Anti-hBD-2 polyclonal antibody was selleck chemical purchased from Peptide International, Inc (Louisville, Kentucky, USA). Lyophilised NVP-AUY922 powder of anti-hBD-2 antibody was reconstituted to the stock concentration of 10 mg/ml with sterile phosphate buffered saline

(GIBCO BRL). Bronchial epithelium medium (BEGM) was obtained from Lonza Group Ltd (Basel, Switzerland). Maintenance of endotoxin-free conditions Experiments were designed to minimise endotoxin contamination by using purchased endotoxin-free plasticware and heating all glassware at 180°C for 4 hours. All solutions used in the experiments contained less then 0.007 endotoxin unit/ml (minimal detectable level) when tested with Limulus amebocyte lysate assay (Sigma). A. fumigatus organisms were washed in

the solution containing Polymixin B during preparation. Patient material Human nasal turbinates of patients undergoing turbinectomy selleck chemicals llc (Pr. G. Lamas, La Pitié-Salpêtrière University Hospital Centre, Paris, France) were used for the preparation of the primary epithelial cells. All patients signed an informed consent form before participating in this research protocol, which was approved by the Institutional Ethics Committee. Fungal strain and growth conditions The A. fumigatus strain, CBS 144.89 (Institut Pasteur, Paris, France), was used throughout this study. A. fumigatus conidia were prepared as previously described [22]. Briefly, conidia of A. fumigatus were obtained from cultures grown on YM agar (0.3% yeast extract, 2% malt extract, 0.5% peptone and 0.5% agar) for three days at 37°C. Conidia were harvested by flooding the plates with sterile distilled water and then suspending the hydrophobic conidia in 0.01% Tween 20 in phosphate-buffered solution (PBS). To remove hyphae and debris, the conidial

suspension was filtered through four levels of gauze. The RC obtained were maintained at 4°C. Preparation of swollen conidia and hyphal fragments SC were prepared as described [47]. Briefly, 5 × 109 of resting A. fumigatus conidia were incubated in 200 ml of Sabouraud medium for 5 hours at 37°C in order to obtain the isodiametric swelling of the conidium resulting in Suplatast tosilate the development of SC. As demonstrated by microscopic examination, the majority of the organisms were single conidia, with a few small clumps containing two to four organisms. To obtain a homogeneous preparation, the suspension was gently sonicated for 10 seconds using a Branson Sonifier 450 (output level 2; Branson Ultrasonics, Danbury, CT, USA). Before exposure of the cells to conidia, the solution was vigorously vortexed and observed microscopically to ensure the absence of clumps. Hyphal fragments (HF) were prepared by incubating 2 × 108 of resting conidium in 200 ml of Sabouraud medium for 18 hours at 37°C with shaking in order to obtain a homogenous solution of the small HF. The tubes were then centrifuged in order to spin down the pellet.

We therefore plated the MC4100-derived Selumetinib research buy strains CT32, containing a single-copy rpoE-lacZ fusion,

JLM164 and JLM165, containing LEE1 lacZ and LEE4 lacZ fusions, respectively, and as a negative control, strain MCamp containing a single-copy bla-lacZ fusion on DMEM agar. Sterile disks containing 15 μl of varying concentrations of zinc acetate were placed on the lawns of bacteria on selective medium containing X-gal, and growth proceeded overnight at 37°C. A relatively small zone of growth inhibition was noted surrounding the disk containing 100 mM zinc acetate for all strains tested (Figure 3). Thus high concentrations of zinc inhibited growth of these MC4100 derivatives. Consistent with our previous assays, we observed decreased β-galactosidase activities, PD0325901 in vivo indicated by a lack of blue color, surrounding the zinc acetate-containing disks on the plates containing the JLM164 and JLM165 strains, demonstrating that LEE1 and LEE4 expression was

down-regulated in the presence of zinc acetate. However, we also observed similar down-regulation of β-galactosidase activity derived from the bla-lacZ negative control fusion from strain MCamp, suggesting that zinc caused a generalized down-regulation of gene expression in E. coli. Figure 3 Zinc downregulates both genes related and non-related to virulence but not  rpoE.  Overnight cultures of single-copy lacZ fusions JLM164 (LEE1−lacZ; A), JLM165 (LEE4−lacZ; B), MCamp (bla−lacZ; C), and CT32 (rpoE−lacZ; D) were spread evenly Aprepitant onto DMEM plates containing 30 mg/ml X-gal. Discs of sterile filter paper were dropped onto the lawn; 15 μl of different concentrations of zinc acetate were placed on each disc RG-7388 order (100 mM, 50 mM, 10 mM, 1 mM, 0.1 mM). These plates were grown for approximately 18 hours and then moved to

4°C for 6 hours to develop the blue color. Virulence genes were downregulated in the presence of zinc (A & B), but so was the bla gene encoding β-lactamase (C). In contrast, rpoE was not downregulated in the presence of zinc (D). Also of note is the small (∼1 mm) zone of growth inhibition around the 100 mM and 50 mM discs. In contrast to these results, we did not observe a down-regulation of the rpoE-lacZ fusion from strain CT32 in the presence of any of the zinc acetate concentrations tested, indicated by blue color directly adjacent to the disks (Figure 3D). Consistent with this observation, by Miller assay [32], β-galactosidase activity derived from the rpoE-lacZ fusion strain CT32 in DMEM increased 1.7-fold from 512±24 to 865±19 Miller units (Student’s t-test; n=3;p< 0.05) in the presence of 0.3 mM zinc acetate. Because rpoE expression occurs via a mechanism whereby the alternate sigma factor rpoE is released from the cytoplasmic membrane upon insult [33], we concluded that E. coli grown in DMEM experiences envelope stress in the presence of zinc acetate, consistent with previously published reports using complex media [30, 31].

The carboxy terminus of CpcA contained a region similar to the basic region of bZIP superfamily of transcription factors with strong sequence similarity to that of the homolog in A. fumigatus or A. nidulans (Figure 2C). selleck chemical In contrast with the Aspergillus homologs, the leucine zipper region contained three conserved leucine residues characteristic of a leucine zipper L-x(6)-L-x(6)-L-x(6)-L (Figure 2C). As expected for a protein with a transcription factor domain, CpcA was predicted by PSORT II to be localised in the nucleus (69.6% probability) and SignalP did not predict the presence of an N-terminal signal peptide (98.7% probability). In A. nidulans, cpcA transcription is autoregulated via cross pathway

regulatory elements (CPRE) 5′ TGA-(C/G)-TCA-3′ in the cpcA promoter [13]. Point mutations in CPRE lead to low levels of cpcA transcripts and CpcA protein, when amino acids are limited. Such an element matching the consensus was present on the minus strand in the promoter region of L. maculans cpcA (-698 to -703). Figure 2 A) The cpcA locus of Leptosphaeria maculans. The conserved leucine zipper region at the C-terminus of cpcA is dark grey. The open boxes indicate the upstream Open Reading Frames uORF1 and uORF2 in the 5′ leader region. The black dot preceding them represents the putative cross-pathway control element (CPRE) whose sequence is 5′TGACTCA3′. B) Alignment

of the deduced amino acid sequence of uORF2 with counterparts in the leader sequences of Aspergillus VX-770 molecular weight fumigatus (Af) cpcA (GenBank XP_751584.1) and A. nidulans (An) cpcA (GenBank

AF302935). Black boxes with white text denote amino acids identical in two of the three fungal species. Grey boxes with white Celecoxib text mark conserved changes. Gaps are introduced to optimize alignment. C) Alignment of the deduced amino acid sequence of the C-terminus conserved leucine zipper region with that of A. fumigatus CpcA and A. nidulans CpcA. The thick black line denotes the bZIP transcription factors basic domain signature (PS00036). Asterisks mark positions where conserved leucine residues characteristic of a leucine zipper (L-x(6)-L-x(6)-L-x(6)-L) should be found. Role of CpcA in sirodesmin PL production in L. maculans Although insertion of the T-DNA downstream of cpcA in mutant GTA7 reduced the transcript size by 127 bp, it did not reduce transcript levels of cpcA compared to those of the wild type (data not shown). Since the efficiency of gene disruption in L. maculans is very low, RNA Selleckchem Ferrostatin-1 mediated silencing was exploited to develop an isolate with extremely low levels of cpcA transcripts in order to study the effect of cpcA on sirodesmin PL production. Several putatively-silenced transformants were analysed and one, cpcA-sil, with 10% transcript level of that in wild type, as seen by q RT-PCR analysis, was chosen for further analysis (data not shown).

Tokyo: Japan Diabetes Society; 2004. 11. Yokoyama H, selleck compound Kawai K, Kobayashi M, Japan Diabetes Clinical Data Management Study Group. Microalbuminuria is common in Japanese type 2 Selleckchem C646 diabetic patients: a nationwide survey from the Japan Diabetes Clinical Data Management Study Group (JDDM 10). Diabetes Care. 2007;30:989–92.PubMedCrossRef 12. Parving HH, Lewis JB, Ravid M, Remuzzi G, Hunsicker LG, DEMAND investigators. Prevalence and risk factors for microalbuminuria in a referred cohort of type II diabetic patients: a global perspective. Kidney

Int. 2006;69:2057–63.PubMedCrossRef 13. Katayama S, Moriya T, Tanaka S, Tanaka Y, Yajima H, Sone S, et al. Low transition rate from normo- and low microalbuminuria to proteinuria in Japanese type 2 diabetic individuals: the Japan Diabetes Complications Study (JDCS). Diabetologia. 2011;54:1025–31.PubMedCrossRef

14. Adler AI, Stevens RJ, Manley SE, Bilous RW, Cull CA, Holman RR, UKPDS GROUP. Development and progression of nephropathy in type 2 diabetes: The United Kingdom Prospective Diabetes Study (UKPDS 64). Kidney Int. 2003;63:225–32.PubMedCrossRef 15. Valk EJ, Bruijn JA, Bajema IM. Diabetic nephropathy in humans: pathologic diversity. Curr Opin Nephrol Hypertens. 2011;20:285–9.PubMedCrossRef 16. Kamijo-Ikemori A, Sugaya T, Yasuda T, Kawata T, Ota A, Tatsunami S, et al. Clinical significance of urinary liver-type fatty acid-binding protein in diabetic nephropathy of type 2 diabetic patients. Diabetes Care. 2011;34:691–6.PubMedCrossRef nearly selleckchem 17. Mima A, Arai

H, Matsubara T, Abe H, Nagai K, Tamura Y, et al. Urinary Smad1 is a novel marker to predict later onset of mesangial matrix expansion in diabetic nephropathy. Diabetes. 2008;57:1712–22.PubMedCrossRef 18. Kimura T, Ikeda H, Fujikawa J, Nomura K, Aoyama T, Wada Y, et al. Usefulness of serum cystatin C in Japanese patients with type 2 diabetes mellitus and nephropathy. Diabetes Res Clin Pract. 2009;83:e58–61.PubMedCrossRef 19. Perkins BA, Ficociello LH, Ostrander BE, Silva KH, Weinberg J, Warram JH, et al. Microalbuminuria and the risk for early progressive renal function decline in type 1 diabetes. J Am Soc Nephrol. 2007;18:1353–61.PubMedCrossRef 20. Levey AS, de Jong PE, Coresh J, Nahas ME, Astor BC, Matsushita K, et al. The definition, classification and prognosis of chronic kidney disease: a KDIGO Controversies Conference report. Kidney Int. 2011;80:17–28.PubMedCrossRef 21. Yokoyama H, Sone H, Oishi M, Kawai K, Fukumoto Y, Kobayashi M, Japan Diabetes Clinical Data Management Study Group. Prevalence of albuminuria and renal insufficiency and associated clinical factors in type 2 diabetes: the Japan Diabetes Clinical Data Management study (JDDM15). Nephrol Dial Transplant. 2009;24:1212–9.PubMedCrossRef 22. Caramori ML, Floretto P, Mauer M. Low glomerular filtration rate in normoalbuminuric type 1 diabetic patients: an indicator of more advanced glomerular lesions. Diabetes. 2003;52:1036–40.PubMedCrossRef 23.

5%. For each pbp gene restriction pattern identified, one isolate was randomly chosen and re-amplified by PCR check details for nucleotide sequencing. Contig assemblages of the DNA sequencing were performed as described above. Nucleotide sequence accession numbers Sequences determined in this study have been deposited in the DBJ/EMBL/GenBank database under accession numbers AM889231 to AM889284 for stkP, AM779386 to AM779409 for penA, AM779338 to AM779361 for pbpX, and AM779362 to AM779385 for pbp1A. Results Influence of stkP mutation on penicillin susceptibility in a model system The role of StkP in penicillin resistance, has

been assessed by genetic analysis in the laboratory transformable strain Cp1015 (Table 1). The penicillin sensitive strain Cp1015 was transformed with DNA from the serotype 9V resistant strain URA1258 related to the international multiresistant clone Spain23F-1 [21]. Penicillin-resistant transformants were selected selleck kinase inhibitor on plates containing

0.1 μg ml-1 of penicillin. One transformant was isolated: strain Pen1, isogenic to Cp1015 but with mutations in PBP2X and 2B and resistant up to 0.125 μg ml-1 of penicillin. Strain Pen1 was then transformed with DNA from URA1258 and transformants were selected on plates containing 0.5 μg ml-1 penicillin; this gave strain Pen2 isogenic to Pen1 but for mutations in pbp1A and resistant to 0.5 μg ml-1 penicillin. Transformation of strains Cp1015, Pen1 and Pen2 with plasmid plSTK (Table 1) and selection on chloramphenicol plates gave the corresponding isogenic strains differing by their PBP and StkP alleles. The MICs of these strains were determined: the StkP- allele significantly and reproducibly increased penicillin susceptibility (Table 3). The StkP- mutations not only increased

the penicillin susceptibility of strain Cp1015 carrying wild-type penicillin binding proteins, but was also epistatic on mutations PBP2B, 2X and 1A; therefore StkP acts 3-MA research buy upstream from the PBPs. Table 3 Resistance phenotype and transformability of RX derivatives with different combinations of PBP and StkP alleles Strain Genotype MIC Pena (μg ml-1) URA1258 Multiresistant strain closely related to Spain 23F-1 clone 0.5–1 Cp1015 PRKD3 Rx derivate, str1; hexA 0.016 Cp7000 Cp1015, stkP::cat 0.008 Pen1 Cp1015, penA and pbpX from URA1258, allelic exchange mutant 0.064 – 0.125 Pen2 Cp1015, penA, pbpX and pbp1A from URA1258, allelic exchange mutant 0.38 – 0.5 Pen1STK Pen1, stkP::cat 0.016 – 0.032 Pen2STK Pen2, stkP::cat 0.032 – 0.125 a: MIC Pen, Minimum inhibitory concentration for penicillin. ND, not determined. Polymorphism of stkP in clinical isolates and relationship to penicillin resistance The effect of the StkP- mutation on penicillin susceptibility, as observed in an isogenic system, led us to question the importance of the stkP gene on penicillin susceptibility among clinical isolates.

The lower capacitance of GO compared to ERGO is also in accordance with previous reports, thus GO is not useful for supercapacitor applications [34–38]. Table 1 Parameters of GO and ERGO obtained using EIS WE Q (S·s

n ) n R 2(Ω·cm2) W (S·s1/2) C (F cm-2) GO 1.5 × 10 -6 0.9096 196.9 1.99 × 10 -3 6.66 × 10 -7 AC220 order ERGO 8.04 × 10 -6 0.9100 32.7 3.47 × 10 -3 3.30 × 10 -6 Cyclic voltammetry in [FeII(CN)6]3-/4- redox couple Cyclic voltammetry with the [FeII(CN)6]4-/[FeIII(CN)6]3- redox couple in 0.1 M KCl supporting electrolyte was done on both the GO and ERGO films with a SCE as the reference. Figure 6a,b shows the voltammetric reponse for GO and ERGO films at 50 mV·s-1. In Figure 6a, the PRT062607 anodic and cathodic currents of the redox couple for the GO film has almost similar baseline currents, as shown by the two straight lines very close to each other. The baseline for the anodic and cathodic currents has larger separation for ERGO films as shown in Figure 6b. This is due to the larger surface capacitance of the highly polarized ERGO surface, which was mentioned earlier in the “FESEM and EIS” section. Both anodic and cathodic currents for the GO film show straight

lines from the plots of I vs. ν 1/2 as shown in Figure 6c. From the Avapritinib concentration Randles-Sevcik equation , the diffusion coefficient (D) of the [FeII(CN)6]4-/[FeIII(CN)6]3- redox couple in 0.1 M KCl was estimated to be 5.9 × 10-10 m2·s-1. Figure 6 Cyclic voltammetry at 50 mV·s -1 with 23 mM [Fe II (CN) 6 ] 4 – / [Fe III (CN) 6 ] 3 -

redox couple and I vs. v 1/2 plots. Cyclic voltammetry in 0.1-M KCl supporting electrolyte (a) GO and (b) ERGO and (c) I vs. ν1/2 plots of GO. Conclusion Solid-phase electrochemical reduction of GO films on graphite in alkaline solution produced ERGO which was confirmed with FTIR and Raman spectra. The EIS results obtained using [FeII(CN)6]4-/[FeIII(CN)6]3- redox couple in 0.1-M KCl supporting electrolyte indicated that the charge transfer resistance for ERGO is lower Sorafenib order than GO and is consistent with the higher electrical conductivity of ERGO. The results also reveal that the capacitance of ERGO is larger than GO, due to its higher polarity of ERGO. This result is also supported by voltammetry of both GO and ERGO in [FeII(CN)6]4-/ [FeIII(CN)6]3- redox couple in 0.1-M KCl supporting electrolyte, where ERGO surface has a larger separation of the anodic and cathodic baseline currents due to the larger capacitance compared to the GO surface. Acknowledgements The authors would like to thank University Malaya and Ministry of Higher Education for providing financial assistance with grant number FP033-2013A and RG181-12SUS for this work. References 1. Becerril HA, Mao J, Liu Z, Stoltenberg RM, Bao Z, Chen Y: Evaluation of solution-processed reduced graphene oxide films as transparent conductors. ACS Nano 2008,2(3):463–470.CrossRef 2.

5, 200 genes were found to be up regulated and 144 genes down regulated by dexamethasone, and 115 genes were up regulated and 137 genes down regulated by Pneumocystis infection. Principle component analyses revealed that the results generated from the twelve microarrays were of excellent quality (Fig. 1). Because of costs, only one time point (eight weeks after organism inoculation) was examined in this IWR-1 datasheet study; this

was a time when the Dex-Pc animals were heavily infected with the organism. An in vitro microarray study had been conducted previously using the human A549 www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html alveolar epithelial cells line [26]. The cells were incubated with P. carinii organisms for 2 hr and then analyzed for global gene expression with the Affymetrix human U95 Arrays. The results showed that some epithelial genes controlling cell cycle progression such as the ras-related rho gene and cyclin G-interacting protein gene were highly up-regulated by P. carinii. TNF-inducible protein and the pim oncogene that are involved in apoptosis signaling as well as inflammatory cytokines and Sepantronium solubility dmso chemokines including Gro-beta, IL-8, ICAM-1, MIP-3 and RANTES were also up-regulated [26]. Another microarray study was conducted by Hernandez-Novoa et al. [27]. They used total RNA from lung cells of wild type and CD40L knockout C57BL/6 mice infected with P. murina for various length of time (7 to 41 days) and found

that 349 genes related to immune responses were up-regulated in wild type mice but not in CD40L-KO mice. The genes involved in innate

response were up-regulated first followed by those involved in adaptive immunity. This study revealed how healthy, immunocompetent hosts respond to Pneumocystis infection [27]. In our study, we used AMs from P. carinii-infected rats to investigate how Pneumocystis affects AM functions by identifying genes that are up- or down-regulated during Pneumocystis infection. IPA analyses showed that many cellular functions of AMs were affected by Pneumocystis infection (Fig. 3). Among them, antigen presentation, cell-mediated immune response, humoral immune Resveratrol response and inflammatory response were most profoundly affected. Up-regulation of genes involved in antigen presentation, such as Tap1, RT1-Bb and RT1-Db1, reflects the attempts AMs make to activate the adaptive immune responses. The observation that most genes involved in both cell-mediated and inflammatory responses were up regulated (Tables 1 and 2) implies that antigen presentation by AMs is functional during PCP. This postulation is consistent with that of Hernandez-Novoa et al. [27]. The fact that PCP progresses despite activation of cell-mediated immune response and inflammatory response indicates that other cellular functions are disabled. Due to the lack of appropriate antibodies, immunosuppression of rats is usually achieved by treatment with dexamethasone which is known to have an anti-inflammatory and a wide range of side effects.

Immunostaining for cytoplasmic

myosin VI and membranous PCI-34051 in vitro E-cadherin was classified as follows: negative and weak positive were considered negative and moderate and strong positive were considered positive. Immunostaining was classified negative and positive for nuclear myosin VI, E-cadherin and beta-catein as well as cytoplasmic beta-catein. The result was considered positive when any staining was detected. Statistical analyses SPSS for Windows 15 (Chicago, IL, USA) was used for statistical analyses. The chi-squared test or Fisher’s exact test was used to study associations between different variables. Survival was analysed with the Kaplan-Meier curve and significance with the log rank test. The Cox regression multivariate model was used for multivariate analysis using Fuhrman grade, stage, tumour GSK2118436 concentration diameter, age or gender as adjusting factors. Results Patient demographics and staining correlation with clinical parameters At the time of diagnosis, the median age of patients was 63 years (range 29-86 years). Seventy-seven (51%) patients were women and 75 (49%) men. The median follow-up time was 90 months (range 0-209 months). During follow-up, 44 (29%) patients Selleck AZ 628 died because of RCCs, 40 (26%) died of other causes and 68 (45%) patients were still alive. The distribution of tumour classes (TNM classification), clinical stages, tumour grades and the histological subtype

of the RCC in comparison to the immunostaining pattern for myosin VI, beta-catenin and E-cadherin are described in Table 1, Table 2 and Table 3, respectively. Table 1 Associations between immunostaining for myosin VI and tumour class, stage, grade and histological subtype of RCC.   Cytoplasmic myosin VI Nuclear myosin VI   positive negative positive negative Tumour class (T)         1 (n = 71) 54 (76%) 17 (24%) 25 (35%) 46 (65%) 2 (n = 11) 6 (55%) 5 (45%) 3 (27%) 8 (73%) 3 (n

= 57) 41 (72%) 16 (28%) 20 (35%) 37 (65%) 4 (n = 6) 3 (50%) 3 (50%) 3 (50%) 3 (50%) Stage         I (n = 66) 50 (76%) 16 (24%) 23 (35%) 43 (65%) II (n = 11) 6 (55%) 5 (45%) 3 (27%) 8 (73%) Dolichyl-phosphate-mannose-protein mannosyltransferase III (n = 49) 35 (71%) 14 (29%) 19 (39%) 30 (61%) IV (n = 19) 13 (68%) 6 (32%) 6 (32%) 13 (68%) Grade         I (n = 5) 5 (100%) 0 (0%) 1 (20%) 4 (80%) II (n = 79) 59 (75%) 20 (25%) 31 (39%) 48 (61%) III (n = 38) 28 (74%) 10 (26%) 10 (26%) 28 (74%) IV (n = 21) 10 (48%) 11 (52%) 8 (38%) 13 (62%) Histological subtype of RCC         clear cell (n = 128) 89 (70%) 39 (30%) 46 (36%) 82 (64%) papillary (n = 10) 9 (90%) 1 (10%) 2 (20%) 8 (80%) chromophobic (n = 5) 4 (80%) 1 (20%) 2 (40%) 3 (60%) undifferentiated (n = 2) 2 (100%) 0 (0%) 1 (50%) 1 (50%) Number of patients with different characteristics and respective cytoplasmic and nuclear myosin VI immunostaining are presented. Table 2 Associations between immunostaining for beta-catenin and tumour class, stage, grade and histological subtype of RCC.

In addition to Salubrinal mouse phenol stress, the colR-deficient bacteria experience serious glucose-related stress resulting in lysis of a subpopulation of cells [10]. Importantly, cell lysis does not occur on medium with gluconate which is degraded like glucose through Entner-Doudoroff pathway. To test whether inactivation of the TtgABC efflux pump would affect phenol stress only on glucose or it would have a more general role in phenol tolerance,

the growth of newly constructed ttgB- and ttgC-deficient strains were examined both on glucose and gluconate minimal media supplemented with different concentrations of phenol (Fig. 1). In accordance with the transposon mutagenesis screen, the disruption of the ttgABC operon made P. putida colR-deficient cells more GSK1904529A ic50 resistant to phenol, and this behaviour was observed on both, glucose and gluconate medium. However, MCC950 manufacturer since the ttgB- and ttgC-deficiency enhanced phenol tolerance also in the wild-type background (Fig. 1), we consider that the TtgABC efflux pump is related to a general

tolerance of bacteria to phenol rather than to a particular phenotype of the colR mutant. Increased phenol tolerance per se does not alleviate the phenol-enhanced autolysis of glucose-grown colR-deficient cells neither does it restore transposition of Tn4652 in the colR mutant In our previous study we showed that phenotypes of the colR-deficient bacteria such as membrane leakiness and cell lysis, which are related with growth on glucose, became more prominent if phenol was added to the medium [10]. For instance, glucose-induced release of cytoplasmic β-galactosidase into the growth medium due to the autolysis of the colR mutant was

significantly enhanced if phenol was supplied [10]. In order to find out whether the increased phenol mafosfamide tolerance can alleviate glucose-induced and phenol-enhanced autolysis of the colR-deficient strain, the ttgC-knockout derivatives were subjected to β-galactosidase assay. To calculate the percentage of unmasked β-galactosidase activity which was used as an indicator of membrane leakiness and cell lysis, the enzyme activity was measured both in suspension of cells permeabilized with SDS and chloroform (total activity), and in that of intact, non-permeabilized cells. In accordance with our previous results only 4% of total β-galactosidase activity was measurable using non-permeabilized wild-type cells regardless of the presence of phenol in the growth medium [10] (Fig. 2). At the same time, about 15% of total β-galactosidase activity was detectable in case of the colR-deficient cells grown on glucose minimal plates, and up to 30% when cells were grown on glucose medium supplemented with 1 mM phenol [10] (Fig. 2). The phenol tolerant ttgC single mutant behaved in this test like the wild-type strain (Fig. 2).

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