scholarly journals Purification and molecular cloning of prostacyclin-stimulating factor from serum-free conditioned medium of human diploid fibroblast cells

1994 ◽  
Vol 303 (2) ◽  
pp. 591-598 ◽  
Author(s):  
T Yamauchi ◽  
F Umeda ◽  
M Masakado ◽  
M Isaji ◽  
S Mizushima ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Sequence Similarity ◽  
Reversed Phase ◽  
Affinity Column ◽  
Fibroblast Cells ◽  
Human Diploid Fibroblast ◽  
Aortic Endothelial Cells ◽  
Diploid Fibroblast ◽  
Sds Page ◽  
Stimulating Factor

We attempted to identify the factor that stimulated prostacyclin (PGI2) production using conditioned medium from cultured human diploid fibroblast cells subjected to a series of purification steps using h.p.l.c. on DEAE-5PW, Heparin-5PW, Protein-Pak 300, and an insulin-like growth factor-1 ligand affinity column. The purified prostacyclin-stimulating factor (PSF) ran as a single band with a molecular mass of 31 kDa by SDS/PAGE. Analysis of the purified PSF by C4 reversed-phase h.p.l.c. showed a single sharp peak in 31% (v/v) acetonitrile. The material was purified 8000-fold with an overall yield of about 18%. The purified PSF stimulated PGI2 production by cultured bovine aortic endothelial cells at a concentration of about 10 ng/ml; maximal stimulation was achieved at a concentration of 25 ng/ml. A cDNA coding for PSF was cloned and sequenced, revealing an apparently novel protein with no obvious sequence similarity to known proteins.

scholarly journals Human platelet glycoprotein IIIb binds to thrombospondin fragments bearing the C-terminal region, and/or the type I repeats (CSVTCG motif), but not to the N-terminal heparin-binding region

1992 ◽  
Vol 284 (1) ◽  
pp. 231-236 ◽  
Author(s):  
B Catimel ◽  
L Leung ◽  
H el Ghissasi ◽  
N Mercier ◽  
J McGregor
Keyword(s):  
Plasmodium Falciparum ◽  
Sequence Similarity ◽  
Terminal Region ◽  
Platelet Glycoprotein ◽  
Affinity Column ◽  
Type I ◽  
Heparin Binding ◽  
Western Blots ◽  
Reducing Conditions ◽  
Sds Page

Major blood membrane platelet glycoprotein IIIb (GPIIIb), also termed GPIV or CD365, has been identified as a receptor for thrombospondin (TSP), collagen and Plasmodium falciparum-infected erythrocytes. The aim of the present study was to identify region(s) of TSP involved in binding of GPIIIb. Proteolytic fragments of TSP (M(r) 140 kDa, 120-18 kDa and 27 kDa on SDS/PAGE under reducing conditions) were purified by f.p.l.c. and identified by N-terminal gas-phase sequencing, e.l.i.s.a. and Western blots using monoclonal antibodies directed against defined domains of TSP. The 140 kDa and 120-18 kDa fragments (C-terminal region), but not the 27 kDa fragment (N-terminal region), were shown to bind to GPIIIb by using e.l.i.s.a. and affinity-chromatography systems. TSP binding to a GPIIIb-affinity column was Ca(2+)-dependent and reduced by 45% in the presence of EDTA. Moreover, TSP was only partially eluted with EDTA from a Ca(2+)-equilibrated GPIIIb column. A fragment of 68 kDa, obtained by further digestion of the 140 kDa fragment, bound to the GPIIIb-Sepharose affinity column. This fragment, or stalk-like region, bears the TSP type I repeats that show sequence similarity to regions on properdin, Plasmodium falciparum proteins and antistasin. Peptides (CSVTCG or SVTCGGGV) representing these repeats bound isolated GPIIIb in a Ca(2+)-independent way, but did not completely inhibit the GPIIIb and TSP interaction. These studies indicate that GPIIIb binds to the TSP via the C-terminal region and/or the CSVTCG motif, but not to the N-terminal region. Interaction between GPIIIb and the TSP C-terminal region or the CSVTCG motif is respectively Ca(2+)-dependent and -independent.

Maintenance of human cytomegalovirus genome in human diploid fibroblast cells

Virology ◽  
1983 ◽  
Vol 130 (1) ◽  
pp. 269-271 ◽  
Author(s):  
Linda J. Bucher ◽  
Brian Wigdahl ◽  
Fred Rapp
Keyword(s):  
Human Cytomegalovirus ◽  
Fibroblast Cells ◽  
Human Diploid Fibroblast ◽  
Diploid Fibroblast

scholarly journals Improved glycine-extracted complement-fixing antigen for human cytomegalovirus

1977 ◽  
Vol 6 (6) ◽  
pp. 647-649
Author(s):  
J D Kettering ◽  
N J Schmidt ◽  
E H Lennette
Keyword(s):  
Human Cytomegalovirus ◽  
High Multiplicity ◽  
Fibroblast Cells ◽  
Human Diploid Fibroblast ◽  
Glycine Buffer ◽  
Diploid Fibroblast

High-titered, sensitive, and stable complement-fixing antigens for human cytomegalovirus were consistently produced from human diploid fibroblast cells infected at a high multiplicity of virus, harvested after 7 days of incubation, and sonically treated immediately in 0.1 M glycine buffer, pH 9.5.

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