Glycosyl transferases in mouse and human milk fat globule membranes

Summary Membranes isolated from mouse and human milk fat globules were found to contain the enzymes responsible for the synthesis of dolichol monophosphate mannose and dolichol monophosphate glucose as well as those involved in the transference of the glycosyl residues from the two dolichol derivatives to dolichol diphosphate oligosaccharides. The levels of most of the enzymes were comparable to those found in mouse mammary gland microsomes. The presence of enzymes involved in protein glycosylation via dolichol derivatives in the milk fat globule membrane provides evidence in favor of an outward flow of membrane components from the rough endoplasmic reticulum, where these enzymes are active in vivo, towards the cell surface.     

Purification and partial characterization of xanthine oxidase from human milk.

Xanthine oxidase was purified from human milk in yields comparable with those obtained from bovine milk. The freshly purified enzyme appeared homogeneous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total M(r) 290,000 +/- 6000. The ultraviolet/visible absorption spectrum differed only slightly from that of bovine milk enzyme and showed an A280/A450 ratio of 5.13 +/- 0.29, indicating a high degree of purity. Xanthine oxidase activities of purified enzyme varied with batches of milk, ranging between 3 and 46 mU/mg protein; values that are some two to three orders of magnitude smaller than those shown by the most highly purified samples of bovine milk enzyme. Direct comparison with commercially-available bovine milk enzyme showed that activities involving xanthine as reducing substrate were 1-6% that of the bovine enzyme, whereas those involving NADH, in contrast, were of the same order for the two enzymes. Anaerobic bleaching experiments indicated that less than 2% of the human enzyme was present as a form active with xanthine. These findings, together with the activity data, are consistent with a very high content, possibly greater than 98%, of demolybdo- and/or desulpho-forms of human enzyme, both of which occur, to a lesser extent, in bovine xanthine oxidase. Molybdenum assay indicated that demolybdo-enzyme could only account for some 26% of this inactive component, suggesting that desulpho-enzyme may account for the remainder.     

Primary structure of the glycans from mouse serum and milk transferrins.

A 'serotransferrin-like' protein was purified from mouse milk. This serotransferrin cross-reacts immunologically with the serotransferrin isolated from mouse plasma and not with the mouse lactotransferrin (lactoferrin). Sugar analysis of the three transferrins, i.e. serotransferrin, milk 'serotransferrin-like' protein and lactotransferrin, revealed that the major difference between the glycan primary structure of mouse serotransferrin and those of mouse milk 'serotransferrin-like' protein and lactotransferrin concerns essentially the presence of one fucose residue in the last two proteins. For structural determination, the N-glycosidically linked glycans were released from the protein by a reductive cleavage of the oligosaccharide-protein linkage under strong alkaline conditions. The primary structure of the released oligosaccharide alditols was determined by methylation analysis and 400 MHz 1H-n.m.r. spectroscopy. The oligosaccharide alditols released from milk 'serotransferrin-like' protein and lactotransferrin were identical and were identified as disialylated biantennary glycans of the N-acetyl-lactosamine type with a fucose residue alpha-1,6-linked to the N-acetylglucosamine residue conjugated to the peptide chain and having the following primary structure: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-N)Asn. The serotransferrin glycan has the same primary structure but is only partially fucosylated (10-15%).     

A chemically derived milk substitute that is compatible with mouse milk for artificial rearing of mouse pups.

The object of this study was to prepare a chemically derived milk substitute that is compatible with mouse-milk. Milk was independently collected from ICR, BALB/c, and FVB/N mice, and analyzed for the protein, fat, and mineral contents to formulate a milk substitute. Thereafter, ICR mouse pups were artificially reared on the milk substitute to evaluate the rate of increase of their body weights. A gastric cannula tube was placed through the esophageal way into 8-day-old ICR pups, and the mice were fed with the milk substitute by computer-regulated infusion pumping by the pup-in-a-cup method. The analytical mean values of total protein and total fat in milk from ICR, BALB/c, and FVB/N mice were 10.23 +/- 0.49% and 21.34 +/- 1.31%, respectively. The milk substitute was constituted from purified bovine casein and whey proteins, five edible oils, including MCT oil, minerals, and vitamins. After 8 days of artificial rearing with the new milk substitute, 36 of the 42 pups had survived, and the growth rate of these mice was not significantly different from that of maternally reared littermate pups. In conclusion, we have succeeded in the preparation of a chemically derived milk substitute for mice pups which is available for clarifying the roles of dietary components such as milk-bone substance during the suckling period in mice pups including those of knockout and transgenic mice.     

Identification of caseins in goat milk

The importance of goat milk in infant diet is growing, because it is reported that goat's milk in some cases is less allergenic than cow's milk. This is due probably to the lower presence of caseins associated with a specific type of alpha(s1)-casein. In caprine breeds, four types of alpha(s1)-casein alleles are identified and associated with various amounts of this protein in milk. The contribution of strong alleles to the goat milk is approximately 3.6 g/L of alpha(s1)-casein, while for middle alleles is only 1.6 g/L, weak alleles 0.6 g/L. The contribution of null allele is very low (or non-existent). The quantity of total caseins in caprine milk is positively correlated with the amount of alpha(s1)-casein. Milk from animals possessing strong alleles contain significantly more total caseins than milk from animals without those alleles. This is important because animals with mild alleles can be employed to produce milk for allergic subjects while the other animals can be used to produce milk for the dairy industry. This work shows casein profiles of two types of classified goat milk (B, strong alpha(s1) allele, 0, null alpha(s1) allele) with two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and it confirms the different polymorphisms at locus alpha(s1) casein.     

Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.     

Quantitative collection of milk and active recombinant proteins from the mammary glands of transgenic mice

Summary

Transgenic mice expressing foreign genes specifically in their mammary glands have been obtained by several groups in the world. The mouse is generally considered as a good reference animal to evaluate the efficiency of gene constructs to be used in larger mammals for the preparation of the corresponding recombinant proteins at an industrial scale. The method described here shows that mammary glands from lactating mice separated from their pups for one day spontaneously released 1.5 ml milk when stored at O'C. The proteins of milk obtained by this method were essentially similar to those obtained after milking. Human growth hormone (hGH) gene under the control of the rabbit whey acidic (WAP) gene promoter was expressed at a high level in the milk of transgenic mice (4 mg/ml milk in the mice examined here). hGH was present in milk obtained after milking or after the incubation of the mammary glands at O'C. In both cases, the hormone was present in essentially similar concentration, undegraded and biologically active (as judged by its prolactin‐like activity). The method depicted here is very simple and can be applied easily to many mice. Its major limitation is that it implies the breeding and the sacrifice of a relatively large number of animals. One gram of crude recombinant protein can be virtually obtained in this way with about 200 lactating mice from their milk containing the proteins at the concentration of 3‐4 mg/ml. The milk of transgenic mice can therefore be considered as a practical source of recombinant proteins for biochemical and pharmaceutical studies.     

Identification, partial purification and biochemical characterization of gamma-glutamyltranspeptidase present as a membrane component in skimmed milk and milk fat-globule membranes, and in mammary-tumour virus from the milk of infected mice.

The enzyme gamma-glutamyltranspeptidase was reproducibly found to be associated with mouse milk particles; it is present in milk fat-globule membranes and mouse mammary-tumour virus of infected Swiss mice, also in particles from the milk of uninfected mice. The enzymatic activities observed range among the highest reported for mammalian tissues. The enzyme was partially purified from mouse mammary-tumour virus, and from milk fat-globule membranes. The molecule requires the presence of detergents to remain soluble, behaves as a high molecular weight component, properties characterizing integral membrane proteins. Kinetics, and the effect of competitors as well as of specific inhibitors show this enzyme to be identical to the well-known kidney gamma-glutamyltranspeptidase ((gamma-glutamyl)-peptide:amino-acid gamma-glutamyltransferase, EC 2.3.2.2). Other oncornaviruses budding from cultured cells originally expressing the enzyme in their plasma membrane also incorporate the enzyme in their structure.     

The molecular manipulation of milk composition

The introduction of cloned genes into the mouse germ line is now routine. Although more difficult technically, gene transfer has been accomplished in farm animals and offers the potential for genetic improvement. In this regard, we have been investigating the use of transgenic animals as production vehicles for high value proteins in milk. We have shown that DNA sequences derived from the gene encoding sheep beta-lactoglobulin mediate efficient and specific expression in the mammary gland. A fusion gene comprising beta-lactoglobulin sequences and those encoding antihemophilic human factor IX has been constructed. This construct has been introduced into sheep; it is expressed in the mammary gland, and the corresponding protein is secreted into milk.     

Observations on the effect of raw milk quality on the keeping quality of pasteurized milk

Raw milk was stored for up to 14 d at 4°C and pasteurized on days 1, 3, 4, 7, 9 and 14. Precautions were taken to eliminate post-pasteurization contamination. The pasteurized milks were stored at 4°C and analysed at weekly intervals for standard plate counts (SPC), psychrotrophic counts (PC) and aerobic spore counts (ASC). The initial raw milk quality was very good and the keeping quality of all the pasteurized milks tested was greater than 22 d. In some cases the milk still had acceptable SPC after 42 d storage, which shows the keeping quality that can be achieved when the process is well controlled. However, the best keeping quality resulted from milk pasteurized on the third and fourth days. Even milk pasteurized on the seventh and ninth had superior keeping quality to that pasteurized on the first day. The lactoperoxidase anti-microbial system in raw milk may be most active around days 3 and 4.     

Bayesian segregation analysis of milk flow in Swiss dairy cattle using Gibbs sampling

Segregation analyses with Gibbs sampling were applied to investigate the mode of inheritance and to estimate the genetic parameters of milk flow of Swiss dairy cattle. The data consisted of 204 397, 655 989 and 40 242 lactation records of milk flow in Brown Swiss, Simmental and Holstein cattle, respectively (4 to 22 years). Separate genetic analyses of first and multiple lactations were carried out for each breed. The results show that genetic parameters especially polygenic variance and heritability of milk flow in the first lactation were very similar under both mixed inheritance (polygenes + major gene) and polygenic models. Segregation analyses yielded very low major gene variances which favour the polygenic determinism of milk flow. Heritabilities and repeatabilities of milk flow in both Brown Swiss and Simmental were high (0.44 to 0.48 and 0.54 to 0.59, respectively). The heritability of milk flow based on scores of milking ability in Holstein was intermediate (0.25). Variance components and heritabilities in the first lactation were slightly larger than those estimates for multiple lactations. The results suggest that milk flow (the quantity of milk per minute of milking) is a relevant measurement to characterise the cows milking ability which is a good candidate trait to be evaluated for a possible inclusion in the selection objectives in dairy cattle.     

Relationship between milk composition and pup's growth in mice

The effect of lactation numbers (first, second, and third) on milk composition and the relationship between milk composition and pup's growth were examined in four strains of mice (SHN, SLN, C3H/He, and GR/A). Although the percentage of milk fat and moisture increased and decreased, respectively, through the first to third lactations in C3H/He mice, no differences in milk composition among lactation numbers were observed in the other mouse strains. In pooled data derived from the four strains of mice, there was no significant correlation between milk composition and body weight or growth rate of pups on Day 12 or 20 of the first lactation. In contrast, the amount of milk fat and moisture was the positively and negatively, respectively, correlated with the pup's growth parameters during the second and third lactations. This was reflected by a lower fat content and a higher moisture content in the milk of GR/A mice, especially evident during the second and third lactations and reduced pup's growth, when compared with the other mouse strains. Milk lactose and ash content was not correlated with pup's growth parameters. The results of our study stress the importance of milk fat for the growth of mouse pups.     

The composition of African lion (Panthera leo) milk collected a few days postpartum

There is very little information on the composition of lion milk. Milk was obtained from all four teats of two African lionesses (Panthera leo) while they were temporarily immobilized; the first lioness was 40 hours postpartum and the second lioness was 10 days postpartum. The lion's milk obtained from the two lionesses in this study contained less solids than reported in literature. The milk of the two females contained, respectively, 192.2 and 166.9 g dry matter/kg milk; 60.2 and 84.6 g protein/kg milk; 113.6 and 136.9 g fat/kg milk; and 68.6 and 77.9 g fat free dry matter/kg milk. Carbohydrate content, analysed only for the first lioness, was 26.5 g lactose/kg milk. These values are much lower than those cited in literature. Electrophoresis and identification of protein bands showed a similar pattern of proteins as seen in cow's milk, but with caseins of less negative charge and whey proteins of smaller molecular size. The milk fat content of the second lioness was higher and varied considerably more among teats. The lipid fraction of lion's milk is characterized by a high content of saturated (palmitic and stearic) and mono-unsaturated (palmitoleic and oleic) fatty acids. Regarding fatty acid composition, lion milk closely resembles human milk. Animal fat plays an important role in the nutrition of African lions. The high fat content of lion milk suggests that the dependency by large predators such as lions on dietary fat is present from birth.     

The presence of cellular metabolites in milk

Cellular metabolites are present in goats' milk. The concentrations of UDPgalactose and some nucleotides were higher in milk than in mammary tissue. Other metabolites were present in milk at similar concentrations to those found in the mammary gland. It is proposed that the concentrations of these metabolites in milk reflect their Golgi-vesicular and cytosolic concentrations.     

The lipid composition of milk from mice fed high or low fat diets.

Total fatty acids and the proportions of methyl esters of individual fatty acids were measured in mouse milk. Pregnant mice were fed either a high fat (HF) diet or a low fat (LF) diet from 14 days of gestation. After parturition, each dam was milked once a day for a period of 18 days. The mean total fatty acid concentration over the entire study period was 110 mg/g of milk (approximately 11.7% fat as triglyceride) for both dietary treatment groups. During days 2 to 6 postpartum, the mean total fatty acid concentration for dams fed HF diet was lower than for the LF group. Although the concentration of total fatty acids of mouse milk was not affected by the level of dietary fat fed to the dam, several variations in the proportions of individual fatty acids were observed.     

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

Feeding low-fat milk during infancy

Despite the widespread agreement that low-fat milk should not be used during infancy, there is a sizable portion of infants in the United States who were fed a diet that included low-fat milk (less than or equal to 2% fat). In 1985, 14% of infants 8 months old, 20% of infants 10 months old, and 32% of infants 12 months old were fed low-fat milk. The reasons given most often by mothers for low-fat milk use was their consideration that low-fat milk has less fat than whole cow's milk and that low-fat milk use was recommended/suggested by their physician. Nutrient intakes of infants fed low-fat milk are compared to those of infants fed whole cow's milk and infant formula. Except for fat, nutrient intakes of infants fed low-fat milk or whole cow's milk were similar. A majority of infants fed either low-fat milk or whole cow's milk received amounts of sodium, potassium, and chloride that exceeded the recommended safe and adequate ranges and amounts of iron below the RDA. These data are considered in relation to dietary requirements during infancy.     

Copeptin,adropin and irisin concentrations in breast milk and plasma of healthy women and those with gestational diabetes mellitus

Copeptin, adropin and irisin are polypeptide hormones implicated in energy homostasis and diabetes. The purposes of this study were (1) to compare the copeptin, adropin and irisin concentrations between colostrum, transitional and mature milk and plasma in lactating women with and without GDM and (2) to compare these values with those from non-lactating women. Venous blood samples were obtained before suckling from 15 healthy lactating women aged 26–30 years, 15 lactating women with GDM aged 26–32 years, and 14 age-matched controls aged 25–31 years. Colostrum, transitional milk and mature milk samples were collected just before suckling. The concentration of copeptin was determined by EIA while the concentrations of adropin and irisin were determined by ELISA. The levels of copeptin, adropin and irisin in the colostrum were significantly higher than those in transitional and mature milk samples from healthy women; also, transitional milk had higher copeptin, adropin and irisin concentrations than mature milk. The amounts of copeptin in the colostrum and transitional milk were significantly higher than in mature milk samples from women with GDM, while the amounts of adropin and irisin were significantly lower. The relative concentrations of copeptin, adropin and irisin in the plasma samples from these groups of women were similar to those in the colostrum, transitional and mature milk samples, but the latter concentrations were higher than those in the plasma. These peptides could influence the regulation of metabolic pathways and the postnatal growth and development of different organs in the newborn.     

Composition of the hydrocarbon fraction of goats' milk

The hydrocarbon fraction of the neutral lipids of goats' milk was chromatographically purified and analyzed by gas-liquid chromatography and mass spectrometry. The goats' milk samples, which were collected during the spring of the year, represent a cross-sectional analysis; the purified hydrocarbon fraction displays a broad spectrum of compounds. The major components of the hydrocarbon fraction identified for the first time in goats' milk were 3,7,11,15-tetramethylhexadec-2-ene (phytene-2) (1.5%), squalene (approximately 2.5%), and n-C29H60 (4.2%); in addition, a series of odd and even carbon number n-alkanes (C15 to C33), a series of alkenes (C16 to C23), and a series of branched chain hydrocarbons were found. The goats' milk hydrocarbon fraction, in comparison to the known distribution from cows' milk, contains a good deal less squalene and phytene, and is more complex. One human milk hydrocarbon fraction isolated from a longitudinal composite sample from one lactation displays a distribution that appears to be more closely related to that of human skin lipids (1983. J. Lipid Res. 24: 120-130) than to those of goats' and cows' milk.     

Androstenedione and testosterone concentrations in plasma and milk of the cow throughout pregnancy

Androstenedione concentrations in both plasma and milk were higher than those of testosterone throughout pregnancy. Testosterone concentration increased during pregnancy; the levels in milk were always lower than in plasma. Androstenedione concentrations increased in plasma and milk during pregnancy but the values were twice as high in milk as in plasma after Day 90. This change in androstenedione distribution suggests that the mammary gland could be active in the uptake of androstenedione from plasma to milk or that the mammary gland itself might synthesize this hormone.