Grant Smolenski

Cloned transgenic cattle produce milk with higher levels of β-casein and κ-casein

To enhance milk composition and milk processing efficiency by increasing the casein concentration in milk, we have introduced additional copies of the genes encoding bovine β- and κ-casein (CSN2 and CSN3, respectively) into female bovine fibroblasts. Nuclear transfer with four independent donor cell lines resulted in the production of 11 transgenic calves. The analysis of hormonally induced milk showed substantial expression and secretion of the transgene-derived caseins into milk. Nine cows, representing two high-expressing lines, produced milk with an 8–20% increase in β-casein, a twofold increase in κ-casein levels, and a markedly altered κ-casein to total casein ratio. These results show that it is feasible to substantially alter a major component of milk in high producing dairy cows by a transgenic approach and thus to improve the functional properties of dairy milk.

Alterations in the salivary proteome associated with periodontitis

AIM To identify changes in the salivary proteome associated with active periodontitis. MATERIALS AND METHODS Quantitative proteomics (two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis) was used to investigate whole saliva from individuals with severe periodontitis and their proteomic profiles before and after periodontal treatment were compared. RESULTS A comparison of 128 proteins across all saliva samples identified 15 protein spots with altered abundance. The predominant alteration observed was an increase in the abundance of the S100 proteins S100A8/A9/A6. Of the remaining proteins with altered abundance, haptoglobin, prolactin inducible protein and parotid secretory protein have previously been associated with host defence. CONCLUSION These results highlight the predominant involvement of S100 proteins in the host response during periodontitis, identify host defence components that have not been linked previously to this disease and suggest new potential biomarkers for monitoring disease activity in periodontitis.

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.

Proteomics data in support of the quantification of the changes of bovine milk proteins during mammary gland involution.

Here we provide data from three proteomics techniques; two-dimensional electrophoresis (2-DE) followed by identification of selected spots using PSD MALDI-TOF MS/MS, one-dimensional gel electrophoresis followed by LC-MS/MS analysis of gel slices (GeLC) and dimethyl isotopic labelling of tryptic peptides followed by Orbitrap MS/MS (DML), to quantify the changes in the repertoire of bovine milk proteins that occurs after drying off. We analysed skim milk and whey sampled at day 0 and either day 3 or day 8 after drying off. These analyses identified 45 spots by MALDI-TOF, 51 proteins by GeLC and 161 proteins by DML, for which the detailed data work-up is presented as three Excel files. The data supplied in this article supports the accompanying publication “Changes in the repertoire of bovine milk proteins during mammary involution” (Boggs et al., 2015) [1]. Data are available via ProteomeXchange with identifiers ProteomeXchange: PXD003110 and ProteomeXchange: PXD003011.

The self-association and thermal denaturation of caprine and bovine β-lactoglobulin

Milk components, such as proteins and lipids, have different physicochemical properties depending upon the mammalian species from which they come. Understanding the different responses of these milks to digestion, processing, and differences in their immunogenicity requires detailed knowledge of these physicochemical properties. Here we report on the oligomeric state of β-lactoglobulin from caprine milk, the most abundant protein present in the whey fraction. At pH 2.5 caprine β-lactoglobulin is predominantly monomeric, whereas bovine β-lactoglobulin exists in a monomer–dimer equilibrium at the same protein concentrations. This behaviour was also observed in molecular dynamics simulations and can be rationalised in terms of the amino acid substitutions present between caprine and bovine β-lactoglobulin that result in a greater positive charge on each subunit of caprine β-lactoglobulin at low pH. The denaturation of β-lactoglobulin when milk is heat-treated contributes to the fouling of heat-exchange surfaces, reducing yields and increasing cleaning costs. The bovine and caprine orthologues of β-lactoglobulin display different responses to thermal treatment, with caprine β-lactoglobulin precipitating at higher pH values than bovine β-lactoglobulin (pH 7.1 compared to pH 5.6) that are closer to the natural pH of these milks (pH 6.7). This property of caprine β-lactoglobulin likely contributes to the reduced heat stability of caprine milk compared to bovine milk at its natural pH.

Increased gene dosage for β- and κ-casein in transgenic cattle improves milk composition through complex effects

We have previously generated transgenic cattle with additional copies of bovine β- and κ casein genes. An initial characterisation of milk produced with a hormonally induced lactation from these transgenic cows showed an altered milk composition with elevated β-casein levels and twofold increased κ-casein content. Here we report the first in-depth characterisation of the composition of the enriched casein milk that was produced through a natural lactation. We have analyzed milk from the high expressing transgenic line TG3 for milk composition at early, peak, mid and late lactation. The introduction of additional β- and κ-casein genes resulted in the expected expression of the transgene derived proteins and an associated reduction in the size of the casein micelles. Expression of the transgenes was associated with complex changes in the expression levels of other milk proteins. Two other major milk components were affected, namely fat and micronutrients. In addition, the sialic acid content of the milk was increased. In contrast, the level of lactose remained unchanged. This novel milk with its substantially altered composition will provide insights into the regulatory processes synchronizing the synthesis and assembly of milk components, as well as production of potentially healthier milk with improved dairy processing characteristics.

Gastric digestion of cow and goat milk: Peptides derived from simulated conditions of infant digestion

Infant formula products are predominantly manufactured using cow milk protein; goat milk also provides a suitable protein source. In this study, we directly compared cow and goat milk protein digestion using pH and enzyme conditions to simulate infant gastric conditions. Generated peptides, identified using liquid chromatography coupled to a mass spectrometer, show both similarities and differences in cow and goat milk post-digestion profiles. The majority of peptides were from casein proteins, 50% representing β-casein, with many peptides unique to each species. Low or no peptides for β-Lactoglobulin and α-Lactalbumin, respectively, suggest these proteins were highly resistant to infant gastric digestion, as reported by others. Minor milk proteins, comprising 5% of peptides, were represented by different proteins from cow and goat. Peptides with known bioactivities were also observed, both in common and unique to each species. Together these data may explain reported differences in digestion characteristics of cow and goat milk.