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Y Tomooka

Publications and source records attributed to Y Tomooka.

53 records · Page 3Linked to original sources
PubMed1987

Evidence for isoaspartyl (deamidated) forms of mouse epidermal growth factor.

A variant form of mouse submaxillary gland epidermal growth factor (EGF) was identified by isocratic reversed-phase HPLC of EGF obtained by Bio-Gel P-10 column chromatography ("culture grade"). The variant form was essentially absent in preparations of EGF further purified by chromatography on DEAE-cellulose ("receptor-grade" EGF). The spectral properties and amino acid composition of the variant form (EGF-I) could not be distinguished from those of the intact polypeptide isolated by HPLC (alpha-EGF). Receptor-binding and mitogenic properties of EGF-I were also equivalent to those of alpha-EGF. These data suggested that EGF-I was structurally very similar to EGF. However, the very low yield (less than 4%) obtained by Edman degradation indicated that the N-terminal (Asn1) of the polypeptide was modified. Isoelectric focusing of EGF-I revealed two major immunoreactive bands: one with a pI equivalent to that of alpha-EGF (pI 4.6) and another at pI 4.1. Alkaline treatment of alpha-EGF (0.1 M NH4OH) yielded peak material by HPLC that coeluted with EGF-I; the alkaline-generated EGF-I yielded bands that also focused at pH 4.6 and 4.1. Ammonium hydroxide treatment of [des-Asn1]-EGF (beta-EGF) did not produce conversion to EGF-I. On the basis of these data, we propose that EGF-I was formed by selective deamidation of the N-terminal Asn of intact EGF. This notion is also supported by liquid secondary ion mass spectrometry, which showed that EGF-I was approximately 1.5 mass units greater than alpha-EGF. The heterogeneity observed by isoelectric focusing supports previous studies which have shown that, following deamidation of N-terminal asparagine, a beta-aspartyl shift can occur, which in the present study might yield succinimido-aspartyl1-EGF and beta-aspartyl1-EGF. Low yields observed during Edman degradation indicate that negligible amounts occur as the alpha-aspartyl1-EGF isomer.

Amino Acid Sequence↗
PubMed1986

Immunological studies on histiocytosis X. I. Special reference to the chemotactic defect and the HLA antigen.

We treated a family with three children with histiocytosis X (H-X). The chemotactic response of the neutrophils in these three patients was depressed and the chemotactic response of the neutrophils of the mother was also depressed compared to that of normal age-matched controls. To elucidate the genetic factors, we examined HLA antigens in five members of this family. All five members had Aw24, B7, Cw7, and DR1. Immunological and genetic studies in an additional 32 patients with H-X were performed. The chemotactic response of 35 patients with H-X (154.9 +/- 58.4/HPF) was significantly depressed in comparison with that of 35 age-matched healthy controls (613.3 +/- 116.7/HPF). In addition, the value of chemiluminescence of 20 of 35 patients (20.5 +/- 6.6 mV) was also significantly depressed in comparison with that of 20 normal controls (45.3 +/- 11.4 mV). The frequencies of Bw61 (54.4%) and Cw7 (45.4%) in 33 patients with H-X were significantly increased in comparison with those of 250 normal healthy controls (20.4 and 18.0%, respectively). Studies of immunoglobulin levels and complement titers of patients with H-X showed no consistent abnormalities. We proposed that defects of polymorphonuclear function may lead to an increased susceptibility to bacterial infections in patients with this disorder.

Adolescent↗
PubMed1986

Proliferation of mouse uterine epithelial cells in vitro.

Although estrogens can stimulate the growth of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro growth of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (21-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal growth factor (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and growth of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by fibroblast growth factor, platelet-derived growth factor, nerve growth factor, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the growth and differentiation of estrogen target cells.

Animals↗
PubMed1985

Growth of seminal vesicle epithelial cells in serum-free collagen gel culture.

Epithelial cells from mouse seminal vesicles were enzymatically dissociated, enriched by gradient centrifugation, and maintained in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morphologically, immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 microgram/ml). The cells required EGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin, and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free media. A time-course study revealed that a lag period preceded rapid growth. The collagen gel, serum-free culture provides a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells.

Animals↗
PubMed1985

Stimulation of mammary epithelial cell growth in vitro: interaction of epidermal growth factor and mammogenic hormones.

A serum-free primary cell culture system was used to examine the direct effects and interactions of mammogenic hormones and epidermal growth factor (EGF) on the growth of mouse mammary epithelial cells. Epithelial cells were isolated by collagenase dissociation followed by Percoll gradient centrifugation and cultured within collagen gels in a mixture of Ham's F-12-Dulbecco's Minimum Essential Medium (1:1) containing insulin (10 micrograms/ml), crude soybean lecithin, trace elements, trypsin inhibitor, and antioxidants. Progesterone (P; 10(-6) - 10(-8) M) or ovine PRL (1 microgram/ml), in the absence of EGF, stimulated the growth of cells from mature virgin mice 2- to 4-fold over that of controls cultured in basal medium only. P and PRL synergized in stimulating growth 3- to 17-fold. 17 beta-Estradiol (10(-7) - 10(-10) M) alone did not stimulate growth or synergize with P and/or PRL. This lack of growth stimulation by 17 beta-estradiol was also observed in medium containing a low concentration of insulin (0.1 microgram/ml). EGF (10 ng/ml) alone stimulated growth to the same extent as the combination of P and PRL. EGF at 1, but not 10, ng/ml when combined with P and PRL could additively stimulate growth. Cells from midpregnant mice were less responsive than cells from virgin mice to the growth-stimulating effects of the combination of P and PRL (2-fold stimulation at most), but not to EGF (3- to 6-fold stimulation). Corticosterone, deoxycorticosterone, and aldosterone, but not cortisol, could synergize with PRL in stimulating the growth of cells from mature virgin mice. However, only deoxycorticosterone could stimulate growth in the absence of PRL. These results suggest that PRL, P, and adrenal corticoids may directly stimulate the growth of mouse mammary epithelial cells. The physiologically relevent adrenal corticoids, corticosterone and aldosterone, only potentiate the stimulatory effect of PRL. The hormonal stimulation of growth in vitro can be obscured by an optimum concentration (10 ng/ml) of EGF. The relative growth responses to mammogenic hormones and EGF may depend on the degree of differentiation of the cells.

Adrenal Cortex Hormones↗
PubMed1984

Collagen gel culture system and analysis of estrogen effects on mammary carcinogenesis.

The results obtained to date from studies dealing with the role of hormones, including estrogen, on growth of mammary epithelial cells inside the collagen gel are described. The collagen gel matrix culture system appears to be a suitable system to obtain in vivo-like effects of hormones on mammary cell in vitro. The results thus far indicate that prolactin along with progesterone or cortisol can stimulate mammary cell proliferation. Thus far, estrogen has not been found to be mitogenic in our in vitro system.

Animals↗
PubMed1983

Growth effect of lithium on mouse mammary epithelial cells in serum-free collagen gel culture.

The effect of lithium on the growth of mammary epithelial cells from adult virgin and midpregnant BALB/c or BALB/cfC3H mice was tested in a serum-free collagen gel culture system. The serum-free medium consisted of a 1:1 mixture of Ham's F12 and Dulbecco's Modified Eagle's medium supplemented with insulin, transferrin, cholera toxin, epidermal growth factor (EGF), and bovine serum albumin fraction V (BSA V). A multifold increase in cell number occurred during 10-12 days of culture in this medium. In dose-response studies in which the concentration of each component of this serum-free medium was varied in turn, the addition of LiCL (10 mM) enhanced growth at most concentrations of each factor. However, LiCl could not enhance growth in the absence of insulin or BSA V, but could replace EGF. The optimal concentration of LiCl was 5-10 mM; higher concentrations (20-80 mM) were toxic. KCl (1-10 mM) when added to the serum-free medium slightly stimulated growth; the addition of NaCl to the medium had little effect on growth. LiCl did not enhance the growth of cells from spontaneous mammary tumors of BALB/cfC3H mice.

Animals↗
PubMed1983

Growth of mammary epithelial cells from neonatally sex hormone-exposed mice in serum-free collagen gel culture.

Neonatal sex hormone treatments are known to cause an increase in mammary tumors in female mice with expressed mammary tumor virus (MTV). The growth of mammary epithelial cells from mice treated neonatally with sex hormones was studied in response to growth-stimulatory factors in a serum-free collagen gel culture system which sustains the growth of normal mammary epithelial cells. Animals were treated with hormones or oil-vehicle for the first 5 days after birth. Cells from control mice at 2 and 3 months of age showed a maximal growth response to insulin at 5-10 micrograms/ml and LiCl at 5-20 mM. Cells responded to epidermal growth factor at all concentrations used (1, 10 and 50 ng/ml). In contrast, mammary epithelial cells from mice treated neonatally with estrogen (estradiol and diethylstilbestrol (DES] showed a reduced growth response to the growth factors tested.

Animals↗
PubMed1982

Serum-free growth of normal and tumor mouse mammary epithelial cells in primary culture.

Freshly isolated normal and tumor mouse mammary epithelial cells embedded within a collagen gel matrix undergo sustained growth when cultured for as long as 3 wk in a serum-free medium composed of a 1:1 (vol/vol) mixture of Hepesbuffered Ham's F12 and Dulbecco's modified Eagle's medium supplemented with insulin, epidermal growth factor (EGF), transferrin, bovine serum albumin fraction V, and cholera toxin. Of these additives, only insulin, EGF, and albumin are required for the growth of most normal cells. Albumin is not always an absolute requirement for growth but greatly enhances it. Lithium has been found to stimulate the growth of normal cells and can replace EGF. The collagen matrix culture system allows sustained growth of primary cultures of both normal and neoplastic mammary epithelium in serum-free conditions. This serum-free system will be useful in identifying and investigating the role of hormones, growth factors, and nutritional factors in regulating the growth of mammary epithelial cells.

Animals↗
PubMed1982

Growth of mouse mammary glands after neonatal sex hormone treatment.

Mammary gland growth was examined in female mice immediately and during 1 month after neonatal treatment with sex hormones. BALB/c mice were given daily injections of hormones for the first 5 days after birth. As a criterion of growth, the number of ductal branchings was counted in stained wholemounts of the fourth (inguinal) pair of glands. At day 6, the growth of the glands was significantly inhibited in animals treated with 17 beta-estradiol (E) or diethylstilbestrol (DES); after treatment with progesterone (P) or androgens, no immediate effect was evident. At day 33, neonatal treatment with E stimulated growth, as did neonatal treatment with testosterone (T) or 5 alpha-dihydrotestosterone (5 alpha-DHT); treatment with a high dose of DES inhibited growth. Treatment with P or 5 beta-dihydrotestosterone had no effect. In conclusion, neonatal treatment with sex hormones has two distinct effects on the growth of mouse mammary gland: 1) inhibition by estrogens, observed immediately after treatment, and 2) stimulation by E, T, and 5 alpha-DHT, observed 4 weeks after treatment.

Animals↗
PubMed1981

Treatment of congenital dilatation of the choledochus- reconstruction of the bile duct after resection of the dilated choledochus.

Reconstruction of the way of bile flow was attempted in 21 cases with the resection of the dilated bile duct, and the post-operative courses were observed over a long period. It was found that ascending cholangitis is seldom caused by the hepaticoduodenostomy. It is important, when hepaticoduodenostomy is performed, that the sound section of the wall of the bile duct without stenosis in the hepatic side should be selected. Reconstruction of the way of the bile flow to prevent ascending cholangitis by leading normal bile flow into the duodenum should be devised.

Adolescent↗
PubMed1978

Electron microscopic study of the response of the adrenocortical X-zone in mice treated with sex steroids.

In the adrenal cortex of 23- to 27-day-old C3H/Tw female mice, the eosinophilic X-zone became increasingly increasingly undetectable after 3 injections of 100 microgram testosterone propionate (TP). Whorls of smooth endoplasmic reticulum (sER) and peculiar complexes of mitochondria and sER, characteristics of X-zone cell, were no longer present in mice given 7 daily injections of TP. The ordinary mitochondria, although reduced in number, became swollen and actually increased in percent area occupied. They had well-developed tubulovesicular cristae. The lipid droplets increased in size and number after 3 daily TP injections, but decreased after 7 daily injections. Rough endoplasmic reticulum and sER were reduced in area in mice receiving 7 daily injections. The X-zone also became indistinguishable from the zona fasciculata after 7 daily injections of 5alpha-dihydrotestosterone propionate. Injections of progesterone or estradiol-17 beta had no effect on the X-zone.

Adrenal Glands↗