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PubMed1988

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization.

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.

Animals↗
PubMed1977

Regulation of casein synthesis by polyamines in mammary gland explants of mice.

Studies were carried out to determine whether the actions of prolactin on the metabolism of the mammary gland may involve polyamines. In mouse mammary gland explants that were preincubated for 2 days with insulin plus hydrocortisone, the rate of [3H]leucine incorporation into casein was enhanced in a prolactin-like manner during a further incubation with spermidine plus cyclic GMP or phospholipase A. Putrescine (0.5 mM) plus PGF2alpha, cyclic GMP or arachidonic acid also enhanced the rate of casein synthesis: but PGF2alpha plus 0.5 mM arginine, ornithine or spermine had no effect. Methyl GAG, an inhibitor of the enzyme S-adenosyl-L-methionine decarboxylase (which is required for the conversion of putrescine to spermidine), abolished the putrescine plus PGF2alpha stimulation of casein synthesis. Since this drug did not affect the action of spermidine plus PGF2alpha on casein synthesis, the specific action of spermidine on casein synthesis is suggested. Neither arginine, ornithine nor the polyamines, by themselves, affected the rate of [3H]uridine incorporation into RNA or the rate of [3H]leucine incorporation into casein. Spermidine levels were elevated within 4 h after adding prolactin to explants which were preincubated for 2 days with insulin plus hydrocortisone; this effect was apparent during incubation periods of up to 48 h with prolactin. Arginase and ornithine decarboxylase activities were also elevated in response to prolactin. Arginase activity was only elevated, however, during long incubation periods with prolactin, i.e., during incubation periods of longer than 2 days. In contrast, ornithine decarboxylase activity was elevated by prolactin within a 30 min incubation period; this effect was maximal after 2 h and persisted during exposure periods of up to 24 h.

Animals↗
PubMed1982

Correlation between prolactin-receptor interaction, down-regulation of receptors, and stimulation of casein and deoxyribonucleic acid biosynthesis in rabbit mammary gland explants.

Increasing concentrations of PRL were added to culture media of rabbit mammary gland explants. After 24 h of culture, free and total PRL receptors, casein synthesis, casein messenger (m) RNA concentrations and DNA synthesis were estimated. In the PRL concentration range of 0-100 ng/ml, receptors were progressively down-regulated by the hormone, but no occupied desaturable receptors could be detected. At concentrations between 1,000 and 20,000 ng/ml, the PRL receptors were increasingly occupied without being quantitatively down-regulated. There exists a reciprocal correlation between the rate of casein synthesis, casein mRNA concentration, DNA synthesis and the down-regulation of the receptors as a function of PRL concentration. The maximal responses were reached around a PRL concentration of 100 ng/ml. Interestingly, with a large excess of PRL a desensitization process was observed, the responses to the hormone being attenuated. This desensitization was more pronounced for DNA synthesis than for casein synthesis or for casein mRNA accumulation. The data suggest that down-regulation of PRL receptors occurs even at physiological concentrations of PRL. These observations are compatible with the hypothesis that each receptor can generate a limited number of intracellular relays eliciting hormonal action before being irreversibly inactivated and degraded. In addition, high concentrations of PRL induce a refractory state in the mammary gland that is possibly related to a low level of peripheral PRL receptors.

Animals↗
PubMed2006

Integration of prolactin and glucocorticoid signaling at the beta-casein promoter and enhancer by ordered recruitment of specific transcription factors and chromatin modifiers.

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent system in which to perform kinetic studies of chromatin remodeling and transcriptional activation. Using HC11 cells as a model, we have investigated the effects of prolactin (Prl) and glucocorticoids both singly and in combination at different time points after hormone treatment. Using chromatin immunoprecipitation analysis, we have determined the dynamics of assembly and disassembly of signal transducer and activator of transcription 5, glucocorticoid receptor, CCAAT enhancer binding protein beta, and Ying Yang-1 at the hormonally activated beta-casein proximal promoter as well as the distal mouse beta-casein enhancer located approximately -6 kb upstream of the transcription start site. Prl alone resulted in a rapid recruitment of both signal transducer and activator of transcription 5 and histone deacetylase 1 to the beta-casein promoter and enhancer, and reciprocally the dissociation of Ying Yang-1 from the proximal promoter. In addition, we have examined the recruitment of coactivator p300 and determined chromatin acetylation status as a function of hormonal treatment. Finally, we have established the time course of RNA polymerase II and phospho-RNA polymerase II accumulation at the beta-casein promoter and enhancer after stimulation with hydrocortisone and Prl. Although glucocorticoids alone led to a rapid increase in histone H3 acetylation, treatment with both hormones was required for stable association of p300 and phospho-RNA polymerase II at both the promoter and enhancer. Collectively, these data suggest a model for the assembly of a multiprotein complex that helps to define how the signaling pathways controlled by these lactogenic hormones are integrated to regulate beta-casein gene expression.

Animals↗
PubMed1990

Prolactin and glucocorticoid hormones control transcription of the beta-casein gene by kinetically distinct mechanisms.

Transcription of the beta-casein milk protein gene in the HC11 mouse mammary epithelial cell line is induced synergistically by the hormones glucocorticoid and PRL. Sequential treatment of HC11 cells with glucocorticoid and PRL demonstrated that the two hormones had different modes of action on beta-casein transcription. Pretreatment with dexamethasone enhanced the response to subsequent induction by PRL, but not vice versa. Dexamethasone increased the sensitivity of the cells to respond to PRL. The increase in sensitivity was slow, extended for 16 days, and could be rapidly reversed by withdrawal of dexamethasone. The dexamethasone-induced sensitivity for the rapid transcriptional regulation by PRL could be observed with transfected rat beta-casein promoter-chloramphenicol acetyltransferase constructs retaining only 175 basepairs upstream from the transcription initiation site. Expression of the endogenous mouse beta-casein gene was regulated identically to that of the promoter constructs with respect to the synergy of the hormones and their different kinetics of action. In contrast to the slow induction of sensitivity toward PRL, dexamethasone rapidly induced the transcription of a mouse mammary tumor virus long terminal repeat controlled gene in HC11. This demonstrated a normal transcriptional activation of the glucocorticoid receptor in this cell line. Thus, glucocorticoid may regulate beta-casein gene transcription indirectly, inducing or repressing other glucocorticoid-regulated genes, whereas the interaction of PRL with its receptor causes a rapid induction of the beta-casein gene promoter.

Animals↗
PubMed1995

Nucleoplasmin associates with and is phosphorylated by casein kinase II.

Nucleoplasmin is a phosphorylated nuclear-accumulating protein. We report herein that the kinetics of its cytoplasm-->nucleus transport are affected by its degree of phosphorylation. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein; (ii) its phospho-transferase activity can be competed out by GTP; (iii) it is stimulated by polylysine; and (iv) it is inhibited by heparin. Moreover, a polyclonal antibody to the alpha (38 kDa) and alpha' (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin's nuclear transport. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II.

Animals↗
PubMed1995

Caseinphosphopeptides (CPP) in feces and contents in digestive tract of rats fed casein and CPP preparations.

A part of caseinphosphopeptides (CPP) formed during the digestion of casein in the small intestine of rats fed casein was not hydrolyzed in the digestive tract, but was excreted into the feces. Amino acid compositions of CPP fraction of feces for wk 1 and 2 were almost identical. The residual CPP in the feces expressed by the rate of bound phosphoserine in the CPP fraction of feces to bound phosphoserine ingested (phosphoserine-CPP/phosphoserine-ingested rate) in the ileum was the highest 4 h after the start of feeding of casein but decreased significantly after 10 h. No significant difference was observed in the rats of contents in jejunum, cecum, and colon between 4 h and 10 h after the start of feeding. No significant difference was observed in the phosphoserine-CPP/phosphoserine-ingested rate in contents of any part of the digestive tract between 50% casein and 50% CPP I (a commercial CPP product with nearly the same amino acid composition as that of casein) 4 h and 10 h after the start of feeding, except for the cecum 4 h after the start of feeding. No significant difference in phosphoserine-CPP/phosphoserine-ingested rate was observed in the contents of any part of digestive tract between the groups fed 50% casein and 5% CPP III +45% soybean protein isolate (SPI) 4 h or 10 h after the start of feeding (CPP III is a commercial CPP product containing nearly 8 times the concentration of phosphoserine as CPP I).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed2004

Effect of beta-casein (1-28) on proliferative responses and secretory functions of human immunocompetent cell lines.

The effect of bovine beta-casein (1-28) purified from commercial casein phosphopeptide preparations on human T, B, and monocyte cell lines was evaluated. Beta-casein (1-28) enhanced the proliferation of the following: T cell lines HUT-78, Jurkat Clone E6-1, and MOLT-4; B cell lines BALL, KHM-1B, and U266B1; and monocyte cell lines U937 and HL-60. Moreover, beta-casein (1-28) stimulated IgA production by KHM-1B over 96 h of culture. Semiquantitative reverse transcriptional-polymerase chain reaction analysis indicated that beta-casein (1-28) enhanced mRNA expression of interleukin (IL)-6 in U266B1 and KHM-1B. These results suggest that beta-casein (1-28) exerts a mitogenic effect on human T, B, and monocyte cells, and an IgA-enhancing effect on B cells.

Animals↗
PubMed1975

[The relations between lactation and cyclic-AMP -- The influences of adenyl cyclase activity on casein biosynthesis ability in the organ culture of the mouse mammary gland (author's transl)].

The relationships between hormonal action and cyclic AMP as the second messenger of hormones have recently been discussed on many hormones. Lactation is influenced by various hormone, especially, insulin, prolactin, and hydrocortisone. Whether adenyl cyclase activity in the mammary gland of mouse epithelial cells has parallel relations with casein biosynthesis ability or not was examined using the mammary gland organ culture method. Female, mid-pregnant (11-14 days), mice of DDY strain were used. Organ culture was done by the Chen's floating lens' paper method, using the hormone-added MEM media and non-added ones. Casein biosynthesis ability was measured by observing 32P incorporation into the casein molecules. Adenyl cyclase activity was estimated by the amount of 14-C-cyclic AMP produced out of adenine-8-14C by the Kuo and Krishna's method. Radio isotope compounds were pulsed for 4 hours in the medium. The experiments revealed that the added hormones had a remarkable effect on caein biosynthesis ability, but none on adenyl cyclase activity. No parallel fluctuation was observed between adenyl cyclase activity and casein biosynthesis ability, that is, the change of adenyl cyclase activity was found to have nothing to do with casein biosynthesis ability. Consequently, the cyclic AMP addition to the media showed no effect on casein biosynthesis ability.

Adenylyl Cyclases↗
PubMed1992

Apparent and true amino acid digestibility of a crystalline amino acid mixture and of casein: comparison of values obtained with ileal-cannulated pigs and cecectomized cockerels.

Six ileal-cannulated pigs that averaged 100 kg BW and 16 adult cecectomized cockerels that averaged 2.35 kg BW were used to determine apparent and true digestibilities of amino acids (AA) in a complete crystalline AA mixture and in casein. A protein-free (PF) diet was included as a treatment to estimate endogenous AA losses. Fasted cockerels were compared to cockerels fed PF diets for calculation of true digestibility of AA in cockerels. For the AA diet, true digestibility of indispensable AA in the pig ranged from a low of 97.2% for leucine to a high of 100.5% for arginine (Arg). True digestibility of indispensable AA in casein ranged from a low of 93.5% for isoleucine (Ile) to a high of 99.9% for Arg. Correcting for endogenous losses increased digestibilities of lysine (Lys) and threonine (Thr) in pigs fed the AA diet by 2.4 and 7.1%, respectively, and increased Lys and Thr digestibility in pigs fed the casein diet by 1.8 and 6.1%, respectively. Feeding a PF diet to chickens to correct for endogenous losses resulted in higher true digestibility values for all AA with the exception of tryptophan, methionine, and Arg than those obtained using fasted animals. True digestibilities of Thr were 88.3 and 86.6% for AA and casein diets, respectively, using fasted controls but were 97.5 and 94.5% when the PF control was used. Proline digestibility was increased (P < .05) substantially in both pigs and cockerels when the PF control was used to correct for endogenous AA losses. Regardless of species, Ile in casein had a lower true digestibility value than any other indispensable AA. The results of this study indicated that true digestibilities of AA in a mixture of crystalline AA and in casein are essentially 100% in both pigs and cockerels.

Amino Acids↗
PubMed2003

Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows.

Milk stasis triggers local stimuli, which make the tight junctions leak and trigger involution. The aim of the study was to test the hypothesis that casein hydrolyzates compromise tight junction integrity and dry-off milk secretion in dairy cows. Six repeated doses of casein hydrolyzates after each milking during 3 d caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion. No such changes were recorded in the control glands that had been treated with nonhydrolyzed casein. Treatment with casein hydrolyzates disturbed tight junction integrity within 8 h (as indicated by changes in Na+ and K+ concentrations), reduced the concentrations of lactose precipitously, activated the plasmin activator-plasminogen-plasmin system, and induced the secretion of immunoglobulin type G and lactoferrin. At the end of the 3-d treatments, we stopped milking the experimental and control glands. Milk composition 19 d later was similar in the experimental and control glands and was consistent with the composition expected in fully involuted glands. We conclude that casein hydrolyzates are among the milk-borne factors that cause the disruption of tight junction integrity and induce involution in cows. The process induced by casein hydrolyzate was more rapid and synchronized than the involution induced at drying-off.

Albumins↗
PubMed2003

Dependence of the interfacial behavior of beta-casein on phosphoserine residues.

The role of the phosphoserine residues on the dynamical and structural properties of beta-casein was studied by molecular dynamics of the protein in water/lipid interfacial regions. The initial protein structure adopted in the modeling was that proposed for bovine beta-casein A2, where the five phosphoserine residues, originally present in its primary structure, were partially or totally substituted by serine residues. The simulations revealed a dependence of the interfacial behavior of beta-casein on the phosphorylation grade. When only partially dephosphorylated, the protein showed a similar behavior as that observed for the original beta-casein reported in previous work. During dynamics, the protein migrated from the aqueous environment towards the lipid medium, and remained attached to the interface separating both media. Quite different was the dynamics of the totally dephosphorylated beta-casein, that did not perceive the interface and immersed incessantly into lipid medium. The results suggest that the phosphoserine residues appear to be, in fact, intrinsically related to the mechanisms of beta-casein emulsion stabilization.

Animals↗
PubMed2004

Influence of residual milk-clotting enzyme on alpha(s1) casein hydrolysis during ripening of Reggianito Argentino cheese.

Milk-clotting enzyme is considered largely denatured after the cooking step in hard cheeses. Nevertheless, typical hydrolysis products derived from rennet action on alpha(s1)-casein have been detected during the ripening of hard cheeses. The aim of the present work was to investigate the influence of residual milk-clotting enzyme on alpha(s1)-casein hydrolysis in Reggianito cheeses. For that purpose, we studied the influence of cooking temperature (45, 52, and 60 degrees C) on milk-clotting enzyme residual activity and alpha(s1)-casein hydrolysis during ripening. Milk-clotting enzyme residual activity in cheeses was assessed using a chromatographic method, and the hydrolysis of alpha(s1)-casein was determined by electrophoresis and high performance liquid chromatography. Milk-clotting enzyme activity was very low or undetectable in 60 degrees C- and 52 degrees C-cooked cheeses at the beginning of the ripening, but it increased afterwards, particularly in 52 degrees C-cooked cheeses. Cheese curds that were cooked at 45 degrees C had higher initial milk clotting activity, but also in this case, there was a later increase. Hydrolysis of alpha(s1)-casein was detected early in cheeses made at 45 degrees C, and later in those made at higher temperatures. The peptide alpha(s1)-I was not detected in 60 degrees C-cooked cheeses. The results suggest that residual milk-clotting enzyme can contribute to proteolysis during ripening of hard cheeses, because it probably renatures partially after the cooking step. Consequently, the production of peptides derived from alpha(s1)-casein in hard cheeses may be at least, partially due to this proteolytic agent.

Aspartic Acid Endopeptidases↗
PubMed1975

Action of rennin on kappa-casein.

The action of rennin on kappa-casein was studied as a function of time using several methods to measure activity. The first indication of rennin cleavage of kappa-casein is precipitability in .1 M acetate buffer at pH 5.2 and 5 C. A longer exposure to rennin is required to alter kappa-casein so that it forms a precipitate with calcium ions and loses its ability to stabilize alpha s-casein. The least sensitive indication of rennin activity is measurement of nitrogen soluble in 2% trichloroacetic acid. Electrophoresis experiments showed that these methods detect various stages in the conversion of kappa-casein para-kappa-casein.

Calcium Chloride↗
PubMed1976

Isolation of kappa-casein-like proteins from milks of various species.

Kappa-Casein-like proteins were isolated from the milks of cow, goat, reindeer, horse, rat, and rabbit. When treated with rennin, all of the isolated kappa-casein components yielded para-kappa-casein-like bands on gel electrophoresis. The rate of cleavage of these components with rennin was determined by measuring material soluble in trichloroacetic acid (macropeptide). The curves were characteristic of a limited, specific attack by rennin on these proteins. The goat and reindeer kappa-caseins were nearly as bovine kappa-casein, but the cleavage of horse, rat, and rabbit kappa-casein-like components was much slower.

Animals↗
PubMed1985

A 20,000-dalton casein fragment in human milk.

A new peptide of 20,000 daltons was found in human milk as a constituent of the casein micelle. Enzymic digestion with plasmin or trypsin revealed that the peptide was identical with a degradation product of human beta-casein. The amino acid composition of the degradation product and the previously reported sequence in the N-terminal region of human beta-casein suggested that the peptide was a fragment of beta-casein lacking the C-terminal region. The thermal sensitivity of this peptide was higher than that of beta-casein, but the peptide lost the property of calcium-dependent precipitation, which intact beta-casein possesses.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of monoclonal antibody directed against bovine alpha s2-casein.

A monoclonal antibody 62-1A of isotype IgM, directed against bovine alpha s2-casein, was isolated and characterized. Monoclonal antibody 62-1A recognized bovine alpha s2-11P- and alpha s2-9P-caseins by indirect solid phase radioimmunoassay and Western blot analysis. Crossreactivity toward native genetic variants (A, B, and C) of alpha s1-casein was similar but lower (approximately 60 to 70%) than that for alpha s2-11P-casein). Little or no crossreactivity was observed for other bovine milk or serum proteins. Antibody affinity for alpha s2-11P-casein was 1.3 x 10(9)/M. As little as 25 ng/ml (.5 ng/well) of alpha s2-11P-casein was detected by solid phase radioimmunoassay.

Animals↗
PubMed1991

Separation of beta-casein A1, A2, and B using cation-exchange fast protein liquid chromatography.

beta-Casein genetic variants A1, A2, and B were separated using cation-exchange fast protein liquid chromatography. beta-Casein from a herd bulk casein sample eluted as a series of three peaks. Casein samples from individual cows containing known combinations of beta-casein A1, A2, and B were used to confirm that the three peaks were beta-casein genetic variants. An acid-PAGE gel confirmed the identity of the peaks that eluted from the column.

Animals↗