Transforming growth factor β in bovine milk: concentration, stability and molecular mass forms

1996 ◽  
Vol 151 (1) ◽  
pp. 77-86 ◽  
Author(s):  
M-L Rogers ◽  
C Goddard ◽  
G O Regester ◽  
F J Ballard ◽  
D A Belford
Keyword(s):  
Growth Factor ◽  
Molecular Mass ◽  
Transforming Growth Factor ◽  
Bovine Milk ◽  
Gel Filtration ◽  
Transforming Growth Factor Β ◽  
Exchange Chromatography ◽  
Epithelial Cell Line ◽  
Acid Activation ◽  
Chromatographic Procedure

Abstract Transforming growth factor β (TGF-β) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-β in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a mink lung epithelial cell line. Most of the TGF-β bioactivity in milk was found to be in a latent form, which was also retained in the whey fraction. After acid activation, the total TGF-β concentration was 4·3 ± 0·8 ng and 3·7 ± 0·7 ng TGF-β per ml of milk and cheese whey respectively. Cation-exchange chromatography at pH 6·5 was used to concentrate latent whey-derived TGF-β, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-β. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-β with a molecular mass of 80 kDa and a smaller peak at 600 kDa. Transient acidification of the cationic whey fraction prior to neutral gel filtration, or gel filtration under acidic conditions, released low molecular mass TGF-β from both high molecular mass peaks. Whey-derived TGF-β was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-β2, which accounted for over 85% of the TGF-β bioactivity in whey. All TGF-β activity in whey could be neutralised by a monoclonal antibody directed against TGF-β1, -β2 and -β3. The results suggest that the majority of TGF-β in bovine milk is present in a small latent complex. Journal of Endocrinology (1996) 151, 77–86

scholarly journals Structural characterization of the latent complex between transforming growth factor β1 and β1-latency-associated peptide

1996 ◽  
Vol 313 (1) ◽  
pp. 343-351 ◽  
Author(s):  
Grainne A. McMAHON ◽  
John D. DIGNAM ◽  
Larry E. GENTRY
Keyword(s):  
Growth Factor ◽  
Cho Cells ◽  
Transforming Growth Factor ◽  
Gel Filtration ◽  
Chinese Hamster ◽  
Transforming Growth Factor Β1 ◽  
Cd Spectroscopy ◽  
Exchange Chromatography ◽  
Lung Epithelial Cells ◽  
Tgf Β1

The formation of a non-covalent complex between mature transforming growth factor β1 (TGF-β1) and its pro region, the β1-latency-associated peptide (β1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian β1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes β1-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between β1-LAP and TGF-β1. The simian β1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-β1 on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with 125I-TGF-β1. Simian β1-LAP was purified to homogeneity by a combination of ammonium sulphate precipitation, gel filtration, dye ligand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa as determined by SDS/PAGE, which is greater than that determined for the transient expression of simian β1-LAP in COS-1 and for the simian precursor of TGF-β1 (pro-TGF-β1) in CHO cells, this major difference being due to more extensive glycosylation of β1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian β1-LAP indicates a mostly β-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-β1 and β1-LAP.

scholarly journals A Novel Transforming Growth Factor-β Receptor-interacting Protein That Is Also a Light Chain of the Motor Protein Dynein

2002 ◽  
Vol 13 (12) ◽  
pp. 4484-4496 ◽  
Author(s):  
Qian Tang ◽  
Cory M. Staub ◽  
Guofeng Gao ◽  
Qunyan Jin ◽  
Zhengke Wang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Light Chain ◽  
Transforming Growth Factor ◽  
Motor Protein ◽  
Transforming Growth Factor Β ◽  
Cytoplasmic Dynein ◽  
Epithelial Cell Line ◽  
Tgfβ Receptors ◽  
Tgfβ Receptor ◽  
Intermediate Chain

The phosphorylated, activated cytoplasmic domains of the transforming growth factor-β (TGFβ) receptors were used as probes to screen an expression library that was prepared from a highly TGFβ-responsive intestinal epithelial cell line. One of the TGFβ receptor-interacting proteins isolated was identified to be the mammalian homologue of the LC7 family (mLC7) of dynein light chains (DLCs). This 11-kDa cytoplasmic protein interacts with the TGFβ receptor complex intracellularly and is phosphorylated on serine residues after ligand-receptor engagement. Forced expression of mLC7-1 induces specific TGFβ responses, including an activation of Jun N-terminal kinase (JNK), a phosphorylation of c-Jun, and an inhibition of cell growth. Furthermore, TGFβ induces the recruitment of mLC7-1 to the intermediate chain of dynein. A kinase-deficient form of TGFβ RII prevents both mLC7-1 phosphorylation and interaction with the dynein intermediate chain (DIC). This is the first demonstration of a link between cytoplasmic dynein and a natural growth inhibitory cytokine. Furthermore, our results suggest that TGFβ pathway components may use a motor protein light chain as a receptor for the recruitment and transport of specific cargo along microtublules.

scholarly journals Identification of betacellulin as a major peptide growth factor in milk: purification, characterization and molecular cloning of bovine betacellulin

1999 ◽  
Vol 344 (3) ◽  
pp. 713-721 ◽  
Author(s):  
Andrew J. DUNBAR ◽  
Ilka K. PRIEBE ◽  
David A. BELFORD ◽  
Chris GODDARD
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Bovine Milk ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Wide Range ◽  
Rapid Amplification

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5ʹ and 3ʹ rapid amplification of cDNA ends (‘RACE’). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.

Isolation and partial characterization of a high molecular weight anionic glycoconjugate from transforming growth factor-β treated bovine articular chondrocyte cultures

1996 ◽  
Vol 74 (2) ◽  
pp. 233-240 ◽  
Author(s):  
Catherine K. T. Chan ◽  
Tassos P. Anastassiades
Keyword(s):  
Molecular Weight ◽  
Growth Factor ◽  
High Molecular Weight ◽  
Transforming Growth Factor ◽  
Sodium Dodecyl ◽  
Amino Sugar ◽  
Transforming Growth Factor Β ◽  
Exchange Chromatography ◽  
Articular Chondrocyte ◽  
Chondrocyte Cultures

A high molecular weight anionic glycoconjugate was isolated from the media of the transforming growth factor-β treated chondrocyte cultures by anion-exchange chromatography on DEAE–Sephacel and Mono Q columns and was partially characterized. This high molecular weight anionic glycoconjugate was not detected in the non-treated (control) cultures. Characterization studies showed that the glycoconjugate is a non-reducible, non-collagenous glycoprotein containing O-linked, N-linked, and sialic acid substituted carbohydrate units. The isolated glycoconjugate stained "blue" with Stains All and migrated as a single band on sodium dodecyl sulfate gradient gels (2.5–10% acrylamide – diallyl tartardiamide) at an estimated molecular weight of 540 000. Amino acid and amino sugar analyses showed that it is rich in aspartic acid – asparagine, glutamic acid – glutamine, alanine, proline, and glycine, and contains galactosamine and glucoasmine. This transforming growth factor-β inducible glycoprotein may be involved in cell differentiation and in the cartilage repair process. It may also be used as a marker to localize the biological activity of transforming growth factor-β in articular cartilage.Key words: transforming growth factor-β, chondrocytes, anionic glycoconjugate synthesis.

scholarly journals Transforming Growth Factor β-Dependent Sequential Activation of Smad, Bim, and Caspase-9 Mediates Physiological Apoptosis in Gastric Epithelial Cells

2005 ◽  
Vol 25 (22) ◽  
pp. 10017-10028 ◽  
Author(s):  
Masatoshi Ohgushi ◽  
Shunsuke Kuroki ◽  
Hiroshi Fukamachi ◽  
Lorraine A. O'Reilly ◽  
Keisuke Kuida ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Gastric Epithelium ◽  
Epithelial Cell Line ◽  
Gastric Epithelial Cells ◽  
Caspase 9 ◽  
Domain 3 ◽  
Induced Apoptosis

ABSTRACT Transforming growth factor β (TGF-β) has been implicated in the maintenance of homeostasis in various organs, including the gastric epithelium. In particular, TGF-β-induced signaling was shown to be required for the differentiation-associated physiological apoptosis of gastric epithelial cells, but its mechanism has not been well understood. In this study, the molecular mechanism of TGF-β-induced apoptosis was analyzed in a human gastric epithelial cell line, SNU16, as an in vitro model. Expression of Smad7 and Bcl-XL, but not viral FLIP, was shown to prevent TGF-β-induced apoptosis, indicating an exclusive requirement of the activation of Smad signaling pathway and mitochondrial dysfunction followed by activation of caspase-9. In addition, treatment with TGF-β induced binding of Bim, a proapoptotic Bcl-2 homology domain 3 (BH3)-only protein, to Bcl-XL, which is dependent on the activation of Smad, and reduction in the expression of Bim by RNA interference decreased the sensitivity to TGF-β-induced apoptosis. Moreover, we found abnormalities in the gastric epithelium of both Bim and caspase-9 knockout mice; these abnormalities were associated with a defect of physiological apoptosis in gastric epithelial cells. These results indicate for the first time that TGF-β is involved in the physiological loss of gastric epithelial cells by activating apoptosis mediated by Smad, Bim, and caspase-9.

Platelet-derived growth factor, insulin-like growth factors, fibroblast growth factors and transforming growth factor β do not account for the cell growth activity present in bovine milk

1997 ◽  
Vol 154 (1) ◽  
pp. 45-55 ◽  
Author(s):  
D A Belford ◽  
M-L Rogers ◽  
G L Francis ◽  
C Payne ◽  
F J Ballard ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Cell Growth ◽  
Molecular Mass ◽  
Inhibitory Activity ◽  
Transforming Growth Factor ◽  
Bovine Milk ◽  
3T3 Cells ◽  
Igf I ◽  
Growth Activity

Abstract Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80–100 kDa. In contrast, acid gelfiltration chromatography resolved two peaks of cell growth activity. A peak at 15–25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bioactivity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15–25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor β (TGF-β), and all inhibitory activity for Mv1Lu cells was immuno-neutralised by an antibody against TGF-β. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-β and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-β; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-β account for the 15–25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk. Journal of Endocrinology (1997) 154, 45–55

Effects of human prostatic mitogens on rat bone cells and fibroblasts

1987 ◽  
Vol 115 (3) ◽  
pp. 447-NP ◽  
Author(s):  
M. Koutsilieris ◽  
S. A. Rabbani ◽  
D. Goltzman
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Bone Cells ◽  
Transforming Growth Factor Β ◽  
Exchange Chromatography ◽  
Mitogenic Activity ◽  
Skin Fibroblasts ◽  
Osteosarcoma Cell ◽  
Fetal Rat ◽  
Interleukin 1Α

ABSTRACT We examined several characteristics of the mitogenic activity of extracts of human prostatic adenocarcinoma and human benign prostatic hyperplasia in fetal rat calvarial cells, cloned rat osteosarcoma cells and rat skin fibroblasts. Prostatic moieties produced maximal stimulation of [3H]thymidine incorporation at 24 h, were mitogenic in the absence or presence of various concentrations of serum, and were active in calvarial cells enriched with osteoblasts, skin fibroblasts and the cloned rat osteosarcoma cell line UMR 108. The known growth-promoting agents insulin, insulin-like growth factor, fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, interleukin-1α and β and transforming growth factor β were also mitogenic in the indicator systems employed; however, maximally stimulating effects of these peptides in cells of the osteoblast phenotype were further enhanced with prostatic material. Prostatic activity was acid-stable and could be enriched with respect to osteoblast stimulation by hydrophobic and carboxymethyl ion-exchange chromatography. The results demonstrate the presence of potent mitogenic activity in hyperplastic and malignant prostatic tissue which appears to include unique osteoblast-stimulating activity. J. Endocr. (1987) 115, 447–454

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