Biomedical subjects

R Grosse

Publications and source records attributed to R Grosse.

At least 55 records · Page 3Linked to original sources

PubMed1989

A mammary-derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells.

The aim of the present study was to investigate the expression of the mammary-derived growth inhibitor (MDGI) and the subcellular localization of MDGI-related antigens in bovine mammary glands. Cell-free translation of poly(A+) = RNA, immunoprecipitation with rabbit anti-MDGI-antibodies, and estimation of the relative contents of MDGI by a radioimmunoassay in mammary tissue of different functional states revealed that the 13 kDa MDGI was dramatically increased in terminally differentiated mammary tissue compared with the proliferating tissue from pregnant animals. To address the question of tissue localization, polyclonal anti-MDGI antibodies and antibodies directed against a synthetic peptide corresponding to residues 69 to 78 of MDGI were used. Western blotting of tissue fractions revealed the cytosolic and microsomal localization of MDGI. Additionally, both types of antibodies detected a 70-kDa antigen in the nuclear fraction of differentiated mammary glands. Salt extraction and DNase I digestion of isolated nuclei, as well as chromatin purification, indicated an association of the 70-kDa antigen with the chromatin. By means of the immunogold technique, MDGI-related antigens were localized within euchromatic nuclear regions of epithelial cells in the intact differentiated mammary gland. The immunostaining was markedly diminished in the proliferating tissue. This finding raises the possibility that MDGI and the 70-kDa antigen influence cell proliferation by acting on gene expression within the nuclei of mammary glands.

Animals↗

PubMed1989

Microangiopathy of endoneurial vessels in hypoxemic chronic obstructive pulmonary disease (COPD). A quantitative ultrastructural study.

A peripheral neuropathy has been reported in patients with chronic respiratory insufficiency due to chronic obstructive pulmonary disease (COPD). It is mainly characterized by axonal degeneration, secondary demyelination and abnormal endoneurial vessels. The pathogenesis of these lesions remains obscure. To investigate whether relationships exist between neuritic and vascular lesions, a qualitative and quantitative ultrastructural study was performed on nerve biopsies in 13 patients with chronic respiratory insufficiency due to COPD, and in 9 normal controls without pulmonary lesions. A computer-assisted multiple regression analysis taking into account clinical, electrophysiological, biological and morphometric parameters was performed. Statistically significant differences in the endoneurial structure of microvessels were: (1) thickening of the basement membrane; (2) narrowing of the lumen; (3) mural pericytic debris deposits, occurring in the COPD group. In the latter, hypercapnia correlated positively with nerve fibers lesions (P = 0.03) and endothelial area (P = 0.03). No correlations were found between age and other parameters. These findings highlight the fact that the microangiopathy in peripheral nerves in patients with COPD may be diffuse and essentially due to hypoxia and reduction in blood flow, as in diabetic neuropathy.

Aged↗

PubMed1989

Purification of a high molecular weight form of epidermal growth factor from urine of breast cancer patients.

A high molecular weight form of epidermal growth factor (EGF) was detected by means of an EGF radio-receptor assay and an anchorage-independent growth assay in the urine of breast cancer patients. Preliminary data indicate that the activity of this growth factor is associated with lymph node status and tumor size and that the activity becomes reduced after removal of the primary tumor. The EGF-related polypeptide was purified to homogeneity by a combination of Sephadex G-25 and Bio Gel P-30 chromatography followed by binding to, and elution from, EGF receptor rich A431 cells. Final purification was achieved after isoelectric focusing by following the biological activity of eluted polypeptides. A polypeptide of a pI of 3.4 was identified to carry EGF-like activity. This polypeptide migrated as a single band of 43 kDa in SDS-PAGE. Its biological activity was neutralized by a specific anti-hEGF-antibody indicating an immunological relationship with hEGF.

Animals↗

PubMed1988

A 13-kilodalton protein purified from milk fat globule membranes is closely related to a mammary-derived growth inhibitor.

With the use of specific antibodies against a previously purified [Boehmer, F.-D., Lehmann, W., Schmidt, H., Lange, P., & Grosse, R. (1984) Exp. Cell Res. 150, 466-477] and sequenced mammary-derived growth inhibitor (MDGI) [Boehmer, F.-D., Kraft, R., Otto, A., Wernstedt, C., Hellmann, U., Kurtz, A., Mueller, T., Rohde, K., Etzold, G., Lehmann, W., Langen, P., Heldin, C.-H., & Grosse, R. (1987) J. Biol. Chem. 262, 15137-15143], the localization and relative amount of immunoreactive 13-kilodalton (kDa) antigen in different fractions of bovine milk were determined. The highest amount of antigen was found to be associated with the milk fat globule membranes (MFGM). As revealed by a dot immunobinding assay, the amount of immunoreactive bovine and human MFGM-associated antigen increased dramatically with the onset of lactation after delivery. This finding corresponds to earlier data obtained for MDGI and indicates a relationship between the proliferative state of mammary epithelial cells and the amount of immunoreactive antigen. The 13-kDa antigen has been purified from MFGM to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution. The MFGM-derived 13-kDa polypeptide was found to be almost identical with MDGI as demonstrated by tryptic digestion and partial amino acid sequence analysis of tryptic fragments of both proteins. The results clearly show the presence of a membrane-bound MDGI-related 13-kDa protein, thus supporting the possible involvement of membrane-associated growth inhibitors in growth regulation of mammary epithelial cells.

Amino Acid Sequence↗

PubMed1988

A polypeptide growth inhibitor isolated from lactating bovine mammary gland (MDGI) is a lipid-carrying protein.

Mammary-derived growth inhibitor (MDGI), a polypeptide growth inhibitor isolated from lactating bovine mammary tissue, previously shown to have extensive sequence homology with fatty acid-binding proteins, was demonstrated to meet the criteria of a fatty acid-binding protein. The protein was found to bind [3H]palmitic acid in a saturable manner and to be complexed with endogeneous free fatty acids. [3H]palmitic acid, when bound to the protein, was more rapidly taken up by the target cells (human mammary carcinoma cells [MaTu]) than was free [3H]palmitic acid, suggesting a lipid carrier function for the inhibitor. It is suggested that the fatty acid-binding properties of MDGI may relate to its ability to inhibit cell growth in vitro and to regulate other cellular functions.

Animals↗

PubMed1988

EGF binding is quantitatively related to growth in node-positive breast cancer.

Number of mitoses and EGF binding were determined in parallel in biopsies of 27 lymph-node positive and of 23 lymph-node negative breast cancer patients. For node-positive patients the parameters for cell growth and EGF binding were quantitatively correlated by the equation y = P3 + P1(1- exp(- P2x]. For node-negative cases neither the non-linear model nor the linear approximation described the data unambiguously. The results strongly suggest that in node-positive patients, growth of breast cancer is related to an EGF-dependent acceleration of cell division.

Adult↗

PubMed1988

Specific effects of nerve growth factor on the differentiation pattern of mouse embryonic stem cells in vitro.

The effect of nerve growth factor (NGF) on the differentiation pattern of pluripotent undifferentiated mouse embryonic stem cells (ESC) was studied. Morphological analysis of differentiating stem cells and histochemical silver-staining analysis of embryonic carcinoma (P19S1801A1) and embryonic stem cells (ESC-BLC 6) permitted the demonstration of nerve cell differentiation. NGF treatment resulted in an accelerated appearance of neuron-like cells in differentiating embryoid bodies of ESC line BLC 6. By use of the silver-staining test NGF dependent induction of nerve cell lineage was demonstrated. The presented embryonic stem cell model allows to study the time course of differentiation and induction of a special cell lineage of undifferentiated embryonic stem cells by growth factors.

Animals↗

PubMed1987

Antibodies against mammary derived growth inhibitor (MDGI) react with a fibroblast growth inhibitor and with heart fatty acid binding protein.

A polypeptide growth inhibitor, designated as mammary derived growth inhibitor, has previously been purified from lactating bovine mammary glands. Polyclonal rabbit antiserum raised against mammary derived growth inhibitor cross-reacts with bovine heart fatty acid binding protein and bovine peripheral myelin P2 protein. These results are consistent with the observation that the amino acid sequence of mammary derived growth inhibitor showed homology to the sequences of these proteins. In a parallel series of immunoblotting experiments, rabbit anti-mammary derived growth inhibitor also reacted specifically with a fibroblast growth inhibitor purified from the conditioned medium of cultured mouse 3T3 fibroblasts. These data suggest that bovine mammary derived growth inhibitor and mouse fibroblast growth inhibitor may share common structural features and raise the possibility that these growth inhibitors may together define a new family of growth regulatory molecules.

Animals↗

PubMed1987

Growth of mammary epithelial cells in breast-cancer biopsies correlates with EGF binding.

In order to understand the role of EGF in the development of human mammary epithelial tissue, we analysed the binding of 125I-EGF to sections of breast cancer biopsies. A mean specific 125I-EGF binding of 8.9 fmol per mg protein was estimated in thin sections of 17 breast cancer biopsies. Microautoradiographic analysis of 125I-EGF binding to the tissue sections was applied to demonstrate that EGF was bound predominantly to mammary epithelial cells. The binding was clearly correlated to the number of mitoses of mammary epithelial cells in the same samples. The highest EGF binding and proliferation rates were found in biopsies from breast cancer with axillary lymph-node metastases.

Animals↗

PubMed1987

Demonstration of an epidermal growth factor-dependent 58 kDa phosphoprotein secreted by rat kidney fibroblasts.

Epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate increased the amount of 32Pi found as phosphoserine in a major, hitherto not described 58 kDa phosphoprotein (pp58) secreted by normal rat kidney fibroblasts. Platelet-derived growth factor, insulin, nerve growth factor and fibroblast growth factor did not affect pp58 while transforming growth factor beta decreased the accumulation of radioactivity into pp58. Cycloheximide, actinomycin D and ammonium chloride suppressed the labelling of pp58.

Animals↗

PubMed1987

EGF receptors on plasma membranes purified from bovine mammary gland of lactating and pregnant animals.

A procedure for purification of plasma membranes from bovine mammary gland has been developed. The binding capacity of EGF to the plasma membranes from mammary tissue of pregnant cows was equal to 335 fmol per mg of protein thus being twofold higher than in membranes from lactating gland. The KD values were not changed. Autoradiographs of the membrane receptor linked to [125J]-EGF revealed three labeled bands corresponding to 160 kDa, 145 kDa and 115 kDa polypeptides. The main band of 145 kDa was labeled stronger in membranes from pregnant animals. The results suggest that the EGF receptor level is enhanced in fast proliferating normal mammary tissue.

Animals↗

PubMed1986

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells.

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.

Animals↗

PubMed1986

Characterization of membrane-associated growth inhibitor activity for Ehrlich ascites mammary carcinoma cells.

Purified plasma membranes obtained from bovine milk or from bovine mammary gland inhibited in vitro growth of Ehrlich ascites mammary carcinoma cells. Plasma membranes derived from tumor cells or from tumor tissue were inactive. Growth inhibition by milk fat globule membranes was shown to be due to release of membrane-associated inhibitory factors into the medium. Gentle removal of proteins from the surface of milk fat globule membranes succeeded in recovery of a fraction inhibiting cell growth specifically. The same fraction contained the antigen cross-reacting with an antiserum raised against the previously purified 13 kD growth inhibitor from bovine mammary gland.

Animals↗

PubMed1986

Inhibition of proliferation of Ehrlich ascites carcinoma cells is functionally correlated with reduced activity of the cytosol to stimulate protein synthesis.

The cytosolic fraction prepared from in vivo stationary phase Ehrlich ascites carcinoma cells (EAC cells) stimulates in vitro protein synthesis by isolated polysomes to a substantially lower extent than the cytosol of exponentially growing cells. The cytosolic fraction of EAC cells treated in vitro for 24 h with a purified 13 kD growth inhibitor from bovine mammary gland was by 20-40% less active in stimulating in vitro protein synthesis in comparison to control cytosols. It could be shown that the growth inhibitor does not act directly on the cytosol but rather exerts its action by (a) plasma membrane mediated mechanism(s). The results are discussed with respect to correlations between regulations of cell proliferation and protein synthesis at the posttranscriptional level.

Animals↗

PubMed1985

Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland.

A recently published method for purification of a new inhibitor of growth of mammary cells in vitro from bovine mammary gland has been modified to yield several hundred micrograms of inhibitor per kg of glandular tissue. The inhibitory effect exerted by this preparation to Ehrlich ascites mammary carcinoma cells fulfilled all biological criteria of specificity established earlier for preparations obtained by other means, the most important being that the inhibitory effect is abolished by the epidermal growth factor and insulin. The preparation is shown to consist mainly of a protein of 13 kDa which appears to be not glycosylated. An antiserum raised in mice against the inhibitor is demonstrated to be specific for the 13 kDa bovine mammary gland protein. Neutralization of the inhibitory activity by the specific antiserum strongly supports the view that the 13 kDa protein is indeed the carrier of inhibitory activity. First data on tissue distribution obtained with an enzyme-linked immunosorbent assay revealed a high concentration of the anti-inhibitor-antiserum-reactive antigen in bovine lactating but not in non-lactating mammary gland tissue and in milk fat globule membranes. Some reactivity was also found in bovine lung. These data are interpreted with respect to a possible physiological significance of the growth inhibitor.

Animals↗

PubMed1985

Secretion of phosphoproteins associated with neoplastic transformation and with the action of transforming growth factors.

Incubation of conditioned media derived from mammary epithelial cells and from fibroblasts with [gamma-32P]ATP revealed much higher intensities of labeled polypeptides in transformed cells compared to normal cells. Conditioned media from human mammary carcinoma cells have in common the presence of characteristic phosphoproteins with molecular masses of about 37,22 and 19.5 kDa. Ehrlich Ascites Mammary Carcinoma Cells secrete a dominant 32 kDa phosphoprotein. Normal fibroblasts secrete elevated levels of phosphoproteins after treatment with transforming growth factors. A phosphoprotein with molecular mass of about 37 kDa becomes secreted preferentially if cell-conditioned media were labeled in vivo. The results indicate that the phosphoproteins comprise a family of secretory polypeptides associated with early steps of transformation.

Animals↗

PubMed1985

Demonstration of transforming growth factor activity in mammary epithelial tissues.

Transforming growth factor (TGF) activity has been demonstrated in acid-ethanol extracts of bovine mammary gland, of Ehrlich Ascites Mammary Carcinoma Cells, and of the ascites fluid. The extracts differ in their activity in the soft agar test when using either human or rat fibroblasts. The most active extract obtained from mammary gland tissue was chromatographed and the TGF activity shown to be coeluting with EGF receptor-competing activity. The present data and our previous reports show that TGFs and a growth inhibitor for mammary epithelial cells coexist in bovine mammary gland as separate growth factors.

Animals↗

PubMed1984

Purification of a growth inhibitor for Ehrlich ascites mammary carcinoma cells from bovine mammary gland.

A growth inhibitor for Ehrlich ascites mammary carcinoma cells in vitro has been purified from bovine mammary gland. The purification procedure involving homogenization and differential centrifugation under hypotonic conditions, ammonium sulfate precipitation, ultrafiltration, gel chromatography and preparative polyacrylamide gel electrophoresis (PAGE) yielded an inhibitor showing half-maximal inhibition of cell proliferation in concentrations of 1-3 ng protein per ml. Upon 125I labelling and analysis by SDS gel electrophoresis, most purified preparations revealed a single band of 12-14 kD, likely to be representative for the inhibitory protein. The inhibitor was shown to affect resumption of proliferation of stationary cells; however, it was inactive towards cells stimulated by incubation with medium before adding the inhibitor. The inhibitor is heat-labile, does not act by exhausting essential components of culture medium, and its action is antagonized by insulin.

Animals↗