Lactating Rabbit Mammary Gland Research Articles

Loss of Membrane Targeting of Vangl Proteins Causes Neural Tube Defects

In the mouse, the loop-tail mutation (Lp) causes a very severe neural tube defect, which is caused by mutations in the Vangl2 gene. In mammals, Vangl1 and Vangl2 code for integral membrane proteins that assemble into asymmetrically distributed membrane complexes that establish planar cell polarity in epithelial cells and that regulate convergent extension movements during embryogenesis. To date, VANGL are the only genes in which mutations cause neural tube defects in humans. Three independently arising Lp alleles have been described for Vangl2: D255E, S464N, and R259L. Here we report a common mechanism for both the naturally occurring Lp (S464N) and a novel ENU-induced mutation Lp(m2Jus)(R259L). We show that the S464N and R259L variants stably expressed in polarized MDCK kidney cells fail to reach the plasma membrane, their site for biological function. The mutant variants are retained intracellularly in the endoplasmic reticulum, colocalizing with ER chaperone calreticulin. Furthermore, the mutants also show a dramatically reduced half-life of ∼3 h, compared to ∼22 h for the wild-type protein, and are rapidly degraded in a proteasome-dependent and MG132-sensitive fashion. Coexpressing individually the three known allelic Lp variants with the wild-type protein does not influence the localization of the WT at the plasma membrane, suggesting that the codominant nature of the Lp trait in vivo is due to haploid insufficiency caused by a partial loss of function in a gene dosage-dependent pathway, as opposed to a dominant negative phenotype. Our study provides a biochemical framework for the study of recently identified mutations in hVANGL1 and hVANGL2 in sporadic or familial cases of neural tube defects.

Human growth hormone: 45-kDa isoform with extraordinarily stable interchain disulfide links has attenuated receptor-binding and cell-proliferative activities

Background Human growth hormone (hGH) is a complex mixture of molecular isoforms. Gaps in our knowledge exist regarding the structures and biological significances of the uncharacterized hGH molecular variants. Mercaptoethanol-resistant 45-kDa human growth hormone (MER-45 kDa hGH) is an extraordinarily stable disulfide-linked hGH homodimer whose biological significance is unknown. Objectives To elucidate the pharmacokinetic abilities of dimeric MER-45-kDa hGH to bind to GH and prolactin (PRL) receptors and to elucidate its abilities to stimulate cell proliferation in lactogen-induced and somatogen-induced in vitro cell proliferation bioassays. Design The binding of MER-45-kDa hGH to GH and PRL receptors was tested in radioreceptor assays (RRAs). Competitive displacements of [ 125I]-bovine GH from bovine liver membranes, [ 125I]-ovine PRL from lactating rabbit mammary gland membranes and [ 125I]-hGH from human IM-9 lymphocytes by unlabelled GHs, PRLs or dimeric MER-45-kDa hGH were evaluated. The abilities of dimeric MER-45-kDa hGH to stimulate proliferation of lactogen-responsive Nb2 lymphoma cells and to stimulate proliferation of somatogen-responsive T47-D human breast cancer cells were assessed by incubation of cells with GHs or PRLs and subsequently measuring growth using the MTS cell proliferation assay. Results Dimeric MER-45-kDa hGH, compared to monomeric hGH, had reduced binding affinities to both GH and prolactin receptors. In a bovine liver GH radioreceptor assay its ED 50 (197.5 pM) was 40.8% that of monomeric hGH. In a human IM-9 lymphocyte hGH RRA its ED 50 (2.96 nM) was 26.2% that of monomeric hGH. In a lactating rabbit mammary gland prolactin RRA its ED 50 (3.56 nM) was 16.8% that of a monomeric hGH. Dimeric MER-45-kDa hGH, compared to monomeric hGH, had a diminished capacity to stimulate proliferation of cells in vitro. In a dose–response relationship assessing proliferation of Nb2 lymphoma cells its ED 50 (191 pM) was 18.0% that of monomeric hGH. While monomeric hGH stimulated a 2.2-fold proliferation of T47-D human breast cancer cells above vehicle control, dimeric MER-45-kDa hGH was unable to stimulate the cells to proliferate and slightly inhibited their proliferation to 77.6% that of control. Conclusions The topological arrangement of monomeric hGHs to form an unusually stable disulfide-linked dimer markedly diminishes hGH's binding affinities to both GH and PRL receptors and also drastically attenuates its ability to stimulate proliferation of cells in vitro.

Purification and characterization of casein kinase II from lactating rabbit mammary gland

1. 1. Highly purified 200 kDa casein kinase II from rabbit lactating mammary gland (MG-CK II) was obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono Q steps. 2. 2. Its K m for ATP was 2.22 μM and 0.57 mg/ml and 0.13 mg/ml for partially dephosphorylated casein and phosvitin respectively. Stathmine was also suitable as substrate. 2-aminopurine and 6-dimethylaminopurine inhibited efficiently MG-CK II K i = 5 and 1 mM respectively). 3. 3. MG-CK II autophosphorylated on its α-, α '- and β-subunits. The β-subunit auto-phosporylation was enhanced in presence of exogenous substrate. Its modulation was highly dependent on ATP concentration. 4. 4. The effects of basic compounds which affected dramatically the phosphorylation of dephosphorylated casein in presence of various ATP concentrations were reported.

Characterization of casein kinase III from lactating rabbit mammary gland : V. Mitev and L.M. Houdebine. Unité de Différenciation Cellulaire, Institut Nationale de la Recherche Agronomique, 78352 Jouy-en-Josas, France

Characterization of casein kinase III from lactating rabbit mammary gland : V. Mitev and L.M. Houdebine. Unité de Différenciation Cellulaire, Institut Nationale de la Recherche Agronomique, 78352 Jouy-en-Josas, France

The possible involvement of protein kinase C(s) and inositol phosphate metabolism in the basal but not in the prolactin stimulated casein release by the lactating rabbit mammary epithelial cell.

The secretagogue effect of prolactin (PRL) on casein release by epithelial mammary cells has been previously related to stimulation of the phospholipase A2-arachidonic acid cascade. In order to determine whether other intracellular pathways are implicated in this secretagogue effect, different agents acting on protein kinase C (PKC) and phospholipase C (PLC) activity have been assessed in vitro in lactating rabbit mammary gland fragments. Phorbol ester (20 nm TPA and 1-oleoyl-2-acetyl-sn-glycerol (10 microM (OAG) stimulated newly synthesized casein secretion and potentiated the PRL secretatogue effect. However, 100 microM quercetin, 100 microM H-7 and 5 and 20 nM staurosporine did not inhibit the latter effect. Exogenous PLC did not stimulate casein secretion. PRL did not affect production of inositol phosphates (IPs) during 10 or 60 min exposure. These results show that PKC activation may increase basal levels of casein secretion, and demonstrate that PRL does not act primarily via PKC activation or by PLC activation to stimulate casein secretion.

Isolation and biochemical properties of four forms of glycosylated porcine prolactin

Four isoforms of glycosylated prolactin (G-pPRL) were isolated from porcine pituitary glands by affinity chromatography and concanavalin A-Sepharose, based upon differences in their affinity for the lectin. Structural analysis indicated differences in the carbohydrate units of the four G-pPRLs. N-glycanase treatment cleaved the oligosaccharide from the G-pPRLs, establishing N-linked glycosylation. The binding of G-pPRLs to receptors from lactating rabbit mammary glands was only 3–8% that of nonglycosylated pPRL (NG-pPRL). The immunological crossreactivity of the G-pPRLs varied from 36 to 65% that of NG-pPRL. When tested in the pigeon crop sac bioassay, G-pPRLs were only 11–40% as active as NG-pPRL. The metabolic clearance rate of one of the G-pPRLs was slower and another faster than that of NG-pPRL. We conclude that there are several forms of G-PRL of variable immuno- and bio-potencies in the porcine pituitary, and that the current radioimmunoassay for the hormone does not measure the actual bioactivity.

Glycosylated Human Prolactin: Alterations in Glycosylation Pattern Modify Affinity for Lactogen Receptor and Values in Prolactin Radioimmunoassay*

The receptor-binding properties of monomeric nonglycosylated human PRL (hPRL), glycosylated hPRL that does not bind to Concanavalin-A-Sepharose (G1-hPRL) and glycosylated hPRL that binds to Concanavalin-A-Sepharose (G2-hPRL) were tested in the lactating rabbit mammary gland RRA for lactogenic hormones. Variations in the glycosylation pattern of G-hPRL altered its receptor-binding properties, suggesting that the site of glycosylation may be proximal to the receptor-binding region. Relative potencies for the displacement of [125I]hPRL by hPRL, G1-hPRL, and G2-hPRL were 100%, 40%, and 26%, respectively. Relative potencies for displacement of [125I]G1-hPRL by hPRL, G1-hPRL, and G2-hPRL were 100%, 44%, and 69%, respectively; however, the displacement curve for G2-hPRL was not parallel to the others. When G2-hPRL was radiolabeled, there was no specific binding to lactogenic receptors. The presence of PRL receptor subtypes and/or kinetic cooperativity was suggested by the complexity of the binding isotherms. The immunoreactivities of the PRLs were tested in a homologous RIA, using polyclonal antiserum. The modification of the glycosylation pattern of hPRL significantly altered the RIA values for PRL. When hPRL was used as the radiotracer, the percent cross-reactivities of hPRL, G1-hPRL, and G2-hPRL were approximately 100%, 23%, and 17%, respectively. When G1-hPRL was used as the radiotracer, the percent cross-reactivities were approximately 100%, 135%, and 54% for hPRL, G1-hPRL, and G2-hPRL; however, the displacement curve for hPRL was not parallel to those of the glycosylated hPRLs. When G2-hPRL was used as the radiotracer, the percent cross-reactivities were approximately 100%, 32%, and 37% for hPRL, G1-hPRL, and G2-hPRL. These data point out that the glycosylation heterogeneity of hPRL is a factor that affects the diagnostic accuracy of hPRL determinations. Specific RIAs for each PRL are needed so that we can have valid and reliable measurements of each PRL isoform and consequently gain a better understanding of PRL's complex biological role.

Biological activity of insulin-like growth factor-I purified from chicken serum

This study describes a rapid purification of insulin-like growth factor-I from chicken serum and the immunological, biological and receptor binding activity of the peptide. It was purified after initial extraction, by cation exchange chromatography, hydrophobic interaction chromatography and reverse phase chromatography up to 1.4 × 10 6-fold with an overall yield ranging from 10–30%. The N-terminal amino acid sequence was the same as predicted from the nucleotide sequence of a chicken IGF-I cDNA and the partial sequence obtained from a previously reported purification. The material was both immunologically and biologically active. It had a 50% potency compared to human IGF-I in a radioimmunoassay using an antiserum raised against human IGF-I, stimulated the incorporation of [ 3H]-thymidine into DNA in cultured chick embryo myoblasts with a half-maximum effective dose of 5 ng/ml and displaced [ 125I]-labelled human IGF-I and IGF-II from binding sites in microsomal membranes prepared from both the chicken liver and the lactating rabbit mammary gland in a dose dependent manner.

Arachidonic acid metabolism and casein secretion in lactating rabbit mammary epithelial cells : Effects of inhibitors of prostaglandins and leukotrienes synthesis

The metabolism of arachidonic acid (AA) in fragments of lactating rabbit mammary glands in vitro was studied by considering the distribution of 13-{ 14C}AA in the cells, and the effects of inhibitors of cyclooxygenase and lipoxygenase pathway on the basal and prolactin (PRL)-stimulated casein secretion. 13-{ 14C}AA was incorporated in all classes of lipids and PRL increased transiently the percentage of free fatty acid after 1 and 5 min. Ten μM ETYA (5,8,11,14-Eicosatetraynoic acid), a tetrayne analogue of AA inhibited prostaglandins F 2α (PGF 2α) production but not leukotrienes B 4 and C 4 (LTB 4 and LTC 4) production and increased basal casein secretion. 10 −4 M DCHA (Docosahexaenoic acid) a competitive inhibitor of prostaglandin-synthetase inhibited PGF 2α production but did not affect basal nor PRL-stimulated casein secretion. Fourteen μM indomethacin inhibited PFF 2α and LTC 4 production and PRL-stimulated casein secretion. Ten μM NGDA (nordihydroguaiaretic acid) an inhibitor of lipoxygenase pathway, inhibited LTB 4 and LTC 4 production, increased basal level of casein secretion and inhibited PRL-stimulated casein secretion. Hundred μM caffeic acid, an inhibitor of glutathion-S-transferase (GST), a class of enzymes implied in the transformation of LTA 4 into LTC 4, had the same effect that NDGA on basal and PRL-stimulated casein secretion. These findings show that inhibitors of AA metabolites alter casein secretion.

Effets de la monensine sur la sécrétion des caséines dans les cellules épithéliales mammaires de lapines en lactation

The effects in vitro of monensin on the secretory pathway of caseins in lactating rabbit mammary gland fragments were investigated. Addition of monensin (0.1 microM or 1 microM) to the incubation medium induced a dilatation of Golgi saccules and vesicles and a decrease of the relative volume of microvesicles. Dilated vesicles did not contain acid phosphatase. Neosynthesized proteins were localized by electron microscope autoradiography near the membranes of dilated vesicles. 1 microM monensin did not inhibit basal secretion of neosynthesized caseins (radioactive caseins, labelled during a 3 min pulse, released into the medium), but increased basal secretion of total beta-caseins (measured by radioimmunoassay). Monensin abolished all the effects of prolactin (increase in the relative volume of Golgi microvesicles and stimulation of secretion of total and neosynthesized caseins). These results show that monensin provokes simultaneously modifications in the Golgi apparatus morphology and inhibition of the prolactin stimulating effect. Moreover, secretion of intracellular caseins is differently modified, depending probably on whether caseins are neosynthesized or stored.

Purification and partial characterization of prolactin from the California ground squirrel (Spermophilus beecheyi).

Prolactin (Prl) secreted by cultured ground squirrel (Spermophilus beecheyi) pituitaries (SbPrl) was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on Polybuffer Exchanger 94. Purification from culture medium from 190 pituitaries yielded 1.1 mg of purified SbPrl. The SbPrl has an apparent molecular weight of 27,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 6.3, and does not contain any asparagine-linked carbohydrate. Purified SbPrl displaces 125I-labeled ovine Prl from binding sites on lactating rabbit mammary gland membranes and stimulates secretion of alpha-lactalbumin by cultured mouse mammary gland epithelial cells.

The effect of goat milk fractions on synthesis of milk constituents by rabbit mammary explants and on milk yield in vivo. Evidence for autocrine control of milk secretion.

Lactose and casein synthesis by rabbit mammary explants in organ culture was inhibited when fractions of goat milk were included in the culture medium. Inhibition was dose-dependent, and readily reversed when milk fractions were removed. The pattern of effects obtained with various fractions of milk indicated that inhibition was caused by a protein of 10,000-30,000 Da, which was present in the milk serum or whey fraction. The inhibitor fraction decreased milk accumulation when injected into lactating rabbit mammary glands via the teat ducts, whereas other milk proteins had no effect. Results are discussed in terms of autocrine regulation of milk synthesis through negative feedback by milk constituents.

Biosynthesis of the IgA antibody receptor: a model for the transepithelial sorting of a membrane glycoprotein.

Secretory IgA dimer antibodies in exosecretions provide the primary immunological defense for mucosal surfaces. Transmission of IgA2 across the epithelia of mucous and exocrine glands is mediated by a receptor called secretory component (SC). Using three antibodies directed against different domains of SC, we examine its processing in the lactating rabbit mammary gland. SC is synthesized as a core glycosylated transmembrane glycoprotein on the rough endoplasmic reticulum. Pulse-chase experiments reveal the time course of SC maturation in the Golgi, as demonstrated by the acquisition of Endo H resistance (30-60 min). The subsequent routing of SC to the basolateral plasma membrane, where IgA2 binding and endocytosis occurs, the cleavage of the membrane anchoring domain of SC, and the exocytosis from the apical plasma membrane of IgA, bound to the ectoplasmic domain of SC takes place rapidly (30-60 min). Thus maturation in the Golgi may represent the rate limiting step in SC routing. We also demonstrate that SC exists in several conformational states that are processed at different rates.

The role of phospholipids in the binding of oxytocin to its receptors in lactating rabbit mammary gland.

Phospholipase C (Clostridium welchii and Bacillus cereus) treatment of lactating rabbit mammary gland membranes (140,000 g pellet and sucrose density gradient purified plasma membranes) resulted in a large decrease in the binding of [3H]oxytocin to these subcellular fractions. This decrease was not due to a solubilization of oxytocin receptors but was the result of the removal of phospholipids which may participate in the hormone-receptor interaction. Phospholipase C treatment of the membrane fractions resulted in a dose-dependent removal of different classes of phospholipids. Sphingomyelin, phosphatidylcholine and phosphatidylethanolamine were removed by phospholipase C (C. welchii and B. cereus) treatment. No significant change was observed in the content of phosphatidylinositol. Phospholipase C from B. cereus reduced the content of phosphatidylserine, while the enzyme from C. welchii did not.

Binding of transferrin-iron to the plasma membrane of a lactating rabbit mammary gland cell

1. 1. Cells isolated from a lactating rabbit mammary gland have been investigated for transferrin-iron receptors. The existence of these structures has been demonstrated through a specific binding with a competitor non-labelled rabbit transferrin. 2. 2. The interaction of iron-receptor complex depends on the concentration of [ 59Fe]transferrin, the number of cells and the time. 3. 3. Scatchard's plot of data indicates two classes of receptor sites: one with a binding capacity 6.48 × 10 −9 g Fe per cell and affinity constant 2.48 × 10 10 M −1 and another with 1.06 × 10 −8 g Fe per cell and 4.66 × 10 11 M −1 respectively. 4. 4. The probable mechanism of the iron transport from blood to milk through the lactating cell was discussed.

Prolactin receptors in the lactating rabbit mammary gland during initiated involution

1. 1. The binding of prolactin to receptors in the mammary gland, liver and kidney was studied during initiated involution in the rabbit. 2. 2. The decline in prolactin receptor numbers at the mammary gland was significant after 10 days of involution although affinity for the hormone remained constant. 3. 3. There were no changes in either receptor number or affinity in the liver and kidney tissue. 4. 4. There was no significant differences in plasma prolactin levels in arterial and venous blood samples taken across the mammary gland.

Effect of prolactin on 86Rb+ uptake, potassium content and [G-3H]ouabain binding of lactating rabbit mammary tissue.

1. Ouabain-sensitive 86Rb+ uptake in slices of lactating rabbit mammary gland significantly increased after 20 min or 1 hr of incubation with ovine prolactin (NIH-P-S12; 1 microgram/ml.). 2. Total K+ content of the tissue significantly increased at 20 min of incubation with prolactin. 3. Neither vasopressin nor oxytocin, at concentrations of 2,20 or 40 mui.u./ml., had a significant effect on ouabain-sensitive 86Rb+ uptake or total K+ of the tissue after 30 min or 1 hr of incubation. 4. Tissue slices incubated in cycloheximide at 10 micrograms/ml. for up to 260 min showed a reduction in ouabain-sensitive 86Rb+ uptake and total K+ content, with half-lives of 115 and 63 min, respectively. 5. No consistent in vitro effect of prolactin on (Na+ + K+)-activated ATPase activity in homogenates, crude microsomal fractions or NaI-activated membrane fractions from lactating rabbit mammary gland was found. 6. After 3 hr of incubation of tissue slices in the presence or absence of prolactin (5 micrograms/ml.), no significant differences in the number of [G-3H]ouabain molecules bound per cell (5.2 X 10(4) and 6.2 X 10(4), respectively) or in the dissociation constant (KD) for ouabain binding (5.4 X 10(-7) M and 5.9 X 10(-7) M, respectively) were observed. 7. Incubation of the tissue with cycloheximide (10 micrograms/ml.) for 1-6 hr caused an exponential decrease in [G-3H]ouabain binding with a half-life of 3 hr. 8. It is concluded that prolactin stimulates the activity of the (Na+ + K+)-activated ATPase in slices of lactating rabbit mammary gland within one hour but over this period does not affect the number of ouabain-binding sites, which are apparently turned over with a half-life of 1-3 hr.

The effect of initiated involution on enzyme activity and substrate uptake by the mammary gland of the lactating rabbit

1. 1. Arteriovenous difference studies across the lactating rabbit mammary gland for glucose, acetate, triacylglycerol and non-esterified fatty acids during initiated involution are reported. 2. 2. A significant reduction in substrate utilisation is paralleled by a decrease in the activities of fatty acid synthetase, acetyl CoA synthetase, citrate synthase and glutamate dehydrogenase in biopsy samples taken from the gland. 3. 3. Results from the analysis of lipid fractions within the gland during this period are discussed in relation to lipid resorption.

Characterization of the translation products of the major mRNA species from rabbit lactating mammary glands and construction of bacterial recombinants containing casein and alpha-lactalbumin complementary DNA.

Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5, 1.7, 3.0 and 3.9 kb. Translation in vitro of total mRNA with [3H]leucine or [35S]methionine as precursor yielded four major bands with apparent Mr values of 16 000, 25 000, 26 000 and 29 000. The four protein bands were identified by immunoprecipitation by using specific antisera as alpha-lactalbumin and x-, kappa- and alpha-caseins, respectively. Labelling with (35S]cysteine followed by immunoprecipitation with anti-transferrin or anti-alpha-lactalbumin sera allowed the identification of two whey proteins. Translated transferrin was resolved as an 80 000-dalton band and alpha-lactalbumin appeared as a 16 000-dalton protein. A library of recombinant plasmids containing cDNA (complementary DNA) sequences representing cytoplasmic polyadenylated RNA was used to isolate clones for the major rabbit caseins and alpha-lactalbumin. A preliminary characterization of these cDNA clones was achieved by colony hybridization with enriched RNA fractions as probes. Positive clones were identified by use of hybrid-promoted translation in vitro and immunoprecipitation of the translation products. The corresponding mRNA species were further identified by hybridizing RNA blots with radioactively labelled cDNA clones. We present the restriction map of alpha-casein and kappa-casein cDNA clones.

Identification of fatty acid synthetase messenger RNA on free polyribosomes isolated from lactating rabbit mammary gland.

The synthesis of fatty acid synthetase on free polyribosomes from lactating rabbit mammary gland was demonstrated by using polyribosomes run-off techniques and immunochemical identification of products with synthetase antiserum. Several reproducible and discrete immunoprecipitable polypeptides were observed which were within the molecular-weight range of the synthetase subunit (235 000--252 000), as well as several of lower molecular weight.