- Email: [email protected]
CSA production in murine long term bone marrow culture after “in vitro” treatment with AM5
Ceil Biology international
Reports, Vol. 14, Abstracts Supplement
MITOGENIC EFFECTS OF BRYOSTATIN 2 IL-3 DEPENDENT CELLS. ON Hagkovec, Mike Lilly', Andrew Inst.Hematol.Blood Transfusion, Kraft3, USA, Prague CSFR, =VA Med.Ctr.,Seattle, sUniv.Alabama, Birmingham, USA. Brvostatins exhibit variety of effects For example, they are on animal cells. mitoqenic for lymphocytes and fibroblasts and they stimulate hematopoietic progenitors. At a molecular level they are stimulators of protein kinase C (PK-C). In our study effects of the bryostatin 2 (Bryo) on in vitro thymidine incorporation of IL-3-dependent mouse cell lines were compared with the effects of rm IL-3, rm GM-CSF and phorbol 12-myristate 13-acetate (PMA). evaluation of the Semiquantitative mitogenic effects: IL-3 Bryo PMA GM-CSF Cells/Stimulator: ++ ++ ++ ++ FDCP-1 4-t + + FDCP-2 ++ + + +++ FDCP-2-1D
1990
97
p73 Cedrik
D-32
++
+
+
-
+ N.T. NFS-60 The table shows that Bryo (lo-*-lo-'M) the lines. mitogenic for most of is Moreover,it shows a very similar effects and PMA (both Bryo stimulators of of with PK-C) does not correlate which the effects of IL-3 and GM-CSF. Preliminary results showed a partial down regulation of PX-C in Bryo-stimulated FDCP-1 cells, which was detected in the western immunoblot.
SOMECONNSGTIONS BRvBIiW CRLLULARACRVI'I'Y MID REDOX CHARACTERISTICS Cristlnsl V. Z&d. Cuelia P&n. Ion I. B%a Center sf Blelegie~l~Remmh, Iaai 6600, ROHMIA The reots of Seealo corealo naintainod in dfffor-t d mditimm, l emred by phjnieal teehnlquea /l/, ware mbjeated to difforont mrlyaia at the naaloar, ~011~1~ and tlamlor 1.~~1~ (the l hremosemial l borretiens fnquenoy (CA) the mlforob tie 1nd.x (DC) ahd th. lot&h of reotm t i)), peetiroly. At an optimum value of r1 for derolepmnt of tlssmo, the ehremesemi~l aborratlrns fnqueney ia the loweat.Uhm the ??I valao is different but near l ptlmm, the ebremeaemial l berratiens frequmand, aa a aempm.sation,the eel16 dll y ineruees ririm rate ia hider. Thla eerrelatien deereases when the rR values are meugh far frar l ptimm.Ie those eases, the @alla divlmim rate la lewor and tissue grerth rat0 i8 elenr. 1. &se&i C. V. l t all., Ror. reum. bioohim., 24, 4, 197, p. 357-360 I I
P75
Almudena Real,Juan A. Bueren,Gabriel Maganto. CIBMAT.Radrid.SPAIN We have previously reported that “in vitro” treatment with At45,a polysaccharide-protein complex,stimulates the hemopoiesis in murine Long Term Bone narrow Cultures (LTRUC). In order to determine whether the hemopoietic stimulation induced with Al45 was mediated by the release of Colony Stimulating Factors (CSPs),we analysed the presence of CSA in the supernatant of these cultures. LTBRCs were stablished according to the technique described by Uexter.Three weeks after the stablishment,different doses of AR5 were added,and seven days later the supernatants were collected. Reasurements of CSA in the supernatants were performed by assessing CPU-GWcolony formation when bone marrow cells were cultured in the presence of different concentrations of supernatants.As a positive control A&conditioned medium was used. The supernatant of LTBHC treated with AR5 induced the formation of CPU-GRcolonies in ausence of exogenous CSPs.The number of CPU-GRdepended on the concentration of supernatant added.Uepending on the dose of AR5 administrated to the LTBRC,the CSA production varied,being higher when the higher dose of AR5 was added. In contrast,no CPU-GR formation occur when supernatants from control LTRWCwere added, even when the concentration used was 30% of a 5 times concentrated supernatant. The results showed that AR5 induce,in a dose dependent qanner,the production of CSA in LTRRCs, what support the hypothesis that AR5 induce hemopoietic stimulation via CSFs production.
CHARACTERIZATION OF THE RECEPTORS TO VASCULOTROPIN ON ENDOTHELIAL AND EPITHELIAL CELLS. Jean Plouist, Hafiia Moukadiri, U 88 INSERM, Paris.
P76
An endothelial cell growth factor has been purified from the conditionned medium of the AtT2g pitukary cell line. It has been characterized as an homodller composed of two subunits wtth Mr of 23 kDa. Since it was angicgenic in vivo and had so far a unique specifW for vascular endothsllal cells in vitro, it Was named v&culotropin. 1251 labelled vaaculotropin binds in a saturable. specific and reversible manner to cell memtxanes. ScGctiard’s analysis revealed two binding sites with apparent Kd of 4 and W-70 pM with respectively 600 and 4000 sites per capillary-derived endothelial cell whereas crosslinking experiments leaded to the detection of a major molecular species of Mr 180 kDa and a minor one of 100 kDa. It was also detected receptors for vascubtropin on non responsive cells such as lens epithefial and cornea1 endothelium cells allthough Scatchard’s analysis showed a single binding site with an apparent Kd of 10 pM. These results suggest that the receptor involved in the mitogenic pathway might be represented by the one binding to cell membranes with the lower affinity.
Reports, Vol. 14, Abstracts Supplement
MITOGENIC EFFECTS OF BRYOSTATIN 2 IL-3 DEPENDENT CELLS. ON Hagkovec, Mike Lilly', Andrew Inst.Hematol.Blood Transfusion, Kraft3, USA, Prague CSFR, =VA Med.Ctr.,Seattle, sUniv.Alabama, Birmingham, USA. Brvostatins exhibit variety of effects For example, they are on animal cells. mitoqenic for lymphocytes and fibroblasts and they stimulate hematopoietic progenitors. At a molecular level they are stimulators of protein kinase C (PK-C). In our study effects of the bryostatin 2 (Bryo) on in vitro thymidine incorporation of IL-3-dependent mouse cell lines were compared with the effects of rm IL-3, rm GM-CSF and phorbol 12-myristate 13-acetate (PMA). evaluation of the Semiquantitative mitogenic effects: IL-3 Bryo PMA GM-CSF Cells/Stimulator: ++ ++ ++ ++ FDCP-1 4-t + + FDCP-2 ++ + + +++ FDCP-2-1D
1990
97
p73 Cedrik
D-32
++
+
+
-
+ N.T. NFS-60 The table shows that Bryo (lo-*-lo-'M) the lines. mitogenic for most of is Moreover,it shows a very similar effects and PMA (both Bryo stimulators of of with PK-C) does not correlate which the effects of IL-3 and GM-CSF. Preliminary results showed a partial down regulation of PX-C in Bryo-stimulated FDCP-1 cells, which was detected in the western immunoblot.
SOMECONNSGTIONS BRvBIiW CRLLULARACRVI'I'Y MID REDOX CHARACTERISTICS Cristlnsl V. Z&d. Cuelia P&n. Ion I. B%a Center sf Blelegie~l~Remmh, Iaai 6600, ROHMIA The reots of Seealo corealo naintainod in dfffor-t d mditimm, l emred by phjnieal teehnlquea /l/, ware mbjeated to difforont mrlyaia at the naaloar, ~011~1~ and tlamlor 1.~~1~ (the l hremosemial l borretiens fnquenoy (CA) the mlforob tie 1nd.x (DC) ahd th. lot&h of reotm t i)), peetiroly. At an optimum value of r1 for derolepmnt of tlssmo, the ehremesemi~l aborratlrns fnqueney ia the loweat.Uhm the ??I valao is different but near l ptlmm, the ebremeaemial l berratiens frequmand, aa a aempm.sation,the eel16 dll y ineruees ririm rate ia hider. Thla eerrelatien deereases when the rR values are meugh far frar l ptimm.Ie those eases, the @alla divlmim rate la lewor and tissue grerth rat0 i8 elenr. 1. &se&i C. V. l t all., Ror. reum. bioohim., 24, 4, 197, p. 357-360 I I
P75
Almudena Real,Juan A. Bueren,Gabriel Maganto. CIBMAT.Radrid.SPAIN We have previously reported that “in vitro” treatment with At45,a polysaccharide-protein complex,stimulates the hemopoiesis in murine Long Term Bone narrow Cultures (LTRUC). In order to determine whether the hemopoietic stimulation induced with Al45 was mediated by the release of Colony Stimulating Factors (CSPs),we analysed the presence of CSA in the supernatant of these cultures. LTBRCs were stablished according to the technique described by Uexter.Three weeks after the stablishment,different doses of AR5 were added,and seven days later the supernatants were collected. Reasurements of CSA in the supernatants were performed by assessing CPU-GWcolony formation when bone marrow cells were cultured in the presence of different concentrations of supernatants.As a positive control A&conditioned medium was used. The supernatant of LTBHC treated with AR5 induced the formation of CPU-GRcolonies in ausence of exogenous CSPs.The number of CPU-GRdepended on the concentration of supernatant added.Uepending on the dose of AR5 administrated to the LTBRC,the CSA production varied,being higher when the higher dose of AR5 was added. In contrast,no CPU-GR formation occur when supernatants from control LTRWCwere added, even when the concentration used was 30% of a 5 times concentrated supernatant. The results showed that AR5 induce,in a dose dependent qanner,the production of CSA in LTRRCs, what support the hypothesis that AR5 induce hemopoietic stimulation via CSFs production.
CHARACTERIZATION OF THE RECEPTORS TO VASCULOTROPIN ON ENDOTHELIAL AND EPITHELIAL CELLS. Jean Plouist, Hafiia Moukadiri, U 88 INSERM, Paris.
P76
An endothelial cell growth factor has been purified from the conditionned medium of the AtT2g pitukary cell line. It has been characterized as an homodller composed of two subunits wtth Mr of 23 kDa. Since it was angicgenic in vivo and had so far a unique specifW for vascular endothsllal cells in vitro, it Was named v&culotropin. 1251 labelled vaaculotropin binds in a saturable. specific and reversible manner to cell memtxanes. ScGctiard’s analysis revealed two binding sites with apparent Kd of 4 and W-70 pM with respectively 600 and 4000 sites per capillary-derived endothelial cell whereas crosslinking experiments leaded to the detection of a major molecular species of Mr 180 kDa and a minor one of 100 kDa. It was also detected receptors for vascubtropin on non responsive cells such as lens epithefial and cornea1 endothelium cells allthough Scatchard’s analysis showed a single binding site with an apparent Kd of 10 pM. These results suggest that the receptor involved in the mitogenic pathway might be represented by the one binding to cell membranes with the lower affinity.