Category Archives: Growth Factor

Differential Distribution of Binding Sites: RESULTS(3)

Cross-linking experiments with basal plasma membranes revealed that l25I-IGF-II bound mainly to the 250-kDa band (C) and to a lesser extent to the 135-kDa (E) and 130-kDa (F) bands. 125I-IGF-II bound to the 250-kDa band was displaced by 200 ng (0.1 |xM) IGF-II but not by IGF-I or insulin (Fig. 2). The l25I-IGF-II bound to the 135-kDa band was displaced by 200 |xg IGF-II and IGF-I, and was completely displaced by 10 (xg (6.7 |xM) insulin. 125I-IGF-II bound to the 130-kDa band was displaced by 200 pug IGFII, IGF-I, and 10 |xg (6.7 jxM) insulin.

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Differential Distribution of Binding Sites: RESULTS(2)

RESULTS(2)

The concentration of IGF-II needed to displace 50% of bound 125I-IGF-II was 0.8 ± 0.2 nM; the IC50 of the Ser29 IGF-II variant was 2.2 ± 0.3 nM, and IGF-I was nearly as effective (IC50: 3.2 ± 0.7 nM). Insulin also displaced 125I-IGF-II but less effectively (/C50: 40 ± 5.0 nM) (Table 1).

The Kd values for the microvillous membranes differed significantly from those for the basal plasma membrane (p < 0.0001), as did the maximum binding of 125I-IGF-II (p < 0.0001).

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Differential Distribution of Binding Sites: RESULTS(1)

Binding Studies

The microvillous membranes bore saturable binding sites for IGF-II. Scatchard analysis revealed a single class of binding sites with a Kd of 0.51 ± 0.04 nM and a maximum binding capacity (Bmax) of 4.4 ± 0.3 pmol/mg protein, with a correlation coefficient of r = 0.90 and a Hill number of 0.84 (n = 5).

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Differential Distribution of Binding Sites: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5)Data Analysis

Results are expressed as the mean ± SEM of at least three single values obtained in separate experiments performed in duplicate. The n values refer to the number of independent membrane preparations from distinct human placentae. The apparent dissociation constant (Kd) and the maximal number of binding sites (Bmax) were obtained from Scatchard and Hill representations. IC50 is the concentration of the antagonist that inhibits labeled ligand-specific binding by 50%. buy antibiotics online

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Differential Distribution of Binding Sites: MATERIALS AND METHODS(4)

Bound IGF-II was precipitated by addition of 1.25 ml 13% polyethylene glycol 6000 in 50 mM Tris-HCl, pH 7.4, plus 0.05% carrier gamma globulins to each tube, and the bound and free materials were separated by centrifugation at 4500 X g for 30 min at 4°C. The supernatant was aspirated off, and the radioactivity in the pellet, membrane-bound 125I-IGF-II, was measured in an Intertechnique gamma counter (Kontron/Intertechnique, St-Quentin-en-Yve-lines, France). Nonspecific binding was determined by incubating 125I-IGF-II with binding buffer without membrane. The results were the same as the nonspecific binding values determined by incubating membranes with excess IGF-II plus the radioligand. Nonspecific binding was 5-8% of total counts (total binding). To perform saturation experiments, increasing amounts of labeled IGF-II (0.002-50 nM) were incubated with 20 (xg membrane protein at 4°C overnight.

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Differential Distribution of Binding Sites: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3)

IGF-Reiated Peptides

Human recombinant (rh) IGF-I and rhIGF-II were obtained from Genzyme (Boston, MA), and bovine pancreatic insulin was from Sigma Chemical Company (St. Louis, MO). The IGF peptides were iodinated by the chloramine T method and separated from aggregated material and free 125-iodine (Amersham, England) on a Sephadex G50 column (Pharmacia LKB, Uppsala, Sweden) eluted with 0.1 M NH4HCO3 containing 0.1% (w:v) IGF-free bovine BSA. The 125I-IGF-specific activity was 80-140 Ci/|xg (6001050 Ci/mmol). Most of the IGF-II used in this study was prepared from an acid placental extract obtained from IME-DEX (Chaponost, France), as was the natural Ser29 IGF-II variant. buy asthma inhalers

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Differential Distribution of Binding Sites: MATERIALS AND METHODS(2)

Preparation of the Syncytiotrophoblast Basal-Side Plasma Membranes

Basal plasma membrane was isolated from human placental trophoblast by a modification of the procedure of Kelley et al. . Villous tissue was cut into small fragments and washed with Earle’s Balanced Salt Solution to remove blood. It was then cut into pieces (1-5 mm3), washed several times with ice-cold 50 mM Tris-HCl (pH 7.4), and collected on nylon mesh (100-|xM pore size). Except where noted, all further steps were performed at 4°C. The tissue (20 g) was cooled in an ice-ethanol bath and sonicated at setting 7 for 20 sec (Branson Ultrasonic Corp. [Danbury, CT] sonicator) in 100 ml 50 mM Tris-HCl buffer, pH 7.4. The sonicated tissue was collected on a nylon mesh, washed three times with 5 mM Tris-HCl (pH 7.4), and stirred in 5 vol 5 mM Tris-HCl buffer (pH 7.4) for 30 min to remove the trophoblast cytoplasm and the microvillous membrane bathing the maternal blood space. The tissue was washed with the same buffer, collected on a nylon mesh, and incubated for 30 min at room temperature in 0.25 mM sucrose, 10 mM EDTA, 50 mM Tris-HCl, pH 7.4. asthma inhalers

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Differential Distribution of Binding Sites: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)

Placenta

Placentae were obtained immediately after elective cesarean section from healthy mothers. The cesarean section was performed without labor under thiopental sodium-suc-cinylcholine anesthesia in the 39th week of pregnancy because of earlier diagnosed cephalopelvic disproportion. No preoperative medication except atropine was given. buy prednisone

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Differential Distribution of Binding Sites for 125I-Insulin-Like Growth Factor II on Trophoblast Membranes of Human Term Placenta: INTRODUCTION(2)

IGF-I has been identified in a variety of fetal tissues, while IGF-II is an important determinant of fetal growth; and IGF receptors of high affinity have been found in many fetal tissues. The human placenta produces and secretes IGFs, which may regulate placental growth throughout gestation. IGF-I and IGF-II stimulate a wide array of reactions in cells, ranging from regulating nutrient transport to stimulating cell replication and inducing cell differentiation. buy asthma inhaler

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Differential Distribution of Binding Sites for 125I-Insulin-Like Growth Factor II on Trophoblast Membranes of Human Term Placenta: INTRODUCTION(1)

 INTRODUCTION(1)

The hemochorial human placenta forms the interface between the maternal and fetal tissues. It changes during pregnancy to become a hemomonochorial-type placenta that provides optimal feto-maternal exchanges. At term, the chorial villi are bathed in the maternal blood in the intervillous space, which is delineated by two epithelia: the maternal decidua and the syncytiotrophoblast, a fetal differentiated, multinucleated epithelium derived from undifferentiated, mononucleated cytotrophoblast cells. The syncytiotrophoblast is bounded by two polar membranes, the basal plasma membrane facing the fetus and the microvillous brush-border membrane facing the mother. These two membranes have different structures, biochemical compositions, and functions, in particular, transport processes and receptor contents. flovent inhaler

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