EoA performance was assessed approximately 5 min after practice with tDCS ended. On Day 2 of the experimental session, the participants were tested for retention of the practiced sequence. Fifty random trials were also presented at baseline, EoA and at Day 2 of retention, buy Torin 1 to control for changes in the reaction time due to changes in visuospatial processing speed over practice. Within these random trials there was no repeating sequence. A more specific measure of implicit

sequence learning was obtained by contrasting the sequential response times against those response times for the random trials. To ensure that the participants did not have explicit knowledge of the motor sequences, they were asked if they noticed any pattern after Day 2 of testing. Three of 12 participants reported that they thought that some pattern was repeating, but could not explicitly recall more than three serial elements of the sequence when asked to reproduce it (i.e. no free recall). One additional participant was able to recall five items of the ten-item sequence on the free-recall test, and was therefore excluded from further analysis. TMS was employed to localize

the M1 location for FDI muscle. Participants were seated in a comfortable chair with the forearm supported in a prone position and hand resting on an arm support. Single TMS pulses were applied over the Selleckchem PD 332991 right motor cortex with a 70-mm figure-of-eight coil attached to a Magstim Rapid Stimulator (The Magstim Company, Wales, UK). The coil was held tangentially to the scalp with the handle pointing posteriorly away from the midline at an angle of ∼45 °. Cortical current induced from this position is directed approximately perpendicular to the central sulcus (Brasil- Neto et al., 1992; Mills et al., 1992). A ‘hot-spot’ for FDI was determined as the site at which the largest motor evoked potential was obtained from FDI at lowest

Etofibrate TMS intensity. This hotspot overlies the area of the M1 that more heavily projects to the FDI, and was the site for M1 tDCS. For the premotor cortex, the tDCS active electrode was positioned 3 cm anterior and 1 cm medial to the hot-spot (Boros et al., 2008). tDCS was delivered at 1 mA current intensity using a constant-current stimulator (Dupel Iontophoresis System, Empi, MN, USA) using an 8-cm2 saline-soaked anode and a self-adhesive carbonized cathode (48 cm2) placed over the forehead above the contralateral orbit. For active tDCS conditions, the current was ramped up over 10 s, held constant at 1 mA for 15 min and then ramped down over 10 s. For the sham tDCS, the current was ramped up for 10 s and then the machine was switched off. All the participants tolerated tDCS very well and there no adverse effects were reported. Only reaction times (RTs) for correct trials were included in the analysis. RTs longer than 2.

, 2009). Thus, the question of arsenite binding is of great interest to synthetic biologists involved in engineering of novel molecular entities that could be used in arsenite detection and decontamination. Most of the molecular models of arsenite binding involve thiol-based chemistry. In fact, most of the proteins that have been identified to bind to arsenite and thus have been inactivated by it do so through Cys residues (Hughes,

2002; Kitchin & Wallace, 2004). However, neither Cys residue nor Tyr residues, which have also been reported to bind arsenite in some proteins (Page & Wilson, 1985), are present within the sensory domain of AroS. Perhaps the difference in the mode of binding arsenite is not too surprising when considering the function that AroS performs. This protein needs to be able to bind arsenite reversibly and to Palbociclib concentration be able to respond to changes in arsenite concentrations. Presently, we cannot provide Venetoclax a definitive answer of what the mechanism of arsenite sensing is; however, our work provides a foundation for further structural and mechanistic analysis of this regulatory system. In addition, not only do arsenite-oxidizing

bacteria need to be able to sense the presence of arsenite in the environment, but they also need means of evading arsenite toxicity. Our studies have demonstrated for the first time that a mutation in aroS has an effect on the growth of NT-26 in the presence of arsenite. Thus, AroS may play an additional role in the regulation of a pathway involved in tolerance to arsenite. T.H.O. is supported by a Natural Environment Research Council studentship (14404A). Fig. S1. 1D 1H NMR spectra for AroS226–490H273N protein that lacks autophosphorylation activity. Please note: Wiley-Blackwell is not responsible for

the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“In this issue, Tordato et al. [1] present interesting findings from the Italian Cohort of Antiretrovial Naive patients (ICONA) study group considering the estimated glomerular 3-mercaptopyruvate sulfurtransferase filtration rate (eGFR) and risk factors for mild renal dysfunction, defined as eGFR<90 mL/min/1.73 m2. Since the introduction of combination antiretroviral therapy and subsequent dramatic improvements in morbidity and mortality [2], patients with HIV infection live longer and research has increasingly been carried out on comorbidities, including renal disease [3]. There have been a number of recent studies focusing on renal function using different definitions and methodologies which can lead to conflicting results and difficulty in interpreting data. GFR is commonly estimated using the Cockcroft–Gault (CG) formula [4], the Modified Diet in Renal Disease (MDRD) equations [5], and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [6].

05 log10 copies/mL (IQR 2.07–5.14 log10 copies/mL)]. The median follow-up time was 2.6 years (IQR 1.1–4.8 years). The majority of patients in the three treatment groups were on an NRTI backbone of zidovudine (ZDV) and lamivudine (3TC): 46%, 46% and 48% on nevirapine, efavirenz and lopinavir, respectively. Twenty-four per cent, 18% and 14%, respectively, were on stavudine (d4T) and lamivudine; this was the second most common NRTI backbone for those on nevirapine and efavirenz. For patients on lopinavir,

the second most common NRTI backbone was tenofovir with one other NRTI. A total of 1417 patients (49%) discontinued nevirapine, efavirenz or lopinavir while under follow-up. Of these, 299 (50%) discontinued nevirapine, Birinapant mouse 748 Fluorouracil molecular weight (51%) discontinued efavirenz and 370 (45%) discontinued lopinavir for any reason while under follow-up. Figure 1 shows the Kaplan–Meier estimation of the probability of all-cause discontinuation of the regimen.

At 24 months after starting the regimen, 30.4% [95% confidence interval (CI) 26.6–34.2%] were estimated to have discontinued nevirapine, compared with 28.1% (95% CI 25.7–30.5%) for efavirenz and 31.7% (95% CI 28.4–35.2%) for lopinavir. The corresponding figures at 48 months were 47.2% (95% CI 42.9–51.5%), 44.3% (95% CI 41.5–47.1%) and 51.2% (95% CI 47.1–55.3%), respectively (P=0.02). In a multivariate Rebamipide Cox proportional hazards model (Fig. 2), stratified by centre, compared with patients starting nevirapine there was no significant difference in the risk of discontinuation of efavirenz [hazard ratio (HR) 1.06; 95% CI 0.91–1.23; P=0.43] or lopinavir (HR 1.14; 95% CI 0.96–1.36; P=0.13). Figures 3(a) and (b) show the Kaplan–Meier estimation of the probability of discontinuation for specific reasons. Seventy-four patients (12%) discontinuing nevirapine, 101 patients (7%) discontinuing efavirenz and 33 patients (4%) discontinuing lopinavir did so because

of reported treatment failure (virological, immunological or clinical). One hundred and fifty-five patients (75%) discontinuing because of reported treatment failure (i.e. on patient follow-up forms) had a viral load >500 copies/mL measured in the 6 months prior to discontinuation. After adjustment, compared with patients starting nevirapine, patients starting efavirenz had a 48% lower risk of discontinuation because of treatment failure (HR 0.52; 95% CI 0.37–0.73; P=0.0002) and those starting lopinavir had a 63% lower risk of discontinuation because of treatment failure (HR 0.37; 95% CI 0.23–0.61; P<0.0001) (Fig. 2). One hundred and thirty-nine patients (23%) discontinuing nevirapine, 436 patients (30%) discontinuing efavirenz and 247 patients (30%) discontinuing lopinavir did so because of reported toxicity or patient/physician choice.

6C–C2; Geula et al., 1993). These

data demonstrate that the cholinergic identity of scgn+ cells is acquired by the third trimester of pregnancy. To evaluate the fate of scgn+ neurons in the EA we analyzed sections from ChAT-EGFP and GAD67-GFP reporter mice, allowing precise delineation of the anatomical boundaries of individual amygdaloid nuclei (Fig. 7A and B). Scgn expression was limited to two morphologically distinct types of neurons in the EA (Fig. 7C): scgn+ neurons with oval perikarya and short ramifying dendrites (Fig. 7Ca) predominate in the CA and IPAC. In contrast, scgn+ neurons learn more as above are intermingled with stellate-like cells with fusiform perikarya and long, smooth primary dendrites in the MA (Fig. 7Cb–Cd). ChAT+/scgn+ neurons were exclusively identified in the VP and dorsal segment of the SI (Mulder et al., 2009b) but not amygdaloid nuclei (Fig. 7D). www.selleckchem.com/products/crenolanib-cp-868596.html GAD67+/scgn+ small-diameter neurons were frequently encountered in the CA and MA (Fig. 7E) but not the IPAC (Fig. 7E1) or the intraamygdaloid segment of the BST (Fig. 7E2). A clear phylogenetic difference in the distribution of scgn+ neurons was the complete absence of scgn immunoreactivity in small-diameter GABAergic neurons of the primate CA and MA.

Instead, fusiform scgn+ neurons populated the MA (Fig. 7F). Based on their cellular origins and connectivity maps, the lateral, basolateral, basomedial and cortical amygdaloid nuclei are classified as pallial structures. In contrast, the BST, CA, MA and SI are of subpallial origin. BST and SI neurons are thought to originate in the pallidal ridge, while neurons inhabiting the CA and MA share their birthplace with striatal neurons (Swanson & Petrovich, 1998). Therefore, the selective expression of scgn in pallidal amygdaloid territories

further illustrates the above developmental dichotomy. Distinct anatomical organization of scgn mRNA expression with heterogeneous signal in a number of structures was evident in the fetal human brain (Fig. 8). Scgn mRNA distribution patterns were similar in all subjects studied. new Invariably strong scgn anti-sense probe hybridization signal was observed throughout the cortical plate of the cerebral cortex (Fig. 8A and B) and in the amygdaloid complex (Fig. 8B). Moderate scgn mRNA expression was observed in the hippocampus, subiculum, thalamic territories and germinal layers, whereas low signal intensity was seen in the caudate nucleus. These data demonstrate that scgn expression in the mammalian amygdaloid complex is phylogenetically conserved. In addition, our results highlight that scgn expression in pyramidal cells is developmentally regulated and can endure into adulthood in this cell type (Attems et al., 2007). Our report identifies the developmental dynamics of scgn expression including the migratory routes and final positions of subpallial neurons expressing this CBP in rodent, primate and human fetal brain.

04% SDS, 20% methanol and Tris-HCl, pH 8.0) at a constant voltage of 30 V for the first hour at 4 °C and then at 80 V for 2 h. The membrane was blocked with 5% skimmed milk in phosphate-buffered saline (PBS), pH 7.4, at 4 °C overnight. The immobilized proteins were probed with rabbit anti-BinB

antibody (1 : 20 000), which was prepared by injecting the fast protein liquid chromatography-purified BinB into a rabbit, for 1 h and goat anti-rabbit IgG conjugated with alkaline phosphatase (1 : 5000) for 1 h. The immunoreactive bands were visualized using an ECL chemiluminescent plus kit (GE Healthcare). Protein inclusions (2–3 mg mL−1) were solubilized by incubation in 25 mM NaOH, 5 mM dithiothreitol at 37 °C for 15 min. Solubilized protein was stepwise dialyzed in 250 vol. of carbonate buffer (50 mM Na2CO3, pH 10.0) with a gradual decrease of NaOH concentrations to 19, check details 13.3, 6.7 this website and 3.4 mM. Each dialysis was performed at 4 °C for 1 h. Finally, the samples were dialyzed three times against 250 vol. of the carbonate buffer. The protein was further purified by gel filtration using a superdex

200HR 10/300 column (Amersham Pharmacia Biotech) and purified protein was kept at −20 °C. In vivo mosquito-larvicidal assays were used to determine the toxicity of mutant toxins against 2nd-instar Culex quinquefasciatus larvae, which were supplied by the mosquito-rearing facility in the Institute of Molecular Biosciences, Mahidol University, Thailand. Equimolar mixtures

of BinA wild-type inclusions and BinB mutant inclusions were diluted in 1 mL of water at several twofold serial dilutions, from 64 μg mL−1 to 0.1250 μg mL−1. Tau-protein kinase These 1-mL dilutions were added to wells in a 24-well tissue culture plate, each well containing 10 larvae. The BinA–BinB wild-type inclusion mixture was used as a positive control, while BinB wild-type inclusions were used as a negative control. After a 48-h incubation period, the mortality of the larvae was recorded. The assays were carried out in duplicate, and at least three independent experiments were performed. The LC50 was then analyzed by Probit analysis (Finney, 1971). A nitrocellulose membrane was cut into strips and was equilibrated in PBS. Various amounts of purified truncated BinA (2.5–20 μg) (Limpanawat et al., 2009) were immobilized on every strip at 25 °C using a Bio-Dot Microfiltration Apparatus (Bio-Rad). The dotted membranes were blocked in 5% skimmed milk at 4 °C overnight. Twenty micrograms per milliliter of purified wild type, or mutant, BinB in 5% skimmed milk was overlaid for 1 h on each strip and subsequently washed with 0.1% Tween-20 in PBS (PBS-T20) three times, for 5 min each time. Bound BinB was detected by probing with rabbit anti-BinB (1 : 20 000) for 1 h.

65 The curse of dual disease during pregnancy is widely studied in the African region.23,59 Recent reports from India also explored the intricate correlation between HIV infection and TB in the context of pregnancy and the post-partum period.61,62 Among

HIV-infected Indian women, Gupta et al. found a high incidence of post-partum TB (five cases per 100 person-years).62 Furthermore, co-infection of TB has substantially increased post-partum maternal death (2.2-fold; 95%CI 0.6–3.8) and death of their infants (3.4-fold; BTK pathway inhibitors 95%CI 1.22–10.59). This raised a serious concern regarding the strategy of screening and managing latent TB during pregnancy in the context of India, and other South Asian countries, where isoniazid prophylaxis is not advocated at present in latent TB. The authors suggested

that active screening and targeted use of isoniazid preventive therapy among HIV-infected women in India should be considered to prevent post-partum maternal TB. In a subsequent article, Gupta et al.61 also reported Bortezomib concentration that maternal TB, mostly detected after delivery, is associated with increased mother-to-child transmission of HIV (30% vs 12%). Therefore, prevention of TB among HIV-infected mothers should be a high priority for communities with significant HIV/TB burden. Dual infection of TB and HIV-infection poses several unique challenges. Its management during pregnancy demands special expertise, judicial sequential combination of anti-TB drugs and anti-retroviral drugs, which is beyond purview of this current review.23,59 This issue was recently addressed elsewhere.59 It is increasingly evident that TB has many adverse effects on maternal and child health in high-prevalent countries, with HIV-infected mothers and their infants being

particularly vulnerable.66 These include not only direct effects, such as morbidity and mortality, but also multiple indirect effects see more that trap the woman in a vicious circle of perpetual poverty and vulnerability.67 As untreated or incompletely treated TB poses a great risk to pregnant women and their fetuses, all women with TB irrespective of sites involved must receive a full course of anti-TB drugs.68 According to the recent World Health Organization (WHO) recommendation, ‘A pregnant woman should be advised that successful treatment of TB with standard regimen is important for successful outcome of pregnancy.’69 Management of active TB during pregnancy is similar to that in non-pregnant women. With the exception of streptomycin, all first-line anti-TB drugs (isoniazid [H], rifampicin [R], ethambutol [E], and pyrazinamide [P]) are considered safe for use in pregnancy, and have no proven teratogenic effects.69–76 Streptomycin-induced fetal ototoxicity leading to hearing impairment and irreversible congenital deafness affects one in six neonates; therefore, it should not be used throughout pregnancy.

For the culture, a cysteine production medium [composition (per liter): a quantity of 12 g of ammonium chloride, 1.5 g of potassium dihydrogenphosphate, 1 g of magnesium sulfate heptahydrate, 0.1 mg of thiamine hydrochloride, 1.7 mg of ferrous sulfate heptahydrate, 0.15 mg of sodium molybdate dihydrate, 0.7 mg of cobalt chloride

hexahydrate, 1.6 mg of manganese chloride tetrahydrate, 0.3 mg of zinc sulfate heptahydrate, 0.25 mg of copper sulfate pentahydrate, 0.6 g of tryptone, 0.3 g of yeast extract, 0.6 g of sodium chloride, 20 g of calcium carbonate, 135 mg of l-histidine monohydrochloride CAL-101 mouse monohydrate, 4 g of sodium thiosulfate, 2 mg of pyridoxine hydrochloride, 40 g of glucose, 12.5 mg of tetracycline] (Nonaka, 2010) was used. For the cultivation with thiosulfate or sulfite as a sulfur source, 8 g L−1 of sodium thiosulfate or 2.6 g L−1 of sodium sulfite was added, respectively.

For the cultivation with sulfate see more as a sulfur source, 15 g L−1 of ammonium sulfate was added instead of ammonium chloride. The BW26424/pACYC-DES and the BW25113/pACYC-DES strains were each applied and spread onto LB agar medium containing tetracycline, and precultured overnight at 34 °C. The streak cells corresponding to about 7 cm on the plate were scraped with an inoculating loop of 10 μL size (NUNC Blue Loop), and inoculated into 2 mL of the cysteine production medium. The culture was grown at 32 °C with shaking for 42 h, and the amount of cysteine accumulated in the medium was quantified. The quantification of cysteine was performed by the method described by Gaitonde (1967). The experiment was performed in hexaplicate for both the strains, and averages and standard

deviations of the accumulated cysteine amounts were calculated. Escherichia coli cells grown in 10 mL of LB medium Bacterial neuraminidase were harvested by centrifugation and resuspended in 0.2 mL 8 M urea/lysis buffer (8 M urea, 50 mM Tris-HCl, pH 8.0 at 4 °C, and 100 mM NaCl), and sonicated. Cell extracts (10 μg) were subjected to 10% SDS-PAGE and blotted on to polyvinylidene difluoride (PVDF) membranes using iBlot semi-dry transfer apparatus (Invitrogen). Membranes were first immuno-detected with anti-β-galactosidase (Progema) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Nacalai tesque) antibodies and then developed with a chemiluminescence kit (Nacalai tesque). The image was analyzed with a LAS-4000 IR multi color (Fuji Film). The transcriptome analysis of E. coli response to metal-shock indicated that the genes for cysteine biosynthesis including cysK are regulated by several metals (Yamamoto & Ishihama, 2005a, b; Hobman et al., 2007). Since a set of the metal stimulon genes are regulated by some of two-component system (TCS) (Yamamoto & Ishihama, 2006; Yamamoto et al., 2008), we tested possible influence of TCS knock-out on cysK expression. For this purpose, we used the collection of TCS deficient E. coli mutants (Oshima et al.

In-hospital costs (for both in-patient and day-care admissions) were based on the DRG system in use in Italy since 1994, in which disease groups Ganetespib mouse are defined according to the hospital discharge form data. Costs

of out-patient consultations and examinations (laboratory and clinical imaging) were calculated based on the official standard costs assigned by the Italian Ministry of Health. According to an agreement between the Italian Ministry of Health and all pharmaceutical companies, hospitals pay half price for antiretroviral drugs, instead of the full cost paid by the public in Italy. All costs in this analysis were annualized and expressed in nominal terms for the year in which they were incurred. Costs incurred by patients (e.g. costs of travel to hospital services or costs of additional services incurred by staying at home), intangible costs (e.g. stress and anxiety) and indirect costs to society (e.g. loss of productivity) were BLZ945 price not estimated, as they did not affect the comparison of medical sector burden between chronic diseases, which was the main objective of the study. For the

same reasons, the cost analysis did not take into consideration the inflation rate, which the Italian National Institute of Statistics confirmed to be 2.1% on average at the time of our study. The criteria for the identification of HIV-infected persons were as follows: a diagnosis of HIV infection based on serological testing; For an HIV-infected person to be considered as having a concomitant chronic disease, they had to satisfy at least one of the above criteria for that chronic disease. HIV-infected persons who were at any time on antiretroviral treatment during the year were classified as ‘on antiretroviral treatment’. A verification procedure to assess the sensitivity of the BLHA database for detecting HIV-infected patients was performed through cross-checking of patients registered in the databases Pyruvate dehydrogenase of the following institutions operating

in the Province: the Institute of Infectious and Tropical Diseases, the Clinic for Sexually Transmitted Diseases, Methadone Dispensing Units, and Primary Health Care Services for HIV Patients. Death certificates were also reviewed. Cross-checking verified that the BLHA database missed only 4% of patients registered in the other available databases. Starting from 2004, all newly identified cases were considered as ‘incident’ cases, by which we mean ‘newly diagnosed’ rather than ‘newly infected’. To calculate a denominator for the annual prevalence and incidence, we used an estimate of the mid-year average number of people who received services from the Brescia Local Health Authority during a calendar year.

Murine innate defence against A. fumigatus is sufficient to prevent infection, even in heavily infected animals, and immunosuppression is required to establish

infection (Lewis & Wiederhold, 2005). In the McDonagh study, both macrophage and neutrophil cell populations were chemotherapeutically targeted, using hydrocortisone acetate and cyclophosphamide, respectively, the former drug administered in a single dose 1 day before infection and the latter periodically administered throughout the duration of the experiment (Lewis & Wiederhold, 2005). Phagocytosis by macrophages harvested from hydrocortisone-treated A. fumigatus-infected mice is known to occur, but fungal killing is compromised (Philippe et al., 2003). It is likely, therefore, that host Selleck AZD9291 cells, predominantly macrophages, are encountered in the alveolar and bronchial spaces (Fig. 2b and c), and encountered macrophages are compromised in their ability to kill A. fumigatus spores. Conversely, the encapsulated facultative intracellular pathogen C. neoformans can establish infection in immunocompetent mice. Moreover, the interaction between macrophages and C. neoformans is critical for containing the dissemination of this pathogenic yeast, whose success is subverted by C. neoformans-derived

factors. Cryptococcus neoformans is capable of replication within the macrophage phagolysosome, a process that ultimately leads to host cell lysis or phagosome extrusion (Tucker & Casadevall, 2002; Alvarez & Casadevall, Ceritinib cost 2006; Ma et al., 2006). As in vitro studies indicate that the time taken to extrude a C. neoformans-containing phagolysosome can be as little as 2 h (Tucker & Casadevall,

2002; Alvarez & Casadevall, 2006; Ma et al., 2006), it is likely that multiple macrophage encounters occurred during the experimental time frame, and, contrary Niclosamide to the A. fumigatus infection model, noninfected macrophages were completely proficient with respect to killing ability. Carbon metabolism was, to varying degrees, commonly implicated among all of the mammalian pathogen datasets with acetyl-CoA synthetase and isocitrate dehydrogenase featuring in all four upregulated genesets. Combined with extant data on fungal carbon-metabolizing gene products and virulence, considerable insight can be gained from our comparative analysis. Firstly, the differential roles of glyoxylate cycle enzymes in virulence, which has been studied in multiple mammalian fungal pathogens, could not have been predicted from our comparative transcriptomic analysis. Glyoxylate cycle gene products are required for full virulence in C. albicans (Lorenz & Fink, 2001; Wang et al., 2003; Barelle et al., 2006) and M. grisea (Wang et al., 2003), but not in A. fumigatus (Schobel et al., 2007; Olivas et al., 2008) or C. neoformans (Rude et al., 2002). Indeed, based on our analysis, one might have predicted the necessity of glyoxylate pathway functionality in C. neoformans and A. fumigatus and nonrequirement in M. grisea (Table 2).

4a and b). The TSP of hutHUI is located 70 nucleotides upstream of the translational start of hutH. For the divergent genes hutG and hutR, TSPs were mapped 24 bp upstream of the start codon of hutG, whereas the TSP of hutR was identical to the first guanine residue of the GTG start codon, indicating the presence of a leaderless transcript (Pátek

et al., 2003). The TSPs were used to deduce the mTOR inhibitor associated promoter regions according to corynebacterial consensus sequences for −10 and −35 regions (Pátek et al., 2003). The transcription of the hut genes is most likely driven by the housekeeping sigma factor SigA. The predicted −10 regions of the hut promoters (TAttgT, TAggaT, TAgggT) contain the typical leading TA and trailing T residues, whereas the predicted −35 regions (TgGtgA, gTGcCA, ccGcgc) showed varying matches to the corynebacterial consensus sequence. To demonstrate the direct interaction of HutR with the upstream regions of the

hut genes, DNA band check details shift assays were performed with Cy3-labeled PCR fragments. For this purpose, the HutR protein was tagged with streptavidin, expressed in E. coli DH5αMCR, and purified by means of Strep-Tactin sepharose-packed columns (data not shown). First, the upstream region of hutH and the intergenic region of hutR-hutG were amplified by PCR (Fig. 4a and b). Retardation of the respective DNA fragments 1 and 4 was observed, as the HutR protein apparently bound to the DNA in vitro (Fig. 4c). A DNA sequence containing a LexA binding site of C. glutamicum (Jochmann et al., 2009) served as a negative control. Subsequently, the DNA fragments were shortened to yield BCKDHA smaller candidate HutR binding regions upstream of hutH (fragments 2 and 3) and in the hutR-hutG gene region (fragments 5 – 7). The results of the respective DNA band shift assays revealed a candidate HutR binding region of 41 bp upstream of

the hutH coding region (Fig. 4a) and a 34-bp region between hutR and hutG (Fig. 4b). In both cases, the deduced HutR binding region is located upstream of the −35 promoter region, suggesting that the HutR regulator might function as an activator (Madan Babu & Teichmann, 2003). To identify the DNA-binding motif of HutR, both DNA regions were aligned, thereby revealing the presence of a common 14-bp motif with the consensus sequence TCTGwwATwCCAGA in front of hutH and in the hutR-hutG gene region (Fig. 5c). This DNA motif contains the 4-bp terminal palindrome TCTG/CAGA. To elucidate whether the 14-bp DNA motif is required for the specific binding of the HutR protein, fluorescein-labeled 40-mers carrying this sequence in the center were used for DNA band shift assays (Fig. 5a and b). Furthermore, mutated versions of the 14-bp motifs were generated by introducing transitions in the four palindromic bases. In these cases, the purified HutR protein failed to shift the mutated 40-mers (Fig. 5a and b).