Lipids noncovalently associated with keratins and other cytoskeletal proteins of mouse mammary epithelial cells in primary culture.

Lipids noncovalently associated with cytoskeletal (CS) proteins of mouse mammary epithelial cells (MMEC) grown in primary culture were analyzed. A CS fraction, prepared by subjecting MMEC to 1.5 M KCl and 1% Triton X-100 in phosphate buffered saline (pH 7.4), was extracted 4-6 times with chloroform/methanol. Thin-layer chromatography (TLC) indicated that in comparison to whole cell lipid extracts, CS lipids consisted mostly of neutral lipids, especially triacylglycerols and, possibly cholesteryl esters. TLC analysis of chloroform/methanol CS extracts prepared from MMEC that had been incubated 4 h in [3H]palmitate revealed similar results, with the majority of label appearing in triacylglycerols and other neutral lipids. By autoradiography of sodium dodecyl sulfate polyacrylamide gels, all of the major CS proteins appeared labelled. The major regions of autoradiographic density of the gel were excised, the protein solubilized, and the lipids extracted and subjected to TLC. Most of the radiolabel appeared at the origin and ion front and resolved as neutral lipids. In contrast, keratins of 54-55 kDa and 46 kDa appeared to be associated noncovalently with a higher ratio of polar lipids (possibly phospholipids) to nonpolar (neutral lipids). Very little radioactivity, mostly neutral lipid, was associated with actin. A previously unidentified CS component of 30 kDa had primarily noncovalently bound neutral lipid. The results are discussed in terms of the apparent interactions of keratin filaments with the plasma membrane, nuclear envelope and cytoplasmic organelles.

Two-dimensional gel analysis of polypeptides from normal, preneoplastic and neoplastic mouse mammary tissues.

High-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) was employed to reveal tumor-associated polypeptide changes, using the BALB/c C4 line mouse mammary model system, for which phenotypic and immunogenic alterations accompanying tumor progression are well defined. In the first set of experiments, polypeptide patterns from 20 micrograms whole tissue lysates of normal mammary gland, C4 preneoplastic hyperplastic alveolar nodule outgrowth (HAN) and spontaneous tumor from C4 HAN were compared. In order to normalize for differential cellularity and extracellular protein content in the whole tissues, our analysis included polypeptide patterns from serum, increased concentration of protein from whole normal mammary gland, and primary cultures of epithelial cells from normal gland, HAN and tumor. Using a computer-based image-analysis system, 90 polypeptides were identified in C4 tumor that were absent in C4 HAN, normal mammary gland and serum. None of the 90 polypeptides could be shown to represent a definite qualitative change in the protein composition of tumor epithelium as they were found to be either present in a higher concentration of protein from whole normal gland, or present in the primary epithelial culture from HAN, or absent in the primary epithelial culture from tumor. Conversely in the second set of experiments, when epithelial cultures were used as the starting point for comparisons to locate tumor-associated polypeptides, none of the 15 polypeptides that were present in cultures from three different tumors, and absent in the culture from normal mammary gland was specific to C4 tumor, as they were present in whole tissues of normal gland. Thus our experimental approach detected significant quantitative but no qualitative polypeptide changes in whole tumor tissue, or in tumor-derived epithelial cell cultures. This finding may reflect the limitations of the two-dimensional PAGE method, and warrants caution in the use of such gel analysis alone to identify tumor-associated proteins.

Prevalence of intramammary infection and teat canal colonization in unbred and primigravid dairy heifers.

Teat canal keratin (n = 461) and mammary gland secretions (n = 370) were collected from 31 unbred and 85 primigravid Jersey heifers from one research and three commercial dairy herds. Of 97 heifers from which secretion samples were obtained, 96.9% had intramammary infections and 29% showed clinical symptoms. Seventy-five percent of quarters were infected. Staphylococcus aureus were isolated from 36 (37.1%) heifers and 55 (14.9%) quarters. One hundred and eight (93.1%) heifers and 326 (70.7%) quarters had teat canals colonized with mastitis pathogens. Staphylococcus aureus were isolated from teat canal keratin samples from 36 (31%) heifers and 57 (12.3%) quarters. The three most common species isolated from secretion and teat canal keratin samples were Staphylococcus chromogenes, Staphylococcus hyicus, and S. aureus. Secretions from infected (n = 240) and uninfected (n = 85) quarters had SCC of 13.6 X 10(6)/ml and 5.7 X 10(6)/ml. Macrophages were the most numerous cell type in secretions of infected and uninfected quarters. Quarters with teat canal colonization, but with no intramammary infections, exhibited higher SCC in secretion (9.3 X 10(6)/ml) than quarters without both teat canal colonizations and intramammary infections (4.9 X 10(6)/ml). Data indicated that intramammary infections and teat canal colonizations were more prevalent and SCC higher than previously realized in dairy heifers.

Methods of collection and lipid composition of teat canal keratin in dry and lactating cows.

Methods for collecting keratin from the teat canal were examined to select a procedure to obtain representative samples for lipid analysis. Data obtained by solvent extraction of excised teats were compared with those obtained by scraping keratin from dissected teats of lactating and dry cows. Solvent extraction with petroleum ether or 2:1 chloroform-methanol yielded similar dry weights of material. However, both solvents removed large amounts of material other than keratin from the teat canal. The lipid class and fatty acid profiles of the material extracted by solvent flushing were not similar to profiles obtained by scraping keratin from the teat canal. A metal tapestry needle was suitable for collection of keratin from the teat canal of living cows. About 78% of the keratin present in the teat was collected with the needle. Lipid composition of keratin collected with the needle was the same as in keratin scraped from excised teats. The tapestry needle was suitable as a tool for collecting repeatable, representative samples of keratin for analysis from single teat canals of living cows.

Milk protein typing of bovine mammary gland tissue used to generate a complementary deoxyribonucleic acid library.

The milk protein genotype of a mammary gland tissue that was used to generate a cDNA library at the University of California, Davis was typed by PAGE. Casein and whey proteins were extracted from the mammary tissue and typed utilizing fast mini-gel procedures that provide excellent resolution of the milk proteins. The genotype of the mammary tissue was classified as kappa-casein AB, beta-casein A2A1, alpha s1-casein BB, beta-lactoglobulin AA, and alpha-lactalbumin BB. The genes kappa-casein A, beta-casein A2, beta-lactoglobulin A, and alpha-lactalbumin B that have been cloned from this cDNA library coincide with those in the above tissue genotype with the exception of the recently reported cloning of the alpha s1-casein A gene. The gene frequencies for the casein genetic variants for three breeds of dairy cattle is presented and discussed in relation to the low frequency (.3%) of the alpha s1-casein A in the population.

Analysis of ecdysteroids in Onchocerca gibsoni, O. volvulus and nodule tissues.

Free ecdysteroids were detected in Onchocerca gibsoni, in tissues constituting O. volvulus and O. gibsoni nodules and in unrelated bovine tissues. Ecdysone and 20-hydroxyecdysone were identified by HPLC-RIA and GC/MS(SIM). The concentration of free ecdysteroids in the nodule tissue immediately surrounding the parasites was at least an order of magnitude higher than that detected in the worms themselves, or in adjacent nodular tissues or other bovine tissues.

Effects of nickel chloride on lactating rats and their suckling pups, and the transfer of nickel through rat milk.

The excretion of nickel into rat milk following subcutaneous (sc) doses of nickel chloride (NiCl2) and the effects on the lactating rat and her suckling pups were determined. Plasma and milk Ni concentrations increased in a dose-dependent manner 4 hr after single doses of 0, 10, 50, or 100 mumol NiCl2/kg to lactating rats, giving milk/plasma Ni ratios of 0.02. Peak plasma Ni concentrations were reached 4 hr after injection, while milk Ni increased until 12 hr and remained elevated at 24 hr. Dosing for 4 days at 50 or 100 mumol NiCl2/kg/day led to higher milk/plasma Ni ratios of 0.10. These doses of NiCl2 had no effect on body weight but caused decreased food consumption, thymic atrophy, and a small increase in hepatic lipid peroxidation in the dams. Significant alterations in milk composition, which were not due to decreased food consumption as determined in pair-fed rats, included increased milk solids (42%) and lipid (110%), and decreased milk protein (29%) and lactose (61%). NiCl2 treatment also caused significant decreases in mammary RNA content and the RNA/DNA ratio compared to both ad libitum-fed and pair-fed rats, indicating that milk synthetic activity was reduced by NiCl2. Pups suckling the NiCl2-treated dams had plasma Ni concentrations of 24 and 48 micrograms/liter in the 50 and 100 mumol/kg dose groups, respectively, and had decreased liver weight but no changes in hepatic lipid peroxidation or thymus weight. The results indicate that high doses of NiCl2 led to the excretion of Ni into rat milk and changes in milk quality and production. Reductions in liver weight in the suckling pups were also observed which may have been due to nickel exposure or to changes in milk composition.

Purification, characterization, and localization of 70 kDa calcium-sensitive protein (calelectrin) from mammary glands.

Mammary glands contain a group of calcium-sensitive proteins that bind to membranes in a calcium-dependent manner. Using the calcium-dependent binding to hydrophobic surfaces in combination with conventional techniques, we have purified the 70 kDa mammary calcium-binding protein (70 kDa M-CBP) to homogeneity. Antisera prepared to the 70 kDa M-CBP or to bovine liver 67 kDa calelectrin reacted in immunoblot analysis with the 70 kDa M-CBP antigen and with several additional mammary CBP species in crude tissue homogenates. Limited proteolysis of the 70 kDa M-CBP produced smaller immunoreactive species; extensive proteolysis resulted in more complete degradation of the protein. Identical data were obtained with digestion of 67 kDa calelectrin. The pl for the 70 kDa M-CBP was determined to be approximately 5.8; the same value reported for 67 kDa calelectrin. Phosphorylation of 70 kDa M-CBP was not detected in epithelial cell culture metabolic labeling. Immunohistochemical localization showed the protein to be located in ductal epithelia of virgin mouse mammary glands with a pattern of increased staining of the basal portions of the cells. Some stromal cells were also reactive. Apparently, the 70 kDa M-CBP and 67 kDa calelectrin are the same protein. Furthermore, like the 32.5 calelectrin (endonexin) and calpactin I/p36/lipocortin II, the 70 kDa protein appears to be a ductal epithelial cell associated protein in the mammary gland.

Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland.

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.

Localization of vimentin in myoepithelial cells of the rat mammary gland.

Myoepithelial cells in the virgin rat mammary gland have been shown to contain vimentin, using a polyclonal antiserum to vimentin purified from hamster fibroblasts. This antiserum has been shown to be specific for vimentin by immunoblotting and ELISA techniques. Similar results were obtained with a monoclonal antibody to vimentin. In the mammary glands of pregnant rats, the staining with vimentin antibodies is much weaker in the myoepithelial cells of the developing alveolar buds than in the main ducts. Similarly, in lactating glands, the staining of myoepithelial cells is much weaker in the secretory alveoli than in lactiferous sinuses. In each case, staining with antivimentin co-localizes with staining with polyclonal antisera to callous keratin (which specifically stain myoepithelial cells in the rat mammary gland).

Effects of a high-linoleate and a high-alpha-linolenate diet on spontaneous mammary tumourigenesis in mice.

SHN mice were fed a high-linoleate diet, a high-alpha-linolenate diet or a control diet. Spontaneous mammary tumourigenesis was significantly inhibited in the high alpha-linolenate group compared to the other two groups, while little difference was observed among groups in the rates of lung metastasis. The dietary alpha-linolenate/linoleate balance affected the fatty acid patterns of tissue lipids. The triacylglycerol/phospholipid ratios and the fatty acid patterns were significantly different between the mammary glands and the mammary tumours. The results indicate that the dietary alpha-linolenate/linoleate balance affects the fatty acid composition and, in turn, spontaneous mammary tumourigenesis in mice.

Cloning and sequencing of a complementary deoxyribonucleic acid coding for a bovine alpha s1-casein A from mammary tissue of a homozygous B variant cow.

A cDNA clone for bovine alpha s1-casein variant A was isolated from a mammary gland cDNA library using a synthetic degenerate oligonucleotide probe. The largest Pst I insert containing an EcoR I site was sequenced. It contained 1090 base pairs, 47 in the 5' noncoding region, 603 in the coding region and 440 in the 3' noncoding region. The nucleotide sequence was compared with three published cDNA sequences for alpha s1-casein variant B. The most obvious difference was the absence of the 39 bases encoding the 13 amino acids that are present in the B variant but absent from the A variant. In addition, five other single base positions differed within individual codons among the four sequences at the third base for each codon, but this did not change the amino acids encoded. There were, however, a number of differences found in the 3' noncoding region. The isolated cDNA was subjected to site-directed mutagenesis to replace a Val-Ile dipeptide with Phe-Phe to increase the chymosin sensitivity of the protein. When the milk proteins from mammary gland tissue extracts were typed, the alpha s1-casein A gene product was not detected.

Suckling-induced prolactin release potentiates mifepristone-induced lactogenesis in pregnant rats.

Suckling, starting at 19:00 h on Day 18 of pregnancy, induced a significant increase in serum prolactin concentration at 20:00 h on Day 19 of pregnancy, but no increase in mammary gland casein or lactose content. Mifepristone (2 mg/kg) injection at 08:00 h on Day 19 of pregnancy induced significant increases in casein, but not in lactose, 24 h after administration. Mifepristone alone did not induce prolactin secretion, indicating that lactogenesis was induced by placental lactogen in the absence of progesterone action. When mifepristone was injected into suckling rats, serum prolactin concentrations were higher than in the untreated suckling rats. Casein in these rats increased significantly 12 h after mifepristone administration and lactose at 24 h after. If the suckling mifepristone-treated rats were given two injections of bromocriptine (1.5 mg/kg) at 12:00 h on Days 18 and 19 of pregnancy, serum prolactin concentrations were not increased by suckling, but casein and lactose concentrations in the mammary gland showed values similar to those obtained in the mifepristone-treated non-suckling rats. Mifepristone can therefore potentiate suckling-induced prolactin release in pregnant rats, demonstrating a direct central inhibitory action of progesterone on prolactin secretion. This suckling-induced prolactin secretion, unable to induce casein or lactose synthesis in the presence of progesterone, enhanced significantly synthesis of these milk components in the absence of progesterone action (rats treated with mifepristone). Fatty acid synthase, which is stimulated by the suckling stimulus in lactating rats, was not modified by mifepristone or suckling in pregnant rats.

Epidermal growth factor precursor in mouse lactating mammary gland alveolar cells.

Previous studies have demonstrated that high levels of epidermal growth factor (EGF) occur in human and rodent milk and that oral administration of this polypeptide stimulates rodent gastrointestinal development. It is not known whether EGF in milk originates from cells of the lactating mammary gland or is sequestered from an extramammary source. In the present study, prepro-EGF mRNA (approximately 4.7 kilobases) was detected in the CD-1 mouse mammary gland throughout the period of lactation; by comparison, negligible levels of this EGF transcript were found in the gland during pregnancy. Low levels of EGF immunoreactivity (4-5 ng/g wet wt tissue) were extracted from lactating (day 18) mammary glands with dilute acetic acid. Immunolocalization was evident with antisera to either EGF or two other regions of the EGF precursor in essentially all alveolar cells of the lactating gland. The most prominent staining with antiserum to EGF was observed along the luminal borders of cells; this pattern of cellular staining required proteolytic pretreatment of tissue sections. Western blot analyses of cell membranes isolated from the day 16 lactating mammary gland revealed an EGF-immunoreactive band at about 145K, which was equivalent in size to the EGF precursor found in mouse kidney cell membranes. Despite these findings, labeling of lactating mammary gland mince with L-[35S]methionine and cysteine for up to 4 h did not reveal any specific bands in immunoprecipitates. These cumulative findings suggest that the precursor form of EGF occurs in alveolar cells of lactating mammary gland and that this protein is translocated to the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Clonal populations of the mouse mammary cell line, COMMA-D, which retain capability of morphogenesis in vivo.

Clonal populations were isolated from the mouse mammary cell line, COMMA-D, by transfection with a dominant-selectable gene, pSV2Neo, which confers resistance to the antibiotic, G418. Seven of twenty-four clones isolated retained the ability of the parental line to repopulate cleared mammary fat pads in vivo as ductal-alveolar hyperplasias. Two sublines designated CDNR2 and CDNR4 retained hyperplastic growth potential after multiple passages in vitro with low incidence of tumor formation. A third subpopulation, CDNR1, contained a single integration site for the pSV2Neo plasmid indicating a bonafide clonal origin for this subline. CDNR1 cells displayed heterogeneous growth phenotypes in vivo including hyperplasia, adenocarcinoma, and bone formation. Functional differentiation of CDNR1 cells organized as alveolarlike structures in vivo or on floating collagen gels in vitro was observed as determined by immunoperoxidase staining for the milk-specific protein, casein. Overall, the results indicate that a subset of cells from the COMMA-D cell line may be functionally analogous to stem cells existing in the mammary gland.

Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase.

Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.

Glucocorticoid receptor from lactating goat mammary tissue comparison of native and activated forms in a cell free system.

Physicochemical properties of native and activated (DNA-binding) forms of the glucocorticoid receptor in cytosol prepared from lactating goat mammary tissue have been examined. Under hypotonic conditions the cytosolic receptor sediments at 8.4 S or 9.9 S in the absence or presence of 10 mM molybdate, respectively. The receptor in cytosol, either with or without molybdate elutes from DEAE-cellulose at approximately 200 mM potassium phosphate concentration. Isoelectric focusing reveals that this form of the receptor focuses at pH 5.5. Further, the cytosolic form of the receptor exhibits minimal binding affinity for polyanions such as DNA-cellulose. Its Stokes radius is 77 A and the mol. wt is approximately 331,000. Following exposure to in vitro activating conditions (including elevated ionic strength or temperature), the liganded receptor exhibits much lower affinity for DEAE-cellulose (elution at 35-55 mM potassium phosphate concentration). Other alterations in properties of the activated receptor, after partial purification, include sedimentation at 3.9 S in hypotonic sucrose gradients, binding to polyanions (DNA-cellulose), and an isoelectric point at pH 7.2. This receptor has a Stokes radius of 58 A and a mol wt of 98,000. A degraded form, with a mol. wt of approximately 57,000 and high affinity for polyanions, was the major form of the receptor obtained if appropriate precautions to prevent or remove proteolytic activity were not observed during purification and/or characterization of the activated receptor.

Release of cholesteryl esters by the mammary gland of the pre-partum cow.

In five cows that were regularly milked before parturition, cholesteryl esters were continuously released into the mammary fluid; their concentration in the fluid was initially high, but decreased a few days before parturition when mammary secretion of fluid and triglyceride was increasing. The composition of fatty acids in the cholesteryl esters of mammary fluid and in blood plasma was different, suggesting mammary synthesis of cholesteryl esters.