The best results were obtained using a fivefold molar excess of b

The best results were obtained using a fivefold molar excess of benzimidazole with respect to quinobenzothiazinium salts 2. It may be assumed that the other reaction product are benzimidazolium salts 5, the structure of which can be stabilized via delocalization

selleckchem of positive charge among the benzimidazole nitrogen atoms. Scheme. 3 Synthesis of compounds 4 Benzimidazolium salts 5 were neither isolated from the reaction mixture nor identified in the course of this study, as the primary objective here was to obtain quinobenzothiazine 4 derivatives as free quinoline bases. Excess benzimidazole and benzimidazolium salts 5 that form during the reaction were separated from quinobenzothiazines 4 by pouring post-reaction mixtures into water. Both benzimidazole and salts 5 are well-soluble in water, whereas

compounds 4 fall out of solution as solids. In order to obtain quinobenzothiazine derivatives 7 containing aminoalkyl substituents at the thiazine nitrogen atom, compounds 4 were transformed, in the presence of sodium hydroxide, selleck products into salts 6, which were then alkylated using aminoalkyl chlorides (Scheme 4). The reaction occurred as N-alkylation at the thiazine nitrogen atom and led to compounds 7. The structure of compounds 7 was confirmed with 1H NMR spectroscopy by performing NOE 1H–1H homonuclear experiment. By irradiating methylene group protons at the thiazine nitrogen atom an enhancement of H1 and H11 proton signals from compounds 7 was obtained (Scheme 5). Scheme. 4 Synthesis of compounds 7 Scheme. 5 NOE 1H–1H homonuclear experiment for compound 7a Antiproliferative activity The activity of the obtained compounds 4 and 7 was investigated in vitro using cultured SNB-19 and C-32 cell lines and cisplatin as a reference. The SB203580 in vitro examined quinobenzothiazines 4 had various substituents (CH3, F, Cl, Br) introduced into 9- and 11-positions of the quinobenzothiazine ring. In Reverse transcriptase addition, they also contain a nitrogen atom in the 8-position

of the quinobenzothiazine ring. Compounds 7 contains aminoalkyl substituents: 2-(N-piperidyl)ethyl (compounds 7(a–d)) and 3-(N,N-dimethylamino)propyl (compound 7e) at the thiazine nitrogen atom. One of the mechanisms involved in antiproliferative effects of chemotherapeutics is DNA intercalation. This mode of action is typical for antiproliferative anthracycline antibiotics (e.g., doxorubicin) that feature planar tetracyclic (aromatic or heteroaromatic) fused rings. This mode of action, affecting cancer cells’ DNA, has been indeed suggested in reports concerning antiproliferative properties of phenothiazine and benzo[a]phenothiazine derivatives (Motohashi et al., 2000; Hossain et al., 2008; Hossain and Kumar, 2009). Structurally, compounds 4 and 7 studied herein are their analogs. The experiments demonstrated that the majority of the investigated compounds 4 and 7 showed antiproliferative activity toward examined cell lines within the 5.6–12.

Authors’ contributions AJM-R and JJF conceived and designed the e

Authors’ contributions AJM-R and JJF conceived and designed the experiments.

AJM-R conducted the experiments. AG-O and AH-C conducted the AFM work and processed the results from AFM measurements. AM conducted the CLSM work. RD-G carried out the statistical analysis. VSM and MN contributed with reagents, materials and valuable advice in the experimental design. AJM-R, AG-O, AH-C and JJF analysed the data. AJM-R and JJF wrote PRN1371 ic50 the paper. All authors read and approved the final manuscript.”
“Background Methanogen diversity has been widely investigated across a range of this website ruminants by using clone library sequence approaches and many unknown methanogen 16S rRNA sequences have been uncovered. Tajima et al. [1] investigated the diversity of bovine rumen fluid using two different

see more archaea-specific primer sets, and for the first time reported the existence of a novel cluster of uncultured archaeal sequences which were distantly associated with Thermoplasma. However, the authors concluded that these novel sequences were likely from transient microbiota contaminating the animal feed, probably scavenging in an ecological niche in the rumen. Wright et al. [2] was the first to verify that these novel Thermoplasma-affiliated sequences were derived from the rumen when they investigated the diversity of rumen methanogens from sheep. The authors suggested a new order of methanogens for these novel sequences in the new cluster. The same authors [3] further found that over 80% of the total methanogen clones (63 of 78 clones) from the rumen of Merino sheep in Australia were 72–75% similar to Thermoplasmaacidophilum and Thermoplasmavolcanium. They [4] also found that about 50% of the total clones from methanogen 16S rRNA gene library Dolutegravir concentration of potato-fed feedlot cattle were present in the new cluster, and 38% for corn-fed feedlot cattle. Huang et al. [5] found that Thermoplasmatales-affiliated sequences dominated in the yak and cattle methanogen clone libraries, accounting for 80.9% and 62.9% of the sequences in the two libraries, respectively. Our previous study [6] on the

diversity of methanogens in the rumen of Jinnan cattle showed that Thermoplasmatales-affiliated sequences were widely distributed in the rumen epithelium, rumen solid and fluid fractions. In addition, ruminant-derived sequences in this new cluster were also found in other studies [4, 7–12]. Based on the analysis of the global data set, Janssenand Kirs [13] placed the majority (92.3%) of rumen archaea detected in total rumen contents into three genus-level groups: Methanobrevibacter (61.6%), Methanomicrobium(14.9%), and a large group of uncultured rumen archaea affiliated with Thermoplasmatales (15.8%), and named the uncultured archaea group in the rumen, for the first time, as Rumen Cluster C (RCC). Using RCC specific DGGE, clone library analysis and quantitative real-time PCR, Jeyanathan et al.

Citrobacter freundii is usually considered

a commensal sp

Citrobacter freundii is usually considered

a commensal species of the human gut, although some isolates have acquired specific virulence traits that enable them to cause diarrhea. Therefore, virulence factors homologous, and some even identical, to those described in E. coli pathotypes were detected in C. freundii strains isolated from sporadic cases of infantile diarrhea [26–29]. Additionally, isolates of C. freundii have been identified as effective recipient strains even since the first articles concerning E. coli conjugation mediated by F pili were published [30]. Reports on check details the transfer of E. coli thermo-stable toxin genes between these species raised considerations about the virulence potential of the bacterial conjugation [29, 31, 32]. A highly conjugative plasmid (pCTX-M3), which is responsible this website for the extensive spread

of extended-spectrum β-lactamase (ESBL) in Enterobacteriaceae, was described in clinical isolates of C. freundii. pCTX-M3 is a 89,468 bp-plasmid belonging to IncL/M group that probability evolved from environmental plasmids through stepwise integration of mobile genetic elements. Moreover, it has been shown that this plasmid is easily transferred to E. coli, Klebsiella sp., Enterobacter cloacae, Serratia marcescens and Salmonella enterica strains [33, 34]. Nowadays, it is known that phenotypic features classically associated with pathogenic E. coli strains are not restricted exclusively to this species. In addition to EAEC, the AA pattern has been recognized in uropathogenic Proteus mirabilis strains [35] and in Klebsiella pneumoniae strains recovered from healthcare-associated infections [36]. In these isolates, the expression of AA pattern has been associated with the ability to form biofilms. Bacterial biofilms found in natural and pathogenic ecosystems are formed in the presence of multiple species Amobarbital and genetically distinct strains. However, the current understanding of these microbial consortia is largely based on single-species models that frequently

use laboratory strains. In this work, wild-type strains of typical EAEC and C. freundii, which were concomitantly recovered from diarrhea, were tested in mixed GSK461364 purchase biofilm assays in order to evaluate the occurrence of synergistic effects on biofilm formation. Firstly, it is shown that the diarrhea-isolated C. freundii strain shared the characteristic AA phenotype displayed by EAEC strains, and henceforth was named aggregative C. freundii (EACF). It follows that EACF strain 205 and diarrhea-isolated typical EAEC strains cooperate to increase bacterial adhesion to HeLa cells and biofilm formation. Moreover, the synergic effect was associated with putative F pili expressed by EAEC strains. Results Aggregative C. freundii During a case-control study of infantile diarrhea, C. freundii strains were isolated from two subjects. The C.

Science 313(5783):58–61PubMedCrossRef PS-341 price Cash DW (2001) ‘In order to aid in diffusing useful and

practical information’: agricultural extension and boundary organizations. Sci Technol Human Values 26(4):431–453CrossRef Cash DW, Clark WC, Alcock F, Dickson NM, Eckley N, Guston DH, Jäger J, Mitchell RB (2003) Knowledge systems for sustainable development. Proc Natl Acad Sci USA 100(14):8086–8091PubMedCrossRef Cash DW, Borck JC, Patt AG (2006) Countering the loading-dock approach to linking science and decision making. Sci Technol Human Values 31(4):465–494CrossRef Cash DW, Moser SC (2000) Linking global and local scales: designing dynamic assessment and management processes. Glob Environ Chang 10:109–120CrossRef Choi BCK, Pang T, Lin V, Puska P, Sherman G, Goddard M, Ackland MJ,

Sainsbury P, Stachenko S, Morrison H, Clottey C (2005) Can scientists and policy makers work together? J Epidemiol Community Health 59(8):632–637PubMedCrossRef Churchman C (1967) Wicked problems. Manage Sci 4(14):141–142 Cortner HJ (2000) Making science relevant to environmental policy. Environ Sci Policy 3(1):21–30CrossRef Demeritt D (2006) Science studies, climate change and the prospects for constructivist critique. Econ Soc 35:453–479CrossRef Dilling L, Lemos MC (2011) Creating usable science: opportunities and constraints for climate knowledge use and their implications for science policy. Glob Environ Chang 21(2):680–689CrossRef Engels A, Hisschemöller M, von Moltke K (2006) When supply meets demand, yet no market emerges: the contribution of integrated Dibutyryl-cAMP environmental assessment to the rationalisation of EU environmental policy-making. Sci Public Policy 33:519–528CrossRef Fairbrass J, Jordan A (2004) Multi-level governance and environmental policy. Bacterial neuraminidase In: Bache I, Flinders MV (ed) Multi-level governance. Oxford University Press, Oxford, pp 147–164CrossRef Farrell K, Van den Hove S, Luzzati T (2013) What lies beyond reductionism?

Taking stock of interdisciplinary research in ecological economics. In: Farrell K, Luzzati T, Van den Hove S (ed) Beyond Reductionism: a passion for interdisciplinarity. Routledge studies in ecological economics. Routledge, London Funtowicz S, Ravetz J (1993) Science for the post-normal age. Futures 25(7):735–755CrossRef Grandjean P (2013) Science for precautionary decision-making in: EEA, Late lessons from early warnings: science, precaution, innovation. EEA Report N 1/13 Gray B (2003) Framing of environmental NVP-BGJ398 cost disputes. In: Lewicki RJ, Gray B, Elliott M (ed) Making sense of intractable environmental conflicts. Island Press, Washington DC, pp 11–34 Guston D (1999) Stabilizing the boundary between politics and science: the role of the office of technology transfer as a Boundary Organization.

The catalytic core was defined

The catalytic core was defined BMS202 purchase by a set of structurally conserved elements, including elements P3 to P8. G391-C277 of intron-F was assumed to be G-binding positions [14]. Extended P5 and P9 stems were displayed in the putative structure of intron-F from PV1. Nine intron-Fs from nine strains (PV2, 3, 28, 33, 34 and 41 and TH9, 31 and 35) of P. verrucosa

were predicted to be the same structures as the putative structure of intron-F derived from PV1 drawn in Figure 4[A], alternatively, shown in Additional file 3. These nucleotide variations among intron-F were observed mainly in the loop and at four positions where one nucleotide of P5a, two of P5.1a and one of P5.2 stem were positioned. The base pairs GU and CG within P6 were

formed in the core region of intron-F [12]. The nucleotides A71, A72, U73 were located in segments J3/4 of PV1 intron-F [15–18]. These predictions of secondary structure revealed that all intron-Fs were IC1 group 1 introns. Figure 4 A-C. – Diagrams for predicted secondary structure of P. verrucosa. [A]: intron-F from rDNA of PV1, [B]: intron-G from PV1 and [C]: intron-G from PV3. Capital letters indicate intron sequences and lowercase letters indicate flanking exon sequences. Arrows point to the 5′ and 3′ splice sites. The guanosin cofactor-binding sites are marked with *. The structure of intron-G (L1921) from PV1 was drawn just as was done for intron-Fs (Figure 4[B]). A G-C pair within P7, i.e. G390-C360, was assumed to be the G-binding positions. The GU-CG pair of P6 and the AAU in J3/4 was the same as in the intron-F core region of PV1. This putative Gilteritinib intron-G exhibited expanded regions of P1 and P5. The three intron-Gs of PV1, PV33 and PV34 were found to be similar among the three strains. Different features were found in PV3 as shown Lck in Figure 4[C] wherein the sequence of PV3 differed in P1 region among four trains; namely, short stems in P1b and P1c and small bulge loops of L1 and L1a (Additional file 4). Moreover, PV3 added P2.0 and P8c, although the other intron-Gs did not. Prediction structures in the remaining two introns of PV33 and PV34 are not shown. Nevertheless, all subgroups

of intron-G were also identified as IC1, based on comparison of tertiary structures across segments P3-7 of the four strains. In conclusion, we have identified that the ten intron-Fs and four intron-Gs of P. verrucosa belong to IC1 group 1 introns. Characterization of intron-H Loss of P5abcd domain in derived S788 introns was correlated with inability to self-splice in vitro in a previous report [19]. Accordingly, we have not confirmed LY333531 datasheet insertion positions of intron-H by RT-PCR. However, we examined PV-28 strain as the representative strain of intron-H by analyzing the sequence alignment of the core region of subgroup IE from other organisms in the database. Moreover, we predicted the secondary structure of this intron-H as shown in Figure 5.

They may combine an affinity for sulfated polysaccharides and oth

They may combine an affinity for sulfated polysaccharides and other polymeric carbon molecules [10, 11] produced by their eukaryote hosts with a resistance to eukaryote chemical defense molecules. The resulting competitive advantage over other bacterial groups that are utilizing the same kind of substrates, for example the Bacteroidetes [35] might be one of the keys to the success of planctomycetes in a wide variety of environments on earth. Our results show differences between the different sampling times (February, July and September), in planctomycete abundance, OTU composition and diversity. For example, in February there is a relatively low abundance of planctomycetes (Figure 1) compared

to July and September. This may be linked to Ganetespib cell line the

age of the kelp tissue, as the kelp lamina is older in February selleck chemicals llc compared to in July Baf-A1 and September due to the seasonal growth cycle of the kelp. Aging of the kelp tissue could be associated with lowered antibacterial chemical defense by the kelp, as the old kelp lamina is to be shed soon after February, and does therefore not need to be defended against microbial colonization. Without the presence of chemical defense substances, the planctomycetes could loose their competitive advantage over other bacterial groups, explaining their lower abundance in February. The senescence of the kelp tissue as it ages could also cause the appearance of new niches involved in degradation of different kelp constituents, thereby enabling the more diverse planctomycete communities that are observed in February compared to July and September (Table 1, Figure 6). Among the different planctomycete lineages that are represented on the kelp, the lineage defined as “”RB1″” in this study appears to be the most abundant, accounting for a majority of the clones at all sampling times (Figure 4). The high abundance of

RB1 planctomycetes may thus be the cause of the observed dominance of planctomycetes on kelp surfaces (Figs. 1 Progesterone and 2). Their high abundance implies a lifestyle that makes them particularly successful on kelp surfaces. Yet the lineage also includes reference sequences from a variety of other marine habitats, indicating that RB1 is not a kelp-specific lineage. The RB1 and RB2 lineages, defined in this study, are clearly related to the “”Pirellulae”", a lineage including the genera Pirellula, Rhodopirellula and Blastopirellula (formerly all included in the genus Pirellula). Yet our phylogenetic analyses did not place them reliably with any of the described genera, indicated by the bootstrap support for the relevant branches in Figure 4. There are no sequences of cultured strains within the RB1 and RB2 lineages available in the databases. Another uncultured lineage, the so-called OM190 planctomycetes (Silva taxonomy) is also represented by clones from kelp surfaces at all sampling times, yet in low numbers.

The DR, together with

The DR, together with Selleck ARN-509 the DL, supported the dorsal-left side of the pocket, and the DMt supported the dorsal-right side. The VR – reinforced by the VL – lined the ventral side of the pocket and was in contact with the IR that lined

the ventral-left side of the flagellar pocket. The microtubules of the DMt and the VR became part of the elements forming the cytostomal funnel and accessory rod (i.e., the C-shape rod apparatus in general), and both the DR and the IR became part of the sheet of microtubules underlining the plasma membrane of the entire cell. Molecular Phylogenetic Position In order to infer the phylogenetic position of B. bacati, we PCR-amplified and sequenced the nearly complete SSU rDNA gene (2057 bp) from two independent isolates. The sequences contained expansions typical of euglenozoan SSU rDNA genes. First, we carried out a 40-taxon Maximum likelihood (ML) analysis that included sequences representing all of the major groups Rigosertib of eukaryotes; the resulting phylogeny showed B. bacati grouped strongly within the

Euglenozoa (not shown). A second analysis included 37 taxa representing all of the major lineages of euglenozoans. The phylogenetic analyses showed that the euglenozoan sequences clustered in five main subgroups with high statistical support (Figure 12): (i) a kinetoplastid clade   (ii) a diplonemid clade   (iii) a bacteriovorous euglenid clade   (iv) a eukaryovorous + phototrophic euglenid clade and   (v) the Symbiontida, a newly named clade that includes Calkinsia aureus and several environmental sequences. Bihospites bacati clustered with the Histone demethylase Symbiontida with extremely high statistical support (ML bootstrap value = 100% and Bayesian posterior probability > 0.95), as the sister lineage to the rest of this group. Calkinsia aureus branched next within the Symbiontida and formed the sister lineage to several environmental sequences (Figure 12). However, the relationship of the Symbiontida to the other main

subgroups within the Euglenozoa was unclear.   Figure 12 Phylogenetic position of Bihospites bacati n. gen. et sp. within the Euglenozoa as inferred from small subunit (SSU) rDNA sequences. Maximum likelihood (ML) analysis of 35 euglenozoan taxa, rooted with two jakobids (Andalucia incarcerata and A. godoyi). Only ML boostraps greater then 50% are shown. Thick branches correspond to Bayesian posterior probabilities over 0.95. Ba, bacterivorous taxa; Eu, eukaryovorous taxa; Ph, photosynthetic taxa. Discussion Bihospites bacati n. gen et sp. possesses all three synapomorphies that unify the Euglenozoa: a tripartite flagellar root system, heteromorphic paraxial rods and tubular extrusomes. Concordantly, our analyses of SSU rDNA sequences clearly places B. bacati within the Euglenozoa, specifically within the Symbiontida. Several studies based on environmental sequences indicated the existence of a novel rDNA clade of euglenozoans [9–11].

Figure 4 Fluorescent microscopy confirmed cell ratios Fluorescen

Figure 4 Fluorescent microscopy confirmed cell ratios. Fluorescent microscopy using labeled antibodies confirmed the presence CA4P price of each species in the community. Samples were stained with DAPI and fluorescently labeled antibodies: green for D. vulgaris and red for C. cellulolyticum. G. sulfurreducens cells were stained blue by DAPI as described in the Materials and Methods section. (A) An artificial mixture of 1:1:1, C. cellulolyticum: D. vulgaris:G. sulfurreducens. Each image was of the same microscopic field. Two separate images taken at different fluorescent wavelengths were 4SC-202 datasheet merged to form the image on the left showing C. cellulolyticum and D. vulgaris. The image in the

center was taken with DAPI and all cells are visible. The image on the right resulted from merging the fluorescent and DAPI images and reveals the G. sulfurreducens cells as stained blue

by DAPI. (B) The three species community culture shown in Figure 2 and described in the text was sampled during steady state growth and stained with DAPI and fluorescently labeled antibodies and merged as described above for (A). For (A) and (B) Arrows indicate the same cells of C. cellulolyticum, C.c., D. vulgaris, DvH, and G. sulfurreducens, G.s., imaged under the different conditions. Proposed Carbon and Electron Flow A model of carbon and electron flow for the three species community was derived from measurements of the three species community selleck chemicals llc steady-state, single culture chemostat experiments, and data from the literature (Figure 5 and Additional File 1 and Table 2). The 640 ml chemostat tri-culture exhibited an ID-8 OD600 of 0.4 with a 236 mg dry weight per liter of biomass. Based on qPCR ratios an approximation was made for each population

and used in the model (Table 2 and Figure 5). The overall carbon recovery was estimated at 93% when including cell mass. When modeled for the three populations the values ranged between 79-112%. Similarly, the overall electron recovery was 112% with the individual population models ranging from 83-122%. There was a larger loss of sulfate than readily accounted for causing a modeled electron recovery greater than 120% for D. vulgaris, while a loss of carbon in the fumarate-malate-succinate pool resulted in a lower carbon and electron recovery for G. sulfurreducens. Because succinate is a readily metabolized end product, 78% of the energy modeled to enter G. sulfurreducens was still in some digestible form that could potentially be available for additional microorganisms representing other trophic groups in future experiments. On the other hand, sulfide generation by D. vulgaris is of little value for other anaerobic trophic groups. Importantly, 71% of the end products from C. cellulolyticum were potentially digestible by other anaerobic trophic groups, and consumption of nearly half of those were evidenced in three-species community described here (Table 2 and Figure 5).

Our data showed that the

Our data showed that the VX-809 mouse expression of btuB was indeed reduced when E. coli cells were grown to stationary phase in an acidic medium as compared to the same cells grown in neutral medium (Table 4). The reduction in the production of btuB in response to acid stress probably represents a physiological regulatory mechanism of bacteria facing environmental challenges such as low pH. Under stress environment, bacteria need to alter their metabolism to adapt to the environmental change. The transportation of cobalamin by BtuB receptor is driven by proton motive force (PMF)[45]. Since the PMF of bacteria is increased at low pH[46], the cobalamin transportation may be

enhanced by increased PMF. The higher concentration of cobalamin in cytoplasm will initiate riboswitch mechanism to repress

the translation Blasticidin S supplier of BtuB receptor, which is in good accord with the repression of btuB transcription by the acid-induced GadX for bacteria to decrease the production of BtuB in response to this acidic stress. Conclusions Through biological and biochemical analysis, we have demonstrated the GadX can directly interact with btuB promoter and affect the expression of btuB. When bacteria were grown to stationary phase in an acidic medium, the increased gadX expression would repress the btuB transcription to help bacteria to adapt to acidic shock. In conclusion, this study provides the first evidence that the expression of btuB gene is transcriptionally repressed by the acid responsive genes gadX and gadY. Methods Plasmid constructions To check details produce the His6-tagged ColE7/Im7 protein complex for the ColE7 resistance assay, pQE30ColE7-Im7 was constructed. The cea7-cei7 genes encoding the colicin E7 and immunity proteins, that form an active ColE7 complex, were amplified from plasmid K317 [47]

by PCR using primers F/cea7-BamHI and R/cei-PstI (Table 5). The 1,996-bp PCR product thus generated was inserted between BamHI and PstI sites of pQE30 (Qiagen), fusing the His6-tag to the N terminus of ColE7. For searching transcriptional regulators of btuB, a genomic library of E. coli K-12 strain constructed with the pGAD10 vector (Figure 1) was purchased from Clontech (catalog number XL4001AB) and transformed into E. coli strain DH5α. The plasmid pGadXY (Figure Sclareol 1) was isolated from the library in this study. To investigate the effect of GadX on btuB expression, pGadX was constructed as follows. A 1,077-bp DNA fragment containing gadX was generated by PCR using pGadXY (Figure 1) as the template and the MATCHMAKER 5′ insert screening sequence 5′-TACCACTACAATGGATG-3′ (Clontech) and R/gadX-PstI (Table 5) as primers. This 1.1-kb PCR fragment was inserted into pGEM-TEasy (Promega) by TA cloning, generating pGEMgadX. The 1.1-kb fragment was then isolated from pGEMgadX by EcoRI digestion and inserted into the EcoRI site of pGAD10, resulting in pGadX (Figure 1).

(A) Total RNA was extracted from Jurkat cells infected with AA100

(A) Total RNA was extracted from Jurkat cells infected with AA100jm, dotO mutant, Corby, or flaA mutant (MOI of 100) for the indicated find more time intervals and used for RT-PCR. (B) Jurkat cells were infected with the indicated concentrations of L. Tozasertib clinical trial pneumophila for 4 h. Total RNA was extracted and used for RT-PCR. (C) Total RNA was extracted from CD4+ T cells infected with Corby (MOI of 50) for 3 h and used for RT-PCR. (D) Jurkat cells were infected with live L. pneumophila Corby or flaA mutant (MOI of 100) for 4 h or incubated with L. pneumophila under the indicated treatment for 4 h. PFA, paraformaldehyde.

Total RNA was extracted and used for RT-PCR. Representative examples of three experiments with similar results. To determine the Bucladesine clinical trial correlation between IL-8 expression level and L. pneumophila bacterial proteins, heat-killed Corby was used to infect Jurkat

cells at a multiplicity of infection (MOI) of 100. At 4 h, IL-8 was not expressed in Jurkat cells infected with the heat-killed strain (Fig. 2D). Furthermore, IL-8 gene expression was not induced when paraformaldehyde-fixed L. pneumophila was used (Fig. 2D). However, bacteria heated at 56°C for 30 min induced IL-8 expression. These results suggest that the surface proteins of bacteria but not lipopolysaccharide are required for IL-8 induction. Considered together, it seems that Legionella flagellin is involved in IL-8 expression in T cells. Flagellin is recognized by toll-like receptor 5 (TLR5) [8]. Thus, we also examined the expression of TLR2, TLR3, TLR4, and TLR5 mRNAs in Jurkat and CD4+ T cells. All TLR mRNAs examined were expressed in Jurkat and CD4+ T cells (Fig. 3A and 3B). Furthermore, their expression levels did not change by L. pneumophila infection in CD4+ T cells

(Fig. 3B) and Jurkat cells (data not shown). PJ34 HCl Figure 3 TLR mRNA expression in T cells. (A) Expression of TLR mRNA in Jurkat cells. Total RNA was extracted from Jurkat cells and used for RT-PCR. (B) CD4+ T cells were infected without or with Corby (MOI of 50) for 3 h. Total RNA was extracted from CD4+ T cells and used for RT-PCR. Representative examples of three experiments with similar results. IL-8 production from Jurkat cells during infection with L. pneumophila We used enzyme-linked immunosorbent assay (ELISA) to determine IL-8 protein levels in culture supernatants of Jurkat cells at 8, 12, or 24 h after infection with either the parental strain Corby or flaA mutant strain at an MOI of 100. IL-8 was induced by Corby in a time-dependent manner. On the other hand, the amount of IL-8 produced by Jurkat cells infected with the flaA mutant strain was significantly less than that by cells infected with the wild-type strain (Fig. 4A). Corby-induced IL-8 production by Jurkat cells was MOI-dependent (Fig. 4B). Corby also induced a significant amount of IL-8 from CD4+ T cells (Fig. 4C). Figure 4 IL-8 production from Jurkat cells during infection with L. pneumophila strains.