Effects

of acute nicotine (500 nm) on DA release probability and its sensitivity to activity were apparent. However, in NAc there was downregulation of the functional dominance of α6-nAChRs (α6α4β2β3), and an emergence in function of Z-VAD-FMK purchase non-α6* nAChRs. In CPu, there was no change in the control of DA release by its α6 nAChRs (α6β2β3) relative to non-α6. These data suggest that chronic nicotine subtly modifies the regulation of DA transmission, which, in NAc, is through downregulation of function of a susceptible population of α6α4β2β3 nAChRs. This imbalance in function of α6:non-α6 nAChRs might contribute to DA dysregulation in nicotine addiction. “
“The orbitofrontal cortex (oPFC) sends substantial projections to the ventrolateral striatum and aspects of the nucleus

accumbens that are, functionally, poorly understood. This is despite probable cortico-striatal involvement in multiple diseases such as addiction and obsessive-compulsive disorder. Here we surgically disconnected the oPFC from the ventrolateral striatum using unilateral asymmetric lesions in mice and classified instrumental decision-making strategies. Mice with symmetric lesions that spared one Everolimus supplier oPFC–striatal network served as controls. As a complementary approach, we selectively knocked down Brain-derived neurotrophic many factor (Bdnf) bilaterally in the oPFC and ascertained behavioral and neurobiological consequences within the downstream striatum. oPFC–striatal disconnection and oPFC Bdnf knockdown blocked sensitivity to outcome-predictive relationships in both food-reinforced and cocaine-associated settings. Bdnf knockdown simultaneously regulated striatal BDNF expression, and striatal c-Fos predicted sensitivity to action–outcome associative

contingencies. Previous evidence strongly implicates the dorsolateral striatum in stimulus–response habit formation. Our findings thus provide novel evidence for functional compartmentalisation within the lateral striatum, with the dorsal compartment subserving classical stimulus–response habit systems and a ventral compartment coordinating outcome-based decision-making via oPFC interactions. This compartmentalisation may apply to both ‘natural’, as in the case of food-reinforced behavior, and ‘pathological’, as in the case of cocaine-seeking, contexts. “
“Metformin is currently the first-line treatment drug for type 2 diabetes. Metformin is a well-known activator of AMP-activated protein kinase (AMPK). In experimental studies, metformin has been shown to exert direct vascular effects by increasing vascular endothelial growth factor expression and improving microvascular density.

glutamicum.

A frequently used method to improve growth of C. glutamicum on a particular metabolite is through selection of fast-growing mutant after serial subculture (Youn et al., 2008). Three cultures of C. glutamicum ATCC 13032 were grown in CGXII medium with 0.5% Neu5Ac and serially subcultured when each culture had reached an OD600 nm of at least 4. This was continued for each culture over 15 days, which was between 8 and 12 subculturing steps. The three resulting evolved strains, Ev1-3, all showed significantly reduced lag phases for growth on Neu5Ac (Fig. 1a open symbols and Supporting Information, Fig. S1), although the final growth yield with 0.5% Neu5Ac is similar to the wild-type strain (Fig. 1b). We investigated the concentration dependence of sialic acid growth in one of these strains, Ev1, and see a quantitative relationship between the selleck chemicals llc starting Neu5Ac concentration and the final growth yield (Fig. 1d). During growth on 0.25% Neu5Ac, growth stops after around 9 h, presumably as the Neu5Ac has been consumed during growth (Fig. 1c). To check the stability of the evolved strains, we subcultured them on BHI medium and then from this further cultured them on CGXII with 1% glucose and then back onto CGXII 0.5% Neu5Ac, upon which the reduced lag phase observed initially was retained

(data not shown). The decreased lag but unaltered growth properties suggests that the regulation of expression of the Neu5Ac uptake/catabolic genes is altered in these mutants. As there was a considerable http://www.selleckchem.com/products/OSI-906.html lag in growth of the wild-type strain when pregrown in CGXII glucose media, we examined the effects of different pregrowth conditions for growth on CGXII Neu5Ac for both the wild-type strain and also for the Ev1 strain. Pregrowth of the wild-type strain in CGXII Neu5Ac yielded

a reduced lag phase compared with pregrowth on CGXII glucose Adenosine (Fig. 2a). In contrast, pregrowth in CGXII medium containing both Neu5Ac and glucose gave a similar growth lag as seen with glucose alone, suggesting that the presence of glucose has a dominant effect over the presence of Neu5Ac (Fig. 2a). When examining the potential of cells pregrown in the same conditions to take up [14C]-Neu5Ac, it is clear that uptake is only detectable in the cells that have been pregrown in CGXII Neu5Ac (Fig. 2c). In contrast to the wild-type strain, the Ev1 strain exhibited similar growth lags on CGXII Neu5Ac, regardless of how the cells had been grown, suggesting that the repressive effect of glucose on expression of the sialic acid utilization genes was lost (Fig. 2a). The sialic acid cluster in C. glutamicum contains a likely ABC transporter for sialic acid, which is homologous to the SatABCD systems from Gram-negative Gammaproteobacterium Haemophilus ducreyi (Post et al., 2005). To test whether this system is also important in C.

The study protocol was approved by the Danish Ethics Committee on clinical research, and written informed consent was obtained from all participants. As most variables, even after log transformation, were not normally distributed, nonparametric statistics were applied; thus, data are presented as medians and interquartile ranges (IQRs). Comparisons between controls and HIV-positive patients were performed using the Mann–Whitney test (unpaired data), and

comparisons between treatment-naïve and treatment-experienced patients were performed using the Wilcoxon signed rank test (paired data). The correlations between variables were determined using Spearman’s correlation coefficient. A value of P < 0.05 was considered significant. The baseline characteristics of learn more the patient and control groups Ixazomib are shown in Table 1. During the first 3 months, 11 patients were treated with boosted indinavir, three of whom were changed to boosted lopinavir because of side effects. One patient left the study because of side effects. Nine patients received boosted lopinavir throughout the first period. One patient was unwilling to change therapy to efavirenz

and was excluded from the second part of the study (Fig. 1). After 3 months, two-thirds of the patients had viral loads (VLs) below 50 copies/mL, and after 6 months all 18 had a VL below this value. CD4 counts find more increased from a median of 160 cells/μL (IQR 125–200 cells/μL) to 220 cells/μL (IQR 160–300 cells/μL) after 3 months of treatment, and to 215 cells/μL (IQR 180–280 cells/μL) after 6 months of treatment. Controls had a median CD4 count of 770 cells/μL (IQR 730–900 cells/μL). At entry and throughout the study period, HIV-positive patients had lower haemoglobin and a lower total leucocyte count compared with controls. Platelet numbers did not differ between patients and controls (Table 2). Total cholesterol, triglyceride, and glucose levels were

normal at baseline (Table 2). During treatment with both a PI and efavirenz, total cholesterol increased significantly compared with baseline. Similarly, PI treatment led to significantly higher triglyceride levels. However, this was negated during treatment with efavirenz, and lowered again to a level comparable to that of the controls (1.47 vs. 0.83 mmol/L, respectively; P = 0.15). Body mass index (BMI) and systolic blood pressure were normal at baseline and did not change during the treatment period. Endothelial function was studied in several ways (Table 3). HIV-positive patients had significantly lower FMD at baseline compared with controls (108 vs. 111%, respectively; P = 0.043) (Table 3). After 3 months of PI-containing HAART, FMD normalized (111%) and did not change significantly after switching from a PI to efavirenz (111 vs. 109% in HIV-positive patients treated with PI vs.

The study protocol was approved by the Danish Ethics Committee on clinical research, and written informed consent was obtained from all participants. As most variables, even after log transformation, were not normally distributed, nonparametric statistics were applied; thus, data are presented as medians and interquartile ranges (IQRs). Comparisons between controls and HIV-positive patients were performed using the Mann–Whitney test (unpaired data), and

comparisons between treatment-naïve and treatment-experienced patients were performed using the Wilcoxon signed rank test (paired data). The correlations between variables were determined using Spearman’s correlation coefficient. A value of P < 0.05 was considered significant. The baseline characteristics of VX-809 nmr the patient and control groups Epigenetics Compound Library research buy are shown in Table 1. During the first 3 months, 11 patients were treated with boosted indinavir, three of whom were changed to boosted lopinavir because of side effects. One patient left the study because of side effects. Nine patients received boosted lopinavir throughout the first period. One patient was unwilling to change therapy to efavirenz

and was excluded from the second part of the study (Fig. 1). After 3 months, two-thirds of the patients had viral loads (VLs) below 50 copies/mL, and after 6 months all 18 had a VL below this value. CD4 counts cAMP increased from a median of 160 cells/μL (IQR 125–200 cells/μL) to 220 cells/μL (IQR 160–300 cells/μL) after 3 months of treatment, and to 215 cells/μL (IQR 180–280 cells/μL) after 6 months of treatment. Controls had a median CD4 count of 770 cells/μL (IQR 730–900 cells/μL). At entry and throughout the study period, HIV-positive patients had lower haemoglobin and a lower total leucocyte count compared with controls. Platelet numbers did not differ between patients and controls (Table 2). Total cholesterol, triglyceride, and glucose levels were

normal at baseline (Table 2). During treatment with both a PI and efavirenz, total cholesterol increased significantly compared with baseline. Similarly, PI treatment led to significantly higher triglyceride levels. However, this was negated during treatment with efavirenz, and lowered again to a level comparable to that of the controls (1.47 vs. 0.83 mmol/L, respectively; P = 0.15). Body mass index (BMI) and systolic blood pressure were normal at baseline and did not change during the treatment period. Endothelial function was studied in several ways (Table 3). HIV-positive patients had significantly lower FMD at baseline compared with controls (108 vs. 111%, respectively; P = 0.043) (Table 3). After 3 months of PI-containing HAART, FMD normalized (111%) and did not change significantly after switching from a PI to efavirenz (111 vs. 109% in HIV-positive patients treated with PI vs.

UDTR employs different rules that converge on specific levels of accuracy. We used a three-up, one-down rule, meaning that for three consecutive hits we adjusted the stimulus one step harder and for any miss we adjusted the stimulus one step easier. This rule necessarily Ku-0059436 nmr converges on an accuracy level of 79.4%. During the experimental session, participants were instructed to respond as quickly and accurately

as possible to the detection of targets within the cued modality and to withhold responses otherwise. Participants were further instructed to refrain from eyeblinks during each trial as much as possible. Each participant completed one visual and one auditory pure-task block of 100 trials, followed by ~20 mixed-task blocks of 30 trials this website each, resulting in the collection of ~300 trials per cue condition. Continuous EEG was recorded, with a bandpass of DC to 134 Hz, from 168 scalp electrodes (Biosemi ActiveTwo System, Amsterdam, Netherlands) at an analog-to-digital sampling rate of 512 Hz. Biosemi replaces the ground electrodes that are used in conventional systems with two separate electrodes: a Common Mode Sense and a Driven Right Leg passive electrode. These two electrodes

create a feedback loop, thus rendering them references. With the Biosemi system, every electrode or combination of electrodes can be assigned as a reference, which is done purely in software after acquisition. EEG data were processed using

the FieldTrip toolbox Masitinib (AB1010) (Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, The Netherlands). This MATLAB (The MathWorks Inc., Natick, MA, USA) toolbox and supporting materials can be accessed at http://www.ru.nl/neuroimaging/fieldtrip. The continuous EEG data were stored and then re-referenced to the average reference and low-pass filtered with a cutoff frequency of 40 Hz. Trials with blinks and excessive eye movements were rejected based on the horizontal and vertical electro-occulogram. Over all other electrodes, a trial rejection threshold of ±100 μV was used. Trials were then epoched from −200 to +1805 ms around the onset of the S1 cue-stimulus. The period of −100 to 0 ms was defined as baseline. To obtain so-called global switching costs, we quantified the difference in reaction times (RTs) and response accuracy (d-prime; see below) between mixed and pure task blocks. To obtain local switching costs, we analysed differences in RT and d-prime between switch and repeat trials within the mixed blocks. The RT was measured from all correct ‘go’ trials (i.e. trials with a target in the cued modality). Responses were only considered valid if they occurred in the window 200–1500 ms following the onset of the gabor in attend-visual conditions and the second tone stimulus in the attend-auditory conditions.

001404). Animals were anesthetized as previously described.[11, 12] Two transplantation surgeries using two monkeys, as shown in Figure 1, was planned. We planned to use the uterine artery and ovarian vein (or, if possible, the uterine vein) for arterial and venous vascularization, respectively, in the transplanted uterus. Because the ovary is removed when the ovarian vein is used, only veins of

a unilateral ovary were used and the contralateral ovary was retained to maintain ovarian function. The uterus of each monkey was removed almost simultaneously from the abdominal cavity (Fig. 1a). Back table preparation was performed as previously described.[9] After back table preparation, the uteri were interchanged and orthotopically transplanted. In case 1, end-to-end anastomosis PS-341 in vitro of the left uterine artery of the host to the left uterine artery of the uterus of case 2 was carried out by interrupted suture with 12-0 nylon thread

(Crownjun). LBH589 solubility dmso Next, end-to-side anastomosis of the right ovarian vein of the host to the right ovarian vein of the uterus of case 2 was carried out by interrupted suture with 9-0 nylon thread (Crownjun). Clamps for vessels were then released and uterine perfusion started. Subsequently, end-to-end anastomosis of the right uterine artery of the host to the right uterine artery of the uterus of case 2 was carried out by interrupted suture with 12-0 nylon thread. Because the uterine vein was extremely thin, no anastomosis was performed. Thus, in case 1, the uterus was perfused using two arteries and one vein (Fig. 1b). In case 2, end-to-side anastomosis of the right uterine artery of the host (vascular diameter, 1.2 mm) to the right uterine

artery of Thiamet G the uterus of case 1 was carried out by interrupted suture with 11-0 nylon thread (Crownjun). Next, end-to-end anastomosis of the left ovarian vein of the host in the mesosalpinx to the left ovarian vein of the uterus of case 1 was carried out by interrupted suture with 11-0 nylon thread. Clamps for vessels were then released and uterine perfusion started. Subsequently, end-to-end anastomosis of the left uterine artery of the host to the left uterine artery of the uterus of case 1 was carried out by interrupted suture with 11-0 nylon thread, and end-to-end anastomosis of the right uterine vein of the host to the right uterine vein of the uterus of case 1 was carried out by interrupted suture with 11-0 nylon thread. Because the left uterine vein was extremely thin, no anastomosis was performed. Thus, in case 2, the uterus was perfused using two arteries and two veins (Fig. 1b). To prevent rejection of each transplanted uterus, immunosuppressants were used in the perioperative and postoperative periods.

Double bands were selected only when two distinct bands could be seen on the gel image and in the bionumerics densitometric curve window. Phylogenetic analyses were performed using the Dice similarity coefficient (Dice, 1945) and the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis based on numbers and positions of bands by bionumerics (Sneath & Sokal, 1973). Gel-purified LpF1 was cloned into the pCR-Blunt II-TOPO vector (Invitrogen) and sequenced using the M13 forward (−20) (5′-GTAAAACGACGGCCAG-3′), M13 reverse

(5′-CAGGAAACAGCTATGAC-3′), P1-FBA1 (5′-CAGATGGTCAATCAACGATC-3′), Alpelisib molecular weight and P2-FBA1 (5′-CCGGGTGGTGGATTTAAACC-3′) primers using a BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems) in a 3730 Genetic Analyzer (Applied Biosystems). LpF1 was subsequently characterized by sequence similarity searches against the GenBank database using the blast

algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1997). The FBA1-specific fragment (LpF2) was amplified using 35 ng of template DNA, P3-FBA1 (5′-TCTATAATTTGTGATACAGGGGTTGCC-3′), and P4-FBA1 (5′-CTCGTAATCACACAGAAATTATGCTGC-3′) under the following cycling conditions: an initial 94 °C for 3 min; 35 cycles at 94 °C for 15 s, 59 °C for 35 s, and AZD2281 68 °C for 2 min; and a final 68 °C for 7 min. Genomic DNA (1 μg) from L. paraplantarum strains digested by Dra I were separated by a 1% agarose gel and transferred to nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany). The LpF2 fragment (946 bp) was purified using a PCR purification kit (Qiagen) and labeled using a Digoxigenin (DIG) High Prime Kit (Roche Diagnostics GmbH) according to the manufacturers’ instructions. Hybridization was carried out at 42 °C. Membrane was washed under conditions of high stringency at 68 °C. Detection was

Fossariinae performed using an anti-DIG antibody alkaline phosphatase conjugate and CSPD. Membrane was activated at 37 °C for 10 min and developed to an X-ray film (Roche Diagnostics GmbH). Strains were preliminarily classified by sequence analyses of pheS, rpoA (Naser et al., 2005), and 16S rRNA genes (Table 1) and further confirmed using PCR-based methods (Berthier & Ehrlich, 1999; Torriani et al., 2001a, b). To discriminate these strains, we evaluated repetitive element sequence-based (REP-) (Jersek et al., 1999), triplicate arbitrarily primed (TAP-) (Cusick & O’Sullivan, 2000), RAPD-, and ERIC-PCRs, but those except ERIC did not yield a band that was specific to L. paraplantarum strains (data not shown). In ERIC-PCR, the L. paraplantarum strains tested had similar band profiles (Fig. 1a, lanes 7–13); the shared bands agreed with the type strain of L. paraplantarum (JCM 12533T, lane 7). The DNA bands of approximately 2.8, 1.1, 0.9, and 0.55 kb generated with the primer set ERIC-1R and ERIC-2 were common to strains of the species L. paraplantarum (Fig. 1a, horizontal arrows).

Factors showing a significant correlation (P ≤ 0.1) were included in a multiple regression model and a backward stepwise procedure removed less significant factors. Analyses were performed using pasw, version 18.0 (IBM/SPSS Inc., Chicago, IL). Apoptosis Compound Library in vitro One hundred and six patients were enrolled in the STOPAR trial and 54 were included in this substudy (34 in the TC arm and 20 in the TI arm) [11]. No differences in baseline

characteristics were found between participants in the general study and in the substudy. Forty-one patients were men (75.9%) with a median age of 42 years, 6.5% had AIDS, and 81.5% were taking NNRTIs at baseline. The median baseline CD4 cell count was higher in the TI arm (939.5 cells/μL; IQR 625, 1817 cells/μL) than in the TC arm (787.5 cells/μL; IQR 523, 1814 cells/μL; P = 0.026).

The median MCP-1 plasma concentration was higher in the TC arm (323.4 pg/mL; IQR 253, 440.9 pg/mL) than in the TI arm (244.6 pg/mL; IQR 184.7, 349 pg/mL; P = 0.039). There were no other www.selleckchem.com/products/ABT-737.html differences between the groups at baseline (Table 1). In the TI arm, median MCP-1 was significantly increased at month 12 (29.3%; IQR −1.9, 108.8%; P = 0.003), month 24 (35.0%; IQR 7.9, 93.0%; P = 0.006) and month 36 (43.2%; IQR 13.9, 82.5%; P < 0.001) compared with baseline, with no changes in the TC arm (P > 0.05 for all comparisons). The median plasma sVCAM-1 concentration was also increased at all three time-points in the TI arm compared with baseline [14.6% (IQR 0.0, 35.9%), P = 0.002;

30.4% (IQR 1.0, 51.5%), P = 0.004; 19.5% (IQR 0.2, 44.7%), P = 0.012, respectively] with no changes in the TC arm (P > 0.05 for all comparisons) (Fig. 1). T-PA was increased in both arms at the three time-points compared with www.selleck.co.jp/products/carfilzomib-pr-171.html baseline (Fig. 1), except at 12 months in the TI arm. A tendency for a greater increase in the TC arm was observed for t-PA at month 36 (P = 0.052). Plasma IL-6-values were under the limit of detection in a high percentage of patients, both at baseline [TC arm, 16 patients (47%); TI arm, 16 patients (80%)] and at month 36 [TC arm, 20 patients (58.8%); TI arm, 16 patients (80%)]; there were no changes in these percentages over the study period (P = 0.566). Plasma IL-8 was also under the limit of detection in a high percentage of patients at baseline [TC arm, 26 patients (76.5%); TI arm, 13 patients (65%)] and at month 36 [TC arm, 23 patients (67.6%); TI arm, 17 patients (85%)], with no changes over the study (P = 1). sP-selectin and sCD40L were under the limit of detection in a high percentage of patients at baseline (Table 1). During follow-up, however, sP-selectin concentrations increased significantly at month 36 compared with baseline in both the TI arm (median 73.8%; IQR 0, 140.5%; P = 0.010) and the TC arm (median 6.9%; IQR −3.1, 70.3%; P = 0.

Further studies

reported that a new galactosaminogalactan and the galactomannan were the major polysaccharides of the in vivo A. fumigatus EPS (Loussert et al., 2010). For A. niger, after germination upon a support, the new hyphae also produce an EPS (Villena & Gutierrez-Correa, 2007b). Singhal et al. (2011) recently reported that primary epithelial cells could support the growth of biofilms under flow conditions that were also associated with significant EPS production compared with biofilms formed under static condition (Singhal et al., 2011). The production of EPS has also been reported elsewhere, where it is shown to be produced on polystyrene and on CF bronchial Selleckchem Pexidartinib epithelial cells (Seidler et al., 2008). This study also reported that biofilm cells attaching to epithelial cells exhibited decreased sensitivity to antifungal drugs. Whilst the precise role of the EPS

buy Stem Cell Compound Library is not known, it is hypothesized that it plays a significant role in antifungal resistance by preventing diffusion. This is supported from data emerging from the C. albicans biofilm field, where it was demonstrated that EPS expression (specifically beta-glucans), encoded through fks1, sequesters antifungal agents and reduces susceptibility (Nett et al., 2010a). Figure 2 illustrates the presence of EPS within A. fumigatus biofilms. Antifungal resistance is a defining characteristic of fungal biofilms. In A. fumigatus, biofilms antifungal resistance has been reported (Beauvais et al., 2007; Mowat et al., 2007; Seidler et al., 2008; Fiori et al., 2011), which has been shown to be phase dependant (Mowat et al., 2008b). Here, three phases of biofilm growth (8, 12 and 24 h) were investigated to assess the effects of antifungal agents on different phases of biofilm. Clear differences in susceptibility were observed in each biofilm population, where younger biofilms (8 h) were significantly Cell press more susceptible than intermediate (12 h) and

mature biofilms (24 h) (Mowat et al., 2008b). Our recent study, supports the concept that this phase resistance is correlated with efflux pump activity. This study reported that efflux activity increases with biofilm maturity, and that sensitivity to voriconazole could be improved through the use of a competitive inhibitor. Transcriptomic analysis showed that maximum activity associated with the early filamentous phase (12 h), and in defined clinical isolates, maximal expression of mdr4 correlated with the highest increase in resistance in 12 h biofilm populations. Conversely, expression of this gene was minimal at 24 h, suggesting phase dependant efflux activity (Rajendran et al., 2011). It was therefore speculated that efflux pump activity plays a contributory role to antifungal resistance. It is conceivable that A.

, 1989). This strategy may be particularly relevant to tetronasin, because it has a much greater affinity for divalent, particularly Ca2+, than monovalent ions, in contrast to other feedlot ionophores, including monensin and lasalocid (Grandjean & Laszlo, 1983). Ca2+ ions are present at much lower concentrations (0.7–11.2 mM) than Na+ (77–157 mM) or K+ (22–68 mM) in the rumen (Durand & Kawashima,

1979); therefore, it seems possible that the potency of an ionophore that carries Ca2+ ions may be more readily enhanced than those that carry the more abundant monovalent ions. The aim of the experiments described in this paper was to determine how varying the ionic composition of the medium affects the toxicity of monensin Bleomycin cost and tetronasin to selected species of ruminal bacteria and ion gradients in sensitive bacteria. Prevotella albensis

M384 (DSM 11370), Lactobacillus casei LB17 and Streptococcus bovis C277 were isolated from the rumen of sheep and are maintained in the culture collection at the Rowett Institute. Eubacterium ruminantium 2388 was originally obtained from the National Collection of Dairy Organisms, Reading. The liquid form of general-purpose, ruminal fluid–containing medium 2 of Hobson (Hobson, 1969) was used as the basal medium for growth experiments with all four bacteria. The C sources contained in this medium are glucose, maltose, cellobiose and lactate. Modifications to the mineral content were made by adding more K+ as phosphate salts and Na+ and Ca+ as chloride salts. The final concentrations of the cations in the control and amended media, OSI-906 mouse respectively, were as follows: Na+, 137 and 172 mM; K+, 19 and 35 mM; Ca2+, 2.8 and 7.4 mM. In experiments to determine Δp and ion gradients in E. ruminantium, cation concentrations

in the medium were DNA ligase 19 mM K+, 149 mM Na+ and 2.8 mM Ca2+. Media were prepared, and cultures were maintained, under O2-free CO2. Growth and incubation temperature was 39 °C. A fresh overnight culture was used to inoculate (7%, v/v) media in Hungate tubes to which ionophores had been added in ethanolic solution (1 μL mL−1) before autoclaving. The concentration of ionophores was serially doubled in these tubes, as described previously (Newbold et al., 1988). Growth was measured by optical density at 650 nm after 48 h. The toxicity of the ionophore was assessed by determining the concentration of ionophore at which growth was inhibited by 50% (IC50). Tetronasin or monensin was added to late-exponential phase cultures of E. ruminantium or cultures that had been in stationary phase for 30 h as ethanolic solutions at 0.064 and 0.256 μg mL−1. Ethanol (1 μL mL−1) was added to control incubations. Intracellular pH was determined 2 h after the addition of ionophore by the distribution of radiolabelled benzoic acid (Rottenberg, 1979). Culture (1 mL) was incubated under CO2 with [carboxy-14C] benzoate (0.25 μCi, 22 mCi mmol−1) and 3H2O (2.