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PubMed1993

Degradation and debittering of a tryptic digest from beta-casein by aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2.

The mode of action of purified aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 on a complex peptide mixture of a tryptic digest from bovine beta-casein was analyzed. The oligopeptides produced in the tryptic digest before and after aminopeptidase N treatment were identified by analysis of the N- and C-terminal amino acid sequences and amino acid compositions of the isolated peptides and by on-line liquid chromatography-mass spectrometry. Incubation of purified peptides with aminopeptidase N resulted in complete hydrolysis of many peptides, while others were only partially hydrolyzed or not hydrolyzed. The tryptic digest of beta-casein exhibits a strong bitter taste, which corresponds to the strong hydrophobicity of several peptides in the tryptic digest of beta-casein. The degradation of the "bitter" tryptic digest by aminopeptidase N resulted in a decrease of hydrophobic peptides and a drastic decrease of bitterness of the reaction mixture.

Amino Acid Sequence↗
PubMed1999

Cell-wall-bound proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: characterization and specificity for beta-casein.

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40 degrees C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes beta-casein mainly and alpha- and kappa-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from beta-casein, which have been identified.

Amino Acid Sequence↗
PubMed2000

The autoproteolysis of Lactococcus lactis lactocepin III affects its specificity towards beta-casein.

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards beta-casein was investigated. beta-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from beta-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.

Amino Acid Sequence↗
PubMed1968

Relief of casein inhibition of Bacillus stearothermophilus by iron, calcium, and magnesium.

Growth of Bacillus stearothermophilus strain NCA 1518 Smooth in Dextrose Tryptone Agar (DTA) was inhibited by sodium caseinate. Binding studies indicated that sodium caseinate, when present in DTA, had the capacity to effect an iron deficiency which could cause inhibition of growth. Additions of essential cations, iron (1 mM), calcium (5 mM), magnesium (10 mM), or hydrogen ion (pH 5.7), relieved inhibition. Responses to and interactions among these relief factors were analyzed statistically. Equations were fitted to the data and were used to estimate responses to all treatment combinations within the ranges tested. Results from these studies indicated that calcium, magnesium, and hydrogen ion acted by decreasing the binding capacity of the protein for iron, rendering this metal available for metabolic needs. Evidence was obtained that ferrous rather than ferric iron was the limiting factor in DTA containing sodium caseinate.

Analysis of Variance↗
PubMed1975

Plaque assay of avian sarcoma viruses using casein.

The caseinolytic activity of several strains of Rous sarcoma virus (RSV), conditional and nonconditional mutants of RSV, and nontransforming avian leukosis viruses was investigated. Only those viruses capable of transforming chick fibroblasts in vitro induced lysis of casein incorporated into an agar overlay. Lysis produced distinct clear areas in the turbid casein-agar gel which allowed a quantitative "plaque" assay of cell transformation. Casein plaque formation could not be separated from morphological conversion in cultures infected by wild-type RSV strains. In plates infected by mutants temperature sensitive for transformation, the caseinolytic activity appeared to be affected by temperature to a lesser extent than morphological conversion.

Avian Leukosis Virus↗
PubMed1990

Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.

The HC11 cell line was isolated from mammary gland cells of pregnant mice. The cells displayed a normal phenotype and retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells expressed the milk protein beta-casein. Various oncogenes were transfected and expressed in HC11 cells. The oncogenes were tested for their transformation ability and for their effects upon the differentiation of the HC11 cells. All of the oncogenes tested, including activated human Ha-ras, human transforming growth factor-alpha, activated rat neuT, and human c-erbB-2 activated by a point mutation in the transmembrane domain, caused transformation of the HC11 cells, as shown by tumor formation in nude mice. HC11 cells expressing the neuT and activated c-erbB-2 genes synthesized beta-casein in response to lactogenic hormones, whereas those expressing the Ha-ras or transforming growth factor-alpha oncogenes were no longer able to respond to the lactogenic hormones. This inhibition of beta-casein production occurs at the transcriptional level and in the transforming growth factor-alpha-transformed cells is due to an autocrine mechanism involving the activation of the epidermal growth factor receptor. This suggests that, although the c-erbB-2 and epidermal growth factor receptors are structurally quite similar, their activation has different effects upon mammary epithelial cell differentiation.

Animals↗
PubMed1994

YY1 represses beta-casein gene expression by preventing the formation of a lactation-associated complex.

Site-specific mutagenesis of the highly conserved milk box (-140 to -110) region suggested that beta-casein expression is regulated by a hormone-mediated relief of repression (M. Schmitt-Ney, W. Doppler, R. K. Ball, and B. Groner, Mol. Cell. Biol. 11:3745-3755, 1991). However, when this sequence was placed upstream of a heterologous thymidine kinase promoter, it activated reporter gene expression. This apparent paradox was resolved when the trans-acting factor YY1, capable of acting as both a positive and negative regulator, was shown to interact with the milk box region, using bacterially expressed YY1 and specific oligonucleotide and antibody competition experiments. Second, it was demonstrated that extracts prepared from several cell types contained a protein(s) interacting with the mammary gland-specific factor (MGF) binding site, previously shown to be required for beta-casein promoter activity (Schmitt-Ney et al., Mol. Cell. Biol. 11:3745-3755, 1991). Sequence analysis of this site revealed similarity to the gamma interferon-activated sequence, suggesting that MGF may be related to the stat91 signaling protein. Finally, using an oligonucleotide encompassing both the YY1 and MGF sites, we detected a slow-mobility complex only in extracts from mammary glands at late pregnancy and lactation (lactation-associated complex [LAC]). Site-specific mutation of the YY1 binding site led to an enhancement in LAC DNA binding activity, while mutation of the MGF site decreased detectable LAC. These results support a model in which lactogenic stimuli lead to a decrease in YY1 binding, and subsequent increased formation of LAC at a nearby binding site, to stimulate beta-casein transcription.

Animals↗
PubMed1989

Net appearance of amino acids in portal blood during the digestion of casein or rapeseed proteins in the pig.

The kinetics of appearance of amino acids (AA) in portal blood following the ingestion of casein or rapeseed protein were compared. Six pigs, fitted with permanent catheters in the portal vein and in the carotid artery, as well as with an electromagnetic flow probe around the portal vein, received three 800 g test meals, one containing 12% rapeseed proteins (RA12) and the others containing 12% and 24% casein (CA12 and CA24), at 1-week intervals and according to a double Latin square design. Portal and arterial blood samples were collected and portal blood flow rate was recorded for 8 h after the test meals. At the end of measurement, an average of 76.1 +/- 5.6% (mean +/- SEM) of total AA from the CA24 diet had appeared in portal blood, compared with 94.3 +/- 10.4% for the CA12 diet and 103.5 +/- 12.6% for the RA12 diet. Similar results were obtained for essential AA. Differences were found in the kinetics of appearance of individual AA. Eight hours after the meal, 79% of lysine, 84% of methionine, and 73% of valine from the CA24 diet had appeared in portal blood compared, respectively, with 100, 89, and 83% from the CA12 diet and 99, 86, and 106% from the RA12 diet. Arginine from rapeseed had a net appearance level lower (82%) than the overall mixture of essential AA. With casein diets, the net appearance of arginine reached 97% (CA12) and 82% (CA24). Following the ingestion of rapeseed proteins, there seemed to be a significant appearance of endogenous AA in portal blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed2000

Kinetics of L-[1-(13)C]leucine when ingested with free amino acids, unlabeled or intrinsically labeled casein.

In two groups of five adults, each adapted to two different dietary regimens for 6 days, the metabolic fate of dietary [1-(13)C]leucine was examined when ingested either together with a mixture of free amino acids simulating casein (extrinsically labeled; condition A), along with the intact casein (extrinsically labeled; condition B), or bound to casein (intrinsically labeled; condition C). Fed state leucine oxidation (Ox), nonoxidative leucine disposal (NOLD), protein breakdown, and splanchnic uptake have been compared using an 8-h oral [1-(13)C]leucine and intravenous [(2)H(3)]leucine tracer protocol while giving eight equal hourly mixed meals. Lower leucine Ox, increased NOLD, and net protein synthesis were found with condition C compared with condition A (19.3 vs. 24.9; 77 vs. 55.8; 18.9 vs. 12.3 micromol. kg(-1). 30 min(-1); P < 0.05). Ox and NOLD did not differ between conditions B and C. Splanchnic leucine uptake calculated from [1-(13)C]- and [(2)H(3)]leucine plasma enrichments was between 24 and 35%. These findings indicate that the form in which leucine is consumed affects its immediate metabolic fate and retention by the body; the implications of these findings for the tracer balance technique and estimation of amino acid requirements are discussed.

Adult↗
PubMed1998

Gut hypertrophy in response to the ratios of casein and whey protein in milk formulas in artificially reared rat pups.

To elucidate the mechanism of gut hypertrophy observed in rats artificially reared (AR) on milk formulas, the effects of four refined formulas with different ratios of casein (C) and whey protein (W), CW 2:8, CW 4:6, CW 6:4 and CW 8:2, on the gut growth of AR rats were examined. Four groups of pups were infused with each formula through an intragastric cannula from age 5 to 15 days. Each of the four milk formulas showed a different character in the stomach, such as no curd, very soft curd, soft curd and hard curd, in response to an increasing ratio of C:W. There were no significant differences in body weight gain among the AR groups and mother-reared (MR) controls. The stomach growth, in weight, of AR rats increased in response to the increasing ratios of C:W. In comparison with MR controls, hypertrophy of the stomach of AR rats appeared within the formulas with higher proportions of casein than whey protein (CW 6:4 and CW 8:2), but not those with lower proportions (CW 2:8 and CW 4:6). The growth of the small intestine was also related to the increasing ratio of C:W in the formulas. A similar pattern of hypertrophy in the hindgut was seen in AR rats. There was no association between hypertrophy of the gut in AR rats and plasma triiodothyronine. The present results clearly demonstrated that the gut growth of AR rat pups was directly influenced by the diet but not by AR per se, and that hard casein-curd in the stomach might be one cause of gut hypertrophy.

Aging↗
PubMed2000

Cloning, expression analysis and chromosome mapping of human casein kinase 1 gamma1 (CSNK1G1): identification of two types of cDNA encoding the kinase protein associated with heterologous carboxy-terminal sequences.

Casein kinase 1 gamma1(CK1 gamma1) is known to be involved in the growth and morphogenesis of eukaryotic cells. We have isolated two types of cDNA for human casein kinase 1 gamma1 (hCK1 gamma1). One of them (hCK1 gamma1S) was found to encode a polypeptide consisting of 393 amino acids, which is highly homologous with already reported rat CK1 gamma1 (rCK1 gamma1). The other type of cDNA (hCK1 gamma1L) encodes a polypeptide consisting of 422 amino acids, which is quite identical in the kinase domain, but different in the C-terminal sequence from hCK1 gamma1S. Namely, hCK1 gamma1L has a characteristic sequence of 50 amino acids at the C-terminal end and this motif was shown to be shared by the casein kinase gamma2 and gamma3 from rat and human, suggesting that it is a signature sequence of the gamma-isoforms. In this sense, newly isolated hCK1 gamma1L might be the original form of CK1 gamma1 subspecies rather than rCK1 gamma1 and hCK1 gamma1S. RT-PCR analysis revealed that hCK1 gamma1S mRNA is predominantly present in the testis, whereas the abundance of hCK1 gamma1L mRNA was nearly the same in the twelve tissues examined. These results suggest that novel hCK1 gamma1L may have a unique functional role different from that of hCK1 gamma1S and rCK1 gamma1. The human hCK1 gamma1 gene (CSNK1G1) was mapped to chromosome 15q22.1-->q22.31 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1993

Localization of the alpha-S2-casein gene (CASAS2) to the homoeologous cattle, sheep, and goat chromosomes 4 by in situ hybridization.

The alpha-S2-casein gene (CASAS2) has been mapped to the homoeologous cattle, sheep, and goat chromosomes 4 and to the long arm of a bovine chromosome translocation, t(4;8), using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The t(4;8) has been characterized by GTG-, GBG-, and RBG-banding and by silver staining of nucleolus organizer regions. These results confirm the previous (ISCNDA, 1989) localization of the casein gene group to chromosome 4 of cattle, sheep, and goats. We propose that the discrepancy between our results and earlier assignments of the casein gene group to chromosome 6 of cattle and sheep can be explained by the fact that the chromosome identified as No. 6 in the Reading Conference (1976) report was renamed chromosome 4 in the ISCNDA (1989) standardized karyotype of both species.

Animals↗
PubMed1975

Effect of chlorogenic acid in a casein diet for rats. Nutritional and pathological observations.

3-week-old male rats were fed on a casein diet containing 1% (w/w) of chlorogenic acid in order to find out if the ingestion of chlorogenic acid may be associated with a reduced utilization of dietary proteins or with causing other adverse effects. The protein efficiency ratio, biological value and digestibility of this diet did not differ from a corresponding casein diet free from chlorogenic acid. No change was observed in the nitrogen balance of the experimental rats. Neither did the rats show any change in hematological values or volumes and pH of the urine. Some deviation was noticed in the weights of kidneys and adrenals of the chlorogec acid-free casein diet. The findings that were made were normal in the microscopic examinations of various organs. Chlorogenic acid did not seem to influence the ultrastructure of the liver as revealed by electron microscopy.

Adrenal Glands↗
PubMed1979

Zinc status and serum levels of retinol-binding protein, tocopherol and lower density lipoproteins in male and female rats fed on semi-purified diets containing rapeseed protein or casein.

Experiments were carried out in order to evaluate the effect of various levels of dietary zinc supplementation on the zinc status, growth and contents of retinol-binding protein (RBP), tocopherol and lower density lipoproteins (VLDL + LDL) in serum of rats fed on diets containing rapeseed protein concentrate (RPC) or casein as the sole source of protein. In male rats fed on RPC diet at a 10% protein 5% fat level, a highly significant correlation was found between dietary zinc content and the total tibia zinc content (r = 0.946) in the dietary zinc range of 35-137 microgram/mg. In contrast, the casein rats attained almost maximal bone zinc contents even at the lower level of dietary zinc. No correlation was found between zinc status and the protein efficiency ratio obtained for the rapeseed protein. In female rats fed on a zinc-supplemented RPC diet at a 20% protein and 10% fat level, the serum levels of tocopherol and VLDL + LDL were reduced in comparison to the levels observed in female rats fed on a corresponding casein diet. No diet-related changes in serum levels of RBP were found. However, male rats showed significantly higher RBP values than the female rats.

Animals↗
PubMed1988

Influence of casein versus soy protein isolate on lipid metabolism of minipigs.

This report describes whether a complete exchange of soy protein isolate for casein affects serum lipid or lipoprotein levels. For this purpose, 10 adult minipigs underwent two 6-week crossover experiments, one with 11 and one with 22 weight % protein in a western-style diet. Cholesterol levels were 2.02 +/- 0.06 and 1.98 +/- 0.04 mmol/l with casein and soy, respectively (22 weight %) and 2.08 +/- 0.06 and 2.04 +/- 0.05 mmol/l, respectively (11 weight %). There was a significant rise of high-density lipoprotein cholesterol by the lower protein intake, both with casein and soy. In all experiments nitrogen balance was positive. Soy feeding was accompanied by a significant rise of fecal bacterial protein excretion, as calculated from diaminopimelic acid. In summary, no statistically significant difference of plasma or low-density lipoprotein cholesterol levels were observed, neither due to the kind nor to the amount of dietary protein.

Animals↗
PubMed1978

Casein and other tumor markers in relation to cancer of the breast.

Five tumor markers can be simultaneously determined in the serum by radioimmunoassay: carcinoembryonal antigen (CEA), alpha-fetoprotein (alpha-FP), human chorionic gonadotropin (HCG), beta-subunit of HCG (beta-HCG) and kappa-casein. In a series of 935 healthy subjects, these antigens remain detectable or are detected within very precise limits. At the start of the clinical evolution of breast cancer, the incidence of pathological concentrations is increased as compared with the highest level observed in normal subjects. This high incidence is mainly due to a concomitant determination of CEA, kappa-casein, HCG and beta-HCG. The alpha-FP test is never positive, while the kappa-casein concentration is particularly high in the first clinical stages of breast cancer and with metastases. The concomitant determination of these tumor markers may be a biological element contributing to the diagnosis of neoplasia, although it is neither an absolute nor a specific criterium. Indeed, a pathological concentration of at least one antigen was observed in 5.5% of the subjects presenting with benign mastopathy. When metastases occur (25 patients), the incidence of pathological concentrations of at least one antigen increases: 88%, the absolute values of these levels increasing simultaneously. The determination of the antigen concentration therefore allows an evaluation of the extension of the disease. Surgical removal reduces the incidence of positivity of these antigens to 34%. Persistence of pathological levels seems to be related to a possibility of relapse or metastatic spreading. Finally, chemotherapy and radiotherapy applied on a tumor which is not excised, does not decrease the incidence of positivity of the tumoral markers, although their levels seem to fluctuate with the clinical evolution.

Adult↗
PubMed1994

Cholesterol-fed and casein-fed rabbit models of atherosclerosis. Part 2: Differing morphological severity of atherogenesis despite matched plasma cholesterol levels.

One-month-old male New Zealand White rabbits were fed either a cholesterol-free casein diet (n = 10) or low-level cholesterol-supplemented chow (n = 10) for 24 weeks, during which total plasma cholesterol levels were matched. After perfusion fixation, aortic tissue samples were taken from six predetermined locations and embedded in epoxy resin for examination by light and electron microscopy. Frozen sections were also obtained for histochemical demonstration of collagen and elastin. Lesion morphology was classified in toluidine blue-stained, semithin epoxy sections as early fatty streaks (round foam cells with little intervening extracellular matrix); advanced fatty streaks (foam cells with extracellular lipid); fibrous plaques (spindle-shaped cells within extracellular matrix); or atheromatous lesions (presence of an atheromatous core). In representative specimens, electron microscopy showed that the ultrastructure of round foam cells was consistent with macrophage derivation, whereas most spindle-shaped cells were clearly smooth muscle cells. Fibrous plaques were more common in the distal than the proximal aorta. Lesions in the casein-fed animals were essentially equally distributed among the four morphological categories, whereas lesions in the cholesterol-fed rabbits were predominantly of the atheromatous type. Thus, cholesterol-fed rabbits had, in general, more advanced lesions than casein-fed rabbits with matched total plasma cholesterol levels. Moreover, the feeding of a low-level cholesterol diet (0.125% to 0.5% by weight) to rabbits for a relatively short time (6 months) led to the development of advanced lesions similar to those seen in humans.

Animals↗
PubMedDate not supplied

Milk casein-based diet containing TGF-beta controls the inflammatory reaction in the HLA-B27 transgenic rat model.

BACKGROUND: A casein-based formula containing TGF-beta has been successfully used in adolescents during acute episodes of Crohn's disease. The role played by this molecule requires confirmation. We have examined the capacity of a TGF-beta containing diet to control the intestinal inflammation in HLA-B27 transgenic rats, and compared its effects with a similar diet devoid of TGF-beta. METHODS: Three groups of rats were studied. HLA-B27/hbeta2M transgenic rats were fed with a casein-based rat-adapted diet containing TGF-beta or a control casein-based diet without TGF-beta. Fischer control animals were fed the latter. Body weight, dietary intake, tissue weights, fecal samples, leukocyte counts, and acute phase response were analyzed. Intestinal inflammation was assessed by histology, myeloperoxidase, and mRNA expression of cytokines. MUC2 protein expression was assessed by immunohistochemistry. Breakdown of muscle protein was examined. RESULTS: The test diet improved diarrhea increasing the fecal dry matter and the colonic inflammation as shown by a lower inflammatory score (2.43 +/- 1.13 vs 4.42 +/- 0.53, p < .05), lower mucosal thickness (431.25 +/- 72.29 vs 508.57 +/- 81.32 microm, p = .08) and decreased IFNgamma mRNA expression. MUC2 protein expression was increased in HLA rats fed the TGF-beta diet compared with HLA rats fed the control diet, but restitution to normal pattern was not observed. The test diet also decreased leukocytosis and the acute phase response and improved the muscle catabolic response. CONCLUSION: The TGF-beta containing diet has a beneficial effect in an animal model of intestinal inflammation. Our observations support a potential role for dietary TGF-beta in the restoration of immune homeostasis.

Acute-Phase Reaction↗