The detection of the messenger ribonucleic acid for the α-lactalbumin of guinea-pig milk (Short Communication)

RNA was extracted from the polyribosomes isolated from the mammary glands of a lactating guinea pig and injected into Xenopus oocytes. On incubation the oocytes effected the biosynthesis of alpha-lactalbumin.     

The presence of the milk protein, alpha-lactalbumin and its mRNA in the rat epididymis

alpha-Lactalbumin, a modifier protein that changes the substrate specificity of galactosyltransferase, to promote the synthesis of lactose, is found in the mammary glands of lactating mammals and in milk. Molecules similar to mammary gland alpha-lactalbumin but distinct in their modifier activity have been found in rat epididymal fluid. We report here, using a rat mammary gland alpha-lactalbumin cDNA clone as a hybridization probe, RNA sequences homologous to alpha-lactalbumin mRNA were detected in total RNA from the rat epididymis. This finding suggests that alpha-lactalbumin or similar molecules, in addition to regulating lactose synthesis in the mammary gland, may have other important functions in mammalian reproduction.     

Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue.

We used 35S-labeled cRNA probes to localize the sites of alpha-lactalbumin, alpha-S1-casein, and lactoferrin mRNA synthesis in sheep and forcibly weaned cattle mammary tissue. Expression of alpha-lactalbumin was absent in three of four "virgin" glands studied, present in some alveoli of "pregnant" glands but not in others, despite a similar histological appearance. In the early lactating gland, expression was high in those alveoli with few fat globules in their cells and lumen and was absent in alveoli with abundant fat globules. These observations suggest either that alpha-lactalbumin gene expression is linked to the long-term secretory activity of cells and falls once cells are resting or regressing, or that there are cyclical variations in expression, or that in the lactating gland some groups of epithelial cells are synthesizing alpha-lactalbumin and some are synthesizing fat. Expression patterns of alpha-S1-casein were similar to those of alpha-lactalbumin. Lactoferrin, in contrast, was expressed almost exclusively in the "fatty alveoli" of both species. Our results show that dramatic variations in milk gene expression can occur throughout the mammary gland of sheep and cattle and that at no stage of pregnancy, lactation, or involution can the gland be considered metabolically homogeneous.     

Guinea-pig milk-protein synthesis. Isolation and characterization of messenger ribonucleic acids from lactating mammary gland and identification of caseins and pre-alpha-lactalbumin as translation products in heterologous cell-free systems.

1. The major milk proteins synthesized by the lactating mammary gland of the guinea pig were identified and designated as caseins A, B and C and alpha-lactalbumin, with estimated mol.wts. of 28000, 25500, 20500 and 14500 respectively. 2. Antisera to the total casein fraction and to alpha-lactalbumin were prepared from rabbits. The milk proteins were also iodinated with either 131I or 125I. 3. A poly(A)-rich RNA fraction was isolated from lactating guinea-pig mammary glands. Isolation was by affinity chromatography on oligo(dT)-cellulose. 4. Examination of this RNA fraction by electrophoresis on polyacrylamide gels containing formamide indicated three major species 1, 2 and 3, with estimated wol.wts. of 5.4 X 10(5) and 3.3 X 10(5), and the apparent absence of rRNA species. 5. The poly(A)-rich RNA stimulated protein synthesis in heterologous cell-free systems based on wheat germ, Krebs II ascites-tumour cells, and the latter supplemented with an initiation factor-3 fraction from rabbit reticulocyte ribosomes. 6. Between 80 and 90% of the protein synthesis directed by the mRNA was for milk proteins. 7. Analysis of the proteins immunoprecipitated by the alpha-lactalbumin antiserum showed in the wheat-germ system that the product was a protein with a molecular weight greater than that of alpha-lactalbumin, whereas in the ascites-tumour-cell systems both this protein and alpha-lactalbumin were found. When the larger protein was treated with CNBr and the resulting peptides were examined, it was shown that the extra peptide was at the N-terminus. This and other evidence is adduced for the initial translation product of alpha-lactalbumin being a precursor with an addition of about ten amino acids at the N-terminus. 8. Similar analysis of the casein immlnospecific proteins produced under the direction of mRNA indicated that the products had a molecular weight that was apparently a littel smaller than that of the caseins synthesized in vivo. This was not consistent with higher-molecular weight casein precursors. 9. Possible explanations for the results obtained are discussed, especially in terms of the physiological significance of the pre-alpha-lactalbumin as a secretory protein.     

Stimulation by insulin of the formation of ribonucleic acid and protein by mammary tissues in vitro

1. Insulin is one of the hormones that are essential for successful tissue culture of explants of the mammary glands of pregnant mice. We report here effects of insulin on RNA and protein formation by mammary tissue from pregnant mice and rats incubated in tissue-culture medium 199. 2. The incorporation of [(14)C]adenine over 3hr. into the RNA of explants of the mammary glands of pregnant mice was increased by an average of 68% when the medium contained 5mug. of insulin/ml. Under similar conditions the incorporation into the RNA of slices of the glands of pregnant rats was increased by an average of 61%. Incorporation into the RNA of slices from lactating rats was stimulated to a smaller extent. 3. Adipose tissue was separated from the glands of pregnant mice and the effect of insulin on the incorporation of adenine into its RNA was studied. In whole explants the incorporation of adenine, both with and without insulin, is almost entirely into the RNA of the mammary parenchyma and not of the adipose tissue. 4. Insulin also stimulated by 38% the incorporation of [(14)C]leucine over 3hr. into the proteins of slices of the glands of pregnant rats. It had no significant effect on slices from lactating rats. 5. Actinomycin D (10mug./ml.) decreased the incorporation of [(14)C]adenine into the RNA of slices of the glands of pregnant rats by an average of 97%. Though it also decreased the incorporation of [(14)C]leucine into the proteins by an average of 25%, the percentage stimulation by insulin of this incorporation remained unchanged.     

Multi-origins of milk serum albumin in the lactating goat.

L-[U-14C]Leucine was infused into the right-hand mammary glands of lactating goats. Milk from both glands of the animals was sampled at intervals for 36 h. After 3 h the specific activity of milk serum albumin from the infused glands was more than six times that from the non-infused glands. The specific activity of milk serum albumin was considerably lower than that of alpha-lactalbumin or beta-lactoglobulin which are exclusively synthesized by mammary secretory cells. Following the intravenous injection of 125I-labeled serum albumin, maximum specific activity of this protein appeared in milk in 12 h. The specific activity of serum albumin in milk attained no more than 45% of the specific activity of the serum albumin in blood. It is concluded that milk serum albumin has multiple origins and that a portion of it, at least (10-20%), is made in the mammary gland.     

Differences in the ribosomes prepared from lactating and non-lactating bovine mammary gland

1. Ribosomes prepared from bovine lactating mammary gland are able to synthesize protein, whereas similar preparations from non-lactating glands are not. Washing the ribosome suspensions through a medium containing 0.5m-ammonium chloride enhanced their ability to incorporate phenylalanine into polyphenylalanine. 2. Ribosomes isolated from non-lactating bovine mammary gland, in contrast with those from rat liver and lactating mammary gland, contained significant amounts of extraneous nucleases. These enzymes could be removed by washing with a medium A buffer containing 0.5m-ammonium chloride. 3. Only those ribosomes from functionally active tissues were able to bind polyuridylic acid and phenylalanyl-tRNA.     

Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300-350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [(3)H]hydroxyproline in scorbutic and recovery wounds showed a 3-4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [(14)C]leucine indicated that general protein synthesis, as measured by (14)C incorporated into non-diffusible material/mug of DNA, was unaltered by ascorbate supplementation. Similar studies of [(3)H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [(3)H]proline incorporated/mug of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (alpha(1))(2)alpha(2). The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.     

Hormonal regulation of retinoic acid-binding proteins in the mammary gland.

A cytosolic retinoic acid-binding (RAB) protein that sediments specifically as a 2S component on sucrose density gradients was detected in the mammary glands of virgin, pregnant and lactating rats. Mammary cytosol from pregnant rats contained significantly higher concentrations of cytosolic RAB protein than did cytosol from either virgin or lactating rats. The glands of pregnant animals exhibited increased concentration of cytosolic RAB protein during the first 5 days of pregnancy, and a steady decline was observed thereafter. The concentration of cytosolic RAB protein dropped to the value observed during lactation on the day 20 of pregnancy. Moreover, throughout lactation, low concentrations of cytosolic RAB protein were maintained. Daily treatment of virgin and lactating animals with 5 micrograms of oestradiol-17 beta for 1 week increased cytosolic RAB protein to concentrations comparable with those seen in pregnant rats. Progesterone, however, did not affect the mammary cytosolic RAB protein content of virgin rats. These results suggest hormonal involvement in the regulation of cytosolic RAB protein concentration of mammary gland during differentiation.     

Effect of a modified separation procedure on the size and protein-synthetic activity of membrane-bound liver polyribosomes

1. Various subcellular fractions containing ribosomes were isolated from rat liver. 2. In the presence of [(14)C]leucine and Sephadex-treated cell sap the radioactivity incorporated into the synthesized protein resulting from the incubation of microsomal preparations or deoxycholate-treated polyribosomes was dependent on the amount of rRNA incubated. In contrast, when Sephadex-treated post-mitochondrial supernatant was incubated, the radioactivity incorporated into the synthesized protein was independent of the amount of rRNA incubated. 3. Microsomal preparations and membrane-bound ribosomes, prepared by the standard procedure, incorporated less [(14)C]leucine into protein, per mg of rRNA incubated, than free or deoxycholate-treated polyribosomes; accordingly, polyribosomes associated with the former fractions were found mainly as monomers. 4. If microsomal fractions or membrane-bound ribosomes were prepared by a simple modification of the standard procedure, i.e. by centrifugation on to a ;cushion' of 2m-sucrose, their protein-synthesizing activity was of the same order as that of the original post-mitochondrial supernatant, and membrane-free and deoxycholate-treated polyribosomes; in this case polyribosome profiles showed that very little degradation had occurred and compared well with those obtained for post-mitochondrial supernatant and isolated polyribosomes. 5. A method is described (Appendix) that provides a rapid and reliable assessment of the concentration of rRNA in subcellular fractions.     

11 beta-hydroxysteroid dehydrogenase activity in the mammary gland

Levels of 11 beta-hydroxysteroid dehydrogenase activity in mammary gland homogenates from pregnant and lactating Sprague-Dawley rats were determined by incubation with [3H]corticosterone under standard conditions, followed by thin-layer chromatography of incubated media. Enzyme activity was high in virgin and pregnant rats, but fell soon after parturition, suggesting a possible role for this enzyme in the co-ordinate regulation of glucocorticoid effects on milk protein synthesis.     

Purification and characterization of mouse alpha-lactalbumin from lactating mammary glands

The whey protein, alpha-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14600). Antibodies prepared against these peptides precipitate newly synthesized and secreted alpha-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse alpha-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine alpha-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of alpha-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect alpha-lactalbumin levels as low as 0.25 ng.     

Expression, activity, and localization of hormone-sensitive lipase in rat mammary gland during pregnancy and lactation

We examined the presence of hormone-sensitive lipase (HSL) in mammary glands of virgin, pregnant (12, 20, and 21 days), and lactating (1 and 4 days postpartum) rats. Immunohistochemistry with antibody against rat HSL revealed positive HSL in the cytoplasm of both alveolar epithelial cells and adipocytes. In virgin rats, immunoreactive HSL was observed in mammary adipocytes, whereas diffuse staining was found in the epithelial cells. Positive staining for HSL was seen in the two types of cells in pregnant and lactating rats. However, as pregnancy advanced, the staining intensity of immunoreactive HSL increased in the epithelial cells parallel to their proliferation, attaining the maximum during lactation. An immunoreactive protein of 84 kDa and a HSL mRNA of 3.3. kb were found in the rat mammary gland as in white adipose tissue. Both HSL protein and activity were lower in mammary glands from 20 and 21 day pregnant rats than from those of virgin rats, although they returned to virgin values on days 1 and 4 of lactation. Mammary gland HSL activity correlated negatively to plasma insulin levels. Immunoreactive HSL and HSL activity were found in lactating rats' milk. The observed changes indicate an active role of HSL in mammary gland lipid metabolism.     

Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

Induction of glutathione S-transferases A, B and C in rat liver cytosol by trans-stilbene oxide

RNAase H, which catalyzes the hydrolysis of the RNA moiety of an RNA-DNA hybrid, was measured in the mammary gland of virgin, pregnant, lactating, and weaning Fischer rats and in the R3230AC mammary tumor grown in the same animals. In the normal mammary gland when DNA levels were low, as in the virgin state or during involution, RNAase H activity was also low. During pregnancy and lactogenesis when DNA levels increased, RNAase H activity, either on the basis of mammary gland weight or DNA content, also increased. During lactation when cellular proliferation ceases but rates of RNA and protein synthesis continue to reach peak values, RNAase H activity decreased. Compared to the corresponding enzyme from host glands, RNAase H from the R3230AC mammary tumor grown in pregnant and lactating hosts changes similarly, but to a lesser extent. The RNAase H activity which, ona tissue weight basis, was higher than in normal tissue also increased during pregnancy and directly after parturition, but decreased during lactation. During pregnancy these changes were accompanied by an increase in tumor DNA values. During lactation the tumor DNA values returned to the level seen in virgin hosts. These results are consistent with a role for RNAase H in DNA replication in rat mammary gland and in R3230Ac mammary tumor.     

Identification of a major N-glycosylated protein of rabbit mammary gland and its appearance during development in vivo

A major N-glycosylated protein was purified from hormonally stimulated pregnant and lactating rabbit mammary tissue. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, immunodiffusion and amino acid analysis showed the protein to be transferrin. The rate of synthesis of the protein during tissue development was studied and found to parallel the whey proteins casein and alpha-lactalbumin. The function of the protein during lactation is discussed.     

Murine mammary gland RNA directed synthesis of casein in a heterologous cell-free protein synthesis system.

A cell-free protein synthesis system derived from Ehrlich ascites tumor cell ribosomes (S30) plus rabbit reticulocyte tRNA was developed and the activity of the system was dependent on rabbit reticulocyte ribosomal salt (0.5 M KC1) wash factors, The exogenous mRNAs from BALB/c mouse liver and the mammary gland were translated with a high efficiency in this heterologous cell-free system. Furthermore, the RNA from the lactating mammary gland faithfully directed the synthesis of casein. The presence of mouse casein in the reaction product was identified by radioimmunoprecipitation with mouse casein antiserum, co-electrophoresis of the reaction product and mouse casein the urea-polyacrylamide gel and by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The major portion of the lactating mammary gland RNA directed synthesis of the milk protein in the cell-free system appeared to be analogous to alphas casein,     

Real-time imaging of beta-lactoglobulin-targeted luciferase activity in the mammary glands of transgenic mice.

This study was aimed at establishing a new platform for real-time monitoring of milk-protein gene expression in the mammary glands. A transgenic reporter composed of the beta-lactoglobulin (BLG)/luciferase hybrid gene was targeted to the mammary glands of pregnant and lactating mice and luciferase activity was imaged in vivo with a low-light imaging system. The mammary glands of a 17-day pregnant mouse occupied an area comparable to that of a 6-day lactating mouse. Nevertheless, the intensity of the luciferase signal was much weaker and confined to regions in the inguinal and thoracic glands. A few small and defined locations of higher expression were also detected, indicating diversity in the initiation of this transgenic milk protein expression. In the lactating mice, high inter- and intra-heterogeneity among regions in a particular gland and among glands was demonstrated, and confirmed by ex vivo analysis of luciferase activity in mammary biopsies. The lack of correlation between luciferase activities and levels of beta-casein accumulation in these biopsies resulted, most probably, from the longer half-life of the native milk protein, compared to the activity of the transgenic marker in the tissue. Unilateral sealing of mammary glands for 4 hr resulted in complete abrogation of luciferase activity, establishing the BLG/luciferase transgene as a reliable tool to follow short-term stimuli. Dispersed mammary epithelial cells preserved luciferase activity in culture, and thus could be used for following mammary gland development after re-implantation. The bioluminescence-based methodology presented here eliminates averaging of heterogeneity in gene expression among glands, and misinterpretations resulting from sampling biopsies taken from inactive regions. Imaging luciferase expression in the mammary glands may enable an accurate monitoring of milk-protein gene expression during cyclic periods of development and apoptosis in a limited number of animals, and could be applied for reporting the consequences of selected drugs on milk-protein gene expression.     

Effects of serum on mammary epithelial proliferation in vitro during mammary gland development

The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed.