In brief, 3-week-old female ICR mice (10-12 g) were anesthetized by ketamine-xylazine injection, and the hair was cut from the left flank using scissors and/or electric shaver to bare the skin, unless otherwise indicated. Bacteria (0.1 ml; 1 × 107 cfu per mouse) grown in BHI-Y were injected with a 27-gauge needle just under the surface of the skin so that a superficial bleb was raised immediately below the skin surface. The number of colony-forming units injected was verified for each experiment by plating bacteria on BHI-Y or sheep blood agar plates (with or without kanamycin) and counting

colony-forming units. The purified recombinant His-IFS or His-TarC was injected as follows: (1) on day 0, 25 μg (per 0.1 ml) was inoculated together with bacteria in the left flank. It was confirmed that both His-IFS and His-TarC had no effect on bacterial viability and growth (data not shown), and (2) on days 2-4, JNK inhibitor 50 μg (per day) was inoculated intraperitoneally. The bacterial viability (and growth) was assessed by incubating the remaining mixture

of bacteria and either His-IFS or His-TarC used on the day 0 for 1 to 6 hours, and counting colony-forming units on BHI-Y or sheep blood agar plates. Because it is difficult to increase injection volume in the skin, we decided to increase the concentration of IFS per ml of injection solution. Preliminary test showed highest concentration (no dilution) was more effective at reducing GAS virulence than any of the IFS dilutions tested (data not shown). Thus, we used the highest concentration MK-1775 purchase to add as much IFS as our possible. Creation of nga mutant of strain GT01 Escherichia coli JM109 was used to propagate plasmid constructions. Non-polar

inactivated mutant of nga was constructed via double-crossover allelic replacement in the chromosome of S. pyogenes GT01. To construct the plasmid for the nga knockout mutant, the 5′ end of nga (fragment 1) was amplified with oligonucleotide primers ngaGT-n1 Liothyronine Sodium (5′-GGCTAGCGAACAGATGTGAAGGTTCTG-3′) with an NheI restriction site and ngaGT-c1 (5′-TCCCCCGGGTTTCTCATGTAAACCACCT-3′) with an SmaI restriction site, and the 3′ end of nga (fragment 2) was amplified with ngaGT-n2 (5′-TCCCCCGGGATAGGAAGTAACAATATGT-3′) with an SmaI restriction site and ngaGT-c2 (5′-GGACTAGTATGTTAGCTTTCAATTGGGT-3′) with an SpeI restriction site. Oligonucleotides ngaGT-n1, ngaGT-c1, ngaGT-n2 and ngaGT-c2 contained a restriction site for NheI, SmaI, SmaI and SpeI, respectively, (shown in bold in the primer sequence). Fragment 2 was digested with SmaI and SpeI for insertion into multi-cloning site 2 of the pFW12 plasmid [22]. The resulting plasmid was then digested with NheI and SmaI, and both the spc2 DNA fragment containing aad9 (promoterless spectinomycin resistant gene), which was obtained from a SmaI digested fragment of pSL60-2 [23], and the NheI-SmaI-digested fragment 1 were inserted.

For those subjects

who chose to add an additional protein supplement to a selected menu, the supplemental protein was included in the calculation of perceived protein needs. Measured Protein Intake Actual protein intake was determined by using 3-day food records and nutrient analysis. Subjects received 3-day food record instruction and education on accurate portion size estimation by a Registered Dietitian (RD). Subjects completed the food record by recording all foods and beverages consumed on two week days and one weekend day. For the follow up visit, subjects met with the same RD and reviewed the 3-day food records to clarify any questions/concerns on portion sizes or food items. Food records were analyzed by the study RD using Food Processor SQL Nutrition

& Fitness software (10.6.0, ESHA Research, Salem, Oregon). Statistical Analyses Single sample t-tests Maraviroc were used to compare measured CHIR-99021 research buy protein intake and perceived protein intake to recommended intakes of 0.8 g/kg/day and 2.0 g/kg/day. A paired t-test was used to compare perceived protein needs from the menu selection to actual protein intake. Data analysis was completed using PASW Statistics 18 software (SPSS Inc., Chicago, IL) and the significance level was set at p ≤ 0.05. Data are presented as means ± standard error unless otherwise noted. Results Subject Characteristics Subjects included men’s basketball (n = 14) and baseball players (n = 28) (Table 1). Mean body fat percentage was in the acceptable range for male athletes and subjects’ BMI averaged in the high end of normal, as expected with lean athletes. Strength exercise frequency (mean ± SD) was 4.0 ± 1.1 days per week, for 2.3 ± 1.4 hours per day at an average intensity of 7.3 ± 1.4, using the 1-10 Borg scale for rating of perceived exertion. Table 1 Subject Characteristics Age (yrs) 19.7 ± 1.2 Height (cm) 188.0 ± 8.2 Weight (kg) 88.0 ± 11.1 BMI (kg/m2) 24.8 ± 2.2 LBM (kg) 78.7 ± 8.7 Body Fat % 10.4 ± 3.1

Energy intake (calories) 3648 ± 1170 % Calories from Carbohydrate 46.4 ± 8.6 % Calories from Fat 33.2 ± 7.6 Body mass index (BMI), Forskolin lean body mass (LBM). Data are presented as means ± standard deviation. N = 42 Perceived Protein Needs The results of the protein survey showed that 67% of the athletes selected “”do not know”" when asked to provide the protein recommendations for athletes in terms of g/kg/d, g/lb/d, or percentage of total calories. The remaining 33% of the athletes indicated that the mean recommended protein intake for athletes was 21.5 ± 11.2 g/kg/d (p = 0.14 vs. 2.0 g/kg/d) or 27 ± 3% of total energy intake. One subject reported the mean recommended protein intake as 200 g/kg/d (i.e. 250-fold greater than the RDI). When this subject was excluded, the mean recommended protein intake reported was 8.7 ± 4.1 g/kg/d. When comparing these numbers to the RDI for protein of 0.8 g/kg/day (p = 0.05), the maximum beneficial level of 2.0 g/kg/day (p = 0.

Thus, even though several reports indicate a

correlation between in vitro growth stimulation and mycorrhiza formation [22, 37] and in vitro growth inhibition and biocontrol [38], the value of tripartite culture systems including the host plant, and a natural substrate, is clear [5, 39]. Plant disease resistance is stimulated by a single Streptomyces strain only Only a single Streptomyces strain isolated from the mycorrhizas, AcM20, stimulated plant photosynthetic yield and plant disease resistance against Alternaria black spot. Non-pathogenic rhizobacteria, including streptomycetes (reviewed in [7]), have been shown to induce resistance in plants both locally and in distal tissues [19]. However, in comparison to Streptomyces GB 4-2, the Norway spruce mycorrhizosphere isolate with positive influence on not only the plants’ disease resistance but also on its photosynthetic yield [20], the response of Arabidopsis thaliana to AcM20 was moderate. Plant growth PFT�� chemical structure promotion and enhancement of photosynthetic capacity is not a general feature among mycorrhiza-associated streptomycetes. This assumption learn more is supported by the fact that

the tested AcM strains, in general, did not affect plant growth. Even the cycloheximide producer AcM11 had only a subtle negative effect on A. thaliana, expressed as lower photosynthetic yield and increased black spot disease index. Conclusions Streptomyces community from mycorrhizal roots may Glutamate dehydrogenase impact the growth of spruce-associated micro-organisms in a strain specific manner. Differential growth-inhibition was related to the metabolite patterns of each strain, indicating that we have found a novel and a potentially interesting niche for small molecule discovery. We suggest that the combination of antifungals produced by the Streptomyces strains from Piloderma mycorrhizas provides a broad spectrum of antifungal activity that protects the mycorrhizal roots from fungal parasites, and selects against mycorrhizal fungal competitors. Methods Isolation of actinomycetes from Norway spruce mycorrhizas Ectomycorrhizas were collected from beneath 10-year-old Norway spruce (Picea abies) trees in a forest stand dominated by Scots pine (Pinus sylvestris)

in Haigerloch, south-west Germany. Mycorrhizal rootlets from the approx. 5 cm thick organic litter layer were excised, transported on ice to the laboratory, pooled, and subsequently immersed in water to remove debris surrounding the hyphal mantle. After washing 10 times with sterile destilled water, the ectomycorrhizas were sorted and white and pale yellow mycorrhizal root tips were pooled for further study. The mycorrhizal sample was used for both bacterial isolation and the analysis of fungal populations in the mantle. First half of the pooled sample of ectomycorrhizas (0.5 g) was used for DNA extraction according to Doyle and Doyle [40] and sequences of fungal internal transcribed spacer regions were obtained from the ectomycorrhizas with ITS1 and ITS4 primers [41].

All authors read and approved the final manuscript.”
“Background Gastric cancer is one of the most formidable cancers [1]. Although therapies have improved over the years, it is still difficult to treat advanced gastric cancer that has metastasized and spread to the lymph glands. Currently, radical surgery is the only treatment with a curative potential for this disease, and adjuvant chemotherapy or radiotherapy have been widely applied. Nonetheless, control of gastric

cancer at an advanced stage still remains difficult [2, 3]. Accordingly, new treatment modalities are worth investment to improve 5-year survival rates of patients. One promising approach is immunotherapy. Dendritic cells (DCs) are professional

antigen presenting cells (APC) with the unique capacity to establish a primary immune response against tumor-associated antigens (TAA) [4, 5]. This essential role of DCs www.selleckchem.com/products/dorsomorphin-2hcl.html in cellular immunity has led to development of feasible and effective DC-based vaccines against tumor Romidepsin mw antigens to eliminate cancer cells. To improve the strategy for DC-based vaccines, it is critical to acquire a large number of appropriate DCs possessing normal function. We have demonstrated that i.v. administration of chemokine ligand 3 (CCL3) or/and CCL20 rapidly recruits a group of F4/80-B220-CD11c+ cells into the peripheral blood. These cells can differentiate into mature DCs [6, 7]. We have reported previously that TAA-loaded DCs can stimulate cytotoxic T lymphocytes (CTL) significantly to lyse gastric cancer cells ex vivo [8]. Moreover, DC vaccination induced protective immunity toward the development of gastric cancer in vivo. However, these

DC vaccines have not been substantially effective in inducing tumor regression in established gastric cancer. Thus, their therapeutic effects are limited. Despite this, DC-based immunotherapy is considered promising for anti-tumor therapy. However, new strategies for improved treatment are necessary. Much research has focused upon finding feasible and effective DC-based vaccines. These include pulsing DC with tumor lysates, tumor antigen peptide, or protein; fusing tumor cells with DC; and transducing genes encoding tumor antigen, cytokines, or chemokines Protirelin into DCs [9]. Melanoma-associated antigen gene-1 (MAGE-1) was initially isolated from the MZ-2 human melanoma cell line [10], which can be recognized by CTL. We and others have previously shown that MAGE-1 is expressed at a high frequency in gastric cancer [11, 12], which suggested MAGE-1 may be a target for anti-tumor immunotherapy. In the present study, we demonstrated that F4/80-B220-CD11c+ DC precursors mobilized by CCL3 and CCL20 can induce tumor-specific CTL and elicit potent, therapeutic effects against solid and metastatic tumors when modified with MAGE-1. Together, our results suggest a promising new immunotherapeutic strategy against gastric cancer.

subtilis vegII promoter in an E. coli – S. aureus shuttle vector constructed in our laboratory. This construct, designated pGMB540, was used for trans-complementation of the nonfunctional endolysin for propagation of the recombinant phage in lytic mode and for their enumeration. Plasmid pGMB540 was introduced into S. aureus strain RN4220 by electroporation according to the protocol described by Schenk and Laddaga [30]. Transformants

were selected on LB medium containing tetracycline (5 μg/ml) and used as bacterial hosts for phage enrichment. Early log phase cells of S. aureus RN4220/pGMB540 grown at 37°C were infected with the recombinant endolysin-deficient phage P954 (MOI = 0.1) and incubated for an additional 3 to 4 hr until the culture lysed. The phage-containing lysate was passed through a 0.2-μm filter, see more and the phages were enumerated on a lawn of S. aureus RN4220/pGMB540 cells. The endolysin-deficient phage P954 was also enriched by induction. Briefly, the lysogen was grown at 37°C until absorbance at 600 nm reached 1.0 and then induced with 1 μg/ml Mitomycin C at 37°C for 4 hr. The cells were pelleted and lysed by vortexing with glass beads. Cell MK-2206 chemical structure debris was removed by centrifugation at 5000 × g for 10 min, and the phage-containing supernatant was passed through a 0.2-μm filter. Comparison of in vitro bactericidal activity of parent and lysis-deficient phage P954 The parent and

recombinant phages were compared for host range and bactericidal activity. Ten MOI equivalent of phage was added to 2 × 108 colony-forming units per ml (CFU/ml) and incubated at 37°C for 90 min. Serial 10-fold dilutions of the mixture were plated on LB agar, and residual viable cells (CFUs) were enumerated. In vivo efficacy of endolysin-deficient

phage P954 in neutropenic mice Animal experiments were performed at St. John’s Medical College and Hospital, Bangalore, India. The experiments were approved by the Institutional Animal Ethics Committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals (registration No. 90/1999/CPCSEA dated 28/4/1999). Healthy male Swiss albino mice (6-8 weeks old, neutropenic) were used to evaluate in vivo efficacy. Neutropenia was induced by intraperitoneal (IP) administration of cyclophosphamide (100 mg/kg). In SB-3CT a preliminary study, the lethality of a clinical MRSA isolate (B911) was determined in the mice (1 × 107 -1 × 108 CFU). We found that 5 × 107 CFU resulted in 80% mortality (LD80), and it was therefore chosen as the challenge dose to evaluate phage efficacy (data not shown). In the efficacy experiment, mice were assigned to six treatment groups (n = 8, each group). Four days after cyclophosphamide treatment, the mice in groups 1-3 were challenged with B911 (200 μl, 5 × 107 CFU). Groups 1 and 4 were then treated with 25 mM Tris-HCl, pH 7.

These or other mechanisms might contribute

to vascular invasion observed in selleck compound this study, which remains to be proven. In man, glypican-3 (GPC3) can be an important aid in the morphologically difficult diagnosis between small HCCs and other small focal lesions. The expression of GPC3 in a small focal lesion present in a cirrhotic liver in man is highly indicative of a HCC, irrespective of the percentage of positive cells. The presence of GPC3 (mRNA and immunohistochemistry) is higher in HCCs compared to cirrhotic tissue or small focal lesions, indicating that the transition from small premalignant lesions to HCC is associated with a sharp increase of GPC3 expression in the majority of cases [21, 28]. Because GPC3 is over expressed in human hepatocellular carcinoma, this marker is used for hepatocellular tumours in human medicine as a marker for malignant change [37–39]. In this study, all the canine tumours with a K19 expression had 30-100% positivity for glypican-3; all the other hepatocellular tumours were negative for glypican-3. Thus, like K19, expression of glypican-3 seems to be linked with a poor prognosis. Therefore, glypican-3 can be used as a marker for hepatocellular malignancy in dogs. In PD0325901 this study, no K19/GPC3 positive hepatocellular tumours express

the hepatocyte marker HepPar-1. This is consistent with a HPC phenotype of these tumours as HPCs/reactive ductules are also negative for HepPar-1. Another explanation could be that these tumours are dedifferentiated to the point where they do not express HepPar-1 anymore. All K19 expressing hepatocellular tumours which are negative for HepPar-1 are categorized in the highest (most malignant) groups of the

grading Chloroambucil and the staging system. This suggests a negative correlation between the expression of HepPar-1 and prognosis. Better characterisation of hepatic tumours by cell surface markers and the use of fluorescence activated cell sorting might in the future contribute to isolation of different tumour cell populations. This will further pave the way for cell-subset-specific gene expression profiling of potential tumour stem cells, other tumour cells and stromal cell populations. In the light of this paradigm, K19 expression in hepatic tumours might correlate with the presence of tumour stem cells deriving from hepatic progenitor cells. If the arising paradigm is verified, a further deepening of our understanding of hepatocellular carcinogenesis is expected. Cell-subset-specific gene expression profiling might indeed uncover specific signalling pathways in tumour stem cells and interactions between tumour stem cells, other tumour cells and stromal cells, which might well be masked in gene expression profiling of the tumour as a whole.

Then, each tomato plant was submerged up to the stem in a 250-ml Erlenmeyer flask filled with 100 ml of liquid Murashige and Skoog (MS) basal medium (Duchefa, Haarlem,

The Netherlands) (MS-P medium). MS is a commonly used medium for plant tissue cultures but it has been also used to analyze Trichoderma secreted proteins in hydroponic systems [8, 14]. Immediately, T. harzianum mycelia obtained as Selleckchem ABT-263 described above were also transferred to the MS-P medium under aseptic conditions. Fungal cultures in MS medium without the presence of tomato plants were used as controls. T. harzianum cultures in rich medium (MS supplemented with 2% glucose: MS-G medium) and in the presence of chitin [MS containing 1% chitin (Sigma, St. Louis, Mo, USA): MS-Ch medium] were also included in the study for comparative https://www.selleckchem.com/products/chir-99021-ct99021-hcl.html purposes. All cultures were maintained at 28°C and 90 rpm for 9 h. After this time, Trichoderma mycelia were harvested by filtration (the mycelium on the plant roots was recovered with a direct water jet, avoiding excessive manipulation). Mycelia were washed twice with sterile

distilled water, frozen in liquid nitrogen, lyophilized, and kept at -80°C until RNA extraction. Microarray design and construction A self-designed Trichoderma high-density oligonucleotide (HDO) microarray was used in this study. A collection Idoxuridine of 14,237 transcript sequences obtained for the “”TrichoEST project”" from ESTs (11,376 singlets and 2,861 contigs provided in additional files 6 and 7, respectively) of twelve strains of eight different Trichoderma spp. [CECT: T. harzianum T34 (CECT 2413); NewBiotechnic S.A. (NBT, Seville, Spain): T. longibrachiatum T52 (NBT52); T. virens T59 (NBT59), T. viride T78 (NBT78); American type Culture Collection (ATCC, Rockville, USA): T. atroviride

TP1 (ATCC 74058), T. harzianum T22 (ATCC 20847); Centraalbureau voor Schimmelcultures (CBS, Baarn, The Netherlands): T. stromaticum TST (CBS 100875); International Mycological Institute (IMI, Egham, UK): T. atroviride T11 (IMI 352941); T. asperellum T53 (IMI 20268); BioCentrum-DTU Culture Collection of Fungi (IBT, Lyngby, Denmark): T. harzianum T3K (IBT 9385); T. aggressivum TH2 (IBT 9394); University Federico II of Naples (UNINA, Portici, Italy): T. harzianum TA6 (UNINA 96)], plus 9,129 transcript sequences predicted from the T. reesei QM 6a genome [38] were used as source sequences to generate probes for the Trichoderma HDO microarray. First, unique sequences were obtained from the whole TrichoEST database by combining ESTs from all twelve Trichoderma strains indicated above in order to minimize redundancy due to transcripts common to different strains.

Cancer Res 2000,60(2):309–20.PubMed Competing interests The authors

declare that they have no competing interests. Authors’ contributions QXP and AWW designed the study, carried out most of the experiments and analyzed the data. JH performed all invasion assays. QXP drafted the original manuscript. AWW and RES equally participated in the critical review and drafting of the final manuscript. KP and ES acquired their authorship for assistance in reviewing the final draft. NPN supervised the project. All authors have read and approved Talazoparib in vivo the final manuscript.”
“Background Glioblastoma is the most common type of malignant brain tumor and its prognosis is very poor. Surgical resection and chemotherapy are common treatments [1]. Despite recent advances

in the understanding of the molecular mechanism of tumorigenesis, the outcome of malignant glioma remains poor [2]. Thus, it is imperative that new effective forms of therapy are developed for its treatment. Statins are cholesterol-lowering agents that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA into mevalonate. Mevalonate is converted into farnesyl pyrophosphate (FPP) or geranylgeranyl Tamoxifen supplier pyrophosphate (GGPP) that can be anchored onto intracellular proteins through prenylation, thereby ensuring the relocalization of the target proteins in the cell membranes [3–5]. Inhibition of HMG-CoA reductase results in alteration of the prenylation of small G proteins such as Ras, which regulates cell growth and survival via the downstream signaling pathways [3–5]. Accordingly, inhibition

of HMG-CoA reductase by statins was found to trigger apoptosis in several cancer cells [3–5]. We recently showed that Axenfeld syndrome statins decreased the activation of the Ras/extracellular regulated kinase 1/2 (ERK1/2) pathway and Ras/phosphoinositol-3 kinase/Akt pathway [3, 4]. In malignant glioma cells, statins induce apoptosis by the activation of c-Jun N-terminal kinase 1/2 (JNK1/2) or by increasing the expression of Bim [6, 7]. However, several aspects of the mechanism by which statins induce apoptosis in glioma cells remain unclear. In the present study, we investigated the mechanism by which statins induce apoptosis in rat C6 glioma cells. Materials and methods Materials Mevastatin was purchased from Sigma (St. Louis, MO, USA), fluvastatin from Calbiochem (San Diego, CA, USA), and simvastatin from Wako (Osaka, Japan). These reagents were dissolved in dimethyl sulfoxide (DMSO) and filtered through syringe filters (0.45 μm; Iwaki Glass, Tokyo, Japan). The dissolved reagents were resuspended in phosphate-buffered saline (PBS, pH 7.4) and used in the various assays described below. Mevalonic acid lactone (MVA), FPP, GGPP, squalene, ubiquinone, isopentenyladenine, and dolichol were purchased from Sigma. These reagents were dissolved in DMSO. These dissolved reagents were then resuspended in PBS (0.05 M; pH 7.4) and filtered through syringe filters (0.

This necessitated reevaluation of the position of the chosen seed points and

repositioning Smad inhibitor into aerated parts of the lungs. This way tumour burden and growth was assessed quantitatively using the decrease in aerated lung volume as a surrogate. The initial increase in lung volume in the first 4 months was attributed to normal growth. In the comparatively small group examined here, tumour growth seemed to occur at a later point of time in male animals as compared to female animals. Female animals showed clinical signs of tumour necessitating sacrifice earlier compared to male animals. Statistical analysis Repeated measurement analysis of the time points 2, 4, 6, 7-13 months showed significant changes of the segmented lung volumes over time (p = 0.009). Interaction of the measurements was rejected (p = 0.035). Testing for group differences did not show significant results, due to the small number of animals and the spread of lung volume at early time points in normal animals. Analysis of time points 8 to 13 months, when tumour progression occurs, showed significant group differences (p = 0.043). Linear regression analysis yielded equation 1 to calculate lung volume. The correlation coefficient was determined as R = 0.538. Alectinib (1) Discussion In this study we examined the tumour growth kinetics of SPC-raf transgenic mice by serial micro-CT examinations.

Small animal imaging allows assessment using each animal as its own control in follow-up examinations. Given the relevant inter-individual spread it has the potential to optimize studies. To prevent intra-individual spread sophisticated imaging and post-processing techniques have to be established as elaborated below. An advantage of imaging especially in diffuse or multifocal pathologies is that the entire volume can be assessed additional to circumscribed areas of sectional histopathology obtained. Very few studies on follow-up micro-CT examination have been performed in transgenic murine models of lung cancer (mainly K-ras transgenic) [12–14]. Other groups performed follow-up examination in single lesions caused by intrapulmonary injection of tumour cells

or several/multiple lesions initiated by intraperitoneal injection of urethane [15–17]. To the best of our knowledge, no report on micro-CT assessment of tumour N-acetylglucosamine-1-phosphate transferase growth kinetics in the SPC-raf transgenic lung tumour mouse model has been published so far. Furthermore, the follow-up exams reported did usually include only a limited number of imaging time points as compared to up to 15 time points in this study, allowing a more detailed assessment of growth kinetics. Further studies have shown the use of micro-CT for the detection of primary lung tumours or pulmonary metastases without a follow-up being performed [7, 18]. All the various imaging approaches of murine animal models of human lung tumour have different advantages and disadvantages.

Taken together, these data demonstrate that ICESt1 and ICESt3 do not share the same transcriptional organization of their regulation module: ICESt1 is organized as two operons, while in ICESt3 the whole module can be co-transcribed. Furthermore, ICESt3 possesses an additional distal promoter upstream the module, which is activated during stationary phase. Growth phase and MMC exposure modulate the transcription of the ICESt1 and ICESt3 core genes Previous analyses showed a derepression of conjugative transfer of ICESt3 but not of ICESt1 after exposure

to mitomycin C (MMC) [10]. In order to explain this difference, we quantified by real-time RT-PCR, Fulvestrant three regions (orfM/orfL junction, orfD/orfC junction and integrase gene) of the conjugation-recombination selleck chemicals transcript of ICESt1 and ICESt3. Quantification was done from cells harvested in exponential growth phase treated or not with MMC at the half of the minimal inhibitory concentration (MIC/2) as well as in stationary phase (Figure 3). Of note, in preliminary experiments, MMC exposure did not affect the transcriptional organization (in particular no activity of ICESt3 Parp2s), cell morphology or chain length but, as expected for a DNA damaging agent, it delayed growth, reduced DNA quantity and increased recA transcript levels (data not shown). Transcription of the ICESt1 conjugation-recombination modules was found up-regulated upon

DNA damage (16-fold for the int gene) and in stationary phase (13-fold for the int gene) compared to exponential growth phase without MMC treatment Methamphetamine (Figure 3A). The same observation was made for ICESt3 with a 84-fold and 11-fold increase of int transcript levels after MMC treatment and stationary phase, respectively (Figure 3B), indicating a probable transcriptional regulation of ICE excision. Whatever the considered region of the conjugation-recombination transcript, higher amounts were found for ICESt3 than for ICESt1 (for example, 16 to 100-fold difference in int gene transcript level depending on the

tested condition). Figure 3 Quantification of the transcripts of the core regions of ICE St1 (A) and ICE St3 (B). Arrows correspond to transcripts. Primer pairs used for cDNA quantification are represented by convergent triangles below the corresponding transcript. Other symbols used in the map are identical to those used in Figure 1. cDNA quantities determined from cells grown in LM17 medium and harvested in exponential growth phase (expo0.6) or stationary phase (stat) or after 2.5 hours of exponential growth with mitomycin C (MMC) at MIC/2 are normalized to the quantity of cDNA of gyrA whose transcription is constitutive [39]. Lack of amplicon is mentioned as non-detected (ND). For each condition, data are average and standard deviation from three independent biological replicates. For both elements, quantitative RT-PCR was also performed on three loci of the regulation module (Figure 3).