[16] In brief, after pretreatment using a microwave with citrate buffer (pH 6), 95°C, for 20 min, blocking endogenous peroxidase, sections were incubated with the primary antibody at Angiogenesis inhibitor 4°C overnight. The Envision+ solution for mouse and rabbit (Dako) was then applied for 30 min at room temperature. The reaction products were visualized using 3-3′-diaminobenizidine tetrahydrochloride (Sigma Chemical, St Louis,

MO, USA) and H2O2. The sections were then lightly counterstained with hematoxylin. Similar dilution of the control mouse or rabbit Immunoglobulin G (Dako) was applied instead of the primary

antibody as a negative control. Positive and negative controls were routinely included. A cut section of the resected tumor showed a nodular lesion of 10 mm in diameter with an ill-defined border (Fig. 2). Color after fixation was light brown, similar to the background liver, and the central part showed congestion. On histology, the nodular lesion had an ill-defined border and hemangiomatous lesions were scattered inside (Fig. 2). Hemangiomatous lesions adjacent to portal tracts were also seen. Sinusoidal dilatation with congestion was observed in the central area of the nodular lesion (Fig. 2). Hepatocytes in the lesion showed a thickened cell layer and increased Lapatinib in vivo cellular density when compared with the background liver, but there was no cellular atypia (Fig. 2). Reticulin fibers were not decreased around hepatic trabeculae. Endothelial cells in the hemangiomatous lesions and dilated sinusoids showed distinct immunoreactivity for CD34. In addition, sinusoidal endothelial cells in the area without sinusoidal

dilatation showed immunoreactivity for CD34, indicating capillarization of sinusoids. The background liver was almost normal and there were few hemangioma-like vessels outside the nodule. There Telomerase were no abnormally thickened arteries or a central stellate scar, so this nodular lesion appeared to be different from the usual FNH. Although the dilated sinusoidal structure resembled inflammatory-type hepatocellular adenoma, immunostaining for SAA was negative. Immunostaining for LFABP and GS did not suggest any other subtypes of hepatocellular adenoma. Taken together, this lesion was diagnosed as a hyperplastic hepatocellular lesion associated with localized hemangiomatosis, including multiple hemangioma-like vessels.

They reported that the atrophic grade improved at the gastric angle in the fifth year and at all locations except for the antrum in the tenth year after H. pylori eradication. On the other hand, the metaplastic score did not change in either the fifth or tenth year after H. pylori eradication. The outstanding

achievements of the current study were summarized as; i) clear documentation supporting the concept that gastric mucosa can be repaired after H. pylori eradication, ii) improved gastric atrophy through eradication of H. pylori infection is associated with prevention of progression to intestinal metaplasia in those aged more than 60 years, iii) there is a long-term beneficial effect of H. pylori eradication leaving room for ultimate hope to witness gastric cancer prevention. However,

similar to the afore-mentioned implication of H. pylori eradication on gastric cancer prevention, studies examining the Alectinib manufacturer reversibility of precancerous selleck inhibitor lesions following eradication of H. pylori also have provided conflicting results. Among earlier studies, several reported that the severity of gastric atrophy and intestinal metaplasia does not change after treatment.12 In contrast, another study has suggested that gastric atrophy and some intestinal metaplasia can improve after H. pylori eradication.13 The contradictory results can be explained by the fact that a significant proportion of eradicated patients have progression of premalignant lesions,

already passing Glutathione peroxidase a ‘point-of-no-return’. Therefore, the eradication of H. pylori infection might not be beneficial if therapy is given passing the ‘point-of-no-return’. However, it is very difficult to discriminate whether the gastric premalignant lesion has passed such a ‘point-of-no-return’ or not by conventional endoscopy or histopathology. Many efforts had been made to find biomarkers or clinical findings that suggest reversibility of gastric atrophy or intestinal metaplasia; ideally, these might adopt high throughput analytical techniques, including microarray or proteomics. Short of this ‘point-of-no-return’, the appropriate time for intervention for H. pylori eradication should be resolved. It is suggested that the sooner H pylori eradication, the higher will be the rate of prevention of H. pylori-related gastric carcinogenesis.14,15 A recent randomized, prospective, placebo-controlled study by Wong et al.3 does suggest that H. pylori eradication reduces the incidence of gastric cancer in patients without pre-existing gastric atrophy and/or intestinal metaplasia stratified to early intervention of H. pylori eradication. Related to the time of the intervention against H. pylori eradication, the article of Toyokawa T et al.11 adopted a stratified rationale for H. pylori eradication even in elderly patients in order to rejuvenate atrophic gastritis. Eradication of H.

Key Word(s): 1. colorectal cancer; 2. autophagy; 3. EZH2; 4. PTEN; Presenting Author: NAN LI Additional Authors: HENG LU, selleck chemicals llc CHUNYAN CHEN, FANGYU WANG Corresponding Author:

FANGYU WANG Affiliations: Nanjing Univ, Sch Med Objective: Fatty acid synthase (FASN) is frequently activated and overexpressed in human cancers, and plays a crucial role in the carcinogenesis of various cancers. But its role in colorectal cancer is still indefinite until now. Therefore, in this study, our aims were to explore the role of FASN in regulating the activity of “HER2-PI3K/Akt axis” and the malignant phenotype in colorectal cancer cells. Methods: Caco-2 cells with high expressions of both FASN and HER2 were selected for the functional characterization. Then, Caco-2 cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid, and followed by RT-qPCR and western blot to examine expressions of FASN, HER2, PI3K and Akt. MTT and colony formation assays were HM781-36B used to assess the proliferation potential. The migration was investigated by transwell, and the apoptosis and cell cycle were assayed by flow cytometry. Results: Notably, expressions of FASN, HER2, PI3K and Akt were downregulated

upon a silence of FASN. The proliferation was decreased after a downregulation of FASN, which was consistent with an increased apoptosis rate. The migration was also impaired in FASN-silenced cells. A downregulation of FASN effectively inhibited the activity of “HER2-PI3K/Akt axis” of Caco-2 cells, and also altered the malignant phenotype of Caco-2 cells. Conclusion: FASN plays a crucial role in the carcinogenesis of colorectal cancer. Key Word(s): 1. selleck Fatty acid synthase; 2. Colorectal cancer; 3. HER2-PI3K/Akt axis; 4. Malignant phenotype; Presenting Author: MINGZHOU GUO Additional Authors: YUNSHENG YANG Corresponding Author: MINGZHOU GUO Affiliations: Chinese PLA General Hospital Objective: It is estimated that up to 90% of the human genome is actively transcribed, but only 2% of the human genome encodes proteins. RNA transcripts that lack protein coding potential

are collectively referred to as non-coding RNAs (ncRNAs). In addition to small regulatory ncRNAs (e.g., microRNAs, small interfering RNAs and others), numerous long non-coding RNAs (lncRNAs) have been identified. LncRNAs are generally defined as non-protein-coding transcripts of more than 200 nucleotides in length. lncRNAs constitute a very heterogeneous group of RNA molecules that allows them to exert multiple functions through different mechanisms. LncRNAs may regulate gene expression in the transcription and post-transcriptional level. Methods: Human esophageal, hepatic, gastric and colonic cancer cell lines, normal tissues from non-cancerous patients, matched primary cancer and adjacent tissues were involved in this study.

Disclosures: The following people have nothing to disclose: Joel P. Wedd, Jane Gralla, Betsy Gans, Sue Dunn, Harvey Solomon, Michael D. Voigt, Scott W. Biggins Introduction: Over the last several years, the number of deceased donor simultaneous Liver-Kidney transplants (SLK) has

increased. However, guidelines for SLK, including when these combined organ transplants are appropriate based on serum creatinine, underlying liver and renal disease, etiology of renal dysfunction and time on dialysis are contentious. Inappropriate SLK removes an organ from the donor pool which would more appropriately be utilized for a patient awaiting kidney transplantation, and failure to provide SLK to a patient who requires prolonged dialysis and kidney transplantation following isolated LT is similarly inappropriate. We hypothesize that Akt inhibitor the use of SLK varies by region, and is unrelated

to mean MELD at the time of transplantation. Our group has previously presented data related to SLK transplants performed between 2002-2010. This data set is herein augmented with additional results from 2011 and 2012. Methods: Utilizing data provided by UNOS, we performed a retrospective review of all SLK performed from 2002-2012, analyzed the percentage of SLK performed in each region based on total number of liver selleck chemicals llc transplants (LT) performed, the ratio of % SLK performed to mean MELD at the time of transplantation, and assessed rate of change in number of SLK by year by region. Results: During this time period, 3,865 SLK and 56, 693 isolated LT were performed. Nationally, the ratio of SLK to LT was 6.7%. This ratio was dramatically different when comparing regions, with the highest ratio in regions 7, 1, and 5 (13, 10, and 9% respectively) and lowest in regions 6, 9, and

11 (3.4, 4.1, and 4.3%). The mean increase per year in number of SLK performed was 22, but also varied dramatically Tolmetin by region, with an increase of 52, 50 and 35 transplants in regions 3, 7, and 5 respectively, and −3, 1, and 2 in regions 1, 6, 8. When analyzing the ratio of % of SLK versus total LT to mean MELD score at the time of transplantation in each region, significant differences were also found, with the highest ratios in regions 7 and 1 (.394, .324) and lowest in regions 9, 6 and 11(.126, .128, .162). Conclusions: 1) Utilization of SLK varies significantly when comparing UNOS regions 2) The increased utilization of SLK does not appear to correlate to increased wait list MELD score at the time of transplantation in regions performing the highest % of SLK. In fact, lowest utilization of SLK is occurring in regions with some of the highest wait list MELD scores. 3) These findings suggest that a uniform policy related to utilization of SLK should be adopted Disclosures: Paul J.

3E). There was a 33% reduction in colony formation in PLC5 cells ectopically expressing dN1, in comparison with wtSHP-1. We also observed an almost 50% reduction

in colony numbers in SK-Hep1 cells, ectopically expressing dN1. Ectopic expression of D61A also exhibited fewer colonies than the control. These results imply that activated SHP-1 protects against tumor cell proliferation. Next, an immunohistochemistry (IHC) study was conducted to examine the role of SHP-1 in tissues from patients with HCC. p-STAT3 was expressed in the majority of HCC tissue, but less SHP-1 was found expressed learn more in the same tissues (Fig. 3F). Further investigation of the role of SHP-1 in HCC tumor progression is warranted. Working upon the assumption that sorafenib relieves

the autoinhibition of SHP-1, we generated a series of sorafenib derivatives to search for potent SHP-1 agonists that may act as better anti-HCC agents than sorafenib. Among the sorafenib analogs generated, we identified two promising new agents, SC-43 and SC-40, the structures of which are shown in Fig. 4A. Both SC-43 and SC-40 had potent effects on induction of SHP-1 activity in vitro and in vivo. SC-43 and SC-40 effectively up-regulated SHP-1 activity at lower concentrations than sorafenib, either in SHP-1-containing cell extract (Fig. 4B) or purified recombinant SHP-1 proteins (Fig. 4C). In addition, both SC-43 and SC-40 did not significantly alert

SHP-2 activity in PLC5 and Hep3B cells. Furthermore, SHP-2 activity was not affected in SC-43- or SC-40-treated recombinant SHP-2 proteins (Supporting Fig. 2). STAT3-related proteins LGK-974 cost Mcl-1, cyclin D1, and survivin were examined in SC-43- and SC-40-treated HCC cells (Fig. 4D,E). Both SC derivatives resulted in substantial apoptosis in HCC cells, as evidenced by sub-G1. SC-43 and SC-40 decreased the viability of HCC cells in a dose-dependent manner (Fig. 5A). Both SC-43 and SC-40 showed lower 50% inhibitory concentration, compared to sorafenib. In addition, Astemizole SC-43 and SC-40 showed more potent inhibition of the p-STAT3-related signaling pathway (Fig. 5B). SC-43 revealed submicromolar inactivation of p-STAT3, relative to sorafenib (Fig. 5C). Furthermore, SC-43 and SC-40 resulted in significant apoptosis in sorafenib-resistant cells at submicromolar concentrations (Fig. 5D). The endogenous induction of p-STAT3 was observed in sorafenib-resistant cells, but not in parental Huh7 cells, which may explain why these cells showed resistance to sorafenib. Our findings provide a molecular rationale for drug optimization on the basis of the crystal structure of SHP-1. We hypothesize that sorafenib binds to the N-SH2 domain and subsequently releases and activates the PTP domain (Fig. 5E). Sorafenib was docked into the pocket between the N-SH2 domain and formed a hydrogen bonding with R44 through the trifluoromethyl group.

24, 25 Indeed, the Kaplan-Meier analysis shows that patients with HCC who had high p28GANK expression in general had worse prognosis than those with low expression. We believe that p28GANK is an attractive candidate gene for risk prognostication and therapy of HCC. However, our data is apparently at odds with a recent

report suggesting that the cumulative survival rate of patients with gankyrin-positive HCC was significantly higher than those patients with gankyrin-negative HCC.26 The discrepancy may be due to different backgrounds of specimens used, including the www.selleckchem.com/products/sorafenib.html proportion of hepatitis C virus and hepatitis B virus infection, sex of patients, and the classification/criteria of tumor-node-metastasis (TNM) staging. Recently, Ortiz and Tang reported that gankyrin messenger Fulvestrant supplier RNA and protein increased in human esophageal squamous cell carcinoma (ESCC) or colorectal cancer (CRC), and its overexpression is poor prognosis of ESCC or CRC due to its significant correlation with TNM stages and metastasis of these tumors, respectively.27, 28 Therefore, p28GANK overexpression may be involved in development of human digestive malignancies such as HCC, ESCC, and CRC. The effect of p28GANK on tumor invasion and metastasis was directly demonstrated in our in vitro and in vivo studies. In both subcutaneous

and orthotopic xenografts, overexpression of p28GANK generated larger primary tumors and more lung metastasis foci, and higher levels of vascularization and angiogenesis, indicating their more aggressive and metastatic properties. Moreover, down-regulation

of p28GANK led to severe suppression of tumor growth and lung metastasis of HCC in mice. To our knowledge, this is the first report that p28GANK expression is critical for HCC metastasis, in addition to tumor proliferation and growth. In this study, we found that TWIST1 is indeed involved in p28GANK-driven EMT. Moreover, p28GANK isothipendyl modulated HIF-1α hyperactivation and expression correlated with TWIST up-regulation and E-cadherin down-regulation. Thus, our data suggest a requirement for HIF-1α in p28GANK-driven EMT. We also observed a role of HIF-1α in p28GANK-regulated VEGF and MMP2 expression, consistent with previous reports that HIF-1α up-regulates VEGF, promoting angiogenesis and invasion of HCC.29–31 Taken together, this study clearly demonstrates a crucial role for p28GANK in induction of EMT and angiogenesis through regulation of HIF-1α, VEGF, and MMP2 expression. An increase in AKT signal is a key tumor survival mechanism, and promotes tumor metastatic processes including EMT, resistance to apoptosis, and angiogenesis.32–34 Previous studies have demonstrated that activated AKT plays a critical role in hematogenous intrahepatic metastasis in an orthotopic implantation model of HCC.35 Our group previously showed a protective role of p28GANK in HCC cells against endoplasmic reticulum stress-induced apoptosis, partially through enhancing AKT phosphorylation.

10 However, the dynamic

relationship between lipid metabolic pathways in hepatocytes and stellate cells is incompletely understood and the elements that regulate LD accumulation upon HSC activation remain obscure.11 Mammalian intracellular fatty acid-binding proteins (FABPs) comprise a superfamily of lipid-binding proteins12 involved in the uptake, transport, and metabolism of FA and other lipid ligands. Liver Fabp (L-Fabp or Fabp1) is abundantly expressed in both hepatocytes and enterocytes and binds multiple ligands, including saturated FA and cholesterol.12 Germline L-FABP−/− mice exhibit decreased hepatic triglyceride content13 with altered FA uptake kinetics. In addition, L-FABP−/− mice fed a high saturated fat, high cholesterol

“Western” diet were protected against diet-induced obesity and hepatic steatosis, likely reflecting altered kinetics of saturated FA utilization.14, https://www.selleckchem.com/products/MK-2206.html 15 Proteomic screens revealed L-Fabp to be overexpressed in obese subjects with simple steatosis, along with paradoxically decreased expression in the progressive versus mild forms Nivolumab concentration of NASH.16 Recent studies have validated new models of diet-induced NAFLD with fibrosis in murine models,17, 18 setting the stage for formal exploration of the role of candidate genes in the progressive forms of murine NAFLD. Here we explore a role for L-Fabp in lipid metabolism in both hepatocytes and stellate cells and report the impact of

L-Fabp deletion in diet-induced HSC activation and hepatic fibrosis in vivo. α-SMA, alpha-smooth muscle actin; C/EBPα, CCAAT/enhancer-binding protein-alpha; Cidec, cell death-inducing DFFA-like effector c; CTGF, connective tissue growth factor; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; ESI-MS, electrospray ionization mass spectrometry; FA, fatty acids; HSCs, hepatic stellate cells; LDs, lipid droplets; L-FABP, liver fatty acid binding protein (Fabp1); NASH, nonalcoholic steatohepatitis; PDGF-βR, platelet-derived growth factor-beta receptor; Plin, perilipin; PPARγ, peroxisome proliferator-activated receptor-gamma; SREBP-1c, sterol regulatory element-binding protein-1c; TFF, trans-fat fructose diet; TG, triglyceride; TGF-βR, transforming growth factor-beta receptor. C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) and congenic L-FABP−/− mice19 were Chlormezanone used in all studies (see also Supporting Methods). Hepatic steatosis with fibrosis was induced by feeding female mice a high trans-fat diet supplemented with high fructose corn syrup, modified from Tetri et al.18 HSCs were isolated by pronase-collagenase perfusion and density gradient centrifugation, with >90% purity.20 For lipidomics analysis, isolated HSCs were subjected to a second gradient purification and frozen immediately at −80°C. Details of protein, immunohistochemical, and lipidomics analyses are provided in Supporting Methods.

Alteration of the mitochondrial membrane potential by CPZ was prevented by cotreatment with NAC, suggesting a role of ROS. Similar alterations were observed in HepaRG cells treated with 5 mM H2O2 starting at 30 minutes (data not shown). F-actin cytoskeleton, which is one of the primary targets of oxidative stress, was visualized by phalloidin fluoprobe labeling. Untreated cells showed pericanalicular location of F-actin and

large bile canaliculi with rounded shape, whereas 50 μM CPZ-treated Selleckchem Alisertib cells exhibited a different distribution with lesser pericanalicular F-actin, retraction, and decreased surface area of bile canaliculi (Fig. 2B). Quantification of bile canalicular surface area and intensity of F-actin in the pericanalicular region showed up to 28% and 26% decrease, respectively, after CPZ exposure. To assess the effect of CPZ on TA efflux, cells were incubated with [3H]-TA for 30 minutes and then treated for another 30 minutes with 50 μM CPZ in standard buffer or Ca2+ and Mg2+-free

buffer. Radiolabeled TA has been measured in these two different buffers and in the cells to determine CFTR activator canalicular and basolateral efflux as well as intracellular accumulation of TA (7). A 25% increase in TA efflux was noticed in untreated HepaRG cells when canalicular tight junctions were disrupted (Ca2+ and Mg2+-free buffer), indicating that bile canaliculi correspond to a delimited closed compartment in these cells (data not shown). After 30 minutes of

treatment with CPZ, a 32% intracellular accumulation of TA was observed; it was associated with 35% decrease in canalicular efflux whereas basolateral efflux remained unchanged (Fig. 3A). These data support the conclusion that intracellular accumulation of TA was caused by a decrease of canalicular efflux rather than a diminution of basolateral efflux. Then the effects of CPZ in TA efflux were measured at different timepoints (0-6 hours) in standard buffer (Fig. 3B). The Ca2+ and Mg2+-free buffer was excluded because an incubation exceeding 30 minutes with this buffer caused increased cell death. No efflux inhibition was observed Glycogen branching enzyme before 30 minutes, whereas maximum inhibition occurred after 2 hours, with a 2-fold TA intracellular accumulation in CPZ-treated HepaRG cells. The CPZ-induced decrease of [3H]-TA efflux was abolished when cells were cotreated with CPZ and NAC; this result showed the involvement of oxidative stress in TA accumulation in CPZ-treated HepaRG cells. Because intracellular accumulation of TA was measured after treatment with CPZ in a standard buffer and that bile canaliculi were at least partially closed in control HepaRG cells, this accumulation could represent bile canalicular storage in addition to intracellular accumulation. To verify this hypothesis, bile canaliculi were disrupted by 5-minute incubation in Ca2+ and Mg2+-free buffer23 after a 2-hour treatment with CPZ.

(1-B) The child’s diagnostic evaluation as it relates CP-690550 in vitro to their primary disease, associated comorbidities, subspecialty consultations, and management strategies should be documented and provided by the primary pediatric specialist responsible for management of the child’s liver disease. These documents should include clinical assessments, results of laboratory and diagnostic studies, medical and nutritional management,

surgical procedures, pathology reports and slides, as well as radiographic reports and copies of the radiographs. Personal communication between a member of the LT evaluation team and the child’s physician will identify clinical, social, and psychological factors that may not be apparent in the medical record. New or worsening comorbidities may be identified during the LT evaluation.[9] 6. A review of the local records by the LT team prior to the LT evaluation will inform the evaluation schedule and enable affirmation

of the primary diagnosis, Torin 1 research buy assessment of comorbidities, and identify technical challenges related to LT. (2-B) 7. In collaboration with the local primary pediatric specialist, management of the primary disease and comorbidities should be reviewed and optimized. (2-B) Complications associated with endstage liver disease include ascites, pruritus,

portal hypertension, malnutrition, vitamin deficiencies, and delayed growth and development.[10] In cirrhosis patients, accumulation of ascites is a result of portal hypertension, vasodilatation, and hyperaldosteronism.[11] Hypoalbuminemia is an additional risk factor for ascites. Ultrasonography is sensitive enough to detect as little as an ounce of intra-abdominal fluid, while significantly more is required for of it to be detected on physical examination. Decisions to initiate diuretic therapy to manage ascites are ill-defined. Abdominal distension alone does not reliably predict ascites, as organomegaly and vascular congestion of the bowel may also contribute to distension. Fluid that is easily palpated between the abdominal wall and the surface of the liver (“ballotable fluid”) would suggest sufficient ascites to warrant therapy; its presence can be used to judge response to therapy. Initial treatment includes spironolactone and a “no-added” salt diet. Loop-diuretics should be used with caution as overaggressive diuresis can precipitate hepatorenal syndrome. For hospitalized patients with significant ascites, intravenous albumin, with or without an accompanying diuretic, can improve diuresis and response to diuretics.

Official French regulations (no.: 87848) for the use and care of laboratory animals were followed throughout, and the experimental protocol was approved by the local ethics

committee for animal experimentation. C57BL/6JRj and C57BL/6J-Lepob/Lepob male mice (Janvier, Le Genest Saint Isle, France) were housed in individual plastic cages and kept on a standard diet (AO4; UAR, Epinay-sur-Orge, France), until the preparation of liver slices. Mice (13 weeks old) were anesthetized with an intraperitoneal injection of ketamine/xylazine (7.5 mg/1 mg for 100 g body weight) and were sacrificed by cervical dislocation. To clear the organ of blood, the liver was immediately rinsed TGF-beta inhibitor by introducing a needle into the heart and perfusing with cold, oxygenated Hanks’ balanced salt solution (HBBS). Then, the organ was removed and sliced using a Brendel/Vitron slicer (Vitron Inc., Tucson, AZ) in the same medium. Slices (approximately 200 μm in thickness and 20 mg in weight) from each liver were rinsed and preincubated for

30 minutes at 37°C in HBBS before being randomly distributed in 15-mL culture tubes (6-7 slices per tube) containing 7 mL of oxygenated William’s Alvelestat medium E (WME), supplemented with heat-inactivated nondelipidated fetal bovine serum (FBS; 10%) and antibiotic-antifongic Phenylethanolamine N-methyltransferase cocktail (1%), as previously described.18 Slices were treated with SR141716

(0-10 μM) and, when specified, with arachidonic acid N-hydroxyethylamide (AEA; 5 μM) or atorvastatin (5 μM). SR141716 and AEA were dissolved in dimethyl sulfoxide and diluted in WME. Atorvastatin was prepared in WME. In each case, a series of liver slices treated with vehicle only was assigned to control assays. Tubes were then installed horizontally on a rocking shaker, pierced on the top to allow gas exchange, and incubated for 21 hours in a 5% CO2 atmosphere at 37°C, under slight agitation. At the end of the incubation period, slices were randomly allocated to the different experiments described thereafter. Chemicals and mediums used in this procedure were supplied by Sigma (Saint-Quentin-Fallavier, France), except SR141716, which was provided by Sanofi Aventis (Paris, France).