Biomedical subjects

J Enami

Publications and source records attributed to J Enami.

31 records · Page 2Linked to original sources

PubMed1993

Mouse gamma-casein cDNA: PCR cloning and sequence analysis.

A partial amino acid sequence of mouse gamma-casein was determined on a gas-phase amino acid sequencer. The N-terminal sequence (23 residues) was obtained using native gamma-casein, and the C-terminal sequence (13 residues) was determined with a corresponding peptide. The C-terminal peptide was isolated by affinity chromatography on an anhydrotrypsin agarose column after digestion of gamma-casein with Achromobacter lysyl-endopeptidase. A cDNA (507 base pairs) encoding the mature protein of gamma-casein was produced by polymerase chain reaction (PCR) amplification of lactating mammary gland first strand cDNA with oligonucleotide primers designed from the N-terminal (KHEIKDK-) and the C-terminal (-AHYTRFY) amino acid sequences. The full-length cDNA including 5'- and 3'-noncoding regions (920 base pairs) was cloned by the rapid amplification of cDNA ends (RACE) method of Frohman et al. Comparison of full-length cDNAs of mouse gamma-casein and rat gamma-casein showed 80% identity at the nucleotide level. The amino acids in the coding region of both gamma-caseins were 75% identical.

Amino Acid Sequence↗

PubMed1992

A single-base-pair substitution abolishes D-amino-acid oxidase activity in the mouse.

Mutant ddY/DAO- mice lacking D-amino-acid oxidase (DAO) activity were examined for the cause of their lack of enzyme activity. Total RNA was extracted from the kidney of the ddY/DAO- mice and cDNA was synthesized. After cDNA encoding DAO was amplified by the polymerase chain reaction it was cloned into a plasmid and sequenced. Comparison of the DAO cDNA sequence with that of normal BALB/c mice revealed the presence of a single-base substitution (G----A) which causes a Gly-181----Arg substitution in the middle of the enzyme molecule. The mutant DAO cDNA was inserted into an expression vector and was expressed in transfected COS-1 cells. The transfected cells synthesized the DAO mutant protein, but they did not show DAO activity. In contrast, when cells were transfected with an expression vector carrying wild-type DAO cDNA, where the substituted base-pair was replaced by a normal base-pair, they showed DAO activity. These results indicate that the single base-pair substitution is the cause of the loss of DAO activity in the ddY/DAO- mice.

Animals↗

PubMed1989

Growth and morphogenesis of mouse prostate epithelial cells in collagen gel matrix culture.

Epithelial cells were isolated from the ventral prostate gland of the mouse after prolonged incubation in a mixture of collagenase, Dispase and hyaluronidase followed by extensive pipetting. The isolated epithelial cells were then embedded in collagen gels. After cultivation in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, epidermal growth factor, 5 alpha-dihydrotestosterone and cortisol, stimulation of growth and branching morphogenesis of the epithelial cells were observed. Under these culture conditions, growth of contaminating fibroblastic cells was rarely seen. These observations suggest that hormones including androgen directly stimulate the growth and morphogenesis of mouse prostate epithelial cells in culture.

Androgens↗

PubMed1983

Growth of normal and neoplastic mouse mammary epithelial cells in primary culture: stimulation by conditioned medium from mouse mammary fibroblasts.

Conditioned medium obtained from mouse mammary fibroblasts markedly stimulated both DNA synthesis and cell proliferation in primary monolayer cultures of mouse mammary tumor cells. Growth of normal mammary epithelial cells from virgin female mice was likewise stimulated by the conditioned medium. The growth-stimulating activity was absent in conditioned media from 3T3 cells, 3T6 cells, mouse embryonic and lung fibroblasts, mammary tumor cells and normal mammary epithelial cells. Stimulation of DNA synthesis in mammary tumor cells was also observed with epidermal growth factor (EGF), although the effect was less than that observed with the conditioned medium. Receptor studies using 125I-labeled EGF further showed that the specific binding of EGF to mammary tumor cells was not inhibited by the conditioned medium. The molecular weight of the growth-stimulating activity in mammary fibroblast-conditioned medium was estimated to be approximately 100,000 daltons by Sephadex G-200 column chromatography. These observations suggest that mammary fibroblasts, which constitute the stroma of the mammary gland, produce a mammary epithelial cell growth factor(s) distinct from EGF.

Animals↗

PubMed1979

Simultaneous production of casein and mammary tumor virus in mouse mammary epithelial cells grown on floating collagen gels.

Simultaneous production of casein and mammary tumor virus (MTV) was analyzed in monolayer cultures of mammary epithelial cells from pregnant BALB/cfC3H mice. A comparison of the 2 cell culture substrata, plastic culture dish and floating collagen gel, showed that the latter supported a much higher degree of simultaneous casein and MTV production in the presence of insulin, cortisol and prolactin in serum-free culture medium. Importance of floating collagen gel was further shown by delaying the flotation of gels. When the release of gels was delayed, there were concomitant delays in the increase of casein and MTV production. These results indicate that hormones, nature of substratum and flotation regulate the degree of differentiation of mammary epithelial cells in vitro.

Animals↗

PubMed1979

Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.

We have developed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H]thymidine incorporation during 8 weeks in culture. An increase in DNA content of the cultured cells as a function of time was observed. Mouse mammary tumor cells cultured in the conventional monolayer system failed to show any significant increase in cell number during a culture period of 6 weeks. In addition, in such monolayer systems, cells progressively became detached from the dishes in long-term culture. The mammary epithelial cell origin of the collagen gel cell outgrowths was shown by electron microscopic demonstration of polarized cells containing tight junctions and budding mammary tumor virus particles. In addition, in vivo transplantation of collagen gel outgrowths resulted in the development of mammary adenocarcinoma histologically similar to the donor tumor. Cellular outgrowth patterns resembling those from tumor cells were also seen in similar collagen gel cultures of normal mammary cells from mouse and human and of hyperplastic alveolar nodule cells from mouse. The significance and usefulness of this system in comparison to the conventional monolayer system are discussed.

Animals↗

PubMed1978

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels.

The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion.

Animals↗

PubMed1978

Prolactin receptor on dissociated mammary epithelial cells at different stages of development.

Properties of prolactin receptors were measured by monitoring [125I]prolactin binding to specific receptor sites on collagenase-dissociated mammary epithelial cells of virgin, pregnant and lactating mice. On a Scatchard plot the data generated a straight line and the estimated dissociation constant (Kd) and number of receptor sites on lactating cells were 0.9 x 10(-9) and 1540 per cell. The [125I]prolactin binding was inhibited in presence of unlabeled prolactin and other lactogenic polypeptide hormones, but not by nonlactogenic polypeptide hormones. The [125I]prolactin binding was sensitive to pronase and trypsin but not to DNAase, RNAase and hyaluronidase. Scatchard plot analysis further showed that while the number of receptors on mammary cells was variable at different stages of endocrine regulated developmental changes of the gland, Kd of the hormone--receptor complex generally remained similar. The high level of prolactin receptors on mammary cells of virgins was reduced during pregnancy and the lactating mammary cells showed a highly elevated level of prolactin receptors. The results demonstrate that specific prolactin receptors can be measured on collagenase dissociated mammary epithelial cells and this method permits a direct assessment of the number of receptors on a per cell basis rather than indirect estimates, based on average DNA or protein content of the tissue, composed of heterogeneous cell types.

Animals↗

PubMed1978

Secretion of casein in cultures of mouse mammary epithelial cells on floating collagen gels.

To study the effect of hormones on the secretion of casein, mammary epithelial cells were obtained from mid-pregnant mice and cultured on floating collagen gels in the presence of combinations of insulin, cortisol, and prolactin in serum-free culture medium. After labeling with phosphorus-32 labeled orthophosphate, the cells which had been cultured in the presence of all three hormones secreted hot-trichloroacetic acid precipitable radioactivity into the medium. The secreted materials were identified as three major mouse caseins by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis followed by autoradiography. The floating collagen gel culture method allows mouse mammary epithelial cells to differentiate and secrete all species of caseins in the presence of hormones. The present culture method and the phosphorus-32 labeling method will be applicable to the study of differentiation of mammary epithelial cells from a variety of mammalian species.

Animals↗

PubMed1977

A sensitive radioimmunoassay for a component of mouse casein.

Mouse casein (m.w. 22,000 daltons) has been purified by employing Sephadex G-100 and DEAE-cellulose column chromatographies. A sensitive radioimmunoassay method has been developed by using [125I]-labelled casein and antiserum elicited in rabbits after injection of glutaraldehyde-treated casein. The assay method is capable of detecting as little as 0.1 ng of casein. The use of the present radioimmunoassay method in detecting casein production in cultured mouse mammary explants has also been demonstrated.

Animals↗

PubMed1977

Hormonal control of milk protein synthesis in cultured mouse mammary explants.

SDS-urea polyacrylamide gel electrophoresis of mouse milk proteins revealed the presence of three major phosphoproteins (caseins) of m.w. 44,000, 26,000 and 22,000 daltons. By using an antiserum against crude casein fraction, an immunoprecipitation method was developed for the quantitative measurement of the rate of milk protein synthesis in the mouse mammary tissue. Cultivation of mammary explants with insulin, cortisol and prolactin resulted in the induction of milk protein synthesis as evidenced by the incorporation of [3H]amino acid and [32P]orthophosphate into immune precipitable materials. The present immunoprecipitation method coupled with a simplified explant culture technique provides a suitable procedure for the study of mouse mammary gland differentiation.

Animals↗

PubMed1977

Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes.

Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level.

Animals↗

PubMed1977

Regulation of mammary tumor virus production by prolactin in BALB/cfC3H mouse normal mammary epithelial cells in vitro.

Hormonal regulation of mammary tumor virus (MTV) production, has been analyzed with normal mammary epithelial cells from chronically infected BALB/cfC3H mice. The effect of prolactin in terms of increased MTV production was not reproducibly observed in cells cultured in tissue culture dishes, whereas the cells grown on floating collagen gels consistently responded to prolactin in a dose-dependent manner. Of the three media tested. Dulbecco's modified Eagle's medium was found to be the best in terms of responsiveness to prolactin and in maximal MTV production. Specificity studies with other pituitary and placental hormones in place of prolactin have shown that both growth hormones and human placental lactogen can replace prolactin, whereas follicle-stimulating hormone, luteinizing hormone, and thyrotrophin were ineffective. Contrary to the mammary tumor systems, where it has been shown that insulin and glucocorticoid can maximally stimulate MTV production, these hormones alone elicited only a small response in the absence of prolactin in normal mammary epithelial cells. Although prolactin alone had very little effect by itself, its presence was necessary (permissive effect) in order for the glucocorticoids to be able to maximally stimulate MTV production in normal cells.

Animals↗