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PubMed2002

Separation and quantification of the major casein fractions by reverse-phase high-performance liquid chromatography and urea-polyacrylamide gel electrophoresis. Detection of milk adulterations.

The separation and quantification of bovine kappa-, alpha- and beta-caseins by HPLC-UV using an RP column which contained polystyrene-divinilbenzene copolymer based packing was optimized and validated. Gradient elution was carried out at a flow-rate of 1 ml/min and a temperature of 46 degrees C, using a mixture of two solvents. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was acetonitrile-water-trifluoroacetic acid (95:5:0.1). The effluent was monitored by a UV detector at 280 nm. The determinations were performed in the linear range of 0.038-0.377 mg/ml for kappa-casein, 0.188-1.883 mg/ml for alpha-casein and 0.151-1.506 mg/ml for beta-casein. The detection limits were 0.006, 0.019 and 0.015 mg/ml for kappa-casein, alpha-casein and beta-casein, respectively. The validity of the method was verified. The recoveries ranged from 91 to 100% for bovine milk. The precision of the method was also evaluated, the RSD being less than 3.67%. The same HPLC procedure was used for the separation of caprine and ovine caseins. Different chromatographic profiles were obtained for bovine, ovine and caprine milks, although it was only possible to detect and quantify additions of 5% or more of bovine milk to caprine milk. With respect to detection of milk adulterations, electrophoresis using urea-polyacrylamide gel electrophoresis (PAGE) analysis was more sensitive. The evolution of casein proteolysis in cheeses made from bovine milk and cheeses made from ovine milk, during 30 days of ripening was followed by HPLC-UV and urea-PAGE methodologies. The results obtained by these techniques were similar.

Animals↗
PubMed1999

Validation of capillary electrophoresis in the analysis of ewe's milk casein.

Recent investigations have shown that capillary electrophoresis (CE) can be an alternative to other techniques such as polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the qualitative analysis and separation of the different casein fractions in cow's and ewe's milk. However, past work has not yet clarified whether that method can achieve good quantifications. The present study has used a commercial whole ovine casein standard and a mixture of the standard and whole casein extracted from ewe's milk cheese to test the reliability of the technique. The results show that CE was able to quantify the ewe's milk caseins. The areas under four of the most representative peaks on the electrophoretogram for two alpha and two beta-caseins (designated alpha-casein1CE, alpha-casein2CE, beta-casein1CE, and beta-casein2CE in order of elution) were used to validate the method. In relation to linearity, coefficient of determination (r2) values greater than 99% were obtained for the regressions of each of the caseins. Moreover, each casein yielded response factors with a relative standard deviation (R.S.D.) of less than or equal to 5. The coefficients obtained in the day-to-day reproducibility analysis were higher than those for the same-day repeatability, but all the values were within acceptable limits. In the study of accuracy, the percentage recovery rates for the alpha-casein fractions were higher than those for the beta-casein fractions, hence quantification of the latter using this technique would appear to be more accurate under the conditions employed.

Animals↗
PubMed1997

Milk-responsive atopic dermatitis is associated with a casein-specific lymphocyte response in adolescent and adult patients.

BACKGROUND: Specific T-cell responses to food antigens have been described in children with atopic dermatitis (AD). However, a subgroup of adolescent and adult patients still experiences food-responsive AD. OBJECTIVE: This study was designed to evaluate lymphocyte responses to bovine casein in adult patients with AD. METHODS: The stimulatory capacity of lipopolysaccharide-depleted bovine casein was tested in proliferation assays and in limiting dilution assays. Casein-specific T-cell clones (TCCs) were generated and characterized. RESULTS: Higher proliferative responses to casein and modulation of cytokine receptors were observed in patients with milk-responsive AD compared with individuals without clinical reactions on milk ingestion. The results did not correlate with the amount of casein-specific IgE in the serum. The frequencies of T cells that grew in the presence of casein or house dust mite antigens were similar. Only 27% of CD4+ casein-specific TCCs from these patients, but the majority of house dust mite-specific TCCs, produced IL-4 on mitogen stimulation. Interferon-gamma, on the other hand, was produced by the majority of TCCs with both specificities. CONCLUSION: A specific T-cell-mediated immune response to casein can be found in the blood of adolescent and adult patients with milk-related exacerbation of AD. In contrast to house dust mite-specific T cells, casein-specific T cells of adult patients who respond to cow's milk with worsening of AD produce little or no IL-4.

Adolescent↗
PubMed1999

Cow's milk casein, a hidden allergen in natural rubber latex gloves.

BACKGROUND: We have previously noted that one natural rubber latex (NRL) glove brand used for skin prick testing in the diagnosis of NRL allergy contained cow's milk casein. OBJECTIVE: We sought to examine whether other commonly marketed NRL glove brands contain cow's milk casein. METHODS: The casein content of 30 NRL glove extracts (1:5 wt/vol) was measured by RAST inhibition and by rocket immunoelectrophoresis and rocket radioimmunoelectrophoresis by using casein-specific rabbit antiserum, a serum pool from patients with cow's milk allergy, and radiolabeled anti-human IgE. The NRL allergen content was measured by ELISA inhibition. RESULTS: The casein content of the glove used in NRL allergy screening (Triflex, Baxter) was rather high (ie, about 400 microgram/g of glove). Its total protein content was 1000 microgram/g of glove. Rocket radioimmunoelectrophoresis detected distinct amounts of casein in 8, minimal amounts in 7, and no casein in the remaining 15 NRL glove brands. CONCLUSIONS: Several brands of NRL gloves were found to contain casein, implying that extracts prepared from such gloves can cause false-positive skin prick test reactions when diagnosing NRL allergy. The use of casein as a stabilizer in glove manufacture without appropriate labeling should be stopped because it can also cause contact urticaria syndrome in individuals with cow's milk allergy.

Allergens↗
PubMed1979

The role of calcium, phosphate and citrate ions in the stabilization of casein micelles.

To obtain greater insight into the interaction of Ca, citrate and phosphate ions with casein, 31PNMR measurements were performed on combinations of these ions with alphas- and kappa-caseins. It was found that addition of alphas-casein to a Ca phosphate solution in D2O at 27 degrees C and pD 6.4 resulted in a downfield shift of the 31P singlet. An almost identical shift was observed with kappa-casein, but no shift was found when only phosphate ions were present or when Ca2+ were added to phosphate ions in the absence of casein. Separate experiments with poly-L-lysine, mol. wt approx. 35,000, resulted in similar downfield 31P chemical shifts of Ca phosphate as with both caseins, whereas no shift was observed when poly-glycine was added. From these results it can be concluded that Ca and phosphate ions associate with casein in a co-operative manner, probably in the way described by ter Horst (1963) as a complex with the NH3+-groups of lysine or arginine in a structure such as: (casein--NH3+)--PO43---Ca2+. The formation of this complex may be enhanced by citrate ions, since preliminary results have shown that addition of Na citrate to a solution of alphas-casein with added Ca phosphate produces a broadening of the 31P signal as well as a chemical shift.

Calcium↗
PubMed1992

New alpha s2-casein variant from caprine milk.

A new alpha s2-casein variant was isolated from goat milk by means of covalent chromatography. With gel electrophoresis at alkaline pH, the alpha s2-casein variant showed a double band with the highest anodic mobility amongst the casein components. The mobility of the isolated alpha s2-casein variant was similar to that occurring in three individual samples of whole casein. Two dimensional electrophoresis revealed a more pronounced heterogeneity of this protein. Developing the two dimensional plate with polyclonal antibodies obtained against the four main bovine casein fractions, we demonstrated that the variant in question belongs to the alpha s2-casein family. Since the alpha s2-casein fractions, immunospecifically developed, spread along two different zones on the gel, it was concluded that a heterozygous alpha s2-casein form was present in the sample.

Animals↗
PubMed1995

Gel electrophoresis and immunoblotting for the detection of casein proteolysis in cheese.

The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immunoblotting with polyclonal antibodies against beta- and alpha s1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4.6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as gamma 3-casein (beta-casein(f108-209)) and alpha s1-PL1 (alpha s1-casein(f80-199). In both cases, a Lys-X bond was hydrolysed, indicating the action of a trypsin-like enzyme in beta- and alpha s1-casein hydrolysis during the ripening of this variety of hard pressed cheese.

Amino Acid Sequence↗
PubMed2003

Accuracy of Fourier transform infrared spectrometry in determination of casein in dairy cows' milk.

For practical purposes the casein content of milk is often estimated from the crude protein content using a general conversion factor. This is done even though it is known that several factors can influence the percentage of casein nitrogen in the total nitrogen (casein number). The uncontrolled variation in casein number affects optimization of cheese production because casein is a limiting factor for cheese yield. Fourier transform infrared spectrometry has recently been introduced for casein determination. Using this technique we obtained a standard error of prediction (SEP) of 0.033% for casein concentrations in the range 2.1-4.0%. The true prediction error was estimated as 0.025%. The correlation between casein numbers obtained by reference analysis and infrared spectrometry could be expressed by an R2 of 0.73 and an SEP of 0.89 for casein numbers in the range 70.7-81.0.

Animals↗
PubMed1999

Effects of dietary protein supply on caseins, whey proteins, proteolysis and renneting properties in milk from cows grazing clover or N fertilized grass.

The objective of this work was to examine whether variation in the amino acid supply to cows could be a reason for the reduced casein content and poorer renneting properties of milk that often occur in late summer, or whether these effects are related to proteolysis in the raw milk. In a 2 x 2 x 2 factorial design, we investigated the effects of sward (clover v. rye-grass) and supplementary feed with a high or low level of rumen-soluble N or of rumen undegradable protein on milk protein composition during the grazing season. A total of 32 Danish Holstein cows were included in the experiment. Milk protein and casein contents and the ratios casein N:total N and casein:true protein were at a minimum in late summer, whereas the contents of urea, non-protein N and whey protein were higher during this period. These seasonal effects were unrelated to either the type of supplementary feed or the type of sward; neither were they clearly related to proteolysis, although casein: true protein was related to the proteose peptone content. The results indicated that whey proteins other than alpha-lactalbumin or beta-lactoglobulin accounted for the higher proportion or concentration of whey protein in late summer. Based on a principal component analysis including variables such as citric acid, lactose and non-protein N, we suggest that the cows' energy supply during this period may be a critical factor in determining the milk protein composition, although our results were not conclusive. There was an interaction between the supplement of rumen undegradable protein and type of sward. When clover was grazed, a high supplement increased the concentrations of protein and casein in milk and the kappa-casein: total casein ratio. When rye-grass was grazed, the opposite response was found, and overall milk protein yield was not affected. The very low N content of clover in early summer reduced milk protein and casein protein during this period.

Animal Feed↗
PubMed2001

Expression and purification of glycosylated bovine beta-casein (L70S/P71S) in Pichia pastoris.

Post-translational glycosylation of bovine beta-casein (L70S/P71S) that results in Asn(68)-linked glycan on the protein was obtained in up to 30% of total beta-casein expressed in the methylotrophic yeast Pichia pastoris. Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine beta-casein, indicating pH-dependent glycosylation. Glycosylated bovine beta-casein (L70S/P71S) expressed in P. pastoris was purified to homogeneity by the combination of ammonium sulfate fractionation, Concanavalin A--Sepharose affinity column, and Mono Q anion-exchange FPLC. The purified glycosylated bovine beta-casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated beta-casein was of high-mannose type. Unlike the hyperglycosylation that occurred in yeast, the majority of bovine beta-casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine beta-casein was normally phosphorylated to the same degree as native bovine beta-casein.

Animals↗
PubMed2004

Ability of alphas-Casein to suppress the heat aggregation of ovotransferrin.

The effects of alphas-casein on heat aggregation of ovotransferrin (OT) were studied by heating at 80 degrees C for 20 min in 10 mM phosphate buffer, pH 7.0. The heat interactions between alphas-casein and OT were followed by turbidity development and polyacrylamide gel electrophoresis. We found that alphas-casein can effectively suppress the heat-induced aggregation of heat-labile OT. The suppressive ability of alphas-casein was reduced by the presence of NaCl on heating. Dephosphorylated alphas-casein had less ability to suppress the aggregation of OT than native alphas-casein. Our results indicate that alphas-casein interacts with the heat-denatured OT through its exposed hydrophobic surface and phosphoserine residue. Such interactions seem to be important in helping to suppress the aggregation of heated OT. The suppressive effects of alphas-casein on heat aggregation of OT would be partially ascribed to the formation of transparent gel from egg white by the addition of alphas-casein.

Caseins↗
PubMed2000

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.

Animals↗
PubMed1978

Separation and partial characterization of guinea-pig caseins.

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

Amino Acids↗
PubMed1994

Specific activation of beta-casein kinase by the inflammatory cytokines interleukin 1 and tumour necrosis factor.

Increases (5-fold) in the rate of phosphorylation of beta-casein were observed in extracts of human gingival fibroblasts that had been stimulated by interleukin 1 (IL-1) or tumour necrosis factor (TNF). The induced kinase was cytosolic and had little activity on alpha-casein. Its chromatographic behaviour on anion-exchange and gel-filtration columns was similar to that of beta-casein kinase, an enzyme detected originally in MRC-5 cells stimulated by IL-1 and TNF. Phosphopeptide maps of beta-casein confirmed that the kinase activated in gingival fibroblasts had the same substrate specificity as beta-casein kinase. In gingival fibroblasts, beta-casein kinase activity was maximum after 15 min of stimulation by IL-1 or TNF, and remained activated for several hours. Activations of small heat-shock protein (hsp27) kinase and mitogen-activated protein (MAP) kinase were also maximum 15 min after stimulation, but decreased to background levels within the next 30 min. Study of the effects of 21 agents other than IL-1 or TNF showed that none activated beta-casein kinase, whereas several activated MAP kinase or hsp27 kinase. beta-Casein kinase was also detected in extracts of bovine articular chondrocytes and human endothelial cells stimulated by IL-1 or TNF. Semi-purified preparations of fibroblast beta-casein kinase were not inactivated by phosphatases in vitro. Our results suggest that it may be involved in responses specific to IL-1 and TNF in a wide range of cell types and that its activation probably involves mechanisms other than its phosphorylation.

Animals↗
PubMed1987

Characterization and hormonal regulation of casein kinase II activity in heterotransplanted human breast tumors in nude mice.

Cytosolic casein kinase type II activity has been identified in MCF-7 and MDA-MB-231 human breast cancer cells heterotransplanted into athymic nude mice. Sephacryl S-300 chromatography of MCF-7 and MDA-MB-231 tumor cytosols revealed a major peak of casein kinase activity with an estimated molecular weight of 150,000. This peak was further characterized and optimal conditions for breast tumor casein kinase activity were established. Polylysine (10 micrograms) acted as a potent stimulator with casein as the phosphate acceptor protein. This enzyme used both ATP and GTP as phosphate donors and the Km for GTP was 10 microM. The rate of phosphorylation with increasing concentrations of [gamma-32p]GTP revealed typical Michaelis-Menten kinetics and Vmax was approached at a concentration of 30 microM GTP. MgCl2 stimulated enzyme activity at concentrations between 10-20 mM. Quercetin, a bioflavonoid, inhibited casein kinase type II activity in a dose dependent manner. MCF-7 (hormone-dependent) human breast cancer cells (2-3 X 10(6)) were inoculated into the mammary fat pads of nude mice, supplemented with a 0.5 mg estradiol pellet. To determine the influence of various regulatory agents on casein kinase activity in vivo, tumor-bearing mice were treated for five days with estradiol, progesterone, dexamethasone or tamoxifen. Casein kinase type II was partially purified by gel filtration on a Sephacryl S-300 column and assayed in the presence of polylysine and casein. Dexamethasone treatment significantly decreased casein kinase II activity in MCF-7 tumors, which are receptor-positive for estrogen, androgen and glucocorticoid receptors.

Animals↗
PubMed1981

Prolactin induces release of a factor from membranes capable of stimulating beta-casein gene transcription in isolated mammary cell nuclei.

Crude microsomes from lactating rabbit mammary gland were incubated with prolactin. The incubation mixture was centrifuged and the supernatant was incubated with isolated mammary cell nuclei from lactating rabbits treated for 4 days by bromocryptin to antagonize prolactin and to deinduce casein gene transcription. Nuclei were incubated with HgCTP, and the newly synthesized mercurated RNA was isolated on SH-Sepharose columns. The content of beta-casein mRNA sequences in the fraction eluted with 2-mercaptoethanol was estimated with a [(3)H]cDNA probe obtained from partially purified beta-casein mRNA. The supernatant markedly stimulated beta-casein gene transcription but not 28S rRNA transcription. The same effect was obtained with other lactogenic hormones such as human growth hormone and ovine placental lactogen but was not observed with bovine growth hormone, insulin, parathyroid hormone, luteotropic hormone, or epidermal growth factor. Prolactin and human growth hormone were totally inactive when added directly to nuclei. The factor stimulating beta-casein gene transcription was also generated by membranes containing prolactin receptors such as those from liver, ovary, adrenals, and brain but not by membranes from heart, lung, and muscle, which do not bind prolactin. The factor stimulated beta-casein transcription when added to mammary nuclei from pseudopregnant or bromocryptin-treated lactating rabbits, in which the transcription rate is submaximal, but was ineffective on mammary nuclei prepared from untreated fully lactating rabbits. The factor was unable to induce beta-casein gene transcription in nuclei isolated from rabbit liver and reticulocytes. The factor did not stimulate the transcription of globin genes in nuclei isolated from reticulocytes or the transcription of mammary "housekeeping" genes evaluated by a cDNA probe prepared from total mRNA isolated from an unstimulated mammary gland. The transcription of beta-casein genes was abolished by adding alpha-amanitin to the medium in the presence or in the absence of the factor, indicating that the generation of mercurated beta-casein mRNA sequences depended upon the transcriptional activity of RNA polymerase II. The addition of the factor to the incubation mixture did not enhance total and alpha-amanitin-sensitive RNA synthesis. These data suggest that the binding of prolactin to its receptor in vitro induces the formation of a second messager, which specifically stimulates the transcription of prolactin-sensitive genes.

Animals↗
PubMed2006

Casein kinase 2 Is activated and essential for Wnt/beta-catenin signaling.

Wnt/beta-catenin signaling is essential to early development. Activation of Frizzled-1 by Wnts induces nuclear accumulation of beta-catenin and activation of Lef/Tcf-dependent gene expression. Casein kinase 2 has been shown to affect Wnt/beta-catenin signaling. How casein kinase 2 exerts an influence in Wnt signaling is not clear; casein kinase 2 has been reported to be constitutively active (i.e. not regulated). Herein we show to the contrary that casein kinase 2 activity is rapidly and transiently increased in response to Wnt3a stimulation and is essential for Wnt/beta-catenin signaling. Chemical inhibition of casein kinase 2 or suppression of its expression blocks Frizzled-1 activation of Lef/Tcf-sensitive gene expression. Treatment with pertussis toxin or knock down of Galpha(q) or Galpha(o) blocks Wnt stimulation of casein kinase 2 activation, as does suppression of the phosphoprotein Dishevelled, demonstrating that casein kinase 2 is downstream of heterotrimeric G proteins and Dishevelled. Expression of a constitutively active mutant of either Galpha(q) or Galpha(o) stimulates casein kinase 2 activation and Lef/Tcf-sensitive gene expression. Thus, casein kinase 2 is shown to be regulated by Wnt3a and essential to stimulation of the Frizzled-1/beta-catenin/Lef-Tcf pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Effect of dietary supplementation with beta-casein A1 or A2 on markers of disease development in individuals at high risk of cardiovascular disease.

The present study is the first to examine the hypothesis that dietary supplementation with beta-casein A1 promotes an increased risk relative to supplementation with beta-casein A2 in patients traditionally at high risk of developing CVD. The study was conducted in fifteen asymptomatic participants (six male; nine female) at high risk of developing CVD. A double-blind cross-over study design was used with a total duration of 24 weeks. Dietary intervention was a daily supplementation (25 g) of either casein A1 or A2 (for 12 weeks each). Surrogate measures of cardioprotection studied included the examination of vascular (endothelium and arterial) function, resting blood pressure, plasma lipids and biochemical markers of inflammation. Total plasma cholesterol levels were significantly lower following 12 weeks of both casein A1 and A2 interventions but the decrease was not different between intervention. Plasma insulin, homocysteine, C-reactive protein, fibrinogen, protein C and S and von Willebrand factor levels were not different between the two casein supplements. Endothelium function, measured as a vascular response using venous occlusion plethysmography to intra-arterial infusions of the endothelium-dependent agonist acetylcholine, were not different between the two casein interventions. Similarly, neither blood pressure nor measures of large artery stiffness were affected by differing the casein variant. We therefore conclude that there is no evidence from the present study that supplementation with casein A1 has any cardiovascular health disadvantage over consumption of casein A2.

Adult↗