Bacillus sp. N16-5, another recalcitrant facultatively alkaliphilic strain, Bacillus agaradhearens DSM 871, was successfully transformed by the above four plasmids with the transformation efficiency of about 103 transformants per µg plasmid DNA using SG25 medium and about 105 transformants per µg plasmid DNA using SA5 medium.Protoplast transformation was carried out with SA5 medium. The transformation efficiency of different plasmids with SA5 medium was about 4–10 times higher than that with SG25 medium , which p4 transformants per µg plasmid DNA was still very low. So we tried to further improve the transformation efficiency. During the genetic transformation of the bacterial strains that possessed the RM system, the transformation efficiency was greatly improved by the employment of methylated plasmid DNA that could resist the digestion of the restriction enzymes in the bacterial strains Bacillus sp. N16-5 that could prevent the incorporation of the exogenous unmodified plasmid DNA and therefore reduce the transformation efficiency.Although we had successfully shortened the transformation time and improved the transformation efficiency by developing SA5 medium as the regeneration medium, the transformation efficiency of about 10Bacillus sp. N16-5, unmodified plasmid pHCMC04 was treated with the whole cell extract prepared from Bacillus sp. N16-5. As shown in 5′-CGCG-3′, which suggested there was a type II REase in Bacillus sp. N16-5 Bacillus sp. N16-5, was constructed. For methylation of plasmid pHCMC04, plasmid pAM1 and pHCMC04 were co-transformed into Escherichia coli (E. coli) Top10. The mixture of plasmid pAM1 and pHCMC04 was extracted and digested by the whole cell extract to determine the effect of the in vivo methylation strategy. As shown in To detect the existence of the RM system in Bacillus sp. N16-5. The highest transformation efficiency of 1.1×105 transformants per µg plasmid DNA was achieved with methylated plasmid pHCMC04 and SA5 medium.Protoplast transformation was carried out with methylated plasmids and SA5 medium. The methylated plasmid DNA was transformed with higher efficiency than the unmethylated, and the transformation efficiency of the methylated plasmid pHCMC04, pBK15 and pMK4 was about 10 times higher than that of the unmethylated . This reBacillus strains, and L colonies often emerged on the soft agar regeneration medium during the protoplast regeneration Bacillus stains Bacillus strains, which needed high temperature Bacillus strains that particularly needed low temperature. Here we developed a hard agar regeneration medium (SA5 medium), which could provide a rather strong substratum for efficient protoplast regeneration of Bacillus sp. N16-5. Furthermore, it was stable at a much broader range of temperature (below 60°C). So SA5 medium might be helpful for the protoplast regeneration and transformation of a greater range of Bacillus stains.Protoplast regeneration was the major difficult step during the protoplast transformation of Bacillus sp. N16-5 and developed an in vivo methylation strategy to methylate the E. coli-Bacillus sp. shuttle plasmids. Unlike some previous in vivo methylation strategy in which the MTase was expressed by an exogenous promoter (araBAD promoter) E. coli for plasmid methylation and the native promoter of the MTase gene might be qualified in an in vivo methylation strategy In this study, we successfully identified a type II RM system in Bacillus sp. N16-5 with the combination of a newly developed hard agar regeneration medium (SA5 medium) and an in vivo methylation strategy. Also the procedure could be used to transform recalcitrant alkaliphilic Bacillus agaradhearens DSM 871 with unmodified plasmid DNA. The method could be helpful for gene inactivation in Bacillus sp. N16-5 by chromosome integration with the thermosensitive plasmid Bacillus strains.This paper described a quick, efficient and highly reproducible procedure for protoplast transformation of recalcitrant alkaliphilic Bacterial strains and plasmids used in this study were showed in 2, 12.5 mM CaCl2, 1% NaCl, 0.5% glucose and 0.04% filter-sterilized bovine serum albumin. SG25 medium (pH 8.0), which was modified from GR medium 2, 12.5 mM CaCl2, 1% NaCl, and 0.5% glucose. SA5 medium (pH 8.0) contained the same components as SA1.2 medium except that the concentration of agar and Tris base was adjusted to 5% and 10 mM and BSA was omitted in SA5 medium. SMM buffer (pH 7.0) contained 0.5 M sucrose, 0.02 M sodium maleate and 0.02 M MgCl2. SMMP buffer (pH 7.0) was prepared by mixing equal volumes of separately sterilized 2×strength SMM and 4×strength Penassay broth. 30% polyethylene glycol (PEG) 8000 was dissolved in SMM buffer.SA1.2 medium (pH 7.5) consisted of 1.2% agar, 0.5 M sodium succinate, 30 mM Tris base, 0.5% casamino acid, 0.5% yeast extract, 30 mM MgClE. coli Top10 was grown aerobically at 37°C in Luria-Bertani medium supplemented, when necessary, with ampicillin (100 µg/ml), tetracycline (40 µg/ml), chloramphenicol (30 µg/ml), or erythromycin (150 µg/ml). Bacillus sp. N16-5 was grown aerobically at 37°C in Horikoshi-I medium with 2% NaCl (pH 10.0) or complex neutral medium (pH 7.7) supplemented, when necessary, with chloramphenicol (2.5 µg/ml), or erythromycin (1 µg/ml). All strains were maintained frozen in 25% glycerol at −80°C.Bacillus sp. N16-5, the bacterial pellet was pretreated with lysozyme of 2 mg/ml at 37°C for 10 min prior to the standard steps of E.Z.N.A Plasmid Extraction Kit. DNA restriction and cloning were carried out according to the standard procedures Pyrobest DNA polymerase, restriction enzymes, T4 DNA ligase, T4 DNA polymerase and pMD18-T vector were purchased from Takara . DreamTaq DNA polymerase was purchased form Fermentas .The genomic DNA preparation, plasmid DNA extraction and DNA purification were carried out with Genomic DNA Extraction Kit , E.Z.N.A Plasmid Extraction Kit  and E.Z.N.A Gel Extraction Kit  following the manufacturer's instructions. For plasmid extraction from Bacillus sp. N16-5 in Horikoshi-I medium with 2% NaCl was inoculated into 100 ml neutral complex medium. After incubation at 37°C, 220 rpm for about 5 h, the culture reached to the early log growth phase with an OD600 of 0.4. Then the culture was collected by centrifugation at 4°C and 5 000×g for 5 min. The collection was washed once with 10 ml ice-cooled SMMP and suspended in 10 ml SMMP. Lysozyme was added to the suspension with a final concentration of 0.2 mg/ml and mixed by gently shaking. The suspension was incubated at 37°C for 60 min. Then 10 ml SMMP was mixed with the suspension before the resulted protoplasts were harvested by centrifugation. The protoplast collection was washed once with 10 ml SMMP and suspended in 2.5 ml SMMP. The suspension was allocated with 100 µl per aliquot and stored at −80°C. To detect the protoplast regeneration, the prepared protoplasts were plated onto the regeneration medium and incubated at 25°C for SG25 medium or at 37°C for the other media.A 1% inoculum of the overnight culture of 100 µl of the prepared protoplast suspension was added with 4 µl of plasmid DNA solution (0.5–1 µg DNA) followed by mixing with 300 µl of 30% PEG 8000 solution. After 3 min incubation at room temperature, the protoplast suspension was mixed gently with 5 ml SMMP. Then the protoplast was recovered by centrifugation at 4°C and 5 000×g for 10 min and suspended in 1 ml SMMP. For expression of the chloramphenicol or erythromycin resistance gene, the suspension was incubated at 28°C and 120 rpm for 2 h. To select the transformants, the incubated suspension was plated onto the regeneration medium (SG25 medium or SA5 medium) supplemented with chloramphenicol (2.5 µg/ml), or erythromycin (0.5 µg/ml).Bacillus sp. N16-5 was grown in 50 ml Horikoshi-I medium to reach to an OD600 of 2.0. Then the culture was collected by centrifugation, washed once with 50 mM K2HPO4-KH2PO4 buffer (pH 7.5) and suspended in 5 ml of the buffer. The suspension was lysed by sonication at 150 W for 25 min and centrifuged at 4°C and 15 000×g for 20 min. The supernatant was stored as the whole cell extract. The whole cell extract was used for the detection of the REase in Bacillus sp. N16-5. The reaction system of the detection contained 1 µg plasmid DNA, 8 µl of the whole cell extract and 2 µl 10×Takara buffer H  in a total volume of 20 µl. And the reaction was performed at 37°C for 8 h.Bacillus sp. N16-5. The resulted DNA fragments were harvested by gel extraction. The recovered DNA fragments were blunt-ended by T4 DNA polymerase and added with an adenine nucleotide tailing by DreamTaq DNA polymerase. Then the modified DNA fragments were ligated with pMD18-T vector for sequencing. The sequences of DNA fragments were aligned with the sequence of plasmid pUC18 or pMK4 to locate the REase cleavage site.Plasmid pUC18 and pMK4 were separately digested by the whole cell extract of E. coli Top10 was used for construction of plasmid pAM1. According to the genome data of Bacillus sp. N16-5 (unpublished data), the gene that might encode a type II DNA MTase (Genbank number: JN381161) was selected and named as bsp165IM. The primer MetEcoRIF (GAATTCCGAATTGTTGCATTTCTTGAT-3′5′-CCG) and MetNdeIR (CATATGCCAGCCTCTTGCAATTATTAT-3′5′-CAGC) were used to amplify the bsp165IM gene together with its own promoter sequence (the underlined letters indicated the restriction enzyme recognition sites). The PCR product was digested by EcoRI and NdeI, and plasmid pAN2 was also digested by EcoRI and NdeI to remove the initial Φ3TI methyltransferase gene. These two digested products were ligated together to generate plasmid pAM1. Plasmid pAM1 had a p15A origin of replication, which was compatible with the ColE1 origin of plasmid pHCMC04, pBK15, pMK4 and pHP13 in E. coli. For methylating the plasmid pHCMC04, plasmid pHCMC04 and pAM1 were co-transformed into E. coli Top10 and maintained in it with ampicillin and tetracycline. The other plasmids were methylated as the same method above with appropriate antibiotics.