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R Grosse

Publications and source records attributed to R Grosse.

At least 37 records · Page 2Linked to original sources
PubMed1993

Effect of mammary-derived growth inhibitor on proliferation of MAC-T bovine mammary epithelial cells.

An established bovine mammary epithelial cell line (MAC-T) was used as a model to examine the effect of mammary-derived growth inhibitor on mammary epithelial cell proliferation. Prior to each proliferation assay, cells were synchronized in G1/G0 by culturing for 24 h without serum. Flow cytometry revealed that 90% of cells were in G1/G0, 4% in G2/M, and 6% in the S-phase after serum deprivation. Approximately 2 x 10(3) cells per well were seeded onto 24-well plates. Cells were incubated for 5 to 6 d with various amounts of mammary-derived growth inhibitor (0 to 100 ng/ml). Mammary-derived growth inhibitor and medium were changed daily. Mammary-derived growth inhibitor decreased mammary epithelial cell proliferation at .1 ng/ml. Synchronization of cells in G1/G0 was necessary for inhibition of cell proliferation. Cells not arrested by serum deprivation were not responsive to mammary derived-growth inhibitor. Inhibition of cell proliferation was transient and observed up to 96 h in culture. Mammary-derived growth inhibitor appears to act in vitro by inhibiting the resumption of stationary cell proliferation following starvation.

Animals↗
PubMed1992

Purification of a mammary-derived growth inhibitor (MDGI) related polypeptide expressed during pregnancy.

The present study was undertaken to screen immunochemically for MDGI-related proteins in the mammary gland. A new form, MDGI 2, not present in lactation could be detected in the bovine gland during pregnancy. It was further distinguished from MDGI by its lower molecular weight, its association with a complex binding to WGA, and by lacking immunoreactivity to an anti-MDGI antibody directed against the C-terminus of MDGI. MDGI 2 was purified by chromatography over DEAE-Sepharose, Bio-Gel P-30 in 1% acetic acid, Sephacryl S-200 in 6 M urea and Mono Q. Final purification included HPLC on TSK G-3000 SW and electroelution from SDS-gels. Cell-free translation of poly (A+)mRNA from glands of pregnant animal yielded one form identical with MDGI. We assume that posttranslational processing of MDGI is involved in its activities.

Animals↗
PubMed1992

A new monoclonal antibody for detection of EGF-receptors in western blots and paraffin-embedded tissue sections.

The prognostic significance of the epidermal growth factor receptor status (EGF-R-status) for certain human tumors requires the development of antibodies useful for clinical application. We used purified receptor preparations to generate monoclonal antibodies immunoreactive with the EGF-R purified from placenta membranes and A431 tumors. Four of the hybridomas contained antibodies (R2, R3, R5, and R9) which recognized both antigens. Antibody R3 was shown to display the following properties: it binds with a KD value of about 10(-9)-10(-10) M to the receptor, a half maximal inhibition of EGF-binding is achieved at 5 x 10(-8) M, and in Western blots of cell membranes R3 specifically detects the EGF-R at 0.1 micrograms/ml. R3 inhibits EGF-dependent clonogenic growth of NRK cells and completely blocks EGF stimulated autophosphorylation of the receptor. Moreover, R3 also detects EGF-R in paraffin-embedded tissue sections taken from human salivary gland, term placenta, and adult skin and mammary carcinomas. Thus, R3 can be used in retrospective diagnostic clinical studies and might help to develop new immunotherapeutic intervention.

Animals↗
PubMed1992

Mammary-derived growth inhibitor in lactation and involution.

Presence of mammary-derived growth inhibitor (MDGI) in mammary tissue of lactating and involuted cows was investigated. Eighteen lactating, non-pregnant high-producing Holstein cows were randomly assigned to 3 experimental groups of 6 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group were slaughtered at 2-3 d, and the others at 4-8 d following sudden cessation of milking. Cessation of milking occurred at approximately 300 d in lactation. Western blot analysis revealed the presence of MDGI in the cytosolic and microsomal fractions of mammary tissue homogenates. High levels of MDGI were detected in mammary tissue obtained from lactating non-pregnant cows. A dramatic reduction in MDGI was observed in early involution (2-3 or 4-8 d following cessation of milking). These data suggest that a relationship exists between MDGI levels and the physiological status of the gland. Lack of MDGI may play a role during the processes of mammary involution and development prior to parturition.

Animals↗
PubMed1992

Mammary-derived growth inhibitor protein and messenger ribonucleic acid concentrations in different physiological states of the gland.

Expression of mammary-derived growth inhibitor in tissue from lactating and involuting bovine mammary glands was investigated. Seventeen lactating, pregnant (220 to 272 d in gestation) cows were divided in two groups of 8 and 9 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group (9 involuting cows) were slaughtered at 13 to 52 d following sudden cessation of milking. High concentrations of mammary-derived growth inhibitor (.63% of the total protein) were detected in mammary tissue of lactating cows. Mammary-derived growth inhibitor (less than .10% of the total protein) was dramatically reduced during most of the involution period (13 to 45 d following cessation of milking). Mammary-derived growth inhibitor was again detected (.28% of the total protein) during the last stage of the involution (46 to 53 d after cessation of milking), which coincided with colostrum formation. When steady state concentrations of mammary-derived growth inhibitor mRNA were examined, the results obtained mirrored those obtained at the protein concentration. These data suggest that regulation of mammary-derived growth inhibitor occurs via modulation of the steady state concentration of its mRNA. Furthermore, there is a strong correlation between mammary-derived growth inhibitor expression and lactation in dairy cows.

Animals↗
PubMed1992

Limited selectivity of a synthetic erbstatin derivative for tyrosine kinase and cell growth inhibition.

The natural tyrosine kinase inhibitor erbstatin and a synthetic analog (1302) were co0pared for their inhibitory activity on EGF receptor kinase, PDGF receptor kinase and a src-type kinase from bovine brain. Erbstatin inhibited both growth factor receptor kinases equally well and had little effect on the src kinase. The analog exhibited similar potency for inhibition of purified EGF receptor tyrosine kinase as erbstatin, however, was clearly less effective for inhibition of purified PDGF receptor kinase as well as the src-type kinase. The selectivity was, however, not seen when the derivative was assayed with respect to inhibition of autophosphorylation of both growth factor receptors in Swiss 3T3 cell membranes. The latter finding might explain a lack of selectivity of the compound for inhibition of DNA synthesis in 3T3 cells when the cells were stimulated comparatively with EGF, insulin, EGF plus insulin or PDGF. The results suggest that the environment of growth factor receptors in the cell membrane can remarkably modify their susceptibility to tyrosine kinase inhibitors.

3T3 Cells↗
PubMed1991

Modulation of the beta-adrenergic-response in cultured rat heart cells. II. Mammary-derived growth inhibitor (MDGI) blocks induction of beta-adrenergic supersensitivity. Dissociation from lipid-binding activity of MDGI.

'Mammary-derived growth inhibitor (MDGI)' is a 14.5 kDa polypeptide with growth-inhibitory activity for various mammary epithelial cells in vitro which is highly homologous to cardiac fatty acid-binding protein (H-FABP). Here we describe a new biological activity of MDGI: Inhibition of L(+)-lactate-, arachidonic acid- and 15-S-hydroxyeicosatetraenoic acid-induced supersensitivity of neonatal rat heart cells for beta-adrenergic stimulation, concerning particularly a small population of beta 2-receptors. Synthetic peptides corresponding to the MDGI-sequence, residue 121-131 mimic the effect of MDGI. Measurements of lipid-binding to MDGI and synthetic peptides excluded the binding of arachidonic acid, 15-S-hydroxyeicosatetraenoic acid or beta-adrenergic agonists to MDGI or the peptides as the mechanism for this effect. Also, no direct interference of MDGI and the synthetic peptides with the binding of the beta-adrenergic agent CGP 12177 to its receptor on A431 cells could be detected. We suggest that MDGI and the peptides act by interference with the function of the beta 2-adrenergic receptor and that this mechanism might also be relevant for the growth-inhibitory activity of MDGI. Furthermore, the data point to a possible function of H-FABP for the modulation of beta-adrenergic sensitivity of cardiac myocytes.

Amino Acid Sequence↗
PubMed1990

A Mr 43,000 epidermal growth factor-related protein purified from the urine of breast cancer patients.

A tumor-associated epidermal growth factor (EGF)-like activity was detected in the urine of breast cancer patients by means of an EGF radioreceptor assay and an anchorage-independent growth assay. The clonogenic growth factor activity of pooled void volume eluate fractions from a Bio-Gel P-30 column was completely neutralized by an anti-human epidermal growth factor antiserum but not by an anti-transforming growth factor alpha antiserum. This activity was determined in the urine of 71 breast cancer patients. A statistically significant correlation was found between EGF-like clonogenic activity and axillary lymph node status, tumor size, stage of disease, and grade of differentiation of the primary tumor. The Bio-Gel P-30 void volume fraction was used to purify the EGF-related polypeptide to apparent homogeneity by subsequent binding to and elution from A431 cells followed by isoelectric focusing. A polypeptide of a pI of approximately 3.4 was identified to be related to EGF by neutralization and immunoprecipitation experiments with anti-human epidermal growth factor antisera. This polypeptide migrated as a single band of Mr 43,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Biological Assay↗
PubMed1990

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells.

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI- and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.

Animals↗
PubMed1990

Developmental regulation of mammary-derived growth inhibitor expression in bovine mammary tissue.

The cDNA for a previously described growth inhibitor, designated as mammary-derived growth inhibitor (MDGI) (Grosse, R., and P. Langen. 1989. In Handbook of Experimental Pharmacology. In press) has been cloned from a plasmid library which was derived from terminally differentiated bovine mammary gland. Sequencing of the cDNA showed an open reading frame coding for a protein of 133 amino acids. In six positions differences were found between the sequence determined from the cDNA and that determined previously by amino acid sequence analysis. Northern blot analysis revealed abundant MDGI mRNA in the terminally differentiated mammary gland, whereas in virgin gland, liver or pancreas transcripts were not expressed. By use of in situ hybridization technique transcription of MDGI in the developing bovine mammary gland was analyzed. Increasing amounts of MDGI mRNA were detected in the epithelial cells of embryonic mammary rudiment, in the epithelium of developing lobules and in terminal parts of ducts and lobuloalveolar epithelial cells of differentiated glands. There was a geographical gradient of MDGI mRNA concentration in bovine mammary gland reaching a maximum in the proximal parts of the tissue. An immunohistochemical analysis with different polyclonal and peptide directed antibodies against MDGI confirmed the in situ hybridization data with respect to the tissue-specific and differentiation-dependent MDGI expression in bovine mammary gland. The results suggest a close relationship between MDGI transcription and developmental processes in the normal bovine mammary gland.

Amino Acid Sequence↗
PubMed1990

Transforming growth factor-beta inhibits lactogenic hormone induction of beta-casein expression in HC11 mouse mammary epithelial cells.

HC11 mouse mammary epithelial cells can undergo a limited functional differentiation in terms of beta-casein synthesis in response to the combined action of dexamethasone and prolactin. Transforming growth factor-beta (TGF-beta) can inhibit beta-casein expression in HC11 cells in a dose-dependent manner. This effect is reversible and specific as shown by comparison with the effect of other growth factors. TGF-beta also inhibits DNA synthesis of HC11 cells. These findings suggest a possible role of TGF-beta as an inhibitor of functional differentiation in the mammary gland.

Animals↗
PubMed1989

Hydrodynamic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI).

The mammary-derived growth inhibitor exists in solution as a monomeric molecule with a molar mass of 14,500 +/- 400 g/mol. The largest diameter and the height of the polypeptide chain were estimated to be 3.75 +/- 0.25 nm and 2.01 +/- 0.13 nm respectively. This is in good agreement with the structurally related bovine peripheral myelin P2 protein (about 70% amino acid sequence homology). CD measurements have revealed MDGI to be a protein with about 50% beta structure and less than 20% alpha helix similarly as in fatty acid-binding proteins. Removal of endogenous long-chain fatty acid by lipidex or storage in the frozen state lead to a destabilization of the active MDGI conformation which is accompanied by a loss of its activity with regard to growth inhibition of Ehrlich Ascites cells.

Animals↗
PubMed1989

Binding properties of biotinylated epidermal growth factor to its receptor on cultured cells and tissue sections.

A biotinylated derivative of murine epidermal growth factor (EGF) was prepared by covalent attachment of the terminal amino group of EGF to N-biotinyl-epsilon-aminocaproyl-N-hydroxysuccinimide. The stoichiometry of biotin incorporation was in the range of one biotin moiety per EGF molecule. The biotinylated EGF (biotinyl-epsilon-caproyl-EGF, BioEGF) binds to EGF receptors on intact Ehrlich ascites carcinoma (EAC) cells with an affinity similar to that of native EGF and displays the same mitogenic activity as EGF in a soft agar test system with normal rat kidney (NRK) cells. BioEGF was visualized on cultured cells and tissue sections of a head and neck tumour by commercial streptavidin/avidin detection systems. Cytochemical analyses of certain tumour forms can be easily performed using the BioEGF probe.

Animals↗