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Abomasal casein infusion and exogenous somatotropin enhance nitrogen utilization by growing lambs.

Growing Dorset wether lambs (23 kg initial body weight) were used to determine whether the magnitude of nitrogen retention response to daily administration of exogenous somatotropin is limited by post-ruminal amino acid availability in growing ruminants. Eight lambs surgically fitted with abomasal cannulae were fed a total mixed ration of 85% of ad libitum intake. All lambs received a continuous abomasal infusion of 2 L of water or casein and twice daily subcutaneous injections of 0 or 100 micrograms recombinant bovine somatotropin (rbST)/kg body wt for 15 d per treatment in a 2 x 2 single reversal design. The casein solution was infused at a rate (4 to 5 g nitrogen/d) to achieve 25% of nitrogen intake observed with ad libitum feeding prior to initiation of treatments. Each lamb received all four treatments. Nitrogen balance was determined on d 8 to 14 of each treatment. Casein infusion increased nitrogen balance 43.4% (P less than 0.001), and rbST increased nitrogen balance 33.5% (P less than 0.001), without significant interaction (P less than 0.88). Combined effects of casein and rbST were additive, resulting in an 89% increase in nitrogen balance when compared with water plus excipient treatment. Results suggest that the quantity or composition of absorbed amino acids, or both, limit nitrogen retention by growing lambs, and that rbST increases the efficiency of utilization of absorbed amino acids for protein deposition.

Abomasum↗

Increasing intake of soybean protein or casein, but not cod meal, reduces nephrocalcinosis in female rats.

Female weanling rats were fed diets with soybean protein, casein or cod meal at 171, 342 or 513 mmol nitrogen/100 g for 3 wk. The diets were isonitrogenous and balanced for fat, cholesterol, calcium, magnesium and phosphorus. Cod meal feeding at 171 and 342 mmol nitrogen/100 g diet produced lower kidney calcium concentrations than the feeding of either soybean protein or casein. Increasing protein intakes were associated with reduced kidney calcium concentrations in the rats fed either soybean protein or casein but not in those fed cod meal. The anti-nephrocalcinogenic effect of increasing intakes of soybean protein may relate to the lowering of urinary phosphorus concentration. Increasing intakes of casein probably inhibited nephrocalcinogenesis by lowering urinary pH and raising urinary magnesium concentration. Increasing cod meal concentrations in the diet lowered urinary pH and raised urinary magnesium and calcium concentrations, but the effects on nephrocalcinogenesis of these changes probably counteracted each other.

Animals↗

Excessive cholesterolemic response in analbuminemic rats fed a cholesterol-rich diet containing casein.

Female Nagase analbuminemic rats and Sprague-Dawley rats were fed purified diets with or without 1% cholesterol and containing either soybean protein or casein. After consuming the cholesterol-free diets, the analbuminemic rats had significantly higher plasma cholesterol and triglyceride concentrations than the Sprague-Dawley rats. The higher plasma cholesterol levels were essentially in the low density and high density lipoproteins. Based on the fact that the analbuminemic rats excreted more bile acids in feces it is possible that the higher baseline plasma cholesterol concentrations in Nagase analbuminemic rats were partly caused by overproduction of cholesterol. The Nagase analbuminemic rats displayed a greater cholesterolemic response to cholesterol feeding than Sprague-Dawley rats, but only if casein was the protein source in the diet. Casein vs. soybean protein in either cholesterol-free or high cholesterol diets reduced bile acid excretion in Sprague-Dawley but not in Nagase analbuminemic rats. The increased sensitivity to casein plus cholesterol feeding in Nagase analbuminemic rats may be caused by a lack of inhibition of de novo cholesterol synthesis.

Animals↗

Inhibitory effect of soybean protein vs. casein on apparent absorption of magnesium in rats is due to greater excretion of endogenous magnesium.

Apparent magnesium absorption is depressed in rats fed diets containing soybean protein or enriched with sodium phytate or phosphate in comparison with casein, whereas it is raised in rats fed lactose. However, the possibility that changes in apparent absorption are caused by changes in fecal excretion of endogenous magnesium cannot be excluded. We studied the effects of casein, soybean protein, sodium phytate, lactose and phosphate on apparent and true absorption of magnesium. True magnesium absorption was measured with the use of oral and intraperitoneal administration of tracer doses of 28Mg. Fecal excretion of endogenous magnesium was calculated from apparent and true absorption. True magnesium absorption was not affected by either substitution of soybean protein for casein or by the addition of sodium phytate to a diet containing casein. Endogenous magnesium excretion in feces was significantly increased by soybean protein and sodium phytate. Thus the observed impairment of apparent magnesium absorption in rats fed soybean protein or sodium phytate is due to enhanced fecal excretion of endogenous magnesium. With the other dietary treatments, enhanced fecal excretion of endogenous magnesium was not associated with a discrepancy in the effects on apparent and true magnesium absorption. Dietary lactose vs. dextrose and supplemental phosphate both stimulated fecal excretion of endogenous magnesium, but lactose raised both true and apparent magnesium absorption, and phosphate depressed both true and apparent magnesium absorption.

Absorption↗

Abomasal casein infusion enhances the mitogenic activity of serum from protein-restricted steers.

Holstein steers fed protein-restricted diets were used to evaluate protein realimentation and site of serum collection on the ability of calf serum to affect proliferation, protein synthesis and degradation in L6 myoblast cell culture bioassay. In Experiment 1, five steers (average weight 227 kg) received continuous abomasal infusion of 4 L of water or casein (50% of basal dietary nitrogen intake) in a switchback design. Serum was collected 2 d before and after 1, 3, 5, 7 and 9 d of infusion. Abomasal casein infusion increased serum mitogenic activity, nitrogen retention (119%) and total post-ruminal amino acid flow (78%). In Experiment 2, serum was collected from the jugular and femoral veins and the carotid artery before and after 7 d of abomasal casein infusion. Serum from calves abomasally infused with casein increased myoblast proliferation (jugular > femoral > carotid) and protein synthesis and decreased protein degradation in cultured myotubes. The addition of test calf serum inhibited the mitogenic activity of control calf serum. Results suggest that post-ruminal amino acid flow and site of serum collection alter the ability of serum to influence cell culture bioassays.

Amino Acids↗

The activities of rat hepatic cysteine dioxygenase and cysteinesulfinate decarboxylase are regulated in a reciprocal manner in response to dietary casein level.

The catabolism of cysteine and cysteinesulfinate, the activities of key enzymes in cysteine catabolic pathways, and the effects of inhibitors of specific enzymes on cysteine catabolism were investigated in hepatocytes isolated from rats fed low (100 g casein/kg diet), moderate (300 g casein/kg diet) or high (600 g casein/kg diet) levels of dietary protein. Cysteine was catabolized predominantly by cysteinesulfinate-dependent pathways. Cysteine dioxygenase activity increased with increases in dietary casein level, and the higher enzyme activity was paralleled by a greater total catabolite production (taurine + hypotaurine + sulfate) from cysteine. However, taurine production did not closely follow cysteine dioxygenase activity. Taurine production doubled with an increase in dietary casein from 100 to 300 g/kg but did not increase with a further increase in dietary casein to 600 g/kg. Taurine production as a percentage of total catabolism decreased progressively with the increases in dietary casein and closely paralleled observed decreases in cysteinesulfinate decarboxylase activity. Thus, taurine production was limited at high protein levels by the decrease in cysteinesulfinate decarboxylase activity such that sulfate production from cysteinesulfinate was favored. D-Cysteinesulfinate inhibited cysteinesulfinate-dependent catabolism of cysteine, but inhibition of cysteinesulfinate decarboxylase was not specific.

Animals↗

Physiological doses of oral casein affect hepatic glycogen metabolism in normal food-deprived rats.

In a previous study, administration of casein hydrolysate to food-deprived rats at a dose of 4 g/kg body wt resulted in an increase in portal plasma glucagon concentration. This was associated with an activation of phosphorylase a and a decrease in hepatic glycogen concentration. The present study was undertaken to determine whether similar results would be obtained with smaller doses. Doses of 1 and 2 g/kg body wt were administered to food-deprived rats. At a dose of 2 g/kg, portal plasma glucagon concentration was significantly elevated. This was associated with a slight increase in phosphorylase a activity (P < 0.05) and a 50% decrease in hepatic glycogen concentration (P < 0.01). At a dose of 1 g casein hydrolysate/kg body wt, changes in portal plasma glucagon concentration, phosphorylase a activity and hepatic glycogen concentration generally were not observed. Hepatic glucose, uridine diphosphoglucose, ATP and glucose-6-phosphate concentrations were unaffected by either dose of casein hydrolysate. The data indicate a dose-response relationship between casein hydrolysate administration and effects on glycogen metabolism in the liver. Protein-induced glycogenolysis is likely to occur when rats ingest a moderate amount of a pure protein meal.

Adenosine Triphosphate↗

Inducibility of hepatic CYP1A enzymes by 3-methylcholanthrene and isosafrole differs in male rats fed diets containing casein, soy protein isolate or whey from conception to adulthood.

Hepatic cytochrome P450 (CYP)1A1 and 1A2 enzymes were studied in male Sprague-Dawley rats derived from 5-7 litters fed diets in which the protein source was casein, soy protein isolate or whey. At age 65 d, rats were gavaged with corn oil (vehicle), 40 mg/kg 3-methylcholanthrene (3-MC) or 75 mg/kg isosafrole (ISO). Hepatic expression of CYP1A1 and CYP1A2 mRNA, apoprotein and associated monooxygenase activities were measured 17 h later. No significant dietary effects were observed on basal expression of either enzyme. However, interactions between diet and the two inducers (3-MC and ISO) were observed in soy-fed rats for ethoxy- and methoxyresorufin O-dealkylase activity, CYP1A1 and CYP1A2 apoprotein and mRNA (P < 0.05). The level of induction of CYP1A1 mRNA and apoprotein was lower in rats fed soy diets than in rats fed casein diets (P < 0.05), and the level of induced CYP1A2 mRNA was lower in rats fed soy or whey (P < 0.05) after treatment with the aryl hydrocarbon (Ah) receptor-dependent inducer 3-MC. This was accompanied by a 50% reduction in constitutive levels of the Ah receptor in liver cytosol of soy-fed, relative to casein-fed rats, and a slightly smaller reduction in whey-fed rats. Expression of the Ah receptor correlated with 3-MC-inducibility of CYP1A1 mRNA in rats fed the three diets. In contrast, in rats induced with ISO, which does not bind to the Ah receptor and induces CYP1As via different mechanisms than 3-MC, ethoxyresorufin O-deethylase activity and levels of CYP1A1 apoprotein and mRNA were elevated to a greater degree in soy-fed than in casein- or whey-fed rats (P < 0.05). Moreover, after ISO treatment, induction of methoxyresorufin O-demethylase activity, CYP1A2 apoprotein and mRNA levels was observed only in rats fed soy (P < 0.05). These data suggest potential effects of dietary protein source on metabolism of a wide variety of CYP1A substrates, including environmental and dietary carcinogens, many of which induce their own metabolism.

Aging↗

Long range restriction analysis of the bovine casein genes.

Pulsed field gel electrophoresis (PFGE) was used to analyse the organization of the bovine alpha s1, alpha s2, beta and kappa casein genes. High molecular weight DNA was prepared from fibroblasts and lymphocytes embedded in agarose and was digested with the restriction endonucleases Clal, Sall, Smal, Xhol. The digestion products were separated by PFGE, transfered to nitrocellulose filters and hybridized to probes corresponding to the cDNAs of the four bovine casein genes. The casein genes were demonstrated to be physically linked within a region of 300 kb, represented by two adjacent Xhol fragments in fibroblasts and by a single fragment in lymphocytes. A restriction map of the casein locus was derived and the order of the genes was shown to be kappa, alpha s2, beta, alpha s1.

Animals↗

Absence of mRNA for casein in free polysomes of lactating ewe mammary gland.

Total polysomal RNA or poly A- containing RNA extracted from free polysomes of lactating ewe mammary gland was unable to direct a significant synthesis of alpha-s- and beta-casein. Polyacrylamide gel electrophoresis of poly A- containing RNA revealed that the two major peaks of mRNA isolated from membrane-bound polysomes were not observed in mRNA isolated from free polysomes. Purified 125-I anti-alpha-s-casein could bind significantly to membrane-bound polysomes only. These data suggest that casein synthesis occurs almost exclusively on membrane-bound polysomes and that this specificity may be attributed to the presence of casein mRNAs on these polysomes and their absence from free polysomes.

Animals↗

Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene.

Using a set of overlapping oligonucleotides from the promoter region of the bovine alpha s2-casein gene we have identified two nuclear factors which probably are involved in expression of this gene and the related calcium sensitive alpha s1- and beta-casein genes. One of these factors which was present in extracts of all tissues that have been tested including Hela cells turned out to be the octamer binding protein OCT-1. Oct-1 binds with different affinity to 4 sites at positions centred around -480, -260, -210 and -50. The strongest of these 4 binding sites, the one around position -50, is highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle. The other nuclear factor (MGF, mammary gland factor) which is specifically expressed in the mammary gland, binds to a site around position -90. This binding site is also highly conserved in all calcium sensitive caseins of mouse, rat, rabbit and cattle.

Animals↗

Modulation of the transferred mouse 26K casein gene in mouse L cells by glucocorticoid hormone.

The cloned 26K casein gene was transferred to mouse L-cells and its expression was measured by Northern blot hybridization. When the lambda clone with 5'- and 3'-flanking sequences was transferred, transcripts were detected without glucocorticoid, but in the presence of glucocorticoid, the level of the transcripts of heterogeneous sizes increased and their pattern was similar to that observed in the mammary glands of non-lactating mice. When the 6.7 kb EcoRI fragment containing most of the coding region was transferred, putative precursor and mature mRNAs were detected without glucocorticoid. Surprisingly, with the addition of glucocorticoid, the level of the transcripts greatly decreased. The presence of multiple sequences responsible for glucocorticoid receptor binding was detected in the 5'-flanking region of the gene in a competition assay using the subfragments of casein gene and on sequence analysis. These results suggest that the 26K casein gene has multiple regulatory domains which interact with glucocorticoid receptors, and these domains may play different roles in the regulation of the casein gene.

Animals↗

Purification and phosphorylation of a M(r) 25,000 protein, an effective phosphate acceptor for casein kinase II and protein kinase C, detected in the cytosolic fraction of Xenopus laevis oocytes.

A common and effective phosphate acceptor protein for casein kinase II and Ca(2+)-phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from the cytosolic fraction of Xenopus laevis oocytes. Its molecular mass was estimated to be approximately 25,000 by SDS-polyacrylamide slab gel electrophoresis and gel filtration analyses. About 1 and 2 mol of phosphate were incorporated per mol of this protein with casein kinase II and protein kinase C, respectively, and the phosphorylated amino acid was identified as serine irrespective of the protein kinase employed. The Km values were calculated to be 1 and 0.5 microM for this M(r) 25,000 protein with casein kinase II and protein kinase C, respectively. However, this protein was a relatively poor substrate for casein kinase I and did not serve as one for cAMP-dependent protein kinase. The amino acid sequence of its amino-terminal region suggests that this protein is a newly identified substrate for these two protein serine/threonine kinases.

Amino Acid Sequence↗

Use of fluorescamine-labeled casein as a substrate for assay of proteinases.

Conditions have been investigated for the use of fluorescamine-labeled casein as a substrate for fluorometric assay of proteinases. Fluorescamine-labeled casein can be prepared simply by mixing solutions of casein and fluorescamine at pH 8.0 and used without removal of the excess reagent or its hydrolysis product. The fluorescence of the labeled casein and its enzymatic digest is moderately stable in the range of pH 7.0 to 10.0. Activities can be determined by measuring the fluorescence of the hydrolysis products soluble in 0.1 M trichloro acetic acid solution at pH 4.0 after adjusting the pH of the acid-soluble fraction to 7.7. This method is suited for assay of proteinases active at neutral to slightly alkaline pH values, and is capable of quantitating about 0.05 microgram of trypsin or 0.5 microgram of alpha-chymotrypsin or papain. The assay can be done in the presence of large amounts of contaminating amino acid, protein and/or exopeptidases which may interfere with the ordinary assay of proteinases.

Caseins↗

Insertion of a casein kinase recognition sequence induces phosphorylation of ovine beta-lactoglobulin in transgenic mice.

We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a beta-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine beta-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel beta-lactoglobulin in their milk. The infrared spectrum, reactivity to antiphosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated beta-lactoglobulin was in the whey and hence not incorporated into casein micelles.

Amino Acid Sequence↗

Primary and predicted secondary structures of the caseins in relation to their biological functions.

In free solution, the caseins behave as non-compact and largely flexible molecules with a high proportion of residues accessible to solvent. Historically, they have been described as random coil-type proteins with only a nutritional function. Nevertheless, secondary structure prediction algorithms indicate that many parts of the (unphosphorylated, unglycosylated) polypeptide chains can form regular structures. In particular, a recurrent motif of the Ca2+-sensitive caseins in man, rat, mouse, guinea pig and ruminant species is an alpha-helix--loop--alpha-helix conformation in which the loop region typically contains a cluster of sites of phosphorylation. The biological function of the caseins is considered and it is suggested that the potential or actual conformations of the group of Ca2+-sensitive caseins are suited to the function of modulating the precipitation of calcium phosphate from solution. Either they can act as sites for nucleation or they can bind rapidly to calcium phosphate nuclei as they form spontaneously from supersaturated solution.

Amino Acid Sequence↗

Incidence of gastroesophageal reflux with whey- and casein-based formulas in infants and in children with severe neurological impairment.

Ten exclusively gastrostomy-fed, neurologically impaired children (4.5-14.5 years old) with gastroesophageal reflux were randomly assigned to receive feedings with either a casein- or a whey-based formula for 48 h each and then crossed over to the other formula. One 24-h pH probe study each was performed while being fed casein- and whey-based formula, respectively. There was a significant reduction in episodes and duration of gastroesophageal reflux while consuming the whey-based formula (p < 0.05). Whey-based feedings should be considered an additional tool in conjunction with other antireflux measures to treat gastroesophageal reflux more effectively in children with severe neurological impairment. A similar study was also conducted involving 14 infants (3-12 months old) with documented gastroesophageal reflux using 24-h pH probe monitoring while consuming a casein-based formula. The formula was changed to a whey-based formula and the pH probe study repeated within 3-5 days. Four infants showed improvement and the rest showed either deterioration (1/14) or comparable results (9/14). The reduction in the mean number of episodes or duration of gastroesophageal reflux with the whey-formula was not significantly different from that with the casein-based formula (p > 0.05). Based on these findings, generalized recommendations for the use of whey-based formula in infants with gastroesophageal reflux cannot be made.

Adolescent↗

Detection of a new kappa-casein variant in cow's milk.

In German Simmental cattle a new kappa-casein variant was detected by isoelectric focusing, which cannot be distinguished from kappa-casein A by polyacrylamide and starch gel electrophoresis. It was named kappa-casein D. The frequencies of kappa-casein alleles were kappa-Cn A 0.75, kappa-Cn B 0.24, kappa-Cn D 0.01.

Alleles↗