scholarly journals The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn.

1993 ◽  
Vol 178 (2) ◽  
pp. 723-730 ◽  
Author(s):  
J L Chu ◽  
J Drappa ◽  
A Parnassa ◽  
K B Elkon
Keyword(s):  
Mrna Expression ◽  
Tumor Necrosis Factor Receptor ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Northern Blotting ◽  
Nucleotide Sequencing ◽  
Mrna Levels ◽  
Wild Type ◽  
Fas Mrna ◽  
A Cell

Fas is a cell surface protein of the tumor necrosis factor receptor, nerve growth factor receptor, CD40 family, and is involved in the control of lymphocyte apoptosis. A mutation in the Fas gene in MRL/lpr mice results in massive lymphoproliferation (lpr) and accelerated autoimmunity. To further study the nature of this defect, Fas mRNA expression was evaluated by reverse transcriptase polymerase chain reaction as well as by Northern blotting. These studies revealed that the wild-type Fas message was produced at approximately 10-fold lower levels in the lpr compared with the ++ substrain of MRL mice. In addition to the wild-type transcript, lpr mice also synthesized chimeric transcripts containing an insertion of the early retrotransposon (ETn). Molecular cloning and nucleotide sequencing of a Fas-ETn chimeric cDNA suggested that the striking reduction in wild-type Fas mRNA levels and the presence of aberrant transcripts in MRL/lpr mice are most likely explained by the insertion of the ETn retrotransposon into an intron of the Fas gene and induction of alternative splicing involving the 5' ETn long terminal repeat.

scholarly journals Stem cell factor and c-kit are involved in hepatic recovery after acetaminophen-induced liver injury in mice

2008 ◽  
Vol 295 (1) ◽  
pp. G45-G53 ◽  
Author(s):  
Bin Hu ◽  
Lisa M. Colletti
Keyword(s):  
Stem Cell ◽  
Liver Injury ◽  
Stem Cell Factor ◽  
Cellular Proliferation ◽  
Hepatocyte Proliferation ◽  
Mrna Levels ◽  
Wild Type ◽  
Hepatocyte Apoptosis ◽  
Large Reservoir ◽  
A Cell

Stem cell factor (SCF) and its receptor c-kit are important in hematopoiesis and cellular proliferation. c-kit has also been identified as a cell surface marker for progenitor cells. We have previously shown that there is a large reservoir of hepatic SCF, and this molecule plays a significant role in liver regeneration after 70% hepatectomy. In the current study, we further examined the expression of SCF and c-kit in acetaminophen (APAP)-induced liver injury in C57BL/6J mice or SCF-deficient sl-sld mice and their appropriate wild-type controls. Following APAP-induced liver injury, c-kit mRNA expression increased, with peak levels detected 48 h postinjury. Hepatic SCF mRNA levels after APAP injury were also increased, with peak levels seen 16 h post-APAP. The mortality rate in SCF-deficient mice treated with APAP was significantly higher than that of wild-type mice; furthermore, administration of exogenous SCF significantly reduced the mortality of APAP-treated wild-type mice. Bromodeoxyuridine incorporation experiments showed that SCF significantly increased hepatocyte proliferation at 48 and 72 h in APAP-treated mice. SCF inhibited APAP-induced hepatocyte apoptosis and increased Bcl-2 and Bcl-xL expression, suggesting that this decrease in hepatocyte apoptosis is mediated through Bcl-2 and Bcl-xL. In summary, SCF and c-kit expression was increased after APAP-induced liver injury. Administration of exogenous SCF reduces mortality in APAP-treated mice, increases hepatocyte proliferation, and prevents hepatocyte apoptosis induced by APAP, suggesting that these molecules are important in the liver's recovery from these injuries.

Expression of Ara-C Metabolizing Enzymes Mediates in Vitro Sensitivity to Ara-C in Primary AML Cells From Patients with Denovo AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3481-3481
Author(s):  
Ajay Abraham ◽  
Savitha Varatharajan ◽  
Ashok kumar Jayavelu ◽  
Shaji R Velayudhan ◽  
Rayaz Ahmed ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Candidate Genes ◽  
P Value ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutation Status ◽  
Expression Levels ◽  
In Vitro Sensitivity ◽  
Mrna Expression Levels

Abstract Abstract 3481 Wide inter-individual variation in terms of treatment outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (ara-C) and daunorubicin. The pre-requisite for the cytotoxic action of pro-drug Ara-C is the enzymatic conversion to its active tri-phosphorylated form ara-CTP. Many drug activating (Deoxycytidine kinase (dCK) and human Equilibrative Nucleoside Transporter 1 (hENT1) and deactivating (Cytidine deaminase (CDA), 5'nucleotidase (NT5C2) genes and ribonucleoside reductase (RRM1), which are involved in transport and biotransformation of cytarabine contribute to the variation in ara-C sensitivity in AML patients. FLT3-ITD and NPM1 mutations act as major poor and good prognostic markers respectively in cytogenetically normal AML. The effect of these mutations in ara-C metabolism remains to be elucidated. The present study aims to determine independent as well as the combined effect of ara-C metabolizing genes mRNA expression on in-vitro ara-C cytotoxicity and the role of FLT3-ITD and NPM mutations on mRNA expression of these genes. Diagnostic bone marrow sample (median blasts 65%; range 21 – 98%) from 98 adult patients with de novo AML (other than AML-M3) were included in this study. mRNA expression levels for each target gene relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. In vitro sensitivity or resistance was classified on the basis of the IC-50 values <6uM and >6uM ara-C respectively. FLT3 ITD and NPM mutation status at diagnosis were determined through PCR followed by Genescan analysis using genomic DNA samples. Type of NPM mutation was identified by sequencing. When ara-C IC-50 values were compared with the mRNA expression levels of these candidate genes, Ara-C sensitive samples (n= 30; IC-50 < 6uM) showed significantly higher mRNA expression of dCK and hENT1 compared to those with Ara-C resistance (n=51) IC50 >6uM (median 314 (61.56 – 1232) vs. 180 (31.87 – 749.2); p = 0.0004 and median 172.1 (44.12 – 657.6) vs. 96.19 (37.49 – 432.4), p= 0.0008 respectively. RRM1 and NT5C2 did not show any association with in vitro Ara-C cytotoxicity, while CDA showed a trend towards association with lower CDA expression in ara-C sensitive samples. Based on these findings we put forward Ara-C resistance index (RI). RI is calculated by the formula RI = ΔCT (dCK X ENT1)/ ΔCT CDA. (Smaller ΔCT value= higher mRNA expression). RI values were significantly higher in resistant (IC50 >6uM) compared to sensitive cells (median: 6.084; range 1.89–11.82) vs. 3.702 (1.89–9.80); p=<0.0001). This association should now be validated in an independent cohort. Effects of NPM and FLT3 mutation status on Ara-C metabolizing genes were then evaluated. No significant association was found between FLT3-ITD status and the mRNA expression of these candidate genes. Interestingly, dCK mRNA levels were significantly higher in samples with NPM mutation (n=39) compared to NPM wild type (n=59); median 272.3 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.01. When analysed separately, patients with NPM type A mutation (n=27) showed significantly higher dCK expression (median 347.4 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.003 compared to those with wild type NPM1. This first report showing an association between expression profiles of ara-C metabolizing genes and NPM mutation should form the basis for evaluating their clinical correlations. Disclosures: No relevant conflicts of interest to declare.

SRC-homology 2 domain-containing phosphatase 2 (SHP2) in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9540-9540
Author(s):  
Rafael Rosell ◽  
Masaoki Ito ◽  
Jordi Codony-Servat ◽  
Ana Giménez-Capitán ◽  
Mireia Serra-Mitjans ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mrna Expression ◽  
Mrna Levels ◽  
Wild Type ◽  
Src Homology 2 ◽  
Src Homology ◽  
Epidermal Growth ◽  
Src Homology 2 Domain ◽  
Egfr Mutant

9540 Background: Epidermal growth factor (EGFR)-mutant lung adenocarcinomas (LUADs) display impaired phosphorylation of extracellular signal-regulated kinase (ERK) and SRC-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. However, the function of SHP2 in early EGFR-mutant LUADs and EGFR wild-type LUADs has not been reported. We posit that SHP2 mRNA expression could be a predictive marker in resected EGFR-mutant LUADs versus EGFR wild-type patients (pts). Methods: We examined 267 resected LUADs from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1 and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction (PCR). EGFR mutant cell lines were investigated for their activity of SHP2. Results: Among the 267 enrolled pts, 100 (37.3%) were EGFR-mutant LUADs. Five-year recurrence-free survival (RFS) and overall survival (OS) were lower for EGFR-mutant LUADs with high SHP2 mRNA levels (hazard ratio = 1.83 and 2.28, respectively. p = 0.03 and p = 0.04). However, SHP2 was not associated with RFS nor OS in the 167 wild-type EGFR LUADs. In EGFR-mutant cells, RMC-4550 (SHP2 inhibitor) plus erlotinib showed synergism via inhibition of AKT (S473) and ERK1/2 (T202/Y204). While erlotinib translocates SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocalizes SHP2 into the cytoplasm membrane, limiting AKT and ERK activation. Conclusions: High SHP2 mRNA is related to shorter RFS and OS in EGFR-mutant LUADs, but not in EGFR wild-type LUADs. The findings indicate that the addition of SHP2 inhibitors could improve adjuvant therapy in EGFR-mutant LUADs.

scholarly journals C-Abl Is Required for the Development of Hyperoxia-Induced Retinopathy

2001 ◽  
Vol 193 (12) ◽  
pp. 1383-1392 ◽  
Author(s):  
Irene Nunes ◽  
Rosemary D. Higgins ◽  
Lucia Zanetta ◽  
Peter Shamamian ◽  
Stephen P. Goff
Keyword(s):  
Mrna Expression ◽  
Retinal Neovascularization ◽  
Mrna Levels ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Vegf Mrna ◽  
Nonreceptor Tyrosine Kinase ◽  
Kinase C ◽  
Endothelial Growth Factor ◽  
Inspired Oxygen

The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl–deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous null for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous null mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

scholarly journals SprB Is a Cell Surface Component of the Flavobacterium johnsoniae Gliding Motility Machinery

2008 ◽  
Vol 190 (8) ◽  
pp. 2851-2857 ◽  
Author(s):  
Shawn S. Nelson ◽  
Sreelekha Bollampalli ◽  
Mark J. McBride
Keyword(s):  
Cell Surface ◽  
Surface Protein ◽  
Gliding Motility ◽  
Wild Type ◽  
Polystyrene Microspheres ◽  
Flavobacterium Johnsoniae ◽  
A Cell ◽  
Transposon Insertions ◽  
Gliding Bacterium ◽  
Surface Adhesins

ABSTRACT Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces by an unknown mechanism. Transposon insertions in sprB resulted in cells that were defective in gliding. SprB is a highly repetitive 669-kDa cell surface protein, and antibodies against SprB inhibited the motility of wild-type cells. Polystyrene microspheres coated with antibodies against SprB attached to and were rapidly propelled along the cell surface, suggesting that SprB is one of the outermost components of the motility machinery. The movement of SprB along the cell surface supports a model of gliding motility in which motors anchored to the cell wall rapidly propel cell surface adhesins.

Foxf1 haploinsufficiency reduces Notch-2 signaling during mouse lung development

2004 ◽  
Vol 286 (3) ◽  
pp. L521-L530 ◽  
Author(s):  
Vladimir V. Kalinichenko ◽  
Galina A. Gusarova ◽  
Il-Man Kim ◽  
Brian Shin ◽  
Helena M. Yoder ◽  
...  
Keyword(s):  
Pulmonary Hemorrhage ◽  
Growth Factor Receptor ◽  
Abnormal Development ◽  
Mouse Lung ◽  
Mrna Levels ◽  
Wild Type ◽  
Downstream Target ◽  
Visceral Mesoderm ◽  
Expression Of Genes ◽  
Affymetrix Microarrays

The forkhead box (Fox) f1 transcription factor is expressed in the mouse splanchnic (visceral) mesoderm, which contributes to development of the liver, gallbladder, lung, and intestinal tract. Pulmonary hemorrhage and peripheral microvascular defects were found in approximately half of the newborn Foxf1(+/-) mice, which expressed low levels of lung Foxf1 mRNA [low- Foxf1(+/-) mice]. Microvascular development was normal in the surviving newborn high- Foxf1(+/-) mice, which compensated for pulmonary Foxf1 haploinsufficiency and expressed wild-type Foxf1 levels. To identify expression of genes regulated by Foxf1, we used Affymetrix microarrays to determine embryonic lung RNAs influenced by Foxf1 haploinsufficiency. Embryonic Foxf1(+/-) lungs exhibited diminished expression of hepatocyte growth factor receptor c-Met, myosin VI, the transcription factors SP-3, BMI-1, ATF-2, and glucocorticoid receptor, and cell cycle inhibitors p53, p21Cip1, retinoblastoma, and p107. Furthermore, Notch-2 signaling was decreased in embryonic Foxf1(+/-) lungs, as evidenced by significantly reduced levels of the Notch-2 receptor and the Notch-2 downstream target hairy enhancer of split-1. The severity of the Notch-2-signaling defect in 18-day postcoitus Foxf1(+/-) lungs correlated with Foxf1 mRNA levels. Disruption of pulmonary Notch-2 signaling continued in newborn low- Foxf1(+/-) mice, which died of lung hemorrhage and failed to compensate for Foxf1 haploinsufficiency. In contrast, in newborn high- Foxf1(+/-) lungs, Notch-2 signaling was restored to the level found in wild-type mice, which was associated with normal microvascular formation and survival. Foxf1 haploinsufficiency disrupted pulmonary expression of genes in the Notch-2-signaling pathway and resulted in abnormal development of lung microvasculature.

scholarly journals Contribution of the Collagen Adhesin Acm to Pathogenesis of Enterococcus faecium in Experimental Endocarditis

2008 ◽  
Vol 76 (9) ◽  
pp. 4120-4128 ◽  
Author(s):  
Sreedhar R. Nallapareddy ◽  
Kavindra V. Singh ◽  
Barbara E. Murray
Keyword(s):  
Enterococcus Faecium ◽  
Multidrug Resistant ◽  
Mrna Levels ◽  
Virulence Determinants ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Fold Reduction ◽  
A Cell ◽  
Peritonitis Model

ABSTRACTEnterococcus faeciumis a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstratingE. faeciumvirulence determinants. Our previous studies showed that some clinicalE. faeciumisolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenicacmdeletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Δacm::catis highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P< 0.0001) and for valve colonization 1 and 3 hours after infection (P≤ 0.0002), making Acm the first factor shown to be important forE. faeciumpathogenesis. In contrast, no mortality differences were observed in a mouse peritonitis model. While 5 of 17 endocarditis isolates were Acm nonproducers and failed to adhere to collagen in vitro, all had an intact, highly conservedacmlocus. Highly reducedacmmRNA levels (≥50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating thatacmtranscription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction inE. faeciumcollagen adherence by affinity-purified anti-Acm antibodies fromE. faeciumendocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

mRNA expression of signal transducer and activator of transcription (STAT5), disheveled-3 (Dvl-3) and dicer in early-stage non-small-cell lung cancer (NSCLC)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 17038-17038
Author(s):  
F. Cecere ◽  
R. Rosell ◽  
M. Taron ◽  
A. Ceribelli ◽  
M. Milella ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Tyrosine Kinases ◽  
Early Stage ◽  
Wnt Signaling Pathway ◽  
Growth Factor Receptor ◽  
Close Correlation ◽  
Mrna Levels ◽  
Internal Reference ◽  
Primary Tumors ◽  
Early Stage Nsclc

17038 Background: STAT5 plays a role in angiogenesis and metastasis. Activation of STAT5 occurs through dysregulation of growth factor receptor tyrosine kinases (TK) (including EGFR TK mutations), non-receptor TKs or Janus kinases. Dvl-3, a critical mediator of the Wingless-type (Wnt) signaling pathway, contributes to self-renewal of stem cells. Dicer has a key role in processing microRNAs required for stem cell regulation. We assessed mRNA expression of STAT5, Dvl-3 and Dicer in early-stage NSCLC. Methods: cDNA was derived from paraffin-embedded primary tumors of 47 surgically resected, early-stage NSCLC patients. Real-time quantitative reverse transcriptase PCR was used to measure STAT5, Dvl-3 and Dicer mRNA levels relative to the internal reference gene β-actin. Results: Median expression levels were: STAT5, 0.66 (0.15–6.03); Dvl-3, 3.69 (1.02–25.83); Dicer, 4.53 (1.22–21.96). STAT5 levels for squamous cell carcinoma (SCC) were 0.57 vs 0.89 for non-SCC (P=0.01), while Dicer levels were higher in SCC (4.25) than in non-SCC (2.60) (P = 0.01). A strong correlation between the levels of Dvl-3 and Dicer was found (rho = 0.49; P < 0.001). 90% of females had low Dicer mRNA levels (P = 0.01). No other correlation between mRNA levels and patient characteristics was found. Conclusions: High levels of Dvl-3 are associated with SCC. The close correlation between Dvl-3 and Dicer levels may indicate that Dicer could process a class of microRNA that activates the Wnt pathway. In non-SCC, overexpression of STAT5 could be involved in tumor angiogenesis. These 3 transcripts could be potential predictors of metastasis and ideal targets for individualized treatment. No significant financial relationships to disclose.

scholarly journals The hypoferremic response to acute inflammation is maintained in thalassemia mice even under parenteral iron loading

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11367
Author(s):  
Chanita Sanyear ◽  
Buraporn Chiawtada ◽  
Punnee Butthep ◽  
Saovaros Svasti ◽  
Suthat Fucharoen ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Serum Iron ◽  
Acute Inflammation ◽  
Iron Homeostasis ◽  
Mrna Levels ◽  
Iron Dextran ◽  
Iron Loading ◽  
Wild Type ◽  
Altered Expression ◽  
Parenteral Iron

Background Hepcidin controls iron homeostasis by inducing the degradation of the iron efflux protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression is influenced by multiple factors, including iron stores, ineffective erythropoiesis, and inflammation. However, the interactions between these factors under thalassemic condition remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of iron homeostasis to acute inflammatory induction by lipopolysaccharide (LPS) in thalassemic (Hbbth3/+) mice with/without parenteral iron loading with iron dextran. Methods Wild type and Hbbth3/+ mice were intramuscularly injected with 5 mg of iron dextran once daily for two consecutive days. After a 2-week equilibration, acute inflammation was induced by an intraperitoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inflammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and Hbbth3/+ mice were analyzed and compared by Kruskal–Wallis test with pairwise Mann–Whitney U test. Results We found the inductive effects of LPS on liver IL-6 mRNA expression to be more pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic protein 6 (BMP6) mRNA levels were decreased under both control and parenteral iron loading conditions. Despite the altered expression of the aforementioned hepcidin regulators, the stimulatory effect of LPS on hepcidin mRNA expression was blunt in iron-treated Hbbth3/+ mice. Contrary to the blunted hepcidin response, LPS treatment suppressed FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels of Hbbth3/+ mice with parenteral iron loading. Conclusion Our study suggests that a hypoferremic response to LPS-induced acute inflammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-independent manner.

scholarly journals Purification and characterization of the Fas-ligand that induces apoptosis.

1994 ◽  
Vol 179 (3) ◽  
pp. 873-879 ◽  
Author(s):  
T Suda ◽  
S Nagata
Keyword(s):  
Cell Surface ◽  
Cytotoxic Activity ◽  
Fas Ligand ◽  
Cytotoxic T Lymphocyte ◽  
Growth Factor Receptor ◽  
Chimeric Protein ◽  
Surface Protein ◽  
Target Cells ◽  
Cell Surface Protein ◽  
A Cell

Fas is a 45-kD cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family, and transduces the signal for apoptosis. The cytotoxic T lymphocyte (CTL) hybridoma, PC60-d10S requires the presence of Fas on target cells to induce cytolysis in target cells. This CTL cell line was weakly but specifically stained by a chimeric protein that consisted of the extracellular domain of mouse Fas and the Fc portion of human immunoglobulin G1 (mFas-Fc). Moreover, mFas-Fc inhibited the cytotoxic activity of PC60-d10S. Sublines of d10S that were stained intensively by mFas-Fc were isolated by repetitive fluorescence-activated cell sorter sorting. A cell-surface protein of about 40 kD was specifically precipitated by mFas-Fc from the lysates of these sublines. This protein was homogeneously purified by sequential affinity chromatographies using mFas-Fc and concanavalin A beads. The purified protein exhibited cytotoxic activity against cells expressing Fas but not to the cells which do not express Fas. These results indicated that the 40-kD membrane glycoprotein expressed on PC60-d10S cells is the Fas-ligand that induces the apoptotic signal by binding to Fas.

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