TUNEL+ cells in the bone marrow were significantly reduced by PTH compared with control (h). *p < 0.05; **p < 0.01; ***p < 0.001 versus control (VC-VC); † p < 0.05; †† p < 0.01 versus the ALN/DEX-VC group PTH promoted tooth BMN 673 order extraction wound healing

after ALN/DEX treatment Next, treatment effects were assessed in the jaw. In the interradicular bone near the tooth extraction site (Fig. 1c), the ALN/DEX treatment resulted in significantly higher bone volume and BMD than control (Fig. 5a). The average bone anabolic effect of PTH was significantly higher in the VC than ALN/DEX treatment group. In the tooth extraction sockets, bone fill and BMD were significantly higher in the ALN/DEX treatment groups than controls (Fig. 5b). PTH significantly enhanced bone fill and BMD regardless check details of the presence or absence of the ALN/DEX treatment. However, the average PTH bone anabolic effect was significantly less in the ALN/DEX vs. VC treatment group, again indicating that the ALN/DEX treatment suppressed bone anabolism by PTH in the tooth extraction wounds. Fig. 5 Treatment effect

on the maxillae. a The result of microCT assessment of undisturbed maxillae. Regardless of treatment, significantly higher bone mass and BMD were found in the treatment groups vs. control. The ALN/DEX treatment significantly increased trabecular thickness and decreased trabecular separation compared with control. No PTH anabolic effect was observed after the ALN/DEX treatment, EPZ015938 cell line while PTH increased bone mass significantly after VC. b The result of microCT assessment of tooth extraction wounds. Both the ALN/DEX and PTH treatments resulted in significantly

higher bone mass, trabecular thickness, and BMD than control. Although PTH significantly increased bone mass and BMD after ALN/DEX, an average increase in bone mass by PTH was significantly smaller after ALN/DEX than VC. *p < 0.05; **p < 0.01; ***p < 0.001 versus control (VC-VC); † p < 0.05 versus the ALN/DEX-VC group PTH rescued ALN/DEX-associated deterred tooth extraction wound healing Tooth extraction wound healing was assessed histomorphometrically. Representative photomicrographs of the trichrome-stained sections of the tooth extraction wounds at 2 weeks post-extractions are shown Mirabegron in Fig. 6a. Open wounds with exposed bone were noted in six rats in the ALN/DEX-VC group and 1 rat in the ALN/DEX-PTH group. In vehicle control (VC-VC), three rats showed open wounds, while no open wound was noted in the VC-PTH group. PTH administration after tooth extractions promoted healing regardless of the presence or absence of the ALN/DEX treatment with significant improvement after the ALN/DEX treatment. TA was defined as the portion of the maxilla surrounding the roots of M2 (Fig. 1d) and bone area (BA/TA) was assessed. The histomorphometric assessment revealed significantly higher bone area in the ALN/DEX-VC, ALN/DEX-PTH and VC-PTH groups vs. control (Fig. 6b), which was consistent with the microCT findings (Fig. 5b).

The criteria of primary systemic vasculitis proposed by the European Medicines Agency (EMEA) algorithm was employed for enrollment [2]. This study was registered on the University Hospital Medical Information Network Clinical Trials Registry (UMIN000001648). Patients were evaluated at 3, 6, 12, 18, and 24 months and at relapse. The primary outcome measure was remission rate, and secondary outcome measures were survival rate, renal survival rate, and relapse. In total, 156 AAV patients were enrolled; all Screening Library observations were completed by March 2013. Final data collection is in progress. Co-RemIT-JAV Based

on our retrospective study elucidating the risk factors for relapse in patients with myeloperoxidase (MPO)-ANCA positive MPA [1], we are conducting an observational cohort study of remission maintenance therapy in Japanese AAV patients (Co-RemIT-JAV) (UMIN000006373). The study objective is to clarify the safety and efficacy of remission maintenance therapy in Japanese AAV patients. At present, 60 of 156 AAV patients registered in RemIT-JAV were extended to follow up every 6 months

up to 48 months after the end of follow-up for RemIT-JAV. The primary outcome measure is relapse rate, and secondary outcome measures are survival and BGB324 chemical structure renal survival rates. The observation stage will be completed by March 2015; data collection is currently in progress. RemIT-JAV-RPGN After RemIT-JAV, we conducted a nationwide, prospective cohort study of remission induction therapy in Japanese patients with ANCA-associated vasculitides and rapidly progressive glomerulonephritis (RemIT-JAV-RPGN) (UMIN000005136) including 47 university hospitals and referring hospitals. Enrollment of consecutive patients

newly diagnosed with AAV began in April 2011 and will CHIR98014 continue till December 2013. The primary and some secondary outcome measures are the same as those in RemIT-JAV, but pathological analysis of renal involvement and radiological analysis of pulmonary involvement will be added. Further, oxyclozanide biological samples (serum, urine, and total RNA) will be collected and offered to the Basic and Pathological Research Subcommittee for Research for identifying candidate biomarkers. Prospective cohort study for large-sized vessel vasculitis We also conducted a nationwide Japanese prospective observational study on the current state and efficacy of therapeutics for large-vessel vasculitis (UMIN000010414). The subjects included patients newly diagnosed with Takayasu arteritis and giant cell arteritis. The study objective was to clarify the current state and efficacy of therapeutics for large-vessel vasculitis in Japan and to evaluate the utility of the current diagnostic criteria and classification for large-vessel vasculitis. The primary outcome measure of this study is remission rate.

In the promoterless BW25113 ΔP relBEF strain, we did not Selleckchem Torin 2 see induction of the relBEF mRNA nor the characteristic accumulation of its

3′ portion (Additional file 1: Figure S3). We still saw a find more transcript that could be detected by the relE and relF probes (Additional file 1: Figure S3B,C) but the level of this transcript did not depend on the RelE production. It might be initiated from a constitutive promoter that was newly created by deletion of P relBEF . Transiently induced smear of RNA that was detected in BW25113 ΔP relBEF with the relB probe (Additional file 1: Figure S3A, lanes 6 and 7) is transcribed from the RelB-expression plasmid pKP3033. That is the reason why we omitted this plasmid when we studied induction see more of relBEF in response to RelE (Figure 1, Additional file 1: Figure S3, lanes 8–11). Thus, we can be sure that the shorter transcripts that massively pile up in response to toxins are indeed cleavage products and are initiated at the genuine P relBEF promoter. Next, we tested whether over-production of the toxin RelE activates other toxin-antitoxin genes in the chromosome. The northern hybridization results show strong induction of the mqsRA, mazEF, dinJ-yafQ, hicAB, yefM-yoeB, and prlF-yhaV TA systems (Figure 2). Similarly to relBEF, the induced transcripts were cleaved and the toxin-encoding parts seem to accumulate preferentially

while the antitoxin-coding parts are more effectively degraded. That appears to be true irrespective of whether the toxin is encoded by the first (mqsRA, hicAB) or the second (mazEF, yefM-yoeB, prlF-yhaV) gene

of the operon (Figure 2). Reliable testing of this phenomenon requires characterization of the cleavage products and additional experiments in the future. Additional experiments indicated that transcriptional cross-activation of TA operons does not occur between all possible TA combinations. Northern hybridization using mqsR probe showed that overproduction of MazF and HicA does not induce the mqsRA promoter while YafQ and HipA induce Fenbendazole it (data not shown), as well as RelE (Figure 2). Activation of mazEF by amino acid starvation is dependent on relBE We wanted to test whether TA cross-activation happens also during natural physiological stresses. Amino acid starvation has been shown to induce transcription of the relBE[14] and mazEF[17] genes. We induced amino-acid starvation by addition of mupirocin to the cultures of BW25113 (wild type) and BW25113ΔrelBEF. Northern analysis indicated that transcription of mazEF is upregulated only in wild type bacteria and not in the relBE deficient strain (Figure 3B). Transcription of mqsRA, the other TA operon that we tested, was induced in both strains, independently of the RelBE system (Figure 3A).

Eur J Agron 18:267–288CrossRef Keating BA, Carberry PS, Bindraban #ATR inhibitor randurls[1|1|,|CHEM1|]# PS, Asseng S, Meinke H, Dixon J (2010) Eco-efficient agriculture: concepts, challenges, and opportunities. Crop Sci 50:S109–S119. doi:10.​2135/​cropsci2009.​10.​0594 Knowler D, Bradshaw B (2007) Farmers’ adoption of conservation agriculture: a review and synthesis of recent research. Food Policy 32:25–48CrossRef Kokic P, Nelson R, Meinke H, Potgieter A, Carter J (2007) From rainfall to farm incomes—transforming advice for Australian drought policy. I. Development and testing of a bioeconomic

modelling system. Aust J Agric Res 58:993–1003. doi:10.​1071/​ar06193 CrossRef Kropff MJ, Bouma J, Jones JW (2001) Systems approaches for the design of sustainable agro-ecosystems. Agric Syst 70:369–393CrossRef Kuratorium für Technik und Bauwesen in der Landwirtschaft (2009) Kalkulationsdaten, Pflanzenproduktion. Kuratorium für Technik und Bauwesen in der Landwirtschaft eV (KTBL) Darmstadt. http://​www.​ktbl.​de Lal R BIBW2992 purchase (2000) Soil management in the developing countries.

Soil Sci 165:57–72CrossRef Lal R (2002) Carbon sequestration in dryland ecosystems of West Asia and North Africa. Land Degrad Dev 13:45–59CrossRef Lampurlanés J, Angás P, Cantero-Martínez C (2002) Tillage effects on water storage during fallow, and on barley root

growth and yield in two contrasting soils of the semi-arid Segarra region in Spain. Soil Tillage Res 65:207–220CrossRef Leenders R, Heydemann S (2012) Popular mobilization in Syria: opportunity and threat, and the social networks of the early risers. Mediterr Politics 17:139–159. doi:10.​1080/​13629395.​2012.​694041 CrossRef López-Bellido L (1992) Mediterranean cropping systems. In: Pearson CJ (ed) Field crop ecosystems. Elsevier, Amsterdam Luo Z, Wang E, Sun OJ, Smith CJ, Probert ME (2011) Modeling long-term soil carbon dynamics and sequestration potential Anacetrapib in semi-arid agro-ecosystems. Agric For Meteorol 151:1529–1544CrossRef McCown RL (2001) Learning to bridge the gap between science-based decision support and the practice of farming: evolution in paradigms of model-based research and intervention from design to dialogue. Aust J Agric Res 52:549–571CrossRef McCown RL (2002) Changing systems for supporting farmers’ decisions: problems, paradigms, and prospects. Agric Syst 74:179–220CrossRef Meinke H, Hammer GL, van Keulen H, Rabbinge R, Keating BA (1997) Improving wheat simulation capabilities in Australia from a cropping systems perspective: water and nitrogen effects on spring wheat in a semi-arid environment.

3). These result indicate that the association of DNT with FN is not related to the intoxication. When human FN was supplied to the culture, FN-null cells showed the colocalization of the toxin and FN. In contrast, DNT did not colocalize with the FN network developed on MRC-5 cells (Fig. 3). These results suggest that DNT does not interact directly with FN, and another cellular component, which is present in the culture of FN-null cells but not MRC-5 cells, is necessary for the

interaction. In fact, MRC-5 cells supplemented with the culture supernatant of FN-null cells showed the colocalization of DNT and the FN network (Fig. 4). Treatment with heat at 95°C or proteinase K abolished the ability of the culture supernatant to recruit DNT to the FN ICG-001 concentration network, which indicates that the unknown

component exists in the culture supernatant of FN-null cells and contains a protein moiety (data not shown). Figure 3 Colocalization of DNT with the FN network on various cells. Cells were incubated with DNT and stained with anti-DNT monoclonal antibody and anti-FN polyclonal antibody. FN-null cells were incubated with or without human FN (hFN) before DNT treatment. Bars, 5 μm. Figure 4 Colocalization of DNT with the FN network on MRC-5 cells supplemented with the culture supernatant of FN-null cells. MRC-5 cells, which were pre-cultured with or without the culture supernatant of FN-null cells (FN-null CS), were incubated with DNT and stained with anti-DNT monoclonal antibody and anti-FN polyclonal antibody. Bars, 5 μm. Screening for a molecule mediating R788 the colocalization of DNT and the FN network We tried to isolate the unknown component from the culture supernatant

of FN-null cells by ion-exchange chromatography (Fig. 5A). Each fraction eluted by Mono Q anion-exchange chromatography was added to the culture of MRC-5 cells, and checked for the ability to recruit DNT to the FN network. second Simultaneously, each fraction was subjected to SDS-PAGE and proteins in the fractions were identified by mass spectrometry. Fraction 4 apparently induced the association of DNT with the FN network on MRC-5 cells (Fig. 5B). Mass AR-13324 manufacturer spectrometry revealed that fraction 4 contains ECM-related proteins such as nidogen-2 in an N-terminally truncated form (open arrowhead), and lysyl oxidase-homolog 2 (LOXL2) and 3 (LOXL3) (Fig. 5C). Similar results were obtained from the culture supernatant of MC3T3-E1 cells: the truncated form of nidogen-2 (open arrowhead) and LOXL3 were found in fraction 4, which induced the association of DNT with the FN network on MRC-5 cells (Fig. 5D). LOXL2 was expressed at neither the mRNA nor protein level in MC3T3-E1 cells, which show intensive colocalization of DNT and the FN network (Fig. 3). LOXL3 supplemented to the culture did not induce the colocalization of DNT with the FN network on MRC-5 cell (data not shown).

Subperithecial tissue a dense homogeneous t. epidermoidea–angularis of variously shaped, thin-walled, hyaline cells (5–)7–26(–36) × (4–)5–11(–13) (n = 30); cells smaller towards the base, and interspersed with thick-walled, yellowish hyphae, (2.0–)2.5–4.5(–6.0) μm (n = 30) wide. Asci (75–)88–106(–117) × (4.0–)4.5–5.5(–6.5) μm, stipe (6–)9–23(–35) μm long (n = 73); no croziers seen. Ascospores hyaline, verruculose; cells dimorphic, but often similar; distal cell (3.5–)3.8–5.0(–6.0) × (3.3–)3.5–4.2(–5.0) μm, l/w 1.0–1.3(–1.7) (n = 72), subglobose or slightly elongated and attenuated upward; proximal cell (3.5–)4.3–6.2(–7.6) × (2.7–)3.0–3.6(–4.7) μm, l/w (1.1–)1.3–1.9(–2.3) (n = 72), oblong, wedge-shaped,

#Selleckchem EPZ5676 randurls[1|1|,|CHEM1|]# or subglobose. Cultures and anamorph: optimal growth

at 25°C on all media; no growth at 35°C. On CMD after 72 h 7–11 mm at 15°C, 22–28 mm at 25°C, 11–21 mm at 30°C; mycelium covering plate after 7–8 days at 25°C. Colony hyaline, distinctly circular with well-defined margin, with little mycelium on surface, forming up to 7 broad and 6 narrow concentric zones. Mycelium radially arranged, with conspicuous difference in width between primary and secondary hyphae. Surface hyphae degenerating, appearing empty. Aerial hyphae scant, short, more frequent and longer mainly at distal margin of the plate Autolytic activity and coilings absent or rare. No distinct odour noted. Sometimes pale yellowish on distal margin from 2 weeks, with minute yellow crystals at the very BIBW2992 in vitro distal margin in densely packed mycelium. Chlamydospores (7–)8–12(–16) × (5.5–)6–11(–14) μm, l/w 0.9–1.5(–2) (n = 32), noted after 50 days, uncommon, terminal and intercalary, globose,

ovoid or clavate. Conidiation from 1 to 2 weeks, macroscopically invisible, scant, effuse, on loosely disposed, minute, simple conidiophores spreading from the plug and proximal margin; at distal margin also on long aerial hyphae; greenish only in the stereo microscope; degenerating from ca 3 weeks; cultures usually sterile after several Thymidine kinase transfers. On PDA after 72 h 4–9 mm at 15°C, 19–26 mm at 25°C, 8–14 mm at 30°C; mycelium covering plate after 8–10 days at 25°C. Colonies circular, dense, compact, indistinctly zonate, mycelium radially arranged, surface hyphae becoming moniliform in the centre due to ?chlamydospores. Aerial hyphae inconspicuous, loosely disposed, short and needle-like, superposed by scant thin and long hyphae, decreasing outwards, forming thin radial strands, soon degenerating, collapsing, giving surface finely downy to granular appearance. Autolytic activity and coilings absent or rare. Odour faint, like fermenting fruits (noted from 1 weeks), colony turning pale or greyish yellow, 3AB3–4, 3B5–6, from the centre. Conidiation from 3 to 5 days, macroscopically invisible, effuse, short, spreading from the plug, becoming farinose in the centre, remaining colourless (1 month). At 15°C conidiation dense in white central area.

Free PHB granules, i.e. PHB granules that were not in contact CBL0137 research buy to the TH-302 chemical structure nucleoid region were not observed. Apparently, constitutive over-expression of phaM resulted in formation of an increased number of small and nucleoid-attached PHB granules. If PhaM is responsible

for the formation of small granules and for the close contact to the nucleoid region, deletion of phaM should have a phenotype. In fact, R. eutropha ∆phaM cells accumulated only very few (0–2) PHB granules that were significantly larger in diameter than those of the phaM over-expressing mutant or of the wild type (Figure 5). Since the diameters of PHB granules of the ∆phaM strain were considerably larger even at early time points a precise analysis whether or not the granules were attached to the nucleoid region was difficult. In most ∆phaM cells the PHB granules were still located close to the nucleoid; however, Buparlisib mw at least in some cells a detachment of PHB granules from the nucleoid region could not be excluded for the wild type or for the phaM over-expressing strain. A clear decision whether the absence of PhaM resulted in detachment from the nucleoid can, however, not be made. Since

R. eutropha expresses at least one other protein with DNA-binding and PHB-binding property (PhaR) [30, 31] it might be that PhaR also contributes to association of PHB with DNA. In summary, our data on mutants with altered expression of PhaM clearly show that number, diameter and subcellular localization of PHB granules depends on the presence and concentration of PhaM. Time course of formation and localization of PHB granules in R. eutropha over-expressing PhaP5 PhaP5 had previously been identified as a phasin in R. eutropha by its in vivo interaction with PhaP2 and other phasins [22]. Remarkably, PhaP5 also interacted

with PhaM. To investigate the influence of PhaP5 on clonidine PHB granule formation the phaP5 gene was cloned in a broad host range plasmid (pBBR1MCS-2) under control of the strong and constitutive phaC1 promotor (PphaC), transferred to R. eutropha H16 and HF39 via conjugation and investigated for PHB granules formation and localization under PHB permissive conditions (Figure 6). In case of strain HF39 a eypf-phaP5 fusion was cloned and used to confirm localization of PhaP5 on the PHB granules by fluorescence microscopy. Controls showed that free eYfp is a soluble protein in R. eutropha (Figure 7). Figure 7 Fluorescence microscopical (FM) investigation of R. eutropha H16 (pBBR1MCS-2-P phaC – eyfp -c1) with over-expression of eYfp (a); R. eutropha H16 (pBBR1MCS-2-P phaC – phaP5 ) with over-expression of PhaP5 (b), and R. eutropha H16 (pBBR1MCS-2-P phaC -eyfp- phaP5 ) with over-expression of eYfp-PhaP5 fusion (c) at various stages of PHB formation.

Based on the ‘+2 rule’ for lipoproteins, which relates the final location of a TGFbeta inhibitor lipoprotein to the amino acid in the

+2 position of the secreted protein [32], the likely cellular location of the Btp zymogens is coupled through a lipid moiety at the post-processing N-terminal Cys residue of the propeptide to the inner leaflet of the outer membrane. They would remain in this inactive form until an activation event occurred. As the proteases would thus have a periplasmic location, for them to contribute to virulence they must come into contact with the host. This could be achieved by a number buy Erismodegib of mechanisms (1) the presence of protease-specific transporters in the outer membrane, (2) by release of the proteases upon bacterial cell death and lysis, or (3) through vesicle-based transport, as previously described for B. fragilis[33]. In the case of the related organism P. gingivalis these vesicles have been associated with proteolytic activity [34, 35]. It is therefore not unlikely that the proteases described in this paper could be exported by vesicles

in a similar manner. The Bti proteins also include predicted leader peptides, and BtiA and BtiB are likely NSC23766 clinical trial to be lipoproteins, which would also most likely be associated with the outer membrane. BtiZ was not predicted to be a lipoprotein (the signal peptide for BtiZ has a signal peptidase I cleavage site) and it is therefore likely targeted to the periplasm of the Bacteroides cell. Having both membrane associated inhibitor and periplasmic inhibitors may be a strategy for maximizing protection afforded by these inhibitors against the C10 protease activity. Another possibility is that the BtiZ molecule is in the process of accumulating mutations

and becoming non-functional in response to loss of BtpZ activity. We have previously demonstrated the transcriptional Tangeritin coupling of B. fragilis C10 protease genes with those for staphostatin-like inhibitors [9]. In the current study transcriptional coupling was also identified for the B. thetaiotaomicron btp and bti genes by Reverse Transcriptase PCR. The btpA gene was found on the same message as btiA. Furthermore, transcriptional coupling was identified for btpB and btiB, and btpZ and btiZ. The btpC gene appears to be transcribed independently of adjacent btp and bti genes. Although, this study does not preclude that the btpA, btpB and btpZ genes could be transcribed independently of the bti genes, the data indicates a similar genetic linkage of these btp genes with staphostatin-like inhibitors as occurs in B. fragilis.

Results were calculated for the distance classes i = 1, 2, 3,…,10. These find more species richness grids S i were combined by performing an inverse distance-weighted approach according to: $$ S_w = \sum\limits_i

= 2^10 \left( d_i^ – p \right. \cdot \left. CHEM1 \right) + S_1 $$ (1)with p > 0, d ≥ 1. S 1 is the original point-to-grid species richness grid, S w is the grid of the resulting weighted species richness and d i is the distance (d 2 = 2, d 3 = 3,…) used as a threshold in the conditional triangulation. For each distance class, the increase in species richness relative to the next smaller distance class was calculated for each quadrat and selleck kinase inhibitor multiplied by a weighting term \(d_i^-p.\) Thereby, p is a tuning parameter of the weighting procedure applied to the quadrats. For each p > 0 and d ≥ 1, the corresponding weighting term lies between 0 and 1. The greater p becomes, the more relative weight is put on species richness calculated for smaller distances. The closer p is to 0, the more relative weight is put on species richness interpolated for larger

distances (see Appendix 2). For the present work, we selected p = 0.5, which resulted in a combination of high weights for small distances and relatively low weights for large distances. The weighted differences between the distance classes were then added to the original point-to-grid data (S 1), yielding the map of weighted species richness S w . Species richness centers were identified as contiguous areas of quadrats with S w  > 100, i.e. more than 100 interpolated species. Adjusting weighted species richness for sampling effort We addressed the impact of uneven spatial sampling effort by incorporating an additional weighting factor. This factor is based on the ratio of the number of species recorded in a quadrat and the maximum number of species reported for each

center of species richness C of the original point-to-grid map [S 1/max C (S 1)]. This relationship between the number of species in a quadrat to the respective reference quadrat is used as a proxy for sampling effort for each quadrat. The PLEKHB2 higher the relative sampling effort in a quadrat, the nearer it will be to 1, hence the smaller the weighting (1—relative sampling effort) for the respective quadrat will be (Eq. 2). The higher the weight (relative sampling effort close to 0), the larger is the fraction of the interpolated species richness that enters the final estimation of species richness for that specific quadrat. The application of this correction factor to the inverse distance-weighted sum of species richness at the distances 2–10, added to the observed point-to-grid species richness S 1 is henceforth referred to as adjusted species richness S adj. $$ S_\textadj = \left( 1 – \fracS_1 \max_C (S_1 ) \right)\,*\,\sum\limits_i = 2^10 \left( d_i^ – p \right. \cdot \left. {\left( S_i \right.

Homologues exist in all species and subspecies of Francisella, however, they are not identical to PdpC of LVS. For example, PdpC in both LVS and SCHU S4 contains 1,328 amino acids, whereas the F. novicida U112 homologue contains 1,325 amino acids. The former two show only 18 amino acid differences, whereas check details 71 and 72 amino acids (95% identity), respectively, are distinct compared to the F. novicida variant. Figure 1 shows a representation of the different genes found in the FPI, and the localization of pdpC at the end of one of the two putative operons. Figure 1 The Francisella pathogenicity island. The two operons are depicted as a sequence of

arrows in the direction of transcription. Arrows in light grey indicate genes with homology to known T6SS core components, while arrows in dark grey represent

genes that lack T6SS VX-689 nmr component homology. To investigate the subcellular localization of PdpC, LVS bacteria were separated into soluble, inner membrane, and outer membrane fractions and the amounts of the protein in each fraction determined learn more by immunoblot analysis. PdpC was found to be predominantly an inner membrane protein, but a small portion was also found in the soluble fraction (Figure 2). It is likely that the transmembrane regions identified in the in silico analysis may contribute to its membrane location. Figure 2 Subcellular localization of PdpC. LVS whole-cell lysate was separated into soluble, inner membrane (IM) and outer membrane (OM) fractions using ultracentrifugation and Sarkosyl treatment. After separation by SDS-PAGE, the presence of PdpC in each fraction was determined by Western blot using polyclonal anti-PdpC antibodies. Antibodies recognizing IglC and PdpB were used as markers for soluble and inner membrane fractions, respectively. mafosfamide Construction and phenotypic characterization of a ΔpdpC null mutant To determine the role of PdpC in F. tularensis LVS, an in-frame deletion mutant was constructed by deletion of both copies of the gene. To verify

the absence of PdpC in the mutant, immunoblot analysis with an anti-PdpC antibody was performed on bacterial pellets and real-time PCR was used to quantify the transcription levels of pdpC. No immunoreactive protein or gene transcript was detected in the mutant, whereas expression of the downstream pdpE gene was not affected (data not shown and Table 1), indicating that the deletion conferred no polar effect. For complementation in cis, the pdpC gene was introduced in the original site of one of the pathogenicity islands of the mutant. Table 1 Differences in FPI mRNA expression between ΔpdpC and LVS   Averagea P value vgrG −1.49 0.176 iglH −3.09 0.119 pdpC −6.8 × 10-6 <0.001 pdpE −0.26 0.913 iglD −3.46 0.010 iglC −3.99 0.055 iglB −2.97 0.040 iglA −3.75 0.080 a The results show the average fold change in gene expression from 7 experiments.