Difference between mammary epithelial cells from mature virgin and primiparous mice. (1/64)

Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.  (+info)

Lactose and major milk proteins are present in secretory vesicle-rich fractions from lactating mammary gland. (2/64)

Preparations enriched in apparently intact secretory vesicles were isolated from homogenates of lactating rat and bovine mammary tissue by differential and density gradient centrifugation in isoosmotic media. Morphologically these preparations consisted nearly entirely of vesicles of varying sizes, at least some of which contained casein micelles. Endoplasmic reticulum vesicles, Golgi apparatus cisterna and dictyosomes, mitochondria, peroxisomes, lysosomes, and nuclei were not observed in secretory vesicle-rich fractions. Vesicle preparations were enriched in lactose relative to total membrane fractions from mammary gland. The galactosyltransferase of lactose synthase (UDPgalactose: D-glucose 4 beta-galactosyl-transferase, EC 2.4.1.22) was also present in secretory vesicle preparations, alphas1- and beta-caseins, alpha-lactalbumin, and beta-lactoglobulin, the major secretory proteins of differentiated mammary epithelial cells, were identified as constituents of vesicle-rich fractions from bovine mammary gland. These observations suggest that the major carbohydrate and major proteins of milk are compartmentalized into secretory vesicles and are secreted by exocytotic fusion of secretory vesicles with the apical plasma membrane.  (+info)

Biosynthesis of beta1,4- and beta1,beta1-galactopyranosyl xylopyranosides in the mammary gland of lactating cow. (3/64)

Lactose is a principal carbohydrate in nearly all species of mammalian milk. In order to examine the acceptor substrate specificity of lactose synthase in vivo, D-xylose as an acceptor substrate was injected into the jugular vein of a Holstein cow during lactation, then a milk sample obtained by milking. beta1, beta1-galactopyranosyl xylopyranoside, a nonreducing disaccharide, was separated from the bovine milk sample after elimination of reducing sugars, identified by fast-atom bombardment (FAB)-MS and 1H-NMR analysis. A mixture of beta1,beta1- and beta1, 4-galactopyranosyl xylopyranoside fractions was also obtained by thin layer chromatography from the milk sample and elucidated by electrospray ionization (ESI)-MS and 1H NMR analysis. Comparison of the integrated intensity of the products shows that the beta1,beta1 and beta1,4 isomers are present in a ratio of 1.0 : 1.4, suggesting that D-xylose, transported from capillary blood across the plasma membrane of the mammary gland, was recognized by lactose synthase in its normal and reverse orientation owing to high symmetry of its structure. While the beta1,4-isomer is known as a fragment of the linkage region between the protein and the polysaccharide chain of proteoglycans, the beta1,beta1-isomer has not been identified in vivo. Here, we demonstrate that galactosylation of D-xylose transported from the capillary blood can occur by lactose synthase catalysis in the mammary gland while the usual galactosylation of D-glucose proceeds. In addition, these results suggest that the possibility of endogenous occurrence of the beta,beta-trehalose type disaccharide in the mammary gland of lactating mammals may not be ruled out.  (+info)

Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones. (4/64)

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.  (+info)

High affinity oestradiol receptors and the activity of glucose-6-phosphate dehydrogenase and lactose synthetase in mammary carcinomata of postmenopausal women. (5/64)

The determination of hormone inducible proteins in endocrine tumours may yield information about the presence of hormone dependent tumour cells. We have estimated the high affinity oestradiol binding capacity in primary mammary carcinomata of 57 postmenopausal patients. Glucose-6-phosphate dehydrogenase and lactose synthetase are known from animal experiments to be hormone inducible. Therefore, in biopsies of sufficient size the activity of glucose-6-phosphate dehydrogenase (47 pateints) and lactose synthetase (23 patients) was also studied. It was found that biopsies with high binding capacity also showed high activities of glucose-6-phosphate dehydrogenase and lactose synthetase A protein (galactosyl transferase). No lactose synthetase B protein (alpha-lactalbumin) has been discovered in the tumours. The present observations may be considered suggestive evidence of a relationship between high oestradiol binding capcity and high activities of the two enzymes on the one hand and hormone dependence of the tumour on the other. However, further clinical studies are required before final conclusions in this respect can be drawn.  (+info)

Characterization of a murine beta 1-4 galactosyltransferase expressed in COS-1 cells. (6/64)

We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGT1-directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGT1-transfected cells was approximately eightfold higher than that in mock- or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGT1-transfected cells and mock- or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal beta-galactosidase, most of the linkage created by this enzyme was in the Gal beta 1-4GlcNAc group. The acceptor specificity of the pCMGT1-directed enzyme was changed from N-acetylglucosamine to glucose by adding alpha-lactalbumin in the reaction mixture. Alpha-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent Km values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP-Gal are 2 mM, 60 microM and 24 microM, respectively. These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a beta 1-4 linkage.  (+info)

Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase). (7/64)

1. Manganese(II) buffers were set up with inorganic triphosphate, trimethylenediaminetetraacetate and tetramethylenediaminetetraacetate to study the Mn dependence of beta 1,4-galactosyltransferase (lactose synthetase) in preparations of rat mammary gland. 2. In intact particulate preparations, treated with the calcium ionophore A23187, lactose synthesis was abolished by chelators and restored by bivalent transition metal ions in a manner characteristic of activation site I of the pure enzyme. Ni(II) also activated, as did Mg at high concentration. 3. Only Mn(II) could restore endogenous rates, giving an apparent Km of 0.1-0.2 microM, and eliciting about 70% full activity without addition of a site II activator. 4. In purified Golgi membrane vesicles, Mn gave an apparent Km of 0.4 microM. This increased sharply to about 10 microM on permeabilization with filipin, lysis with detergents, solubilization with Triton X-100, or in the pure enzyme. Preparations of chemically undamaged Golgi vesicles, known to include a proportion of the enzyme on exposed membranes, exhibited both low-Km and high-Km components. 5. The response of particulate galactosyltransferase to apparently physiological concentrations of free Mn(II) ion is interpreted as either due to a sensitizing factor within the Golgi lumen, or to the accumulation of Mn at elevated concentrations. Alternatively, the high Km of the soluble enzyme may reflect proteolytic damage.  (+info)

Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA. (8/64)

Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.  (+info)