umfree culture medium for 15 min at 37uC. The pinhole was set to give an optical slice of,1 mm. CoroNa Red was excited at 543 nm and fluorescence emission was measured from 560 nm to 615 nm. ROIs excluding nuclei with a high density of mitochondria were selected in individual cells and the average fluorescence signal within these regions was analyzed over time. For each data point we obtained SEM values that were smaller than 7% of the corresponding fluorescence mean values. Analysis of mitochondrial calcium. Ca2mit changes were monitored in cells permeabilized as described above. Briefly, get AZ-6102 SH-SY5Y and C6 cells were loaded with 5 mM Rhod-2 AM in culture medium for 60 min at 37uC. To remove the fluorescence contribution of the cytosolic component, cells were permeabilized with digitonin 5 mM in intracellular buffer containing 300 nM or 400 nM CaCl2. Rhod-2 was excited at 543 nm and fluorescence emission was measured from 560 nm to 600 nm. Real-time confocal imaging Analysis of mitochondrial inner membrane potential. SH-SY5Y and C6 cells grown for 18 h on poly-L-lysine-coated glass coverslips were loaded at 37uC with 150 nM tetramethylrhodamine ethyl ester . In these conditions, after mitochondrial depolarization, TMRE is released from the quenched matrix to the cytoplasm, resulting in an increase in cytoplasmic fluorescence. After 20 min cells were washed and transferred to a ” microscopy chamber in standard buffer solution in the presence of 150 nM TMRE. Confocal images were obtained using the 510 LSM microscope equipped with a META detection system and a 406oil immersion objective. Illumination intensity was kept to a minimum to avoid phototoxicity; the pinhole was set to give an optical slice of,1 mm. TMRE was excited at 543 nm and fluorescence was measured from 580 nm to 700 nm in cytoplasmic regions of interest. For data analysis fluorescence was expressed as ratios of fluorescence counts relative to Analysis of ATP production ATP production was evaluated using 18264101
a commercially available luciferase-luciferin system. Isolated mitochondria. Mitochondria were incubated in a solution containing: KCl, 100; NaCl, 5; CaCl2, 0.0001; mannitol, 75; sucrose, 25; KH2PO4, 10; Tris-HCl, 10; and ADP, 0.1, with or without 0.51 mM glutamate. In preliminary experiments these glutamate concentrations gave the maximal response in terms of stimulation of ATP synthesis without toxic effects in our systems. The tested drugs or the respective vehicles were added to mitochondrial suspensions 15 min before glutamate stimulation and throughout the experiments. ” Luminescence was measured with a luminescence counter. All experiments were performed using,60 mg of mitochondria, an amount that in preliminary tests ensured strong and reproducible ATP signal, and 1 h incubation, which in the same preliminary tests provided the best Mitochondrial NCX1/EAAC1 Sustain Brain Metabolism compromise between mitochondrial viability and ATP accumulation. The magnitude of ATP response to glutamate alone was found to vary between mitochondrial preparations. Therefore, for each set of experiments, for each experimental session, every batch of mitochondrial preparations was used and divided in the following groups: unstimulated, stimulated with glutamate, treated with tested drugs 6 glutamate, as indicated. Cultured cells. Cells, 24 h after been plated in 96 multiwell plates, were transfected with ODNs for additional 24 h for NCX1 or EAAC1 and 48 h for Citrin/AGC2 knock-down experiment

xist. E-mail: Yuanan Lu [email protected] Introduction Sewage-contaminated recreational water can pose numerous health risks to the public; effective water quality monitoring is therefore absolutely essential. Currently, microbiological water quality is primarily assessed via bacterial indicators such as enterococci, fecal coliform, and total coliform bacteria. However, these indicators often fail to reflect the presence of important hazardous viruses. This is of important concern, as viral pathogens shed in human feces may compromise public safety by polluting recreational waters that meet bacterial indicator standards. Additionally, these bacterial indicators may grow naturally in tropical environments, resulting in inaccurate assessment of water pollution levels. Therefore, alternative monitoring systems are needed to improve the surveillance of recreational waters and secure public protection from waterborne disease. Human enteric viruses, represented by the astroviruses, rotaviruses, noroviruses, adenoviruses, and picornaviruses, have been associated with many waterborne outbreaks and are suggested as alternative indicators of microbial water quality. Enteric viruses are primarily transmitted via the fecal-oral route, and viral particles are shed in extremely high numbers from ONX-0914 web infected individuals. Although most enteric virus infections are primarily associated with diarrhea and self-limiting gastroenteritis, they may also cause hepatitis, conjunctivitis, and respiratory infections. Additionally, in immunocompromised persons, enteric Detection of Enterovirus from Environmental Water negative results, presents an additional barrier. Detection challenges may be overcome by improved methods for viral concentration from water samples and by efficient inhibitor removal during nucleic acid extraction. Here, we have developed a highly optimized molecular protocol for the effective detection of enteroviruses from Hawaiian environmental waters. Enteroviruses, RNA viruses belonging to the Picornavirus family and consisting of coxsackievirus, poliovirus, echovirus, and the numbered enteroviruses, are the most commonly detected enteric viruses in polluted waters and are estimated to cause 30 50 million infections in the US annually. The EnV disease spectrum is wide, including gastroenteritis, respiratory infection, diabetes, heart disease, bronchiolitis, conjunctivitis, meningitis, paralysis, and the common cold. Because these viruses are common, fecally shed in extremely high numbers from infected individuals, highly tolerant to salinity and temperature fluctuations, and stable in the environment for extended time periods, they have been suggested as a parameter for evaluating viral pollution of environmental waters. The availability of permissive cell lines for determining EnV infectivity greatly enhances the attractiveness of using this important enteric virus subset as an ” alternative indicator of water quality. Additionally, in order to enhance viral concentration from environmental water samples, we briefly report the potential utilization of marine bivalves as bioindicators of water quality. ” Because these animals are filter feeders, they process large volumes of water daily, which causes viruses to accumulate within their tissues at a concentration higher than that in the surrounding water. Combining this natural bioconcentration phenomenon with our highly optimized RT-PCR protocol for EnV detection shows promising potential to aid in ef

that activation of GCGR by glucagon also induced the b-catenin signaling pathway. Importantly, we found that Lrp5/6 is required for glucagon-induced b-catenin signaling. These results may help to explain the pleiotropic phenotypes of Lrp5 and 6 mutations and have important implications in understanding the role of Lrp5/6 in metabolic syndrome. Results Glucagon agonist induced the cAMP/PKA pathway in GCGR-expressing cells As a classical GPCR, activation of the glucagon receptor causes an increase of intracellular cAMP level, which in turn activates the PKA signaling pathway to activate cAMP-response element -mediated gene expression. Using the CRE-Luc reporter construct, we found that HEK293 without GCGR transfection did not respond to GCG1-29 stimulation. After transfecting with GCGR, HEK293 cells became responsive to GCG1-29, but not to Oleandrin GCG9-29 . As a control, forskolin, a direct PKA activator, activated CRE luciferase activity independent of GCGR expression. Using western blot, we confirmed that HEK293 cells have no detectable expression of GCGR until after transfection with a GCGR expression plasmid. These experiments suggest that HEK293 cells can be used to model GCGR signaling after ectopic expression of the receptor. We 22314911 also asked if we could detect CRE luciferase activity in cells with endogenous GCGR expression. Primary liver hepatocytes are known to have endogenous GCGR expression. We found that the GCG1-29 could directly activate CRE luciferase activity in primary liver cells without the need to transfect with 9336340 a GCGR plasmid. catenin protein levels relative to a control, non-treated sample or that treated with the antagonist GCG9-29. As a positive control, treatment with lithium chloride also caused an increase in b-catenin levels, an indication of activation of the Wnt/b-catenin signaling pathway. To confirm this result, we also examined cells with endogenous GCGR expression, including the hepatocarcinoma cell line Hep3B and primary liver cells. Treatment of Hep3B cells with GCG1-29 caused a rapid increase of b-catenin protein levels within 15 minutes. Treatment of primary hepatocytes also caused an increase in b-catenin protein. These experiments demonstrate that activation of the GCGR receptor in cell lines and primary cells leads to bcatenin stabilization. Activation of the b-catenin pathway leads to stabilization of bcatenin in the cytosol, which can translocate into the nucleus and associate with TCF transcription factors to activate TCF promotermediated gene expression. Because we observed the stabilization of b-catenin protein upon activation of GCGR receptor, we next examined whether activation of GCGR stimulated TCF promotermediated luciferase activity, an indicator for an active b-catenin signaling pathway. 293STF cells were transfected with the GCGR receptor and then treated with GCG1-29 or GCG9-29 peptides. We observed a small but statistically significant increase in TCF-mediated luciferase activity upon treatment with GCG1-29, but not with GCG9-29. Treatment with LiCl also caused an increase in TCF luciferase activity. Similarly, we observed a dose-dependent increase in TCF luciferase activity in primary hepatocytes treated with GCG1-29, but not with GCG9-29 or PTH1-34 peptides. These experiments demonstrate that activation of the GCGR receptor increases TCF promoter activity. Together with the western results, they demonstrated that activation of GCGR receptor leads to active b-catenin signaling. Coexpression o

m of ECAM could correspond to a change in the position of the TED domain, which, in C3b, is located between 75 and 100 A away from its position in C3. In order “7901789 to gain further insight into this possibility, we manually fitted the structure of C3b onto the electron microscopy 3D model of methylamineactivated ECAM. This analysis reveals two important points. First, it corroborates the location the TED domain in the activated form of the bacterial protein. In addition, this analysis suggests that the C-terminal region of C3b could be fitted into two different regions of density; only one was modeled, but the other potential conformation of the C-terminus of ECAM is indicated with red arrows. Thus, both SAXS and EM techniques point to the fact that the C-terminus of ECAM is potentially solvent-exposed and flexible. In eukaryotic a2Ms, the C-terminal, receptor-binding domain is exposed when the molecules are reacted with either methylamine or proteases, thus requiring a conformational modification for solvent accessibility . This is also confirmed by the elegant docking of the structure of C3 and C3b onto electron microscopy maps of eukaryotic a2M, performed by Janssen and coworkers, as well as the recent 4.3 A crystal structure of methylamine-activated human a2M. This suggests that proteins of the a2M family share a number of overall structural similarities that include overall conformational modifications upon activation. It is of interest that inhibition of C3b by a Staphylococcal inhibitor protein occurs through the generation of an `open’ conformer of the former, which subsequently blocks formation of the C3 convertase, underlining the importance of complex conformational changes not only for C3 518303-20-3 manufacturer function but also for its targeting by pathogens. The level of circulating a2M-protease complexes in humans is low, as a consequence of the recognition of the C-terminus of a2M by lipoprotein receptors and their subsequent internalization and degradation. Thus, the C-terminal region of eukaryotic a2M plays a key role in its recognition of partner macromolecules, leading to its eventual clearance. The flexible C-terminal end of ECAM, described here, could also potentially serve as a binding region for partners. This could include PBP1c, whose gene cooccurs with that of a-macroglobulin in a number of bacterial species. PBP1c is a periplasmic molecule that is anchored to the inner membrane through a single transmembrane region. The concerted action of PBP1c and ECAM could favor protection of cell integrity in the presence of foreign proteases, potentially through the involvement of a direct interaction between the PBP and the C-terminal region of the a-macroglobulin. This could reflect a novel bacterial defense mechanism that implicates the action of both protease inhibition and cell wall biosynthesis processes. On the other hand, pathogens have also been shown to encode proteins that mimic components of the complement system in order to manipulate the host inflammatory response; thus, due to their similarity to “9357531 C3/C3b, it is conceivable that bacterial a-macroglobulins could also play yet undefined roles in the disruption of the complement amplification pathway in situations where the outer cell wall is weakened. Either one of these potential mechanisms could represent unexplored targets for the development of novel antibacterials. Materials and Methods Materials Porcine pancreatic elastase was dissolved in 0.2 M Tris-HCl pH 8.0. HisTrap HP, Superd

the animals returned to the rearing room to continue development. Early results suggested that higher single doses sometimes produced less effect than lower single doses, possibly indicating that at high doses the drug, which is dissolved in ” DMSO, precipitated out as it was injected into the aqueous hemolymph. For this reason, and because we were concerned that newly expressed FGFRs might overwhelm single drug injections, two or three 0.5 mg injections spaced 24 hr apart were used rather than a single, larger injection. We found no difference in phenotype between animals receiving 2 vs 3 injections. Immunocytochemistry Animals at various stages of metamorphic adult development were anesthetized by cooling on ice. Brains were dissected under insect saline solution at 100 mm. The final step in all protocols, also unless noted, was clearing the brains or sections for 15 min each first in 50% glycerol in water, then in 80% glycerol in water, and finally mounting on slides in 80% glycerol. For some preparations, glial cell nuclei also were labeled with the nucleic acid stains Syto 13 or Syto 59. Sections were washed in 10 mM Tris, then incubated in the Syto dye 1:10,000 in Tris for 60 min, washed in Tris, and mounted in H2O/glycerol.Fixed brains were washed in PBS, cryoprotected in 10, 20, and 30% sucrose in 0.1 M phosphate buffer, pH 7.4, at 4uC, flashfrozen in liquid propane, and cryosectioned at 20 mm. Sections were then processed according to the apoptosis detection kit instructions, using propidium iodide to counterstain nuclei. Western blot Antennal lobes of three female animals at stage 7 of adult development were removed and solubilized in Novex lithium dodecyl sulfate sample buffer containing protease-inhibitor and phosphatase-inhibitor cocktails. Solubilized samples were run on a Novex NuPAGE 4 12% Bis-Tris gel and transferred to a PVDF membrane as described previously. Blots were incubated for 1 hr at RT in blocking solution, then ON at 4uC in blocking solution with anti-pFGFR antibody. Blots were then washed in TBS-Tween and incubated 4 hr at RT in blocking solution plus horseradish peroxidase-conjugated goat antirabbit antibody. Blots were washed again and developed using the Opti-4CN kit. An additional blot to compare pFGFR labeling of antennal lobes treated with DMSO or buy PBTZ 169 PD173074 was done as described above using lobes and attached nerves from two animals for each treatment. Because immunocytochemistry suggested residual labeling in AL neuron cell bodies following PD173074 treatment, these cell body clusters were removed from the tissue prior to processing in order to assess solely the effect on glia. Glial FGFRs in Glia-Neuron Signaling Confocal microscopy and image processing Sections were viewed on a Nikon PCM 2000 or a Zeiss 510 Meta laser scanning confocal system using Simple 32 software or LSM software, respectively. Optical sections were acquired at 1- to 5-mm intervals through the depth of the antennal lobe and saved as three-dimensional stacks. To examine the ” localization of FGFRs, HSPGs, and Syto dyes to cellular subcompartments, we used a 406, oil immersion EC PLANNEOFLUAR, N.A. 1.3 lens. Vehicle controls were always imaged along with experimental brains and imaging parameters were always held constant when comparing between controls and experimental brains or across developmental stages. Confocal image stacks were projected and merged in false color using Confocal Assistant or the Zeiss LSM image browser, and then i

hanks ” to Adriaan van Aelst and 8733580 Tiny Franssen-Verheijen for assistance with electron microscopy and Jacques Meis for XAV-939 strains. Anidulafungin was contributed by Pfizer, NL. Author Contributions Conceived and designed the experiments: CJI PMS. Performed the experiments: CJI. Analyzed the data: CJI PMS. Contributed reagents/ materials/analysis tools: CJI. Wrote the paper: CJI PMS. As one of the major cell types comprising alveolar epithelial tissue, the alveolar type II epithelial cells play an important role in maintaining alveolar integrity by forming the key alveolar barrier, repairing damaged type I cells, and being the source of alveolar surfactant. Increasing studies also suggest a critical role for alveolar type II epithelial cells in regulating local lung inflammatory response. For example, our previous study and others have suggested that alveolar type II epithelial cells may play special roles in counteracting microbes by releasing cytokines and chemokines that recruit both dendritic cells and alveolar macrophages to the site of infection. However, the potential role of alveolar type II epithelial cells in lung innate immunity and the molecular mechanisms whereby the expressions of inflammatory mediators are regulated in alveolar type II epithelial cells remain largely unknown. IL-1b is one of the most biologically active cytokines in edema fluid and bronchoalveolar lavage fluid from patients at an early stage of acute respiratory distress syndrome. Moreover, IL-1b has been shown to affect the function of the lung epithelial barrier. IL-1b is known to modulate the activity of many transcription factors including NF-kB and C/EBPs. However, the role of C/EBPs in IL-1b-mediated inflammatory responses in alveolar type II epithelial cells remains unknown. The goal of the current study was to investigate the role of C/EBPc in IL-1bstimulated IL-6 production from alveolar type II epithelial cells. C/EBPa, -b, -d, -e, -c, and -f comprise a family of basic regionleucine zipper transcription factors that dimerize through a leucine zipper and bind to DNA through an adjacent basic region. All C/EBP members can form homo- and hetero-dimers with other family members. C/EBPs can activate transcription from promoters that contain a consensus binding site: 59-TNNGNAA-39. Among them, C/EBPb and -d appear to be effectors in the induction of genes responsive to LPS, IL-1b or IL-6 stimulation, and have been implicated in the regulation of inflammatory mediators as well as other gene products associated with the activation of macrophages and the acute phase inflammatory response . C/EBPc is a ubiquitously expressed member of the C/EBP family of transcription factors that has been shown to be an inhibitor of C/EBP transcriptional activators. Different from C/EBPb and -d, C/EBPc was proposed to act as a buffer against C/EBP-mediated activation because of the fact that C/EBPc lacks known activation domains. C/ EBPc-deficient mice showed a high mortality rate within 48 h after birth. Although C/EBPc chimeras showed normal T and B cell development, the cytolytic functions of their splenic natural killer cells after stimulation with cytokines such as IL-12, IL-18 and IL-2 were significantly reduced. However, the role C/EBPc Suppresses IL-6 Production of C/EBPc in inflammation remains largely unknown. In the current study, we demonstrate that C/EBPc expression is induced by IL-1b in lung epithelial cells, and apparently contributes to the inhibition of IL-1b-

and higher risk of death. Several strategies, including using iso-osmolar contrast, limiting the amount of administered contrast media and volume expansion have become well established methods for the prevention of CIN. The pathophysiological mechanisms of CIN is not well known. However, multiple studies have suggested that renal vasoconstriction, oxidative stress, inflammation and direct tubular cell damage by contrast media may play crucial important roles in the renal injury process. Statins, drugs primarily associated with lowdensity lipoprotein Butein chemical information cholesterol-lowering effects, have been shown to possess pleiotropic effects that include enhancement of endothelial nitric oxide production, anti-inflammatory and antioxidative actions. Therefore, statins are considered as promising candidate agents for the prevention of CIN. A few studies focused on statin therapy as specific prophylactic measures of CIN have been published with conflicting results. In this meta-analysis of randomized controlled trials, we aimed to assess the effectiveness of short-term high-dose statin treatment for the prevention of CIN and clinical outcomes and reevaluate of the potential benefits of statin therapy. The literature search was performed on PubMed, OVID, EMBASE, Web of science and the Cochrane Central Register of Controlled Trials. We derived three comprehensive search themes that were then combined using the Boolean operator “AND”. For the theme “contrast media”, we used combinations of MeSH, entry terms and text words: contrast, radiocontrast, contrast 17125260” medium, contrast media, contrast dye, radiographic contrast, radiocontrast media, radiocontrast medium and contrast agent. For the theme “renal insuficiency”, we used: renal insufficiency, renal failure, diabetic nephropathies, nephritis, nephropathy, nephrotoxic, and, contrastinduced nephropathy and contrast-associated nephropathy. For the theme “statin”, statin, atorvastatin, rosuvastatin, cerivastatin, simvastatin, pravastatin, lovastatin, Hydroxymethylglutaryl-CoA reductase inhibitors and HMG-CoA reductase inhibitors were used. Appendix S1 shows the detailed search method. We did not restrict by language or type of article. To identify other relevant studies, we manually scanned reference lists from identified trials and review articles, and we also searched conference proceedings. We requested original data by directly contacting authors. dose of 80 mg or ” 40 mg) versus low-dose statin treatment or placebo. Studies that incorporated NAC were included only if both arms were administered NAC; studies reported the incidence of contrast-induced nephropathy in both arms. We did not restrict eligibility according to kidney function. The primary outcome measure was the development of contrast-induced nephropathy, defined as an increase in baseline serum creatinine level of 25% or an absolute increase of 44 mmol/L within 2 to 5 days after the exposure to contrast medium. Secondary outcome measures were need for dislysis, in-hospital mortality and length of hospital stay. Data extraction and quality assessment Data were collected independently by 2 reviewers. Extracted data included patient characteristics; inclusion criteria; type and dose of contrast media; protocol for the treatment of statins; periprocedural hydration protocol and specific definition of CIN. Quality assessment was judged on concealment of treatment allocation; similarity of both groups at baseline regarding prognostic factors; eligibil

buffer. The enzyme activities were then calculated by measuring the optical density of precipitation, colorimetric enzyme reaction products, using the NIH ImageJ software. Standard curves were created from multiple determinations of complex activities in cultured 3T3-L1 cell extracts. Transmission electron microscopy Three days post transfection of siRNA, 3T3-L1 cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate LY341495 site buffer for two hours at 4uC, and post-fixed with 1% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer for one hour at 4uC. The specimens were then incubated in 0.5% aqueous uranyl acetate for 2 hours at room temperature for en bloc staining, and in a graded series of ethanol for dehydration. Thereafter, the specimens were embedded in Embed 812 resin. Ultrathin sections were cut and post-stained with uranyl acetate and lead citrate. These sections were examined using a JEOL 1200EX transmission electron microscope. Statistics All samples were at least prepared in triplicate. Results from the quantitative studies are expressed in terms of mean and standard deviation of three independent experiments. Statistical analyses were performed by one-way ANOVA and comparisons between groups were performed using the Student’s t test. Differences were considered significant at p,0.05. Mitotracker staining and confocal microscopy MitoTracker Red CMXRos, a mitochondriaspecific cationic fluorescent dye, was used to label mitochondria. 3T3-L1 cells in Lab-Tek chamber slides were stained with 250 nmol/L MitoTracker in serum-free DMEM for 15 minutes at 37uC according to the manufacturer’s instructions. A Leica TCS SP5 Confocal Microscopy System, consistent with a prior report of a microarray analysis showing that the expression levels of PHBs are increased during 3T3-L1 cell adipogenesis. The sequentially induction of the adipogenic markers, CCAAT/ enhancer-binding protein beta, peroxisome proliferator-activated receptor gamma and adipocyte Protein 2, were observed in these conditions. Additionally, we determined ” that the alterations of protein levels of PHB1 and PHB2 followed 11118042” a similar pattern during adipogenesis in human ASC compared to mouse 3T3-L1 cells. When we examined the mRNA levels of PHBs in differentiating 3T3-L1 cells, we found that both PHB1 and PHB2 were significantly increased as early as six hours post adipogenic induction and peaked at day two and fell to basal levels by one to two weeks, suggesting post-translational protein stabilization. Among the three hormone ingredients in the adipogenic cocktail, the PHBs expression was mainly induced by IBMX and insulin rather than dexamethasone in 3T3-L1 cells. In addition, the levels of PHBs in white adipose tissue from wild-type, heterozygous and homozygous obese mice, both female and male, were compared. Interestingly, the expression levels of PHBs in WAT from obese mice were not higher than that from wild type ones. Actually, even decrease of the expression of adipogenic genes in obesity has been observed, which is considered as the consequence of the dedifferentiation in WAT from obese mice. PHBs are required for PPARc expression and adipocyte differentiation in 3T3-L1 cells To investigate the possible roles of increasing PHBs during adipogenesis, we screened siRNA for effective knockdown of PHBs expression in this model. Three different siRNA oligonucleotides, siPHB1-1, siPHB1-2 and siPHB1-3, were used to target PHB1; while siPHB2-1, siPHB2-2 and siPHB2-3 w

virtually 100% viability of both the control and MeCP2-null astrocytes after 24 h incubation with 10 mM Glu. Glu-induced gliotoxic effects have been previously reported by Chen et al., and are probably due to distinct differences in culture conditions, specifically the presence of glucose. These results showed that H2O2 and NH4Cl had a similar effect in both strains of astrocytes. There was no significant difference in viability between the control and MeCP2-null astrocyte cultures, indicating that MeCP2 deficiency did not affect astrocyte viability upon treatment with H2O2 and NH4Cl. Effects of glutamate on glutamate transporters and glutamine synthetase transcripts in MeCP2-null astrocytes High extracellular Glu interferes with the expression of the astrocyte transporter subtypes, excitatory amino acid transporter 1/glutamate/aspartate transporter and EAAT2/glutamate transporter-1 . To explore the effects of Glu on the expression of Glu transporter genes in cultured astrocytes from wild-type and MeCP2-null mouse brains, we asked whether treatment with 1.0 mM Glu altered expression of EAAT1 and EAAT2 mRNA, using a semi-quantitative RTPCR assay. EAAT1 and EAAT2 mRNA were expressed in both wild-type and MeCP2-null astrocytes, and were slightly higher in controls than in MeCP2-null astrocytes. Both EAAT1 and EAAT2 mRNA levels were altered in the control astrocytes after treatment with 1.0 mM Glu. EAAT1 mRNA levels decreased significantly in the wild-type astrocytes, both 12 h and 24 h after treatment with Glu. In contrast, EAAT1 decreased significantly in the MeCP2-null astrocytes, at 12 h but not 24 h after treatment. As with EAAT1, EAAT2 mRNA levels also decreased significantly in the control astrocytes, both 12 h and 24 h after treatment. However, EAAT2 decreased significantly in MeCP2-null astrocytes, 24 h but not 12 h after treatment. In addition, the effects of Glu on EAAT1 and EAAT2 relative fold expression at 12 h were altered in 23416332” the MeCP2-null astrocytes. These results suggest that the loss of MeCP2 leads to transcriptional dysregulation of these genes, either directly or indirectly. One important enzyme that plays a role in the Glu metabolic pathway is glutamine synthetase . GS is mainly located in astrocytes; cultured astrocytes response to Glu with increased GS expression. Consistent with this, 1.0 mM Glu treatment stimulated GS mRNA expression in both the wildtype and MeCP2-null astrocytes about 1.2-fold after 12 h but not 24 h. In addition, MeCP2 deficiency did not modify the Results 10460232” Characterization of MeCP2-null astrocytes It was recently reported that MeCP2 is normally present not only in neurons but also in glia, including astrocytes, oligodenrocytes, and microglia. To determine the roles of MeCP2 in astrocytes, we cultured cerebral cortex astrocytes from both wild-type and MeCP2-null mouse brains. MeCP2-null astrocytes exhibited a large, flattened, polygonal shape identical to that of the wild-type astrocytes, suggesting that normal patterns of cellular recognition and contact were present. Semi-quantitative RT-PCR using primer sets that specifically amplify two splice variants, Mecp2 e1 and e2, showed that control astrocytes expressed Mecp2 e1 and e2, whereas neither Mecp2 variant was detectable in MeCP2-null astrocytes. We further confirmed expression of MeCP2 by immunocytochemical staining of astrocytes. In control samples, almost all Scopoletin web GFAP-positive cells exhibited clear nuclear MeCP2 immunoreactivity in astrocytes,

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