Marita K. Broadhurst

Stat5 phosphorylation status and DNA-binding activity in the bovine and murine mammary glands

The transcription factors Stat5a and Stat5b are mediators of prolactin signalling in mammary epithelial cells, and are thought to play a role in lactogenesis. In cultured cells, activation of Stat5 activity through phosphorylation results in Stat5 binding to the promoters of at least some of the milk protein genes, thereby stimulating their transcription. However, the mammary biology of Stat5 differs between species, and the role of Stat5 in the bovine mammary gland is not fully understood. We have generated an antibody that specifically recognises the phosphorylated forms of Stat5a and Stat5b and used it to compare the levels of phosphorylated Stat5 with Stat5 DNA-binding activity in bovine and murine mammary tissue. Both Stat5 DNA-binding activity and phosphorylation status in the bovine mammary gland were at near-maximal levels at late pregnancy (27-35 days prior to calving), when at least three of the major milk proteins are not highly expressed. In addition, these studies revealed significant animal-to-animal variation in the level of Stat5 activity in both species. The results are consistent with a role in terminal differentiation of mammary epithelial cells. They also suggest that the stimulation of high-level expression of milk protein genes in the bovine mammary gland is not through activation of the prolactin receptor-Jak2-Stat5 pathway.

Host-defence-related proteins in cows' milk

Milk is a source of bioactive molecules with wide-ranging functions. Among these, the immune properties have been the best characterised. In recent years, it has become apparent that besides the immunoglobulins, milk also contains a range of minor immune-related proteins that collectively form a significant first line of defence against pathogens, acting both within the mammary gland itself as well as in the digestive tract of the suckling neonate. We have used proteomics technologies to characterise the repertoire of host-defence-related milk proteins in detail, revealing more than 100 distinct gene products in milk, of which at least 15 are known host-defence-related proteins. Those having intrinsic antimicrobial activity likely function as effector proteins of the local mucosal immune defence (e.g. defensins, cathelicidins and the calgranulins). Here, we focus on the activities and biological roles of the cathelicidins and mammary serum amyloid A. The function of the immune-related milk proteins that do not have intrinsic antimicrobial activity is also discussed, notably lipopolysaccharide-binding protein, RNase4, RNase5/angiogenin and cartilage-glycoprotein 39 kDa. Evidence is shown that at least some of these facilitate recognition of microbes, resulting in the activation of innate immune signalling pathways in cells associated with the mammary and/or gut mucosal surface. Finally, the contribution of the bacteria in milk to its functionality is discussed. These investigations are elucidating how an effective first line of defence is achieved in the bovine mammary gland and how milk contributes to optimal digestive function in the suckling calf. This study will contribute to a better understanding of the health benefits of milk, as well as to the development of high-value ingredients from milk.

The abundance of milk cathelicidin proteins during bovine mastitis

Current on-farm methods for detecting mastitis in dairy cows have limitations with their specificity and sensitivity, particularly at an early stage of infection. There is therefore a need to explore new approaches for detecting early and subclinical mastitis. This study examined the expression of a group of neutrophil-specific proteins, the cathelicidins, in milk samples from naturally occurring as well as experimentally induced mastitis infections. Immunoblot analysis indicated that cathelicidin proteins are only observed in infected quarters and demonstrate a high correlation with somatic cell count (SCC) during the onset of infection. In most of the infections examined, cathelicidin was detected prior to the observation of clinical symptoms and at SCC counts as low as 6.2 × 10(3)cells/mL. In naturally occurring mastitis the correlation between cathelicidin and infection status is not as strong, with 25% of pathogen-positive milk samples containing no detectable cathelicidin. This may reflect the varying levels of neutrophil concentration and activity at different stages or severities of infection. Our results indicate that milk cathelicidin levels increase following intramammary infection and cathelicidin-based biomarkers may assist in the detection of preclinical mastitis or determining the stage of infection.

Host defence related responses in bovine milk during an experimentally induced Streptococcus uberis infection

BackgroundMilk contains a range of proteins of moderate or low abundance that contribute to host defence. Characterisation of these proteins, the extent to which their abundance is regulated by pathogenic stimuli, and the variability of their response between and within individual animals would facilitate a better understanding of the molecular basis for this important function of milk.ResultsWe have characterised the host defence proteins in bovine milk and their responses to intra-mammary infection by a common Gram positive mastitis pathogen, Streptococcus uberis, using a combination of 2D gel electrophoresis and GeLC mass spectrometry. In total, 68 host defence-associated proteins were identified, 18 of which have a direct antimicrobial function, 23 of which have a pathogen-recognition function, and 27 of which have a role in modulating inflammatory or immune signalling. The responsiveness of seven proteins was quantified by western blotting; validating the proteomic analyses, quantifying the within- and between animal variability of the responses, and demonstrating the complexity and specificity of the responses to this pathogen.ConclusionsThese data provide a foundation for understanding the role of milk in host-microbe interaction. Furthermore they provide candidate biomarkers for mastitis diagnosis, and will inform efforts to develop dairy products with improved health-promoting properties.

Effect of gestation length on the levels of five innate defence proteins in human milk

BACKGROUND Human milk contains a range of host defence proteins that appear to contribute to health and wellbeing, but their variability in abundance among individuals has not been very well characterised. Milk from mothers of premature infants has altered composition, but the effect of gestation length on the host-defence properties of milk is not known. A study was therefore undertaken to determine the variability and effect of gestation length on the abundance of five host-defence proteins in milk; lactoferrin, secretory IgA, IgG, secretory component, and complement C3. METHODS Milk was obtained from 30 mothers at their second and fifth week of lactation. These were from three groups of ten mothers having had very premature (V; 28-32 weeks gestation), premature (P; 33-36 weeks) or full term deliveries (T; 37-41 weeks). The concentration of each of the five proteins was measured in each milk sample by either ELISA or quantitative western blotting. RESULTS The concentration of IgG, and complement C3 ranged 22- and 17-fold respectively between mothers, while lactoferrin, secretory IgA, and secretory component ranged 7-, 9-, and 4-fold, respectively. The V group had significantly lower concentrations of four of the five proteins, the exception being IgG. Levels of these four proteins also decreased between weeks 2 and 5 of lactation in the P and T groups. Significant correlation was found between the concentrations of the host defence proteins within individual mothers, indicating some degree of co-ordinate regulation. CONCLUSIONS Mothers vary widely in the levels of host defence proteins in milk. Very short gestation length results in decreased abundance of host-defence proteins in milk. This may have functional implications for very premature infants.

Comparative analysis of human milk and infant formula derived peptides following in vitro digestion

It has long been recognised that there are differences between human milk and infant formulas which lead to differences in health and nutrition for the neonate. In this study we examine and compare the peptide profile of human milk and an exemplar infant formula. The study identifies both similarities and differences in the endogenous and postdigestion peptide profiles of human milk and infant formula. This includes differences in the protein source of these peptides but also with the region within the protein producing the dominant proteins. Clustering of similar peptides around regions of high sequence identity and known bioactivity was also observed. Together the data may explain some of the functional differences between human milk and infant formula, while identifying some aspects of conserved function between bovine and human milks which contribute to the effectiveness of modern infant formula as a substitute for human milk.

Gastric digestion of cow and goat milk: impact of infant and young child in vitro digestion conditions.

Many infants and young children are fed nutritional milk formulas. Although products are commonly based on cow milk, goat milk provides an alternative. We directly compared digestion of cow and goat milk proteins, varying pH, enzyme concentrations and incubation times to simulate infant and young child gastric conditions. Protein digestion and peptide formation were evaluated using electrophoresis and chromatography. Digestion of higher molecular weight whey proteins increased with decreased pH and higher enzyme concentrations of young child gastric digestion conditions compared to infant conditions. β-lactoglobulin was poorly digested under all gastric digestion conditions. Caseins reacted to pH changes differently compared to whey proteins, with less digestion of casein at pH 3.0 than at pH 5.0. Caseins from goat milk tended to be more efficiently digested compared to caseins from cow milk and peptide profiles from goat milk were distinct from cow milk.