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PubMed1998

An ELISA for a multiphosphorylated peptide, alpha(s1)-casein(59-79).

Multiphosphorylated segments of proteins are predicted to be in or near epitopes. However, due to the very hydrophilic nature of multiphosphorylated peptides, epitope mapping by ELISA using conventional microtitre plates can produce false negatives due to poor antigen adsorption. We have developed a sensitive ELISA for a multiphosphorylated peptide alpha(s1)-casein(59-79) containing five phosphoseryl residues using Nunc-Immuno Maxisorp modules for antigen adsorption. The peptide alpha(s1)-casein(59-79) was detected in the ELISA at antigen coating concentrations of 1.0 microg/ml and above, using rabbit anti-casein antibodies at a dilution of 1/10,000 in Tris-buffered saline containing 0.05% (w/v) Tween 20 and 1.0% (v/v) normal goat serum. At an antigen coating concentration of 10 microg/ml, anti-casein antibodies bound to alpha(s1)-casein(59-79) and produced an absorbance of more than 100 times background. Using conventional polyvinyl chloride and polystyrene plates the peptide alpha(s1)-casein(59-79) could only be detected at very high antigen coating concentrations of 1-10 mg/ml. The addition of 0.05% (w/v) Tween 20 to the blocking, antibody diluting and wash buffers of the ELISA was shown to significantly reduce nonspecific binding of the primary antibody. Further, the inclusion of normal goat serum in the blocking and antibody diluting buffers resulted in a small but significant increase in absorbance. The ELISA developed in this study has been used successfully with a range of enzymatically derived and synthetic peptides containing one to five phosphorylated residues such that it should have general applicability to the study of antigenicity of multiphosphorylated peptides.

Amino Acid Sequence↗
PubMed2000

Stress down regulates milk yield in cows by plasmin induced beta-casein product that blocks K+ channels on the apical membranes.

Stress and stress related hormones such as glucocorticoids inhibit lactation in cows. In the present study we propose a novel mechanism connecting stress with plasminogen-plasmin system (PPS) (an enzymatic mechanism in milk, which leads to the breakdown of the major milk protein casein). We show that stress activates the PPS leading to an increase in plasmin activity, and that a distinct plasmin-induced beta-casein breakdown product (fraction 1-28) is a potent blocker of potassium channels in mammary epithelia apical membranes. The reduction in milk production due to dehydration stress or glucocorticoid (dexamethsone) was correlated with the activities of plasmin and channel blocking activity in the milk of the tested cows. The notion that the axis Stress-PPS-beta-casein fraction 1-28 is responsible for the reduction in milk yield is supported by the results of experiments showing that injecting solution composed of casein digest enriched with beta-casein fraction 1-28 to the udder lumen leads to a transient reduction in milk production. Furthermore, injecting a pure beta-casein fraction 1-28 to the udder lumen of goat's lead also to a transient reduction in milk production with kinetics that was similar to the kinetics observed in cows.

Animals↗
PubMed1996

Establishment and characterization of alpha s1-casein-specific T-cell lines from patients allergic to cow's milk: unexpected higher frequency of CD8+ T-cell lines.

To study cow's milk allergy at the cellular level, we assessed the reactivity of peripheral blood mononuclear cells from patients allergic to cow's milk to alpha s1-casein, which is one of the major allergens in cow's milk. Proliferation of the cells to alpha s1-casein activation showed a rather weak response. Therefore to understand T-cell reactivity to alpha s1-casein in more detail, we prepared alpha s1-casein-specific T-cell lines from patients allergic to cow's milk and established 26 T-cell lines. These T-cell lines could be classified into three groups by analyzing their surface marker expression: those containing predominantly CD4+ CD8- T cells, those containing both CD4+CD8- and CD4-CD8+ T cells, and those containing predominantly CD4- CD8+ T cells. The CD8+ T cells were obtained at an unexpectedly higher frequency from the patients. These T-cell lines produced interferon-gamma and IL-4. These results suggest that CD8+ T cells specific for alpha s1-casein and CD4+ T cells were primed by the stimulation with alpha s1-casein in patients allergic to milk and that both T cells may play a key role in the onset, progression of, or recovery from cow's milk allergy.

CD8-Positive T-Lymphocytes↗
PubMed1996

Cell-mediated immune response to beta casein in recent-onset insulin-dependent diabetes: implications for disease pathogenesis.

BACKGROUND: The cows' milk hypothesis for the cause of insulin-dependent diabetes (IDDM) is based on the concept that early consumption of cows' milk may expose the immune system to a foreign protein possessing immunological cross-reactivity with an antigen present on pancreatic beta-cells. METHODS: We measured in-vitro peripheral lymphocyte response to beta casein, a protein in cows' milk, in 47 patients with recent-onset IDDM, in 36 healthy people and, to test disease specificity, in 10 patients with autoimmune thyroid disease. Other antigens tested for were bovine serum albumin, purified protein derivative, human serum albumin, and phytohaemagglutinin. RESULTS: Specific proliferation of T lymphocytes with bovine beta casein was seen in patients with IDDM (mean [SD] age 18.7 [9]) with a significant difference in mean stimulation index (SI) versus healthy people (p < 0.00001) or patients with autoimmune thyroid disease (p < 0.002). 24 of 47 (51.1%) patients with IDDM versus 0/10 patients with thyroid disease and 1/36 (2.7%) healthy people had a positive response to beta casein defined as a SI above the mean value +2 SD of healthy people (p < 0.00001). No significant differences were observed between the groups of subjects with respect to other antigens tested. INTERPRETATION: The association between IDDM and early consumption of cows' milk may be explained by the generation of a specific immune response to beta casein. Exposure to cows' milk triggers a cellular and humoral anti-beta casein immune response which may cross-react with a beta-cell antigen. It is of interest that sequence homologies exist between beta casein and several beta-cell molecules.

Adolescent↗
PubMed1999

Comparative aspects of milk caseins.

The caseins comprise the major protein component of milk of most mammals and are secreted as micelles that also carry high concentrations of calcium. They are phosphoproteins that represent the products of four genes, equivalent to those that encode the bovine alpha s1, alpha s2, beta, and kappa-caseins. There is considerable variation in the relative proportions of the particular caseins across species. The primary sequences of the alpha s1, alpha s2, and beta-caseins also show considerable species variation consistent with rapidly evolving genes that are proposed to have a common precursor. In contrast, the kappa-caseins exhibit features that demonstrate a separate origin and function where they are proposed to stabilise the micelle structure. This review focuses on comparative aspects of the caseins across a number of species for which information is now available.

Amino Acid Sequence↗
PubMedDate not supplied

Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin.

BACKGROUND: Urokinase (uPA) and the urokinase receptor (uPAR) form a multifunctional system capable of concurrently regulating pericellular proteolysis, cell-surface adhesion, and mitogenesis. The role of uPA and uPAR in directed proteolysis is well established and its function in cellular adhesiveness has recently been clarified by numerous studies. The molecular mechanisms underlying the mitogenic effects of uPA and uPAR are still unclear, however. RESULTS: We identified mechanisms that might participate in uPA-related mitogenesis in human vascular smooth muscle cells and demonstrated that uPA induces activation of a unique signaling complex. This complex contains uPAR and two additional proteins, nucleolin and casein kinase 2, which are implicated in cell proliferation. Both proteins were isolated by affinity chromatography on uPA-conjugated cyanogen-bromide-activated Sepharose 4B and were identified using nano-electrospray mass spectrometry and immunoblotting. We used laser scanning and immunoelectron microscopy studies to further demonstrate that nucleolin and casein kinase 2 are located on the cell surface where they colocalize with the uPAR. Moreover, the proteins were co-internalized into the cell as an entire complex. Immunoprecipitation experiments in combination with an in vitro kinase assay demonstrated a specific association of uPAR with nucleolin and casein kinase 2 and revealed a uPA-induced activation of casein kinase 2, which presumably led to phosphorylation of nucleolin. Blockade of nucleolin and casein kinase 2 with specific modulators led to the inhibition of uPA-induced cell proliferation. CONCLUSIONS: We conclude that in human vascular smooth muscle cells, uPA induces the formation and activation of a newly identified signaling complex comprising uPAR, nucleolin, and casein kinase 2, that is responsible for the uPA-related mitogenic response. The complex is not a unique feature of vascular smooth muscle cells, as it was also found in other uPAR-expressing cell types.

Amino Acid Sequence↗
PubMed2001

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes.

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.

Animals↗
PubMed1979

The formation of gamma-caseins during cooling of raw milk.

It has been shown that there is a time-dependent transfer of beta-casein and the milk serine proteinase system from micelles to milk serum with change of temperature from 38 to 4 degrees C. It has been established that the gamma-caseins can be formed by proteolytic degradation of beta-casein. By a simple extraction technique, the very hydrophobic gamma-casein fraction was separated from stored milks (26 and 4 degrees C) and estimated quantitatively. The results showed that the proteolytic degradation of beta-casein is faster at 4 degrees C than at room temperature and this can be explained by the immobilization of enzyme and substrate at the micelle surface at higher temperatures (26 degrees C). The results indicate that irreversible changes during cooling for short periods do not cause problems in milk processing, but the formation of gamma-caseins and phosphopeptides may influence the technological properties of raw milk stored for more than 48 h.

Animals↗
PubMed1982

Hormonal control of casein synthesis in organ culture of the bovine lactating mammary gland.

Explants from lactating bovine mammary gland were cultured in vitro in serum-free medium though 1-9 d. casein synthesis was determined by [32P] incorporation into newly synthesized Ca rennin precipitable fraction. High correlation (r = 0.98) was found between incorporation of [32P] and [3H]amino acids in explants cultured under different hormonal regimes, thus indicating that post-translational phosphorylation is not a rate-limiting step in casein synthesis. Hormonal effects on casein synthesis were studied by supplementing the incubating medium with insulin (I), prolactin (PRL), cortisol (F), thyroxine (T4) and triiodothyronine (T3). It was found that both PRL and I were required absolutely for maximal synthesis and almost maximal effect was achieved with 50 ng/ml. The effect of F was less clear, but some increase was achieved at the 200-1000 ng/ml range. T4 and T3 did not affect casein synthesis at a range of 10(-11)-10(-7) M while a significant inhibition was observed at 2 X 10(-5) M. A time-course study of casein synthesis further substantiated the dominant role of PRL in maintenance or even elevation of the initial rate of casein synthesis in the explants, through the first 4 d of incubation.

Animals↗
PubMed1986

Fractionation of bovine caseins by reverse phase high performance liquid chromatography: identification of a genetic variant.

Samples of reduced whole casein from genetically typed individual cows were quantitatively separated into their main components, alpha s1-, alpha s2-, beta- and kappa-caseins by reverse phase high performance liquid chromatography (RP-HPLC) using a mobile phase of phosphate-buffered aqueous propan-2-ol containing sodium dodecyl sulphate and an octadecylsilyl stationary phase. One casein sample was found to give two peaks of beta-casein of approximately equal areas. RP-HPLC of tryptic digests of the two separated peak fractions gave identical patterns with the exception of one peptide peak with different retention times. Amino acid analyses performed on both fractions showed that they corresponded to peptide 114-169 of beta-casein A1. The atypical beta-casein differed from the typical one by a Pro----Leu substitution in region 114-169.

Amino Acids↗
PubMed1993

Comparative study of the separation of casein from bovine, ovine and caprine milks using HPLC.

The separation of cows', sheep's and goats' milk casein components by HPLC on a strong anion-exchange (P.L-SAX 8 mu 1000A) column is described. During HLPC, whole caseins of the three kinds of milk behaved differently from conventional separations. The casein components of the three kinds of milk were well resolved under the chromatographic conditions used. HPLC resolved the kappa-caseins better than did ion-exchange chromatography on DEAE cellulose, and was particularly efficient in the case of goats' milk. Goats' and sheep's milks had almost similar chromatographic profiles but these differed considerably from that of cows' milk. Caseins from the sheep and the goat were also similar in that a shallower NaCl gradient was required for the separation of casein components than for cows' milk.

Animals↗
PubMed1989

Opioid antagonist peptides derived from kappa-casein.

Opioid antagonists were sought in the fragments of kappa-casein which were obtained by chemical synthesis and enzymic digestion. A synthetic bovine kappa-casein peptide (35-41), Tyr-Pro-Ser-Tyr-Gly-Leu-Asn (casoxin A) showed opioid antagonist activity at 200 microM in the guinea pig ileum assay. A synthetic peptide Tyr-Pro-Tyr-Tyr (casoxin B) which is found in bovine and human kappa-casein, also showed opioid antagonist activity at 100 microM. Another opioid antagonist peptide (casoxin C) was isolated from tryptic digests of bovine kappa-casein by reverse-phase HPLC. The structure of the peptide was Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg, which corresponded to kappa-casein (25-34). Casoxin C was active at 5 microM in the guinea pig ileum assay. Thus, bovine kappa-casein contains three potential opioid antagonist sequences.

Amino Acid Sequence↗
PubMed1992

Determination of ovine casein heterogeneity using gel electrophoresis and immunochemical techniques.

Discontinuous PAGE at alkaline and acid pH, polyacrylamide gel isoelectric focusing, two dimensional electrophoresis and immunoblotting have been used to study the heterogeneity of sheep caseins. Three main phenotypes were selected either because of mobility at alkaline and acid pH of the individual alpha s components or because of their relative intensity. On electrophoresis at alkaline pH, one phenotype showed two distinct bands of lower electrophoretic mobility than beta 1- and beta 2-caseins. A detailed study of these components using immunospecific detection with beta-casein antiserum showed that these minor components of ovine casein may have a polypeptide chain similar to that of beta 1- and beta 2-caseins. Complete electrophoretic patterns of the casein components in some individual milks are also presented.

Animals↗
PubMed1983

Proteinases in normal bovine milk and their action on caseins.

Native proteolytic enzymes in good quality normal bovine milk readily hydrolysed the caseins during incubation or storage, producing the gamma-caseins, proteose-peptone components 5 (PP5) and 8-fast (PP8F) and a considerable number of other unidentified fragments, many of which were also subsequently found in the proteose-peptone fraction. The rate of casein hydrolysis was greater in pasteurized than in raw milk, with beta-casein being slightly more susceptible to attack than alpha S1-casein. Measurements of gamma-casein and proteose-peptone formation have been made and it was found that PP5 was an intermediate product that was subject to further proteolysis while PP8F was a stable end-product. With the exception of component 3 (PP3), virtually all constituents of the proteose-peptone fraction increased during storage and appeared to be products of the action of proteolytic enzymes. Further evidence was obtained from the effects of various inhibitors that the principal proteinase of normal milk is plasmin, but slight differences were apparent between the protein breakdown patterns induced by storage and by added plasmin, which was consistent with the presence of more than one proteinase. Incubations in the presence of soya bean trypsin inhibitor to prevent plasmin action clearly revealed that another enzyme(s) was also involved.

Animals↗
PubMed2002

Determination of the casein content in bovine milk by 31P-NMR.

The relative proportion of caseins to total protein is a parameter that can be used to control the protein quality in standardised milk, an increasing tendency in dairy industries. 31P-NMR was used to analyse the casein content of milk, by the quantitation of the area under the resonances belonging to SerP, and using methylenediphosphonic acid as internal standard. This procedure yielded good results, as similar values of caseins were obtained from N Kjeldahl and NMR analysis for slightly heated milk samples. Heating at 95 degrees C for 15 min did not alter the casein content results. Casein content of raw, pasteurised and UHT milks (25.6 +/- 1.4, 26.4 +/- 1.8, 25.5 +/- 1.6 g casein/l milk, respectively), obtained by NMR, were not significantly different, giving an average of 25.8 +/- 1.6 g casein/l for bulk liquid milk. This work concluded that 31P-NMR could be used as an alternative method to determine casein in raw, pasteurised, dry and UHT milks.

Animals↗
PubMed2005

Growth hormone acts on the synthesis and secretion of alpha-casein in bovine mammary epithelial cells.

To study the effect of growth hormone (GH) on the functions of mammary epithelia, we examined the effect of GH on the synthesis and secretion of alpha-casein in a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-d-pregnant Holstein heifer. GH receptors (GHR) were observed in the BMEC on the membrane as well as in the cytoplasm. After BMEC were plated onto cell culture inserts, GH stimulated alpha-casein release in both the presence and absence of the lactogenic hormone complex, which included dexamethasone, insulin and prolactin (DIP). DIP enhanced the effect of GH on alpha-casein release. Although alpha(s1-) casein mRNA expression was not detected in untreated control cells, its expression was observed in BMEC in response to the GH, DIP and GH + DIP treatments. Expression was greater for GH and GH + DIP than for just DIP. Expression of GHR mRNA was increased by DIP treatment, while the mRNA expression was little changed by GH treatment. We conclude that GH acts on BMEC and induces the expression of both the alpha-casein gene and protein. GHR gene expression was shown to be regulated by DIP and GHR. GHR may be involved in a synergic effect between GH and DIP on casein secretion. These results suggest that GH, in addition to its widely accepted homeorhetic role in vivo, also can act on the mammary parenchyma, and that the effects of GH on mammary epithelial cells could partly account for the clear galactopoietic effect of recombinant bovine GH seen in lactating dairy cows.

Animals↗
PubMed1998

Factors contributing to variation in the proportion of casein in cows' milk true protein: a review of recent INRA experiments.

The aim of this study was to identify and rank the various factors, in particular those involving feeding, that affect the proportion of caseins in milk true protein. Twenty-nine feeding trials involving 821 lactations were assessed, and lactoprotein genetic variants were known for 551 of these. The main factor affecting the casein: protein ratio was the genetic variant of beta-lactoglobulin: once corrected for other factors, the milk of BB type animals had a ratio nearly 30 g/kg total protein higher than AA animals. kappa-Casein variant B also had a positive effect (+12 g/kg in favour of BB relative to AA animals). Except in the last weeks of pregnancy and the first weeks of lactation, the casein: protein ratio varied little during lactation. It was significantly reduced when milk cell count exceeded 200,000 cells/ml, even in the absence of clinical mastitis. It also decreased slightly with parity. Among the various dietary factors studied (level and type of nitrogen and energy supplies, forage type and preservation method), none had any significant effect on the milk casein: protein ratio, except in drastic dietary situations. That ratio increased very slightly in parallel with the animals' milk yield and milk protein content. In practice, measuring the milk protein content in animals free from clinical mastitis remains a very precise predictor of casein content, accounting for 93% of its variation.

Animal Nutritional Physiological Phenomena↗
PubMed1999

Effects of sheep alpha s1-casein CC, CD and DD genotypes on milk composition and cheesemaking properties.

The effects of sheep alpha s1-casein CC, CD and DD genotypes on milk composition and cheese yield were studied. Processed bulk milk was collected from three groups of 15 ewes, carrying alpha s1-casein CC, CD and DD genotypes. CC milk was higher in casein content than CD or DD milk (+3.5 and +8.6% respectively), and had a higher protein: fat ratio and a smaller casein micelle diameter. In addition, DD milk had a significantly lower alpha s1-casein content. The main differences were in curd formation: CC milk had better renneting properties. Cheesemaking trials, carried out in a pilot plant, showed that CC milk had better cheesemaking characteristics than DD milk, while CD milk was intermediate. Both 1 d old and fully ripened cheeses had different fat: dry matter ratios and alpha s1-I-casein electrophoretic mobilities: these were lower for DD cheese. As a consequence, these genotypes could be considered as markers of milk and/or cheese quality.

Animals↗