Glycoprotein synthesis in explants of developing rabbit mammary gland in culture.
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ABSTRACT:
SUBMITTER: Bradshaw JP
PROVIDER: S-EPMC1163318 | biostudies-other | 1981 Sep
REPOSITORIES: biostudies-other
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Project description:1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.
| S-EPMC1161419 | biostudies-other
Project description:1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.
| S-EPMC1183894 | biostudies-other
Project description:1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37 degrees C in air for 24-48h in Medium 199 buffered with 20mm-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na(+)/K(+)-dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K(+), and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na(+) and K(+) concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37 degrees C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K(+) in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10mum) and increased Na(+) and decreased K(+) concentrations in the tissue. 3. Ouabain at concentrations up to 1mum did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1mum-ouabain. 4. Explants cultured in the presence of 2mum-ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na(+)/K(+)-dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.
| S-EPMC1185725 | biostudies-other
Project description:1. A cannulation technique is described for measuring arteriovenous differences across the lactating-rabbit mammary gland. 2. Analysis of milk obtained before and after surgery shows no effect of cannulation on milk constituents. 3. Results of blood analysis show significant net changes in the concentrations of glucose, acetate, 3-hydroxybutrate, triacylglycerols and non-esterified fatty acids across the mammary gland. 4. The molar proportions of individual fatty acids in both the triacylglycerol and non-esterified fatty acid fractions did not alter between the arterial and venous samples. 5. The extraction rates are compared with those obtained from other species.
| S-EPMC1185909 | biostudies-other
Project description:Extensive data suggest that estradiol contributes to the development of breast cancer by acting as a mitogen and exerting direct genotoxic effects after enzymatic conversion to 4-hydroxyestradiol (4-OHE2) via cytochrome P450 1B1 (CYP1B1). The mammary gland, ovary, and uterus all express CYP1B1. Overexpression of this enzyme has been associated with an increased risk of breast cancer and blockade might reduce this carcinogenic effect. For this reason, we conducted systematic in vitro and in vivo studies of a CYP1B1 inhibitor, TMS (2,3',4,5'-tetramethoxystilbene). We found that TMS blocked the enzymatic conversion of radiolabeled estradiol to both 2-hydroxyestradiol (2-OHE2) and 4-OHE2, but did not inhibit Cyp1b1 message formation. In vivo studies using mass spectrometry showed that TMS inhibited formation of 2-OHE2 and 4-OHE2 and the resulting estrogen-DNA adducts. To examine its biologic actions in vivo, we investigated whether TMS could block the hyperplastic changes that occur in the developing breast of aromatase-transfected mice. We found that TMS induced a significant reduction of ductal structures in mice less than 6 months in age. In older mice, no reduction in breast morphology occurred. These latter studies uncovered unexpected estrogen agonistic actions of TMS at high doses, including a paradoxical stimulation of breast ductal structures and the endometrium. These studies suggest that the enzyme inhibitory properties of TMS, as well as the effects on developing breast, could implicate a role for TMS in breast cancer prevention, but only in low doses and on developing breast.
| S-EPMC4443710 | biostudies-literature
Project description:The biosynthesis of fatty acids has been studied in lactating rabbits at 6h after intravenous injection of sodium [1-(14)C]acetate. The specific radioactivities of the individual fatty acids (C(6:0) to C(14:0)) and the proportions of these fatty acids synthesized were similar in mammary tissue and milk. Hexanoic acid had the highest specific radioactivity, and the C(8:0)-C(14:0) fatty acids had similar specific radioactivities, which were about five times those of C(16) and C(18) acids. No radioactivity was detected in fatty acids of chain length <C(14) in the liver, blood or adipose tissue and the specific radioactivities of fatty acids of chain length >C(14) in these tissues were similar to those of the long-chain fatty acids in the milk and mammary gland. The results show that the C(4:0)-C(14:0) fatty acids are synthesized within the mammary gland rather than by fatty acid uptake from circulating blood or by oxidation of long-chain fatty acids within the gland. We conclude that de novo synthesis of esterified fatty acids in vivo by this tissue has a high degree of chain-length specificity.
| S-EPMC1178508 | biostudies-other
Project description:1. Explants of mammary glands of mid-pregnant rabbits that had been cultured for 18h in the presence of insulin, prolactin and cortisol were incubated at 37 degrees C for 2h in Medium 199 containing l-[4,5-(3)H]leucine. After a wash procedure at 4 degrees C, explants were re-incubated at 37 degrees C in fresh medium and the radioactivity of casein polypeptides isolated by isoelectric focusing (at pH 4.6) was followed with time. Casein radioactivity rose during the first hour of re-incubation, but fell markedly during the subsequent hour. 2. Loss of radioactivity represented casein degradation, since less than 10% of newly synthesized casein was found in the incubation medium. 3. Such a loss of radioactivity was not due solely to hydrolysis of signal peptides, since similar results were obtained when l-[5-(3)H]proline, which is not part of casein signal peptides, was the radiolabelled precursor. 4. A dual-isotope experiment using l-[U-(14)C]proline and N-[(3)H]acetyl-d-mannosamine gave similar profiles of radioactivity loss from isoelectrically focused casein, indicating that degradation of mature casein was occurring. 5. Analysis of total pellet and particle-free-supernatant fractions prepared by centrifugation of explant homogenates at 115000g(av.) for 1h did not show loss of radioactivity on re-incubation. Total pellet-protein radioactivity remained constant, whereas total soluble-protein radioactivity increased during the 2h re-incubation period. 6. Radioactivity in a specific particle-free-supernatant polypeptide, the subunit of fatty acid synthetase, mimicked that of the total soluble protein. 7. Addition of cycloheximide (20mug/ml) during the re-incubation period completely blocked the incorporation of radioactivity from l-[5-(3)H]proline into casein and the subsequent fall, indicating that observations were being made on newly synthesized casein. 8. Addition of chloroquine (50mum) did not prevent the increase in radioactivity from l-[5-(3)H]proline into casein during the first hour of re-incubation, but did prevent the loss of radioactivity in the second hour. 9. The intracellular degradation of a newly synthesized milk protein is discussed in relation to the known intracellular degradation of other secretory polypeptides.
| S-EPMC1158082 | biostudies-other
Project description:1. Excellent precipitating antibodies to rabbit recombined casein polypeptides were obtained in a sheep after 8 weeks of immunization with rabbit recombined polypeptides coupled to Sepharose-albumin. 2. The antiserum was assessed for specificity by several immunochemical techniques and was monospecific when tested against acid-precipitated casein, recombined casein and extracts of lactating rabbit mammary tissue. 3. A specific anti-casein immunoglobulin fraction was prepared by immunoadsorption of the antiserum by using Sepharose-recombined casein as immunoadsorbent. 4. The specific anti-casein immunoglobulin was used to prepare a Sepharose-anti-casein immunoadsorbent for the isolation of casein from extracts of rabbit mammary tissue.
| S-EPMC1185854 | biostudies-other
Project description:The proportion of C(8:0) and C(10:0) fatty acids synthesized by the microsomal plus particle-free supernatant fraction from lactating rabbit mammary gland is enhanced at high protein concentrations. This fraction appears to contain a soluble high-molecular-weight factor that modifies the specificity of the fatty acid synthetase complex for termination of the growing acyl chain.
| S-EPMC1177567 | biostudies-other
Project description:The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast tumors compared to normal breast tissue. Cdc42 regulates key processes that are critical for mammary gland morphogenesis and become disrupted during the development, progression, and metastasis of breast cancer. However, the contribution of Cdc42 to normal and neoplastic mammary gland development in vivo remains poorly understood. We were therefore interested in investigating the effects of Cdc42 overexpression on mammary gland morphogenesis as a first step toward understanding how its overexpression may contribute to mammary tumorigenesis.We developed a tetracycline-regulatable Cdc42 overexpression mouse model in which Cdc42 can be inducibly overexpressed in the developing mammary gland. The effects of Cdc42 overexpression during postnatal mammary gland development were investigated using in vivo and in vitro approaches, including morphometric analysis of wholemounted mammary glands, quantification of histological markers, and primary mammary epithelial cell (MEC) functional and biochemical assays.Analysis of Cdc42-overexpressing mammary glands revealed abnormal terminal end bud (TEB) morphologies, characterized by hyperbudding and trifurcation, and increased side branching within the ductal tree. Quantification of markers of proliferation and apoptosis suggested that these phenotypes were not due to increased cell proliferation or survival. Rather, Cdc42 overexpressing MECs were more migratory and contractile and formed dysmorphic, invasive acini in three-dimensional cultures. Cdc42 and RhoA activities, phosphorylated myosin light chain, and MAPK signaling, which contribute to migration and invasion, were markedly elevated in Cdc42 overexpressing MECs. Interestingly, Cdc42 overexpressing mammary glands displayed several features associated with altered epithelial-stromal interactions, which are known to regulate branching morphogenesis. These included increased stromal thickness and collagen deposition, and stromal cells isolated from Cdc42 overexpressing mammary glands exhibited elevated mRNA expression of extracellular matrix proteins and remodeling enzymes.These data suggest that Cdc42 overexpression disrupts mammary gland branching morphogenesis by altering Rho GTPase and MAPK signaling, leading to increased MEC contractility and migration in association with stromal alterations. Our studies provide insight into how aberrant Cdc42 expression may contribute to mammary tumorigenesis.
| S-EPMC3978759 | biostudies-literature