Xanthine oxidase,an indicator of secretory differentiation in mammary cells

Elevated levels of xanthine oxidase were found in (1) lactating mouse mammary glands, compared with virgin and midpregnant glands; and (2) primary mouse mammary cells cultured on floating collagen gels, compared with non-secretory cells on attached gels. In primary culture, increase in xanthine oxidase activity above a basal level coincided with secretory activity as measured by casein production; intracellular levels of casein and xanthine oxidase showed a high degree of correspondence. It is suggested that xanthine oxidase levels can be used as an indicator of in vivo and in vitro secretory differentiation in mammary epithelial cells.     

The effect of pre-partum milking on the transfer of immunoglobulin into mammary secretion of cows.

The significance of local effects associated with mammary involution on transfer of immunoglobulin and especially on the selective transfer of IgG1 into mammary secretion of cows approaching parturition has been determined. This was carried out by measuring the changes in the concentration of IgG1, IgG2, IgM and IgA in serum and mammary secretion of 5 cows in which two mammary glands were milked continuously (twice daily) during the period preceding parturition, while the other two glands were allowed to undergo normal involution. In the secretion of unmilked glands of all cows there was a substantial increase in the concentration of IgG1 as cows approached parturition. In contrast, the increases in the concentration of IgG1 and in the selective index for IgG1 of milked glands were either virtually non-existent (1 cow) or generally reduced in magnitude and delayed in time of onset (4 cows). It is clear from the results that continued milking of a mammary gland throughout pregnancy tends to maintain milk production in the milked gland and at the same time reduces the massive selective transfer of IgG1 into secretion of that gland.     

Casein-derived phosphopeptides disrupt tight junction integrity,and precipitously dry up milk secretion in goats

Mammary involution is triggered by local stimuli, but the precise mechanism has not been defined. Milk stasis accumulate local signals, which makes the tight junctions (TJ) leaky. The aim of the study was to check the hypothesis that casein hydrolyzates (CNH) compromise TJ integrity and dry up milk secretion. A single dose of CNH transiently (12 to 24 h) compromised TJ integrity in the treatedudder. This was associated by a transient (12 to 96 h) decline in milk secretion. No such changes were recorded in the contralateral gland that served as a control. Four repeated doses of CNH after each milking caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion within 96 h. No such changes were recorded in goats treated with de-phosphorylated casein (control). We conclude that CNH are the milk-borne factors that cause the disruption of TJ integrity and induction of involution, and that the serine-Ps in the CNHs are essential for the excretion of biological activity.     

The effect of immune antibodies and the xanthine oxidase-xanthine enzymatic link on Vibrio cholerae

As revealed in experiments on V. cholerae, highly diluted cholera antiserum enhanced the inhibitory action of the enzymatic link xanthine oxidase-xanthine-Fe2+ on the multiplication of V. cholerae, while low dilutions of the antiserum weakened this action. Normal rabbit serum produced no such effect. The antivibrionic effectiveness of the immune molecular cycle, viz. antiserum--the xanthine oxidase enzymatic link, was found to depend also on the concentration of xanthine. Immune antibodies to cholera antigens activated the bacteriostatic action of the enzymatic link at the concentration of xanthine oxidase equal to 0.0125 g/l and its bactericidal action at the concentration of xanthine oxidase equal to 0.025 g/l. In this article the values of the specificity indices of immune interaction and immunological effectiveness, characterizing the effectiveness of immune molecular cycles (antibodies--the xanthine oxidase enzymatic link), are presented.     

Regulation and functional relevance of milk fat globules and their components in the mammary gland

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.     

Action of the lactoperoxidase system on Salmonellae and Shigellae

In vitro experiments have demonstrated that the lactoperoxidase system produces a bactericidal effect on salmonellae and shigellae. The physiological concentrations of the components of this system, making it possible to obtain a pronounced bactericidal effect, have been established. Lactobacilli have been shown to potentiate the effect of the lactoperoxidase system. The possibility of realizing the bactericidal properties of the lactoperoxidase system with respect to salmonellae and shigellae in the preparations of immune lactosera, intended for passive enteral immunization against intestinal infections, has been suggested.     

Regulation of casein messenger RNA during the development of the rat mammary gland.

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion.     

Bactericidal Activity of Lachrymal Secretion and Complement System in Copper Deficient Bovines

Copper deficiency in humans and animals has been related to increased susceptibility to infections. Neutrophils are one of the most studied components of the immune response; however, to the best of our knowledge, other defenses of the innate immune system have not been analyzed in copper-deficient animals. Our previous studies in copper-deficient bovines have shown increased susceptibility to infectious keratoconjunctivitis, an ocular disease caused by Moraxella bovis. The objective of this work was to evaluate the bactericidal activity of lachrymal secretion and complement system, two main mechanisms of the innate immune response against M. bovis, in copper-deficient cattle. Our results indicate that copper deficiency has no effect on bactericidal activity of complement system and lachrymal secretion against M. bovis in calves. Other components of local and systemic ocular defense mechanisms that could explain the increased susceptibility to infectious keratoconjunctivitis observed in copper-deficient bovines should be investigated.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrate reduction: evaluation of its role in nitrite and nitric oxide generation in anoxic tissues

In addition to nitric oxide (NO) generation from specific NO synthases, NO is also formed during anoxia from nitrite reduction, and xanthine oxidase (XO) catalyzes this process. While in tissues and blood high nitrate levels are present, questions remain regarding whether nitrate is also a source of NO and if XO-mediated nitrate reduction can be an important source of NO in biological systems. To characterize the kinetics, magnitude, and mechanism of XO-mediated nitrate reduction under anaerobic conditions, EPR, chemiluminescence NO-analyzer, and NO-electrode studies were performed. Typical XO reducing substrates, xanthine, NADH, and 2,3-dihydroxybenz-aldehyde, triggered nitrate reduction to nitrite and NO. The rate of nitrite production followed Michaelis-Menten kinetics, while NO generation rates increased linearly following the accumulation of nitrite, suggesting stepwise-reduction of nitrate to nitrite then to NO. The molybdenum-binding XO inhibitor, oxypurinol, inhibited both nitrite and NO production, indicating that nitrate reduction occurs at the molybdenum site. At higher xanthine concentrations, partial inhibition was seen, suggesting formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. The pH dependence of nitrite and NO formation indicate that XO-mediated nitrate reduction occurs via an acid-catalyzed mechanism. With conditions occurring during ischemia, myocardial xanthine oxidoreductase and nitrate levels were determined to generate up to 20 microM nitrite within 10-20 min that can be further reduced to NO with rates comparable to those of maximally activated NOS. Thus, XOR catalyzed nitrate reduction to nitrite and NO occurs and can be an important source of NO production in ischemic tissues.     

The onset of mammary extraction of plasma triglycerides and circulating levels of progesterone during lactogenesis in the cow and goat

In untreated cows and goats, the onset of mammary extraction of plasma triglyceride was sometimes detected several hours before parturition, prior to removal of secretion from the mammary glands by offspring and when circulating progesterone concentrations were low. In cows and goats that had secretion regularly removed from the mammary glands late in gestation, the onset of triglyceride extraction occurred up to several days before parturition, when circulating progesterone concentrations were moderately high (up to 3.9 ng/ml in the cow and 6.2 ng/ml in the goat).     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

Summary The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion. This investigation was supported by Grant No. CA 05388 awarded by the National Cancer Institute, DHEW, and by Cancer Research Funds of the University of California.     

Light and electron microscopy of cells in pig colostrum, milk and involution secretion

Cells in pig colostrum, milk and involution secretion were identified using light and electron microscopy. Cell types identified were neutrophils, macrophages, epithelial cells, eosinophils and lymphocytes. The neutrophils predominated in colostrum and involution secretion, whereas in milk it was the epithelial cell. Macrophages and lymphocytes were present throughout lactation and so too were eosinophils which were always present in lower concentrations. Both neutrophils and macrophages were seen with phagocytic vacuoles containing either lipid, casein or cellular debris. The possible roles played by the phagocytic and lymphoid cells in the protection of the mammary gland of the sow and the gut of the neonate from pathogenic microorganisms is discussed.     

The reactions of horseradish peroxidase, lactoperoxidase, and myeloperoxidase with enzymatically generated superoxide

The formation and decay of intermediate compounds of horseradish peroxidase, lactoperoxidase, and myeloperoxidase formed in the presence of the superoxide/hydrogen peroxide-generating xanthine/xanthine oxidase system has been studied by observation of spectral changes in both the Soret and visible spectral regions and both on millisecond and second time scales. It is tentatively concluded that in all cases compound III is formed in a two-step reaction of native enzyme with superoxide. The presence of superoxide dismutase completely inhibited compound III formation; the presence of catalase had no effect on the process. Spectral data which indicate differences in the decay of horseradish peroxidase compound III back to the native state in comparison with compounds III of lactoperoxidase and myeloperoxidase are also presented.     

Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows’ mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down‐regulation and location of bta‐miR‐26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real‐time PCR, in situ hybridization and dual‐luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta‐miR‐26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage.     

All three promoters of the acetyl-coenzyme A-carboxylase alpha-encoding gene are expressed in mammary epithelial cells of ruminants.

The activity of the enzyme acetyl-CoA-carboxylase alpha (ACC-alpha) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PI-PIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-alpha-related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-alpha synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution.     

SYNTHESIS OF LACTOFERRIN AND CASEIN BY EXPLANTS OF BOVINE MAMMARY TISSUE

Synthesis of lactoferrin and casein by the bovine mammary gland was determined in an experimental model where lactation was maintained in one mammary half, while involution was induced in the contralateral half. Culture of explants with prolactin had no consistent effect on synthesis of casein or lactoferrin in tissue from either mammary half. Endotoxin and tumor necrosis factor-α generally decreased synthesis of casein and lactoferrin, suggesting that these inflammatory mediators are not directly responsible for increasing lactoferrin synthesis during mammary inflammation or involution. Synthesis of lactoferrin was increased and casein decreased in the involuting mammary half vs. the lactating half. These results suggest that local factors in the mammary gland play a role in the regulation of lactoferrin synthesis during involution.     

A note on the inhibition of Listeria monocytogenes NCTC 11994 in milk by an activated lactoperoxidase system

A lactoperoxidase system activated by glucose oxidase was bacteriostatic to Listeria monocytogenes inoculated into UHT milk supplemented with glucose. The incorporation of urea peroxide as an additional hydrogen peroxide-generating agent did not enhance the inhibitory effect.     

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.