, Chicago, IL, USA) software was KPT-330 research buy used for all statistical analyses. A total of 782 arriving pilgrims were examined before the 2009 Hajj season with 432 questionnaires filled and 519 nasal and throat swabs examined. A total of 2,768 pilgrims were examined after the 2009 Hajj season with 2,730 questionnaires filled

and 2,699 nasal and throat swabs examined. Table 1 shows the demographic and clinical characteristics of arriving and departing pilgrims in the survey samples. The mean age of the two groups combined was 49.4 years (SD ± 13.5 y). The mean age of pilgrims in the arrival survey (44.7 y) was significantly less than among pilgrims in the departure survey. Those aged >60 years represented 24% of the samples of arriving pilgrims and 11% of the sample of departing pilgrims. The majority of pilgrims were male (58%); this proportion was higher among arriving pilgrims (75%) than among departing pilgrims (56%). Arriving pilgrims were mainly

(63%) Middle Eastern (including 10% Saudi); 37% were Asian or African. Table 2 shows that the majority of arriving pilgrims described their health as excellent (49%) or at least very good (33%). Only 13% stated they had a chronic disease, namely hypertension, diabetes, heart disease, or asthma. None of the pre-Hajj population was a current smoker and the majority (85%) stated they had never smoked. Table 2 also shows the vaccination status of arriving pilgrims. The majority (84%) stated that they had received at least one vaccine before the Hajj. CYTH4 Coverage for meningococcal and seasonal influenza vaccine in both groups combined was relatively high (73% and 53%, respectively), PF-562271 mw but coverage for pandemic influenza A(H1N1) vaccine was considerably lower (30%). The reasons reported for not getting the seasonal influenza vaccine in the past year were lack of knowledge about the vaccine (41%), did not know it was required (20%), did not know where to get it (15%), felt healthy and was not worried about influenza (14%), and did not think influenza is a serious illness (9%). In all, 35% of arriving pilgrims reported wearing

a face mask. Although meningococcal vaccination is a Hajj requirement for all pilgrims arriving into the Kingdom of Saudi Arabia (KSA), unfortunately compliance with this requirement is not 100%. The government of KSA does not send back pilgrims who are found not to be vaccinated; instead they are administered prophylactic antibiotics and allowed to complete the Hajj ritual. Table 3 shows the knowledge of H1N1 among arriving pilgrims. The majority of pilgrims believed that H1N1 is a serious disease (76%). However, they were roughly split in expressing their worry about catching pandemic influenza A(H1N1) during Hajj, with 47% worried and 53% not worried. More than half (56%) of pilgrims were aware of fever as a main symptom of H1N1 influenza. However, not more than a quarter were aware that sore throat (26%), cough (24%), and headache (22%) were also main symptoms of H1N1 influenza.

(1994), Carmichael & Price (1995), Freedman et al. (2000) and Paxinos et al. (2000). Digital image files were imported into Adobe Photoshop 7 or CS3 and Small molecule library were processed routinely for grey/colour levels, brightness and contrast before being composed into figure illustrations for publication. The data were obtained in two behaving unanaesthetized young adult macaque monkeys (BM, BQ). A total of 249 neurons were screened in both animals [172 (69%) in BM and 77 (31%) in BQ] using a selection of visual, auditory, gustatory, somatosensory and olfactory stimuli (Rolls, 2008). In addition, the firing rates of each cell were assessed

to see if they were influenced by eye-closure during periods when the animals were not being actively tested. Figure 1A illustrates the wide areal distribution of the 249 electrophysiologically sampled cells in the PFC. The single neuron recordings were made from mPFC areas – BAs 9, 10, 13 m, 14c, 24b (dorsal anterior cingulate cortex) and 32 (pregenual area; Fig. 1B). The anterior–posterior extent of the recordings ranged from + 10 mm to + 14 mm anterior to the posterior lip of the sphenoid bone (Fig. 1C–E). After a period without behavioural testing and interaction with the experimenter, the subjects would adopt a relaxed position in their chairs in which the arms and legs

became motionless, and the eyelids would gradually droop and eventually close. When closed, the eyes showed a slow drift Pirfenidone typical of drowsiness

prior to entry into SWS. These behavioural criteria for the animals being ‘awake’ (BS3 – eyes-open), ‘drowsy’ (BS2 – partial eye-closure) or ‘asleep’ (BS1 – eyes-closed) were made from live images of the monkeys displayed on a video monitor placed outside the hexagonal recording chamber (Balzamo et al., 1998). ECG evidence obtained during the initial recording sessions in both animals confirmed that when the animals were in BS1 they were most probably in a state of SWS (Fig. 2). Several distinct types of neuronal responses were observed as the animals passed between BS1, 2 and 3 (see Table 1 and Figs 5 and 6). As a result, a preliminary cell classification Niclosamide based on significant changes in firing rates associated with BS1, 2 and 3 was defined (see Figs 3-7 and Tables 1 and 2): Type 1 cells (28.1% of the screened population) significantly increased (+ 329 ± 26%; mean ± SEM, n = 70; P ≪ 0.01) their firing rate from the spontaneous rate when the subjects closed their eyes and went to sleep (mean ± SEM, n = 70; Awake = 3.1 ± 0.4 spikes/s; Asleep = 10.2 ± 0.8 spikes/s; P ≪ 0.01; P = 3.4 × 10−15). Type 2 cells (6.0% of the screened population) significantly decreased (−68 ± 7.2%; mean ± SEM, n = 15; P < 0.01) their firing rate on eye-closure, returning to their former level of activity with eye-reopening (mean ± SEM, n = 15: Awake = 7.7 ± 1.7 spikes/s; Asleep 2.5 ± 0.9 spikes/s; P < 0.05; P = 1.1 × 10−2). Type 3 cells (65.

(1994), Carmichael & Price (1995), Freedman et al. (2000) and Paxinos et al. (2000). Digital image files were imported into Adobe Photoshop 7 or CS3 and this website were processed routinely for grey/colour levels, brightness and contrast before being composed into figure illustrations for publication. The data were obtained in two behaving unanaesthetized young adult macaque monkeys (BM, BQ). A total of 249 neurons were screened in both animals [172 (69%) in BM and 77 (31%) in BQ] using a selection of visual, auditory, gustatory, somatosensory and olfactory stimuli (Rolls, 2008). In addition, the firing rates of each cell were assessed

to see if they were influenced by eye-closure during periods when the animals were not being actively tested. Figure 1A illustrates the wide areal distribution of the 249 electrophysiologically sampled cells in the PFC. The single neuron recordings were made from mPFC areas – BAs 9, 10, 13 m, 14c, 24b (dorsal anterior cingulate cortex) and 32 (pregenual area; Fig. 1B). The anterior–posterior extent of the recordings ranged from + 10 mm to + 14 mm anterior to the posterior lip of the sphenoid bone (Fig. 1C–E). After a period without behavioural testing and interaction with the experimenter, the subjects would adopt a relaxed position in their chairs in which the arms and legs

became motionless, and the eyelids would gradually droop and eventually close. When closed, the eyes showed a slow drift buy Y-27632 typical of drowsiness

prior to entry into SWS. These behavioural criteria for the animals being ‘awake’ (BS3 – eyes-open), ‘drowsy’ (BS2 – partial eye-closure) or ‘asleep’ (BS1 – eyes-closed) were made from live images of the monkeys displayed on a video monitor placed outside the hexagonal recording chamber (Balzamo et al., 1998). ECG evidence obtained during the initial recording sessions in both animals confirmed that when the animals were in BS1 they were most probably in a state of SWS (Fig. 2). Several distinct types of neuronal responses were observed as the animals passed between BS1, 2 and 3 (see Table 1 and Figs 5 and 6). As a result, a preliminary cell classification Megestrol Acetate based on significant changes in firing rates associated with BS1, 2 and 3 was defined (see Figs 3-7 and Tables 1 and 2): Type 1 cells (28.1% of the screened population) significantly increased (+ 329 ± 26%; mean ± SEM, n = 70; P ≪ 0.01) their firing rate from the spontaneous rate when the subjects closed their eyes and went to sleep (mean ± SEM, n = 70; Awake = 3.1 ± 0.4 spikes/s; Asleep = 10.2 ± 0.8 spikes/s; P ≪ 0.01; P = 3.4 × 10−15). Type 2 cells (6.0% of the screened population) significantly decreased (−68 ± 7.2%; mean ± SEM, n = 15; P < 0.01) their firing rate on eye-closure, returning to their former level of activity with eye-reopening (mean ± SEM, n = 15: Awake = 7.7 ± 1.7 spikes/s; Asleep 2.5 ± 0.9 spikes/s; P < 0.05; P = 1.1 × 10−2). Type 3 cells (65.

CCA-containing precursor tRNA (pre-tRNAs) are processed exonucleolytically (Schurer et al., 2001). In cyanobacteria, the processing of CCA-containing pre-tRNAs has not been characterized. All tRNA precursors are processed at the 5′ side by RNase P. We have studied the expression and processing of the tRNAs encoded in the delta plasmid of Anabaena 7120, and we have determined that they are correctly processed and aminoacylated. During the study of the tRNA cluster, we have identified a variant tRNASerGCU that was not LY2157299 purchase annotated in the database. A structural analysis of this tRNA shows that it presents a tRNA-like structure, with a serine GCU codon, and other determinants of a

tRNASer. We demonstrate

that this newly identified tRNA is aminoacylated in vitro and in vivo. Anabaena 7120 (Rippka et al., 1979) was grown photoautotrophically p38 MAPK assay at 30 °C under white light (65–100 μE m−2 s−1). Medium used for growth on plates was BG11 (NaNO3 as the nitrogen source) or BG110 (N2 as the nitrogen source; Rippka et al., 1979). Liquid cultures were grown in the same media supplemented with 10 mM NaHCO3 and bubbled with 1% CO2-enriched air. Cells from cultures grown to 5 μg chlorophyll mL−1 were collected by filtration (filter type: 0.45 μm HA; Millipore HAWP05000) and washed in RNase-free TE buffer [10 mM Tris–HCl (pH 7.5), 1 mM EDTA]. Pelleted cells were reduced to dust after freezing in liquid nitrogen and resuspended in a buffer containing 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 5 mM CaCl2 and 20% glycerol, and the samples were centrifuged Nabilone at 2500 g for 10 min at 4 °C. Protein was quantified by Lowry’s method (Lowry et al., 1951). Cells pellets prepared as described above were resuspended in 100 μL of a lysozyme solution (3 mg mL−1) and subjected to three freeze–thaw cycles to facilitate cell lysis. RNA was isolated with 1 mL of Trizol reagent (Invitrogen), using manufacturer instructions. RNA was

extracted with phenol and with chloroform/isoamyl alcohol (24 : 1), precipitated with absolute ethanol and washed with 70% ethanol. Finally, RNA was resuspended in 30 μL of RNase-free water. To isolate RNA under acidic conditions, we used the method described by Varshney et al. (1991). Briefly, cells from a 25-mL culture were collected by filtration and resuspended in 300 μL of 0.3 M sodium acetate (pH 4.5) and 10 mM EDTA and subjected to two extractions with phenol equilibrated with the same buffer. The aqueous phase was then precipitated with absolute ethanol and resuspended in 60 μL of 0.3 M sodium acetate (pH 4.5) and 1 mM EDTA. The RNA was again precipitated with absolute ethanol and resuspended in the same buffer. A total of 10 μg of total RNA was treated with 2 units of RQ1 DNase (Promega), in 20 μL, for 1 h at 37 °C. Reaction was stopped with 2 μL of the stop buffer provided and heated for 10 min at 70 °C.

, Tokyo, Japan) with the significance criteria of the program (P<0.05). Real-time PCR was performed using a 7900HT Fast real-time PCR system Ponatinib chemical structure (Applied Biosystems). Reactions containing cDNAs from 100 ng total RNA and gene-specific primers were prepared with SYBR Green Realtime PCR Master Mix (Toyobo) according to the manufacturer’s protocol. The primers used are listed in Table S1. The thermal cycle settings used were as follows: initial denaturation at 95 °C for 1 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 56 °C for 15 s, and extension at 72 °C for

1 min. The expression of target genes was normalized to the endogenous 16S rRNA gene in each strain. The relative quantification of target gene expression was performed using the comparative cycle threshold (CT) method (Livak & Schmittgen, Alvelestat solubility dmso 2001). blast searches revealed that the TF0022 ORF encodes a HTCS protein that shares homology with GppX from P. gingivalis. We sequenced a fragment containing TF0022 and the upstream

flanking region from the ATCC 43037 genome and compared the data with the existing database sequence (http://www.oralgen.lanl.gov). Although some minor differences were found at the nucleotide level, none altered any functional domains or conserved motifs in the encoding protein (Fig. 1a, DDBJ/GenBank ID: AB587729). Alignment of the TF0022 and GppX polypeptides Org 27569 revealed a notable structural difference: the TF0022 protein lacks the N-terminal portion containing a transmembrane region and part of a putative periplasmic/sensor domain with a TPR motif (Fig. 1b). However, the immediate upstream ORF, TF0023, is predicted to encode a small polypeptide containing an N-terminal transmembrane region and a C-terminal TPR motif of almost the exact length needed to complement the ‘lost’ N-terminus of the TF0022 polypeptide. Indeed, both TF0022 and TF0023

were found to share homology with GppX, yielding similar blast scores (Fig. 1b). A single TPR motif typically consists of 34 amino acids (Das et al., 1998), and GppX from P. gingivalis harbors three tandem repeats of TPR (ranging from residues 155 to 254) at the center of the putative periplasmic domain (Fig. 1b). Interestingly, the TF0023 and TF0022 genes are in the same reading frame, and translation of the nucleotide sequences across the two ORFs uncovered an additional TPR motif when an 18-bp intergenic region was included (Fig. 1a). In P. gingivalis, one of the characteristic phenotypes of the disrupted gppX locus is enhanced autoaggregation (K. Nishikawa, unpublished data). In T. forsythia, ATCC 43037 wild-type cells gradually autoaggregate in broth cultures and eventually precipitate to the bottom of the test tubes. However, we noticed that the broth cultures of TF0022-ko mutant tended to precipitate faster than those of the wild-type strain.

Instead,

they still persisted with a dolichofacial pattern when compared with nasal breathers. “
“International Journal of Paediatric Dentistry 2010; 20: 458–465 Aim.  To compare subjective symptoms among three diagnostic subgroups of young patients with temporomandibular disorders (TMDs). Design.  We comprehensively examined 121 patients with TMDs (age ≤20 years; 90 female patients and 31 male patients) who completed self-reported forms for assessing subjective symptoms, which consisted of five items on pain intensity in the orofacial region and six items on the level of difficulty in activities of daily living (ADL) (rating scale, 0–10). They were divided into three diagnostic subgroups: temporomandibular see more joint (TMJ) problem (JT) group, masticatory muscle pain (MM)

group, and the group with a combination of TMJ problems and masticatory muscle pain (JM group). Their symptoms were compared using the Kruskal–Wallis and Mann–Whitney U-tests. Results.  The intensity of jaw or face tightness and difficulty in talking and yawning were not significantly different among the groups. However, the MM and JM selleck inhibitor groups had a significantly higher rating for jaw or face pain, headache, neck pain, tooth pain, and difficulty in eating soft foods (P < 0.01). Conclusions.  Young patients with MM or JM report more intense pain in the orofacial region and have more difficulties in ADL than those with JT problems alone. "
“Trauma to primary teeth may have consequences. Cobimetinib To study frequency of enamel defects

in permanent successors after luxation injuries, and to report carers’ experiences. Children 8–15 years (n = 170) suffering luxation injury to primary dentition in 2003 were reexamined in 2010. Permanent successors (n = 300) were clinically examined and photographed. Data from dental records, registration form and a questionnaire were analysed by cross-tabulation and tested by chi-square and t-test. Enamel defects were registered in 130 successor teeth, 22% due to trauma, 21% due to other aetiological factors (MIH, dental fluorosis, idiopathic). Successors with enamel defects were after concussion 8%, subluxation 18%, lateral luxation 41%, intrusion 38% and avulsion 47%. Enamel defects were associated with the child’s age and severity of the injury (P < 0.05). Six children had enamel defects in successors of non-injured primary teeth. Anxiety recorded by carers was associated with severity and number of injured teeth (P < 0.05). According to carers eight children developed dental fear, seven were younger than 3.5 years and had had their injured teeth removed. Minor luxation injuries and indirect trauma may cause enamel defects in permanent successors. Lower age at injury, severity and number of injured teeth affect carer and child negatively.

2 mg/kg (maximum dose 200 mg) twice

a day (bid) plus OBR. Sixty-seven per cent of patients had previously used efavirenz or nevirapine. At week 48, the most common treatment-related grade ≥ 2 adverse event (AE) was rash (13%); 12% experienced grade 3 AEs. Only two grade 4 AEs occurred (both thrombocytopaenia, not etravirine related). At week 48, 56% of patients (68% children; 48% adolescents) achieved Staurosporine a virological response (VL<50 copies/mL; intent-to-treat, noncompleter=failure). Factors predictive of response were adherence > 95%, male sex, low baseline etravirine weighted genotypic score and high etravirine trough concentration (C0h). Seventy-six patients (75%) completed the trial; most discontinuations occurred because of protocol noncompliance or AEs (8% each). Sixty-five per cent of patients were > 95% adherent by questionnaire and 39% by pill count. Forty-one patients experienced virological failure (VF; time-to-loss-of-virological-response

non-VF-censored algorithm) (29 nonresponders; 12 rebounders). Of 30 patients with VF with paired baseline/endpoint genotypes, 18 (60%) developed nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations, most commonly Y181C. Mean etravirine area under the plasma concentration–time curve over 12 h (AUC0–12h; 5216 ng h/mL) and C0h (346 ng/mL) were comparable to adult target values. Results with etravirine 5.2 mg/kg bid (with OBR) in this treatment-experienced paediatric population and etravirine Teicoplanin 200 mg bid in treatment-experienced adults were comparable. Etravirine is an NNRTI option for treatment-experienced paediatric patients. “
“Kaposi’s sarcoma (KS),

Selleckchem Dabrafenib invasive cervical carcinoma (ICC) and non-Hodgkin lymphoma (NHL) have been listed as AIDS-defining cancers (ADCs) by the Centers for Disease Control and Prevention since 1993. Despite this, HIV screening is not universally mentioned in ADC treatment guidelines. We examined screening practices at a tertiary centre serving a population where HIV seroprevalence is 0.4%. Patients with KS, ICC, NHL and Hodgkin lymphoma (HL), treated at Lausanne University Hospital between January 2002 and July 2012, were studied retrospectively. HIV testing was considered part of the oncology work-up if performed between 90 days before and 90 days after the cancer diagnosis date. A total of 880 patients were examined: 10 with KS, 58 with ICC, 672 with NHL and 140 with HL. HIV testing rates were 100, 11, 60 and 59%, and HIV seroprevalence was 60, 1.7, 3.4 and 5%, respectively. Thirty-seven patients (4.2%) were HIV-positive, of whom eight (22%) were diagnosed at oncology work-up. All newly diagnosed patients had CD4 counts < 200 cells/μL and six (75%) had presented to a physician 12–236 weeks previously with conditions warranting HIV testing. In our institution, only patients with KS were universally screened. Screening rates for other cancers ranged from 11 to 60%.

The statistical parameters were presented based on missing data of each variable. For categorical variables, the

differences in patient characteristics and risk factors were tested using chi-square or Fisher’s exact test. Comparison of means between groups was analyzed by independent t-test. Mann–Whitney test was used for nonparametric analysis. Some continuous variables were grouped together and analyzed as categorical variables. p Value of < 0.05 was considered to be statistically significant. Of 394 pilgrims who returned the questionnaires, 219 were males and 173 were females. Two persons did not state their gender and were excluded from the analysis. Five other forms were grossly incomplete and were also dropped from the analysis. The mean age was 50.4 Angiogenesis inhibitor ± 11.0 years. Seventy-three (19.7%) hajj pilgrims went for hajj using private travel package. In descending order the prevalence of symptoms among Malaysian hajj pilgrims were: cough 91.5%

(95% CI 88.7–94.3); runny nose 79.3% (95% CI 75.3–83.4); fever 59.2% (95% CI 54.3–64.1); and sore throat 57.1% (95% CI 52.2–62.1). The symptoms lasted less than 2 weeks in the majority of cases (Table 1). Only 3.6% (95% CI 1.8–5.5) of Malaysian pilgrims did not suffer from any of these symptoms throughout their stay in the RG7204 nmr holy land. About 87.1% (95% CI 83.7–90.4) of Malaysian hajj pilgrims had more than one respiratory symptom and 58.9% (95% CI 54.0–63.8) had fever with other symptoms. Besides cough that occurred significantly more common in older age, there was no other influence of age and gender to the respiratory symptoms among Malaysian pilgrims in 2007 Sulfite dehydrogenase (Table 2). As

protective measures, 72.8% of hajj pilgrims received influenza vaccination before departure and 72.9% wore facemasks. In terms of specific respiratory symptoms, influenza vaccination did not have a significant increase in any of the respiratory symptoms but it was significantly associated with longer duration of sore throat (Table 3). Wearing a mask was significantly associated with sore throat (OR 1.89; 95% CI 1.20–2.97) and longer duration of sore throat and fever (Table 4). The prevalence of hajj pilgrims with triad of cough, subjective fever, and sore throat were 40.1% (95% CI 35.2–45.0). ILI cases were not influenced by age, as the age of ILI cases was 49.8 ± 10.6-year-old and non-ILI cases was 50.7 ± 11.2-year-old (p = 0.422). It was also not influenced by gender as male gender was 54.8% in ILI versus 56.5% in non-ILI (p = 0.752). There was no significant association between ILI with influenza vaccination and those wearing a facemask (Table 5). Respiratory symptoms are one of the most common problems faced by pilgrims in Mecca.12 Besides low returned survey form, the major limitation of the study was the definition of acute respiratory infection.

Cross-linked peptidoglycan synthesis has been monitored in Escherichia coli (Eco) membranes by incubation with the two sugar precursors UDP-N-acetyl-muramylpentapeptide [UDP-MurNAc(pp)] and UDP-GlcNAc, one of which is radiolabelled (Chandrakala et al., 2001). In the membranes, the disaccharide unit of peptidoglycan is synthesized on a lipid carrier by the MraY and MurG enzymes and subsequently polymerized by the transglycosylase and cross-linked to pre-existing peptidoglycan by the transpeptidase (Fig. 1). The radiolabelled,

newly synthesized cross-linked peptidoglycan formed can be monitored by paper this website chromatography or a microplate scintillation proximity assay (SPA) using wheat germ agglutinin (WGA)-coated SPA beads (Chandrakala et al., 2001, 2004). To monitor MurG activity,

the pathway of reactions must be stopped at lipid II (Mengin-Lecreulx et al., 1991) (Fig. 1a) using an inhibitor of the transglycosylase (Ravishankar et al., 2005). Typically, in a first step, the MurG substrate is synthesized in situ; in a second step, transfer of radiolabelled GlcNAc by MurG occurs (Fig. 1b). The product lipid II can be separated from UDP-GlcNAc by paper chromatography (Mengin-Lecreulx et al., 1991) or by an SPA (Ravishankar et al., 2005) (Fig. 1b). We intended setting up an assay GW-572016 mw for Mycobacterium tuberculosis (Mtu) MurG by introducing it into an E. coli background, so that an established SPA (Ravishankar et al., 2005) could be used. Strain OV58 has an amber mutation in murG and a temperature-sensitive amber suppressor, so that practically no E. coli protein is made at 42 °C (Salmond et al., 1980; Mengin-Lecreulx et al., 1991). A key question was

whether the Mtu murG would functionally replace the E. coli homologue. Wheat germ agglutinin-coated (WGA) beads for the SPAs were from Amersham International plc. U.K. UDP-[3H]-N-acetyl glucosamine was from NEN Dupont, USA. Moenomycin was gifted by Hoechst India. Ni-NTA resin was from Qiagen, USA. Other chemicals were from Sigma-Aldrich. Thiamine-diphosphate kinase UDP-N-acetyl muramyl pentapeptide [UDP-MurNAc(pp)] was purified from Bacillus cereus 6A1 (Chandrakala et al., 2001) and radiolabelled by incubation with [3H]-NHS-propionate (Solapure et al., 2005). Escherichia coli murG(Ts) (Salmond et al., 1980) was a gift from W.D. Donachie. pRSETA and E. coli BL21(DE3) were from Novagen; pBAD/Myc-HisA and PMOSBlue were from Stratagene. L-broth (LB) was used for bacterial growth medium, and ampicillin was added at 50 or 100 μg mL−1 when required (LB-amp). The murG gene was PCR-amplified from Mtu genomic DNA with forward (5′- AAG GAC ACG GTC AGC CAG CC -3′) and reverse primers (5′- TCT AAA GCT TCG TCG TTG TCC TGG CAC CGG -3′) and cloned into pBAD/Myc-His A (Guzman et al., 1995) between the NcoI and HindIII sites. The resulting plasmid pAZI8952 has Mtu murG gene under the control of BAD promoter.

Cross-linked peptidoglycan synthesis has been monitored in Escherichia coli (Eco) membranes by incubation with the two sugar precursors UDP-N-acetyl-muramylpentapeptide [UDP-MurNAc(pp)] and UDP-GlcNAc, one of which is radiolabelled (Chandrakala et al., 2001). In the membranes, the disaccharide unit of peptidoglycan is synthesized on a lipid carrier by the MraY and MurG enzymes and subsequently polymerized by the transglycosylase and cross-linked to pre-existing peptidoglycan by the transpeptidase (Fig. 1). The radiolabelled,

newly synthesized cross-linked peptidoglycan formed can be monitored by paper this website chromatography or a microplate scintillation proximity assay (SPA) using wheat germ agglutinin (WGA)-coated SPA beads (Chandrakala et al., 2001, 2004). To monitor MurG activity,

the pathway of reactions must be stopped at lipid II (Mengin-Lecreulx et al., 1991) (Fig. 1a) using an inhibitor of the transglycosylase (Ravishankar et al., 2005). Typically, in a first step, the MurG substrate is synthesized in situ; in a second step, transfer of radiolabelled GlcNAc by MurG occurs (Fig. 1b). The product lipid II can be separated from UDP-GlcNAc by paper chromatography (Mengin-Lecreulx et al., 1991) or by an SPA (Ravishankar et al., 2005) (Fig. 1b). We intended setting up an assay Raf inhibitor for Mycobacterium tuberculosis (Mtu) MurG by introducing it into an E. coli background, so that an established SPA (Ravishankar et al., 2005) could be used. Strain OV58 has an amber mutation in murG and a temperature-sensitive amber suppressor, so that practically no E. coli protein is made at 42 °C (Salmond et al., 1980; Mengin-Lecreulx et al., 1991). A key question was

whether the Mtu murG would functionally replace the E. coli homologue. Wheat germ agglutinin-coated (WGA) beads for the SPAs were from Amersham International plc. U.K. UDP-[3H]-N-acetyl glucosamine was from NEN Dupont, USA. Moenomycin was gifted by Hoechst India. Ni-NTA resin was from Qiagen, USA. Other chemicals were from Sigma-Aldrich. Protein kinase N1 UDP-N-acetyl muramyl pentapeptide [UDP-MurNAc(pp)] was purified from Bacillus cereus 6A1 (Chandrakala et al., 2001) and radiolabelled by incubation with [3H]-NHS-propionate (Solapure et al., 2005). Escherichia coli murG(Ts) (Salmond et al., 1980) was a gift from W.D. Donachie. pRSETA and E. coli BL21(DE3) were from Novagen; pBAD/Myc-HisA and PMOSBlue were from Stratagene. L-broth (LB) was used for bacterial growth medium, and ampicillin was added at 50 or 100 μg mL−1 when required (LB-amp). The murG gene was PCR-amplified from Mtu genomic DNA with forward (5′- AAG GAC ACG GTC AGC CAG CC -3′) and reverse primers (5′- TCT AAA GCT TCG TCG TTG TCC TGG CAC CGG -3′) and cloned into pBAD/Myc-His A (Guzman et al., 1995) between the NcoI and HindIII sites. The resulting plasmid pAZI8952 has Mtu murG gene under the control of BAD promoter.