Although HPV + tumours typically present at a more advanced stage, they are associated with a more favourable prognosis. Tumour hypoxia has been associated with radioresistance but direct measurement of tumour oxygenation has practical limitations. Consequently, candidate endogenous markers of hypoxia (EMH) (e.g. Glucose Transporter 1 (GLUT1) and Carbonic Anhydrase IX (CAIX)) have been evaluated. No previous studies have stratified EMH analysis by HPV status. Moreover, there have

been no previous studies quantifying EMH expression within the stromal compartment of these tumours. Methods: Ninety-two patients diagnosed with locally advanced HNSCC and treated with concurrent cisplatin and radiotherapy between 2000 and 2005 were identified. Fifty-five patients LY2606368 nmr had pre-treatment FFPE tumours available for analysis. Triplicate 0.6 mm cores were assembled into TMAs. Semi-quantitative p16 immunohistochemistry (IHC) staining was used as a surrogate for HPV status. Automated, quantitative IHC (AQUA HistoRx™) was used to quantify staining for CAIX and GLUT1, as candidate EMH. We analysed the tumour and stromal expression of each

candidate EMH, stratified by tumour p16 status. Overall survival was estimated from Kaplan-Meier method and curves compared using a log rank test. Results: 53% of tumours were p16+ and 47% were p16-. For https://www.selleckchem.com/products/Cyt387.html patients with p16- tumours

and high stromal CAIX expression, 2-year overall survival was 33%, compared to 91% with low stromal CAIX expression (p < 0.05). At 5 years, this overall survival difference remained significant (42% vs 22%, respectively, p < 0.05). Epithelial CAIX expression was not a statistically significant Branched chain aminotransferase predictive factor. Conclusion: High stromal CAIX expression is a significant negative predictive factor for survival in locally advanced HNSCC patients with p16- tumours. This finding may impact therapeutic targeting for this patient group, including use of hypoxic radiosensitizers. Poster No. 7 Avastin Has a Direct Deleterious Effect on Multiple Myeloma Cell Lines Oshrat Attar1,2, Michael Lishner1,2,3, Shelly Tartakover Matalon1,2, Liat Drucker 1,2 1 Oncogenetic Laboratory, Meir Medical Center, Kfar Saba, Israel, 2 Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel, 3 Internal Medicine Department, Meir Medical Center, Kfar Saba, Israel Introduction: Multiple myeloma (MM) is an incurable malignancy of plasma cells.

In this work, the In desorption behavior in the molecular-beam-epitaxy (MBE) growth of InGaAs/AlGaAs MWIR QWIP was studied. With low-temperature capping technology of a thin AlGaAs,

the In composition can be well controlled. Methods The samples in this work were grown on a Si-GaAs substrate by a VG-80H MBE system and divided into two groups assigned as groups I and II. The growth rates were firstly determined by reflection high-energy electron diffraction and finely calibrated by X-ray diffraction (XRD) and photoluminescence MDV3100 chemical structure (PL) measurement. Group I including samples A, B, and C were used for observing the In composition-losing behavior in InGaAs. Figure 1a,b shows the growth procedure illustrations of samples A and B, respectively. Sample C was a complete replica of sample A. Figure 1 Procedure schematics GSK1120212 price of (a) sample A, (b) sample B, (c) sample D, and (d) sample E. In order to demonstrate the effect of a low-temperature thin AlGaAs capping layer on suppressing the In desorption, group II including samples D, E, and F were grown. All the three samples were designed to have the same structure with a peak

absorption wavelength from the inter-subband transition in InGaAs/AlGaAs quantum wells around 4.3 μm. The layer sequence of the sample structure is 1 μm Si-GaAs bottom contact layer, 20 periods of quantum wells consisting of 50 nm Al0.4Ga0.6As barrier, 0.5 nm GaAs, 2.7 nm In0.3Ga0.7As, and 0.5 nm GaAs. Then, 500 nm Si-GaAs top contact layer was grown to finish the structure. The growing details of samples D and E were displayed in Figure 1c,d, respectively. Finally, we prepared

another sample F using the same growth procedure of sample E. Results and discussions Considering the obvious growth temperature difference between InGaAs and AlGaAs, it is wise to grow InGaAs quantum well under low temperature, then increase the growth temperature to grow the AlGaAs barrier in order to achieve best crystal quality. It is without a doubt that such growth procedure will decrease the In composition in the InGaAs quantum well. However, if the In FER atom desorption behavior is predictable and repeatable, it is still possible to grow a determined In composition quantum well through the growth of an InGaAs layer with higher In composition to compensate the In loss during the increase of substrate temperature. So, we firstly design an experiment to study such phenomenon. Because both the quantum well composition and thickness contribute to the inter-band transition energy, it is impossible to determine the structural characteristics of the quantum well by PL directly. Besides, the InGaAs quantum well is as thin as around 2.7 nm in the 4.3 μm QWIP, it is also very hard to measure the precise composition by XRD technology. In order to quantitatively explore the In composition-losing behavior in the quantum well, samples A and B were grown.

for 60 min. The eluted ions were analyzed by one full precursor MS scan (400–1500 m/z) followed by four MS/MS scans of the most abundant ions detected in the precursor MS scan while operating under dynamic exclusion or direct data acquisition system. Spectra were obtained in the positive ion mode with a nano ESI-Q-Tof micro mass spectrometer (Micromass,UK), deconvoluted, and analyzed using the MassLynx software 4.1 (Micromass, UK). A peak list (PKL format) was generated to identify +1 or multiple charged precursor ions from the mass spectrometry data file. The instrument was calibrated in MS/MS

mode using 500 fmole of (Glu1)-Fibrinopeptide B human with a RMS residual of 3.495 e-3 amu or 7.722 e0 ppm. Parent mass (MS) and fragment mass (MS/MS) peak ranges were 400–1500 Da and 65–1500 Da, respectively. Mascot server v2.3.0 and Mascot Daemon Toolbox v2.3.0 (http://​www.​matrix-science.​com, UK) MEK inhibitor drugs in MS/MS ion search buy LY3009104 mode (local licenses) were applied to conduct peptide matches (peptide masses and sequence tags) and protein searches against NCBInr v20110707 (14605097 sequences; 4996850242 residues) using taxonomy filter S. pyogenes (24089 sequences, 6976687 residues). The following parameters were set for the search: carbamidomethyl (C) on cysteine was set as fixed; variable

modifications included asparagine and glutamine deamidation and methionine oxidation. One missed cleavage was allowed; monoisotopic masses were counted; the precursor peptide mass tolerance was set at 2 Da; fragment mass tolerance was 0.3 Da. The MS/MS spectra were searched with MASCOT using a 95% confidence interval (C.I.% ) threshold (p < 0.05), with which a minimum score of 36 was used for peptide identification (identity or extensive homology). The protein redundancy that appeared at the database under different gi and accession numbers were limited to S. pyogenes with the first priority assigned to NZ131. All proteins identified were found within these domains. Enzymatic activity assays To measure extracellular DNase activity, the wild-type and codY mutant strain were cultured for

24 h with CDM. Sterile CSPs were prepared exactly as was done for the protein analysis. CSPs were incubated Reverse transcriptase for various times at 37°C with PCR-generated DNA from S. pyogenes and 1X New England Biolabs buffer 2. The CAMP test was done by inoculating Staphylococcus aureus RN6390 onto agar plates containing sheep blood and then inoculating the wild-type and codY mutant strains perpendicular to RN6390. The plates were incubated for 18 h at 30°C. Cfa activity is indicated by increased hemolysis at the intersection of S. aureus and CAMP factor-producing strains of S. pyogenes. Biofilm assays Biofilm formation on polystyrene microtiter plates (Becton Dickinson, Lincoln Park, NJ) was done essentially as previously described [11]. Briefly, the strains were incubated with either CDM or THY for 24 h at 37°C in 5% CO2.

In order to determine whether the Tunisian PVL positive strains also carried the same PVL phage as phi7401PVL, we conducted two PCR studies to identify the regions in common with two PVL phages (phi7401PVL and phiSA2mw): a long range PCR study identifying gene linkage lukS and the tail gene that can identify find more PVL phages of the elongated head type and another PCR study identifying the region related to lysogeny (Additional file 1: Table S1 and Figure 1a). In our experiments, all the PVL positive strains were positive in both PCR studies. Discussion Antibiotic resistance to agents other than β-lactams The majority of the HA-MRSA isolates were resistant to kanamycin, amikacin

and tetracycline. Although the ratio was slightly low (25~55%), these strains were also frequently resistant to tobramycin, gentamicin, erythromycin, quinolones and rifampicin. Recently, it has been reported that rifampicin resistance is related to glycopeptides resistance [25, 26]. Since the ratio of rifampicin resistant

strains was relatively high, there is a possibility that there might be glycopeptides related to low resistance strains, e.g., hetero-VISA strains. However, glycopeptide resistance is beyond SC79 the focus of this study, so we did not examine the details for these findings. Similar to HA-MRSA isolates, the majority of CA-MRSA isolates were resistant to kanamycin, amikacin and tetracycline, but were susceptible to other antibiotics, except for erythromycin and ciprofloxacin. These data suggest that Tunisian CA-MRSA strains were more resistant to kanamycin, tetracycline and erythromycin than U.S. and Oceanian isolates [27]. In our study, only four CA-MRSA strains were resistant to clindamycin, thus suggesting that clindamycin can be used for the treatment of CA-MRSA infections in Tunisia. The PVL phage carried by Tunisian MRSA PDK4 The phi7401 carried by a ST80 Tunisian MRSA was highly homologous to phiSa2mw

carried by ST1-SCCmecIVa MRSA. Only two ORFs, TUP03 and TUP16, showed a lower identity to those of phiSa2mw. Interestingly, TUP03 was identical to ORFs in phi12, phi13, and the bacteriophage in MRSA strains JH1 and JH9, and TUP16 was highly homologous to dUTPase in phiSLT and phi108PVL, with nucleotide identities of 97%. These data suggest that the components of phages were chimeric. Numerous lysogenized phages were induced from the cells of four strains, including JCSC7401 by mitomycin C induction. However, a hybridization experiment with a PVL probe showed that no plaque of the PVL phase was observed. This might have been due to the carriage of a truncated int. It seems that lysogenization of the phage occurred early to thus cause a mutation in the phage genome or that the ST80 strains might have an ability to cause a mutation in the int to keep the inserted phage genome in the chromosome in a stable form.

This principle simply states that if protein A is homologous to protein B, and protein B is homologous to protein C, then protein A must be homologous to protein C, regardless of whether significant sequence similarity

can be documented for proteins A and C. Homology by definition means derived from a common ancestral protein. It is thus unnecessary to identify regions of high sequence similarity between two proteins if one or more sequences of adequate sequence similarity can be found that interlinks the aforementioned two sequences. To establish homology between repeat elements in the transmembrane domains of ABC importers, we used the Superfamily Principle as defined above to extend the significant internal homology decisions to other evolutionarily selleck inhibitor related proteins (e.g., derived from a common ancestor) [17, 18]. This principle has been used to establish homology for distantly

related members of extensive superfamilies [13, 19–21]. As documented in this communication, we have used statistical means to establish homology for all ABC uptake transporters except for TC family 3.A.1.21 which clearly belongs to the ABC1 family. Additionally, we have established homology for internal repeat elements in representative transmembrane domains [4, 17, 18]. Finally, we have obtained preliminary evidence that two of the six primordial TMSs in ABC2 protein (TMSs 3 and 4) gave rise to the 2 TMS repeat elements in ABC1 porters, suggesting that the evolution of ABC2 porters 4-Hydroxytamoxifen preceeded that of ABC1 porters. Many families

of integral membrane transport proteins evolved independently of each other following different evolutionary pathways [19]. These pathways involved intragenic multiplication events where the primordial genes presumably encoded channel-forming peptides, usually with one, two or three α-helical TMSs [19]. They duplicated, triplicated or quadruplicated—sometimes in a single step, sometimes in more than one step [19, 22, 23]. The bacterial maltose transport system proteins, MalF (P02916) and MalG (P68183) are two distinct membrane proteins that together comprise the channel of an ABC superfamily member. High resolution structural information Thiamine-diphosphate kinase is available for this system (TC# 3.A.1.1.1). Consequently, it is known that these two proteins differ in their TMS architecture. MalF has a 3 + 5 TMS structure whereas MalG has a 3 + 3 TMS structure. We here propose that these proteins, and almost all integral membrane constituents of ABC uptake systems, are of the ABC2-type as noted above, arising from a 3 + 3 repeat topology. This raises the question of how the MalF protein arose from a MalG-like precursor. The MalF protein contains a long hydrophilic sequence insert between TMS 3 and TMS 4.

Thank you, Andy, for the insights you have given us into topics that will be of broad interest to many people and I believe will benefit from

those for years to come.   Epilogue In his closing months in Berkeley, Benson worked feverishly with Jacques Mayaudon, a Belgian postdoc, in identifying S. Wildman’s Fraction I protein NCT-501 chemical structure as Rubisco. Benson left the manuscript with Calvin before departing for Penn State in 1954. Calvin presented the results in 1955 at the International Congress of Biochemistry, mentioning Mayaudon but not Benson (Cavin 1955). The critical findings were published in 1957 with Mayaudon as sole author (1957). It is not clear who submitted the Mayaudon manuscript. Benson became aware of these publications after Calvin’s death more than 40 years later. END OF VIDEO Acknowledgments A number of colleagues helped make this video possible. We wish to acknowledge our science advisers: Roland Douce (Grenoble), Hartmut Lichtenthaler (Karlruhe), George Lorimer (College Park) and Roger Summons (Cambridge); technical adviser,

Marie Felde (UC Berkeley); video production personnel, Mike Fausner and Matt Hale (Creative Services and Publications, UC San Diego); and the sponsor of the video, Energy Biosciences Institute (UC Berkeley). We also thank H. Lichthenthaler for comments on the manuscript. References Calvin M (1955) The photosynthetic carbon cycle. In Liébecq C (ed) Proceedings of the third international congress of biochemistry, Brussels, Academic press, New York, pp 211–225 Mayaudon J (1957) Study of association between the main nucleoprotein of green leaves and carboxydismutase. Enzymologia Proteases inhibitor 18:343–354PubMed”
“Introduction Photosynthetic acclimation to different levels of growth irradiance has been studied extensively (Boardman 1977; Anderson et al. 1995; Walters 2005). The same is true for growth temperature (Berry and Björkman 1980; Hikosaka

et al. 2006; Sage and Kubien 2007). Acclimation to irradiance and temperature is achieved by similar changes in the photosynthetic apparatus, associated metabolism and possibly shared sensory systems (Huner et al. 1998). The two environmental factors could thus interact in their ultimate effect on the photosynthetic apparatus. However, the combined effect of growth irradiance and temperature on photosynthesis has received much less attention in higher plants (Hikosaka 2005; before Muller et al. 2005). Reduced growth irradiance typically causes a reduction in the amount of Rubisco, other Calvin cycle enzymes and components of the electron transport chain, all expressed per unit leaf area. However, chlorophyll content remains generally rather constant (Hikosaka and Terashima 1996), causing a change in the balance between light harvesting and photosynthetic capacity in favor of the former. The change in the balance is achieved by an increase in light harvesting complex (LHC) relative to core chlorophyll, which is reflected in a lower chlorophyll a/b ratio (Anderson et al.

Western blotting Preparation of nuclear extracts for NF-κB 4T1 and NMuMG cells treated under various conditions were washed with cold PBS and suspended JAK2 inhibitors clinical trials for 30 min in 0.4 ml of a hypotonic lysis buffer (20 mM Tris–HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA, 2 mM Na3VO4,) containing protease inhibitors (10 μg/ml leupepton, 1 μM pepstatin). The cells were then lysed with 12.5 μl of 10% nonyl phenoxylpolyethoxylethanol (NP-40). The homogenate was centrifuged, and the supernatant, which contained the

cytoplasmic extracts, was stored at −80°C. The nuclear pellet was resuspended in 25 μl of ice-cold nuclear-extraction buffer for 30 min, with intermittent mixing. Then, the extract was centrifuged, and the supernatant containing the nuclear extract was obtained.

The protein content was measured by using the BCA protein assay kit (Pierce, Rockford, IL, USA). The nuclear and cytoplasmic extracts (40 μg of protein) were fractionated on polyacrylamide-sodium dodecyl sulfate (SDS) gels and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham, RAD001 cell line Arlington Heights, IL, USA). The membranes were blocked with a solution containing 3% skim milk and incubated with the anti-NF-κB p65 antibody (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C. Subsequently, the membranes were incubated with anti-rabbit IgG sheep antibody coupled to horseradish peroxidase (Amersham) for 1 h at room temperature. The reactive proteins were visualized by using ECL-plus (Amersham) according to the manufacturer’s instructions. Anti-lamin A antibody (Santa Cruz Biotechnologies, CA, USA) was used as the internal standard; it was used as the primary antibody to detect lamin Non-specific serine/threonine protein kinase A. Preparation of whole-cell lysates 4T1 and NMuMG cells treated

under various conditions were lysed with a lysis buffer containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 100 mM NaF, 1% NP-40, 1 μg/ml leupeptin, 1 μg/ml antipain, and 1 mM phenylmethylsulphonyl fluoride. The protein content in the cell lysates was determined using a BCA protein-assay kit. The extracts (40 μg of protein) were fractionated on polyacrylamide-SDS gels and transferred to PVDF membranes (Amersham). The membranes were blocked with a solution containing 3% skim milk and incubated overnight at 4°C with each of the following antibodies: anti-NF-κB p65, anti-phospho-extracellular signal-regulated kinase (ERK) 1/2 antibody, anti-phospho-Akt antibody, anti-phospho-mammalian target of rapamycin (mTOR) antibody, anti-phospho-c-Jun N-terminal kinase (JNK) antibody, anti-phospho-signal transducers and activator of transcription 3 (STAT3) antibody, anti-ERK1/2 antibody, anti-Akt antibody, anti-mTOR antibody, anti-JNK antibody, and anti-STAT3 antibody (Cell Signaling Technology).

Šmarda), E. coli pCol5 and E. coli pCol10 (H. Pilsl). As microcin control selleck kinase inhibitor producers, the following bacterial strains were used: E. coli 449/82 pColX (microcin B17); E. coli 313/66 pColG (microcin H47); E. coli 363/79 pColV (microcin V, original source: H. Lhotová); E. coli TOP10F’

pDS601 (microcin C7); E. coli D55/1 (microcin J25); E. coli B1239 (microcin L, D. Šmajs). Cultivation conditions The ability to produce bacteriocins of all the strains was tested in parallel on 4 different agar plates containing (i) TY medium, (ii) nutrient broth, (iii) TY medium supplemented with mitomycin C, and (iv) TY medium supplemented with trypsin. The rich TY medium consisted of yeast extract (Hi-Media, Mumbai, India) 5 gl-1, tryptone (Hi-Media) 8 gl-1, sodium chloride 5 gl-1; the TY agar consisted of a base layer (1.5%, w/v, solid agar) and a top layer (0.7%, w/v, soft agar). As a relatively unenriched medium, a Difco™nutrient broth (Difco Laboratories, Sparks, MD) 8 gl-1, NaCl 5 gl-1, was used for 1.5% (w/v) agar

plates. For induction of colicin production, the base agar layer was supplemented with 0.01% selleck chemicals llc (w/v) mitomycin C. To test protease sensitivity of the inhibitive agents, 0.1% (w/v) trypsin was added to the base layer of agar. Detection of colicin producers The agar plates were inoculated by needle stab with fresh broth cultures and the plates were incubated at 37°C for 48 hours. The bacteria were then killed using chloroform vapors and each plate was then overlaid with

a thin layer of soft agar containing 107 cells ml-1 of an indicator strain. The plates were then incubated at 37°C overnight. All 772 E. coli strains of clinical origin were tested on four parallel plates against all 6 indicators, i.e. each strain underwent 24 individual tests. Identification Ribonucleotide reductase of colicin and microcin types and determination of E. coli phylogenetic group Identification of individual colicin types (colicins A, B, D, E1-E9, Ia, Ib, Js, K, M, N, S4, U, Y, 5 and 10) was performed using PCR with primers designed using the Primer3 program [42] or with previously published primers [26]. The list of primer pairs and the corresponding length of PCR products are listed in Additional file 1. Total bacterial DNA was isolated using DNAzol (Invitrogen, Carlsbad, CA) reagent according to the manufacturer’s protocol. After 100-fold dilution, this DNA was used as a template for PCR reactions. Alternatively, all producer strains were tested with colony PCR. A bacterial colony was picked with a sterile inoculation loop and resuspended in 100 μl of autoclaved water. For each individual PCR reaction, 1 μl of cell suspension was added to the reaction. The PCR detection protocol was as follows: 94°C (2 minutes); 94°C (30 seconds), 60°C (30 seconds), 72°C (1 minute), 30 cycles; 72°C (7 minutes). For DNA amplification directly performed from lysed whole cells (colony PCR), the initial step was extended to 5 minutes (94°C, 5 minutes).

Biodiv Conserv 20. doi:10.​1007/​s10531-011-0133-x Engelbrecht I, Prendini L (2011) Assessing the taxonomic resolution of southern African trapdoor spiders (Araneae: Ctenizidae; Cyrtaucheniidae; Idiopidae) and implications for their conservation. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0115-z Hassall C, Hollinshead J, Hull A (2011) Environmental correlates of plant and invertebrate species richness in ponds. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0142-9 Hsieh Y-L, Linsenmair KE (2011) Underestimated spider diversity in a temperate beech forest. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0158-1 Hawkswoth DL (2011) Books on insect biodiversity and conservation. Biodiv

Conserv 20. doi:10.​1007/​s10531-011-0177-y Hébert C, Janssen P, Fortin D (2011) Biodiversity conservation www.selleckchem.com/products/BIBW2992.html in old-growth boreal forest: black spruce and balsam fir snags harbour distinct assemblages of saproxylic beetles. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0127-8 Ileana T. Galanes, John R. Thomlinson (2011) Soil millipede

diversity in tropical forest patches and its relation to landscape structure in northeastern Puerto Rico. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0128-7 Januschke K, Brunzel S, Haase P, Hering D (2011) Effects of stream restorations on riparian mesohabitats, vegetation and carabid beetles. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0119-8 Lichtwardt RW (2012) Trichomycete gut fungi from this website tropical regions of the world. Biodiv Conserv 21: in press, Biodiv Conserv 20. doi:10.​1007/​s10531-011-0146-5 Medan D, Torretta JP, Cyclooxygenase (COX) Hodara K, de la Fuente EB, Montaldo NH (2011) Effects of agriculture expansion and intensification on the vertebrate and invertebrate diversity in the Pampas of Argentina. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0118-9 Ödman AM, Mårtensson L-M, Sjöholm C, Olsson PA (2011) Immediate responses in soil chemistry, vegetation and ground beetles to soil perturbation when implemented as a restoration measure

in decalcified sandy grassland. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0108-y Péré C, Bell R, Turlings TCJ, Kenis M (2011) Does the invasive horse-chestnut leaf mining moth, Cameraria ohridella, affect the native beech leaf mining weevil, Orchestes fagi, through apparent competition? Biodiv Conserv 20. doi:10.​1007/​s10531-011-0134-9 Ranius T, Roberge JM (2011) Effects of intensified forestry on the landscape-scale extinction risk of dead wood dependent species. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0143-8 Ranius T, Martikainen P, Kouki J (2011) Colonisation of ephemeral forest habitats by specialised species: beetles and bugs associated with recently dead aspen wood. Biodiv Conserv 20. doi:10.​1007/​s10531-011-0124-y Samways MJ (2005) Insect diversity conservation.

Importantly, because the centrifugation assay is so rapid (~25 min duration), the observed effects must be due to existing efflux pumps and membrane fatty acid (FA) composition rather than being Torin 1 influenced by induction of emhABC transcription or long-term membrane modifications through de novo synthesis of FA. Because incubation temperature affects FA composition and fluidity of membranes, which in turn can affect protein-lipid interactions and integral membrane protein activity [11], we determined the effect of growth

temperature over a 25°C range on subsequent phenanthrene efflux activity. The cell-associated phenanthrene prior to azide addition was 1.34 ± 0.19 μmol/g, 1.93 ± 0.34 μmol/g and 2.30 ± 0.36 μmol/g in cLP6a cells grown at 10°C, 28°C and 35°C respectively, indicating

reduced efflux activity with increasing growth temperature. Consistent with previous work [18], cLP6a cells grown at 28°C exhibited active efflux of phenanthrene (Figure 2a): the steady state concentrations of phenanthrene associated with the cell pellet before (1.93 ± 0.34 μmol/g ) and after (5.28 ± 0.56 μmol/g ) azide addition were significantly different (P < 0.0001). Figure 2 Phenanthrene partitioning into P. fluorescens strains cLP6a and cLP6a-1. Partitioning of phenanthrene into the cell pellet of P. fluorescens strains, determined using a rapid efflux assay: (a) strain cLP6a grown at 10°C, 28°C or 35°C; (b) strain cLP6a-1 grown at 10°C, 28°C or 35°C. The vertical dashed line indicates Selleck BMS-936558 the addition of azide (120 mM). Each data point is the mean of three independent experiments, and error bars, where visible, indicate the standard deviation. Efflux assays were also performed with the emhB disruption strain cLP6a-1 (Figure 2b) to determine the steady state concentration of phenanthrene in the absence of efflux in the cells. As expected, there was no evidence of phenanthrene efflux by mutant

cLP6a-1 BCKDHB at 28°C and 35°C, as the steady state concentrations of cell-associated phenanthrene were unchanged before and after azide addition. Notably, the cell-associated phenanthrene prior to azide addition was significantly greater in cLP6a-1 cells grown at 28°C (6.60 ± 0.50 μmol/g) than in the parallel cLP6a cells (1.93 ± 0.34 μmol/g; P < 0.0001) (Figure 2). Thus, EmhABC is the sole efflux system responsible for phenanthrene efflux in cLP6a cells grown at 28°C and 35°C. The cell-associated phenanthrene concentration in cLP6a-1 cells grown at 35°C before azide addition (4.32 ± 0.19 μmol/g) was significantly lower (P < 0.0001) than in cells grown at 28°C (6.60 ± 0.50 μmol/g; Figure 2b), suggesting that phenanthrene partitioning into the cells was affected by changes in membrane FA composition induced by the incubation temperature.