Peptides generated from milk proteins in the bovine mammary gland during involution

OBJECTIVE: To determine if the interaction between isoniazid and hydralazine, consisting of increased hypotension accompanied by bradycardia, occurs with other vasodilators. METHODS: Blood pressure and heart rate responses to a number of vasodilators were determined in rats under chloralose-urethane, pretreated or not with 250 mg/kg of isoniazid. The influence of this dose of isoniazid on GABA levels in the hypothalamus and pons-medulla was assessed in other groups of rats. RESULTS: Increased hypotension and bradycardia following i.p. isoniazid were observed with dipyridamole, prazosin, pinacidil and hydralazine given i.v. Bradycardia without increased hypotension appeared with papaverine and verapamil, while increased hypotension with unchanged heart rate was observed with minoxidil and captopril. Isoniazid decreased GABA in the hypothalamus and pons-medulla. CONCLUSIONS: At the high dose used, isoniazid interacts with various vasodilators, irrespective of their mechanism of action. The interaction could be due to the influence of the drug on GABA levels at cardiovascular regulatory sites.     

Programmed cell death in bovine mammary tissue during lactation and involution

Cessation of milk removal causes mammary tissue involution, which in rodents is characterized by extensive tissue degeneration and loss of the majority of luminal epithelial cells by apoptosis. In contrast, bovine mammary tissue shows little histological evidence of tissue remodelling between lactations. In this study, we combined histology with molecular biology to examine the cellular and molecular changes in bovine mammary tissue on cessation of milking. Oligonucleosomal laddering of genomic DNA extracted from lactating tissue indicated that a proportion of cells were dying by apoptosis. This was confirmed by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labelling of apoptotic cells in lactating tissue sections (TUNEL). One week after cessation of milking, alpha-lactalbumin and alpha S1-casein messenger RNA (mRNA) abundance had decreased by 99 and 85%, respectively, whereas lactoferrin mRNA had increased 20-fold. Drying off was also accompanied by an increase in oligonucleosomal laddering of genomic DNA, and by an increase in the proportion of TUNEL-positive cells, which were localized preferentially in regions where alveolar structure had deteriorated. Therefore, termination of lactation was associated with partial loss of the mammary cell population and dedifferentiation of the remainder.     

60K gelatinase involved in mammary gland involution is regulated by beta-oestradiol

Imperfect reference standards (alloyed standards) often hinder evaluation of new diagnostic tests. Discrepant analysis, a two-stage modification of the standard evaluation of diagnostic tests, has been used to circumvent this problem. In discrepant analysis, additional testing is performed to resolve discrepant results of the new diagnostic test and the alloyed standard. This article demonstrates algebraically that sensitivity and specificity will be overestimated by discrepant analysis, even when a perfect gold standard is used to resolve the discrepant results. The magnitude of the bias depends on the true sensitivity and specificity of the new test and initial alloyed standard, the prevalence of disease in the study population, and the proportion of concordant errors between the two tests. The article also demonstrates substantial bias associated with the use of alloyed standard tests in both stages of discrepant analysis. This procedure should not be used routinely for evaluation of diagnostic tests.     

Environmental streptococcal intramammary infections of the bovine mammary gland

Characteristics of environmental streptococcal IMI were investigated over a 7-yr period for a herd in total confinement. A total of 374 new environmental streptococcal IMI was detected. Approximately 50.5% of IMI were new in the dry period, and 49.5% were new in lactation. The rate of new IMI was .00312 IMI/cow day during the dry period and .00054 IMI/cow day during lactation. The percentages of cows and quarters with an environmental streptococcal IMI present at calving were 10.6 and 3.2%, respectively. The percentage of heifers with an environmental streptococcal IMI at calving was similar to that for cows. The rate of new environmental streptococcal IMI was greater during the 1st mo of lactation than during the remainder of lactation. The rate of IMI during late lactation was higher for older cows than for either heifers or cows in second lactation. The rate of environmental streptococcal IMI during the dry period and during lactation was greatest during summer. The mean days of lactation that cows were infected for all IMI was 12.3 d. Approximately 41% of IMI had a duration of < 8 d. Stage of lactation, season of the year, and parity influenced the rate of new IMI.     

Distribution of annexins I, II, and IV in bovine mammary gland

Annexins belong to a family of proteins that are characterized by their ability to bind phospholipids in a Ca(2+)-dependent manner that is thought to be involved in a variety of biological processes. The present study determined the localization of annexins in subcellular fractions, nuclei in particular, of cow mammary gland by immunoblot analysis using monoclonal antibodies to annexins I, II, IV, and VI. The analysis revealed that annexins I, II, and IV were present in cytosol, but VI was not. Annexins I and IV were found in the nuclear fraction, but annexin II was only faintly present. Annexin VI was also undetectable in this fraction. Cytosolic annexin I had a molecular mass of 36 kDa. The 36-kDa annexin I was also found in the nuclear fraction. A 38-kDa annexin I was additionally detected in nuclei. The cytosolic and nuclear 36-kDa annexin I and the nuclear 38-kDa annexin I showed different isoelectric points, as revealed by two-dimensional PAGE. Annexin IV from cytosolic and nuclear fractions had similar molecular masses and isoelectric points.     

Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland

The involvement of glycoprotein CD36 and fatty-acid-binding protein (FABP) in cellular growth, differentiation, lipid transport and metabolism led us to examine the possible biochemical and physiological relationship(s) between these two proteins. We investigated three aspects of this relationship. We first attempted to identify any physical complex formed between CD36 and FABP in bovine milk fat globule membranes. These membranes are the product of mammary gland secretory epithelial cells. The second aspect studied was the effect of synthetic peptide analogs to the C-terminus (amino acid residues 121-131) of bovine mammary gland FABP on cell proliferation, as a result of the interaction of these peptides with the ectodomain of CD36. Finally, mammary gland CD36 and FABP coexpression was defined at different stages of lactation and during involution. Immunoprecipitation, Western immunoblotting with anti-FABP and anti-CD36, Northern-blot analysis and a mammary epithelial cell proliferation assay demonstrated that: (a) bovine milk fat globule membranes contain the complex of CD36 and FABP, and that this complex is, most likely, formed as a result of FABP binding to the cytoplasmic segments of CD36; (b) synthetic analog of the C-terminus of FABP with the sequence Val-Thr-Cys, identical to the sequence found in the CD36-binding domain of thrombospondin, was a more potent inhibitor of bovine mammary gland epithelial cell proliferation than a synthetic peptide with the Val-Cys-Thr sequence; (c) the expression of FABP and CD36 is related to the state of mammary cell differentiation, since it reaches its maximum during lactation and declines during the involutionary period.     

Antibiotic properties of bovine lactoferrin on Helicobacter pylori

To investigate a potential new treatment for gastric Helicobacter pylori infection, we have examined the use of the natural antibiotic lactoferrin, found in bovine milk, for activity against Helicobacter species both in vitro and in vivo. Lactoferrin was bacteriostatic to H. pylori when cultured at concentrations > or =0.5 mg/ml. Growth of H. pylori was not inhibited by another milk constituent, lysozyme, or by a metabolite of lactoferrin, lactoferricin B, but growth was inhibited by the iron chelator deferoxamine mesylate. Lactoferrin inhibition of growth could be reversed by addition of excess iron to the medium. Lactoferrin in retail dairy milk was found to be more stable intragastrically than unbuffered, purified lactoferrin. Treatment of H. felis-infected mice with lactoferrin partially reversed mucosal disease manifestations. It is concluded that bovine lactoferrin has significant antimicrobial activity against Helicobacter species in vitro and in vivo. Bovine lactoferrin should be further investigated for possible use in H. pylori infections in man.     

Functional receptors for atrial natriuretic peptide in the rat mammary gland during lactation

The present study was undertaken: 1) to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat mammary gland; and 2) to elucidate ANP-induced cellular formation of cyclic GMP (cGMP) and alterations in alveolar morphology during both early and late lactation. Receptor autoradiography, employing rat-specific [125I]ANP as radioligand, demonstrated binding sites in the secretory tissue and larger blood vessels of the mammary gland. Binding of [125I]rANP to membrane fractions was completely displaced by unlabeled ANP and brain natriuretic peptide. C-type natriuretic peptide and cANP(4-23) revealed limited competition with radiolabeled ANP only during early lactation, indicating a more heterogeneous receptor population at that time. Systemically administered ANP induced cGMP formation in the alveolar epithelium, as shown with immunohistochemistry, and increased mammary tissue cGMP concentrations in vivo throughout the lactation period. Image analysis revealed enlargement of alveolar (but not epithelial) cell area after ANP stimulation in late lactation, suggesting altered alveolar filling or myoepithelial cell relaxation. These results indicate that ANP induces biological effects in the rat mammary gland through specific ANP-A receptor interaction with subsequent intracellular cGMP formation. ANP may therefore play a regulatory role in the control of mammary gland blood supply and secretory function.     

Properties of a heparin-binding peptide derived from bovine lactoferrin

A heparin-binding peptide was isolated from a proteolytic hydrolysate of bovine lactoferrin by affinity chromatography using an immobilized heparin column. Analysis of amino acid sequences at the N-terminus showed that this heparin-binding peptide is derived from the region beginning at the 17th amino acid residue of the bovine lactoferrin sequence. The molecular mass of this peptide was 3195.5 as measured by matrix-assisted laser desorption-time of flight mass spectrometry. This peptide is the same as the bactericidal peptide lactoferricin B. In an aqueous environment, this peptide displays mainly a beta-sheet structure and an unordered structure as assessed by measurements of circular dichroism spectra. When this peptide was mixed with heparin, a distinct spectral change was induced because of conformational alteration of the peptide. This spectral change was reversible. Analysis of data from peptide synthesis indicated that binding by the sequence Arg28-Met29-Lys30-Lys31 of bovine lactoferrin is significant and that there is a synergistic contribution from Lys18-Cys19-Arg20-Arg21, and Arg38-Arg39.     

Delayed involution of the mammary epithelium in BALB/c-p53null mice

In mammals, weaning of neonates and subsequent milk stasis initiates removal of the secretory epithelium of the mammary gland by apoptosis. The p53 tumor suppressor gene is induced rapidly following weaning of neonates, but its role in the process of involution has not been defined. Therefore, experiments were performed to identify the cell types in which the p53 gene is expressed during involution and determine the consequences of its absence in BALB/c-p53null mice. Both p53 mRNA and protein were detected in the mammary epithelium within 48 h following weaning and resulted in an eightfold increase in levels of p21WAF1 mRNA. Induction of p21WAF1 mRNA was absent in BALB/c-p53null mice, and therefore, was shown to be p53-dependent. The BALB/c-p53null mice exhibited delayed involution of the mammary epithelium, as measured by 60% greater epithelial area compared to BALB/c-p53(wt) mice through 5 days post-weaning. The delay was transient with no differences being apparent at 7 days post-weaning. Expression of the stromal protease stromelysin-1 was unaffected by the absence of p53 suggesting that stromal responses were intact. These data demonstrate that p53 participates in the first stage of involution initiated by the epithelium itself, but does not affect the second phase during which stromal proteases are induced.     

Nutritional control of amino acid supply to the mammary gland during lactation in the pig

The paucity of data relating to lactation physiology of the sow has frustrated researchers in estimating nutrient needs for production and mammary maintenance functions. The nutritional control of amino acid supply for milk synthesis is influenced by factors that have yet to be measured, such as blood flow and amino acid contribution from the body protein pool. The interaction or role of hormones such as insulin, glucagon or prolactin in amino acid dynamics and inter-organ exchange during lactation in the sow, are not well understood. The discrepancy existing between milk and mammary amino acid uptake profiles relative to lysine may be indicative of mammary metabolism and possibly maintenance requirements for specific amino acids. Hence, amino acid metabolism in the mammary gland, regardless of arterial blood substrate supply, may play an important role in a factorial approach to determining requirements. Mammary amino acid uptake ratios rather than milk amino acid ratios should provide a better tool to estimate amino acid requirements relative to lysine. Although lysine has typically been limiting in maize-soyabean-meal-based diets fed to lactating sows, current production trends are bringing a new dimension to the formulation of lactating-sow diets. Other amino acids may become limiting if dietary crystalline lysine is added without concern for the whole essential amino acid profile. Formulations based on an ideal amino acid profile for the lactating sow will, therefore, become critical.     

Three-dimensional structure of diferric bovine lactoferrin at 2.8 A resolution

The three-dimensional structure of diferric bovine lactoferrin (bLf) has been determined by X-ray crystallography in order to investigate the factors that influence iron binding and release by transferrins. The structure was solved by molecular replacement, using the coordinates of diferric human lactoferrin (hLf) as a search model, and was refined with data to 2.8 A resolution by simulated annealing (X-PLOR) and restrained least squares (TNT). The final model comprises 5310 protein atoms (residues 5 to 689), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), 2 Fe3+, 2 CO32- and 50 water molecules. This model gives an R-factor of 0.232 for 21440 reflections in the resolution range 30.0 to 2.8 A. The folding of the bLf molecule is essentially the same as that of hLf, but bLf differs in the extent of closure of the two domains of each lobe, and in the relative orientations of the two lobes. Differences in domain closure are attributed to amino acid changes in the interface, and differences in lobe orientations to slightly altered packing of two hydrophobic patches between the lobes. Changed interdomain interactions may explain the lesser iron affinity of bLf, compared with hLf, and two lysine residues behind the N-lobe iron site of bLf offer new insights into the "dilysine trigger" mechanism proposed for iron release by transferrins. The bLf structure is also notable for several well-defined oligosaccharide units which demonstrate the structural factors that stabilise carbohydrate structure. One glycan chain, attached to Asn545, appears to contribute to interdomain interactions and may modulate iron release from the C-lobe.     

Osteopontin expression in mammary gland development and tumorigenesis

Osteopontin (OPN) is a secreted phosphoprotein that binds to cells via an Arg-Gly-Asp sequence and to mineralized surfaces. The protein can mediate cell adhesion and is strongly implicated in transformation and tumorigenesis. We have examined the expression pattern of OPN in mouse mammary glands at different stages of postnatal development. Whereas OPN is expressed at low-to-moderate levels in mammary glands from virgin and pregnant mice, the levels of OPN mRNA are extremely high in the lactating gland, consistent with the presence of the protein in milk. Expression is highest at 2 days of lactation and declines thereafter, but it remains high through involution. OPN expression is restricted to small nests or groups of cells at 9 days of involution. These results suggest that OPN may play a specific role in the process of involution that may be distinct from its role during lactation. In mammary tumors arising spontaneously in transgenic mice expressing the oncogenes c-myc and/or v-Ha-ras under the control of the mouse mammary tumor virus promoter, the level of OPN expression is increased dramatically over that in the normal gland in these same animals. Numerous cells expressing OPN mRNA are widespread throughout the tumors. OPN protein is detectable by Western blotting in extracts from the mammary gland at 2 days of lactation and from the tumors, but not in mammary glands at other stages of development. We hypothesize that OPN is exported from most tissues and that the protein is only detectable in tissues elaborating fluids, such as the lactating mammary gland, or in pathological situations when expression of OPN is abnormally high, such as in tumors.     

Lymphocyte subpopulations in the mammary gland of the goat

Mammary glands of pregnant, lactating and resting goats were studied by immunohistochemistry for lymphocyte subpopulations using a panel of monoclonal antibodies. All T lymphocyte subpopulations that may have a role in the immune response, CD2+, CD4+, CD8+ and gamma delta T cells and subsets, were present in the mammary gland and were noted to increase in number progressively during pregnancy, decrease significantly during lactation, and then moderately increase during the resting period. CD4+ cells, the predominant cell type in the mammary gland, were located mainly in the connective tissue, whereas CD2+, CD8+ and TcR1-N24+ cells were predominant in the intraepithelial areas. TcR1-N6+ cells were detected almost exclusively during pregnancy, being localized mainly in the connective tissue. Their proportion decreased markedly following parturition. Very few WC1-N3+ and -N4+ cells were detected in the mammary gland. It is suggested that the majority of gamma delta T lymphocytes in the mammary gland of the goat are CD2+ CD8+ WCl-, a distinctive subset from that of the WCl+ subset in peripheral blood.     

Application of a mechanistic model to study competitive inhibition of amino acid uptake by the lactating bovine mammary gland

A mathematical model is used to describe uptake by a countertransport system and subsequent flow of three amino acids (AA), Phe, Val, and Met, from arterial blood to milk protein in the mammary gland of a lactating cow. The model suggests that total uptake of all AA is higher than net uptake and that a large proportion of the incoming AA is released from the cell directly back to blood. The model is used to predict which of the three AA is limiting the rate of milk protein synthesis and the response to increased arterial concentration of the first-limiting AA. Simulations are performed to predict possible outcomes of several experimental protocols to AA infusion, which might be used to test in vivo the responsiveness of the bovine mammary gland to an altered arterial concentration of AA. Of the three AA considered, arterial Met concentration appears to be first-limiting. The infusion profile that gives the greatest response in milk protein synthesis rate alters the arterial profile of AA such that it is identical to that of proteins originating in the mammary gland. Model construction can be simplified by acknowledging normal biological constraints.     

Characterization of the protein and glycan moieties in different forms of bovine lactoferrin

The BrCN cleavage of lactoferrin-a or -b (LF-a or LF-b) led to the observation of four fragments by SDS-PAGE, whose molecular masses were 77, 58, 52, and 30 kDas, or 74, 54, 47, and 30 kDas, respectively. N-Terminal amino acid sequence analyses show that the sequences of 58, 52, and 30 kDa fragments (residues 64-471, 130-471, and 472-689) of LF-a coincide with those of the 54, 47, and 30 kDa fragments of LF-b. respectively. All these fragments, which were positive by PAS staining, were not stained after being treated with glycopeptidase F. This treatment changed the 58 and 52 kDa fragments of LF-a to the 54 and 47 kDa fragments, respectively, whose molecular masses were the same as those of the treated fragments of LF-b. The 58 and 52 kDa fragments of LF-a bound to the lectin, Ricinus communis agglutinin, while the 54 and 47 kDa fragments of LF-b hardly bound to it.     

Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats

The influence of bovine lactoferrin (bLF) on colon carcinogenesis was investigated in male F344 rats treated with azoxymethane (AOM). Following three weekly injections of AOM, the animals received 2 or 0.2% bLF for 36 weeks. No effects indicative of toxicity were noted, but significant reduction in both the incidence and number of adenocarcinomas of the large intestine was observed with both doses. Thus, the incidences of adenocarcinomas in the groups receiving 2% and 0.2% bLF were 15% and 25%, respectively, in contrast to the 57.5% control value (P < 0.01 and P < 0.05, respectively). The results indicate that bLF might find application for chemoprevention of colon cancer.