spinosa trans1

compared with 100 (± 77) mg L−1 in the pa

spinosa trans1

compared with 100 (± 7.7) mg L−1 in the parental strain. Quantitative real time polymerase chain reaction analysis of three selected genes (spnH, spnI, and spnK) confirmed the positive effect of the overexpression of these genes on the spinosyn production. This study provides a simple avenue for enhancing spinosyn Copanlisib production. The strategies could also be used to improve the yield of other secondary metabolites. Saccharopolyspora spinosa was originally isolated in 1982 from a soil sample collected in a Caribbean island (Mertz & Yao, 1990). Fermentation broth extracts from this strain contain a series of spinosyn factors that are highly efficient against a broad range of pests, and appear to INCB018424 manufacturer have little or no effect on non-target insects and mammals (Sparks et al., 1998). Previous studies showed that spinosyns are derived from nine acetate and two propionate units, which produce a cyclized polyketide molecule; three carbon–carbon bonds are soon formed to obtain the tetracyclic aglycone (AGL). The rhamnose is subsequently attached and is tri-O-methylated to yield the intermediate pseudoaglycone (PSA), followed by the incorporation of forosamine sugar, giving the final spinosyns product. The most active and abundant spinosyns from S. spinosa fermentation broth are spinosyn A and spinosyn D. They differ from each

other by a single methyl substituent at position 6 of the polyketide. Other factors of the spinosyn family, produced as minor components, exhibit different methylation patterns and are significantly less active (Crouse et al., 2001). A naturally occurring mixture of spinosyn A (c. 85% of spinosad) and spinosyn D (c. 15%

of spinosad) is called spinosad (Waldron et al., 2001). The c. 74-kb spinosyn biosynthetic Thalidomide gene cluster contains 23 open reading frames (ORF) including five genes encoding a type I polyketide synthase (PKS) (spnA, B, C, D, and E); four genes involved in intramolecular C–C bond formation (spnF, J, L, and M); four genes responsible for rhamnose attachment and methylation (spnG, I, K, and H); six genes participating in forosamine biosynthesis (spnP, O, N, Q, R, and S) and four genes (ORF-L15, ORF-L16, ORF-R1, and ORF-R2) with no proven role in spinosyn biosynthesis (Waldron et al., 2001). The genes involved in rhamnose biosynthesis (gtt, gdh, epi, and kre) are not linked to this cluster (Madduri et al., 2001b). Traditionally, improvement of secondary metabolite-producing strains is achieved by random mutagenesis and selection techniques (Parekh et al., 2000). Although these techniques have succeeded in generating many industrial strains, they are time-consuming and costly. Rational strain improvement strategies overlap with classical approaches in generating a mutant population (Adrio & Demain, 2006).

, 2009) Dilutions of the stock solution were then used in the as

, 2009). Dilutions of the stock solution were then used in the assays, to yield a final concentration of 192 μg mL−1, 384 μg mL−1 of extract in samples. The concentration of methanol in a sample was never >0.8% and it had no visible effect on the cell lines. The extract was assayed in a susceptibility test according to the M27-A2 method of NCCLS (National Committee Etoposide mw for Clinical Laboratory Standards, 2002). None of the tested concentrations influenced C. albicans growth. Caco-2 and Intestin 407 cells were obtained from the Polish Academy of Science Culture Collection (Wrocław). Both lines were cultivated at 37 °C in 5% CO2 in MEM+GlutaMAX™-I containing Earle’s

and 25 mM HEPES (Gibco) and 1% antibiotic/antimycotic solution (Gibco) supplemented with heat-inactivated fetal bovine serum (FBS, Gibco) at a final concentration of 20% for Caco-2 and 10% for Intestin 407. To obtain a fully confluent and enterocyte-like morphology in the case of the Caco-2 monolayer, Caco-2 and Intestin 407 cells were grown for 21 and 2–3 days, respectively. GSK2126458 research buy For the adhesion experiment, Caco-2 and Intestin 407 cells were seeded onto 96-well tissue microplates (Nunc) at a density of 2.0–2.5 × 104 cells per well at a final volume of 100 μL. Cells were grown

to ∼85–95% confluence with media refreshment every 2nd day. Subsequent, cells were washed to rinse out antibiotics and resuspended in the same medium supplemented with 2% FBS without antibiotics. Subsequent experiments were set up with the addition of: (1) C. albicans, (2) C. albicans with various concentrations of S. boulardii extract, and (3) C. albicans and S. boulardii at ratios of 1 : 1 (corresponding to 2 × 106 CFU mL−1 for both strains) and 1 : 10 (corresponding to 2 × 106 and 2 × 107 CFU mL−1 for C. albicans and S. boulardii, respectively). Control with S. boulardii extract contained 0.8%

methanol. The adhesion test was carried out for 3 h at 37 °C. After incubation, the wells were gently washed with PBS and cells were fixed with 4%p-formaldehyde. For a check details quantitative assessment of binding, cells were stained with 0.5% crystal violet (Freshney, 2002; Noverr & Huffnagle, 2004). The OD595 nm was read in a spectrophotometer (Asys UVM 340, Biogenet). Results are expressed as the percent of C. albicans adhesion inhibition with respect to 100% adhesion of C. albicans to the relevant cell line in the control well. Experiments were repeated eight times, with six repetitions in each. For microscopic analysis, plates stained with 0.5% crystal violet were observed under the inverted microscope CKX41 (Olympus) using × 40 objective and photographed using an ARTCAM-300MI camera. For assessment of the cytokine mRNA response of Caco-2 line, cells were typically cultured in 6 mL standard medium supplemented with 10 mM butyric acid in 40-mL flasks (Nunc), as described previously (Saegusa et al., 2004).

Results  Of the 157 children in the baseline sample, 144 (917%)

Results.  Of the 157 children in the baseline sample, 144 (91.7%) were followed up.

The overall P-CPQ score showed a large decrease following treatment, along with an increase in the number scoring 0 (no impact). Similar relative HKI-272 concentration changes were observed in the oral symptoms and emotional well-being subscales, whereas the other two subscales showed moderate decreases. All post-treatment FIS scores were lower than pre-treatment ones; all showed moderate effect sizes. The greatest relative changes were seen in the parental/family activity and parental emotions subscales. Conclusions.  The dental treatment of young children under GA is associated with considerable improvement in their OHRQoL. The P-CPQ and the FIS are valid and responsive to treatment-associated changes in young children with early childhood caries (ECC). “
“International Journal of Paediatric Dentistry 2010; 20: 242–253 Aim.  This study aimed to investigate the role of dental fear (DF) and other personal characteristics in relation to dental behaviour management problems (DBMP). Design.  A study group of 230 patients

(7.5–19 years old; 118 girls), referred because of DBMP, was Forskolin compared to a reference group of 248 same-aged patients (142 girls) in ordinary dental care. Patients and their parents independently filled in questionnaires including measures of fear and anxiety, behavioural symptoms, temperamental reactivity, and emotion regulation. Results.  Study group patients referred because of DBMP differed from the reference group in all investigated aspects of personal characteristics. In the multivariate analyses, DF was the only variable with consistent discriminatory capacity through all age and gender subgroups. Aspects of anxiety, temperament, and behavioural symptoms contributed, but differently for different subgroups and at different levels of dental fear. Conclusions.  Among older children and adolescents, DF deserves to be re-established as the single most important discriminating variable for DBMP at clearly lower scores than commonly used. Further research should focus on the different patterns of DBMP development, considering Florfenicol various personal characteristics that may trigger, maintain,

or exacerbate young patients’ vulnerability to DF and DBMP. “
“Singapore is unique in that it is a 100% urban community with majority of the population living in a homogeneous physical environment. She, however, has diverse ethnicities and cultures as such; there may be caries risk factors that are unique to this population. The aims were to assess the oral health of preschool children and to identify the associated caries risk factors. An oral examination and a questionnaire were completed for each consenting child–parent pair. One hundred and ninety children (mean age: 36.3 ± 6.9 months) were recruited from six community medical clinics. Ninety-two children (48.4%) were caries active. The mean d123t and d123s scores were 2.2 ± 3.3 and 3.0 ± 5.6, respectively.

Reduced treatment intensity in patients with early-stage Hodgkin’

Reduced treatment intensity in patients with early-stage Hodgkin’s lymphoma. N Engl J Med 2010; 363: 640–652. 35 Radford J, Barrington S, Counsell N et al. Involved field radiotherapy versus no further treatment in patients with clinical stages IA and IIA Hodgkin lymphoma and a ‘negative’ PET scan after 3 cycles ABVD. Results of the UK NCRI RAPID Trial. 54th

ASH Annual Meeting and Exposition. Atlanta, GA, December 2012 [Abstract 547]. 36 Hentrich M, Berger M, Wyen C et al. Stage-adapted treatment of HIV-associated Hodgkin lymphoma: results of a prospective multicenter study. J Clin Oncol 2012; 30: 4117–4123. buy IWR-1 37 Eich HT, Diehl V, Gorgen H et al. Intensified chemotherapy and dose-reduced involved-field radiotherapy in patients with early unfavorable Hodgkin’s lymphoma: final analysis of the German Hodgkin Study Group HD11 trial. J Clin Oncol 2010; 28: 4199–4206. 38 Hoskin PJ, Lowry L, Horwich A et al. Randomized comparison of the Stanford RG7204 concentration V regimen and ABVD in the treatment of advanced Hodgkin’s lymphoma: United Kingdom National Cancer Research Institute Lymphoma Group Study ISRCTN 64141244. J Clin Oncol 2009; 27: 5390–5396. 39 Viviani S, Zinzani PL, Rambaldi A et al. ABVD versus BEACOPP for Hodgkin’s lymphoma when high-dose salvage is planned. N Engl J Med 2011; 365: 203–212. 40 Carde PP, Karrasch M, Fortpied C et al. ABVD (8 cycles) versus BEACOPP (4 escalated cycles => 4 baseline) in stage III-IV high-risk Hodgkin lymphoma (HL): First results of

EORTC 20012 ifenprodil Intergroup randomized phase III clinical trial. ASCO Annual Meeting. Chicago, IL, June 2012 [Abstract 8002]. 41 Bauer K, Skoetz N, Monsef I et al. Comparison of chemotherapy including escalated BEACOPP versus chemotherapy including ABVD for patients with early unfavourable or advanced stage Hodgkin lymphoma. Cochrane Database Syst Rev 2011; 8: CD007941. 42 Xicoy B, Ribera J-M, Miralles P et al. Results of treatment

with doxorubicin, bleomycin, vinblastine and dacarbazine and highly active antiretroviral therapy in advanced stage, human immunodeficiency virus-related Hodgkin’s lymphoma. Haematologica 2007; 92: 191–198. 43 Spina M, Gabarre J, Rossi G et al. Stanford V regimen and concomitant HAART in 59 patients with Hodgkin disease and HIV infection. Blood 2002; 100: 1984–1988. 44 Hartmann P, Rehwald U, Salzberger B et al. BEACOPP therapeutic regimen for patients with Hodgkin’s disease and HIV infection. Ann Oncol 2003; 14: 1562–1569. 45 Shah BK, Subramaniam S, Peace D, Garcia C. HIV-associated primary bone marrow Hodgkin’s lymphoma: a distinct entity? J Clin Oncol 2010; 28: e459–460. 46 Tsimberidou AM, Sarris AH, Medeiros LJ et al. Hodgkin’s disease in patients infected with human immunodeficiency virus: frequency, presentation and clinical outcome. Leuk Lymphoma 2001; 41: 535–544. 47 Hessol NA, Pipkin S, Schwarcz S et al. The impact of highly active antiretroviral therapy on non-AIDS-defining cancers among adults with AIDS. Am J Epidemiol 2007; 165: 1143–1153.

Sixteen percent took acetazolamide preventively (median dose 250

Sixteen percent took acetazolamide preventively (median dose 250 mg/d or 3 mg/kg/d, range 1–7 mg/kg/d) for a median of 4 days (range

1–21 d). Those who took acetazolamide preventively spent the same number of nights between 1,500 and 2,500 m, but their mean-maximum overnight altitude was slightly higher than of those who did not take it preventively: 4,178 m versus 3,917 m (p = 0.000). Five hundred and thirty-one (74%) travelers MAPK inhibitor had physical complaints on the first days at or above 2,500 m: headache (47%), shortness of breath (44%), fatigue (23%), nausea/vomiting (14%), sleeping disorders (14%), and dizziness (4%). Other complaints included diarrhea, epistaxis, palpitations, and edema of the fingers. One person was “talking GSK458 molecular weight nonsense” for 20 minutes without any other complaint. Seven individuals had tingling sensations, six of whom took acetazolamide as prevention or treatment. One hundred and eighty-four responders (25%) had complaints that met the definition of AMS. Most (76%) of these complaints disappeared within 3 days. Some travelers with AMS adapted their travel schedule, while about half of them climbed higher despite symptoms (Table

2). Of the latter a quarter experienced worsening of symptoms. Of those who did not climb higher with symptoms, 64% were free from symptoms within 2 days, compared with 48% for those who continued to climb, but the difference was not significant (p = 0.655). The majority took medication, mostly analgesics, followed by acetazolamide and coca-leaves or -tablets. Thirty-four percent took acetazolamide for treatment at a median dose of 375 mg/d or 5 mg/kg/d (range 1–11 mg/kg/d) and a median duration of 3 days (range 1–15 d). Several travelers remarked that they did not know when to start taking acetazolamide exactly and that traveling companions had received different advice on its use. Four travelers received oxygen; all reported dyspnoea but only one met the AMS criteria. Those who did not take any medication often argued Tacrolimus (FK506) that their symptoms

were not severe enough to start acetazolamide. Those who took coca preparations often remarked that the guides had recommended coca or “soroche-pills” over acetazolamide. The majority climbed higher after the AMS symptoms disappeared; 26% of them reported that the AMS symptoms recurred. In univariate analysis, previous AMS (p = 0.014), gender (p = 0.030), age (p = 0.037), maximum overnight altitude (p = 0.015), average altitude increase in meter per day above 2,500 (p = 0.046), and number of nights between 1,500 and 2,500 m at the beginning of the journey (p = 0.000) were associated with the development of AMS (Table 3). There was no association between AMS and destination (p = 0.

Six months post-cART, of the 3745 HIV-1 RNA measurements between

Six months post-cART, of the 3745 HIV-1 RNA measurements between 1001 and 10 000 copies/mL and 7150 HIV-1 RNA measurements >10 000 copies/mL, Selleck Alectinib 31% and 55%, respectively, coincided with a treatment interruption. Figure 2 shows CD4 cell count trajectories predicted by

the best-fitting model, separately in participants without (Fig. 2a) and with (Fig. 2b) virological failure 6 months after starting cART. In participants without virological failure, CD4 cell counts continued to increase up to 8 years after the start of cART, in all baseline CD4 cell count groups, with little evidence that between-group differences in CD4 cell count reduced over time. In contrast, among participants with at AZD6244 order least one episode of virological failure 6 months after starting cART, predicted CD4 counts either declined (baseline CD4 count ≥200 cells/μL) or increased at a slower rate (baseline CD4 count <200 cells/μL). Table 2 shows estimated geometric mean CD4 cell counts at 4 and 8 years after initiation of cART, according to baseline CD4 cell count and whether participants experienced virological failure from 6 months post-cART. At 8 years, geometric mean CD4 counts among

participants who did not experience virological failure varied between 415 cells/μL [95% confidence interval (CI) 386, 443 cells/μL] and 897 cells/μL (95% CI 812, 981 cells/μL) in participants with baseline CD4 counts of 0–24 and ≥500 cells/μL, respectively. Geometric mean CD4 cell counts in participants who experienced virological failure were approximately half those in participants who did not. Among participants who did not experience virological failure, there was clear evidence of continuing rises during this period. In contrast, for participants who experienced virological failure, and whose baseline CD4 count was >200 cells/μL, CD4 counts declined between 4 and

8 years. Table 3 shows estimated effects of virological failure, treatment interruption and patient characteristics on geometric mean CD4 cell counts. In model 1, which estimated effects of mafosfamide virological failure before adjusting for treatment interruptions, virological failure of >10 000 copies/mL was associated with lower subsequent CD4 cell counts, with the greatest adverse effects occurring within the first 44 days. For all time periods since the occurrence of a virological failure, viral loads >10 000 copies/mL had a greater adverse effect on subsequent CD4 cell counts than viral loads >1000 to ≤10 000 copies/mL. The size of these adverse effects decreased as time since virological failure increased. Unadjusted geometric mean ratios were almost identical to those adjusted for age, sex, ethnicity and risk group (data not shown). The crude geometric mean CD4 count ratios for the effect of cumulative years with viral load >1000 to ≤10 000 and >10 000 copies/mL were 0.83 (95% CI 0.81, 0.84) and 0.79 (95% CI 0.78, 0.

The new construct, pER164 (Fig 1), was conjugated into B fragil

The new construct, pER164 (Fig. 1), was conjugated into B. fragilis 638R and IB263 strains by triparental mating to construct BER-96 and BER-105, respectively. For the microscopy slides, 1 mL of bacterial cultures

grown to mid-log phase in BHIS under conditions described above were centrifuged at 3000 g for 3 min and washed once in 1 mL of phosphate buffer saline (PBS) ATR inhibitor (4.3 mM dibasic sodium phosphate, 1.47 mM monobasic potassium phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4). Bacteria were suspended in 1 mL PBS and a drop of this suspension was added to each slide and allowed to air-dry. The coverslips were mounted with glycerol and the slides were analyzed with a Confocal Microscope Zeiss LSM 510 using an excitation of 450 nm Venetoclax nmr and an emission filter in the range of 475–525 nm. For dual channel fluorescent color detection in slides stained with Alexafluor-546-phalloidin

conjugate (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction, an excitation at 556 nm and emission at 573 nm were also used. The J774.1 macrophage cell lines was grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum and 2 mM l-glutamine. The cells were grown over sterile coverslips placed inside six-well microplates at 37 °C in a 5% CO2 humidified atmosphere. For all assays, six-well plates were seeded with approximately 2 × 105 macrophages mL−1 and incubated until confluence was reached. The bacterial cell numbers were determined spectrophotometrically at 600 nm. The assay was carried out by inoculating B. fragilis at an approximate multiplicity of infection of 100 into the six-well plates under anaerobic conditions. Infected monolayers were incubated for 1 h http://www.selleck.co.jp/products/AG-014699.html inside the anaerobic incubator to allow phagocytosis and internalization to occur. Then, the monolayers were

washed three times with PBS without an antibiotic to remove unbound bacteria. The cells were then fixed with 3.7% formaldehyde for 10 min and washed three times with PBS. Macrophages were stained with Alexafluor-546-phalloidin conjugate. The coverslips were removed from the wells and placed on top of glass slides for laser confocal microscopy analysis as described previously. In this study, we show the use of the fluorescent protein BS2 as a reporter for in vitro and in vivo gene expression studies in the anaerobe B. fragilis. When the promoterless bs2 gene was cloned in fusion with the starch/maltose and oxygen inducible promoter osu (Spence et al., 2006), addition of maltose was able to induce expression of fluorescent BS2 under anaerobic conditions compared with uninduced culture controls. These results clearly demonstrate that expression of BS2 in cultures of B. fragilis BER-85 in the absence of oxygen yield an intense fluorescence characteristic of BS2.

All reactions were conducted in 50 μL volume containing PCR buffe

All reactions were conducted in 50 μL volume containing PCR buffer with 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 μM each of primers pA (AGAGTTTGATCCTGGCTCAG) and pH (AAGGAGGTGATCCAGCCGCA) according to Edwards et al. (1989), 0.6 μL of dimethyl sulfoxide and 1.25 U of Taq polymerase (Qiagen TAQ PCR Core Kit). PCR was performed using a Mastercycler ep S gradient thermocycler (Eppendorf, Canada) Selleckchem Cabozantinib with the following conditions: 5 min at 94 °C, followed by 29 cycles of 30 s at 94 °C, 30 s at 58 °C and 1 min at 72 °C, and finally one cycle of 7 min at 72 °C. PCR amplicons were

sequenced at the Genome Quebec Innovation Center (Montreal, Canada). Escherichia coli cells and sterile water were, respectively, used as positive and negative controls. Sequences were identified by blast nucleotide searches in the NCBI website, and the seven different sequences obtained were deposited in the EMBL database under accession numbers FN668006–FN668012. The phylogenetic tree was inferred using the maximum likelihood method based on the Tamura–Nei model in mega software with 1000 bootstrap replicates (Tamura et al., 2007). Three washed G. irregulare spores

isolated from soil were individually directly placed in a PCR tube, and the 16S rRNA gene was amplified using a nested-PCR protocol. A first round using pA and pH primers (Edwards et al., 1989) and a second round using primers 968-GC/1378 (Heuer et al., 1997) amplified approximately a 500-bp fragment corresponding to the hypervariable regions V6–V9. Bacterial biodiversity was assessed by running the amplicons through denaturing gradient gel electrophoresis (DGGE), as described RNA Synthesis inhibitor in Yergeau et al. (2007) with a 45–65% denaturant gradient using a DCode Universal Mutation Detection System (BioRad). Based on the approach described in St-Arnaud et al. (1995), two-compartment Petri dishes were prepared as described above, except that after solidification of the gel, sterile microscope coverslips were placed along

the central wall, and a further 3 mL of gellified sterile Fossariinae water was poured over the edge of coverslips to form a bridge helping the fungus to cross (Fig. 1). Plates received transformed carrot roots inoculated with G. irregulare in the compartment filled with M medium and the roots were regularly trimmed to avoid any crossing into the second compartment, where only the hyphae were allowed to grow. When hyphae grew over the coverslip, they were inoculated with various bacterial isolates from cultures grown in a liquid tryptic soy broth medium for 24 h. All bacterial cells were rinsed and the concentrations were adjusted to 106 CFU mL−1 with a sterile 0.9% NaCl solution before use. A 150-μL aliquot of each bacterial suspension was deposited directly on the top of the coverslip where hyphae were growing. Each bacterial isolate was replicated five times and the controls included E.

No institution

except the Zurich centre offered structure

No institution

except the Zurich centre offered structured programmes during the study period. Nearly all institutions reported providing – in addition to ‘standard care’ – ‘frequent short counselling’, half of the institutions reported offering ‘detailed counselling’ if indicated, and around half reported handing out information booklets. Also, institutions reported using nicotine substitution, or prescribing bupropion or varenicline in some patients. All institutions reported referring patients to specialized addiction treatment institutions if the patient so wished. During the intervention at the Zurich centre from November 2007 to December 2009, 1689 participants had 6068 cohort visits, and 46% smoked at their last visit (Table 1). Smoking status checklists were not available for 739 of 6068 visits (12%) and incomplete find protocol for 208 (3.4%), so that 5121 (84%) completed checklists were available. Visits with missing checklists were more likely to arise for nonsmoking participants (56%) than for currently smoking participants (44%). There was variation in the number of missing checklists between physicians (data not shown). Current smoking was

declared in 44.5% of the completed checklists. Among the 2374 checklists for those currently smoking, motivation was assessed as: 85 (3.6%) intended to stop immediately; 262 (11%) intended to stop within 6 months; 804 (33.9%) would stop later; 784 (33%) did not intend to stop; and 439 (18.5%) did not answer. Smoking cessation counselling was carried out in 1888 of 2374 visits (80%) for current smokers. Reasons for not counselling were: other priorities (50%), patient refusal (19%), lack of time (12%) selleck chemical and other reasons (18%). Among counselled participants, the following types of Baf-A1 chemical structure additional support were given (multiple types per patient possible): distribution of handout (8.1%), detailed counselling (6.5%), varenicline prescription (3.8%), nicotine substitution

(2.5%), follow-up date arranged (2.4%), agreed upon stop date (1.5%), bupropion prescription (0.9%), and referral to specialized institution (0.2%). Changes in motivation were very common (Table 2), with the exception of persons who did not smoke, of whom 95% remained nonsmokers. In smokers, the probability of a change in motivation level between two visits was more than 50% (diagonal elements in Table 2). The probability of changing from smoking to not smoking between two visits strongly depended on the motivation level, with 14% among persons ready for an immediate stop and 13% among those intending to stop within the next 6 months, but only 5.3% for persons who indicated to stop later, and 5.1% for those who were not motivated at all. When compared with ‘no motivation’, the odds ratios (95% confidence intervals) for not smoking at the next visit were 1.9 (0.85–4.2) for ‘immediate stop’, 2.1 (1.2–3.8) for ‘stop within 6 months’, and 1.0 (0.61–1.7) for ‘stop later’.


This HDAC inhibitor is consistent with real-time RT-PCR results, where the transcription level of katA in the ahpC mutant was 29.6 ± 0.6 times greater than that of the wild-type strain. The investigation was extended to examine whether alkyl hydroperoide reductase could functionally replace catalase activity in protecting X. campestris pv. campestris from the lethal heat treatment. The pAhpC expression plasmid

containing ahpC (Patikarnmonthon et al., 2010) was transferred into a double katA-katG mutant and transformants were tested for their ability to survive the lethal heat treatment. The results showed that the high-level expression of ahpC could not restore the reduction in the survival rate after the heat treatment of the double mutant (Fig. 1). Similar to catalases, alkyl hydroperoxide reductase could metabolize H2O2, albeit at a different rate and Km. The inability to protect the double mutant from lethal heat treatment suggested that either the treatment generated H2O2 at a nonoptimal level for AhpC to work or the enzyme was heat sensitive and

itself see more was heat inactivated. Catalases catalyze the conversion of H2O2 to water and oxygen. The results in the current study suggest that in X. campestris pv. campestris, the lethality of heat treatment in part could be due to the accumulation and subsequent toxicity of H2O2. Several lines of evidence point to the enhanced production and accumulation of ROS, including a superoxide anion, and peroxides resulting from heat treatment are one of the factors contributing to cell death in both eukaryotic and

prokaryotic cells (Martin & Chaven, 1987; Benov & Fridovich, 1995; Noventa-Jordao et al., 1999; Abrashev et al., 2008). The efficient degradation of H2O2 not only ameliorates Selleckchem RG7420 its toxicity but also prevents the formation of hydroxyl radicals that are the most reactive radicals. The reduced heat survival observed in the X. campestris pv. campestris kat mutants likely arises from the reduced bacterial ability to cope with H2O2 generated from heat shock. While the precise mechanism is unclear, phenotypic data indicate a critical role of catalases in heat shock protection for this bacterium. Experiments were extended to test the effects of ROS scavengers on the protection of X. campestris pv. campestris from heat treatment. The addition of 10 mM pyruvate, a H2O2 scavenger, or 1 M glycerol, a hydroxyl radical scavenger (Patikarnmonthon et al., 2010), before heat treatment resulted in a subsequent 10-fold increase in the survival of the katA katG double mutant compared with the untreated conditions. This protective effect was also observed in the wild-type strain as it showed five- and 10-fold increased survival in cells pretreated with pyruvate and glycerol, respectively (data not shown). These observations support the idea that the killing effects of heat shock involve the generation of ROS.