1 M phosphate buffer (pH 7 2) for 2 h at 4°C, and then post-fixed

1 M phosphate buffer (pH 7.2) for 2 h at 4°C, and then post-fixed in 1% osmium tetroxide at 4°C for 2 h. The specimens were dehydrated with a series of ethanol solutions (30%-100%) and treated with hexamethyldisilazane LEE011 price twice for 15 min. The specimens were mounted on metal stubs, coated with a thin layer Akt inhibitor platinum under argon using a sputter-coater (SCD 005; BAL-TEC, Bannockburn, IL, USA), and then visualized by field emission-scanning electron microscopy (FE-SEM) (Supra 55VP; Carl Zeiss, Oberkochen, Germany) at the accelerating voltage of 2 kV at the National Instrumentation Center for Environmental Management (NICEM; Seoul, Korea). Images were captured

in TIFF format. Confocal microscopy To determine membrane

integrity, bacterial cells were stained with membrane-permeant and -impermeant fluorescent selleck products dyes according to the manufacturer’s instructions (Live/Dead BacLight Bacterial Viability Kit; Molecular Probes, Eugene, OR, USA) followed by confocal microscopy. Hp cells from BB agar plates were inoculated (OD600, 0.01 or 0.1) into BB-NBCS media and grown under various gas conditions. Aliquots were taken at 12 or 36 h, stained with SYTO 9 and propidium iodide (PI) for 15 min, and washed twice with phosphate buffered saline (PBS). Cells were then spread on slide glasses, covered with mounting medium and cover slips, and visualized by confocal microscopy (Leica TCS SP5; Leica Microsystems GmbH, Wetzlar, Germany). SYTO 9 is a green fluorescent membrane-permeant dye that labels all bacteria by staining nucleic acid, whereas PI is a Ribose-5-phosphate isomerase red fluorescent membrane-impermeant dye that labels only bacteria with damaged membranes. High performance liquid chromatography analysis of organic acid metabolites The concentrations of fermentation products in the Hp culture media were determined by high

performance liquid chromatography (HPLC) using the HP1100 system (Hewlett Packard, Palo Alto, CA, USA) at NICEM. Hp cells grown on agar plates were collected, washed, and inoculated into 20 ml of fresh media (OD600, 0.1). Cells were cultured under various gas conditions for 36 h, and the culture medium was collected and divided into two aliquots (one of which was spiked with 15 mM pyruvate as internal control for quantification), which were processed simultaneously. The culture medium was extracted twice with phenol/chloroform to remove proteins and then passed through a 0.45-μm syringe filter. The samples were injected into an ion exchange column (Aminex HPX-87H, 300 × 7.8 mm; Bio-Rad, Richmond, CA, USA), and eluted at 40°C with 0.01 N H2SO4 at a flow rate of 0.5 ml/min. Organic acids were analyzed with a refractive index detector HP1100 (Hewlett Packard). Solutions containing glucose and organic acids including acetate, formate, propionate, lactate, pyruvate, succinate, and butyrate were used as standards.

2 2 Study Design The study subjects were randomly assigned to one

2.2 Study Design The study subjects were randomly assigned to one of six administration sequences, each consisting of three treatment periods separated by a washout period of

at least 7 days in duration. The subjects were allocated a 4-digit randomization number, starting at 1001, immediately prior to the predose pharmacokinetic blood draw after eligibility was determined. At least six subjects were to be randomized to each of the six possible treatment sequences (1: GXR, MPH, GXR + MPH; 2: GXR, GXR + MPH, MPH; 3: MPH, GXR, GXR + MPH; 4: MPH, GXR + MPH, YM155 GXR; 5: GXR + MPH, GXR, MPH; 6: GXR + MPH, MPH, GXR). The study medication was administered at a clinical research center that was supervised by clinical staff. The subjects were required to fast for approximately 10 h prior to the administration of each dose of study medication. All study medication was given with water in the

morning. A moderate-fat lunch was provided 4 h after dose administration. The subjects were confined at the center Saracatinib concentration during each treatment period and remained there until all discharge procedures were completed, approximately 72 h after the subjects received the treatment. 2.3 Pharmacokinetic and Safety Assessments Vital signs were monitored, blood samples collected, and ECG data obtained before administration of the study medication for each treatment period. Guanfacine, dexmethylphenidate (d-MPH), and l-methylphenidate (l-MPH) levels were measured in plasma produced from blood samples BIBF 1120 research buy collected predose and at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12, 24, 30, 48, and 72 h postdose. Immediately after blood collection, the blood samples were kept on ice until they were centrifuged, within 30 min following the blood draw. Plasma concentrations

of guanfacine, d-MPH, and l-MPH were measured using liquid chromatography with tandem mass spectrometry (LC–MS/MS) detection methods that were validated for the quantitation of guanfacine, d-MPH, and l-MPH in human K3-EDTA plasma. The method utilized a liquid-liquid extraction procedure prior to LC–MS/MS analysis. The stable isotope-labeled compounds guanfacine (13C15N3) and MPH-D9 were used below as the internal standards for guanfacine and d/l-MPH, respectively. For guanfacine, the LC–MS/MS analysis was carried out with a Sciex 4000 mass spectrometer coupled with a Shimadzu liquid chromatography (LC) pump (model LC-10AT) and Perkin-Elmer 200 series autosampler. The chromatographic separation was achieved on a XBridge phenyl, 3.5 μm, 4.60 × 50 mm LC column, with a mobile phase. The mass spectrometer was operated in positive electrospray ionization mode, and the resolution settings used were unit for Q1 and low for Q3. The multiple reaction monitoring (MRM) transition was m/z 246 → 60 for guanfacine, and the MRM transition was m/z 250 → 159 for the internal standard, guanfacine (13C15N3).

cDNA was prepared according to standard methods: RNA was reverse-

cDNA was prepared according to standard methods: RNA was reverse-transcribed with oligo(dT) primer using 1 μg total RNA in a total volume of 20 μl containing transcription buffer, RNase Inhibitor, Prime Script™ RTase. For PCR, 30 cycles of denaturation (94°C for 45s), annealing (60°C for 45s), and elongation (72°C for 1 min) was performed using the following primer pairs for HIF-1α [19]: BLZ945 clinical trial forward: 5′-TGGACTCTGATCATCTGACC-3′, reverse: 5′-CTCAAGTTGCTGGTCATCAG-3′, which yielded a 434-bp product. 30 cycles of denaturation (95°C for 1 min), annealing (55°C for 60s), and elongation (72°C for 1 min) were performed using the following primer pairs for

MDR1 [20]: forward: 5′-GAATCTGGAGGAAGACATGACC-3′, reverse:5′-TCCAATTTTGTCACCAATTCC-3′, which yielded a 259-bp product.35 cycles of denaturation

selleck products (95°C for 30s), annealing (50°C for 1 min), and elongation (72°C for 1 min) were performed using the following primer pairs for MRP1 [21]: forward: 5′-TCAGCCCTTCCTGACAAGCT-3′, reverse: 5′-TCTCTGCTGCAGGAGGTCCG-3′, which yielded a 318-bp product. The GAPDH [22] control PCR was performed using the following primer pairs: forward: 5′-ACCACCATGGAGAAGGCTGG-3′, reverse: JQEZ5 5′-CTCAGTGTAGCCCAGGATGC-3′, which yielded a 527-bp product. For negative controls, the PCR reaction was performed without prior reverse transcription. Amplified cDNA was visualized by ethidium bromide staining on 1.5% agarose gels on a Bio-Rad gel scanner (Bio-Rad, USA). Western Blot The chordoma cell line CM-319 and frozen nucleus pulposus tissues were harvested and lysed with a cold RIPA protein lysis buffer for 30 minutes on ice. The lysates were transferred to Eppendorf tubes and clarified by centrifugation at 12,000 g for 10 minutes at 4°C. The supernatant was kept in -80°C for future use. The BCA method was performed to determine

the protein concentration in the supernatant. Samples (30 μg of total protein each) were boiled at 95°C for five minutes and loaded onto SDS-PAGE (5% stacking gel and 8% separating gel), followed with a separation at 80 volts for about two hours and subsequent transferred onto a nitrocellulose membrane. The membrane was blocked in 5% defatted milk for 1 hour at room temperature, and was then incubated in the primary antibodies diluted in 5% defatted milk/TBST overnight at 4°C (MDR1 1:200, mouse Thiamet G anti-human, Santa Cruz; MRP1, 1:200, rabbit anti-human, Santa Cruz; HIF-1α, 1:200, rabbit anti-human, Santa Cruz). The membrane was washed three times with TBST and incubated with the second antibodies for an hour at room temperature, then washed three times with TBST again. The enhanced chemiluminescene (ECL) system (Piece) was used for detection of MDR1, HIF-1α and MRP1. Protein bands were visualized and quantified using Quantity-One software (Bio-Rad USA). The MDR1, HIF-1α and MRP1 bands were visualized at an apparent molecular weight of 170, 120 and 190 kDa, respectively.

Association of Oct-4 expression with survival in all cases and in

Oct-4 expression in tumor Navitoclax tissue and differentiation of tumor cells were strongly associated with cancer-associated death. Notably, an Oct-4 expression level less than the median histoscore (25.80) was associated with improved survival (HR, 1.011), whereas elevated Oct-4 expression was associated with shorter cumulative survival (p = 0.009). A Kaplan-Meier plot showed a prominent difference in survival estimates for patients with high versus low Oct-4 expression in tumor tissue; this difference corresponded to a median survival of 18.2 ± 6.0 months for patients with high GW786034 order Oct-4 expression compared with a median survival of more than 24.7 ± 9.1 months for patients with low Oct-4 expression (Figure 3A). 27.3 ± 9.6 months; Figure 3B) and the www.selleckchem.com/products/Temsirolimus.html squamous cell carcinoma subset (20.7 ± 9.5 vs. Table 2 Univariate and multivariate analyses of individual variables for correlations with overall survival: cox proportional hazards model Variables Univariate Multivariate   HR 95%CI P HR 95%CI P Age 0.988 0.969-1.008 0.231 1.001 0.978-1.025

0.922 Gender 0.852 0.517-1.405 0.530 0.525 0.305-0.906 0.121 Smoking 1.179 0.740-1.880 0.489 1.277 0.743-2.195 0.376 Histological type 1.087 0.697-1.695 0.713 1.168 0.706-1.932 0.546 Histological differentiation 3.727 2.139-6.495 < 0.001 3.666 1.937-6.939 0.001 Local advance 1.282 0.920-1.731 0.149 1.222 0.928-1.609 0.153 Lymph node metastasis 1.487 1.148-1.927 0.003 1.042 0.743-1.461 0.813 Oct-4 expression 1.105 1.007-1.024 < 0.001 1.011 1.003-1.020 0.009 Age 0.990 0.963-1.018 0.482 1.014 0.978-1.051 0.450 Gender 0.786 0.408-1.512 0.470 0.296 0.087-1.008 0.052 Smoking 1.231 0.646-2.346 0.527 0.733 0.237-2.265 0.590 Histological type 0.785 0.408-1.512 0.470 0.869 0.386-1.956 0.735 Vasopressin Receptor Histological differentiation 1.428 0.701-2.910 0.327 1.418 0.591-3.405 0.434 Local advance 1.191 0.780-1.817 0.418 0.934 0.560-1.558 0.793 Lymph node metastasis 1.217 0.833-1.778 0.310 1.560 0.976-2.495 0.063 Oct-4 expression 1.014 1.002-1.025 0.021 1.024 1.007-1.042 0.005 Age 0.994 0.965-1.024 0.688 1.005 0.967-1.044 0.801 Gender 0.790 0.395-1.580 0.505 0.401 0.166-0.966 0.096 Smoking 1.232 0.635-2.389 0.537 0.921 0.382-2.219 0.855 Histological type 1.439 0.767-2.700 0.257 1.247 0.598-2.600 0.556 Histological differentiation 1.925 0.934-3.969 0.076 1.962 0.791-4.868 0.146 Local advance 1.

4 mM 4-hydroxybenzoate A1501 showed a shorter lag phase and a hi

4 mM 4-hydroxybenzoate. A1501 showed a shorter lag phase and a higher growth rate when cells were grown on the mixture than when benzoate was supplied alone (Figure 8A). Sepantronium concentration Furthermore, under the latter growth conditions, the culture gradually became dark brown in color because of autoxidation of the accumulated catechol (data not shown). However, when the 4-hydroxybenzoate concentration increased to 0.8 mM, growth of A1501

was completely inhibited (Figure 8A). These results indicate that 4-hydroxybenzoate at low concentrations can enhance the ability of A1501 to grow on benzoate. We then evaluated the effect of 4-hydroxybenzoate on the metabolism of benzoate using HPLC. When 4 mM benzoate alone was provided to the culture, it was completely

VX-770 purchase consumed within 26 h, and metabolic intermediates were present. When 4 mM benzoate and 0.4 mM 4-hydroxybenzoate were provided together as growth substrates, benzoate was completely SP600125 research buy consumed within 18 h, while no discernible loss of 4-hydroxybenzoate was detected (Figure 8B). Additionally, analysis of the intracellular metabolites by HPLC revealed accumulation of catechol derived from benzoate both in the presence and absence of 4-hydroxybenzoate in the growth medium. The concentration of catechol reached 0.28 mM when A1501 grew on benzoate alone, whereas the concentration of catechol reached approximately 0.12 mM when both benzoate and 4-hydroxybenzoate were in the growth medium (Figure 8C). Collectively, these results suggest that 4-hydroxybenzoate can significantly enhance the ability of A1501 not only

to degrade benzoate, but also to remove the catechol accumulated from benzoate. Discussion The data presented here reveal that the sequence and organization of the ben, pob, cat, and pca genes in A1501 are very similar to those within other well-studied Pseudomonas strains, raising the question of whether these genes have common origins. Increasing evidence indicates that horizontal gene transfer is an efficient mechanism for introducing catabolic pathways into different bacterial genomes [37]. In general, Protein kinase N1 recently acquired transferable genomic regions are associated with insertion sequence elements and mobility-related genes, whereas anciently acquired genomic regions may lose these genetic elements [38]. Furthermore, horizontally acquired DNA regions are usually chromosomally inserted in the vicinity of tRNA or rRNA genes [38]. We also discovered that an rRNA operon is located directly downstream of the ben gene cluster and that a tRNA-Gly gene is located downstream of the pca gene cluster.

Mutants were confirmed by PCR and Southern hybridization Tests o



dnd-1 AGAGATCACCACATATGCACCTGAGCACC NdeI dnd-2 CAGCCGGATCCTGATCTCAG BamHI dndE-L CACATATGCCGTCTGAGATCACC NdeI dndE-R TAAGGCCTATTCGGCGGTGA   Intensity of DNA bands was quantified from the fluorescence intensity using GeneTool software (Syngene). Refinement of the limits of the dnd gene cluster pHZ1900: a 10-kb BamHI fragment from

pHZ825 was cloned Selleckchem GNS-1480 into pSET152. Farnesyltransferase pJTU1203 or pJTU1204 (with opposite direction): a 7.9-kb MluI-EcoRI fragment from pHZ1904 was blunt-ended and cloned into the EcoRV site of pSET152. pJTU1208: the 1.0-kb BglII fragment from Hippo pathway inhibitor pHZ1900 was inserted into the BamHI site of pBluescript II SK (+). Then a 0.3-kb SalI fragment of this plasmid was replaced with a 1.3-kb SalI fragment from pHZ1904 to generate pHZ2850, in which dndA accommodated in a 2.0-kb BamHI/BglII-SacI region. A 1.4-kb fragment from pHZ2850 generated by complete digestion with EcoRI and partial digestion with BglII was inserted into the EcoRI and BamHI sites of pSET152 to give pHZ2851. Finally, a 2.1-kb XbaI-SfiI fragment of pJTU1204 was replaced with a corresponding 0.8-kb fragment from pHZ2851, generating pJTU1208. Thus, in pJTU1208, the dnd gene cluster was shortened to the BglII site near the end of dndA, covering a 6,665-bp region. pHZ2862 (also the vector for dndA deletion): a 2.0-kb PvuII fragment from pHZ1900 was cloned into the SmaI site of pBluescript II SK(+) to give pHZ2853, then a 6.5-kb SmaI-EcoRI fragment from pHZ1900 was used to replace the 0.7-kb corresponding fragment in pHZ2853 to give pHZ2861, in which dndB-E lay in a 7.8-kb SmaI/PvuII-EcoRI region. A 7.8-kb BamHI fragment from pHZ2861 was cloned into pSET152 to give pHZ2862.

The chromatographic data processing was performed by the Agilent

The chromatographic data processing was performed by the Agilent Chemstation Software (GC-MS Data Analysis from Agilent, Waldbronn, Germany) while detected compounds were identified firstly by matching with the mass spectrum library NIST 2008 (Gaithersburg, MD, USA) and additionally confirmed with retention time of standardized reference materials. All compounds used

for identification and quantification (calibration) were purchased from Sigma Aldrich (Sigma-Aldrich, Steinheim, Germany). Sampling procedure for human breath samples A cohort of 55 selleck chemicals llc individuals (32 non-smokers, 23 active-smokers) www.selleckchem.com/products/salubrinal.html was recruited for this study. Amongst smokers, 12 males were in the age range from 22 to 64 years and 11 females were in the age range from 21 to 65 years. The cohort of non-smokers this website comprised 12 males and 20 females in the age range from 22 to 87 years. All individuals gave informed consent to their participation. The volunteers completed a questionnaire describing their current smoking status (active smokers, non-smokers, ex-smokers) and the time elapsed since last smoking (if applicable). No special dietary regimes were applied. All volunteers recruited to this study were healthy, especially in respect to lung diseases caused by bacterial infections but also asthma, chronic obstructive pulmonary disease (COPD) and lung cancer. The samples were collected at different times of the day

at least 2 hours after last meal and were processed within 6 hours after sampling. Volunteers were asked to rest for at least 5 minutes before sampling. The alveolar air samples were collected into Tedlar bags (SKC Inc, Eighty Four, PA) by means of an in-house produced breath sampler, allowing also the collection of ambient air (also in Tedlar bags). The device operated C1GALT1 in two different sampling modes based on the CO2-content. Digitally controlled electronic valves switched to sampling mode if (a) the absolute level of CO2 in the breath exceeded 3% or (b) the

relative level of CO2 in the breath was above 80% of the maximal CO2-level in previous exhalation. Two breath samples and respective indoor-air were collected in described above way from each subject. Before use, all bags were thoroughly cleaned to remove any residual contaminants by flushing with nitrogen 6.0 (purity of 99.9999%), heating at 85°C (while filled with N2) for more than 8 hours and subsequent secondary flushing. The study was approved by the local ethics committee of Innsbruck Medical University. Preparation of breath samples Tedlar® bags filled with breath samples were thermostated for few minutes in an incubator at 40°C (to prevent condensation) and connected by means of Teflon tubes to a multibed sorption tube. The sample flow rate of 20 ml/min was diluted with additional flow (40 ml/min) of dry nitrogen 6.0 (additionally purified with Carboxen 1000) in order to avoid excessive adsorption of water vapor.

V cholerae has been proposed to be a useful prokaryotic model of

V. cholerae has been proposed to be a useful prokaryotic model of alterations in L-tyrosine catabolism and has been used to study the molecular basis of diseases related to L-tyrosine catabolism [15]. However, to date, all the research on melanogenesis in V. cholerae has been based on chemically

induced mutants or mutants generated using transposons. During our cholera surveillance, some O139 and O1 strains that produced soluble brown pigments were isolated from environmental water samples and patients. Unusually, these strains can produce pigment under the normally used experimental growth conditions [Luria-Bertani (LB) nutrient agar or broth without temperature limitation].

Using transposon click here mutagenesis, we determined that the p-hydroxyphenylpyruvate dioxygenase (HPD; VC1344 in the N16961 genome) in the tyrosine catabolic pathway was responsible for the pigment production in these strains [24]. Further, the three genes in a cluster QNZ supplier selleck chemicals downstream of VC1344 were found to correspond to the other three enzymes involved in tyrosine catabolism [23, 24]. In this study, we analyzed the sequence variance of the four genes involved this website in tyrosine catabolism and the functions of the mutant genes to determine the possible mechanism of pigment production in these

isolates. We also found a close relationship of clonality among these strains, even though they were isolated in different years and from different areas. The potentiality of clone selection and pathogenicity of such strains should be considered. 2. Methods 2.1 Strains In this study, 22 V. cholerae O1 and O139 toxigenic and nontoxigenic strains were used (Table 1). Among these isolates, 95-4, 98-200, JX2006135, JX2006136, JX2006175, GD200101012, and 3182 are pigment-producing strains. These strains were isolated in different years and from different provinces of China. The El Tor strain 3182 was isolated from patients and the other six O139 strains were isolated from environmental water. In addition to the reference strains, including N16961, 569B, and MO45, the controls included other non-pigment-producing strains that were isolated in the same province or at the same time as the pigmented strains. Strains were cultured in LB liquid medium shaking at 37°C or on LB agar plates (1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 1.5% agar).

Since the pH of the

Since the pH of the RF-preparations used in this study did not reach extreme acidic levels, the Gad system may not have been induced. In the Arg system, decarboxylation (speA) of arginine via proton consumption resulting in the formation of agmatine stabilizes the cytoplasmic pH. Agmatine is either

exported via the arginine-agmatine antiporter (aidC) or converted (speB) to putresceine as part of the polyamine biosynthetic pathway. Considering that O157 is exposed to heat-shock, starvation and stationary-phase-like growth in the rumen, it is possible that these factors enhance acid-tolerance in the bacteria through other mechanisms such as outer membrane changes and synthesis of proton https://www.selleckchem.com/products/AG-014699.html transport-related protective proteins, as well [49, 50]. Several stress (acid, low oxygen, osmolites, stationary phase)-responsive genes were expressed by O157 in this study, and included genes associated

with the metabolism of arginine (speA, speB), lysine (lysU), formate (hyC), tryptophan (tnaA) and maltoporin (lamB), catalase (katG), DNA polymerase-1 (polA) and AidA-1 adhesin-like protein (aidA) [49–51]. Flagellar genes are differentially NCT-501 ic50 expressed under varying acid-stress conditions [51–53], and in our study, these genes were up-regulated in dRF and fRF but not uRF, suggesting less pH variation in the course of growth in uRF and limiting the role of flagella to motility alone. Stressed bacteria have been shown to be more adherent [35, 40, 53]; proteins associated with adherence (AidA-1 adhesin-like) and biofilm formation (BssR, CsgG, CsgB) were identified after 48 h incubation and not after longer incubation periods. Interestingly, several ‘resistance’ related proteins were up-regulated in RF-preparations, a subset of which (tellurite resistance, serine protease) have also been shown to contribute towards O157 adherence

[54, 55]. This suggests that adherence may be critical during the initial phase of O157 colonization and although LEE is suppressed, the bacteria rely on other mechanisms to adhere or form biofilms in the rumen. It has been observed that bacteria and protozoa in the rumen tend to adhere to the fibrous mat layers comprising of plant material to remain in the rumen and assist in the digestion of insoluble feed materials Clomifene [56]. While this may not be in the case of O157, initial adherence to or biofilm formation on available surfaces may give the bacteria time to adapt and survive the rumen environment [34]. It appears that much of the adaptive changes are initiated early in colonization as reflected in more stress-induced, structural integrity-related outer membrane proteins (AsmA, LptE, Lpp, NagA, SlyB, OmpA, BamA, BamD, TolC, OmpW, ElaB, YbjP, LppC, YqjD), and cell AZD1480 division and growth proteins, being expressed at 48 h. This supports the observation that O157 is maintaining slow growth in the RF-preparations as well.

Thus, electrostatic repulsions between

Thus, electrostatic repulsions between Ganetespib N- and C-terminal domains force the protein into the “”open”" position. This in turn releases the N-terminal domain,

forming a stable complex with KdpE~P and the DNA [25] initiating kdpFABC expression. Replacement of the KdpD-Usp domain with UspF or UspG results in inversion of the surface net charges. The negative net surface charge of these two proteins forces electrostatic attraction between the N- and the C-terminal regions, leaving KdpD in the “”OFF”" state under all conditions. Conclusion The Usp domain within KdpD is important for proper KdpD/KdpE signaling. Alterations within this domain can completely prevent the response towards K+ limitation as well as salt stress. The KdpD-Usp domain surface contains numerous positively charged amino acids. Electrostatic repulsion and attraction between the N-terminal and C-terminal domain are supposed to be important for KdpD (de)activation. Therefore, selleck the KdpD-Usp domain not only functions as a binding surface for the native scaffold UspC, but also seems to be crucial

for internal KdpD signaling, shifting the protein from an “”OFF”" into an “”ON”" state. Methods Materials [γ32-P]ATP and NAP-5 gel filtration columns were purchased from Amersham GE Healthcare. Goat anti-(rabbit IgG)-alkaline phosphatase was purchased from Biomol. All other reagents were reagent grade and obtained from commercial sources. Baf-A1 cell line Bacterial strains and plasmids E. coli strain JM 109 [recA1 endA1 gyrA96 thi hsdR17 supE44λrelA1 Δ(lac-proAB)/F'traD36 proA + B + lacI q lacZΔM15] Progesterone [30] was used as carrier for the plasmids described. E. coli strain TKR2000 [ΔkdpFABCDE trkA405 trkD1 atp706] [31] containing different

variants of plasmid pPV5-3 encoding the different KdpD-Usp derivatives (see below) was used for expression of the kdp-usp derivatives from the tac promoter. E. coli strain HAK006 [ΔkdpABCD Δ(lac-pro) ara thi] [32] carrying a kdpFABC promoter/operator-lacZ fusion was used to probe signal transduction in vivo. E. coli LMG194 [F- ΔlacX74 galE galK thi rpsL ΔphoA (PvuII) Δara714leu::Tn10] [33] was used for expression of the kdp-usp derivatives from the araBAD promoter. To replace the Usp domain in E. coli KdpD with the E. coli Usp protein sequences, the corresponding usp genes were PCR amplified using genomic DNA of E. coli MG1655 [34] as a template. The uspA, uspD, uspE, uspF, and uspG genes were amplified with primers complementary at least 21 bp to the 5′ or the 3′ ends of the corresponding genes with overhangs for a 5′ NsiI site and a 3′ SpeI site, respectively. uspC was amplified similarly, but with a 5′ terminal SacI site.