Ultracentrifugal and Electrophoretic Studies on Fetuin.

Pedersen, Kai O.

doi:10.1021/j150451a012

Cited by 100 publications

(33 citation statements)

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“…The concentration was nearly optimal; however, at 2 mg/ml, a slight increase (5%) in cell attachment was observed (unpublished results). These concentrations are within the concentration range of fetuin in medium containing 10% fetal calf serum (1-2 mg/ml) (21). The optimal concentrations of insulin and transferrin were 10 ,gg/ml and 5 gg/ml, mally to insulin and transferrin at similar concentrations (11,12).…”

Section: Methodsmentioning

confidence: 63%

Growth of embryonal carcinoma cells in serum-free medium.

Rizzino¹,

Sato²

1978

Proc. Natl. Acad. Sci. U.S.A.

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Two mouse embryonal carcinoma cell lines, PCC.4 aza-1 and F9, have been grown in serum-free F-12 medium supplemented with Pedersen fetuin, insulin, transferrin, and 2-mercaptoethanol. This medium supports longIterm growth of both cell lines. When these cells are transferred from medium containing serum to this serum-free medium, growth continues without any detectable lag. PCC.4 aza-1 grown in this medium for over 20 generations retains the capacity to differentiate in vivo. This medium appears to be a general serum-free medium for the growth of emb onal carcinoma cells. Little is known about the hormonal requirements of early mammalian development. One approach to this problem has been the cultivation of mouse embryos in a variety of defined media. Unfortunately follicle stimulating hormone, triiodothyronine, and parathyroid hormone (11, 12). These results argue that one of the major roles of serum in cell culture is to provide hormones. Furthermore, these studies suggest that it is feasible to develop serum-free media for other cell lines, including embryonal carcinoma cells. In this article we report that two different embryonal carcinoma cell lines (PCC.4 aza-1 and F9) grow for extended periods of time in a serum-free medium supplemented with insulin, transferrin, 2-mercaptoethanol, and Pedersen fetuin. PCC.4 aza-1 cells maintained in this serum-free medium retain the ability to differentiate in vivo. METHODS AND MATERIALSCell Culture. PCCA4 aza-1 and F9 were obtained from M. Sherman at the Roche Institute of Molecular Biology. These cells were maintained on Falcon tissue culture flasks and kept under a mixture of 5% CO2 in air. The medium was composed of 45% Dulbecco's modified Eagle's medium (Gibco HG-21), 45% nutrient mixture F-12 (Gibco H-17), and 5% each horse and fetal calf serum. When cells were switched from serumcontaining to serum-free medium, the cells were briefly washed with serum-free F-12 medium. This was done by first removing the serum-containing medium from stock plates and replacing it with serum-free F-12 medium. This medium was then removed, and fresh F-12 medium was added. Cells were removed from the plates by pipetting the medium up and down over the cells with a sterile pasteur pipette. When cells were grown for prolonged periods in EM-1 medium (F-12 medium containing 500 ,g of fetuin per ml, 10 1Ag of insulin per ml, 5 ug of transferrin per ml, and 10,uM 2-mercaptoethanol), the medium was changed nearly every day. Cell growth was determined by counting cells with a Coulter counter.Preparation of Serum-Free Growth Media. F-12 medium was prepared from powdered nutrient mixture F-12

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Section: Methodsmentioning

confidence: 63%

Growth of embryonal carcinoma cells in serum-free medium.

Rizzino¹,

Sato²

1978

Proc. Natl. Acad. Sci. U.S.A.

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“…One-fifth of every other fraction was used for soft agar assays on AKR-2B cells (e) and one-fifth fractions were supplemented with EGF at 2 ng/ml and assayed on NRK cells (0). Protein content per tube (x) was determined by the dye-binding assay (12).…”

Section: Resultsmentioning

confidence: 99%

“…Soft agar assays using nontransformed anchorage-dependent mouse AKR-2B and rat NRK cells were performed as described (6,10). Colonies were quantitated after [5][6][7][8][9][10][11][12][13][14] To test for stimulation of colony formation by known growth factors, the following factors were added to the soft agar assay in the concentration ranges indicated: EGF, 1-800 ng/ml; multiplication-stimulating activity (Collaborative Research, Wal-tham, MA), 1-100 ng/ml; bovine insulin (Sigma), 1 ng/ml to 1 mg/ml; somatomedin C (obtained from Judson Van Wyk, University of North Carolina at Chapel Hill), 1-100 ng/ml; and PDGF (obtained from W. Jackson Pledger, University of North Carolina at Chapel Hill), 1-100 units/ml. Serum and Plasma.…”

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confidence: 99%

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Serum contains a platelet-derived transforming growth factor.

Childs¹,

Proper²,

Tucker³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

273

112

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High concentrations of fetal bovine serum induced colony formation in soft agar by anchorage-dependent, nontransformed mouse AKR-2B and rat NRK cells. The colony-stimulating activity in fetal bovine serum was precipitated by 45% saturated ammonium sulfate and migrated in molecular sieve chromatography as a single peak of activity in the 10,000-15,000 molecular weight range. The colony-stimulating activity was heat and acid stable and was destroyed by trypsin and dithiothreitol, indicating the activity is due to a polypeptide that requires disulfide bonds for biologicalactivity. No competition for binding to the epidermal growth factor receptor was associated with the colonystimulating activity. Isoelectric focusing revealed activity in the pI 4-5 range. The colony-stimulating activity in serum appeared to be of platelet origin because platelet-poor plasma and plateletpoorplasma-derived serum containedlittle activity, whereas acid/ ethanol extracts ofbovine and human platelets had potent colonystimulating activity. Chromatography of platelet extracts on BioGel P-60 revealed peaks of AKR-2B colony-stimulating activity in the 12,000 and 20,000 molecular weight ranges. The other biological and chemical properties of the platelet colony-stimulating activity were the same as those for the serum activity. The data indicate the presence in serum of a platelet-derived growth factor(s) with properties similar to those of the transforming growth factors.Cells that are normally anchorage dependent can be stimulated to form colonies when suspended in semisolid medium in the presence of higher than usual concentrations of serum. O'Neill et al. (1) reported that anchorage-dependent NIL-8 hamster fibroblasts exhibited a serum concentration-dependent growth in soft agarose and that the rate of growth of attached and suspended cells was approximately the same when the concentration of fetal bovine serum was raised to 66%. Peehl and Stanbridge (2) found that normal human fibroblasts would grow suspended in methylcellulose in the presence of 20% fetal bovine serum but not in medium with 10% fetal bovine serum; this anchorage-independent growth was enhanced by the addition of hydrocortisone.

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“…897 ing protein as thyroxine binding fetal protein (TBFP) and preliminary studies indicate that it is, in fact, fetuin, an a-1-glycoprotein unique to fetal and neonatal serum of several species, including the calf, foal, sheep, pig, and chick [10]. Fetuin has a molecular weight of about 50,000 [16]. The T4-binding affinity of fetuin is less than that of TBG and the maximal binding capacity is very high (>600 ^g/100 ml) [10].…”

Section: Resultsmentioning

confidence: 99%

Thyroxine and Triiodothyronine Metabolism in Maternal and Fetal Sheep

et al. 1972

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ExtractKinetic studies of thyroxine (T4) and triiodothyronine (T3) and measurements of hormone turnover were conducted in chronically catheterized maternal and fetal sheep during the last trimester of gestation. Mean serum total T4 and free T4 (FT4) concentrations were higher in fetal than in maternal serum (7.5 versus 5.4 /ig/100 ml and 5.1 versus 2.4 ng/100 ml, respectively), whereas total T3 and free T3 (FT3) concentrations were higher in maternal than in fetal serum (74 versus <18 ng/100 ml and 176 versus <90 pg/100 ml, respectively). Mean maximal thyroxine-binding globulin (TBG) binding capacity was higher in maternal than in fetal blood (16.5 versus 8.1 ng T4/100 ml, respectively). Fetal serum was observed to contain a T4-binding a-1-globulin, which probably is identical with fetuin, a glycoprotein unique to sera of fetuses and neonates of several species including the calf, foal, sheep, pig, and chick. Mean T4 clearance values were 0.614 and 0.107 liters/kg/24 hr, respectively, in fetal and maternal sheep and mean T3 clearance values were 8.52 versus 1.66 liters/kg/24 hr, respectively. Mean T4 turnover values were 46.1 and 5.59 ^g/kg/24 hr, and mean T3 turnover values were < 1.50 and 1.22 Mg/kg/24 hr in fetal and maternal sheep, respectively. No placental transfer of T4 occurred and net maternal to fetal transfer of T3 amounted to only about 1% of total turnover of fetal thyronine.These data confirm the autonomy of the fetal hypothalamic-pituitary-thyroid axis and they indicate that hormone utilization is much higher in fetal sheep (per kilogram body weight) than in maternal sheep. In addition, the higher T4/T3 serum concentration ratio and T4/T3 turnover ratios in the fetus (>471/1 versus 74/1 and >31/1 versus 4.6/1) indicate that the relative rates of T3 secretion and/or T3 conversion from T4 in peripheral tissues are less in fetal than in maternal sheep. Finally, the data indicate a poor correlation between free hormone concentrations and either hormone clearance or hormone turnover rates, which suggests tissue binding and/or metabolism are important determinants of thyroid hormone turnover in the fetus. SpeculationThe present data regarding thyroid hormone metabolism in fetal sheep are consistent with present evidence about thyroid hormone metabolism in the human fetus and suggest that the sheep can serve as a useful model for the human system. Furthermore, data in both species support the concept of autonomy of fetal thyroid function. The ob- 894Thyroxine and triiodothyroxine metabolism in sheep 895 servation of high T4/T3 concentration and turnover ratios in the fetus was unexpected and might be explained on the basis of a high T4/T3 secretion ratio, a low rate of conversion of T4 to T3, or a high rate of biliary T4 excretion in the fetal sheep.

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Ultracentrifugal and Electrophoretic Studies on Fetuin.

Cited by 100 publications

References 0 publications

Growth of embryonal carcinoma cells in serum-free medium.

Growth of embryonal carcinoma cells in serum-free medium.

Serum contains a platelet-derived transforming growth factor.

Thyroxine and Triiodothyronine Metabolism in Maternal and Fetal Sheep

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