Agglutination was examined by dark-field microscopy and titres were measured as the last dilution where at least 50% of the leptospires were agglutinated (Cole et al., 1973). MAT results were read blind by two expert operators in two different centres; the reactions with LaiWT and LaiMut were tested at the same time. Twenty millilitres of 14-day cultures of LaiWT Crizotinib or LaiMut were centrifuged (3500 g at 4 °C) for

6 min. The pellet was washed twice with sterile distilled water and then resuspended in 700 μL of distilled water, to which 350 μL of 3 × treatment buffer [0.125 M Tris-HCl (pH 6.8); SDS (sodium dodecyl sulphate, 4%); glycerol (20%); 2, β-mercaptoethanol (15%); bromophenol blue (0.1%)] was added. The mixture was heated to 100 °C for 5 min, 1 μg proteinase K was added, and incubated overnight at 60 °C (Hitchcock & Brown, 1983), and the solution was stored frozen until analysis. A discontinuous SDS-PAGE (gradient 6–15%) was used to analyse lipopolysaccharide molecules from LaiWT and LaiMut (Laemmli, 1970). For the

visualization of lipopolysaccharide, polyacrylamide gels were stained using the procedure described by Tsai & Frasch (1982) as modified by Hitchcock & Brown (1983). Following electrophoresis, lipopolysaccharide was transferred to Immobilon-P membranes (Millipore, St. Louis, MO) (Towbin et al., 1979) and probed with mAb F70C7 at a 1 : 100 dilution as the primary antibody and selleck chemicals llc alkaline phosphatase-labelled goat anti-mouse immunoglobulin G (Kirkegaard and Perry, MD) at a 1 : 5000 dilution as the secondary antibody. Reactions were visualized colourimetrically with a solution containing 90 μL of NBT (75 mg mL−1 of nitroblue tetrazolium in 70% dimethylformamide), 70 μL of BCIP Glycogen branching enzyme (50 mg mL−1 of 5-bromo-4-chloro-3-indolyl phosphate), and 20 mL of alkaline buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5). Each sequence read was trimmed for quality and mapped to a region of the reference genome sequence of serovar Lai (Ren et al.,

2003) representing the lipopolysaccharide biosynthesis locus using sequencher 3.1 (Gene Codes, Ann Arbor, MI). An escape mutant strain of LaiWT was obtained after serial subculture in the presence of mAb F70C7. Strains LaiWT and LaiMut had identical RFLP patterns using either EcoRI or BamHI (data not shown). This near genetic identity was further confirmed by sequencing of the secY gene, which was identical in both strains. The MAT titre of F70C7 against LaiWT was 1280, whereas LaiMut was not agglutinated by F70C7. Additional MAT testing with a set of mAbs and polyclonal sera revealed that the agglutination of LaiMut was decreased by all reactive mAbs and polyclonal sera against serovar Lai, except for mAb F20C4-1, which showed an increased titre.

48, P = 0.03, Bonferroni corrected). Analysis of ipsilateral electrodes showed no P100 attention effect. A correlation of the ERP attention modulation and behavioural effect showed no significant relationship (r = 0.25, n.s). Analysis of the endogenous counter-predictive task showed no significant effects involving the factor Cue. There was a Task

× Cue × Hemisphere interaction (F2,22 = 7.05, P = 0.004,  = 0.39), as well as a main effect of Cue (F1,11 = 20.87, P = 0.001,  = 0.66) and Cue × Hemisphere interaction (F1,11 = 16.27, P = 0.002,  = 0.60). The significant interaction was further broken down into separate analysis for each task. Exogenous task analysis of the N140 showed a significant Cue × Electrode site × Hemisphere ALK activation interaction (F5,55 = 3.34, P = 0.029, mTOR inhibitor  = 0.23), which was broken down into separate analyses for each hemisphere. However, there were no significant effects including the factor Cue at electrodes ipsilateral

or contralateral to the target presentation, indicating no attention modulation at the N140 in the exogenous task. Analysis of the endogenous predictive task revealed a significant main effect of Cue (F1,11 = 16.95, P = 0.002,  = 0.61), and also Cue × Hemisphere interaction (F1,11 = 21.53, P = 0.001,  = 0.66). The interaction was broken down revealing a significant effect of Cue, both for ipsilateral (F1,11 = 26.66, P < 0.001,  = 0.71) and contralateral

electrodes (F1,11 = 8.77, P = 0.013,  = 0.44), and both these effects showed enhanced negativity for expected compared with unexpected trials (the interaction was driven by larger effect size over ipsilateral compared with contralateral hemisphere; Fig. 4). That is, the N140 attention effect in the endogenous predictive task was present over both hemispheres. Moreover, and importantly, there was a significant correlation between the ERP attention modulation and the behavioural RT effect, with larger amplitude difference between expected Nabilone and unexpected conditions for each participant relating to larger RT attention effect (r = 0.69, P = 0.013; see Fig. 7 for a scatterplot of this relationship). The endogenous counter-predictive task revealed the attention effect was, similar to the endogenous predictive task, bilateral as there was a significant effect of Cue (F1,11 = 5.16, P = 0.044,  = 0.32). There was no significant correlation between ERP attention modulation and RT effect (r = 0.32, n.s.). At this last analysed time window the overall task analysis demonstrated a Task × Cue × Hemisphere interaction (F2,22 = 8.29, P = 0.002,  = 0.43) and also Cue (F1,11 = 11.02, P = 0.007,  = 0.50), and subsequently each task was analysed separately. The exogenous task revealed a Cue × Hemisphere interaction (F1,11 = 8.57, P = 0.014,  = 0.44).

2) is essential. Mycobacterium smegmatis is unique among Mycobacteria in having

a third chaperonin gene, cpn60.3. The cpn60.1 gene has a gene upstream (cpn10) that is homologous to the gene for the E. coli co-chaperonin GroES. Phylogenetic analysis of the mycobacterial homologues suggests that early gene duplication and sequence divergence gave rise to the cpn60.1 and cpn60.2 genes found in all Mycobacteria species, while cpn60.3 appears to have been acquired by horizontal gene transfer. Here, we show that cpn60.2 and cpn10 are expressed more strongly than cpn60.1, while Selleckchem MK 2206 cpn60.3 shows very low levels of expression. The expression of all the genes, except cpn60.3, is significantly induced by heat shock, but much less so by other stresses. We mapped mRNA 5′-ends for the cpn10 and cpn60.1 genes, and measured the promoter activity of the upstream regions of both genes. The results show that the mRNA for this operon is cleaved between the cpn10 and cpn60.1 genes. These results are consistent with the evolution of a distinct function for the cpn60.1 gene. Protein structures are fully determined by their E7080 datasheet amino acid sequences

(Anfinsen, 1973). However, in vivo, molecular chaperones are required to assist the folding of many proteins to their native state under normal conditions, where a high protein concentration can lead to aggregation unless transiently exposed hydrophobic regions are protected (Lin & Rye, 2006; Ellis, 2007; Horwich

et al., 2007). Chaperones also play a key role during stresses such as heat shock, which can lead to the partial unfolding of proteins. One group of chaperones, the chaperonins (Hemmingsen et al., 1988), is typified by the Escherichia coli GroEL protein, which is the only essential chaperone in that buy Decitabine organism (Fayet et al., 1989). Chaperonins are tetradecamers made up of 60 kDa subunits arranged in two heptameric rings, each with a central cavity where protein folding can occur. Each subunit has three domains referred to as the apical, intermediate and equatorial domains (Braig et al., 1994). Bacterial chaperonins interact with a separate heptameric co-chaperonin. In E. coli, the co-chaperonin (GroES) is also essential (Fayet et al., 1989). Generically, chaperonins are referred to as Cpn60 proteins, and the co-chaperonins as Cpn10 proteins (Coates et al., 1993). Chaperonins bind their client proteins by hydrophobic interactions, initially to the apical domain (Fenton et al., 1994). Binding of the co-chaperonin displaces the bound protein into the cavity, where it can fold without interacting with other proteins with which it might aggregate. The cycle of binding and release of co-chaperonin and client protein is mediated by ATP binding and hydrolysis, via a complex set of allosteric interactions within and between the two rings (reviewed in Saibil et al., 2001; Horwich et al., 2007).

, 2008). The remaining substrates arabinose and maltose caused the RAD001 order efficient phosphorylation of Crh~P (80%) but no comparable accumulation of HPr(Ser)~P (21% and 13% of total HPr, respectively; Singh et al., 2008). Therefore, CCR caused by these substrates is weak. How can this discrepancy be explained? When arabinose or maltose is utilized, more than 60% of all HPr molecules are

phosphorylated either at His15 or at both sites (Singh et al., 2008). Neither of these forms, HPr(His)~P or doubly phosphorylated HPr, is active in CCR because phosphorylation at His15 impedes complex formation with CcpA (Schumacher et al., 2004). It would appear that the phosphorylation at His15 provides an additional level of control that allows integration of information about the phosphorylation status of the PTS into the global mechanism of CCR. Evidence is accumulating that Crh has no dedicated role in CCR. However, it appears to regulate glycolytic flux through interaction with two metabolic enzymes, methylglyoxal synthase (MgsA) and glyceraldehyde-3-phosphate dehydrogenase (GapA). Non-phosphorylated Crh inhibits MgsA (Landmann et al., 2011), whereas phosphorylated Crh~P, in concert with HPr(Ser)~P, inhibits GapA activity (Pompeo et al., 2007). Non-phosphorylated Crh accumulates when bacteria grow on less favorable (gluconeogenic) SAHA HDAC carbon sources or

when carbohydrates become exhausted and cells enter the stationary growth phase (Figs 2-4). Consequently, MgsA activity and concomitantly flux through the methylglyoxal pathway is expected to be inhibited by Crh under these famine conditions. Under feast conditions, Crh is predominantly phosphorylated.

Thus MgsA gains activity, whereas GapA is repressed, leading to re-direction of flux from the EMP pathway towards the methylglyoxal pathway. This mechanism may prevent accumulation of sugar-phosphates when there is an excess of sugars and uptake rates exceed the capacity of EMP pathway. We thank Sabine Lentes for excellent technical assistance. We are grateful to Gerald Seidel for providing information on the Crh antiserum and for insightful discussion. This work (-)-p-Bromotetramisole Oxalate was supported by the Federal Ministry of Education (Research SYSMO network) to J.S. and W.H., and by grant GO1355/7-1 of the Deutsche Forschungsgemeinschaft to B.G. J.J.L. was supported by a stipend of the Fonds der Chemische Industrie. Wolfgang Hillen passed away on 17 October 2010. “
“Thermophilic bacteria have recently attracted great attention because of their potential application in improving different biochemical processes such as anaerobic digestion of various substrates, wastewater treatment or hydrogen production. In this study we report on the design of a specific 16S rRNA-targeted oligonucleotide probe for detecting members of Coprothermobacter genus characterized by a strong protease activity to degrade proteins and peptides.

tumefaciens (Zhang et al., 2002). In the case of the bacteroidete learn more T. maritimum, the presence of a QQ enzyme for long AHLs may represent an exclusion mechanism to interfere with the QS systems of competitors (Dong

& Zhang, 2005). Evidence is beginning to accumulate indicating that QS and QS inhibition processes, including enzymatic degradation of the signal or QQ, are important in the marine environment. Besides the well-characterized phenomenon of the production of furanones by the red alga D. pulchra to avoid surface colonization by Gram-negative biofilm formers (Givskov et al., 1996), QS systems mediated by AHLs have been found in many species of marine pathogenic bacteria (Bruhn et al., 2005). AHLs also seem to play an important role in the eukaryotic–prokaryotic interactions in the marine environment, as demonstrated by the importance of the production of AHLs by marine biofilms for the surface selection and permanent attachment of zoospores of the green alga Ulva (Tait et al., 2005), for spore release of the red alga Acrochaetium sp. (Weinberger et al., 2007), and for some initial larval settlement behaviours in the polychaete Hydroides elegans (Huang et al., 2007). As most of the isolates involved in algal morphogenesis belong to the CFB group (Hanzawa et al., 1998; Matsuo et al., 2003), the discovery of the production

and degradation of AHLs by members of this group provides the possibility of new interactions Everolimus datasheet many between bacteria and eukaryotes in the marine environment. For the first time, the production of AHL-type QS signals and QQ activity has been demonstrated simultaneously in a pathogenic member of the CFB group. Because of the ecological significance of the Cytophaga–Flavobacterium cluster, especially in the marine environment, the discovery of AHL-mediated QS processes among

their members will advance our understanding of the microbial interactions in complex ecosystems. Moreover, cell-to-cell communication phenomena should be reconsidered in other habitats in which the Bacteroidetes play an important role, such as intestinal flora or dental plaque. As QS controls the expression of important virulence factors in many pathogenic bacteria, the disruption of QS mechanisms in T. maritimum and other fish pathogenic bacteria may represent a new strategy for the treatment of infections in aquaculture. This work was financed by a grant from Consellería de Innovación e Industria, Xunta de Galicia, Spain (PGIDIT06PXIB200045PR). M.R. is supported by an FPU fellowship from the Spanish Ministry of Science and Education. We would like to thank Noemi Ladra (University of Santiago) and Catherine Ortori (University of Nottingham) for LC-MS analysis. The sensor Chromobacterium violaceum VIR07 was kindly provided by Prof. T. Morohoshi. “
“Biofilm detachment is a physiologically regulated process that facilitates the release of cells to colonize new sites and cause infections.

Both human and agricultural diseases are treated with azole antifungals. These compounds target 14 sterol demethylase and interfere with ergosterol production in the fungus (Yoshida & Aoyama, 1984, 1987; Wright et al., 1990). Recent testing of A. fumigatus clinical isolates has identified cases of azole resistance (Sanglard et al., 1995, 1997; Kelly et al., 1997; Nolte et al., 1997; Bafetinib manufacturer Franz et al.,

1998; Gupta et al., 1998; Schaller et al., 1998; Marichal et al., 1999; Calabrese et al., 2000; Moore et al., 2000; Yang & Lo, 2001; Verweij et al., 2002; Gomez-Lopez et al., 2003; Drago et al., 2004; Hsueh et al., 2005; Pfaller et al., 2006) some of which have been shown to be resistant to treatment with itraconazole (ITR) in murine models of infection (Denning et al., 1997a; Dannaoui et al., 1999, 2001). Long-term

treatment for patients with ITR (Chryssanthou, 1997; Denning et al., 1997a; Dannaoui Entinostat concentration et al., 2001; Warris et al., 2002; Chen et al., 2005) appears to lead to azole resistance in infecting fungi, although these cases were not always associated with ITR treatment failure. Two per cent of 913 A. fumigatus isolates in the literature published before 2000 were found to be resistant (Dannaoui et al., 2001). Recent surveys have reported frequencies ranging from 1% to 6% (total number of isolates surveyed was 357; Verweij et al., 2002; Gomez-Lopez et al., 2003; Drago et al., 2004; Hsueh et al., 2005), although other surveys have reported no resistant isolates out of a total of 2100 isolates (Verweij et al., 1998; Chandrasekar et al., 2001; Diekema et al., 2003; Balajee et al., 2004; Dannaoui et al., 2004; Kauffmann-Lacroix

et al., 2004; Guinea et al., 2005; Pfaller et al., 2005). Recent estimates from our laboratory suggest higher levels of azole resistance (Howard et al., 2009) and similar studies in the Netherlands have also shown high levels of azole resistance. A number of mutations in the cyp51A lanosterol 14α-demethylase gene have been associated with azole resistance (Denning et al., 1997a; Diaz-Guerra et al., 2003, 2004; Ferreira et al., 2004; Mellado et al., 2004, 2005; Chen et al., 2005). Other mechanisms for azole resistance include from increased expression of efflux pumps. The increased expression of an ATP-binding cassette (ABC) transporter (AtrF) in the presence of ITR has been shown in a clinical isolate with reduced drug accumulation (Slaven et al., 2002), and possible transporters have been implicated in azole resistance of laboratory selected mutants (Denning et al., 1997a; Nascimento et al., 2003; Ferreira et al., 2004). Restriction enzyme-mediated integration (REMI) was employed, as it increases the transformation efficiency and promotes single-copy, non-rearranged integrations of the transforming DNA (Lu et al., 1994; Bolker et al., 1995; Brown et al., 1998; de Souza et al., 2000).

Defects in flagellar function were identified by the absence of outward migration on the media; this was then confirmed by direct observation under a light microscope.

An inverse PCR method was used to amplify the sequence flanking the inserted mini-Tn5 transposon in the chromosome of the swarming-defective click here mutants. Genomic DNA from each mutant was isolated according to the cetyltrimethylammonium bromide protocol and completely digested with TaqI. The DNA fragments were self-ligated with T4 DNA ligase and then used as templates for inverse PCR with the primers P904 (5′-GGAGAGGCTATTCGGCTATG-3′) and P194c (5′-GTAAGGTGATCCGGTGGATG-3′), which were designed according to the motile sequence of Mini-Tn5-Km plasmid. The PCR products were separated by agarose gel electrophoresis and then purified using a gel extraction kit (Watson Biotechnologies Inc.). The PCR products were directly sequenced at Shanghai GeneCore BioTechnologies.

If sequencing failed, the PCR products were ligated to PMD18-T vector (Takara Co. Ltd, Dalian, China) and the sequencing was attempted again. To identify the mutant genes, nucleotide sequence databases were searched with the blastn and blastx programs developed by the National Center for Biotechnology Information (NCBI). Flagellin was isolated from bacterial cells according to the method described by DePamphilis & Adler (1971). The bacterial cells were suspended in 0.1 M Tris-HCl Belnacasan purchase buffer (pH 7.5). Flagellar filaments were sheared with a tissue homogenizer at maximum speed for 30 s. The bacteria were observed microscopically to ascertain loss of motility. Cell debris was removed from the flagella by centrifugation at 15 000 g for 15 min, and flagellar filaments were then pelleted from

the supernate by ultracentrifugation and then suspended in 0.1 M Tris-HCl buffer (pH 7.5). Sodium dodecyl sulfate-polyacrylamide Metalloexopeptidase gel electrophoresis (SDS-PAGE) was used to analyze the purity of samples. Flagellin protein dissolved in Tris-HCl buffer was emulsified in Freund’s incomplete adjuvant (1 : 3). One rabbit was immunized three times at intervals of 2 weeks. Serum was collected 1 week after the final injection and stored at −20 °C. Bacteria were suspended in Tris-HCl buffer and then adjusted to 1 OD600 nm. All the cell lysates were subjected to SDS-PAGE electrophoresis and then transferred onto a nitrocellulose membrane. The flagellin was visualized via Western blotting with enhanced chemiluminescence detection (Pierce). Rabbit polyclonal antiflagellin serum was used as the primary antibody. The secondary antibody was a goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. Detection was performed according to the protocol of the supplier. The surface hydrophilicity of bacterial cells was quantified using bacterial adherence to hydrocarbon (BATH) test, originally described by Rosenberg et al. (1980).

To identify gene candidates involved in the spatially protective effects produced by early-life conditioning seizures we profiled and compared the transcriptomes of CA1 subregions from control, 1 × KA- and 3 × KA-treated animals. More genes were Quizartinib in vivo regulated following 3 × KA (9.6%) than after 1 × KA (7.1%). Following 1 × KA, genes supporting oxidative stress, growth, development, inflammation and neurotransmission were upregulated (e.g. Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12 and Grik1). After 3 × KA, protective genes were differentially over-expressed

[e.g. Cat, Gpx7, Gad1, Hspa12A, Foxn1, adenosine A1 receptor, Ca2+ adaptor and homeostasis proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp and suppressor of cytokine signaling (Socs3)]. Distinct anti-inflammatory interleukins (ILs) not observed in adult tissues [e.g. IL-6 transducer, IL-23 and IL-33 or their receptors (IL-F2 )] were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1 × KA but reduced after 3 × KA; the pro-inflammatory gene Cox1 was either upregulated or unchanged after 1 × KA but reduced by ~70% after 3 × KA. Enhanced GFAP immunostaining

following 1 × KA was selectively attenuated in the CA1 subregion after 3 × KA. The observed differential transcriptional responses may contribute to early-life seizure-induced pre-conditioning and neuroprotection CYC202 mouse by reducing glutamate receptor-mediated Ca2+ permeability of the hippocampus and redirecting

inflammatory Flavopiridol (Alvocidib) and apoptotic pathways. These changes could lead to new genetic therapies for epilepsy. “
“It has recently been suggested that learning signals in the amygdala might be best characterized by attentional theories of associative learning [such as Pearce–Hall (PH)] and more recent hybrid variants that combine Rescorla–Wagner and PH learning models. In these models, unsigned prediction errors (PEs) determine the associability of a cue, which is used in turn to control learning of outcome expectations dynamically and reflects a function of the reliability of prior outcome predictions. Here, we employed an aversive Pavlovian reversal-learning task to investigate computational signals derived from such a hybrid model. Unlike previous accounts, our paradigm allowed for the separate assessment of associability at the time of cue presentation and PEs at the time of outcome. We combined this approach with high-resolution functional magnetic resonance imaging to understand how different subregions of the human amygdala contribute to associative learning.

To identify gene candidates involved in the spatially protective effects produced by early-life conditioning seizures we profiled and compared the transcriptomes of CA1 subregions from control, 1 × KA- and 3 × KA-treated animals. More genes were ALK inhibitor regulated following 3 × KA (9.6%) than after 1 × KA (7.1%). Following 1 × KA, genes supporting oxidative stress, growth, development, inflammation and neurotransmission were upregulated (e.g. Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12 and Grik1). After 3 × KA, protective genes were differentially over-expressed

[e.g. Cat, Gpx7, Gad1, Hspa12A, Foxn1, adenosine A1 receptor, Ca2+ adaptor and homeostasis proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp and suppressor of cytokine signaling (Socs3)]. Distinct anti-inflammatory interleukins (ILs) not observed in adult tissues [e.g. IL-6 transducer, IL-23 and IL-33 or their receptors (IL-F2 )] were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1 × KA but reduced after 3 × KA; the pro-inflammatory gene Cox1 was either upregulated or unchanged after 1 × KA but reduced by ~70% after 3 × KA. Enhanced GFAP immunostaining

following 1 × KA was selectively attenuated in the CA1 subregion after 3 × KA. The observed differential transcriptional responses may contribute to early-life seizure-induced pre-conditioning and neuroprotection Dasatinib mouse by reducing glutamate receptor-mediated Ca2+ permeability of the hippocampus and redirecting

inflammatory Janus kinase (JAK) and apoptotic pathways. These changes could lead to new genetic therapies for epilepsy. “
“It has recently been suggested that learning signals in the amygdala might be best characterized by attentional theories of associative learning [such as Pearce–Hall (PH)] and more recent hybrid variants that combine Rescorla–Wagner and PH learning models. In these models, unsigned prediction errors (PEs) determine the associability of a cue, which is used in turn to control learning of outcome expectations dynamically and reflects a function of the reliability of prior outcome predictions. Here, we employed an aversive Pavlovian reversal-learning task to investigate computational signals derived from such a hybrid model. Unlike previous accounts, our paradigm allowed for the separate assessment of associability at the time of cue presentation and PEs at the time of outcome. We combined this approach with high-resolution functional magnetic resonance imaging to understand how different subregions of the human amygdala contribute to associative learning.

tumefaciens GV3101∷pMP90 to obtain the strain GV3101∷pMP90(pPZP-eGFP). The vector pRK415 Selleckchem MAPK Inhibitor Library (Keen et al., 1988) or the plasmid pRKLACC (Shah et al., 1998), which is pRK415 containing the acdS gene from Pseudomonas putida UW4 under the control of a lac promoter, was electroporated into A. tumefaciens GV3101∷pMP90(pPZP-eGFP) to obtain strain YH-1, which is GV3101∷pMP90(pPZP-eGFP)(pRK415), and strain YH-2, which is GV3101∷pMP90(pPZP-eGFP)(pRKLACC). Agrobacterium strains were grown in Luria–Bertani (LB) (Miller, 1976) or M9 medium (Atlas, 1993) (for ACC deaminase

activity assay) at 28 °C. When required, antibiotics were added at the following concentrations: rifampicin, 50 μg mL−1; gentamicin, 50 μg mL−1; spectinomycin, 50 μg mL−1; streptomycin, 20 μg mL−1; and tetracycline, 2 μg mL−1. An ACC deaminase activity assay was performed as described by Hao et al. (2007). The infection and regeneration protocols were modified from Cardoza & Stewart (2003). The media used are listed in Table 1. Seeds of B. napus cv. Westar, B. napus cv. Hyola 401 and B. napus cv. 4414 RR were surface sterilized by soaking in 70% ethanol for 1 min, followed by 20% commercial bleach for 20 min, and were then

rinsed four times with sterilized distilled water and planted at a density of 10–12 seeds per Petri dish (100 × 25 mm) (Fisher Scientific, Ottawa, ON) on seed germination medium. Seeds were germinated at 22–25 °C in the dark for about 1 week. The seedling find more hypocotyls were cut into about 1-cm pieces and preconditioned for 3 days on a cocultivation medium. Agrobacterium tumefaciens strains YH-1 and YH-2 were grown in 50 mL LB medium until the culture reached OD600 nm≈1. The cells were pelleted, resuspended in the infection medium and normalized to OD600 nm=1 to obtain a 1 × dilution. Serial dilutions were then performed using the infection medium to obtain 10−1× and 10−2× dilutions. The preconditioned explants were infected by soaking in A. tumefaciens culture suspensions for 30 min at room temperature with gentle shaking. The infected hypocotyls were first

cocultured on a cocultivation medium for 48 h, then transferred to a callus induction medium for 2 weeks, Fossariinae then to an organogenesis medium with (OA) or without AgNO3 (OB) for another 2 weeks, and then to a shoot induction medium for 3–6 weeks until shoots appeared. The induced shoots were transferred to a shoot elongation medium for 2 weeks and then to a rooting medium for another 2 weeks, and finally, the transgenic plants were transferred to soil and grown in a greenhouse. Plant tissue cultures were maintained in a growth chamber at 25 °C with 16 h of light and 8 h of dark, with a light intensity of 40 μmol m−2 s−1 from cool-white fluorescent lamps. The stable transformation frequency was calculated using the following formula: transformation frequency=the number of transgenic plants obtained/the number of explants used for transformation. After infection with various dilutions of A.