scholarly journals The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally.

1990 ◽  
Vol 10 (12) ◽  
pp. 6454-6459 ◽  
Author(s):  
T J Fahrner ◽  
S L Carroll ◽  
J Milbrandt
Keyword(s):  
Growth Factor ◽  
Molecular Mass ◽  
Pc12 Cells ◽  
Receptor Gene ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Carboxy Terminal

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.

The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally

1990 ◽  
Vol 10 (12) ◽  
pp. 6454-6459
Author(s):  
T J Fahrner ◽  
S L Carroll ◽  
J Milbrandt
Keyword(s):  
Growth Factor ◽  
Molecular Mass ◽  
Pc12 Cells ◽  
Receptor Gene ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Carboxy Terminal

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.

Mitogenesis by serum and PDGF is independent of PI degradation and PKC in VSMC

1989 ◽  
Vol 256 (4) ◽  
pp. C886-C892 ◽  
Author(s):  
M. Kihara ◽  
P. J. Robinson ◽  
S. H. Buck ◽  
R. C. Dage
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Growth Control ◽  
Calcium Ionophore ◽  
Rat Aorta ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Pi Turnover ◽  
Fetal Bovine ◽  
20 Nm

Capacities of serum, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) on phosphatidylinositol (PI) degradation and cell growth were compared in cultured vascular smooth muscle cells (VSMC) from rat aorta. The role of protein kinase C (PKC) in growth control was also evaluated using polymixin B, a selective inhibitor of PKC. Both dialyzed and nondialyzed fetal bovine serum (FBS) in concentrations from 2 to 20% stimulated [3H]thymidine incorporation into DNA and cell growth without producing corresponding increases in PI turnover. Moreover, both PDGF (40-160 ng/ml) and FGF (6.25-150 ng/ml) also stimulated mitogenesis, but PDGF was more effective although less potent. Mitogenic amounts of PDGF did not stimulate PI turnover, whereas a maximally mitogenic amount of FGF (50 ng/ml) did produce a slight increase. Polymixin B inhibited PKC activity (IC50, 32 microM) from these cells but failed to suppress DNA synthesis produced by 10% FBS or PDGF (50 ng/ml). However, it did suppress that by FGF (50 ng/ml). Angiotensin II (10(-11)-10(-7) M) and phorbol 12,13-dibutyrate (PDB, 1-20 nM) were not mitogenic in the presence or absence of insulin (10 micrograms/ml) or the calcium ionophore A23187 (0.25-4 microM), under serum-free conditions. Instead, PDB inhibited mitogenesis of cells maintained under 0.2% FBS or stimulated with insulin (10 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Human platelet fibrinogen: purification and hemostatic properties

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 808-815 ◽  
Author(s):  
TJ Kunicki ◽  
PJ Newman ◽  
DL Amrani ◽  
MW Mosesson
Keyword(s):  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Calcium Ionophore ◽  
Fluid Phase ◽  
Calcium Ionophore A23187 ◽  
Plasma Fibrinogen ◽  
Ionophore A23187 ◽  
Significant Difference

Abstract Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.

245 ANALYSIS OF CORTICAL GRANULE EXUDATE OBTAINED BY CHEMICAL OOCYTE ACTIVATION IN PIGS

2011 ◽  
Vol 23 (1) ◽  
pp. 221
Author(s):  
R. Romar ◽  
M. J. Izquierdo-Rico ◽  
H. Funahashi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Isolation And Identification

Cortical granules (CG) are clue organelles in the mammalian oocyte because once released, their content modifies the zona pellucida (ZP) and oolema, thus preventing polyspermy. However, research on putative CG proteins has progressed slowly because of the picogram amount of proteins contained in CG. Isolation and identification of CG contents in porcine oocytes would help to elucidate the molecular mechanism involved in blocking polyspermic fertilization. Our objective was to study the contents of CG from in vitro-matured (IVM) porcine oocytes, and to achieve this objective, CG exudate was collected after its release from chemically activated oocytes. Oocytes were subjected to IVM in porcine oocyte medium supplemented with 50 μM β-mercaptoethanol for 44 h. After the IVM period, the ZP was removed by protease treatment (0.5% pronase in PBS), and the ZP-free oocytes were activated with calcium ionophore A23187 (6.5 μM, 2 min) in a medium consisting of 114.06 mM NaCl, 3.20 mM KCl, 0.50 mM MgCl2·6H2O, 10.00 mM sodium lactate, 0.35 mM NaH2PO4, 5.00 mM glucose, 25.07 mM NaHCO3, and 8.00 mM calcium lactate·5H2O. After activation, oocytes were transferred to fresh medium without calcium ionophore and kept for 30 min to allow release of the CG content. After this time, medium containing the CG exudate was collected, as well as the activated oocytes, and both samples were stored at –80°C until analysis. Samples were thawed and the CG proteins were concentrated by centrifugation in 10-kDa centrifugal devices (Microcon, Millipore, Billerica, MA) following the manufacturer’s instructions. The CG exudates from activated oocytes (n = 300) and activated oocytes (n = 125) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In brief, 4% stacking and 12% separating gel was used and run using 25 mM Tris–0.2 M glycine buffer, pH 8.6, containing 0.1% SDS for 1.5 h at 150 V and room temperature. After electrophoresis, the gel was silver stained. Thirteen strong bands were identified in the CG exudate lane, with an approximate molecular mass from approximately 45 to 105 kDa. However, the lane for activated oocytes showed faint protein bands. The presence of well-defined bands in the CG exudate lane might correspond to different CG-derived proteins. These preliminary results show a new approach for studying CG content. Further proteomic analysis of the bands will help to describe specific proteins contained in these organelles, shedding light on the role of the cortical reaction in pigs. Supported by MEC and FEDER (AGL2009-12512-C02-01) and Okayama Universit R. R. was granted funding by JSPS (Ref. S-09210).

scholarly journals Human platelet fibrinogen: purification and hemostatic properties

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 808-815
Author(s):  
TJ Kunicki ◽  
PJ Newman ◽  
DL Amrani ◽  
MW Mosesson
Keyword(s):  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Calcium Ionophore ◽  
Fluid Phase ◽  
Calcium Ionophore A23187 ◽  
Plasma Fibrinogen ◽  
Ionophore A23187 ◽  
Significant Difference

Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.

Platelet-derived growth factor-stimulated c-myc RNA accumulation in MG-63 human osteosarcoma cells is independent of both protein kinase A and protein kinase C

1990 ◽  
Vol 10 (1) ◽  
pp. 184-192
Author(s):  
K K Frick ◽  
C D Scher
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Phosphodiesterase Inhibitor ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Platelet Derived Growth Factor ◽  
Ionophore A23187 ◽  
Simultaneous Treatment ◽  
Human Osteosarcoma Cells

Treatment of quiescent MG-63 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF) stimulates the rapid accumulation of c-myc RNA. We have now determined that a similar effect can be induced by cAMP. Treatment with forskolin (an activator of adenylate cyclase), IBMX (a phosphodiesterase inhibitor), PGE1, and isoproterenol stimulated accumulation of both cAMP and c-myc RNA, but no increase in either cAMP or c-myc RNA was seen with the inactive forskolin analog 1,9-dideoxyforskolin. Forskolin and IBMX acted synergistically in stimulating accumulation of both cAMP and c-myc RNA. However, three lines of evidence indicated that PDGF action is not mediated by cAMP. First, PDGF treatment caused no elevation of cAMP within 1 h, even in the presence of IBMX. Second, the kinetics of c-myc RNA elevation after treatment with PDGF or forskolin were similar, ruling out delayed onset of cAMP stimulation. Finally, simultaneous treatment with forskolin and the calcium ionophore A23187 enhanced the elevation of c-myc RNA levels; no such effect was seen with PDGF. We had previously shown that PDGF action is not affected by prior treatment of MG-63 cells with TPA, a treatment which desensitizes the c-myc response to TPA. Similarly, TPA pretreatment had minimal effect on forskolin or IBMX-induced c-myc expression. These data suggest that cAMP, phorbol esters, and PDGF act independently to stimulate c-myc RNA expression in MG-63 cells. However, nuclear runoff experiments and RNA half-life measurements demonstrated that PDGF, phorbol ester, and cAMP all act to increase the transcription of the MYC gene.

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