4.1 (WKM Business Software BV, Assen, The Netherlands), which is routinely used to register vaccination and chemoprophylaxis prescription at the pre-travel clinic. The second was Norma EMD/EPD (MI Consultancy, Katwijk, The Netherlands), which

is used as the electronic patient record for daily clinical care at the AMC and includes medical details of patients. Orion Globe 7.4.1 was used to collect information on travel and demographic details (age, gender, country of destination, travel period and duration, pre-travel vaccinations, and antibiotics prescribed). Norma EMD/EPD was used to collect information on clinical specifics such as patient history, medication, and relevant laboratory parameters: eg, CD4+ count in HIV positive patients. Through telephone questionnaires, we obtained details selleck chemicals llc on the Selleck MK0683 occurrence of health problems during or after travel: type of illness, timing, self-medication, contact

with local medical facilities (including hospital admission), and disease outcome. Additionally, we questioned participants about the nature of their travel (whether visiting friends or relatives, vacation, internship, or business). Travel destinations: We reported a maximum of three countries of destination. If patients visited more than three countries, we specified the region as described by Freedman and colleagues.10 If a patient had visited three continents or more, we defined the journey as a world trip. In our statistical analysis, we defined the region where exposure most likely happened, deduced from timing of TRD, as the travel destination. Medication: We documented both name and dosage of immune-suppressive agents used. Additionally, we documented use of other medication (only the drug, not the dosage). A minimum of 10 mg prednisolone per day or an equivalent was noted, based on the LCR statement that this is the minimum dose to exert a relevant

effect on the immune system.9 For chemotherapy among cancer patients, we only included patients who had their last course <3 months prior to inclusion, as no significant effect on the immune system is expected after this period.6,9 Reported health problems: Health problems were divided in syndrome categories as described by Freedman and colleagues.10 If available, we documented a diagnosis. Relevant TRD: We defined relevant TRD as self-reported fever Cyclic nucleotide phosphodiesterase (measured temperature above 38°C); self-reported diarrhea with or without blood (acute: frequent loose stools lasting >1 d; persistent to chronic: frequent loose stools lasting >14 d), infectious dermatological disorders, respiratory problems, and fatigue/overall malaise lasting over 7 days resulting in a physician’s consultation. We excluded health problems that did not potentially have an infectious cause from the definition of TRD (eg, traumatic injuries). If more than one health problem was reported in the same time period (<3 d between the onset of the two symptoms), we recorded the predominant symptom.

4,8 Clinically, both can be present in an insidious manner with chronic abdominal and systemic symptoms.10 However, a previous or family history of TB, history of chronic immunosuppression, and an origin from a country of high TB endemicity are all suggestive of TB rather than Crohn’s disease. Fistulizing disease is one of the hallmarks of Crohn’s disease but this is also well described in intestinal TB.10 Histologically, both Crohn’s disease and intestinal TB are characterized by granulomatous inflammation but multiple large confluent caseating granulomas which may be submucosal and associated with disproportionate submucosal inflammation, caseous necrosis, and ulcers lined with epithelioid histiocytes are

more commonly seen in intestinal TB.10,12–14

Once a definitive or presumptive diagnosis has been made of TB, treatment with standard regime antituberculous MDV3100 price drugs is highly effective.4 Our case illustrates the importance of considering intestinal TB as a significant differential to Crohn’s disease, especially in patients with high-risk demographics. The overlapping clinical features and lack of rapid and specific diagnostic tests highlight the diagnostic challenge posed by intestinal TB. The current TB incidence in Nepal is 163/100,000 which contrasts markedly to Australia’s 6.4/100,00015 highlighting the burden of disease that is transferable with the advent of rising migration from countries of high TB endemicity. It is therefore more ABT-199 clinical trial likely that local clinicians will face the diagnostic

dilemma of differentiating intestinal TB from PJ34 HCl Crohn’s disease. The importance of this is further emphasized by the significant differences in treatment of the two diseases and the potentially dire consequences that may ensue in misdiagnosing intestinal TB for Crohn’s disease. The authors state they have no conflicts of interest to declare. “
“Diagnostic confusion may occur between dengue and malaria when febrile patients with thrombocytopenia return from travel to previous malaria endemic areas. Laboratory tests should include blood smear examination for malaria parasites even though current malaria endemicity in Sri Lanka is low. Sri Lanka has been able to significantly reduce its malaria burden since the year 2000. The overall reduction in the reported positives is 99%.1 In contrast there has been an exponential increase in the incidence of dengue fever since 2004.2,3 In the wake of this epidemic, during the year 2010, the number of dengue infections reported in the country was 34,105 while the malaria incidence has remained low at 703 (of which 52 cases were imported malaria originating in other countries).4,5 In addition to the similar clinical expression of the two diseases there is also an overlap of the dengue and malaria endemic regions in the country with malaria–dengue coinfections being reported during the past 2 years.

[1] First, schistosomiasis is associated with eosinophilia in approximately 60% of cases; in fact, eosinophilia in a returning traveler from a Schistosoma-endemic region should be sufficient to suspect infection. Second, Dogon Country has a high prevalence of schistosomiasis, as a result, 44% of cases reported by TropNetEurop since 1999 (412 cases)[2] have come from Dogon Country in Mali

and Lake Tanganyika in Malawi. Third, the febrile episode experienced by the patient during the final part of the trip was likely an acute schistosomiasis. Artemisinin has been reported to be partially effective against Schistosoma and schistosomules.[3] PARP inhibitor Eradication has been achieved in 25% of chronic infections, together with >95% reduction in ova production.[4] Artemisinin is not active in adult schistosomes older

than 6 weeks (given 3 weeks after exposure in our case); however, it may have some activity against immature worms. Thus, artemisinin exposure may have reduced the adult worm burden in our patient resulting in late seroconversion and absence of parasites in the urine microscopies. Serology is more sensitive in returning travelers than urine or stool microscopy. LBH589 Indeed, series describe up to 88% of imported cases of schistosomiasis as being diagnosed with serology, of whom only 44% had parasites in stool or urine.[5] Seroconversion typically occurs from 6 weeks onwards,[6] although late seroconversions (6 months after exposure) have been reported.[7] In this case, the negative IHA serology 5.5 months after exposure together with persistently negative urine microscopy and denial of the epidemiological factor made us question a parasitic etiology, and led us to perform a diagnostic cystoscopy while waiting for the second serology result. Although not a first line diagnostic tool, invasive techniques such as cystoscopy or rectal snips can be helpful in diagnosis of difficult cases; these tests are highly sensitive and typically Amisulpride demonstrate ova invading the mucosa with the characteristic submucosal granulomatous reaction.[8] In this case, cystoscopy was decisive to reach the final diagnosis, as ova were only released into the urine

after the biopsy, resulting in a pathogen-directed treatment. Despite reasonable doubts about parasitic infection, we are aware that cystoscopy could have been avoided by waiting for the second serology or simply by administering empirical treatment, especially if eosinophilia after returning from an endemic region was assumed to be schistosomiasis, despite the patient’s denial of water exposure. Different techniques were used for the first and second serological determination (IHA and ELISA, respectively). The sensitivity of the techniques varies according to the type of antigen and the stage of the infection. IHA is generally more widely available and recommended as first line assessment, although it is less sensitive than ELISA.

This construct was amplified by PCR using the primers EapXhoRev2 (5′-GGGCTCGAGGCTAATGTTGTTGTAATCAATGAC-3′) and EAPXhoFwd2 (5′-GGGCTCGAGAGTATTTAAAGCAACTGACATTAAAAAG-3′) to delete eap Bleomycin ic50 before an erythromycin resistance cassette restricted with XhoI was ligated. For nptase, primers NPtaseDelFwd (5′-GGATCCCAGCAATACTTAATAGAGCGACC-3′) and NPtaseDelRev (5′-GGATCCGAATTTGACAGGTACTGCATCAGG-3′) were used to clone the surrounding sequence and primers NPtaseXhoFwd

(5′-GGGCTCGAGGTATGGTAGTTGGGAAGCTACG-3′) and NPtaseXhoRev (5′-GGGCTCGAGGCTGTAGAATTTGTCGTTTGTGG-3′) were used to delete nptase before an erythromycin resistance cassette restricted with XhoI was ligated. Once gene replacements in RN4220 were confirmed, the mutations were transduced to S. aureus strains SA113 and 10833 using phage 80, and transductants were selected for on TSA containing 10 mg erythromycin mL−1 (Kasatiya & Baldwin, 1967). The eap and nptase genes were cloned into the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible vector pCL15 (kindly provided by Dr Chia

Lee, University of Arkansas) (Luong & Lee, 2006). For complementation, initial cloning was performed in E. coli. The primer set KT488 (5′-GACATGGATCCGAGAAAGTCTGGCTATAATAAAG-3′) and KT489 (5′-GACAGTGAATTCCTACAAAATGTAAAAGGCACCCCAC-3′) was used to amplify eap and the primer set KT490 (5′-GACATCTGCAGAATCATGAGGTGATAAGATG-3′) and KT491 (5′-GACATGGATCCTTATTTAACTTCGCCTGTTTTAGGATCG-3′) was used to amplify nptase from genomic DNA from strain SA113. The KU-60019 manufacturer eap product was restricted with BamHI and EcoRI

and ligated to pCL15 to produce pCL15-eap. The nptase PCR product was restricted with BamHI and PstI and ligated to pCL15 to produce pCL15-npt. Plasmids purified from E. coli and confirmed by sequencing were transformed into RN4220 and transformants were selected on TSA containing chloramphenicol before the constructs were transduced to SA113 and 10833 by phage 80. The biofilm phenotype was restored at 1 mM IPTG, and so this concentration IMP dehydrogenase was used for complementation. To confirm complementation of Nptase, we performed phosphatase assays by extracting surface proteins from the different strains in 10 mM Tris (pH 7.0)+1 mM MgCl2+1 mM ZnCl (TMZ) by sonication. Reactions containing 1-mg protein and 0.2 mg para-nitrophenyl phosphate in TMZ were incubated at 37 °C for 2 h and the OD405 nm was determined. RNA was isolated from exponentially growing bacteria following induction with 1 mM IPTG for 2 h using the FastRNA Pro Blue Kit (MP Biomedicals, Solon, OH) according to the manufacturer’s instructions, and contaminating DNA was digested with Turbo DNAse (Ambion, Austin, TX). Expression levels of eap and nptase were measured by quantitative reverse transcriptase (RT)-PCR.

For example, ERP studies show that, while spatial attention typically leads to clearly lateralized amplitude enhancements of the P1 and N1 components

of the attended stimuli (e.g. Eimer & Schröger, BGB324 manufacturer 1998; Teder-Sälejärvi et al., 1999; Eimer & Driver, 2000; McDonald et al., 2005; Störmer et al., 2009), temporal attention reflects in a spatially widespread, less lateralized activation pattern on the scalp (Griffin et al., 2002). In terms of ERP components, both temporal and spatial attention have been shown to affect the P1 component (Correa et al., 2006) and the N1 component (Miniussi et al., 1999; Griffin et al., 2002; Lange & Röder, 2006; Sanders & Astheimer, 2008; Astheimer & Sanders, 2009; Lampar & Lange, 2011; Lange, 2012), but only temporal attention has been shown to modulate the N2 component (Griffin et al., 2002; Sanders & Astheimer, 2008) and the P3 component (Miniussi et al., 1999; Griffin et al., 2002; Lampar & Lange, 2011). At the level of oscillatory dynamics, it is well known that orienting spatial and temporal attention both lead to amplitude modulations of EEG activity at low frequency bands in the pre-stimulus period (Foxe & Snyder, 2011; Händel et al., 2011; Hanslmayr et al., 2011). For instance, the deployment of visual spatial attention leads to contralateral power decreases and ipsilateral

power increases in the alpha band over the occipital cortex (e.g. Worden et al., 2000; Sauseng et al., 2005; Thut et al., 2006;

Trenner et al., 2008; Gould et al., 2011; small molecule library screening Händel et al., 2011; Capilla et al., 2012). In cross-modal studies, the low-frequency spatial modulation, due to orienting attention to the expected location and away from STK38 the unexpected one, does also occur between the corresponding sensory cortices of the expected vs. unexpected modality (Bauer et al., 2012). However, it is less clear how this inter-hemispheric modulation could encompass a function of temporal selective attention. Instead, because brain oscillations represent events in time itself (Buzsáki, 2006), one could assume that temporal attention might modulate the readiness towards an attended modality by modulating the ongoing oscillatory patterns within sensory-specialised brain regions (van Ede et al., 2011). Further evidence for distinct underlying neural mechanisms comes from more spatially precise imaging models. Studies conducted with fMRI (Brunetti et al., 2008; Silk et al., 2010; Li et al., 2012; Yang & Mayer, 2014) and PET (Corbetta et al., 1993; Nobre et al., 1997) show that spatial and temporal attention orienting engages some common brain areas (such as the prefrontal cortex, the insular cortex, the dorsolateral premotor cortex or the inferior parietal lobe), though there are also some important differences.

Uptake of [14C]-Neu5Ac was not stimulated by Na+ for cells expressing NanT and in fact was inhibited slightly (Fig. 4a). In contrast, uptake in the absence of Na+ was minimal for cells DAPT ic50 expressing the STM1128 and SiaPQM transporters, but was stimulated by the addition of Na+ (Fig. 4b and c), demonstrating

Na+ dependence for these two transporters. For both the SSS and TRAP transporters, the specificity for Na+ was demonstrated by observing that neither Li+ nor K+ could restore Neu5Ac uptake (not shown). However, the presence of added Li+ or K+ had the same effect on NanT-mediated transport as that observed for Na+, suggesting that the increased ionic strength is the most probable cause of the apparent inhibitory effect of Na+. We were able to demonstrate the obligate Na+ requirement of the SSS and TRAP transporters by comparing cultures on solid minimal Fulvestrant supplier medium containing Neu5Ac and either sodium or potassium salts (Fig. 4d). Secondary carriers are driven by gradients and hence are, by definition, reversible. One frequently observed phenomenon of uptake via secondary carriers is that cells can be

forced to exchange a preinternalized substrate upon addition of excess extracellular substrate (Poolman & Konings, 1993). Examination of this phenomenon, the so-called ‘cold chase’ experiment, revealed Glutamate dehydrogenase that preinternalized [14C]-Neu5Ac was removed from

SEVY1 pES41 (STM1128+) cells by addition of 1 mM exogenous Neu5Ac, but not by a similar addition of water (Fig. 5). This is consistent with the behaviour of a secondary carrier such as NanT and differs from the SBP-dependent secondary carrier SiaPQM (Mulligan et al., 2009). Bacterial genome sequencing has revealed the presence of sialic acid utilization genes in a wide range of bacteria from human pathogens to marine bacteria. In this study, we have used a ΔnanT strain of E. coli to characterize two known and one putative sialic acid transporter genes from bacterial genomes, providing for the first time experimental evidence that a member of the SSS family of transporters, the STM1128 protein, can transport Neu5Ac. The STM1128 transporter appears to be a typical member of the SSS (TC 2.A.21) family of secondary carriers in that its activity is dependent on Na+ and it is a reversible transporter. Although we have not investigated the exact specificity of this particular SSS transporter in detail, the observations that homologous SSS transporters are predicted to be the only route for sialic acid acquisition in some bacteria (Fig.

Uptake of [14C]-Neu5Ac was not stimulated by Na+ for cells expressing NanT and in fact was inhibited slightly (Fig. 4a). In contrast, uptake in the absence of Na+ was minimal for cells Antiinfection Compound Library research buy expressing the STM1128 and SiaPQM transporters, but was stimulated by the addition of Na+ (Fig. 4b and c), demonstrating

Na+ dependence for these two transporters. For both the SSS and TRAP transporters, the specificity for Na+ was demonstrated by observing that neither Li+ nor K+ could restore Neu5Ac uptake (not shown). However, the presence of added Li+ or K+ had the same effect on NanT-mediated transport as that observed for Na+, suggesting that the increased ionic strength is the most probable cause of the apparent inhibitory effect of Na+. We were able to demonstrate the obligate Na+ requirement of the SSS and TRAP transporters by comparing cultures on solid minimal selleck compound medium containing Neu5Ac and either sodium or potassium salts (Fig. 4d). Secondary carriers are driven by gradients and hence are, by definition, reversible. One frequently observed phenomenon of uptake via secondary carriers is that cells can be

forced to exchange a preinternalized substrate upon addition of excess extracellular substrate (Poolman & Konings, 1993). Examination of this phenomenon, the so-called ‘cold chase’ experiment, revealed FER that preinternalized [14C]-Neu5Ac was removed from

SEVY1 pES41 (STM1128+) cells by addition of 1 mM exogenous Neu5Ac, but not by a similar addition of water (Fig. 5). This is consistent with the behaviour of a secondary carrier such as NanT and differs from the SBP-dependent secondary carrier SiaPQM (Mulligan et al., 2009). Bacterial genome sequencing has revealed the presence of sialic acid utilization genes in a wide range of bacteria from human pathogens to marine bacteria. In this study, we have used a ΔnanT strain of E. coli to characterize two known and one putative sialic acid transporter genes from bacterial genomes, providing for the first time experimental evidence that a member of the SSS family of transporters, the STM1128 protein, can transport Neu5Ac. The STM1128 transporter appears to be a typical member of the SSS (TC 2.A.21) family of secondary carriers in that its activity is dependent on Na+ and it is a reversible transporter. Although we have not investigated the exact specificity of this particular SSS transporter in detail, the observations that homologous SSS transporters are predicted to be the only route for sialic acid acquisition in some bacteria (Fig.

J Clin Oncol 2012; 30: 4297–4301. 52 Alfa-Wali M, Allen-Mersh T, Antoniou A et al. Chemoradiotherapy for anal cancer in HIV patients causes prolonged CD4 cell count suppression. Ann Oncol 2012; 23: 141–147. 53 Mistrangelo M, Conte ID, Cassoni P et al. Anal cancer: differences between HIV+ and HIV- patients. Colorectal Dis 2011; 13: 20. 54 Takahashi T, Braghiroli MI, Souza CE et al. Concurrent chemoradiation as definitive treatment in anal squamous cell carcinoma – Efficacy and safety Selleckchem Alectinib in HIV+

patients under HAART. Eur J Cancer 2011; 47: S448. 55 Salama JK, Mell LK, Schomas DA et al. Concurrent chemotherapy and intensity-modulated radiation therapy for anal canal cancer patients: a multicenter experience. [Erratum appears in J Clin Oncol 2008; 26: 694]. J Clin Oncol 2007; 25: 4581–4586. 56 DeFoe SG, Beriwal S, Jones H et al. Concurrent chemotherapy and intensity-modulated radiation therapy for anal carcinoma–clinical

CFTR modulator outcomes in a large National Cancer Institute-designated integrated cancer centre network. Clin Oncol (R Coll Radiol) 2012; 24: 424–431. 57 Azria D, Vieillot S, Lemanski C et al. Clinical outcome of patients treated with IMRT for locally advanced anal canal cancer. Int J Radiat Oncol Biol Phys 2011; 81: S377. 58 Kachnic LA, Tsai HK, Coen JJ et al. Dose-painted intensity-modulated radiation therapy for anal cancer: a multi-institutional report of acute toxicity and response to therapy. Int J Radiat Oncol Biol Phys 2012; 82: 153–158. 59 Hoffman R, Welton ML, Klencke B et al. The significance of pretreatment CD4 count on the outcome and treatment tolerance of HIV-positive patients with anal cancer. Int J Radiat Oncol Biol Phys 1999; 44: 127–131. 60 Peddada AV, Smith DE, Rao check details AR et al. Chemotherapy and low-dose radiotherapy in the treatment of HIV-infected patients with carcinoma of the anal canal. Int J Radiat Oncol Biol Phys 1997; 37: 1101–1105. 61 Place RJ, Gregorcyk SG, Huber PJ, Simmang CL. Outcome analysis of HIV-positive patients with anal squamous cell carcinoma. Dis Colon Rectum 2001; 44: 506–512. 62 Blazy A, Hennequin

C, Gornet JM et al. Anal carcinomas in HIV-positive patients: high-dose chemoradiotherapy is feasible in the era of highly active antiretroviral therapy. Dis Colon Rectum 2005; 48: 1176–1181. 63 Wexler A, Berson AM, Goldstone SE et al. Invasive anal squamous-cell carcinoma in the HIV-positive patient: outcome in the era of highly active antiretroviral therapy. Dis Colon Rectum 2008; 51: 73–81. 64 Fraunholz I, Weiss C, Eberlein K et al. Concurrent chemoradiotherapy with 5-fluorouracil and mitomycin C for invasive anal carcinoma in human immunodeficiency virus-positive patients receiving highly active antiretroviral therapy. Int J Radiat Oncol Biol Phys 2010; 76: 1425–1432. 65 Ajani JA, Winter KA, Gunderson LL et al. Fluorouracil, mitomycin, and radiotherapy vs fluorouracil, cisplatin, and radiotherapy for carcinoma of the anal canal: a randomized controlled trial.

We argue that this implicit measure was accessible to visuo-vestibular modulation of the sense of self, possibly mediated by shared neural processes in the insula involved in vestibular and interoceptive signalling, thermoregulation and multisensory integration. “
“The developing brain is not a small adult brain. Voltage- and transmitter-gated currents, like network-driven patterns, follow a developmental sequence. Studies initially performed in cortical structures and subsequently in subcortical structures have unravelled a developmental sequence of events in which intrinsic voltage-gated

calcium currents are followed by nonsynaptic calcium plateaux and synapse-driven giant depolarising potentials, orchestrated by TGF-beta inhibitor depolarizing actions of GABA and long-lasting NMDA receptor-mediated currents. The function of these early patterns is to enable heterogeneous neurons Roxadustat purchase to fire and wire together rather than to code specific modalities. However, at some stage, behaviourally relevant activities must replace these immature patterns, implying the presence of programmed stop signals. Here, we show that the developing striatum follows a developmental sequence in which immature patterns are silenced precisely when the pup starts locomotion. This is

mediated by a loss of the long-lasting NMDA-NR2C/D receptor-mediated current and the expression of a voltage-gated K+ current. Oxymatrine At the same time, the descending inputs to the spinal cord become fully functional, accompanying a GABA/glycine polarity shift and ending the expression of developmental patterns. Therefore, although the timetable of development differs in different brain structures,

the g sequence is quite similar, relying first on nonsynaptic events and then on synaptic oscillations that entrain large neuronal populations. In keeping with the ‘neuroarcheology’ theory, genetic mutations or environmental insults that perturb these developmental sequences constitute early signatures of developmental disorders. Birth dating developmental disorders thus provides important indicators of the event that triggers the pathological cascade leading ultimately to disease. “
“In the published manuscript of Garcia-Lazaro et al. (2007) there were some mistakes in Figure 6 and the text due to a programming mistake the data analysis routine which attributed data points (firing rates) to the wrong stimulus parameters. In the article, it was stated that neural response gain appeared to be increasing with increased stimulus variance, whereas in reality it decreased. Corrections have been marked in bold in the text below. Last paragraph of the introduction Response level functions tended to become systematically steeper if the mean of the stimulus distribution was held approximately constant but stimulus variance was decreased.

RSV strains Long and A2, human type II pulmonary epithelial cell line A549, S. pneumoniae strain R6, and H. influenzae strain Rd (KW20) were obtained from the American Type Culture

Collection (Manassas, VA). Clinical isolates of S. pneumoniae and H. influenzae were described previously (Yokota et al., 2004; Ohkoshi et al., 2008). RSV was grown in HEp-2 cells. The virus titer of RSV was determined by a plaque-forming assay using HEp-2 cells as an indicator (Okabayashi et al., 2009). Fosfomycin was obtained from Meiji Seika Kaisha (Tokyo, Japan). A PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N,-trimethyl)- hexanolamine, was purchased from Calbiochem-Merck KGaA (Darmstadt, Germany). An NF-κB inhibitor, PDTC, was purchased from Sigma-Aldrich (St. Louis, MO). A phosphocholine-deficient mutant was isolated by serial passage of S. pneumoniae strain R6 in a chemically defined LGK-974 molecular weight medium (CDM) containing decreasing concentrations of ethanolamine with each passage according to Yother et al. (1998). Briefly, approximately

106 cells were cultured in 2 mL of CDM containing 200 μg mL−1 ethanolamine for 12 h at 37 °C and then diluted 100-fold into the same medium. Following five 12-h passages in CDM containing 200 μg mL−1 ethanolamine, similar passages were performed in successively lower concentrations of ethanolamine (20, 2, 0.2, and then 0 μg mL−1). The resulting mutant was capable of growth in CDM without choline

or ethanolamine. The cell wall fraction was prepared as follows: cells grown to Caspase cleavage the mid-log phase were harvested and immediately boiled with saline containing 4% SDS for 20 min. The boiled cells were disrupted by Glutathione peroxidase sonication and then centrifuged at 20 000 g for 15 min. The pellet was washed extensively with saline, and then used as a cell wall fraction. The content of choline in the cell wall preparation was determined using an enzymatic method (Assmann & Schriewer, 1985). The choline contents of cell wall fractions from R6 and the mutant were 435 nmol mg−1 and undetectable, respectively. Cell surface expression of the PAF receptor was examined by flow cytometry. A549 cells were harvested from culture flasks using a cell scraper, and then incubated with 2.5 μg mL−1 of mouse anti-PAF receptor monoclonal antibody [11A4 (clone 21); Cayman Chemical, Ann Arbor, MI] or mouse IgG2a,κ isotype control antibody (eBioscience, San Diego, CA). After incubation at 4 °C for 30 min, cells were collected by centrifugation and washed once with Dulbecco’s phosphate-buffered saline [PBS(−)]. Cell suspensions were incubated with a phycoerythrin-conjugated goat anti-mouse IgG F(ab)2 fragment antibody (1 : 100 dilution) (Abcam, Cambridge, UK) at 4 °C for 30 min, and the stained cells were assessed with a FACSCalibur (BD Bioscience, San Jose, CA). A bacterial suspension in 0.1 M NaCl–50 mM sodium carbonate buffer (pH 9.5) at 1 × 108 CFU mL−1 was prepared.