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The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, β-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a β-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Background: The aim of the study was to investigate bioactive whey protein concentrations in cow's milk collected in late pasture (LP) and early indoor feeding (EIF) season from conventional and organic farms in Poland., Results: Results showed that in the LP somatic cell count (SCC) was higher under organic farming conditions. However, percentages of protein and fat were higher under conventional farming conditions. In EIF, milk from conventional dairy farms had a higher percentage of fat and lactose and a lower concentration of protein and SCC in comparison to milk from organic farms. Organic milk in LP had higher concentrations of beneficial whey proteins than conventional milk, including β-lactoglobulin (β-Lg, 4.12 vs. 2.68 g L⁻¹), lactoferrin (Lf, 334.99 vs. 188.02 mg L⁻¹), and lysozyme (Lz, 15.68 vs. 12.56 µg L⁻¹). However, conventional milk in EIF had higher concentrations of bovine serum albumin (146.47 vs. 118.65 mg L⁻¹), Lf (49 vs. 185.27 mg L⁻¹), and Lz (16.63 vs. 13.22 µg L⁻¹)., Conclusions: The results show significant differences in the investigated parameters between organic milk and milk from conventional system during EIF and LP. Moreover, extending the pasture season during EIF in organic farms decreases concentration of bioactive compounds of milk., (Copyright © 2012 Society of Chemical Industry.)

The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were 2.1- or 3.1-fold higher, respectively, than in their bovine L-1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase (S6K1) on Thr(389) but not on Ser(411) was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E-BP1 or S6K1 phosphorylation than deprivation of all AA. In AA-deprived CID-9 cells, Leu specifically restored BLG synthesis from pre-existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E-BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu- and AA-stimulated BLG translation initiation in a dose-dependent manner. A further reduction was observed for Leu-treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells., ((c) 2006 Wiley-Liss, Inc.)

The regulation of milk constituents, synthesis and secretion in tissue cultures of the bovine mammary gland was altered by a whey fraction of bovine milk. alpha-Casein gene expression, casein secretion and fatty acid synthesis were inhibited by the whey fraction in a dose-dependent manner. The whey fraction inhibited the enhancement activity of prolactin on alpha-casein gene expression and fatty acid synthesis, and also inhibited casein secretion to the medium, in explants cultured in a medium with or without prolactin. No effect on the expression of the beta-lactoglobulin gene was found.

Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine beta-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of beta-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with gamma-interferon activation site-related motifs within the beta-lactoglobulin promoter, which we show are required for matrix dependence of beta-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.

To explore the possibility that genes might compete for expression, we have studied transgenic mice producing high levels of the sheep milk protein, beta-lactoglobulin (BLG), in the mammary gland. Mice carrying one or more transgene loci expressed BLG in milk at levels ranging from 7 to 33 mg/ml. The effects of BLG synthesis on the levels of endogenous milk gene expression were examined. No significant increase in total milk protein concentration was recorded even in mice expressing the largest amounts of BLG. Measurement of individual milk proteins showed that transgene protein was manufactured at the expense of host protein synthesized in the gland. Whey acidic protein production was more suppressed than casein production. Suppression of endogenous proteins was matched by a reduction in the corresponding steady-state mRNA levels; in double-transgenic mice, which expressed the largest amounts of BLG, beta-casein and whey acidic protein mRNA populations were reduced to 75 and 56% of control levels respectively. We demonstrate that an exogenous gene competes effectively for expression with endogenous genes. Possible mechanisms of competition are discussed.

Mammary development and milk secretion were studied in transgenic mice which exhibited mammary tissue-specific expression of the sheep beta-lactoglobulin gene, and secreted significant quantities of the foreign protein in milk. Mammary development was unaffected by transgenesis. Tissue DNA content and the activities of several key enzyme markers of cell differentiation were similar in transgenic mice and non-transgenic controls. Milk yield, whether estimated by pup weight gain or measured by a 3H2O-dilution method, was unchanged by foreign gene expression. Gross milk composition, including milk protein concentration, was also similar in transgenic and non-transgenic animals, even though beta-lactoglobulin accounted for 29% of total milk protein. Therefore the foreign gene product was synthesized at the expense of endogenous milk proteins. However, transgenic mammary tissue in vitro exhibited a significantly higher rate of total protein synthesis than did control tissue. This suggested that a factor limiting milk protein synthesis or secretion in transgenic mice in vivo may have been removed by short-term explant culture of mammary tissue. The results emphasize that the use of transgenesis for manipulating milk composition may depend not only on high-level mammary-specific expression of the foreign gene, but also on the biosynthetic capacity of the mammary gland itself.

Five month-old transgenic female pigs from three lines carrying the mouse whey acidic protein (WAP) gene and nontransgenic female littermates were implanted with slow-release estrogen and progesterone pellets. Histological analysis of biopsies taken at the time of implantation and 4 weeks later revealed that mammary alveolar development had occurred upon hormonal stimulation in vivo. beta-Casein and beta-lactoglobulin mRNA was found in all induced animals, and WAP mRNA was detected in two of the three transgenic pigs. Differential hormonal regulation between the transgenes in the three lines and also between endogenous milk protein genes was observed in induced mammary tissue cultured in vitro. In the presence of insulin, hydrocortisone, and PRL, beta-casein and WAP mRNA levels increased in all transgenic pigs. In contrast, beta-lactoglobulin mRNA had reached or exceeded lactational levels in response to the in vivo induction, and no further increase was observed in vitro. This suggests that the regulation of the beta-lactoglobulin gene is distinct from that of beta-casein and WAP. Differences were also observed during pregnancy; whereas beta-lactoglobulin gene expression was induced in early pregnancy, a time when PRL levels are low, WAP mRNA levels increased sharply around parturition. Finally, the observation that hormonal regulation of WAP transgenes greatly differed between the three lines suggests that chromatin surrounding the integration site can modify the response of transcription elements.

The amino acid requirement of rat mammary cells for milk protein synthesis was investigated in dispersed cell culture. A three-dimensional central composite design utilizing three variables (X1 = lysine; X2= methionine, valine, and arginine; X3 = isoleucine, tryptophan, threonine, phenylalanine, and histidine) at five concentrations each, was duplicated twice with mammary cells from lactating Sprague-Dawley rats. The optimum combination of amino acids for maximum milk protein synthesis from multiple regression models was X1 15.0-, X2 4.5-, and X3 1.5-fold their quantities in Eagle's minimal essential medium with leucine, tyrosine, cystine, and glutamine at the base 1-fold in the medium.

The radiolabelled primary translation products of ovine mammary mRNAs synthesized in a wheat germ cell-free system were isolated by immunoprecipitation and analysed by automated Edman degradation. The 3 'Ca-sensitive' caseins (alpha S1, alpha S2 and beta), kappa-casein, beta-lactoglobulin and alpha-lactalbumin were found to be synthesized as precursors with amino terminal extensions of 15, 21, 18 and 19 amino acid residues respectively. The extra pieces of these various lactoproteins are similar to 'signal' peptides of other secretory proteins in their length and hydrophobicity. The occurrence of an alanyl residue at the C-termini of the extra pieces of the 6 ovine prelactoproteins suggests that the mammary proteinase responsible for the cleavage of the signal peptides may have an elastase-like specificity. When mammary mRNAs were translated in a wheat germ cell-free system in the presence of mammary microsomal membranes, pre-beta-casein was converted into authentic beta-casein as demonstrated by amino terminal sequence analyses. Additionally, pre-beta-casein was post-translationally converted into authentic beta-casein by a specific proteinase(s) extracted from rough microsomes with Na deoxycholate.

This review, written for non-specialists, describes briefly the steps of protein biosynthesis from their precursors in the blood to the excreted molecules, taking rat gamma-casein as an example. A schematic description is given of the procedures employed for preparing cDNAs which can provide the sequences of the corresponding mRNAs and proteins. Recent findings concerning milk proteins are briefly mentioned, and in particular those dealing with sequence determinations of mRNAs coding for milk proteins from several species.

Effect of labeled precursor amino acids, varying cell densities, graded quantities of amino acid complement, and incubation environment on milk protein synthesis were studied with cultures of mammary cells isolated from Sprague Dawley rats. The essential amino acid complement of Eagle's minimal essential medium was used as base. Protein synthesis, measured by incorporation of labeled lysine, leucine, and phenylalanine, was affected by source of label for the "beta-lactoglobulin fraction" and beta-casein but not alpha-lactalbumin. Cell numbers between 6 X 106 and 6 X 107 per 5 ml of culture medium did not significantly alter rates of synthesis. Increasing amounts of amino acid concentration from one to three-fold increased synthesis of "beta-lactoglobulin fraction" and alpha-lactalbumin regardless of cell population. Response to addition of essential amino acid for "beta-lactoglobulin fraction" synthesis was linear over one to five-fold with 30.9 mug/flask per fold addition (linear regression coefficient; squared correlation = .91). Results were similar for beta-casein synthesis; 25.9 mug/flask and squared correlation = .91. No culturing effects between carbon dioxide and conventional incubators were significant.

Experiments are presented to determine the effect of labeled precursor amino acid on synthesis of beta-lactoglobulin and beta-casein and to identify the most limiting amino acids in Eagle's minimal essential medium for synthesis of milk protein. Protein synthesis was measured by incorporation of carbon-14 labeled amino acids into milk protein from bovine mammary cell culture. Mammary tissue from 13 Holsteins (12.7 kg milk/day) was dispersed and cultured for 18 h. In Experiment 1, three replications (cows) with three amino acid mixtures, utilizing labeled lysine or leucine as the marker amino acid, showed that labeled lysine overestimated the effect of medium lysine, and labeled leucine overstimated the effect of leucine on beta-lactoglobulin and beta-casein synthesis. Subsequently Experiment 2 utilized 10 replications (cows) to determine the effect of a three-fold increase of each of 13 amino acids in Eagle's medium. Increased threonine or methionine improved beta-lactoglobulin synthesis and cystine increased beta-casein. Second and third best response in beta-casein were from addition of methionine and threonine. Combined analysis from 10 cows and two protein responses by multivariate procedures identified, in order of decreasing magnitude, cystine, threonine, and methionine as limiting amino acids in the dispersed cell culture.

To identify the limiting amino acid in the minimal essential medium as published by Eagle (Science 130:432, 1959) for milk protein synthesis in rat mammary cells in tissue culture, two different experimental approaches were used. The first study involved the reduction of amino acids singly from the total amino acid complement of the medium for milk protein synthesis. The second study was to investigate the effect on milk protein synthesis of single amino acid addition to the basic complement of amino acids. Order of limiting amino acids was lysine (first) and possible methionine, valine, or arginine (second).