3%. When ARMS was used, 6 more patients were defined as mutation positive, with the ORR of the 22 patients at 72.7%. For patients who provided plasma, 5 mutation positive patients were detected only by ARMS, with the ORR at 80%. Generally, our result was consistent with that of OPTIMAL and IPASS research, SB202190 mouse both using tumor tissue for EGFR

mutation analysis [5, 9]. The ORR for mutation positive patients in OPTIMAL using direct sequencing was 83%, higher than that of IPASS using ARMS strategy (71.2%). Interestingly, such difference also occurred in our study using pleural fluid samples (81.3% Vs 72.7%). The results implied that, more sensitive methods such as ADx-ARMS may find more positive patients, but for them, mutative cells may represent a minority of the whole tumor, which may influence the final clinical outcome of TKIs. The explanation is consistent with the work of Qing Zhou et al. which found that the relative

MEK phosphorylation EGFR mutation abundance could predict benefit from EGFR-TKIs treatment for advanced NSCLC [19]. Our data emphasized that, for mutation positive results, the predictive effect of body fluid was no less than that of tumor tissue. As considered for the two problems mentioned above, our research agreed with former reports that more sensitive method such as ARMS would be one of the feasible solutions [14, 20]. Compared with direct sequencing, ADx-ARMS assay found 18.8% (6/32) and 27.8% (5/18) more patients to be mutation positive for pleural fluid and plasma, respectively. Direct sequencing is currently the routine method used to detect EGFR mutations. The merits of this method are readily available and economic, but the procedure is complicated and time-consuming. Meanwhile, the sensitivity of sequencing is about 30%, which tends to cause false negative result [21]. Given the poor sensitivity of DNA sequencing, many patients and physicians opt to start TKIs treatment even if the sequencing results were Ribonucleotide reductase negative for EGFR mutation. If the tumor does not contain

activating mutations on EGFR, treatment with TKIs will most likely be ineffective. In our study, 11 former negative patients (6 pleural fluids, 5 plasmas) defined by sequencing were proved to be positive at last, and the clinical outcome for them was quite satisfactory. If the treatment plan was made according to the result of direct sequencing, those patients may lose the chance of TKIs therapy. Besides, by using ARMS, we also found 7 samples which harbouring double mutations (2 patients with 19 del and L858R, 1 with L858R and L861Q or S768I, 4 with 19 del and T790M). The clinical evaluations for the former 3 patients were all PR. This result was consistent with the study of Zhang et al. [22] which showed that patients with double activating mutations involving both exons 19 and 21 tend to respond well to TKIs and the sensitivity to TKIs was enhanced compared with either single mutant. As demonstrated by Qing Zhou et al.

J Biol Chem 2000, 275:25262–72.PubMedCrossRef 51. Zagorska A, Pozo-Guisado E, Boudeau J, Vitari AC, Rafiqi FH, Thastrup J, Deak M, Campbell DG, Morrice NA, Prescott AR, Alessi DR: Regulation of activity and localization of the WNK1 protein kinase

by hyperosmotic stress. J Cell Biol 2007, 176:89–100.PubMedCrossRef 52. Cheng CJ, Huang CL: Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1. J Am Soc Nephrol 2011, 22:460–71.PubMedCrossRef 53. Xu BE, Stippec S, Chu PY, Lazrak A, Li XJ, Lee BH, English JM, Ortega B, Huang CL, Cobb MH: WNK1 activates SGK1 to regulate the epithelial sodium channel. Proc Natl Acad Sci USA 2005, 102:10315–20.PubMedCrossRef 54. Xu BE, Stippec S, Lenertz L, Lee BH, Zhang W, Lee YK, Cobb MH: WNK1 activates ERK5 by an MEKK2/3-dependent mechanism. J Biol Chem 2004, 279:7826–31.PubMedCrossRef 55. Ellinger-Ziegelbauer H,

Brown Belinostat solubility dmso K, Kelly K, Siebenlist U: Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by an inducible mitogen-activated protein kinase/ERK kinase kinase 3 (MEKK) derivative. J Biol Chem 1997, 272:2668–74.PubMedCrossRef 56. Yang J, Lin Y, Guo Z, Cheng J, Huang J, Deng L, Liao W, Chen Z, Liu Z, Su B: The essential role of MEKK3 in TNF-induced NF-kappaB activation. Nat Immunol 2001, 2:620–4.PubMedCrossRef 57. Sun W, Li H, Yu Y, Fan Y, Grabiner BC, Mao R, Ge N, Zhang H, Fu S, Lin X, Yang J: MEKK3 is required for lysophosphatidic acid-induced NF-kappaB activation. Cell Signal 2009, 21:1488–94.PubMedCrossRef 58. Barroga CF, Stevenson JK, Schwarz EM, Verma Semaxanib supplier IM: Constitutive phosphorylation of I kappa B alpha by casein kinase II. Proc Natl Acad Sci USA 1995,

Prostatic acid phosphatase 92:7637–41.PubMedCrossRef 59. Lin R, Beauparlant P, Makris C, Meloche S, Hiscott J: Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Mol Cell Biol 1996, 16:1401–9.PubMed 60. Wang D, Westerheide SD, Hanson JL, Baldwin AS Jr: Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem 2000, 275:32592–7.PubMedCrossRef 61. Razani B, Reichardt AD, Cheng G: Non-canonical NF-kappaB signaling activation and regulation: principles and perspectives. Immunol Rev 2011, 244:44–54.PubMedCrossRef 62. Jiwani S, Wang Y, Dowd GC, Gianfelice A, Pichestapong P, Gavicherla B, Vanbennekom N, Ireton K: Identification of components of the host type IA phosphoinositide 3-kinase pathway that promote internalization of Listeria monocytogenes . Infect Immun 2012, 80:1252–66.PubMedCrossRef 63. Cowan C, Jones HA, Kaya YH, Perry RD, Straley SC: Invasion of epithelial cells by Yersinia pestis : evidence for a Y. pestis -specific invasin. Infect Immun 2000, 68:4523–30.PubMedCrossRef Competing interests The authors declare that they have no competing interests.

However, other studies suggested that GKN1 may be secreted from epithelial cells, and have functions in both paracrine and autocrine systems [6] in control of normal cell growth, differentiation, and apoptosis. In addition, this study demonstrated that GKN1 was able to increase the sensitivity of gastric cancer cells to 5-FU treatment. This finding suggested that p38 MAPK inhibitor GKN1 may be useful as an adjuvant target in combination with other chemotherapeutical agents in the treatment of gastric cancer. 5-FU has been a widely used as a chemotherapeutic agent in treating

patients with gastric cancer. It is a pyrimidine analogue and can incorporate into DNA or RNA for the induction of cell cycle arrest and apoptosis through inhibition of DNA duplication in tumor cells. In this regard, GKN1 could induce cell apoptosis, thus GKN1 could enhance 5-FU antitumor activity in gastric cancer cells. This result may

partially explain the reason that patients who have lost GKN1 expression have shorter overall survival [20]. However, it remains to be determined how GKN1 is able to induce apoptosis in gastric cancer cells. Our preliminary data revealed that GKN1 expression was able to modulate expression of several apoptosis-related genes using a cDNA microarray click here analysis. Of the 112 genes covered by the Oligo GEArrays Human Apoptosis Microarray, the expression of 19 genes may directly affect by GKN1. However, some of these screening genes (such as BAX and BCL2A1) may be indirectly or even not affected or regulated by GKN1 protein [14, 21]. Considering limitations of the microarray analysis, these screening genes need to be verified by qRT-PCR or western blot analyses in the further study. Conclusions In summary, expression of GKN1 mRNA and protein was progressively downregulated from the

normal mucosa, precancerous to cancerous gastric tissues. Restoration of GKN1 expression Farnesyltransferase induced gastric cancer cells to undergo apoptosis, and enhanced sensitivity to 5-FU-induced apoptosis. These data indicate that GKN1 plays a role in regulation of gastric epithelial homeostasis and that lost GKN1 expression could contribute to gastric cancer development. Acknowledgements This study was supported in part by grants from The National Natural Science Foundation of China (No. 81072048 and No. 30871145), from the Natural Science Foundation of Guangdong Province (No. 7001641), from the Junior Teacher Cultivation Project of Sun Yat-sen University (No. 09ykpy22), and (No. 10ykjc23). References 1. Talamonti MS, Kim SP, Yao KA, Wayne JD, Feinglass J, Bennett CL, Rao S: Surgical outcomes of patients with gastric carcinoma: the importance of primary tumor location and microvessel invasion. Surgery 2003, 134:720–727. discussion 727–729PubMedCrossRef 2. Jemal A, Thomas A, Murray T, Thun M: Cancer statistics, 2002. CA Cancer J Clin 2002, 52:23–47.PubMedCrossRef 3. Krejs GJ: Gastric cancer: epidemiology and risk factors. Dig Dis 2010, 28:600–603.

The algorithm to predict hearing damage in the first 10 years is interpolated from the predicted median NIHL after 10 years of exposure and the assumed hearing threshold of 0 dB HL at buy MDV3100 the beginning of exposure (ISO 1990), resulting in a steep linear increase in hearing loss during the first years of exposure. A study of NIHL in railway workers showed that 20% of final hearing loss at 2 and 4 kHz was already

established after the first year of noise exposure. This highly exceeded the predictions of the ISO model, yet after 3–4 years of exposure data and model are in close agreement (Henderson and Saunders 1998). On the contrary, another study found only a slight increase in hearing threshold levels (HTLs) of construction apprentices after the first

3 years of employment in construction industry (Seixas et al. 2005), which was much smaller than predicted by ISO-1999. Because NIHL is preventable, hearing conservation programmes are established, often relying on employee’s use of hearing protection devices (HPDs) rather than on controlling the noise exposure at its source (Neitzel and Seixas 2005). Protection from HPDs depends largely on the consistency of usage, because noise exposure during non-use greatly reduces their effectiveness (Neitzel and Seixas 2005). Discomfort, hinder to communication and highly variable noise levels, which GSK1120212 datasheet are common in construction, can cause irregular use of HPDs (Suter 2002; Neitzel and Seixas 2005). Several studies focusing on the use of hearing protectors in construction demonstrated low level of HPD usage; Lusk et al. (1998) found that workers in different construction trades reported to wear protection during only

18–49% of the time exposed to self-reported high noise. In a more recent study, this percentage was 41% (Edelson et al. 2009). Neitzel and Seixas (2005) reported an even lower percentage of usage of less than 25% of the time that combined with the amount of attenuation resulted in negligible effective protection (Neitzel and Seixas 2005). Nevertheless, a study examining hearing loss in Canadian construction workers showed that FER HPD usage was common (>90%) and resulted in a protective effect on hearing (Hessel 2000). These different findings underline the complicating effects of the consistency of HPD usage in assessing the relationship between occupational noise exposure and NIHL. In addition, there is also a great variability in individual susceptibility to hearing loss (Henderson et al. 1993; Sliwinska-Kowalska et al. 2006), partly explained by other possible causes of hearing loss. These are both intrinsic and external factors (Sliwinska-Kowalska et al. 2006; Prince et al. 2003). Intrinsic factors are for example gender, race, genetics, medical history and hypertension (De Moraes Marchiori et al. 2006). External factors concern ototoxicity, leisure noise exposure, HPD usage and smoking (Mizoue et al. 2003; Wild et al.

The drugs of nitrosourea type, such as FM, express high cytotoxicity through the formation of interstrand cross-links in DNA [33]. The

dominating mechanism of chemoresistance to alkylating agents is the repair of DNA adducts by the enzyme O6-methylguanine DNA-methyltransferase [3]. Ionizing radiation also induces activity of this enzyme [34]. In melanoma cells exposed to the alkylating agents or ionizing radiation the level of O6-methylguanine DNA-methyltransferase may increase, resulting in a resistance to such treatments. Some melanoma cell lines inherently express high level of O6-methylguanine MK-8931 clinical trial DNA-methyltransferase [5]. The weak effect of combined treatments is due to the relatively high level of O6-methylguanine DNA-methyltransferase that might be intrinsically present in the HTB140 cells and/or triggered by proton irradiation. Another possible reason for such a limited effectiveness Selleck MLN2238 of the combination of protons and drugs is the nuclear transcription factor kappa B (NF-κB) that is constitutively expressed in melanoma cells [35]. NF-κB is an important feature in the development and progression of malignancies

by targeting genes that promote cell proliferation, survival, metastasis and angiogenesis. NF-κB also regulates apoptosis by controlling the transcription of genes that block cell death. Activation of NF-κB induces overexpression of bcl-xl, bcl-2, vascular endothelial growth factor and interleukin-8. This may affect resistance to apoptosis induced by radiation and chemotherapy [36]. Alkylating agents as well as ionizing radiation can induce cell death through the activation of apoptosis [21, 28,

37]. However, the described mechanism can cause defects in apoptotic pathways, leading to a high cellular resistance [35]. In the HTB140 cells proton irradiation induced G1 phase arrest, while FM as well as combined treatments provoked significant G2 arrest (Figure 3A). After ionizing radiation a delay in G2 phase is the most frequent event, but significant delays could also occur in G1 and S phase [38]. These results are in agreement very with the high radioresistance of HTB140 cells [16]. FM generally produces a G2/M block in the cell cycle, while higher drug concentrations could induce S phase accumulation [39]. In samples exposed to FM or in combined treatments the cell proliferation (Figure 1B) was in agreement with the S phase (Figure 3A). Combined treatment with protons and DTIC, did not induce major changes in the cell cycle as compared to the control or single DTIC treatment (Figure 3B). Similar cell cycle arrest in S and G2/M phase caused by DTIC was also reported for other melanoma cells [40]. Compared to protons, after combined treatment there was a slight reduction of G1 phase and an increase of S phase. Most of the analysed cells were in G1/S phase, thus being viable and able to replicate DNA.

The reaction was initiated by addition of the enzyme, and at 0, 5, 10, and 15 min intervals, 10 μl reaction mixture was withdrawn and spotted onto the DE81 filter paper and dried. The unreacted substrate was washed and the products were eluted and counted in a liquid scintillation counter. With [3H]-Gua GDC-0449 order as substrate

the reaction (in a total of 25 μl) was initiated by addition of the enzyme (10 μl), incubated at 37°C for 2 min, stopped by addition of 1 M HCl (10 μl), and placed immediately on ice. After neutralization, 15 μl of the mixture was spotted onto the DE81-filter paper. The filters were then washed, and the products were eluted and counted by liquid scintillation. IC50 values for purine analogs were determined for both Mpn HPRT and human HPRT using fixed concentrations of [3H]-Hx (10 μM) or [3H]-Gua (10 μM) and variable concentrations of the inhibitors.

Thymidine kinase assay was performed using tritium labelled thymidine ([3H]-dT) as substrate and various concentrations of the inhibitors essentially as previously described [40] to determine the IC50 values of TFT and 5FdU. Kinetic parameters for TFT were determined by using the phosphoryl transfer assays as previously described [52]. Briefly, each reaction was performed in a total volume of 20 μl containing 50 mM Tris/HCl, pH 7.5, 0.5 mg/ml BSA, 5 mM DTT, 2 mM MgCl2, 15 mM NaF, variable concentrations of TFT, 0.1 mM [γ-32P]-ATP, and 50 ng purified enzyme at 37°C for 20 min, and stopped by heating at 70°C for 2 min. After brief centrifugation, 1 μl supernatant was spotted onto a TLC plate this website (PEI-cellulose, Merck) and dried. The TLC plates were developed in isobutyric acid/ammonia/H2O (66:1:33). The reaction products were visualized and quantified by phosphoimaging analysis (Quantity One, Bio-Rad). Statistical analysis The data were analysed by unpaired student’s t-test (two tailed) using GraphPad Prism 5 software. P < 0.05 is considered as significant. Acknowledgements This work was supported by a grant from the Swedish Research Council for Environment, Agricultural Sciences, and

Spatial Planning. We thank Professor Pär Nordlund, Karolinska Institute, Stockholm, for providing the nucleoside and nucleobase analogs. References 1. Razin Phospholipase D1 S, Yogev D, Naot Y: Molecular biology and pathogenicity of Mycoplasmas . Microbiol Mol Biol Rev 1998, 62:1094–1156.PubMed 2. Waites KB, Talkington DF: Mycoplasma pneumoniae and its role as a human pathogen. Clin Microbiol Rev 2004, 17:697–728.PubMedCrossRef 3. Narita M: Pathogenesis of extrapulmonary manifestations of Mycoplasma penumoniae infection with special reference to pneumonia. J infec Chemother 2010, 16:162–169.CrossRef 4. Lenglet A, Herrado Z, Magiorakos A, Leitmeyer K, Coulombier D: Surveillance status and recent data for Mycoplasam pneumoniae infection in the European Union and European Economic area, January 2012. Euro Surveill 2012, 17:2–7. 5.

pylori at the air-liquid interface. The formation of biofilms was initiated by inoculating 10 μl of pre-cultured cell suspension (approximately 5 × 105 cells in Brucella broth) into each well. The cultures were incubated under microaerobic conditions at 37°C for 1 to 6 days with shaking (80-100 rpm). After incubation, the coverslips were removed and washed with phosphate-buffered saline (PBS). The samples were then air

dried and stained with crystal violet for 30 s. After being stained, the coverslips were rinsed with distilled H2O to remove excess dye and then air LGX818 price dried for 30 min. All dye associated with the biofilms was dissolved with 1 ml of ethanol and 200 μl of the ethanol solutions were used to measure the absorbance at 594 nm with a microplate reader to determine the amount of biofilm formation. Confocal laser scanning microscopy (CLSM) and measurement of biofilm thickness For visualization, the

biofilms of H. pylori strains on the coverslips were stained with a BacLight LIVE/DEAD bacterial viability kit solution (Molecular Probes, Leiden, The Netherlands) according to the directions of the supplier. Confocal images were collected by using a Zeiss LSM 510-META confocal laser scanning microscope (Carl Zeiss, Jena, Germany). To determine biofilm thickness, a series of horizontal (xz) optical sections at 0.5 μm intervals were taken through the height of the biofilm for measurement. Each biofilm was scanned selleck chemicals at five randomly selected positions. Each sample was observed independently more than three times. Confocal images of green and red fluorescence were constructed simultaneously using a multitrack mode. Cell viability assay To determine the numbers of viable bacteria, biofilm cells on the coverslips were scrapped into PBS. The optical densities and colony-forming units (CFU) of the cell suspensions were quantitated as the mean of three independent observations. As controls, standard cell broth cultures were used. Electron microscopic studies To observe the biofilm ultrastructure, the biofilms formed on the coverslips were examined by scanning electron microscopy (SEM). The biofilms on the coverslips

were fixed with 2% glutaraldehyde for 3 h at room temperature and the samples were observed using a JSM-5600LV electron microscope (JEOL, Tokyo, Japan). To observe Cyclin-dependent kinase 3 the OMV-like structures, the biofilms of strain TK1402 on the glass slides were examined by using transmission electron microscopy (TEM). Glass slides cut in half were placed into 6-well microtiter plates and the biofilms were allowed to form as described above. The biofilms were fixed with 2% glutaraldehyde for 3 h at room temperature. The samples were then dehydrated and embedded in Epon 813 embedding solution (Chemische Werke Lowi GmbH, Waldkaraigurg, Germany). The sections were finally observed with a JEM-100 electron microscope (Jeol). Isolation of outer membrane vesicles Isolation of OMV was performed as described previously [30]. Briefly, H.

Protein Kinase Activity Eighty cell permeable and ATP competitive protein kinase inhibitors were purchased from EMD (San Diego). Each compound in the InhibitorSelect protein kinase library was screened at 10 μM (unless otherwise noted) in an in vitro PknD autophosphorylation assay. Briefly, each reaction contained 100 ng GST-PknD KD, 20 μM ATP,

5 mM MnCl2 and 3 μCi [γ-32P]-ATP in 25 mM HEPES buffer Epigenetics inhibitor (pH 7.1) supplemented with 1× complete EDTA-free protease inhibitors, unless otherwise noted. Reactions were incubated for 90 min. at 33°C, terminated with SDS-PAGE loading buffer, separated by 10% SDS-PAGE and transferred to polyvinyldinedifluoride (PVDF) membrane. Membranes were exposed to Kodak X-OMAT film for 1-12 hours at -80°C and subsequently developed using an X-ray processor. ATPase Activity ATP hydrolysis by GST-CdsN purified from glutathione-agarose beads was measured using a malachite green assay (R & D Systems). Reaction mixtures contained 100 ng of GST-CdsN, 4 mM ATP, 50 mM Tris-HCL pH 7.0, 5 mM MgCl2, and 10 mM KCl. Compound D7 was added to final concentrations of 1 μM, 5 μM, 10 μM and 100 μM. The reaction mixture (50 μL) was incubated at 37°C for 30 min. The reaction was stopped by the addition of 10 μL of Malachite Green Reagent A followed by 10 μL of Malachite Green Reagent B and incubated at room temperature for one minute before an OD610 reading was taken, according

to the manufacturer’s instructions. Immunofluorescent Microscopy Epigenetics Compound Library cell assay and Chlamydia Growth Experiments HeLa cells

(1 × 105) on coverslips in shell vials were infected with C. pneumoniae CWL029 (MOI of 1) using centrifugation, and replacement media containing 2 μg/mL cycloheximide was added Resminostat at 1 hpi. Protein kinase inhibitors (compounds D4, D5, D6 and D7) were added to the replacement media to a final concentration of 10 μM (unless otherwise noted), for the duration of the Chlamydia developmental cycle (72 hours). For time course immunofluorescence (IF) experiments, compound D7 was added at 1, 15 and 24 hpi. For IF staining cell monolayers were fixed in methanol for 10 minutes at 72 hpi for C. pneumoniae and at 48 hpi for C. trachomatis. Inclusions were stained with the Pathfinder reagent, a FITC-conjugated anti-LPS monoclonal antibody (Bio-Rad, Mississauga) containing Evan’s Blue counterstain. Images were captured at 400× magnification using an Olympus BX51 fluorescent microscope equipped with a color camera (Q color 5; Olympus). To determine the infectivity of Chlamydia grown in the presence of inhibitors, HeLa cells were infected with C. pneumoniae CWL029 and grown for 72 or 84 hrs in the presence of various compounds (used at 10 μM) or vehicle (DMSO 0.1%) then cells were lysed with glass beads into fresh MEM. Serial dilutions of lysates were used to infect fresh HeLa cells and inclusions were stained at 72 hr as described above.

To separate cells in pellicle and underneath, cultures were withdrawn carefully for collecting planktonic cells and the left pellicles. For growth measurement, 27 parallel starting cultures were used and 3 were collected at each

time point and the rest remained undisturbed. The cell density (OD600) of cultures containing planktonic cells was measured first as the planktonic cell density and measured again as the overall cell density after cells from pellicles were added and extensively vortexed. To quantify the pellicles formed by the S. oneidensis wild-type and mutant strains, cells from pellicles P505-15 were collected, suspended in 30 ml fresh LB, violently vortexed, and applied to the spectrometer at 600 nm. Proteinase K and DNase I treatment of S. oneidensis pellicles S. oneidensis was statically cultured in LB broth with the addition of proteinase K (0 μg/mL, 100 μg/mL, and 500 μg/mL) or DNase I (Qiagen, 0U/mL, 100U/mL, 500U/mL and 1000U/mL) for 3 days [55]. We also investigated whether these 3 enzymes could dissolve established pellicles. 2-day old pellicles were rinsed Quisinostat concentration with 20 mM Tris-HCl (pH = 8.0) and incubated in the same buffer supplemented with proteinase K at 37°C for 2 days. Similarly, 2-day old pellicles were incubated with DNase I to examine

the DNA content at room temperature for 2 days. Mutagenesis, physiological characterization and complementation of the

resulting mutants Deletion mutation strains were constructed using the fusion PCR method illustrated previously [56]. Primers used for mutagenesis were listed in Additional file 1. In brief, two DNA fragments flanking selleck kinase inhibitor the target gene were generated from S. oneidensis genomic DNA by PCR with primers 5F/5R and 3F/3R, respectively. Fusion PCR was then performed to join these two DNA fragments with primers 5F/3R. The resulting single fragment was digested with SacI and ligated into the SacI-digested and phosphatase-treated suicide vector pDS3.0. The resultant vectors were electroporated into the donor strain, E. coli WM3064 and then moved to S. oneidensis by conjugation. Integration of the mutagenesis construct into the chromosome and resolution were performed to generate the final deletion strains. The deletion was verified by PCR and DNA sequencing. For complementation, DNA fragments containing aggA or flgA were generated by PCR amplification with MR-1 genomic DNA as the template using primers SO4320-COM-F/SO3988-COM-R and SO3253-COM-F/SO3253-COM-R, respectively as listed in Additional file 1. These fragments were digested with SacI and ligated to SacI-digested pBBR1MCS-5 to form pBBR-AGGA and pBBR-FLGA, which was electroporated into WM3064.

* Statistically

significant (P < 0.05, t-test) as compared with NP69 group. The values are expressed as means ± SD of six repeated experiments. TGF-β type II receptor and Smads in CNE2 cells To investigate alterations of the TGF-β/Smad signaling pathway in CNE2 cells, the TGF-β type II receptor (TβR-II) and the TGF-β/Smad signaling components-Smads signal transduction were explored at both mRNA level and protein level by real time RT-PCR, using specific primers according to GenBank database sequences, western blotting and immunocytochemical analysis, respectively. First, we investigated TβR-II mRNA expression which is an upstream signaling partner of the TGF-β/Smad signaling pathway, while the normal nasopharyngeal epithelial cells were used as control. Under the same culture conditions, we found that TβR-II was significantly up-regulated in CNE2 cells compared to the levels observed in NP69 cells. We further evaluated the Smads which are the principal Selleckchem OSI 906 intracellular components of the TGF-β signaling pathway, and the results showed that Smad2, Smad3 and Smad4 mRNA all increased significantly in CNE2 cells compared to the levels observed in NP69 cells. However, the mRNA level of smad7, known as an inhibitory Smad, remained at same level as that observed for the

normal nasopharyngeal cells (Figure 2A, 2B). To investigate the protein expression of the TβR-II receptor and Smads, buy FK228 western blotting was performed in NP69 and CNE2 cells. We found that Smad2, Smad3, Smad4 and TβR-II were also up-regulated in protein levels, but Smad7 protein level were no different compared to that observed in NP69 cells (Figure 3). To further see more localize the expression of the above signaling components in CNE2 cells, immunocytochemical staining was conducted. A positive staining of TβR-II was found in most CNE2 cells,

and the cell membrane was the main localization of the protein. The positive staining of Smad2, Smad3 and Smad4 was found in regions of both the cytoplasm and nucleus, while the staining of Smad7 was mainly in the nucleus (Figure 4A). Figure 2 The mRNA level of the TGF-β receptor II and the Smads in CNE2 and NP69 cells. (A) Expression level of the TβRII, Smad 2, Smad 3, Smad 4, Smad 7 in CNE2 cells and NP69 cells by RT-PCR using specific primers. β-actin was used as a control and was further to normalize. (B) Bar diagram of the TβRII, Smad 2, Smad 3, Smad 4, Smad 7 mRNA level from densitometric measurement of three real-time quantitative PCR from three separate treatments. * Statistically significant (P < 0.05, t-test) as compared with NP69 group.** Statistically significant (P < 0.01, t-test) as compared with NP69 group. Figure 3 The expression of the TGF-β receptor II and the Smads in CNE2 and NP69 cells. Expression level of the TβRII, Smad 2, Smad 3, Smad 4, Smad 7 in CNE2 cells and NP69 cells by western blot. Actin was used as a protein loading control and was further to normalize.