摘要:

AbstractUsing polyclonal antibodies which recognize 18 kDa BMP-2, we have previously demonstrated that the major BMP-2 protein inXenopusembryo extracts is monomeric and not dimerized whereas, the mammalian counterpart derived from bone extract was purified as a dimeric protein. In this study, the same antibodies (Ab383) were used to detect high molecular weight forms ofXenopusBMP-2. Partial purification of the immunoreactive 18 kDa BMP-2 fromXenopusembryo extract by gel filtration, heparin-Sepharose affinity chromatography and preparative SDS-PAGE resulted in co-purification and identification of higher molecular forms of BMP-2 of 110 kDa and 36 kDa. Diagonal SDS-PAGE analysis suggested that they are homodimer and multimer of the 18 kDa species, respectively, linked through disulfide bridge(s).

ISSN:0897-7194    

DOI:10.3109/08977199309011019

出版商:Taylor&Francis    

年代:1993

数据来源: Taylor

摘要:

AbstractSpecific antibodies toXenopus laevisbone morphogenetic protein-4 (xBMP-4) were raised by immunizing rabbits with a fusion protein of bacterialβ-galactosidase and xBMP-4. The antibodies were used to detect xBMPs expressed in mammalian cells by Western blotting. The antibodies were found to recognize xBMP-4 specifically and not to cross-react with either xBMP-2 or xBMP-7 which are similar to xBMP-4. In addition, the antibodies recognized dimeric xBMP-4 whereas our previous antibodies recognized the reduced form only. The present antibodies detected an immunoreactive 27 kDa protein in extracts of developingXenopusembryos from oocyte to tailbud embryo. The xBMP-4 peptide appeared to be monomeric in structure because the molecular weight did not shift upon reduction of disulfide bond(s).

ISSN:0897-7194    

DOI:10.3109/08977199309011020

出版商:Taylor&Francis    

年代:1993

数据来源: Taylor

摘要:

AbstractWe showed in previous work that LAK cell supernatants contain a large molecular weight factor with toxic activity for A375 melanoma and other cell lines. The factor, Fr1, was identified tentatively as TGFβ-related, since its activity was abolished by anti-TGFβserum. This relatedness is further confirmed in the present work, which demonstrates that, like TGFβ, Fr1 stimulates the release and deposition of fibronectin and induces morphological changes indistinguishable from those induced by TGFβ. The TGFβderived from LAK cells, although associated with a large carrier molecule, is directly acting and does not dissociate from its carrier following gel filtration in acetic acid. Its carrier is different from alpha-2 macroglobulin. SDS-PAGE and immunoblotting showed that FR1 contains TGFβcomplexed with large molecules (150 and<200 kDa), which dissociate in reduced gels to molecules of 60-67 kDa. We interpret these data as showing that TGFβsecreted by LAK cells is, presumably, covalently linked to monomeric carrier molecules of approximately 60 kDa, which, in turn, are S-S bonded to form multimeric molecules of 150 kDa and<200 kDa.

ISSN:0897-7194    

DOI:10.3109/08977199309011021

出版商:Taylor&Francis    

年代:1993

数据来源: Taylor

摘要:

AbstractPhophatidylinositol (PI) anchored TGF-β2 binding proteins of approximately 60 and 140 kDa were recently identified in MG-63 human osteogenic sarcoma and fetal bovine heart endothelial cells (FBHEC) (Cheifetz, S., Massagué, J. (1991) J. Biol. Chem.266, 20767-20772). The relationship between these two PI-anchored TGF-β2 binding proteins was investigated. Specifically labeled bands of approximately 60 and 110 kDa were observed when125I-TGF-β2 labeled FBHEC and MG-63 cells were separated by SDS-PAGE under non-reducing conditions. Partial proteolysis of the affinity labeled 60 and 110 kDa species yielded similar peptides. The integrity of the 110 kDa species under denaturing conditions is dependent on both disulfide bonds and chemical cross-linking: reduction of the 110 kDa species yielded an affinity labeled species co-electrophoresing with the 60 kDa band; cleavage of chemical cross-links in the 110 kDa complex yielded a labeled 60 kDa component. These results indicate that the 110 kDa affinity labeled species in FBHEC and MG-63 cells is a homodimer. Within this complex the binding protein monomers can be chemically cross-linked to opposite arms of the disulfide-linked TGF-β2 homodimer.

ISSN:0897-7194    

DOI:10.3109/08977199309011022

出版商:Taylor&Francis    

年代:1993

数据来源: Taylor

摘要:

AbstractRetroviral insertion mutagenesis has been used extensivelyin vivobut notin vitroto induce and identify critical mutations during oncogenic progression and differentiation. We have developed a tissue culture system using the human, growth factor-dependent, hematopoietic precursor cell line TF-1 that permits the use of retroviral vectors to induce a large (up to 28-fold) increase in the mutation frequency to growth factor independence and thus the isolation of many mutants. The mutation frequency, as expected, is directly proportional to the number of retroviral insertions (2.2×10−7mutants per insertion). The mutant phenotypes can be subdivided into mutants that release growth factors and those that do not (“autonomous”mutants). The majority of growth factor-producing mutants release an unidentified ligand. A subset of the autonomous mutants shows alterations in expression of theαsubunit of either the GM-CSF or the IL-3 receptor. One mutant expresses neither GM-CSF nor IL-3αreceptor chains, thus showing coordinate regulation of theαreceptor subunits.

ISSN:0897-7194    

DOI:10.3109/08977199309011023

出版商:Taylor&Francis    

年代:1993

数据来源: Taylor

摘要:

AbstractAcidic Fibroblast Growth Factor (aFGF) binds on two classes of fibroblast growth factor receptors, the high affinity receptors (HAR) a family of four known transmembrane tyrosine kinases and the low affinity receptors (LAR), related to cell surface heparan sulfate proteoglycan (HSPG). We analysed the relationship between the binding of aFGF on the HAR and on the LAR in bovine lens epithelial (BEL) cells in the presence of heparin or suramin.Through Northern blotting analysis we demonstrated that the three immunoglobulin-like transcript of FGF receptor type 1 (FGF-R1) is the major expressed high affinity receptor in BEL cells. On the contrary, HAR-aFGF complexes are present in two forms (150 kDa and 135 kDa) revealed by cross-linking experiments with125I aFGF. Moreover125I aFGF binding to BEL cell surface induces the spontaneous formation of a125I aFGF dimer (31 kDa) which is then internalized and degraded in the cells as the 15.5 kDa aFGF native form is.It has been observed that heparin at 10μg/ml (1) in cross-linking experiments, reduces by half the total number of HAR complexes by preventing the formation of the 150 kDa complex but does not affect the 135 kDa complex, (2) in binding experiments, suppress the spontaneous formation of thel25I aFGF dimer bound to LAR, and then its internalization and degradation in the cells.Moreover, we demonstrate that (1) - only HAR contributes specifically and directly to the aFGF internalization process, (2) - HAR internalization is ligand concentration and time saturable, (3) - there is no desensitization of aFGF internalization induced by Iigand binding to HAR, (4) - a FGF dimerization process is highly dependent on the apparent affinity of FGF for heparin, since aFGF mutant with a reduced affinity for heparin does not promote the dimerization.These data strongly suggest that a heteroreceptor-aFGF complex (150 kDa) is formed by one molecule of HAR (FGF-R1) associated to one molecule of LAR through their respective interactions with a very stable aFGF homodimer. Such a three component receptor induced by FGF dimerization may be a process involved in the mechanism of action of FGFs which could explain the diversity of the biological response of FGF depending on the presence of the HSPG on the extra cellular matrix.In addition prebinding of unlabelled aFGF to the cells induces a 4 fold increase in the affinity of HAR tol25IaFGF concomitant with its down regulation by 80% and initiates the formation of the HAR homodimer.All these data obtained with FGF-R1 are consistent with the general allosteric oligomerization model of the growth factor receptor tyrosine kinases.

ISSN:0897-7194    

DOI:10.3109/08977199309011024

出版商:Taylor&Francis    

年代:1993

数据来源: Taylor

摘要:

AbstractIn order to obtain information on the possible role of epidermal growth factor (EGF) in rat prenatal development, we tested the effects of a neutralizing antiserum to rat EGF and of recombinant human EGF on the growth and development of transplanted rat embryos and fetal structures. Ten-day embryos or 16-day fetal intestines (ileum and jejunum) or paws were transplanted under the capsule of both kidneys of young adult syngeneic host rats. Osmotic minipumps were used to infuse antiserum to rat EGF or normal rabbit serum (NRS) into the renal artery of the right kidney to ascertain direct effects on development. The transplants on the contralateral side served as internal controls. Infusion of the NRS did not affect growth of any of the fetal structures or of the embryo transplants. The antiserum to rEGF did not affect growth of the fetal ileum or paw transplants, but it inhibited growth of the fetal jejunum by 38%, and suppressed differentiation of hair follicles in the paws by∼90%. Tissue differentiation in the two segments of the intestine was unaffected by the antiserum. By contrast, growth of embryo transplants wasstimulatedby approximately 60% by the anti-EGF serum. Infusion of antiserum did not affect the growth of the kidneys upon which the transplants were grown, and infusion of different doses of recombinant human EGF had no effect on growth of embryo transplants.Our data suggest that EGF may function as a negative growth regulator during the embryonic period, but it becomes a growth stimulator for specific tissues during the fetal period. These results are consistent with our previous findings with basic fibroblast and insulin-like growth factors in illustrating that the regulatory functions of growth factors may be more general during embryogenesis but they become more organ or tissue specific in the fetal period of development.

ISSN:0897-7194    

DOI:10.3109/08977199309011025

出版商:Taylor&Francis    

年代:1993

数据来源: Taylor