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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.

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.

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 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 lipid globules of various species are surrounded by a membrane structure that is separated from the triglyceride core of the globule by a densely staining fuzzy coat layer of 10- to 50-nm thickness. This internal coat structure remains attached to the membrane during isolation and extraction with low- and high-salt buffers, is insoluble in nondenaturing detergents, and is enriched in an acidic glycoprotein (butyrophilin) with an apparent Mr of 67,000. Guinea pig antibodies against this protein, which show cross-reaction with the corresponding protein in some (goat) but not other (human, rat) species, have been used for localization of butyrophilin on frozen sections of various tissues from cow by immunofluorescence and electron microscopy. Significant reaction is found only in milk-secreting epithelial cells and not in other cell types of mammary gland and various epithelial tissues. In milk-secreting cells, the staining is restricted to the apical cell surface, including budding milk lipid globules, and to the periphery of the milk lipid globules contained in the alveolar lumina. These findings indicate that butyrophilin, which is constitutively secreted by surface budding in coordination with milk lipid production, is located at the apical surface and is not detected at basolateral surfaces, in endoplasmic reticulum, and in Golgi apparatus. This protein structure represents an example of a cell type-specific cytoskeletal component in a cell apex. It is suggested that this antigen provides a specific marker for the apical surface of milk-secreting cells and that butyrophilin is involved in the vectorial discharge of milk lipid globules.

Amino acids and peptides of albumin hydrolyzates from the mammary gland and blood serum were studied for lactating and nonlactating (dry, pregnant 1-4.5 and 4.5-9 months) black-and-white cows. Most pronounced difference between the content of certain amino acids of the mammary gland and blood serum albumins are established for lactating cows and least pronounced for nonlactating dry cows. Dactylography detected 55-57 fragments of products resulted from trypsin hydrolysis of the mammary gland and blood serum albumins of the animals under study. Differences are found in the content and mobility of certain peptides.

RNA complexity analysis of poly(A)-containing RNA isolated from the lactating guinea-pig mammary gland demontrates that the complexity within the nuclear population was three--four-fold greater than that in the equivalent post-nuclear polyribosomal population [see Craig et al., Biochem. J. (1979) 181, 737-756]. Up to 21 000 different sequences distributed in two abundance groups were detected in the nucleus, whereas 5000 sequences distributed in three abundance groups were present in the post-nuclear population. All poly(A)-containing RNA sequences present in the post-nuclear fraction could be detected in the nuclear poly(A)-containing population. Analysis of the relative distribution of the three post-nuclear abundance populations within the nuclear poly(A)-containing RNA population demonstrates that the abundant and moderately abundant post-nuclear sequences were present a similar concentrations within the nucleus, and comprised the abundant nuclear population. The abundant and moderately abundant post-nuclear sequences were present at 62200 and 785 copies of each sequence/cell in the post-nuclear fraction respectively, and 44 and 118 copies of each sequence/cell in the nuclear fraction respectively. The scarce post-nuclear sequences (18.5 copies of each sequence/cell) were also present at low levels in the nuclear fraction (0.1 copy of each sequence/cell). the results are discussed in terms of the post-transcriptional regulation of gene expression in the lactating guinea-pig mammary gland.

A linear pathway of morphologically intermediate cells has been identified between the cuboidal epithelial stem cells and the doming alveolar-like cultures of the cell line Rat Mammary (Rama) 25 in the order: cuboidal----grey----dark----dark droplet cell----doming cultures. The overall process can be accelerated by dimethyl sulfoxide (DMSO) or retinoic acid (RA) in the presence of mammotrophic hormones. From 400-450 [35S]methionine-labeled polypeptides that are routinely separated by two-dimensional gel electrophoresis approximately only 3% change during this process. As the Rama 25 cultures become confluent, three polypeptides of molecular weights (MW) 35 kD (pl = 7.7), 45 kD (pl = 7.5) and 33 kD (pl = 7.7) increase dramatically in radioactive abundance. These increases correspond to increases in numbers of grey cells for the 35 kD polypeptide, to increases in numbers of dark cells together with increases in peanut lectin-binding-ability for the 45 kD polypeptide, and to increases in the numbers of dark cells and in the numbers of droplet cells for the 33 kD polypeptide. After treatment with DMSO, RA or in spontaneously doming cultures, a second set of four polypeptides of MW 26 kD (pl = 5.9), 27 kD (pl = 6.2), 30 kD (pl = 7.2), and the same 33 kD polypeptide as above increase with the increase in numbers of droplet cells, domes, and increase in casein secretion. A variant of Rama 25, Rama 259, which fails to produce droplet cells, domes, or to secrete casein with DMSO and hormones also shows the same changes in the first set but not in the second set of polypeptides. The elongated, myoepithelial-like cell line derived from Rama 25, Rama 29, which cannot undergo any of the above intercellular conversions, fails to show changes in any of these polypeptides. Major changes in radioactive polypeptides have been confirmed for nonradioactive polypeptides and for polypeptides labeled for 4 hr with [35S]methionine. The synthesis of these novel polypeptides thus marks specific morphological stages of the differentiation of mammary epithelial stem to alveolar-like cells in culture, and as such may mark similar differentiation stages in vivo.

Oxytocin-receptor concentrations in the rat mammary gland were determined by Scatchard analyses with [3H]oxytocin. There was about a 100-fold increase in the number of receptors per mammary gland between the 1st day of pregnancy and late lactation. The number of receptors then fell markedly during postweaning mammary regression, but rose again during a second pregnancy and lactation cycle. The changes in oxytocin-receptor number corresponded to changes in alkaline phosphatase activity per mammary gland. These results strongly support data suggesting that alkaline phosphatase, like oxytocin receptors, is a specific marker for mammary myoepithelial cells. Despite the fall in oxytocin-receptor number per mammary gland during postweaning regression, the concentration of receptors, expressed per milligram of protein, increased 10-fold over lactating levels on the 6th day of regression. Thereafter, receptor concentrations declined, but were still elevated about fivefold over lactating levels on the 15th day of regression. It is likely that the increased concentration of receptors was due to a decrease in the relative amount of nontarget secretory cells. The factors that regulate the concentration of oxytocin receptors on mammary myoepithelial cells are presently unknown; however, the involuting mammary system may be practical for obtaining enriched populations of oxytocin-sensitive myoepithelial cells.

Mammary development was assessed in lactating goats using a combination of biopsy (for analysis of nucleic acids) and udder volumes (for determination of gross size). Single biopsies were shown to be highly representative of the composition of the whole gland provided that they were taken from carefully selected sites. Results indicated an increase in both milk yield and the size of the mammary cell population ( DNAt ) over the first three weeks of lactation. Yield, but not DNAt , continued to increase until peak lactation at around week eight. As milk yield fell between weeks eight and twenty-three the size of the cell population also decreased; beyond week twenty-three and until week thirty-six DNAt stabilized but yield continued to fall. It is concluded that the first part of the increase in milk yield during ascending (early) lactation in goats can be attributed to proliferation of secretory cells, but subsequently there is an increase in the amount produced by each cell. Likewise, declining lactation is initially characterized by a loss of cells, and yield per cell falls later.

A factor which inhibits the resumption of proliferation of stationary Ehrlich ascites mammary carcinoma (EAC) cells in vitro was isolated from the normal, lactating bovine mammary gland. After fractionation of the 50,000 g supernatant of the tissue homogenate by ammonium sulfate precipitation and gel filtration, it was further purified 2600-fold by ultrafiltration and lectin affinity chromatography. Polyacrylamide electrophoresis revealed one band containing all the activity. SDS gel electrophoresis showed a main band corresponding to a protein with a molecular weight of about 15,000 dalton. At this state of purity the factor concentration for half-maximal inhibition of proliferation was 12 ng/ml or 8 X 10(-10) M. The effects of factor concentrations of 4 X 10(-9) or 4 X 10(-8) M are abolished by 5 X 10(-9) M insulin almost completely or to 70%.

Four doses (5 to 100 micrograms, 1 dose/quarter) of Escherichia coli endotoxin were introduced into lactating mammary glands of 2 cows. There was no effect on milk prostaglandin (PG) E2 concentration, except that the concentration was increased from 200 pg/ml of milk to 1,060 pg/ml at post-treatment hour (PTH) 8 in cow 1 and from 75 to 420 pg/ml at PTH 4 in cow 2 after the highest dose 100 micrograms. Endotoxin caused a dose-dependent increase in milk PGF2 alpha concentrations in both cows. After the highest dose, PGF2 alpha was maximally increased from 200 to 3,500 pg/ml at PTH 4 in cow 1 and from 250 to 2,000 pg/ml in cow 2 at PTH 8. The instillation of 50 micrograms of endotoxin in all 8 quarters of 2 more lactating cows caused no significant (P greater than 0.05) changes in milk PGE2 and thromboxane B2 concentrations, whereas milk PGF2 alpha was significantly increased from the base-line value of 642 to 2,683, 1,189, and 2,281 pg/ml at PTH 4, 8, and 12, respectively. The 6-keto-PGF1 alpha was also significantly increased from the base-line value of 305 to 871, 631, and 600 pg/ml at the corresponding times, respectively. A marked increase in vascular permeability, as judged by high concentrations of serum albumin in the whey, was observed as early as PTH 4 and peaked at PTH 12 followed by a gradual decline, although it remained significantly increased over the control for 48 hours after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The amino acid fucosides of tumorigenic and nontumorigenic mouse mammary gland-derived cells were studied. The cells examined included tumorigenic cell lines derived from mammary carcinomas of the following etiologies: induction by hormone; virus; and chemical carcinogen. Also studied were cells derived from normal mammary glands and several clones of cells, which were derived from a mammary carcinoma but were not demonstrably tumorigenic at lower passage levels after cloning, while they were highly tumorigenic at higher passage levels. Cells were cultured in medium supplemented with radiolabeled fucose and extracted, and extracts were analyzed for the amino acid fucosides. Radiolabeled compounds which comigrated with the amino acid fucosides glucosylfucosylthreonine, fucosylthreonine, and fucosylserine were observed. There was a distinctive difference between the tumorigenic and nontumorigenic cells; the ratio of fucosylthreonine plus fucosylserine to glucosylfucosylthreonine was higher in all tumorigenic cells as compared to the ratio observed for the nontumorigenic cells.

The distribution pattern of immunoreactive insulin-like growth factor-I in normal lactating bovine mammary tissue and in tissue obtained after bovine somatotropin treatment was determined by indirect immunofluorescence. In normal tissue, insulin-like growth factor-I immunoreactivity was observed almost exclusively associated with stromal elements. Intralobular stromal cells, small blood vessels, and capillaries all expressed moderate to high immunoreactivity. In contrast, mammary epithelial cells displayed only sparse cytoplasmic immunoreactivity. Immunoreactive material was also present in the periductular connective tissue area, possibly associated with the basal plasma membrane of epithelial cells. Somatotropin treatment of animals resulted in elevated serum insulin-like growth factor-I concentrations and altered the distribution of insulin-like growth factor-I-stainable material in mammary tissue. After somatotropin treatment, immunoreactivity was still detected in mammary stroma; however, prominent staining was also observed in the cytoplasm of mammary epithelial cells. Given the possible role of insulin-like growth factor-I in the regulation of bovine mammary epithelial cell growth and function, our findings raise the possibility that somatotropin may induce insulin-like growth factor-I production in mammary tissue, or other tissues, to influence indirectly the growth or function of the epithelial cells. This offers a possible mechanism for bovine somatotropin stimulation of lactation.

The abilities of the two different mol wt forms of rat placental lactogen (rPL) to maintain luteal steroidogenesis in pregnant rats in the absence of PRL were determined. The two forms of rPL present in day 12 pregnant rat serum were separated by gel chromatography, lyophilized, and reconstituted in a volume of saline equal to the original volume of serum. Rats were injected with 0.4 mg ergocryptine (ECO) on day 6 of pregnancy to suppress PRL and were then treated with a preparation of the large mol wt (LMW) hormone, the small mol wt (SMW) hormone, or both molecules (2 ml/day). Control animals received saline. Twice daily injections of the amount of LMW hormone contained in 1 ml day 12 pregnant rat serum reversed the abortifacient effect of ECO. In contrast, administration of the SMW hormone did not maintain either pregnancy or progesterone levels. Administration of both mol wt forms of rPL was also capable of maintaining luteal function. Sera obtained from day 18 pregnant rats containing only the SMW hormone had no luteotropic activity when administered, yet treatment with sera of day 12 pregnant rats sustained progesterone synthesis and fetal survival after ECO treatment. rPL were measured in the peripheral circulation and in the uterine vein throughout pregnancy by radioreceptor assay (RRA) using particulate membranes from either rabbit mammary gland or rat ovaries. Two peaks of activity were observed in the peripheral circulation by the two RRAs: one between days 11-14 and another between days 17-21, with a decline in activity between days 14-16. In contrast, levels of rPL in the uterine vein remained elevated throughout pregnancy. Concentrations of the LMW placental luteotropin were 5-10 times higher by ovarian RRA (O-RRA) than by mammary gland RRA (MG-RRA) between days 11-13, but concentrations of the SMW placental lactogen were found by the two RRAs to be similar in the later stages of pregnancy. Gel filtration of day 12 pregnant rat serum revealed two peaks of PRL-like activity. The O-RRA detected 19 times more LMW placental luteotropin than did the MG-RRA in the first peak of activity, yet measured equivalent amounts of the SMW placental lactogen in the second peak. Similar to results found in the peripheral circulation, levels of LMW placental luetotropin in the uterine vein measured by MG-RRA were significantly lower than those determined by O-RRA until day 14 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

The process of growth of the mammary gland and change in the plasma concentration of progesterone were investigated throughout the course of pregnancy (30 days) in the home musk shrew, Suncus murinus L. Development of the mammary gland of the musk shrew was limited during the first half of pregnancy. Extensive branching of ducts and conspicuous alveolar formation and a marked increase in the DNA content of the gland started between day 15 and 20 of pregnancy, and continued until term. Milk synthesis indicated by accumulation of the secondary fluid in the alveolar lumen and a sudden rise in the RNA/DNA ratio of the gland tissue seemed to be initiated 1 or 2 days before parturition. No lactose was detected in either the mammary tissue or milk of the house musk shrew. Plasma concentration of progesterone was very low until mid-pregnancy and began to rise after day 15, reaching a peak of 10--13 ng/ml around day 25. The steroid level started to fall shortly before parturition and returned to the basal level in post-parturient animals. Ovariectomy interrupted pregnancy in some animals, but not in others. When pregnancy was maintained, the mammary development and the plasma level of progesterone were normal.

Regression analysis related simulated production to simulated wet weight of mammary gland, weight of dry fat-free tissue, deoxyribonucleic acid content, and ribonucleic acid content for the first 21 days of lactation of the guinea pig. Coefficients of determination were .52 for wet weight, .03 for dry fat-free tissue, .84 for deoxyribonucleic acid, and .20 for ribonucleic acid. Coefficients of determination were larger when ascending and descending phases of lactation were considered separately. Milk production per unit mammary wet weight, dry fat-free tissue, deoxyribonucleic acid, and ribonucleic acid were related to day of lactation by an equation of the form Y = aXbecX, where Y was milk production per unit mammary gland growth, X was day of lactation, e was the base of natural logarithms, and a, b, and c were constants. Efficiency of milk production, defined as milk production per unit mammary size, increased with increasing milk yield.

The insoluble cytoskeletal material remaining after detergent lysis of 'Normal' Murine Mammary Gland (NMuMG) cells, growing on plastic or collagen gel substrata, was analyzed by two-dimensional gel electrophoresis. The identity of the cytoskeletal elements was determined by their solubility properties, electrophoretic separation pattern, and immunoreactivity using monoclonal antibodies against intermediate filament proteins (AIF), keratins (AE1 and AE3) and actin. The electrophoretic pattern of the cytoskeletal elements from the NMuMG cell strain was found to be very similar to that of primary mouse mammary epithelial cells. Both NMuMG and primary mammary epithelial cells when grown on collagen exhibited an increased expression of a 49 kD protein with a pI of 5.6, that appeared to be a cytokeratin. Many of the cytoskeletal proteins remained tightly attached to the collagen gel substratum after cell lysis. These results demonstrate that the NMuMG cell strain has retained a stable expression of cytokeratins that remains responsive to the presence of extracellular matrix material.

The specific activity of aryl hydrocarbon hydroxylase in the mammary gland has been measured in six different mouse strains comprising both mammary cancer susceptibility and resistance, under basal conditions, and after pretreatment of the mice with 3-methylcholanthrene (3MC). No correlation was observed between the ease of 3MC-enhanced mammary tumorigenesis and the basal or induced specific activity of aryl hydrocarbon hydroxylase. Furthermore, it was shown that in the AKR/J and A+/Ki mice, only minor enhancement in aryl hydrocarbon hydroxylase was seen upon injection of 3MC. The C57BL/6J and C3Hf/He Tex mammary glands responded the most in this regard. It was also shown that the inability of the AKR/J or A+/Ki mice to respond was not due to their inability to absorb 3MC from the peritoneal cavity and transmit the polycyclic hydrocarbon to the mammary gland.

1. Calf mammary-gland cytosol apparently has a single oestrogen receptor capable of auto- and/or hetero-association of varying complexity. Computation of the dissociation constant for oestradiol-17beta gives Kd = 0.5 nM. The number of binding sites is 40 fmol/mg of cytosol protein. The oestrogen receptor in the presence of NaBr, a chaotropic salt that inhibits the interaction of receptor with other cytosol components, sediments through sucrose density gradients as a single sharp peak at 4S, and it has a Stokes radius of 3.4 nm measured by gel filtration. 2. A large-scale purification procedure of the calf mammary-gland oestrogen receptor based on the inhibition of receptor aggregation by NaBr and interaction with heparin-Sepharose is reported. The receptor is purified more than 1500-fold over that in the 27,000g supernatant of the homogenate, with a 30% yield. In 'low-salt' buffer the purified receptor sediments through sucrose gradients at 4S and the Stokes radius, measured by gel filtration in the presence of heparin, is 3.4 nm. The mol.wt computed from these values is about 60,000, and the frictional ratio is 1.3.

Cultured mouse mammary (NMuMG) cells produce heparan sulfate-rich proteoglycans that are found at the cell surface, in the culture medium, and beneath the monolayer. The cell surface proteoglycan consists of a lipophilic membrane-associated domain and an extracellular domain, or ectodomain, that contains both heparan and chondroitin sulfate chains. During culture, the cells release into the medium a soluble proteoglycan that is indistinguishable from the ectodomain released from the cells by trypsin treatment. This medium ectodomain was isolated, purified, and used as an antigen to prepare an affinity-purified serum antibody from rabbits. The antibody recognizes polypeptide determinants on the core protein of the ectodomain of the cell surface proteoglycan. The reactivity of this antibody was compared with that of a serum antibody (BM-1) directed against the low density basement membrane proteoglycan of the Englebarth-Holm-Swarm tumor (Hassell, J. R., W. C. Leyshon, S. R. Ledbetter, B. Tyree, S. Suzuki, M. Kato, K. Kimata, and H. Kleinman. 1985. J. Biol. Chem. 250:8098-8105). The BM-1 antibody recognized a large, low density heparan sulfate-rich proteoglycan in the cells and in the basal extracellular materials beneath the monolayer where it accumulated in patchy deposits. The affinity-purified anti-ectodomain antibody recognized the cell surface proteoglycan on the cells, where it is seen on apical cell surfaces in subconfluent cultures and in fine filamentous arrays at the basal cell surface in confluent cultures, but detected no proteoglycan in the basal extracellular materials beneath the monolayer. The amino acid composition of the purified medium ectodomain was substantially different from that reported for the basement membrane proteoglycan. Thus, NMuMG cells produce at least two heparan sulfate-rich proteoglycans that contain distinct core proteins, a cell surface proteoglycan, and a basement membrane proteoglycan. In newborn mouse skin, these proteoglycans localize to distinct sites; the basement membrane proteoglycan is seen solely at the dermal-epidermal boundary and the cell surface proteoglycan is seen solely at the surfaces of keratinocytes in the basal, spinous, and granular cell layers. These results suggest that although heparan sulfate-rich proteoglycans may have similar glycosaminoglycan chains, they are sorted by the epithelial cells to different sites on the basis of differences in their core proteins.

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.

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

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.

Gestation- and lactation-dependent variations of magnesium concentrations were measured in the maternal and fetal body fluids and tissues of rabbits. Samples of serum, milk, and mammary gland were investigated. A decrease in magnesium in the maternal serum during the early gestation period from 0.80 mmol/l in the first week to 0.73 mmol/l in the third week was observed. In the last week of gestation a rapid increase of magnesium concentrations both in maternal (0.83 mmol/l) and fetal (2.84 mmol/l) serum was determined. An assumed active diaplacental transfer of magnesium from the doe to the fetus was indirectly established. An active transport mechanism for magnesium from the maternal serum into the mammary glands was enhanced by strong concentration gradients of approximately 1:33 between serum and milk.

Experiments were carried out to identify progestin-binding receptors in the mammary gland where casein synthesis is known to be inhibited by this hormone. A progestin-binding component with high affinity, low capacity and a sedimentation coefficient of 8.8 S was isolated from the cytosol of lactating rat mammary glands. This component strongly bound [3H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) with a dissociation constant of 3.9 . 10(-9) M under low-salt conditions and with that of 8.2 . 10(-10) M in the presence of 0.3 M KCl. Specificity studies showed a high degree of progestin specificity under high salt conditions. In the absence of KCl, binding of [3H]-R5020 was inhibited by unlabeled glucocorticoid in the same degree as unlabeled progestin, but the inhibition by glucocorticoid was greatly diminished by the presence of 0.3 M KCl. These observations suggest that the [3H]R5020-binding-component is the progestin receptor and that its function may be regulated by the concentration of glucocorticoid and salt.

The nucleosides of tRNA(IAGLeu) (with a long variable loop) from the cow mammary gland included in formation of the three-dimensional structure have been analysed by the chemical modification methods. Exposed guanosine and cytidine residues were detected by means of dimethylsulfate, whereas diethylpyrocarbonate was used to detect exposed adenosine residues. The low level of the modification was characteristic of guanosine residues in positions 10 (m2G), 13, 15, 23, 24, 29, 30, 47 H, 51, 52, 53, 57; of cytidine residues in positions 48 (m5C), 56 and those involved in Watson--Crick pairing; of adenosine residues in positions 14, 22, 31, 42, 59, 64. Most bases of tRNA(IAGLeu) thus detected are similarly located in the yeast tRNA(Phe) molecule, which suggests a common role of these bases in the formation of the spacial structure of both tRNAs.

The expression of the mouse mammary tumor virus (MMTV) in hormone-induced mammary tumors was investigated by means of a radioimmunoassay for two major MMTV proteins, gp52 and p27. MMTV proteins were isolated on lectin affinity- and ion-exchange chromatography columns. The purified viral proteins were electrophoretically homogeneous and retained immunoreactivity after labelling with 125iodine. Standard competition assays showed that group-specific antigenic determinants were reacting. Mammary tumors were induced in three strains of mice with a low natural incidence of mammary tumors, C57BL, O20 and C3Hf, by a combined hormone treatment, consisting of hypophyseal isografts and administration of progesterone and estrone. Mammary tumors and mammary glands of hormone-treated animals were extracted and used for competition radioimmunoassays. In general, the tumorigenic hormone treatment resulted in enhanced amounts of MMTV proteins in the mammary glands, compared to the amounts found in lactating mammary glands of untreated animals. The levels of MMTV proteins in the mammary tumors were lower than in the mammary glands.

The regulatory role of thyroid hormones in the synthesis of milk proteins and functional differentiation of the murine mammary gland has been demonstrated. Further, T4 and T3 and their metabolites are found in both rat and human milk. Since the enhanced metabolic demands of mammo- and lactogenesis may result in increased local hormonal requirements, we examined breast tissue from normal (virgin), pregnant, and lactating female rats, as well as postmenopausal human breast tissue for the presence of a thyroid hormone-binding inhibitor (THBI). This substance, which decreases binding of T4 to serum T4-binding globulin, has been described in rat liver, kidney, muscle, and intestine, and in the circulation of patients with nonthyroidal illnesses. THBI activity in the present study was assessed by equilibrium dialysis in whole tissue homogenates; the ether-soluble fraction was also analyzed for THBI activity by RIA. In a range of 0.8-8.0 mg tissue protein, we found lactating rat breast to contain elevated THBI levels when compared to those found in normal, virgin breast tissue. Half-maximum binding inhibition was achieved at 0.15 mg lactating breast tissue protein, vs. 0.23 mg breast tissue protein and 0.35 mg liver protein in homogenates from normal resting animals. Expressed in tissue milligram equivalents, half-maximum binding inhibition of lactating, 18-day pregnant, and normal rat breast tissue was 0.7, 2.6, and 4.8 mgeq, respectively. Thus, the THBI activity of lactating rat breast tissue was more than 3-fold greater than that of the pregnant rat and 7-fold greater than that found in resting breast tissue. Binding inhibition in postmenopausal human breast tissue was comparable to that of rat breast and liver tissue at low protein concentrations, but the maximum inhibition attainable (205 +/- 6%, n = 3) was significantly lower than that achieved by normal rat breast tissue (338 +/- 6%, n = 6; means +/- SEM). The ether-soluble fraction of breast tissue homogenates was also analyzed employing a THBI-RIA. THBI activity, expressed as percent binding inhibition, was elevated in extracts from lactating rat breast tissue in comparison to tissue from pregnant or normal animals at 6.0, 12.5, and 25 mgeq; the largest differences were observed at 25 mgeq tissue: lactating (57.8 +/- 2.3%) vs. pregnant (46.5 +/- 3.1%, P less than 0.01) vs. normal (41.7 +/- 2.3%, P greater than 0.05); n = 3 in all cases.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Poly(A)-containing RNA was isolated from the nuclei of mammary gland, liver and brain of lactating guinea pigs. 2. Total nuclear poly(A)-containing RNA from mammary gland inhibited mRNA-directed protein synthesis by a wheat-germ cell-free system. It also inhibited the endogenous activity of the wheat-germ and other cell-free systems. It did not inhibit a wheat-germ cell-free system directed by poly(U). 3. Total nuclear poly(A)-containing RNA from liver and brain did not inhibit the mRNA-directed wheat-germ system. 4. Fractionation of the nuclear poly(A)-containing RNA revealed inhibitory activity in the less than 10 S fraction from mammary gland as well as that from liver and brain. 5. The mechanism of protein-synthesis inhibition appeared to be at the level of elongation. 6. The inhibitory activity could be reversed in a wheat-germ system by increasing the amount of S-30 supernatant. 7. The mechanism of inhibition of protein synthesis is discussed in relation to other RNA species known to inhibit such systems.

Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain soluble glucocorticoid receptors. The kinetics of binding of dexamethasone reveal a saturable binding site [dissociation constant (Kd), approximately 1 nM], and the binding site obeys a steroid specificity characteristic of a glucocorticoid receptor. As with the receptor isolated from intact glands, the receptor from the cultured cells also requires the addition of dithiothreitol for maximal binding of dexamethasone. The receptors are maintained at in vivo levels (approximately 1.3 pmol/mg DNA) for at least a period of 10 days in culture. However, the presence of both cortisol and PRL is required for the maintenance of the receptors, and the effect of both these hormones is dose dependent.

Saturable, high-affinity binding sites for 3,5,3'-triiodo-L-thyronine (T3) were identified in isolated nuclei and solubilized chromatin extracts of mammary glands, spontaneous mammary tumors, and liver from C3H/HeN mice. Receptor concentration in whole mammary gland nuclei (254 fmol/mg DNA) was only about one-half that of mouse liver nuclei (536 fmol/mg DNA), but in molecular weight (55,000) and in their affinity for various thyroid hormone analogues, the binding was essentially identical. Saturation analysis of T3 binding in a series of individual spontaneous mammary tumors and pooled lactating mammary glands indicated that the concentrations of T3-binding sites of the mammary gland are conserved in the transition to neoplasms and are somewhat increased in the largest tumors. Thyroxine binding was identical in capacity to T3 binding in mammary gland nuclei and nuclear extract but showed a higher binding capacity than did T3 in the largest tumors. High-performance molecular exclusion chromatography did show a difference between mammary gland and liver in the distribution of competible [125I]T3 binding between two macromolecular forms; the excluded peak (Mr greater than 450,000) comprised 56% of the T3 binding in the liver but only 9% in the mammary gland with the included peak (Mr 55,000) contributing the balance of binding in each case. Spontaneous mammary tumor resembled the mammary gland in the macromolecular distribution of specific T3 binding (16% excluded). Thymidine uptake showed only a modest decrease in the larger tumors (greater than 2.0 g), while nuclear histone acetylase activity was significantly decreased in this group. Neither measurement showed a significant correlation with T3 or thyroxine binding capacity.

The distribution and metabolism of an ip dose of [14C]aflatoxin B1 (AFB1) were studied in lactating Sprague-Dawley rats fed for the previous 13 days on a diet containing 0.5% butylated hydroxytoluene (BHT). Compared with ingestion of a BHT-free diet, treatment with BHT increased the biotransmission of AFB1 metabolites, predominantly aflatoxin M1 (AFM1), into the mammary gland and its content of milk, decreased AFB1 binding to liver nuclear DNA and enhanced the excretion of water-soluble metabolites of AFB1, all measured 6 hr after an oral dose of [14C]AFB1. These changes are related to the induction by BHT of hepatic enzymes involved in the transformation and detoxification of AFB1. The results suggest that exposure to BHT may protect the lactating animal from the carcinogenic effect of AFB1 but may increase the risk of exposure of the newborn infant to the carcinogenic metabolite AFM1.

tRNA2Leu from cow mammary gland has been degraded with pancreatic ribonuclease, and the fragments obtained were separated by DEAE-cellulose micro-column chromatography in 7 M urea at pH 7,5. Rechromatography was performed on a DEAE-cellulose micro-column at pH 3,7 and also on Dowex 1 X 2 in a formiate system. Nucleotide analysis was carried out with the aid of T2-RNase hydrolysis followed by chromatography on anion-exchanger AG 1 X 8. Nucleosides were separated on Aminex A-6 at pH 9,8. 15 minor components were shown to be present: T, 2 psi, 2Um, 2D, m5C, ac4C, m1G, 2m2G, m22G, m1A and N, the N is not identified so far. The structure of oligonucleotides was established by terminal analysis, hydrolysis with T1-RNase and also using incomplete hydrolysis by the snake venom phosphodiesterase.

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.

Butylated hydroxytoluene (BHT) is a phenolic antioxidant which is widely used in foods and has been shown to inhibit chemical carcinogenesis in the mammary gland induced by 7,12-dimethylbenz(a)anthracene. However, its mechanism of action as a tumor inhibitor is unclear. The purpose of this work was first to develop a method for extracting and quantitating BHT and then to determine the amounts that accumulate in the tissues and serum of rats as a starting point for looking at mechanistic possibilities in the inhibition of mammary carcinogenesis. Methodology of extracting BHT from rat tissues and serum was developed using a modified lipid extraction procedure. The sensitive nature of reverse-phase high-performance liquid chromatography proved useful in detecting and quantifying BHT after its extraction from biological tissues. All tissues were taken from animals consuming semipurified diets with and without 0.3% BHT for various periods of time (weeks). BHT was found in much higher levels in mammary tissue than in the liver and serum of rats. The lipid content in mammary tissue appears to be predictive of the amount of BHT found in this tissue, presumably because of the lipophilic character of the antioxidant.

Lactating rats have been fed either a protein-restricted diet (10 vs. 20% casein in the control diet) or the control diet at 80, 60 and 40% of the voluntary intake for 7 d from d 7 of lactation. Food consumption, changes in maternal live weight, litter live weight gain and the mass of several maternal tissues were determined together with the activity of several mammary and liver enzymes, including 10 that are essential for fatty acid and complex lipid synthesis. Milk production was estimated from the litter weight gain and litter weight. Lactating rats fed the 20% protein diet ad libitum consumed three times that of nonlactating rats; their liver and kidney masses were significantly higher and their adipose mass was lower. The livers of the lactating rats were fatty, containing 118 mg lipid/g compared with 42 mg/g for the nonlactating rats. Lactating rats fed either the protein-restricted diet or the control diet at 40 and 60% of the ad libitum intake of the control diet had lower mammary, liver and kidney masses than rats consuming the control diet ad libitum. Both protein and food restriction led to lower rates of milk production than those of ad libitum-fed control rats as evidenced by the decrease in litter live weight gains. The concentrations of total lipid, total protein and lactose in milk were not affected by these dietary treatments. The concentration of alpha-lactalbumin in milk of rats fed the low protein diet was, however, lower than that in the milk of all rats receiving the control diet, irrespective of intake. Consumption of the restricted diets resulted in only small changes in specific activities (mu/mg protein) of 15 mammary enzymes. In the livers, lactation led to higher specific activities of all four soluble lipogenic enzymes examined but did not affect the particulate enzymes involved in complex lipid synthesis. The dietary restrictions resulted in lower specific activities of the soluble enzymes compared with those of the lactating rats consuming the control diet ad libitum without affecting the particulate enzymes. Total activities of these enzymes were, however, lower than those for the control rats as a result of the smaller liver mass in the rats receiving the restricted diets.

The extracellular matrix protein tenascin (previously described as myotendinous antigen) is selectively present in the mesenchyme surrounding fetal rat mammary glands, hair follicles, and teeth, three organ anlagen where the mesenchyme is essential for development. No tenascin is detectable in the normal adult mammary gland. Carcinogen-induced mammary tumors contained tenascin in their fibrous tissue. As reported for the molecule described as a "hexabrachion," tenascin contaminates so-called "cell-surface fibronectin," where it accounts for most of the detectable hemagglutinating activity. Of the extracellular matrix proteins compared, tenascin is the least effective substrate for attachment of primary mammary tumor cells, but the most effective in promoting cell growth after serum is removed from the culture medium.