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Resistant starch prevents colonic DNA damage induced by high dietary cooked red meat or casein in rats.

In a previous study we have shown that high levels of dietary protein (as casein) result in increased levels of colonic DNA damage, measured by the comet assay, and thinning of the colonic mucus layer in rats when dietary resistant starch (RS) is negligible. Feeding RS abolishes these effects. This study aimed to establish whether a diet high in protein as cooked red meat would have similar effects and whether RS was protective. Rats were fed a diet containing 15% or 25% casein or 25% cooked lean red beef, each with or without the addition of 48% high amylose maize starch (a rich source of RS) for four weeks. As expected, high dietary casein caused a 2-fold increase in colonic DNA damage compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% greater DNA damage than the high casein diet but reduced mucus thickness to a similar degree to casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. Cecal and fecal short chain fatty acid pools were also increased by inclusion of RS in the diet. Because DNA damage is an early step in the initiation of cancer, these findings suggest that increased DNA damage due to high dietary protein as cooked red meat or casein could increase colorectal cancer risk but inclusion of resistant starch in the diet could significantly reduce that risk.

Amylose↗

Identification of antibacterial peptides from bovine kappa-casein.

The objective of the present study was to identify antimicrobial peptides present in several digests of commercial caseins with gastric enzymes. The most active hydrolysate against Escherichia coli ATCC 25922 and Listeria innocua CECT 910T corresponded to a pepsin digest of bovine kappa-casein. The protein digest was first separated by semipreparative high-performance liquid chromatography (HPLC), and the most active fractions were again subjected to a second chromatographic step. Finally, identification of the active peptides was carried out by online and offline HPLC-electrospray ionization-tandem mass spectrometry. By means of this technique, 21 peptides were identified in the active HPLC fractions. Although most were derived from bovine kappa-casein, some of the identified fragments corresponded to beta-casein and alpha(s)-casein fragments, a result of the presence of small amounts of these proteins in the preparation of kappa-casein. Some of the peptides identified were chemically synthesized and showed antibacterial effects against several gram-positive and gram-negative bacteria. Among the synthesized peptides, kappa-casein f(18-24), f(30-32), and f(139-146) were most effective against all bacteria tested. The antibacterial effect of these peptides is discussed in relation to their amino acid sequences.

Amino Acid Sequence↗

Ischaemic heart disease, Type 1 diabetes, and cow milk A1 beta-casein.

AIM: To test the correlation of per capita A1 beta-casein (A1/capita) and milk protein with: 1) ischaemic heart disease (IHD) mortality; 2) Type 1 (insulin-dependent) diabetes mellitus (DM-1) incidence. METHODS: A1/capita was estimated as the product of per capita cow milk and cream supply and its A1 beta-casein content (A1/beta) (calculated from herd tests and breed distribution, or from tests of commercial milk), then tested for correlation with: 1) IHD five years later in 1980, 1985, 1990 and 1995, in 20 countries which spent at least US $1000 (purchasing power parities) per capita in 1995 on healthcare; 2) DM-1 at age 0-14 years in 1990-4 (51 were surveyed by WHO DiaMond Project; 19 had A1 data). For comparison, we also correlated 77 food, and 110 nutritive supply FAO (Food and Agriculture Organization)-based measures, against IHD and DM-1. RESULTS: For IHD, cow milk proteins (A1/capita, r = 0.76, p <0.001; A1/capita including cheese, r = 0.66; milk protein r = 0.60, p = 0.005) had stronger positive correlations with IHD five years later, than fat supply variables, such as the atherogenic index (r = 0.50), and myristic, the 14-carbon saturated fat (r = 0.48, p <0.05). The Hegsted scores for estimating serum cholesterol (r = 0.42); saturated fat (r = 0.37); and total dairy fat (r = 0.31) were not significant for IHD in 1995. Across the 20 countries, a 1% change in A1/capita in 1990 was associated with a 0.57% change in IHD in 1995. A1/capita correlations were stronger for male than female mortality. On multiple regression of A1/capita and other food supply variables in 1990, only A1/capita was significantly correlated with IHD in 1995. DM-1 was correlated with supply of: A1/capita in milk and cream (r = 0.92, p <0.00001); milk and cream protein excluding cheese (r = 0.68, p <0.0001); and with A1/beta in milk and cream (r = 0.47, p <0.05). Correlations were not significant for A2, B or C variants of milk beta-casein. DM-1 incidence at 0-4, 5-9 and 10-14 years was equally correlated (r = 0.80, 0.81, 0.81 respectively) with milk protein supply. A 1% change in A1/capita was associated with a 1.3% change in DM-1 in the same direction. CONCLUSIONS: Cow A1 beta-casein per capita supply in milk and cream (A1/capita) was significantly and positively correlated with IHD in 20 affluent countries five years later over a 20-year period--providing an alternative hypothesis to explain the high IHD mortality rates in northern compared to southern Europe. For DM-1, this study confirms Elliott's 1999 correlation on 10 countries for A1/capita,1 but not for B beta-casein/capita. Surveys of A1 beta-casein consumption in two-year-old Nordic children, and some casein animal feeding experiments, confirm the A1/capita and milk protein/capita correlations. They raise the possibility that intensive dairy cattle breeding may have emphasised a genetic variant in milk with adverse effects in humans. Further animal research and clinical trials would be needed to compare disease risks of A1-free versus 'ordinary' milk.

Adolescent↗

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs. Immunoblot analysis utilizing anti-casein kinase II antiserum of cytosolic extracts prepared from COS-1 cells co-transfected with pCMV-alpha and pCMV-beta showed 2- and 4-fold increases in immunoreactive alpha and beta subunit protein, respectively, relative to vector-transfected cells. These same cytosolic fractions exhibited an average 5-fold increase in casein kinase II catalytic activity. COS-1 cells transfected with pCMV-alpha alone exhibited a 3-fold increase in immunoreactive alpha subunit protein and a nearly 2-fold increase in cytosolic casein kinase II catalytic activity. Transfection with the cDNA coding for the noncatalytic beta subunit alone also caused a near doubling of cytosolic casein kinase II catalytic activity. No increase in immunoreactive alpha subunit protein was observed in pCMV-beta-transfected cells, and no increase in immunoreactive beta subunit protein was observed in pCMV-alpha-transfected cells. These results indicate that a portion of the endogenous cellular casein kinase II protein is not fully active and that raising the concentration of the alpha or beta subunit stimulates this latent activity.

Amino Acid Sequence↗

Regulation of casein kinase II activity by epidermal growth factor in human A-431 carcinoma cells.

In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivo and serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor.

Alkaline Phosphatase↗

Casein and histone kinases of a rat ascites hepatoma as compared with those of rat liver.

Seryl/threonyl-protein kinases in cytosolic and particulate fractions from rat liver and AH-13, a rat ascites hepatoma, have been studied by chromatographing these fractions on DEAE-cellulose and assaying the eluates with casein, phosvitin, histone and protamine as substrates. Liver cytosolic fraction contains a group of well-characterized seryl/threonyl-protein kinases, namely, casein kinases I and II and histone kinases I and II. Liver particulate fraction, on the other hand, is almost totally devoid of casein kinase I and histone kinase I but contains an additional peak of casein kinase tentatively designated casein kinase III. In AH-13, cytosolic casein kinase I is markedly increased and particulate-associated casein kinases II and III are moderately increased as compared with liver. Moreover, it was found that in AH-13, the histone kinase I level is high in the particulate fraction but markedly decreased in the cytosolic fraction. It is suggested that particulate-associated histone kinase I may be of cytosolic origin.

Animals↗

Insulin-like growth factor I and insulin rapidly increase casein kinase II activity in BALB/c 3T3 fibroblasts.

We have tested whether growth factors added to serum-deprived BALB/c 3T3 fibroblasts alter the casein kinase II activity measured in cell extracts. A rapid phosphocellulose chromatography method was developed that provides a 40-fold partial purification of casein kinase II activity assayed with the specific substrate peptide Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. Using this technique, kinase activity is stimulated 1.6-2.5-fold when isolated from fibroblasts treated with insulin or insulin-like growth factor I (IGF-I). The activated kinase activity exhibits the specific properties of casein kinase II such as the ability to utilize [gamma-32P]GTP as phosphate donor and marked inhibition by low concentrations of heparin. Activation of casein kinase II appears specific for these hormones because epidermal growth factor and platelet-derived growth factor have no effect on the kinase activity when added to fibroblasts under conditions where they markedly stimulate [3H]thymidine incorporation into DNA. Increases of casein kinase II activity by insulin and IGF-I were detected within 1 min of their addition to cell cultures. IGF-I is more potent in stimulating casein kinase II than insulin in mouse fibroblasts. These results demonstrate that casein kinase II is a selective target for insulin and IGF-I action in BALB/c fibroblasts, consistent with the hypothesis that this kinase plays a role in cellular signaling by these hormones.

Animals↗

[Effects of monensin on the secretion of caseins by mammary epithelial cells from lactating rabbits].

The effects in vitro of monensin on the secretory pathway of caseins in lactating rabbit mammary gland fragments were investigated. Addition of monensin (0.1 microM or 1 microM) to the incubation medium induced a dilatation of Golgi saccules and vesicles and a decrease of the relative volume of microvesicles. Dilated vesicles did not contain acid phosphatase. Neosynthesized proteins were localized by electron microscope autoradiography near the membranes of dilated vesicles. 1 microM monensin did not inhibit basal secretion of neosynthesized caseins (radioactive caseins, labelled during a 3 min pulse, released into the medium), but increased basal secretion of total beta-caseins (measured by radioimmunoassay). Monensin abolished all the effects of prolactin (increase in the relative volume of Golgi microvesicles and stimulation of secretion of total and neosynthesized caseins). These results show that monensin provokes simultaneously modifications in the Golgi apparatus morphology and inhibition of the prolactin stimulating effect. Moreover, secretion of intracellular caseins is differently modified, depending probably on whether caseins are neosynthesized or stored.

Animals↗

Casein kinase II is a major protein phosphorylating activity in the nuclei of Xenopus laevis oocytes.

The nuclei of Xenopus laevis oocytes contain kinases capable of phosphorylating endogenous and exogenous proteins using either ATP or GTP as phosphoryl donors. These enzymes are much more active with casein and phosvitin as substrates than with histones or protamines. The protein phosphorylating activity of oocyte nuclear extracts is not regulated by cyclic nucleotides, phorbol esters, calmodulin and calcium, or phospholipids. However, the casein phosphorylating activity can be greatly enhanced by the polyamines spermine or spermidine and drastically inhibited by heparin. Fractionation of the nuclear casein kinase activities by DEAE-Sephadex chromatography and glycerol gradient centrifugation indicate that the nuclei contain enzymes with the properties of casein kinases I and II as characterized in other species. Oocyte casein kinase I (Mr 37,000) is specific for ATP as phosphoryl donor, is only slightly inhibited by 10 micrograms/ml heparin, and is not significantly stimulated by polyamines. Casein kinase II (Mr 135,000) can use both ATP and GTP as substrates, and is very sensitive to heparin inhibition and polyamine stimulation. The fact that low concentrations of heparin (10 micrograms/ml) can inhibit a large percentage of the endogenous phosphorylation of nuclear extracts or of whole nuclei indicates that casein kinase II is probably the major protein phosphorylating activity of these oocyte organelles.

Animals↗

Abnormal responses to ingested substances in murine systemic lupus erythematosus: apparent effect of a casein-free diet on the development of systemic lupus erythematosus in NZB/W mice.

To assess the development of oral tolerance to casein in NZB/W female mice, they must be bred and raised on a casein free diet. We examined the specific immune responses of the mice to the long term experimental feeding of casein. Twelve of fifteen casein free mice were still alive at 10 months of age, although by this age only 1/10 mice eating the normal diet was still alive. The casein free mice had markedly less anti-DNA antibody, their IgM to IgG antinative DNA switch was delayed and deposits of immunoreactants in the glomeruli were greatly decreased. The reason for this apparent effect of the removal of casein from the diet is unknown; however, immunostimulatory and endorphin-like regions have recently been reported in casein.

Animals↗

Inhibition of macrophage accessory cell function in casein-treated B6C3F1 mice.

Humoral immunity of casein-treated B6C3F1 mice was evaluated. Splenocytes from casein-treated mice sensitized in vitro exhibited a marked suppression in their antibody response to the T-dependent antigen, SRBC (82%) and T-independent antigen, DNP-Ficoll (80%). In contrast, a control response to the polyclonal antigen, lipopolysaccharide (LPS), was observed. Cell fractionation and crossover reconstitution assays of adherent (ADH) and nonadherent (NAD) splenocyte populations from vehicle and casein-treated mice indicated that: 1) ADH splenocytes from casein-treated mice were responsible for suppression of humoral responses, 2) NAD splenocytes from casein-treated mice reconstituted with vehicle or naive ADH cells abrogated the immunosuppression, and 3) suppression of humoral responses in cultures containing casein ADH splenocytes was due to this cell populations inability to function as accessory cells in humoral responses rather than induction of suppressor macrophages. Results from in vivo studies with casein-treated mice sensitized with sheep red blood cells or dinitrophenyl-Ficoll paralleled the in vitro results.

Amyloidosis↗

[In vitro proteolysis of casein and gluten by pancreatic enzymes].

The time-course of proteolysis of casein and gluten by pancreatic enzymes (trypsin, chymotrypsin, pancreatic juice) was studied in vitro with various concentrations of enzyme or substrate. The effect of a previous peptic proteolysis, with or without sodium phytate, on proteolysis by pancreatic juice was also studied. Proteolysis was evaluated by measuring the nitrogen in products solubilized in trichloroacetic acid (peptides + amino acids) and in phosphotungstic acid (amino acids). Without a previous peptic proteolysis, at a low enzyme/substrate ratio, the release of peptides and amino acids was greater for casein than for gluten. At a high enzyme/substrate ratio, peptide release by pancreatic juice and chymotrypsin was quite similar for casein and gluten but amino acid release was a little less for gluten. After a previous high peptic proteolysis without sodium phytate, pancreatic juice proteolysis, at a low enzyme/substrate ratio, was increased. Peptide release was similar for casein and gluten but amino acid release was a little less for the gluten. With sodium phytate, the release of peptides was quite similar for casein but was less for gluten. The release of amino acids was diminished in both cases. Thus, gluten was more resistant than casein to the action of pancreatic enzymes. Nevertheless, this was not necessarily observed when those enzymes were used for proteolysis at a high enzyme/substrate ratio or following a high peptic proteolysis. The differences between casein and gluten proteolyses have been discussed according to the specific action of the pancreatic enzymes. In conclusion, in comparing the proteolyses of various foods, it is important to use various enzyme/substrate ratios.

Amino Acids↗

Nitrogen movements in the upper jejunum lumen in humans fed low amounts of casein or beta-lactoglobulin.

OBJECTIVES AND METHODS: To compare the progression of milk proteins in the upper part of the digestive tract, gastro-jejunal nitrogen movements were studied in 6 healthy human volunteers after beta-lactoglobulin and casein ingestion. 400 mL of water (control), purified beta-lactoglobulin (20 g/L) or casein (20 g/L), each adjusted to 25 microCi with 14C-polyethylene glycol, were given per os. Samples were collected in the stomach and 20 cm below the Treitz ligament every 20 min for 2 hours and measured for volume, osmolarity, ions and nitrogen content. RESULTS: The jejunal flow rate peaked in the 0-20 min period following water and beta-lactoglobulin ingestion, and in the 20-40 min period after casein ingestion. The gastric half-emptying time (T1/2 min) of the liquid phase was significantly different (P < 0.05) for water (12.1 +/- 0.8), beta-lactoglobulin (14.5 +/- 3.3) and casein (26.5 +/- 9.3). Before ingestion of the test meals, the basal rate of nitrogen was 9.14 +/- 4.09 mmol/h in the jejunum. The total nitrogen content in the jejunum peaked significantly in the 0-20 min period after beta-lactoglobulin ingestion and the 20-40 min period after casein ingestion. The apparent gastro-jejunal protein absorption values were 63% for casein and 66% for beta-lactoglobulin in the 120 min period. CONCLUSIONS: These results show that beta-lactoglobulin and casein behave differently in the upper part of the digestive tract due to different gastric emptying rates.

Adult↗

Mouse gamma-casein cDNA: PCR cloning and sequence analysis.

A partial amino acid sequence of mouse gamma-casein was determined on a gas-phase amino acid sequencer. The N-terminal sequence (23 residues) was obtained using native gamma-casein, and the C-terminal sequence (13 residues) was determined with a corresponding peptide. The C-terminal peptide was isolated by affinity chromatography on an anhydrotrypsin agarose column after digestion of gamma-casein with Achromobacter lysyl-endopeptidase. A cDNA (507 base pairs) encoding the mature protein of gamma-casein was produced by polymerase chain reaction (PCR) amplification of lactating mammary gland first strand cDNA with oligonucleotide primers designed from the N-terminal (KHEIKDK-) and the C-terminal (-AHYTRFY) amino acid sequences. The full-length cDNA including 5'- and 3'-noncoding regions (920 base pairs) was cloned by the rapid amplification of cDNA ends (RACE) method of Frohman et al. Comparison of full-length cDNAs of mouse gamma-casein and rat gamma-casein showed 80% identity at the nucleotide level. The amino acids in the coding region of both gamma-caseins were 75% identical.

Amino Acid Sequence↗

Assessment of the interaction between calmodulin and casein kinase II.

Calmodulin is an in vitro substrate for casein kinase II and is also reported to inhibit casein kinase II activity in rat liver nuclear extracts. Here we demonstrate that in the presence or absence of Ca2+, calmodulin did not significantly alter either in vitro casein kinase II activity or autophosphorylation of its beta-subunit. In contrast, Ca2+ inhibited in a dose-dependent manner both casein kinase II activity and casein kinase II-catalysed calmodulin phosphorylation. These data indicate that calmodulin does not directly inhibit casein kinase II activity, but casein kinase II is sensitive to physiologic Ca2+ concentrations.

Adenosine Triphosphate↗

Phosphorylation of the insulin receptor substrate IRS-1 by casein kinase II.

IRS-1, a principal substrate of the insulin receptor, is phosphorylated on serine, threonine, and tyrosine residues in a variety of tissues during insulin stimulation. Casein kinase II, an insulin-sensitive serine/threonine kinase, catalyzed the in vitro incorporation of 1 to 2 mol of phosphate/mol of recombinant rat IRS-1. Two-dimensional phosphopeptide mapping of IRS-1 phosphorylated by casein kinase II in vitro and IRS-1 immunoprecipitated from intact Chinese hamster ovary cells demonstrated multiple common phosphopeptides, suggesting that overexpressed IRS-1 is a substrate for casein kinase II in these cells. Moreover, the common phosphopeptides that appeared to be insulin-sensitive in intact cells comprised 22% of casein kinase II-catalyzed 32P incorporation into IRS-1 in vitro. These data suggest that casein kinase II mediates a portion of the insulin-stimulated serine/threonine phosphorylation of overexpressed IRS-1 in vivo. By using phosphoamino acid analysis, strong cation exchange analysis, manual Edman degradation, and automated amino acid sequencing, Thr-502 was identified as the major casein kinase II-catalyzed phosphorylation site in rat IRS-1. Furthermore, Ser-99, an additional site labeled at low yield, appeared to be contained in an insulin-sensitive phosphopeptide. Thus, casein kinase II-catalyzed phosphorylation of IRS-1 may be a component of the intracellular insulin signalling cascade.

Amino Acid Sequence↗

The comparison of pepsin and trypsin action on goat, cow, mare and human caseins.

The degree of proteolysis of micellar caseins of human, goat's, mare's and two breeds (Black&White and Red Polish) of cow's milk was compared for pepsin and trypsin action in vitro. Human and goat's caseins were hydrolysed in 100% and 96%, respectively, mare's casein--92%, Black&White cow's casein--90%, Red Polish cow's casein--76%. The differences can be related to the micelle structure, especially to the prevalence of beta casein in the human and goat's casein. The significant dissimilarity between the two breeds of investigated cows is surprising and indicates a different geometry of micellar aggregates.

Animals↗

Creaming and Rheology of Oil-in-Water Emulsions Containing Sodium Dodecyl Sulfate and Sodium Caseinate.

The creaming and rheology of fine n-tetradecane oil-in-water emulsions at pH 6.8 containing the commercial protein sodium caseinate and the ionic surfactant sodium dodecyl sulfate (SDS) have been studied, and an overview diagram relating surfactant composition and creaming stability has been constructed. The presence of both SDS and sodium caseinate in an emulsion system increases the overall stability with respect to creaming. Excess SDS promotes destabilization through fast creaming; this can be attributed to depletion flocculation brought about by unadsorbed surfactant micelles. Addition of sodium caseinate was found to reduce this effect, even at relatively high SDS concentrations. The behavior of the caseinate + SDS emulsions is thus different from the behavior of the previously reported caseinate + Tween 20 systems, where the combination of the two surface-active agents was found to reduce the emulsion stability, as indicated by fast creaming and shear-thinning rheology. Addition of sodium chloride was found to increase the extent of non-Newtonian behavior and to enhance the degree of creaming for SDS-containing emulsions. Increased caseinate levels in these systems seem to offer some stabilization through reduction of the shear-thinning character and improvement in creaming stability. These phenomena can be explained in terms of a considerable amount of SDS binding to the protein, which reduces the amount of SDS available to promote protein displacement and depletion flocculation. In contrast to the SDS systems, the properties of equivalent emulsions containing caseinate + nonionic surfactant Tween 20 are relatively insensitive to salt content. Copyright 2000 Academic Press.

Journal Article↗