메뉴 건너뛰기




Volumn 285, Issue 5428, 1999, Pages 736-739

Cell activation and apoptosis by bacterial lipoproteins through Toll- like receptor-2

Author keywords

[No Author keywords available]

Indexed keywords

IMMUNOGLOBULIN ENHANCER BINDING PROTEIN; LIPOPROTEIN; MEMBRANE RECEPTOR;

EID: 0033618630     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5428.736     Document Type: Article
Times cited : (1304)

References (35)
  • 3
    • 0029782133 scopus 로고    scopus 로고
    • M. V. Norgard et al., Infect. Immun. 64, 3845 (1996).
    • (1996) , vol.64 , pp. 3845
    • Norgard, M.V.1
  • 10
    • 0032541661 scopus 로고    scopus 로고
    • R. B. Yang et al., Nature 395, 284 (1998).
    • (1998) Nature , vol.395 , pp. 284
    • Yang, R.B.1
  • 12
    • 0032509295 scopus 로고    scopus 로고
    • A. Poltorak et al., Science 282, 2085 (1998).
    • (1998) Science , vol.282 , pp. 2085
    • Poltorak, A.1
  • 15
    • 0344760065 scopus 로고    scopus 로고
    • note
    • 3Cys was from Novabiochem. MLP (12) contained <25 pg of LP5 per milligram of protein. Unless indicated, other reagents were from Sigma.
  • 17
    • 0031757986 scopus 로고    scopus 로고
    • Base treatment was done as in V. Vidal et al., Nature Med. 4, 1416 (1998). The products were resolved by reversed-phase high-performance liquid chromatography with a C4 column (Vydac) and a linear acetonitrile gradient. Fractions were analyzed by electrospray ionization mass spectrometry. A fraction was isolated that contained (i) a compound that has lost two of the three palmitate moieties (molecular weight = 1032.6, major peak) and (ii) a compound that has lost the entire S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl] moiety (molecular weight = 924.7, minor peak). The concentration was determined by ultraviolet absorption.
    • (1998) Nature Med. , vol.4 , pp. 1416
    • Vidal, V.1
  • 20
    • 0344327473 scopus 로고    scopus 로고
    • note
    • mAb 2392 was generated by immunization of BALB/c mice with the purified, extracellular domain of hTLR2 (9). The specificity of mAb 2392 for hTLR2 was confirmed by the following methods: (i) An enzyme-linked immunosorbent assay which demonstrated that mAb 2392 reacted with hTLR2-Fc but not hTLR4-Fc. (ii) A flow cytometric analysis with 293 cells expressing gD epitope-tagged versions of hTLR2, hTLR4, and hTLR6. Although each cell line was labeled with mAb 5B6, which recognizes the gD epitope, only 293hTLR2 cells were labeled with mAb 2392. (iii) An immunoprecipitation (24), followed by protein immunoblotting with mAb 5B6, from the membrane extracts of 293 cells transiently transfected with gD epitope-tagged versions of hTLR2, hTLR1, hTLR4, and murine TLR2. mAb 2392 only precipitated hTLR2. However, equivalent amounts of each receptor were obtained when mAb 5B6 was used for the immunoprecipitation. Supplemental data illustrating the specificity of mAb 2392 can be seen at www.sciencemag.org/feature/data/1040361. shl.
  • 21
    • 0344759494 scopus 로고    scopus 로고
    • note
    • The test compounds were added to the 293 stable cell lines (9) in serum-free Dulbecco's modified Eagle's medium, 0.05% HSA, 5% fetal bovine serum (FBS). Cells were either photographed or stained with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) by using the Apoptosis Detection System (Promega) and analyzed by flow cytometry. At least 5000 events were counted per sample. Results are presented as specific apoptosis (%) determined as [(% TUNEL-positive cells in experimental - % TUNEL-positive cells in control)/(100 - % TUNEL-positive cells in control] × 100. The % TUNEL-positive cells in controls was always <5%.
  • 26
    • 0345189890 scopus 로고    scopus 로고
    • note
    • Plasmids pKRN and phTLR2 (9) were prepared with the Endotoxin-free Plasmid Maxi kit (Qiagen). 293 cells were transfected with the Effectene Transfection reagent (Qiagen). Thirty hours after transfection, an equal volume of diluent or diluent containing sBLP (200 ng/ml) was added. Thirty-six hours later the cells were stained with Annexin V-biotin (Pharmingen) and Streptavidin Tri-Color (Caltag) and analyzed by flow cytometry.
  • 27
    • 0344759493 scopus 로고    scopus 로고
    • note
    • THP-1 cells (American Type Culture Collection) were maintained in RPMI 1640, 10% FBS, penicillin, streptomycin, and 2 mM L-glutamine. Six hours after seeding THP-1 cells in RPMI 1640, the indicated compounds diluted in RPMI 1640, 0.05% HSA were added. Six hours later, cell death was quantified by a lactate dehydrogenase release assay (Promega). Where indicated, cells were first treated with 32 nM PMA for 6 hours or cotreated with cycloheximide (50 μg/ml). PMA and cytcoheximide diluents (dimethyl sulfoxide and ethanol) were not cytotoxic.
  • 35
    • 0345189888 scopus 로고    scopus 로고
    • note
    • We thank D. Littman, V. Dixit, C. Scharff, and J. Moss for critical reading of the manuscript, M. Garabedlan for discussions, and T. Neubert for mass spectrometry analysis. pCDNA3 and pGFP were a gift of the Littman Lab. A.O.A. was supported by a grant from the Ufe and Health Insurance Fund. This work was supported by grants from the National Institute of Allergy and Infectious Diseases (AI 37720-04 to A.Z. and AI-38394 to J.D.R.).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.