For the detection of hup transcripts the RNA was extracted from cells grown under N2-fixing conditions (BG110) and collected in the transition between the light and the dark phase. Reverse transcription (RT) reactions were performed with 1 μg of total RNA, following the protocol of the ThermoScript™ RT-PCR System (Invitrogen Corporation, Carlsbad, CA), and using LmhoxHR, GWhoxW1R or LmhupW2R as hoxH-, hoxW-, or

hupW-specific antisense primers, respectively. The three different cDNAs produced were used as templates in PCR amplifications for the detection of the cotranscription of hoxEF, hoxF-hcp, hoxUY, hoxYH (cDNA generated using LmhoxHR), ORF16-hoxW (cDNA generated using GWhoxW1R), and hupSL and hupL-W (cDNA generated using LmhupW2R). The cDNAs produced were used in PCR amplifications performed with the primer pairs RThoxE1F-GWhox8R, Metabolism inhibitor RThoxF1F-LmHCPR, LMhoxUF1-GW5Lmhox2R, LmhoxYF-LmhoxHR, LmhoxWorfF1-LmhoxWR2, LMS3′A-LMH2B, and LMH5A-GW3LmhupWR1, for hoxEF, hoxF-hcp, hoxUY, hoxYH, ORF16-hoxW, hupSL, and hupL-W detection, respectively (Table 2).

The PCR program profiles were as follows: 94°C for 2 selleck chemicals llc min followed by 35 cycles of 45 s at 94°C, 45 s at 50°C (hox), 55°C (hupSL) or 64°C (hupL-W) and 1 to 2 min at 72°C, concluding with a 7 min extension at 72°C. Negative controls included the omission of reverse transcriptase in the RT reaction prior to the PCR, and a PCR to which no template was added. Genomic DNA was used as a Selleckchem MK-0457 positive control. Generated PCR products were analyzed on a 1% (w/v) agarose gel. Identification of transcription start points (tsp) by Rapid Amplification of cDNA Ends (5′-RACE) The RNA used to establish the localization of the transcription start points was extracted from cells grown in the same conditions and collected

at the same time points as for the cotranscription experiments (see above). 5′-RACE was carried out using the FirstChoice® RLM-RACE Kit (Ambion, Inc., Austin, TX) following the instructions of the manufacturer. see more For the identification of the tsp upstream hoxE, hoxW and hupW the gene-specific antisense primers RChoxE1R, RChoxE2R, RChoxE3R, and RChoxE4R (hoxE), LmxisHR4, LmxisHR3, LmxisHR2, and LmxisHR1 (xisH), or LmhupW3R, LmhupW2R, LmhupW1R, and GW3LmhupWR1 (hupW) (Table 2) were used together with the kit adaptor-specific primers. PCR amplifications were carried out with the following profiles: 94°C for 3 min followed by 35 cycles of 30 s at 94°C, 30 s at 55°C (hoxE and xisH) or 58°C (hupW), and 1 min at 72°C, and concluding with 7 min extension at 72°C. The obtained PCR products were cloned into the pGEM®-T Easy vector (Promega, Madison, WI), and subsequently sequenced at STAB Vida (Lisbon).

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EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM: Common virulence factors for bacterial pathogenicity in plants and animals. Science 1995,268(5219):1899–1902.PubMedCrossRef 63. Sabat A, Krzyszton-Russjan J, Strzalka W, Filipek R, Kosowska K, Hryniewicz W, Travis J, Potempa J: New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates. J Clin Microbiol 2003,41(4):1801–1804.PubMedCrossRef 64. Massey RC, Buckling A, Peacock SJ: Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus . Curr Biol 2001,11(22):1810–1814.PubMedCrossRef 65. Schaaff F, Bierbaum G, Baumert N, Bartmann P, Sahl HG: Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus . Int J Med Microbiol 2003,293(6):427–435.PubMedCrossRef 66. Clinical and Laboratory Standards Institute (CLSI): Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically:

Approved Standard. 2006. 67. Besier S, Smaczny C, von selleck compound Mallinckrodt C, Krahl A, Ackermann H, Brade V, Wichelhaus TA: Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease. J Clin Microbiol 2007,45(1):168–172.PubMedCrossRef 68. Zaborina O, Lepine F, next Xiao G, Valuckaite V, Chen Y, Li T, Ciancio M, Zaborin A, Petrof EO, Turner JR, et al.: Dynorphin activates quorum sensing quinolone signaling in Pseudomonas aeruginosa . PLoS Pathog 2007,3(3):e35.PubMedCrossRef Authors’ contributions GM, DLS and AEA carried out the experiments. GM, DLS, ED, AMC, EHF, SM and FM designed and conceived the study. GM and FM wrote the paper. All authors read and approved the final manuscript.”
“Background Typhoid and paratyphoid fever, due to infection with Salmonella

enteric serovar Typhi (S. typhi) and selleck kinase inhibitor Paratyphi (S. paratyphi), are major global problems. Nalidixic acid-resistant (NAR) S. typhi and S. paratyphi are endemic to many Asian countries [1]. NAR isolates have reduced susceptibility to fluoroquinolones, which is associated with higher rates of morbidity and mortality, particularly prolonged fever clearance time and increased need for retreatment [2]. Quinolone resistance in Salmonella is usually associated with mutations of the target site, DNA gyrase, most commonly in the quinolone resistance-determining region (QRDR) of the A subunit. Plasmid mediated quinolone resistance genes of qnr (qnrA, qnrB, qnrS, and qnrD) and aac(6′)-Ib-cr has also been described in quinolone-resistant non-Typhi Salmonella[3, 4].

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32. Wirth T, Falush D, Lan R, Colles F, Mensa P, Wieler L, Karch H, Reeves P, Maiden M, Ochman H, Achtman M: Sex and virulence in Escherichia coli : an evolutionary BIBW2992 datasheet perspective. Mol Microbiol 2006, 60:1136–1151.PubMedCrossRef

33. Toledo-Arana A, Dussurget O, Nikitas G, Sesto CFTRinh-172 in vivo N, Guet-Revillet H, Balestrino D, Loh E, Gripenland J, Tiensuu T, Vaitkevicius K, Barthelemy M, Vergassola M, Nahori MA, Soubigou G, Régnault B, Coppée JY, Lecuit M, Johansson J, Cossart P: The Listeria transcriptional landscape from saprophytism to virulence. Nature 2009, 459:950–956.PubMedCrossRef 34. Chen Y, Ross WH, Gray MJ, Wiedmann M, Whiting RC, Scott VN: Attributing risk to Listeria monocytogenes subgroups: does response in relation to genetic lineages. J Food Prot 2006, 69:335–344.PubMed through 35. Sabet C, Lecuit M, Cabanes D, Cossart P, Bierne H: LPXTG protein InlJ, a newly identified internalin involved in Listeria monocytogenes virulence. Infect Immun 2008, 73:6912–6922.CrossRef 36. McLauchlin J: The identification of Listeria species. Int J Food Microbiol 1997, 38:77–81.PubMedCrossRef

37. Geoffroy C, Gaillard JL, Alouf JE, Berche P: Production of thiol-dependent haemolysins by Listeria monocytogenes and related species. J Gen Microbiol 1989, 135:481–487.PubMed 38. Liu D: Listeria monocytogenes : comparative interpretation of mouse virulence assay. FEMS Microbiol Lett 2004, 233:159–164.PubMedCrossRef 39. Tamura K, BAY 63-2521 datasheet Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 1596–1599. 40. Rozas J, Sánchez-DelBarrio J, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 2003, 19:2496–2497.PubMedCrossRef 41. Tajima F: Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 1989, 123:585–595.PubMed 42. Zhang W, Jayarao BM, Knabel SJ: Multi-virulence-locus sequence typing of Listeria monocytogenes . Appl Environ Microbiol 2004, 70:913–920.PubMedCrossRef 43. Milkman R, Bridges M: Molecular evolution of the Escherichia coli chromosome. III. Clonal Frames. Genetics 1990, 126:505–517.PubMed 44.

TRITC (tetramethyl rhodamine isothiocyanate)-labeled wheat germ agglutinin (Molecular Probes, Eugene, OR) was used at a concentration of 0.1 mg/mL to stain the PIA in biofilms [17]. Hemoglobin was purchased from Sigma and used as indicated concentrations. The Ethics Committee of the Zhongshan Hospital of Fudan University and the East Hospital of Tongji University both exempted this study from review because the current study only focused on bacteria. Cultivation of bacterial biofilms Biofilm cultivation in polystyrene microtitre plates was carried out as described previously [11]. Briefly, overnight cultures of Se strains grown in TSB (0.25% glucose) medium were diluted 1:200.

The diluted cultures were transferred

to wells of polystyrene microtitre plates (200 μL per well) and incubated at 37 °C for 24 h. After washing, the wells find more were stained with 2% crystal violet for 5 min. Then, the plate was rinsed, air-dried, redissolved in ethanol and the absorbance was determined at 590 nm. For cultivation of Se biofilms in the flow-chamber system, the flow-chamber system was first assembled and selleck chemicals prepared as described previously [18]. Briefly, the flow chambers were inoculated by injecting 350 μL overnight culture diluted to OD600 = 0.001 into each flow channel with a small syringe. After inoculation, flow channels were left without flow for 1 h, after which medium flow (0.2 mm/s) was started using a Watson-Marlow 205 S peristaltic pump. Microscopy All microscopic observations and image acquisition were performed Edoxaban using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena) equipped with detectors and filter sets for monitoring SYTO 9, PI, DDAO and TRITC fluorescence. Images were obtained using an x63/1.4i objective or an x40/1.3i objective. Simulated 3D images and sections were generated using the IMARIS software

package (Bitplane). Bacterial C59 wnt attachment assays Initial cell attachment was tested as described previously [11]. Briefly, cell suspensions from the mid-exponential phase of bacterial growth were diluted to OD600 = 0.1 in PBS, and then incubated in wells (1 mL per well) of cover-glass cell culture chambers (Nunc) for 30 min at 37°C, after which attached cells were calculated by microscopy. Quantification of extracellular DNA Extracellular DNA was quantified as described previously [11]. Overnight cultures were diluted to OD600 = 0.001 in AB medium supplemented with 0.5% glucose, 0.05 mM PI and 10% TSB. The diluted cultures were transferred to wells of polystyrene microtitre plates (150 μL per well) and incubated for 24 h at 37°C, upon which PI absorbance was measured at 480 nm and cell density was measured by OD600 using a Wallac microtitre plate reader. Relative amounts of extracellular DNA per OD600 unit were calculated.

Materials and methods Cell lines The T-ALL cell lines, Molt-4 (GC resistant) and Jurkat (GC resistant) were kindly provided by Dr. Stephan W. Morris (St. Jude Children’s Research Hospital). CEM-C1-15 (GC resistant) and CEM-C7-14 (GC sensitive) were kindly provided by Dr. E. Brad Thompson (University of Texas Medical Branch). Cytoskeletal Signaling inhibitor All cell lines were maintained in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, St Louis, MO, USA), 2 mM L-glutamine (Gibco),

and antibiotics (penicillin 100 U/mL and streptomycin 50 μg/mL) at 37°C in a humidified 5% CO2 in-air atmosphere. Reagents and antibodies Rapamycin (Calbiochem, La Jolla, CA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma) and used at the concentration of 10 nM. Dex (Sigma) was dissolved in ethanol and used at the concentration of 1 μM. The final concentrations of DMSO and ethanol

in the medium were 0.05% and 0.1%, respectively, at which cell proliferation/growth or viability was not obviously altered. MTT and Propidium iodide (PI) were Citarinostat supplier purchased from Sigma. Annexin V-PI Kit was purchased from Keygen (Nanjing, China). Antibodies to phospho-4E-BP1, phospho-p70S6K, cyclin D1, p27, Bax, and Bcl-2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to p21 was purchased from BD Bioscience (San Jose, CA, USA) Montelukast Sodium and antibodies to Bim, Mcl-1,

cyclin A, caspase-3 (cleaved at Asp175), NF-κB, and secondary antibodies of horseradish peroxidase (HRP)-conjugated donkey anti-rabbit antibody and HRP-conjugated sheep anti-mouse antibody were all obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Anti-GAPDH antibody was obtained from Kangchen Bio-Tech (Shanghai, China). Cell treatment Logarithmically growing cells were harvested and replaced in 96- or 6-well sterile plastic culture plates (see more Corning Inc., Acton, MA, USA), to which 10 nM rapamycin (Rap group), 1 μM Dex (Dex group), 10 nM rapamycin plus 1 μM Dex (Rap+Dex group), and 0.05% DMSO plus 0.1% ethanol (Control group) were added respectively. At the end of the incubation period, cells were transferred to sterile centrifuge tubes, pelleted by centrifugation at 400 g at room temperature for 5 min, and prepared for analysis as described below. Proliferation assay MTT assay is based on the conversion of the yellow tetrazolium salt to purple formazan crystals by metabolically active cells and provides a quantitative estimate of viable cells. Cells were seeded in 96-well plates (20,000/mL) and incubated for 48 h. 0.5 mg/mL MTT (final concentration) was added to each well for 4 h at 37°C. Then, 100% (v/v) of a solubilization solution (10% SDS in 0.01 M HCl) was added to each well, and the plates were re-incubated for 24 h at 37°C.

Furthermore, fifty-five out of the 147 ArcA-activated genes (37%), and 100 out of the 245 ArcA-repressed genes (41%) contained at least one putative ArcA-binding site (Additional file 1: Table

S1). Figure 2 Logo of the information matrix obtained from the alignment of ArcA sequences for S . Typhimurium. Sequences were obtained by searching the S. Typhimurium LT2 genome [Accession #: AE006468 (chromosome) and AE606471 (plasmid)] with known ArcA sequences derived from the corresponding ArcA-regulated genes in E. coli. A total of 20 E. coli sequences were used to obtain the logo shown. The total height of each column of characters represents the amount of information [measured in bits, which is the maximum entropy for find more the given sequence buy SBI-0206965 type (ex. Log2 4 = 2 bits for DNA/RNA and log2 20 = 4.3 bits for proteins)] for that specific position and the height of each individual character represents the frequency of each nucleotide. ArcA as a repressor Transcription of the genes Belnacasan order required for aerobic metabolism, energy generation, amino acid transport,

and fatty acid transport were anaerobically repressed by ArcA (Additional file 1: Table S1). In particular, the genes required for cytochrome-o-oxidase, succinyl-CoA synthetase, glutamate/aspartate transport, trehalose-6-phosphate biosynthesis, long-chain fatty acids transport, spermidine/putrescine transport, dipeptide transport, the genes encoding the two-component tricarboxylic transport system and the site-specific DNA factor for inversion stimulation (fis) were among the

highest repressed by ArcA. Genes required for L-lactate transport and metabolism, phosphate transport, acetyl-CoA transferase, APC family/D-alanine/D-serine/glycine transport, putative cationic amino acid transporter, peptide methionine sulfoxide reductase, multiple antibiotic resistance oxyclozanide operon, as well as many poorly characterized genes were also repressed by ArcA (Additional file 1: Table S1). Additionally, some genes related to Salmonella virulence were repressed by ArcA. For example, the expression of the mgtCB operon (member of SPI-3) that is required for Mg2+ transport/growth in low-magnesium and involved in systemic infections in mice/intramacrophage survival [37–40], genes constituting the lambdoid prophage Gifsy-1 that contributes to the virulence of S. Typhimurium [41], and genes coding for a leucine-rich repeat protein (sspH2) that is translocated by and coordinately regulated with the SPI-2 TTSS [42] were highly repressed by ArcA (Figure 3A and Additional file 1: Table S1). Figure 3 Organization of major genes for (A) SPI-3, (B) ethanolamine utilization, (C) propanediol utilization, and (D-F) flagellar biosynthesis and motility.

In this work, the excitation wavelength of 400 nm is used as the excitation source with photon of 3.10 eV, which is higher than the band gap of Cu2O. Room temperature FL spectra results for samples deposited at the different applied Selleck PRIMA-1MET potentials are individually presented in Figure 5. The FL signals of the samples are quite similar. The primary FL

spectral characteristics for all samples include an emission peak centering at about 603 nm (2.06 eV). As the band gap of Cu2O is about 2.0 eV, the emission at 603 nm can be attributed to near band-edge emission from free exciton recombination [30]. Figure 5 FL spectra of Cu 2 O thin films. Conclusions In summary, Cu2O thin films were deposited on Ti sheets in a solution consisting of cupric acetate and sodium acetate by electrodeposition method. XRD measurement shows the existence EX 527 cell line of Cu2O with cubic structure and the peak of Cu only at −0.5 V. SEM images reveal that the applied potential has significant influence on the surface morphology. The morphology of Cu2O films turns octahedral into cubic and agglomerate as the applied potential becomes more cathodic. Band gap values of the films vary from 1.83 to 2.03 eV. The emission at 603 nm (2.06 eV) of FL spectra

can be caused by near band-edge emission from free exciton recombination. Acknowledgements This work is supported by the National Natural out Science Foundation of China (No. 51072001 and 51272001), National Key Basic

Research Program (2013CB632705), the National Science Research Foundation for Scholars Return from Overseas, Ministry of Education, China, and Science Foundation for The Excellent Youth Talents of Chuzhou University (2013RC007). The authors would like to thank Yonglong Zhuang and Zhongqing Lin of the Experimental Technology Center of Anhui University for electron microscope test and discussion. References 1. Hiroki N, Tatsuya S, Hiroki H, Chihiro M, Ichiro T, Tohru H, Mitsunobu S: Chemical fabrication of p-type Cu 2 O transparent thin film using molecular precursor method. Mater Chem Phys 2012, 137:252–257.CrossRef 2. Ho JY, Huang MH: Synthesis of submicrometer-sized Cu 2 O crystals with morphological evolution from cubic to hexapod structures and their comparative photocatalytic activity. J Phys Chem C 2009, 113:14159–14164.CrossRef 3. Park JC, Kim J, Kwon H, Song H: Gram-scale synthesis of Cu 2 O ACY-1215 supplier nanocubes and subsequent oxidation to CuO hollow nanostructures for lithium-ion battery anode materials. Adv Mater 2009, 21:803–807.CrossRef 4. Sharma P, Sharma SK: Microscopic investigations of Cu 2 O nanostructures. J Alloy Comp 2013, 557:152–159.CrossRef 5. Miyake M, Chen YC, Braun PV, Wiltzius P: Fabrication of three-dimensional photonic crystals using multibeam interference lithography and electrodeposition. Adv Mater 2009, 21:3012–3015.CrossRef 6.

miR-302b is a member of the miR-302 cluster, which is specifically expressed in pluripotent human embryonic stem cells but not in #AUY-922 mw randurls[1|1|,|CHEM1|]# differentiated embryoid bodies or adult tissues [30]. This miR-302 family is also able to reprogram human skin cancer cells into a pluripotent ES cell-like state [22]. It was found that overexpression of miR-302b induced caspase-3-mediated apoptosis in the human neuroblastoma SH-SY5Y cell line [23]. But, a recent report found that miR-302b is overexpressed in primary

human tumors specimens, and the down-regulation of miR-302b effectively decreased tumor cell growth in human head and neck squamous cell carcinoma patients [31]. Our results showed that miR-302b is down-regulated in tumor tissues compared to paired normal adjacent learn more tissues. There were significant correlations between the expression of miR-302b and lymph node metastasis and differentiation.

Furthermore, a low expression level of miR-302b was an independent factor that indicated poor prognosis in ESCC patients. This evidence suggests that down-regulation of miR-302b in tumor cells may play roles in the development of ESCC and may have prognostic value. We then investigated whether ErbB4 could be regulated by miR-302b and the effect that miR-302b had on ESCC cell behaviors. Our study documented that ErbB4 protein expression was negatively regulated by miR-302b both in cell and tissue analysis. The overexpression of miR-302b significantly decreased the ErbB4 protein level but not mRNA level in ESCC cells, indicating the post-transcriptional down-regulation of ErbB4 by miR-302b. Moreover, the overexpression of miR-302b significantly decreased the luciferase activity of pmirGLO that contained the ErbB4 3′-UTR sequence, while it did not decrease the activity of pmirGLO that contained the ErbB4 3′-UTR mutant sequence, indicating that the target site was specific. Furthermore, to reveal the exact role of miR-302b in ESCC, we tested the effect of miR-302b on proliferation, apoptosis, and invasion by up-

and down-regulating PIK3C2G the expression level of miR-302b. The results suggested that miR-302b acted as a tumor suppressor gene in ESCC by inhibiting proliferation, inducing apoptosis, and repressing invasion. Contrary to our observations, Murray et al. showed that miR-302b was overexpressed in malignant germ cell tumors compared to normal gonad and benign germ cell tumors [32]. But miR-302b function as a tumor suppressor gene both in gastric cancer by targeting EGFR [33]. These results indicate that onco-miRNAs and suppressor-miRNAs can regulate two different roles of the same gene, behaving as oncogenes or tumor suppressors, depending on the tissue type and specific targets [34]. We will carry out further in vivo experiments to confirm the role of miR-302b and its target genes in ESCC. Conclusions This was the first study to evaluate the relationship between ErbB4 and miR-302b in ESCC.

Several new chemotherapy agents are being tested in combination with radiation, but the best chemotherapy remains to be determined. The fate of irradiated cells is believed to be controlled by the network of signaling elements that lead to different modes of cell death or survival. Many stress-responsive genes are inducible by IR [18, 19]. These radiation-inducible genes are believed to have effects on the chemosensitivity IACS-10759 of tumor cells [13, 20]. To determine the correlation between radio-resistance and sensitivity to chemotherapeutic drugs in esophageal cancer cells, we then analyzed the chemosensitivity of

EC109 and EC109/R cells with chemotherapeutic drugs cisplatin, 5-fluorouracil, doxorubicin, MK 8931 in vivo paclitaxel or etoposide. EC109/R, which survived 80 Gy irradiation, became more sensitive to different concentrations of 5-fluorouracil, doxorubicin, paclitaxel and etoposide, but maintained tolerance to cisplatin, as assessed by MTT assay (Figure 4). These findings suggest that cellular resistance to ionizing radiation have effects on the chemotherapeutic drug sensitivity in esophageal cancer cells. Several genes associated with cellular sensitivity to anticancer drugs have been selected for esophageal cancer. They were B4GALT5 (UDP-Gal: βGlcNAc β1,4-galactosyltransferase, polypeptide 5 gene), UGCG (UDP-glucose ceramide

glucosyltransferase gene), and XBP1 (X-box binding protein 1 gene) for 5-fluorouracil, Captisol mouse NRCAM (neuronal cell adhesion molecule gene) for doxorubicin, ARFRP1 (ADP-ribosylation factor related protein 1 gene), IFITM1 (interferon induced transmembrane protein 1 gene), KIAA0685, and SIPA1L2 (signalinduced proliferation-associated 1 like 2 gene) for cisplatin [14]. Fractionated irradiation might induce cellular sensitivity related gene and protein expression in human tumor cell lines. The fact

that drug Interleukin-3 receptor sensitivity is determined by multiple genes required a better understanding of the intricate network of the selected genes in the expression levels. Fractionated radiation treatment has also been reported to cause drug resistance in ovarian carcinoma cells [21] and ascites tumor cells [22]. It can induce functionally relevant multidrug resistance gene and protein expression in human tumor cell lines [13]. There are multiple factors that contribute to cisplatin resistance, but alterations of DNA repair processes have been known for some time to be important in mediating resistance [23, 24]. The most important DNA repair pathways involved in the cisplatin response are nucleotide excision repair (NER) and mismatch repair (MMR). MSI, which results from disorder of the MMR system and loss of MLH1 protein, is frequently induced during cisplatin-based chemotherapy [25]. Data have shown that suppression of ERCC1 expression enhances or restores cisplatin sensitivity, and combination of p53 inactivation and MMR deficiency results in cisplatin resistance [26].

The individual molecular mechanisms of resistance have been identified for all first-line drugs and the majority of second-line drugs [7]. In M. tuberculosis, resistance to RMP results from mutations in the β-subunit of RNA polymerase, which is encoded by the rpoB gene [8]. Approximately 95% of RMP-resistant strains carry mutations within an 81-bp region containing codons

507 through 533 of the rpoB gene [8–10]. The single mechanism of resistance and narrow distribution of mutations make rpoB-81 bp region very attractive for selleck compound molecular detection of resistance to RMP [11, 12]. However, within several dozen different mutations detected in the rpoB-81 bp region of RMP-resistant M. tuberculosis strains [for review see [13]], very few were tested by cloning and complementation assays. Mutated rpoB genes (S531L; H526Y; D516V) were introduced into the RMP sensitive M. tuberculosis H37Rv strain, resulting click here in acquired drug resistance of the host strain [14]. These authors observed that the level of acquired resistance was higher for mutants carrying mutations in codons 531 and 526 compared to mutation in codon 516. In this paper a genetic model was constructed allowing for a relatively simple verification of the relationship between the presence of a given mutation in rpoB-81 bp region and the RMP resistance of the host

strain carrying such a mutation. Some rpoB mutations revealed drug-resistance only in selected M. tuberculosis strains suggesting that genetic background of the host is important for the development of resistance to RMP. Methods Bacterial strains and growth conditions The M. tuberculosis strains examined for this study were isolated from TB patients in Poland in 2000 during the second national survey of drug resistance [12, 15]. Eight clinical strains identified as drug resistant, carrying different mutations in the

rpoB gene, and two susceptible strains identified as drug sensitive, which did not carry any mutation in rpoB, were selected. Moreover, a control laboratory strain M. tuberculosis H37Ra, was included in this study. Primary isolation, differentiation, and drug susceptibility testing were performed with 3-mercaptopyruvate sulfurtransferase Lowenstein-Jensen (LJ) medium and the BACTEC 460-TB system (Becton-Dickinson, Sparks, Md.), as reported earlier [15]. All mycobacterial strains used in this study were cultured in Middlebrook 7H9 broth supplemented with OADC (albumin-dextrose-sodium chloride) and with kanamycin (25 μg/ml), or hygromycin (10 μg/ml), when required. Mycobacterial transformants were selected on Middlebrook 7H10 agar plates find more enriched with OADC containing kanamycin (Km) or hygromycin (Hyg). Gene cloning strategies Standard molecular biology protocols were used for all cloning procedures [16].