Proteomic analysis and immunodetection of the bovine milk osteopontin isoforms.

The aim of this study was to characterize the osteopontin (OPN) secreted in bovine milk and to determine whether the different forms are the product of spliced variants. Spliced variants of the human gene and secreted osteopontin isoforms have been reported in human tumor tissue. In bovine milk, we identified 2 major forms: one corresponding to the full-length coding transcript and a truncated version of this form. No alternative spliced transcripts were detected in the lactating mammary gland tissue, in milk somatic cells, or in peripheral blood immune cells. The 60-kDa bovine osteopontin (bOPN) and a truncated 40-kDa protein isoform were confirmed by mass spectrometry and further characterized by immunoblotting using a panel of 6 antibodies targeting different domains of the protein. Of the 3 human anti-OPN antibodies targeting the N-terminal segment of the protein, only one detected all forms on sodium dodecyl sulfate-PAGE; one human anti-OPN antibody failed to detect bOPN, whereas the other detected only the 60-kDa protein, albeit barely in its phosphorylated form. Detection was generally more sensitive when the 60-kDa protein was dephosphorylated. Two polyclonal antibodies raised against bOPN were tested: one targeting the milk-purified bOPN (bOPN-121) and one targeting a bovine epitope (synthetic peptide) corresponding to a carboxy-terminal domain of the protein (bOPN-117). The bOPN-121 antibody detected all forms irrespective of the phosphorylation status of bOPN. The bOPN-117 and the mouse anti-human OPN (hOPN-4) antibodies, which recognized different domains of the carboxy-terminal segment of the protein, also preferentially detected the dephosphorylated 60-kDa protein. Whereas phosphorylation had a major effect on detection for several antibodies, deglycosylation slightly decreased immunodetection for the tested antibodies. In particular, phosphorylation is the major posttranslational modification that influenced the weak detection capacity of several antibodies. This fact needs to be taken into account for immunodetection of milk content. In conclusion, the OPN forms secreted in bovine milk are not the product of alternative splicing. The 40-kDa protein appears to be a truncated hypophosphorylated variant of the full-length 60-kDa form, which is highly phosphorylated. Together, the proteomic and immunoblotting analyses used to characterize bovine milk OPN revealed the complex nature of the bovine milk OPN forms.

Effect of pepsin-treated bovine and goat caseinomacropeptide on Escherichia coli and Lactobacillus rhamnosus in acidic conditions.

Caseinomacropeptide (CMP) is a 7-kDa phosphoglycopolypeptide released from κ-casein during milk digestion and in the cheesemaking process. The objective of the study was to analyze the effect of pepsin-treated CMP from cow and goat milk on the resistance of Escherichia coli and Lactobacillus rhamnosus during acid stress. Bacterial cells in the exponential growth phase were suspended in acidified phosphate buffered saline with or without pepsin-treated CMP. Viability was determined during a 90-min incubation period. Pepsin-treated CMP exhibited bactericidal activity at pH 3.5 when added in a dose-dependent manner to E. coli, decreasing survival by more than 90% within 15 min at 0.25 mg/mL. At pH >4.5, the bactericidal activity disappeared, indicating that pepsin-treated CMP was efficient at low pH only. The effectiveness of pepsin-treated CMP at pH 3.5 was not affected by the presence of glycoconjugates linked to CMP or by the bovine or caprine origin of milk. In contrast, L. rhamnosus, a probiotic, was more resistant to acid stress when pepsin-treated bovine or caprine CMP was added to the media. Viability reached 50% after 60 min of incubation at pH 3 compared with 5% survival in the media without added pepsin-treated CMP. Neither glycosylation extent nor sequence variations between CMP from bovine milk and caprine milk affected the protective activity of hydrolyzed CMP toward L. rhamnosus. This suggests that encrypted bioactive peptides released by the pepsin treatment of CMP had an antibacterial effect on E. coli in acidic media, but improved the resistance of L. rhamnosus to acid stress. The peptide fragment accountable for bactericidal activity is the N-terminal region κ-casein f(106-124).

Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus.

To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS(+) Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS(+) strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS(-)) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.

Short communication: extraction of beta-casein from goat milk.

Peptides derived from milk beta-casein have potential biological activities, such as antihypertensive and immunostimulating properties. These biological properties increase the demand for the production of specific bioactive peptides. beta-Casein can be isolated directly from renneted skim milk, based on the preferential solubilization of beta-casein at low temperature. This study was conducted to compare the recovery and purity of beta-casein extracted from goat and cow milks. Rennet casein was prepared from both milks, heat treated, and dispersed in demineralized water at various temperatures. beta-Casein recovery in the soluble phase increased with decreasing incubation temperature. Concentration of beta-casein was 43% higher in goat milk than in cow milk, which had a direct effect on beta-casein recovery. Furthermore, beta-casein was extracted more efficiently from goat rennet casein. As a result, the extraction yield of beta-casein was 53% higher in goat milk than in cow milk. The purity of beta-casein extracted from both milks reached approximately 90% after incubation at 0 degrees C.

Galactose metabolism and capsule formation in a recombinant strain of Streptococcus thermophilus with a galactose-fermenting phenotype.

The capsule-producing, galactose-negative Streptococcus thermophilus MR-1C strain was first transformed with a low-copy plasmid containing a functional galK gene from Streptococcus salivarius to generate a recombinant galactose-fermenting Strep. thermophilus strain named MR-AAC. Then, we compared the functional properties of Strep. thermophilus MR-AAC with those of the parent MR-1C strain when used as starter for fermented products and cheese. In lactose-supplemented laboratory medium, MR-AAC metabolized galactose, but only when the amount of lactose was less than 0.1% (wt/vol). After 7 h of fermentation, the medium was almost depleted of galactose. The parent strain, MR-1C, showed the same pattern, except that the concentration of galactose decreased by only 25% during the same period. It was found that, during milk fermentation and Mozzarella cheese production, the galactose-fermenting phenotype was not expressed by MR-AAC and this strain expelled galactose into the medium at a level similar to the parent MR-1C strain. In milk and in lactose-supplemented medium, capsular exopolysaccharide production occurred mainly during the late exponential phase and the stationary growth phase with similar kinetics between MR-1C and MR-AAC.

Detection and quantification of capsular exopolysaccharides from Streptococcus thermophilus using lectin probes.

The aim of this work was to use fluorescently labeled lectins to develop a convenient and reliable method to determine the relative abundance of capsular polysaccharides (CPS) at the surface of Streptococcus thermophilus MR-1C cells. Fluorescein isothiocyanate-labeled peanut agglutinin isolated from Arachis hypogaea was found to interact specifically with the CPS of Strep. thermophilus MR-1C. This labeled lectin was then used as an effective probe to detect and quantify CPS. A fluorescence-based lectin-binding assay was successfully applied to follow the accumulation of CPS during the growth of Strep. thermophilus MR-1C in milk and in M17 broth supplemented with lactose. Our results showed that in both media, CPS production by Strep. thermophilus MR-1C began during the exponential phase of growth and continued for several hours after the culture reached the stationary growth phase.

Proteases involved in mammary tissue damage during endotoxin-induced mastitis in dairy cows.

During and after diapedesis, milk polymorphonu-clear neutrophils (PMN) release many proteases that have the potential of degrading extracellular matrix proteins and milk proteins. However, the kinetics of milk proteolysis during inflammation and the underlying mechanisms are poorly defined. The enzymes involved in bovine mammary tissue destruction were investigated in this study using an endotoxin-induced mastitis model. Using zymography techniques, the proteolytic activity of milk and mammary tissue during mastitis was examined. Mastitic milk produced 6 caseolysis bands, 4 of which differed from the ones produced by plasmin. Peak proteolytic activity, bovine serum albumin contents, and mammary tissue damage occurred between 6 and 12 h postchallenge. Mastitic milk proteases hydrolyzed casein, gelatin, collagen, hemoglobin, mammary gland membrane proteins, and lactoferrin. These results confirm that mastitic milk proteases have a broad spectrum of activity. The hydrolytic activity of mastitic milk was partially inhibited by aprotinin, EDTA, 1,10-phenanthroline, leupeptin, and pefabloc. When cocultured with normal mammary tissue, mastitic milk, but not normal milk, caused mammary tissue degradation. In situ zymography of mammary gland showed increased proteolytic activity in mastitic tissue compared with normal tissue. The similarity of zymograms of mastitic milk, blood PMN, milk somatic cells, and PMN strongly suggests that proteases in mastitic milk mainly originate from milk PMN. These results suggest that proteases released by PMN are actively involved in udder tissue damage during mastitis.

Levels of vascular endothelial growth factor (VEGF) in serum of patients with endometriosis.

BACKGROUND: Elevated concentrations of vascular endothelial growth factor (VEGF) have been detected in the peritoneal fluid of patients with endometriosis. Furthermore, it was postulated that VEGF is involved in the development of endometriotic lesions. The present study is aimed at determining whether high levels of VEGF could also be found in the serum of patients with endometriosis. METHODS: VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA) in serum from 131 subjects with surgically confirmed endometriosis and 146 controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. Parameters such as demographics, personal habits, menstrual characteristics and clinical profile were collected from each subject included in this study. RESULTS: The mean VEGF levels were not significantly modulated in serum samples of cases compared with controls in a crude general linear model and in a model adjusted for possible confounders. VEGF serum levels did not correlate with the score, stage of endometriosis or the presence of benign gynaecological disorders. However, a correlation was found between circulating concentrations of VEGF and body mass index. CONCLUSION: Although VEGF seems to play a pivotal role locally in the implantation and development of endometriotic lesions, the disease is not associated with a significant modulation in the levels of circulating VEGF.

Changes in ABA and gene expression in cold-acclimated sugar maple.

To determine if cold acclimation of sugar maple (Acer saccharum Marsh.) is associated with specific changes in gene expression under natural hardening conditions, we compared bud and root translatable mRNAs of potted maple seedlings after cold acclimation under natural conditions and following spring dehardening. Cold-hardened roots and buds were sampled in January when tissues reached their maximum hardiness. Freezing tolerance, expressed as the lethal temperature for 50% of the tissues (LT(50)), was estimated at -17 degrees C for roots, and at lower than -36 degrees C for buds. Approximately ten transcripts were specifically synthesized in cold-acclimated buds, or were more abundant in cold-acclimated buds than in unhardened buds. Cold hardening was also associated with changes in translation. At least five translation products were more abundant in cold-acclimated buds and roots compared with unhardened tissues. Abscisic acid (ABA) concentration increased approximately tenfold in the xylem sap following winter acclimation, and the maximum concentration was reached just before maximal acclimation. We discuss the potential involvement of ABA in the observed modification of gene expression during cold hardening.

Effects of soil freezing and drought stress on abscisic acid content of sugar maple sap and leaves.

In 1991 and 1992, mature maple trees (Acer saccharum Marsh.) were freeze-stressed or drought-stressed by preventing precipitation (snow or rain) from reaching the forest floor under selected trees. Lack of snow cover caused a decrease in soil temperature to well below 0 degrees C from December to April and a lowering of the soil water content to 10%. The abscisic acid (ABA) concentration in the spring sap of deep-soil frost-stressed trees was significantly higher than in control or drought-stressed trees. The increase in ABA concentration in the xylem sap in the spring of 1991 and 1992 preceded symptoms of canopy decline and a decrease in leaf area that were observed during the summers of 1991 and 1992. These results suggest a role for ABA in root-to-shoot communication in response to environmental stress. The largest differences in ABA concentration induced by the treatments was found in sap collected at the end of sap flow. The increase in ABA concentration in spring sap at the end of the sap flow could be used as an early indicator of stress suffered by trees during the winter. Not only did the increase in ABA concentration occur before any visible symptoms of tree decline appeared, but the trees that showed the most evident decline had the highest ABA concentrations in the spring sap. Leaf ABA concentration was not a good indicator of induced stress.

Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation.

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic "cobblestone" morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in beta-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) alpha s- and beta-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.

Association of kappa-casein glycosylation with milk production and composition in Holsteins.

A total of 545 milk samples were collected from 53 Holstein cows for 1 yr. On test day, morning milk yields were recorded; milk samples were analyzed for N-acetylneuraminic acid, protein, fat, casein, kappa-casein, and SCC. the relationship between the degree of glycosylation of kappa-casein and morning milk yield and composition was investigated. Data were analyzed using least squares procedures with a model that included test day, parity, stage of lactation, SCC, and phenotype for kappa-casein as fixed effects and N-acetylneuraminic acid content of kappa-casein as covariate. After adjustments were made for effects of environmental and genetic factors, the degree of glycosylation of kappa-casein was associated with morning milk yield, protein, and casein content. Milk yield increased linearly with an increase of N-acetylneuraminic content up to approximately 70 micrograms/mg of kappa-casein.

Identification of epididymal proteins associated with hamster sperm.

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.

Heterogeneity of epididymal spermatozoa of the hamster.

Epididymal transit is involved in the acquisition of fertilizing ability by mammalian spermatozoa. The epididymis is implicated in the addition and/or modification of sperm surface proteins functionally involved in the processes leading to fertilization. In order to characterize these sperm components, we have studied the modifications of sperm membrane proteins during epididymal maturation. Hamster spermatozoa were collected from the caput, corpus, and proximal and distal cauda of the epididymis and submitted to a continuous Percoll gradient centrifugation. Independently of their epididymal origin, the spermatozoa were distributed into two bands of buoyant densities of 1,045 and 1,088 on the gradient. However, the proportion of more dense spermatozoa increased progressively along the epididymis. This proportion is 87-fold higher in the distal cauda compared to the caput. SDS-PAGE electrophoresis demonstrated that although they originate from different regions of the epididymis, spermatozoa of the same density exhibited similar membrane protein patterns. In contrast, the electrophoretic patterns of spermatozoa with densities of 1,045 and 1,088 were very different. One of these differences involves a 26 kD protein that is implicated in zona pellucida recognition (Sullivan and Bleau, Gamete Res 12:101-116, 1985). This protein is present in dense sperm obtained from all regions of the epididymis but is absent in spermatozoa of low density. If we consider that this 26 kD protein is a "marker" of surface changes occurring during sperm maturation, we can hypothesize that in the proximal segments of the epididymis a certain proportion of spermatozoa are indeed mature but that this proportion increases considerably along the epididymis.

Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte.

Hamster oviducts in culture incorporate [35S]-methionine into secretory proteins. One of these proteins is immunoprecipitated by a monoclonal antibody specific to an antigen found in oviductal oocytes but not in ovarian oocytes. This antigen, called oviductin, is progressively added to the oocyte during its transit through the oviduct. Oviductin migrates as a diffuse band with a molecular mass between 160 and 250 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The electrophoretic behavior of this protein suggests the presence of polysaccharide side chains. Chemical deglycosylation causes a decrease in molecular mass and removes the antigenic determinant originally present on the glycoprotein. By using the radiation inactivation method, the molecular mass of the core protein has been found to be approximately 44 kDa. These results indicate that the oviduct is an actual site of synthesis of the oviductin. This glycoprotein contains a high proportion of sugar residues, which account for antigenic determinant recognized by the monoclonal antibody.