Therefore, to better understand the mechanism by which APF regula

Therefore, to better understand the mechanism by which APF regulates T24 bladder carcinoma cell proliferation, we determined the effect of as -APF on the expression or activation of enzymes involved in wingless-int (Wnt)/frizzled signaling (including AKR-transforming enzyme (Akt), glycogen MLN4924 chemical structure synthase kinase-3 beta (GSK3β), β-catenin, and matrix metalloprotease 2 (MMP2), as well as the role of CKAP4 in mediating as -APF activity in T24 cells. Methods Cell Culture T24 human

urinary bladder cancer cells (ATCC HTB-4) were grown to 60-80% confluence in McCoy’s 5A medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS), 1% antibiotic/antimycotic solution, 1% L-glutamine (all Savolitinib order from Sigma, St. Louis, MO) and 2.2 grams/L sodium

bicarbonate (Invitrogen), in a 37°C/5% CO2 atmosphere. siRNA Transfection AZD8931 cell line double-stranded siRNA corresponding to nucleotides 594-616 of CKAP4 (5′-AACUUUUGAGUCCAUCUUGAGAA-3′ sense strand) and a scrambled double-stranded negative control siRNA (5′-AAUUCUGUAUGCUACCUGUAGAA-3′ sense strand) were prepared by preincubating single-stranded sense and antisense strands prepared with double A overhangs in serum-free McCoy’s 5A medium at 37°C for 1 hour. T24 human bladder cancer cells were trypsinized for 10 minutes at room temperature, centrifuged in growth medium (as defined above), and the cell pellet was resuspended in serum-free medium at a density of 1 × 106 cells/ml. Two hundred microliters of the cell suspension were then transferred to a sterile 2-mm cuvette with 14 μg of CKAP4 siRNA, scrambled non-target siRNA, or no siRNA, and electroporated at 160 V/500 microfarad capacitance using a Bio-Rad Gene find more Pulser Xcell. The cells were then immediately

transferred to T75 cell culture flasks (Corning Incorporated, Corning, NY) (for extraction of RNA and protein) or to 96 well tissue culture plates (Corning Incorporated) (for the cell proliferation assay) and incubated in growth medium overnight in a 37°C/5% CO2 atmosphere. APF Treatment (for RNA and Protein Extraction) Following overnight incubation in growth medium, transfected T24 human bladder cancer cells were further incubated with serum-free McCoy’s 5A medium for the next 24 hours, after which they were treated with 500 nM as -APF or 500 nM inactive nonglycosylated peptide control (both from PolyPeptide Laboratories, Incorporated, San Diego, CA). Cells were then incubated for an additional 48 hrs in a 37°C/5% CO2 atmosphere prior to RNA and protein extraction. RNA Extraction Following cell incubation with as -APF or its control peptide/diluent, the culture medium was removed, T24 cells were washed with 1× PBS, and RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA concentration was measured at 260 nM in a UV/VIS spectrophotometer from Perkin Elmer. Extracted RNA was stored at -80°C.

Restriction enzyme (Thermo Scientific) and T4 DNA ligase (Thermo

Restriction enzyme (Thermo Scientific) and T4 DNA ligase (Thermo Scientific) reactions were performed as per the manufacturer’s

instructions at the appropriate temperature where all ligation reactions were incubated at room temperature. DNA purifications were either performed using the GeneJET PCR purification kit (Thermo Scientific) or the GeneJET Gel extraction kit (Thermo Scientific) following the manufacturer’s instructions. selleck inhibitor Protein purification was carried out using the Ni-NTA Spin Kit (Qiagen) following the manufacturer’s instructions. Construction of the E. amylovora acrD-deficient mutant A 1058-bp fragment located in the acrD gene was amplified using the primer pair acrD_ko_fwd and acrD_ko_rev and verified by AG-881 order sequencing. A chloramphenicol cassette flanked by Flp-FRT sites was cut from plasmid pFCM1 and inserted into BamHI-digested pJET.acrD-ko, yielding pJET.acrD-ko.Cm. A 2.2-kb EcoRI fragment cut from pJET.acrD-ko.Cm was ligated into EcoRI-digested pCAM-Km,

yielding the final replacement plasmid pCAM-Km.acrD-Cm. The plasmid was transformed into electrocompetent cells of E. amylovora Ea1189, which subsequently were grown for 3 h at 28°C in dYT broth. Putative mutants were screened for homologous recombination events by testing their antibiotic resistance. Mutants that resulted from single crossover events were identified by their ability to grow on plates containing Km. In order to confirm selleck kinase inhibitor gene disruption through a double crossover event in Cm-resistant and Km-sensitive colonies, primers acrD_fwd and acrD_rev were designed, which bind upstream and downstream, respectively, of the 1058-bp acrD fragment used for generation of the gene replacement vector. PCRs were done using these locus-specific primers

with primers binding in the Cm cassette (cat_out2, cat_out3, cat_out4, cat_out5). Amplified PCR products were verified by sequencing. The Cm-FRT cassette was finally N-acetylglucosamine-1-phosphate transferase excised using the temperature-sensitive plasmid pCP20 that carries the yeast Flp recombinase gene [43, 45]. Briefly, Cm-resistant mutants of Ea1189 were transformed with pCP20 and selected at 28°C on LB plates containing Ap. Subsequently, Ap-resistant transformants were streaked on non-selective agar plates and incubated at 43°C for 1 h; following incubation at 28°C for 48–60 h. Single colonies were selected and tested on agar plates containing Cm or Ap to confirm successful excision of the Cm cassette and loss of plasmid pCP20. Construction of acrD overexpression plasmids A 3.06-kb fragment containing acrD was amplified from E. amylovora Ea1189 using the primer pair acrD-ApaI and acrD-SacI. The PCR product was sequenced and further cloned into ApaI-SacI-digested pBlueScript II KS(+) and pBlueScript II SK(+), respectively (pBlueKS.acrD, pBlueSK.acrD).

Vector Borne Zoonotic Dis 2005,5(4):315–323 PubMedCrossRef 55 Ha

Vector Borne Zoonotic Dis 2005,5(4):315–323.PubMedCrossRef 55. Hardestam STAT inhibitor J, Karlsson M, Falk

KI, Olsson G, Klingstrom J, Lundkvist A: Puumala hantavirus excretion kinetics in bank voles. Emerg Infect Dis 2008,14(8):1209–1215.PubMedCrossRef 56. Kallio ER, Begon M, Henttonen H, Koskela E, Mappes T, Vaheri A, Vapalahti O: Hantavirus infections in fluctuating host populations: the role of maternal antibodies. Proc Roy Soc Lond, B 2010, 277:3783–3791.CrossRef 57. Kuenzi AJ, Douglass RJ, Bond CW, Calisher CH, Mills JN: Long-term dynamics of Sin Nombre viral RNA and antibody in deer mice in Montana. J Wildl dis 2005,41(3):473–481.PubMed 58. Kallio ER, Poikonen A, Vaheri learn more A, Vapalahti O, Henttonen H, Koskela E, Mappes T: Maternal antibodies postpone hantavirus infection and enhance individual breeding

success. Proc Biol Sci 2006,273(1602):2771–2776.PubMedCrossRef 59. McSorley HJ, Loukas A: The immunology of human hookworm infections. Parasite Immunol 2010,32(8):549–559.PubMed 60. Schoenrich G, Rang A, Lütteke N, Raftery MJ, Charbonnel N, Ulrich RG: Hantavirus-induced immunity in rodent reservoirs and humans. Immunol Rev 2008, 225:163–189.CrossRef 61. Morimoto M, Zhao AP, Sun R, Stiltz J, Madden KB, Mentink-Kane M, Ramalingam T, Wynn TA, Urban JF, Shea-Donohue T: IL-13 Receptor alpha 2 Regulates the Immune and Functional Response to Nippostrongylus brasiliensis Infection. J Immunol 2009,183(3):1934–1939.PubMedCrossRef 62. Reece JJ, Siracusa MC, Southard TL, Brayton CF, Urban JF, Scott AL: Hookworm-induced persistent changes to the immunological environment of the lung. Infect Immun 2008,76(8):3511–3524.PubMedCrossRef 63. Erb KJ, Trujillo C, Fugate M, Moll H: Infection with the helminth Nippostrongylus brasiliensis does not interfere with efficient elimination

of Mycobacterium bovis BCG from the lungs of mice. Clinic Diagn Lab Immunol 2002,9(3):727–730. 64. Guivier E, Galan M, Male PJ, Kallio ER, Voutilainen L, Henttonen H, Liothyronine Sodium Olsson G, Lundkvist A, Tersago K, Augot D, et al.: Associations between Major Histocompatibility Complex genes and PUUV infection in Myodes glareolus are detected in wild populations but not from experimental infection data. J Gen Virol 2010, 91:2507–2512.PubMedCrossRef 65. Kloch A, Babik W, Bajer A, Sinski E, Radwan J: Effects of an selleckchem MHC-DRB genotype and allele number on the load of gut parasites in the bank vole Myodes glareolus . Mol Ecol 2010, 19:255–265.PubMedCrossRef 66. Guivier E, Galan M, Ribas Salvador A, Xuéreb A, Chaval Y, Olsson G, Essbauer S, Henttonen H, Voutilainen L, Cosson JF, et al.: Tnf-α expression and promoter sequences reflect the balance of tolerance/resistance to Puumala virus infection in European bank vole populations. Infect Genet Evol 2010,10(8):1208–1217.PubMedCrossRef 67.

In contrast, the Euro-African “”strain cluster C”" has a low freq

In contrast, the Euro-African “”strain cluster C”" has a low frequency of cognate sites for RMS in cluster 1, but high for “”RMS cluster 2″” (Figure 2). The cognate sites for RMS cluster 1 have a significantly lower G + C content compared to the cluster 2 cognate sites (59.4 ± 17.4 and 91.6 ± 20.4%, respectively. T-test = 0.002). “”Strain cluster B”" includes hspEAsia as well as hpEurope and hpAfrica1

from Mestizo and African hosts and shows a mosaic profile BYL719 of the cognate recognition sites, consistent with the mosaic genetic structure shown in their MLS (Additional file 1: Figure S1). Figure 2 Heatmap of the profile for 15 RE recognition sites on MLS DNA sequences for 110 H. pylori strains. Higher and lower frequencies of the cognate recognition sites are represented by red and blue, respectively. The upper tree showed three main strainclusters: A) Includes hspAmerind Pevonedistat in vivo (N=25), hspEAsia (N=5), and hpEurope (N=7) strains; B) Mostly hpEurope (N=21), but also hspEAsia (N=6), and hpAfrica1 (N=2) strains; and C) hpAfrica1 (N=23), and hpEurope (N=20) strains. The hpEurope Selleck RG-7388 strains studied were mostly recovered from Mestizo hosts. The phylogeny

on the left shows two enzyme clusters, that correlate with the A, B and C cluster-strains. Strain-specific methylase representation Differences in transformation rates might be due to differences in the frequency of cognate restriction sites, but also to variation in the protection conferred by active methylases belonging to the RMS. We tested the hypothesis that cognate restriction sites are more

protected by active methylases in hpEurope than in hspAmerind strains. Cell press We selected 18 representative H. pylori strains; 9 were hpEurope recovered from European (n = 4), Mestizo (n = 4), and Amerindian (N = 1) hosts, and 9 were hspAmerind from Amerindian hosts (Additional file 1: Table S2). To determine methylase protection, genomic DNA from each strain was subject to digestion by each of 16 restriction endonucleases (Additional file 1: Table S3). Susceptibility to digestion indicated lack of an active methylase. The restriction results showed a range of 5–14 active methylases (average = 8.6 ± 2.6) per H. pylori strain of the 16 examined. There were non-significant differences in the number (Wilcoxon test, p > 0.05; Figure 3, Additional file 1: Table S3) or variances (F test, p > 0.05) of active methylases between hpEurope and hspAmerind strains. The only exception was the enzyme HpaII, to which DNA from the hspAmerind strains was significantly more resistant (83%) than DNA from the hpEurope strains (42%; Wilcoxon test; p < 0.05). Overall, the results confirm that H. pylori strains conserve similar active methylase protection, regardless of their population assignment. Figure 3 Total number of active methylases per strain.

The synchronization of cells in S phase by MTX was reversible as

The synchronization of cells in S phase by MTX was reversible as the pattern of cell cycle progression of MTX-treated cells was similar to that of untreated cells 48 hr after drug removal (Figure 1A). Our results thus this website suggest that MTX is more effective in synchronizing DHDK12 cells in S phase than ara-C or aphidicolin. Consequently, the efficacy of MTX in synchronizing

cells in S phase was then tested in the HT29 cell line. Figure 1 Distribution in cell cycle-phase after MTX treatment. Cell cycle phases of DHDK12 cells (A) and HT29 cells (B) were obtained by uniparametric flow cytometry analysis of DNA content (propidium iodide red-fluorescence intensity in fluorescence units) at various time after MTX removal. On the ordinate is shown the number of cells corresponding Palbociclib clinical trial to the fluorescence units. In HT29 cell line, the effect of MTX on cell cycle progression was slightly different. As illustrated in Figure 1B, cells began to accumulate in S phase almost immediately after MTX removal. While the rate of cells in S phase was 18% without JQ-EZ-05 concentration treatment (Figure 1B), this rate reached 55% 6 hr after MTX removal and decreased thereafter to

reach the ratio of untreated cells 24 hr after MTX removal. Taken together, these observations indicate that the pattern of cell cycle synchronization after MTX removal is specific for each cell line. Because we hypothesize that gene transfer efficiency is improved by potent cell cycle synchronization, the time window for transduction experiments with the β-gal reporter gene should be different between the two cell lines. Improvement of gene transfer efficiency in synchronized cell To determinate the optimal period for gene transfer in synchronized cells, we used the β-gal reporter

gene. The rate of DHDK12 cells transduced with the β-gal gene was 3% with X-Gal staining while it was 10% with FDG in flow cytometry (data not shown). The treatment of DHDK12 cells with MTX improved retroviral gene transfer ADP ribosylation factor efficiency. Figure 2 shows that the level of transduction increased in cells synchronized in S phase. The highest level of transduction was obtained in the cells infected 20 hr after MTX removal. At that time, the proportion of transduced cells was 26% for cells treated with MTX, while it was 11% in untreated cells (Figure 2A). In the MTX-treated cell population, 44% of cells were in S phase. When the cell cycle distribution of MTX-treated cells returned to the control value 54 hr after drug removal, the efficiency of transduction became similar to that of control cells (Figure 2A). Thus, the optimal period to improve transduction efficiency of reporter gene in synchronized cells was obtained between 12 and 32 hr after drug removal. Figure 2 Infection efficiency of the β- gal retroviral vector. DHDK12 cells (A) and HT29 cells (B) were treated for 24 hr with (filled circle) or whithout (open circle) MTX. Cells were transduced with TG 5391 at the indicated times after MTX removal.

In the IPC+IPO group HIF-1α mRNA expression was significantly low

In the IPC+IPO group HIF-1α mRNA expression was significantly lower compared

to the IRI group (IRI vs. IPC+IPO, p ≤ 0.01). The HIF-1α mRNA levels were comparable between group CG, IPC, IPO and IPC+IPO (Figure 3) Figure 3 Expression of HIF-1α mRNA. Expression after 30 min of reperfusion. CG, Control group. IRI, 30 min of ischemia. IPC, IPC + 30 min of ischemia. IPO, 30 min www.selleckchem.com/products/mk-4827-niraparib-tosylate.html ischemia + IPO. IPC+IPO, IPC + 30 min of ischemia + IPO. * indicates p ≤ 0.01 compared to group IRI. ¤ indicates p = 0.065 compared to group IRI. VEGF expression As shown in Figure 4, VEGF mRNA expression was significantly increased in the IRI group compared to the control group (p ≤ 0.01). When applying IPC+IPO VEGF mRNA expression was also increased compared to the control group (p ≤ 0.038). No significant differences were LDN-193189 observed between groups IPC, IPO and the control group (IPC vs. CG, p ≤ 0.067) and (IPO vs. CG, p ≤ 0.067). Figure 4 Expression of VEGF mRNA. Expression PCI32765 after 30 min of reperfusion. CG, Control group. IRI, 30 min of ischemia. IPC, IPC + 30 min of ischemia. IPO, 30 min ischemia + IPO. IPC+IPO, IPC + 30 min of ischemia + IPO. *indicates p ≤ 0.01 compared to group CG. **indicates p ≤ 0.038 compared to group CG. TGF-β1 expression No differences in TGF-β1 mRNA expression were observed between the five groups (Figure 5). Figure 5 Expression of TGF-β1 mRNA. Expression after

30 min of reperfusion. CG, Control group. IRI, 30 min of ischemia. IPC, IPC + 30 min of ischemia. IPO, 30 min ischemia + IPO. IPC+IPO, IPC + 30 min of ischemia + IPO. Discussion As expected HIF-1α mRNA expression was increased significantly in rats subjected to 30 minutes of warm liver ischemia and 30 minutes of reperfusion compared to the control group. The main finding of this study was an absent of HIF-1α induction in IPC or IPC+IPO treated animals. In both of these groups, the expression levels were similar to that of CG. In the IPO group the same tendency towards an absent induction of HIF-1α was observed although not significant. VEGF mRNA expression increased significantly when applying 30 min of ischemia without ischemic conditioning compared to sham operated controls. IPC+IPO also showed

increased VEGF mRNA expression compared to sham operated controls, whereas neither ischemia nor ischemic conditioning affected hepatic TGF-β expression. The cytoprotective effects of IPC, GBA3 defined as brief periods of ischemia and reperfusion prior to prolonged ischemia, on I/R injuries to the liver have become indisputable with an increasing number of studies supporting this fact [12–14]. The IPC protocol used in this study has previously been shown to induce hepatoprotection against I/R injuries. We choose circulating ALAT as marker of hepacellular injuries, as this parameter is well established and known to correlate to the degree of injury [28–30]. However, we were unable to see any hepatoprotective effects as assessed by changes in liver parameters.

trachomatis strains Statistical significance is indicated with t

trachomatis strains. Statistical significance is indicated with the asterisk above the individual

treatment groups, as compared to pcDNA-transfected cells (Student’s t-test, p < 0.01). The multinuclear phenotype was manifested by the carboxy-terminal 179 amino acids of Entospletinib supplier CT223p (Fig. 4). A reduced but still significant level of multinuclear cells were identified in cells transfected with a plasmid encoding only the carboxy-terminal 56 amino acids of CT223p, but, transfection of a plasmid encoding an internal fragment of CT223p (CT223/CT91p) did not lead to a significant level of multinuclear cells. These data suggest that the YH25448 purchase domain of the protein responsible for blocking cytokinesis was present in the carboxy-terminal 56 amino acids. Cytosolic expression of other incs The orf encoding CT223p is within a likely operon encoding known and candidate inc genes CT223-CT227, and is adjacent to a very early operon containing two inc genes (CT228 and CT229). We tested each orf in these operons for an association with a polynuclear phenotype. Each orf was expressed in transfected cells and there was no

apparent difference in expression level, based on fluorescence microscopy of transfected cells (not shown). Orfs CT224 and CT225 also were associated with a reduced but still statistically significant percentage of polynuclear cells Cyclooxygenase (COX) in a transfected population (Fig. 4). None of the other tested orfs were associated with an increased number of polynuclear cells. The same approach was used for testing the effects check details on cell cytokinesis of other Inc proteins. HeLa or McCoy cells transfected with plasmids encoding each protein from C. trachomatis incA and incC, and C. caviae incA, incB and incC were compared with cells expressing full length and truncated CT223. None of these plasmids led to an increase in polynuclear cells relative to controls (Fig. 4). The CT223 coding sequence

from different C. trachomatis strains encode proteins with up to 5% difference in amino acid sequence (22). We therefore tested plasmids encoding CT223p from strains with known amino acid sequence differences for their ability to block cytokinesis. Transfection of plasmids encoding each CT223p sequence was associated with an increase in multinucleate cells (Fig. 5). In contrast, transfection of a plasmid expressing C. muridarum TC0495, which is a syntenous, apparent CT223 homolog (less than 30% predicted amino acid sequence identity), did not lead to an increase in the number of multinucleate cells relative to controls (Fig. 5). Cells producing CT223p and CT223/179p have increased numbers of centrosomes To further explore the multinuclear phenotype, cells expressing CT223 were labeled with antibodies specific against γ-tubulin.

The growth rate of the culture at pH 5 5 was almost half of that

The growth rate of the culture at pH 5.5 was almost half of that at pH 6.0. The expression pattern at pH 5.5 was different from the patterns at the higher pH levels studied, in that it lacked the sharp expression peak in the transitional phase. At pH levels below 6.0, low amounts of SEA were produced. This supports the LY2835219 nmr theory that pH 5.5 is close to the limiting pH of the bacterium. The SEA levels remained constant at pH 5.0 and pH 4.5 during the cultivation of Mu50, with a final SEA concentration of 62 ng/ml for both pH levels, indicating that no SEA production occured GDC-0449 price ≤ pH 5.0. This observation is supported by Barber and Deibel [32]. Using hydrochloric

acid, they found that the lowest pH values that supported SEA biosynthesis in buffered BHI medium incubated aerobically was 4.9. SFP can be caused by as little as 20-100 ng of enterotoxin [33]. Levels higher than 100 ng/ml were detected at pH levels 7.0-5.5 in the mid-exponential growth phase. Conclusions This study has shown that

the food preservative acetic acid increases sea gene expression in S. aureus. At pH 6.0 and 5.5, maximal sea expression was observed. At pH 6.0 there was a marked shift in growth rate and phage production peaked at pH 5.5. These findings suggest prophage induction. At pH 5.0 and 4.5, the sea gene BMN 673 ic50 copy numbers increased dramatically during late stages of cultivation, but SEA levels and phage copy numbers were low indicating that protein synthesis was affected. It is our hypothesis that the acetic acid lowers the intracellular pH of S. aureus, activating the temperate phage and, as a consequence, boosts the sea expression. Our results support the theory proposed by other research groups that

prophages not only facilitate the dissemination of virulence genes, but also take part in the regulation of the expression of the genes. Methods Culture conditions The S. aureus strains used in this study were Mu50 (LGC Promochem, London, UK), MW2 (donated by Dr. T. Baba, Juntendo University, Tokyo, Japan), Newman (donated by Dr. H. Ingmer, Copenhagen University, Copenhagen, Denmark), RN4220 (Culture Collection University of Göteborg, Göteborg, Sweden), RN450 (donated by Dr. J. R. Penadés, Instituto Valenciano de Investigaciones Agrarias, Castellón, Spain), SA17 and SA45 (donated by the Swedish Institute for Cediranib (AZD2171) Food and Biotechnology, SIK, Göteborg, Sweden). All cultivations were performed in BHI (Difco Laboratories; BD Diagnostic Systems, Le Point de Claix, France) broth (with agitation) or agar at 37°C. S. aureus was transferred from glycerol stock to broth for overnight cultivation prior to the experiments. Broth (300 ml) was inoculated with a sufficient volume of S. aureus overnight culture to give an OD value at 620 nm (OD620) of 0.1 at the start of cultivation. Batch cultivations were then performed at different pH levels (pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5) using in-house fermentors.

Figure 1 Complete set of PBPs identified with Boc-FL in whole cel

Figure 1 Complete set of PBPs identified with Boc-FL in whole cells of L. monocytogenes. Samples of whole cells (100 μg of selleck kinase inhibitor total protein) were labeled with Boc-FL at concentrations of 0 (1), 0.5

(2), 1 (3), 2.5 (4), 5 (5), 10 (6), 50 μM (7) and 50 μM plus 100 μg/ml ampicillin (8). Labeled bands were detected directly on the gel, quantified, and their selleckchem molecular mass estimated. The affinity of each band for Boc-FL (ID50) was estimated from their fluorescence as a function of the concentration of Boc-FL. The name of the PBP corresponding to each band is indicated on the right, while the positions of molecular weight markers (bars) and unspecific bands (arrowheads) are shown on the left. Table 2 Competition binding assay and affinity of different PBPs of L. monocytogene s for Boc-FL PBP Boc-FL Kd50 a Ampicillin c PPBA1 (PBP1) >10 μM 95 PBPB2 (PBP2) 0.25 μM 90 PBPB1 (PBP3) 0.25 μM 0 PBPA2 (PBP4) 0.25 μM 90 PBPB3 >20 μM 95 PBPD1 (PBP5) 5.0 μM 0 PBPC1 >20 μM 100 PBPC2 >20 μM 100 PBPD3 n.a. n.a. PBPD2 2.5 μM b 0 b a affinity of the respective bands for Boc-FL Epacadostat ic50 estimated from their fluorescence as a function of the concentration of Boc-FL (Kd50) b obtained with purified recombinant Lmo2812 c percentage of Boc-FL binding capacity remaining after sample was preincubated with 100 μg/ml ampicillin Characterization of protein Lmo2812 (PBPD2) Gene lmo2812 was amplified by PCR

from the wild-type EGD strain and cloned in vector pET30a without

its putative lipobox signal peptide. Expression of the His-tagged fusion protein in E. coli BL21(DE3) cells was induced with IPTG and it was purified from cell lysates on a nickel affinity column. The recombinant Lmo2812 protein was eluted from the column by washes with 250 and 500 mM imidazole. These two fractions were combined and further purified on a desalting Y-27632 2HCl column, yielding 4 mg/ml of pure protein. The purified protein was incubated with different concentrations of Boc-FL (0.25, 0.5, 2.5, 5 and 10 μM). Saturation binding studies showed that Lmo2812 covalently bound Boc-FL, indicating that the recombinant protein retained its authentic activity. Lmo2812 was the major band on gels, with a slower migrating minor band thought to represent a dimeric form (Figure 2). Figure 2 Purified recombinant L. monocytogenes Lmo2812 (PBPD2) identified with Boc-FL. Samples of purified recombinant Lmo2812 (10 μg) were labeled with Boc-FL at concentrations of 0 (1), 0.25 (2), 0.5 (3), 2.5 (4), 5 (5) and 10 μM (6). Labeled bands were detected directly on the gel, quantified and their molecular mass estimated. The affinity of the bands for Boc-FL (Kd50) was estimated from their fluorescence as a function of the concentration of Boc-FL. The names of the bands are indicated on the right, and the positions of the molecular weight markers are shown on the left.

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