We found that the preferred spatial and temporal frequencies, spatial resolution and high temporal frequency cutoff of area MT neurons were reduced in aged monkeys, and were accompanied by the broadened tuning width of spatial frequency, elevated spontaneous activity, and decreased

signal-to-noise ratio. These results showed that, for neurons in area MT, aging significantly changed both the spatial and temporal frequency response tuning properties. Such evidence provides new insight into the changes occurring at the electrophysiological level that may be related to the aging-related visual deficits, especially in processing spatial and temporal information. Selleckchem Natural Product Library “
“During neuronal maturation, the neuron-specific K–Cl co-transporter KCC2 lowers the intracellular chloride and thereby renders GABAergic transmission hyperpolarizing. Independently of its role as a co-transporter, KCC2 plays a crucial role in the maturation of dendritic spines, most probably via an interaction with the cytoskeleton-associated protein 4.1N. In this study, we show that neural-specific overexpression of KCC2 impairs the development of the neural tube- and neural crest-related structures in mouse embryos. At early

stages (E9.5–11.5), the transgenic embryos had a thinner Y-27632 neural tube and abnormal body curvature. They displayed a reduced neuronal differentiation and altered neural crest cell pattern. At later stages (E11.5–15.5), the transgenic embryos had smaller brain structures and a distinctive cleft

palate. Similar results were obtained using overexpression of a transport-inactive N-terminal-deleted variant of KCC2, implying that the effects were not dependent on KCC2′s role as a K–Cl co-transporter. Interestingly, the neural tube of transgenic embryos had an aberrant cytoplasmic distribution of 4.1N and actin. This was corroborated in a neural stem cell line with ectopic expression of KCC2. Embryo phenotype and cell morphology were unaffected by a mutated variant of KCC2 which is unable to bind 4.1N. These results point to a role of KCC2 in neuronal differentiation Morin Hydrate and migration during early development mediated by its direct structural interactions with the neuronal cytoskeleton. KCC2 is a neuron-specific isoform of the K–Cl co-transporters. Its developmental upregulation is temporally associated with maturation of postsynaptic GABAergic inhibition in central neurons (Rivera et al., 1999; reviewed in Blaesse et al., 2009). Functional expression of KCC2 during neuronal development leads to a decrease in the intraneuronal Cl− concentration and, consequently, to a hyperpolarizing shift in the reversal potential of GABAA receptor-mediated currents (EGABA) from depolarizing values that are characteristic for immature neurons.

5, containing 150 mM NaCl and the recombinant proteins were then purified using a one-step affinity chromatography. The diluted crude extract (5 mL) was applied to a 5 mL Strep-Tactin Superflow cartridge (IBA GmbH, Göttingen, Germany). The purification was performed according to the manufacturer’s protocol. The MT I enzyme assay was performed in anaerobic quartz cuvettes with N2 as the gas phase. The total volume Pirfenidone was 100 μL. The activity was determined by the formation of methylcobalamin (ɛ528nm=7.9 mM−1 cm−1; Friedrich, 1975). The enzyme assay contained 50 mM Tris-HCl, pH 7.5,

2 mM ATP, 10 mM MgCl2, 5 mM substrate, 50 mM dithiothreitol, 0.5 mM titanium(III) citrate, 20 μM CP and crude extract with recombinant

AE; AE in the enzyme assay was estimated to be about 1 μg. MT Ivan activity was determined with vanillate (4-hydroxy-3-methoxybenzoic acid) and MT Iver activity with veratrol (1,2-dimethoxybenzene) as a substrate. The assay was started by adding MT I. All enzyme activities were the result of at least duplicate HTS assay determinations. The SDs were ≤10%. The protein determination was performed according to the method of Bradford (1976) with bovine serum albumin as a standard. The zinc content was determined photometrically using the method described by Zhou et al. (1999). To remove unspecifically bound zinc, the proteins were incubated with 2.5 mM EDTA in 25 mM Tris-HCl, pH 7.5, for 15 min at room temperature and were then applied onto a gel

filtration column Superdex 75 (16/60) equilibrated with 50 mM Tris-HCl pH 7.5. The same buffer was used as an eluent at a flow rate of 1 mL min−1 to separate the proteins from EDTA. Enzyme-containing fractions were pooled and subsequently concentrated using Vivaspin 50 centrifugation units (Vivascience, Hannover, Germany). The Niclosamide protein and the zinc contents of the mutated enzymes were determined as described above. Structure predictions of the methyltransferases were performed using the quickphyre program (Bennett-Lovsey et al., 2008). A crystal structure of MT I is not yet available. Attempts to crystallize the enzymes have resulted in nondiffracting crystals so far. In addition, the enzyme appeared to be rather unstable under the experimental conditions applied. Therefore, we attempted to identify the zinc-binding amino acids using site-directed mutagenesis. Potential zinc-binding partners are histidine, glutamate, aspartate and cysteine. Plenty of these amino acids are present in both MT I. The alignment of the amino acid sequences with other zinc-containing enzymes (Vallee and Auld, 1990b) did not provide a clue about the amino acids involved, indicating an unusual type of binding motif. Therefore, we exchanged several amino acids to alanine and tested the resulting recombinant enzymes for activity and zinc content.

This is a golden age for microbial ecology. We are generating datasets that could lay the foundation of the next phase in microbial ecosystem modeling. As greater spatial and temporal resolution is achieved, the finer details of community structure will be elucidated, enabling biological, chemical, and physical relationships to be described with mathematical formalisms. The next generation of microscale,

bottom-up models will focus on imposing more accurate metabolic models to define flux rates of enzymatic reactions for biological learn more units that interact in massively parallel computational arrays (e.g. http://systems.cs.uchicago.edu/projects/bhive.html). These systems, built of cellular and biochemical components, rely on a mechanistic understanding, which must be a focus for future microbial research. Without an improved knowledge of the biochemical nature of metabolism, metabolic interactions cannot be accurately described. A challenge for such systems will be to integrate physical and chemical disturbance into the model environment. As has been shown with macroscale models of the global ocean, the physical currents, once modeled, enable significantly improved accuracy of prediction for community structure and biomass of individual taxonomic units. It may be Target Selective Inhibitor Library manufacturer that microbial ecosystems, similar to life at macroscales, are fundamentally fractal in

nature (Gisiger, 2001; Brown et al., 2002), displaying statistical self-similarity across multiple scales. If everything were in fact everywhere, then Casein kinase 1 every sampled microbial population would contain a representation of the whole. Patterns of changing abundance in a milliliter of seawater might then mimic the patters observed in entire oceans. Fractal and multifractal systems have been applied to ecological patters in the past (Borda-de-Agua et al., 2002; Brown et al., 2002), and these tools may be valuable in modeling microbial systems as well. As understanding of microbial ecosystems continues

to grow, the connections between the micro and the macroscales will become more apparent. The ability to observe the taxonomic and functional diversity of microbial systems is still a very new technology, and microbial ecosystems are ancient. For a largely immortal organism that takes only 10 000 years to move across the globe and can be safely embedded in solid rock to await the geochemical conditions suitable to resume growth, a few years of observations might be insufficient to grasp the true dynamics of these ecosystems. Perhaps for some microbial taxa, the passing of the seasons are less important than the cycles of El Niño/La Niña, or even the coming and going of ice ages. Microbial ecosystem models are the only lens through which the full scope of microbial ecology can be observed, and provide opportunities for researchers to make predictions of microbial taxonomic and functional structure that extend far beyond the current range of possible observations. Funding for S.M.G.

, 1998; Cantarel et al., 2009). Genomic DNA from E. faecalis V583 and pBAD/HisB expression plasmid (Invitrogen, Karlsruhe, Germany) from Escherichia coli were isolated, using the E.Z.N.A.® Bacterial DNA Kit (Omega Bio-Tek Inc., Norcross, GA), and the E.Z.N.A.® Plasmid Miniprep kit I (Omega), respectively. The gene corresponding to EF2863 (without the part encoding a predicted N-terminal signal peptide) was amplified by PCR (forward primer, 5′-AGATCTGCATCAACTGTTACACC-3′; reverse primer, 5′-GAATTCTTAAGGTGTTGGAACAGTT-3′;

restriction sites are underlined). Amplified fragments were digested with BglII and EcoRI and cloned into a BglII/EcoRI-digested pBAD/HisB-vector (Invitrogen) using Quick Ligation Kit (New England Biolabs, selleck compound Ipswich, MA). Transformation of the ligation mix into E. coli TOP10 compentent cells followed by selective plating on brain heart infusion (BHI) plates containing 0.1 mg mL−1 ampicillin yielded transformants containing the pBad/HisB-EF plasmid for EfEndo18A expression. Bafilomycin A1 cost The gene sequence was verified by DNA sequencing using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Perkin Elmer/Applied Biosystems, Foster City,

CA). A 10-mL overnight culture of E. coli harbouring pBAD/HisB-EF was added to 500 mL fresh BHI broth (Oxoid Ltd., Hampshire, UK) containing 0.1 mg mL−1 ampicillin, and the culture was incubated at 37 °C with shaking. At an OD600 nm of 0.7, expression was induced by the addition of l-arabinose to a final concentration of 0.002% (w/v). The

culture was further incubated at 30 °C overnight, after which the cells were harvested by centrifugation (7700 g, 10 min, 4 °C) and resuspended in 20 mL Buffer A (100 mM TrisHCl Monoiodotyrosine pH 8, 20 mM imidazole). The cells were lysed by sonication, using a Vibra cell Ultrasonic Processor converter (Sonics, Newton, CT), at 20% amplitude with 5-s pulses (with a 5-s delay between pulses) for 15 min on ice. The sonicated cells were centrifuged (17 400 g 15 min, 4 °C), and the supernatant was applied to a Ni-NTA column equilibrated with Buffer A. EfEndo18A was eluted with Buffer B (100 mM TrisHCl pH 8, 100 mM imidazole) and concentrated using a centricon Plus-20 unit (Millipore, Billerica, MA). Protein purity was analyzed by SDS-PAGE, and the protein concentration was determined using the Bradford micro-assay (Bio-Rad Laboratories Inc., Hercules, CA) according to the suppliers’ procedure. Purified EfEndo18A was stored in 20 mM Tris-HCl pH 8 at 4 °C until use. The enterococcal chitinase EF0361, cloned and purified by nickel affinity chromatography in the same way as EfEndo18A (G. Vaaje-Kolstad, L.A. Bøhle, G. Mathiesen, V.G.H. Eijsink, unpublished results), was used as negative control. Glycosidase activity was measured by incubating 500 μg fetuin (Sigma, St.

Therefore, we investigated whether these strains possessed the 3-hydroxyl-3-methylglutaryl coenzyme A reductase (hmgr) gene, which indicates the presence of the mevalonate pathway. As a result, six strains belonging to the genera Streptomyces (SpC080624SC-11, SpA080624GE-02, and Sp080513GE-23), Nocardia (Sp080513SC-18), and Micromonospora (Se080624GE-07 and SpC080624GE-05) were found to possess the hmgr gene, and these genes were highly similar to hmgr genes in isoprenoid biosynthetic gene clusters. Among the six strains, RO4929097 mw the two strains

SpC080624SC-11 and SpA080624GE-02 produced the novel isoprenoids, JBIR-46, -47, and -48, which consisted of phenazine chromophores, and Sp080513GE-23 produced a known isoprenoid, fumaquinone. Furthermore, these compounds showed cytotoxic activity against human acute myelogenous leukemia HL-60 cells. Isoprenoids are the largest family of compounds found in nature. With over 30 000 known examples, isoprenoids include industrially useful compounds such as flavors, antibiotics, and plant hormones MLN0128 (Bohlmann & Keeling, 2008). Isoprenoids are composed of units of isopentenyl diphosphate (IPP), which can be synthesized by two independent pathways: the mevalonate pathway and/or the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (Kuzuyama & Seto, 2003). All Actinobacteria, including Streptomycetes,

use only the MEP pathway for the formation of IPP as a primary metabolite. On the other hand, some Actinobacteria strains possessing the MEP pathway have been reported to use the mevalonate Mannose-binding protein-associated serine protease pathway for the production of isoprenoids

as secondary metabolites. These strains include Kitasatospora griseola (terpentecin producer; Isshiki et al., 1986), Actinoplanes sp. A40644 (BE-40644 producer; Seto et al., 1998), Streptomyces sp. CL190 (naphterpin A producer; Shin-ya et al., 1990; Takahashi et al., 1999; Takagi et al., 2000), Streptomyces sp. KO-3988 (furaquinocin A producer; Funayama et al., 1990), Streptomyces griseolosporeus MF730-N6 (terpentecin, Dairi et al., 2000; Hamano et al., 2001), Chainia rubra (napyradiomycin A producer; Shiomi et al., 1986), and Streptomyces cinnamonensis (furanonaphthoquinone I and endophenazine A producer; Bringmann et al., 2007). Because these isoprenoids show interesting biological activities, including antitumor, antibacterial, and antioxidative properties, novel isoprenoids produced by Actinobacteria are expected to be promising candidates for drug discovery. In addition, it has been reported that Actinobacteria possessing a key enzyme gene, the 3-hydroxyl-3-methylglutaryl coenzyme A reductase (hmgr) gene, in the mevalonate pathway produce terpenoids (Kuzuyama et al., 2002). Therefore, we screened isoprenoids from Actinobacteria possessing the hmgr gene.

Outside HIV infection, studies show an independent association between higher total bilirubin and better endothelial function as well as a lower prevalence of coronary heart disease, possibly as a consequence of the anti-inflammatory and antioxidant effect of bilirubin. The aim of this study was to determine whether such an association exists in HIV-infected individuals. A cross-sectional study was performed in HIV-1-infected adults on stable antiretroviral therapy (ART) to determine if a relationship exists between total bilirubin and endothelial function [flow-mediated dilation (FMD) of the brachial artery], inflammation

[interleukin-6 (IL-6), soluble tumour necrosis factor receptors, C-reactive protein, and adhesion molecules], coagulation markers Ixazomib in vitro (fibrinogen and D-dimer) and oxidative stress (F 2-isoprostanes). Endpoints were compared based on total bilirubin levels and atazanavir status using distributionally appropriate, two-sample tests. Correlation coefficients were determined between Protease Inhibitor Library screening total bilirubin and endpoints. Linear regression was used to model the relationship between total bilirubin (and atazanavir status) and FMD. A total of 98 adults were included in the study. Total bilirubin was higher in the atazanavir group when compared to the non-atazanavir

group [median (interquartile range) 1.8 (1.1–2.6) vs. 0.6 (0.4–1.4) mg/dL; P < 0.01] as were insulin, the homeostasis model assessment of insulin resistance (HOMA-IR) and fibrinogen. Total bilirubin was positively correlated with fibrinogen and was not correlated with other outcomes. After adjustment, neither total bilirubin nor atazanavir status was associated with FMD. In virologically suppressed,

HIV-infected adults on stable ART, neither total bilirubin nor atazanavir use was associated Histone demethylase with improved endothelial function as measured using FMD, inflammation or oxidative stress as measured using biomarkers. The important role of inflammation in atherosclerosis and atherothrombosis is increasingly recognized [1], and in HIV-infected patients, it may be the principal driver of increased risk of subclinical atherosclerosis [2] and cardiovascular events [3]. This has spurred interest in the development of anti-inflammatory therapeutics to reduce cardiovascular risk. Bilirubin, an endogenous product of haemoglobin catabolism, has antioxidant and anti-inflammatory properties that attenuate endothelial activation and dysfunction in response to pro-inflammatory stress [4]. It has been shown to prevent oxidation of low-density lipoproteins and to inhibit vascular cell adhesion molecule-1 (sVCAM-1)-dependent migration of leucocytes into the endothelium [5]. Epidemiological studies in HIV-uninfected populations have associated elevated serum bilirubin levels with better endothelial function [6] and lower prevalences of coronary heart disease [7], stroke [8] and lower-extremity peripheral arterial disease [9].

The tissue was centrifuged again, HBSS was removed, and the tissue immediately frozen at

−80 °C and stored until used for Western blot analysis. Reelin-treated and control spinal cord tissue was dissected and lysed in ice-cold RIPA lysis buffer containing 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, pH 7.4, 1 mm EDTA, 1% NP-40, 0.5% Na-deoxycholat, 0.1% SDS, 0.004% NaN3 with protease inhibitor and phosphatase inhibitors. The lysates were centrifugated at 10 000 g twice for 20 min at 4 °C. The resulting crude supernatants were taken, and protein concentration was measured by using the DC Protein Assay (BioRad, Munich, Germany). Equal amounts BIBF 1120 of protein in sample buffer were loaded and separated by SDS polyacrylamide gel electrophoresis. Proteins were transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare, Munich, Germany). The membranes were blocked in Tris-buffered solution (TBS), pH 7.4, with 0.05% Tween20 (TBS-T) and 5% non-fat dry milk. Membranes were washed three times using TBS-T and incubated overnight at 4 °C with primary antibodies diluted in TBS-T containing 5% BSA. Membranes were washed three times for 5 min with TBS-T following incubation with the secondary antibody diluted in TBS-T containing 5% BSA for 1 h at room temperature.

Signals were detected by enhanced chemiluminiscence with SuperSignal West Pico Chemiluminiscent Substrate (Pierce Protein Research Products, Thermo Fisher Scientific, Rockford, IL, USA) on Fuji Super RX film. Photographs were either taken with an Olympus BX 61 or Zeiss LSM 510 NLO confocal microscope. Images were processed using Adobe Photoshop 5.5. As a first step in our study of a BIBW2992 mw potential role of Reelin-induced cofilin phosphorylation for normal arrest of SPNs in the IMLC, we retrogradely traced these cells by labelling them with DiI in embryonic tissue from wild-type animals,

reeler mutants and mutants lacking the Reelin receptor VLDLR. As shown next previously (Yip et al., 2003, 2007a,b, 2009), retrogradely labelled SPNs in wild-type animals were found in ventral and dorsolateral positions at E13.5, reflecting their migratory route from the neuroepithelium near the central canal to ventrolateral and then dorsolateral locations, eventually assembling in the IMLC (Fig. 1A). In reeler mice, DiI-labelled SPNs were similarly observed in ventrolateral positions; however, their assembly in the IMLC was incomplete, as reflected by the weak fluorescence staining of the IMLC (Fig. 1B). Instead, many SPNs could be traced to more medial positions (Fig. 1B, arrow), suggesting an ‘over-migration’ of SPNs towards the central canal. A much less pronounced phenotype was observed in vldlr mutants of this embryonic stage (Fig. 1C). In adult mice, the normal assembly of SPNs in the IMLC and the result of aberrant migration in reeler and Reelin receptor mutants were visualized by retrograde labelling with FG (Fig. 2A–D).

The paired t-test was used to identify differences between two study time-points, when ANOVA showed statistically significant results. All tests were two-tailed with an alpha level of 0.05. All statistical analyses were performed using GraphPad Prism Version 5.0a (GraphPad Software, La Jolla, CA). Baseline characteristics for the 56 patients enrolled in this study are summarized in Table 1.

At the time of enrolment, there learn more were no differences between the two groups regarding sex, age, route of infection, or immunological or virological determinants. Time since HIV viral load undetectable (months) [mean ± SD (range)] Of the 56 patients enrolled, five participants withdrew during the first 6 weeks of the study: three were on VPA therapy and withdrew because of adverse events and two subjects withdrew during the observation period, one for compliance reasons and the other because of HAART-related adverse events. Seven additional participants withdrew between 16 and 48 weeks, PR-171 cell line six while on VPA therapy and one during the observation period. Among patients receiving VPA, five participants withdrew because of adverse events (mood changes and/or gastrointestinal side effects and, in one patient, pulmonary emboli) and the other was a compliance dropout. A total of 24 patients in arm 1 and 20 patients

in arm 2 completed the study follow-up period (Fig. 1). All patients had undetectable viral load (<50 copies/mL) at the screening visit, but three subjects showed a blip at baseline prior Diflunisal to starting VPA therapy. One patient in arm 1 had a viral load of 55 copies/mL when starting the trial, while two patients in arm 2 had viral loads of 77 and 156 copies/mL, respectively, at baseline. However, these patients

showed no blips at follow-up visits. All participants were on stable HAART. Fifty-two per cent of subjects in arm 1 and 48% in arm 2 were taking nucleoside reverse transcriptase inhibitors (NRTIs) with protease inhibitors (PIs), while 37% in arm 1 and 38% in arm 2 were taking NRTIs with nonnucleoside reverse transcriptase inhibitors (NNRTIs). Only a few study participants were taking NNRTIs with PIs or the three drug classes. A total of two patients (7%) in arm 1 and six patients (20%) in arm 2 had to change their medication during the 48-week period for tolerance reasons. No significant differences in HAART regimens between the two groups were noted during the study period. Overall, VPA therapy was relatively safe and well tolerated with only minor side effects. Circulating VPA levels were adjusted and maintained at the therapeutic range throughout the study period for all participants. Over the study period, CD4 and CD8 cell counts did not change and no significant differences were observed between the two groups (P = 0.17). Similarly, no significant changes in viral loads were observed over time in both groups (data not shown).

Thus, it was postulated that inhibitors of HDACs could induce HIV-1 gene expression in latently infected cells, thereby leading to a reduction in the size of the latent HIV-1 reservoir if HAART is maintained [7]. Among several HDAC inhibitor drugs, valproic acid (VPA) was found to reactivate the transcription of HIV-1 genes in latently infected CD4 T cells isolated from successfully treated subjects, without inducing T-cell activation [8]. In an early small study testing the ability of VPA to reduce the HIV-1 reservoir, three of four HIV-1-infected

patients exhibited a substantial decline in the number of latently infected cells after 16–18 weeks of VPA therapy [9, 10]. Although these results were encouraging, recent findings indicate that VPA has no ancillary effect on latent HIV reservoirs [11-15]. However, all these studies Osimertinib in vivo examined a limited number of patients, ranging from nine to 11, and were retrospective and not randomized. In addition, the duration of VPA therapy varied among studies, making comparisons difficult. Furthermore, plasma VPA levels were not usually adjusted to therapeutic values. To overcome these limitations, a prospective cross-over, open-label, randomized clinical trial was designed to investigate the effectiveness of VPA in reducing the size

of the HIV reservoir in HIV-infected patients receiving HAART. We conducted a multicentre, randomized, open-label cross-over study, in which 56 chronically HIV-1-infected patients with undetectable viral load (<50 copies/mL) GSK-3 cancer under HAART for at least the previous 12 months were enrolled. This study design allows us to compare two different time periods of VPA exposure within the same study. Study participants were randomly assigned, in equal numbers, either to receive VPA plus HAART for 16 weeks followed by HAART alone for 32 weeks (arm 1) or to continue to receive HAART alone for 16 weeks and then VPA plus the HAART regimen for 32 weeks (arm 2). Randomization was

stratified by site using permuted blocks of size two and four. Computer-generated treatment allocation lists were prepared at the national data centre of the Canadian Institutes of Health Research-Institute (CIHR)/Canadian Methocarbamol HIV Trials Network (CTN) in Vancouver. When a patient was deemed eligible, the site coordinator accessed the randomization code through an interactive telephone line connected to the randomization computer. Study participants were followed every 4 weeks for a total of 48 weeks. Patients were enrolled from seven HIV-1 hospital or private medical centres throughout Canada between November 2006 and January 2009. All patients signed an ethics board-approved informed consent form. Adult male and female patients with confirmed HIV-1 infection were included in the study.

However, the physiological and pathological significance of the interaction remains to be elucidated. High-density lipoprotein (HDL), one of the major lipoproteins, enables lipids such as cholesterol and triglycerides to be reversely transported. A high level of HDL-associated cholesterol seems to protect against cardiovascular diseases (Kapur et al., 2008). It has also been demonstrated

that HDL may participate in innate immunity by protecting against some infections (Grunfeld & Feingold, 2008). When infection and inflammation induce the acute-phase response, the level of HDL in plasma is decreased (Khovidhunkit et al., 2004). HDL can bind the endotoxin (lipopolysaccharide) of Gram-negative bacteria and LTA Pirfenidone research buy of Gram-positive bacteria, and neutralize their toxic effects (Grunfeld et al., 1999; Khovidhunkit et al., 2004). In addition, HDL possesses a broad antiviral activity (Singh et al., 1999). Notably, trypanosoma lytic factors, one subset of HDL, protect humans from Trypanosoma brucei (Raper et al., 2001) and Leishmania infections (Samanovic et al., 2009). Interestingly, the serum GSK J4 clinical trial opacity factor (SOF) expressed by class II GAS strains interacts with ApoAI and ApoAII of HDL, subsequently causing the disrupture of HDL, which may attenuate the anti-inflammatory functions of HDL and contribute to the pathogenesis of GAS infection (Courtney

et al., 2006). The present study demonstrates that Scl1 from M41-type GAS ATCC12373 specifically binds HDL. The

interaction mechanism was also studied. Two strains of GAS, M6 (CMCC32175, obtained from the China Medical Culture Collection Center) and M41 (ATCC12373, obtained from the American Type Culture Collection), were used in this study. Cultivation of GAS was performed as reported previously (Han et al., 2006a, b). The GAS was grown in Todd–Hewitt broth (Becton, Dickinson and Company, MD) supplemented with 0.2% yeast extract (Oxoid, Hampshire, England) (THY medium). Nutrient broth agar containing 5% sheep blood was used as a solid medium. GAS was incubated in THY broth, 5% CO2, 37 °C to the Urocanase mid-logarithmic phase (OD600 nm∼0.5). GAS cells were collected by centrifugation at 6000 g for 10 min at 4 °C, and the cell pellet was washed twice with PBSA [phosphate-buffered saline (PBS) containing 0.02% NaN3], and the GAS cell suspensions in PBSA were used for the following experiments. Escherichia coli was grown at 37 °C in Luria–Bertani (LB) broth (tryptone 10 g L−1, yeast extract 5 g L−1 (Oxoid), and LB agar was used as a solid medium. Ampicillin (100 μg−1 mL) (Bio Basic Inc., ON, Canada) and 0.2 μg mL−1 anhydrotetracycline (IBA-GmbH, Göttingen, Germany) were used as selection markers. Recombinant Scls (rScl) were produced in E. coli using the Strep-tag II expression and purification system (IBA-GmbH), as described previously (Xu et al., 2002; Han et al., 2006b).