Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

peer-reviewedBackground: About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results: The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified. Conclusions: Transcriptional diversity can potentially be generated in poly-Q encoding genes by the impact of CAG repeat tracts on mRNA alternative splicing. This effect, combined with the physical interactions of the encoded proteins in large transcriptional regulatory complexes suggests that polymorphic variations of proteins in these complexes have strong potential to affect phenotype.Dairy Australia (through the Innovative Dairy Cooperative Research Center

15-Deoxy-Δ-prostaglandin J2 induces chemokine expression, oxidative stress and microfilament reorganization in bovine mammary epithelial cells

The roles of the pro-adipogenic ligands of the transcription factor Peroxisome Proliferator Activated Receptor gamma (PPARG) in regulating innate immune responses in bovine mammary epithelial cells (bMEC) were investigated using quantitative real-time PCR. The analyses revealed that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) enhanced the expression of Interleukin 8 (IL-8) and Chemokine (C-X-C motif) ligand 6 (CXCL6) in these cells in a dose-dependent manner. 15d-PGJ2 also induced the expression of transcripts encoding proteins involved in oxidative stress, including Ferritin heavy chain and Superoxide dismutase 1, as well as substantial microfilament reorganization. In contrast, synthetic PPARG agonists displayed a different and much smaller range of effects on the cells, causing down-regulation of Interleukin 1-beta, Interleukin 6 and IL-8 and increased expression of Chemokine (C-C motif) ligand 2 (CCL2) and Tumour necrosis factor alpha (TNFα). In an independent analysis, the cells were pre-incubated with PPARG agonists followed by lipopolysaccharide stimulation. This study revealed that troglitazone increased the responsiveness of the cells to lipopolysaccharide resulting in up-regulation of Interleukin 1-beta, TNFα, IL-8, CCL2 and CXCL6 while 15d-PGJ2 caused down-regulation of TNFα, CCL2 and CXCL6. These findings are relevant to understanding the anti-inflammatory potential of the PPARG ligands and underline different mechanisms of action of 15d-PGJ2 and troglitazone in bMEC. Furthermore, the present results demonstrate that the generation of pro-inflammatory mediators can be modulated by currently available therapeutic agents and may therefore be of value in the treatment of mastitis in ruminants

Identification of immune genes and proteins involved in the response of bovine mammary tissue to Staphylococcus aureus infection

Background Mastitis in dairy cattle results from infection of mammary tissue by a range of micro-organisms but principally coliform bacteria and Gram positive bacteria such as Staphylococcus aureus. The former species are often acquired by environmental contamination while S. aureus is particularly problematic due to its resistance to antibiotic treatments and ability to reside within mammary tissue in a chronic, subclinical state. The transcriptional responses within bovine mammary epithelial tissue subjected to intramammary challenge with S. aureus are poorly characterised, particularly at the earliest stages of infection. Moreover, the effect of infection on the presence of bioactive innate immune proteins in milk is also unclear. The nature of these responses may determine the susceptibility of the tissue and its ability to resolve the infection. Results Transcriptional profiling was employed to measure changes in gene expression occurring in bovine mammary tissues sampled from three dairy cows after brief and graded intramammary challenges with S. aureus. These limited challenges had no significant effect on the expression pattern of the gene encoding β-casein but caused coordinated up-regulation of a number of cytokines and chemokines involved in pro-inflammatory responses. In addition, the enhanced expression of two genes, S100 calcium-binding protein A12 (S100A12) and Pentraxin-3 (PTX3) corresponded with significantly increased levels of their proteins in milk from infected udders. Both genes were shown to be expressed by mammary epithelial cells grown in culture after stimulation with lipopolysaccharide. There was also a strong correlation between somatic cell count, a widely used measure of mastitis, and the level of S100A12 in milk from a herd of dairy cows. Recombinant S100A12 inhibited growth of Escherichia coli in vitro and recombinant PTX3 bound to E. coli as well as C1q, a subunit of the first component of the complement cascade. Conclusion The transcriptional responses in infected bovine mammary tissue, even at low doses of bacteria and short periods of infection, probably reflect the combined contributions of gene expression changes resulting from the activation of mammary epithelial cells and infiltrating immune cells. The secretion of a number of proinflammatory cytokines and chemokines from mammary epithelial cells stimulated by the bacteria serves to trigger the recruitment and activation of neutrophils in mammary tissue. The presence of S100A12 and PTX3 in milk from infected udder quarters may increase the anti-bacterial properties of milk thereby helping to resolve the mammary tissue infection as well as potentially contributing to the maturation of the newborn calf epithelium and establishment of the newborn gut microbial population

Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection-2

Ol for each cow to identify differentially expressed transcripts. The figure shows the mean gene expression cluster profiles for the significantly differentially expressed elements that were either up-regulated (a) or down-regulated (b) relative to the control in response to intramammary infusion of . The clusters only contain significantly differentially expressed elements that were expressed in all samples and showed at least 2 fold change relative to the control in at least one sample. RNA samples were obtained from mammary tissue of three Holstein Friesian cows at peak lactation that had been infused with pyrogen-free PBS (dose 0, = 3) in one quarter (intra-animal controls) and in other quarters with at dose rates of 500 (= 1), 10,000 (= 2), 100,000 (= 3) or 1,000,000 (= 2) bacteria. Data are expressed as log(fold change) ± 1 S.D. for the centroid of the cluster.<p><b>Copyright information:</b></p><p>Taken from "Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection"</p><p>http://www.biomedcentral.com/1746-6148/4/18</p><p>BMC Veterinary Research 2008;4():18-18.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2430192.</p><p></p

Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection-3

) and (b) were monitored using qRT-PCR over a 24 h period. Data are expressed as mean fold change (= 3) compared to expression in unstimulated cells. Significance was determined by ANOVA compared to unstimulated bMEC. P < 0.01 (*) was considered significant.<p><b>Copyright information:</b></p><p>Taken from "Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection"</p><p>http://www.biomedcentral.com/1746-6148/4/18</p><p>BMC Veterinary Research 2008;4():18-18.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2430192.</p><p></p

Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection-4

reducing conditions. Lanes 1–4: 1, 2.5, 5 and 12.5 μg of rS100A12, respectively. (b) SDS-PAGE analysis of rPTX3 purified using Ni-NTA affinity chromatography. The samples were reduced and alkylated, and run under reducing conditions. Lanes 1–3: 1, 2 and 4 μg of rPTX3, respectively. (c) SDS-PAGE (lane 1) and immunoblot (lane 2) analyses of purified rtnPTX3 secreted by cells. The samples were reduced and alkylated and run under reducing conditions. 10 μg of rtnPTX3 was used for the SDS-PAGE analysis and 5 μg for the immunoblot.<p><b>Copyright information:</b></p><p>Taken from "Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection"</p><p>http://www.biomedcentral.com/1746-6148/4/18</p><p>BMC Veterinary Research 2008;4():18-18.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2430192.</p><p></p

Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection-5

. Each sample comprised 25 μl of milk whey and was run under reducing conditions. (a) Representative analysis of S100A12 expression in milk whey from cow 1419. Lane 1, control quarter pre-infection; lane 2, control quarter post-infection; lane 3, pre-infection control from the quarter receiving 1 × 10; lane 4, post-infection sample (1 × 10bacteria); lane 5, pre-infection control from the quarter receiving 1 × 10; lane 6, post-infection sample (1 × 10bacteria). Arrows denote the positions of S100A12 immunoreactive oligomers (monomer, dimer, trimer) detected using the immunoaffinity purified antibodies raised to rS100A12. (b) Analysis of PTX3 expression in milk whey. Lane 1, cow 1419 pre-infection sample from the quarter receiving 1 × 10; lane 2, post-infection sample (1 × 10bacteria); lane 3, cow 1419 pre-infection sample from the quarter receiving 1 × 10; lane 4, cow 1419 post-infection sample (1 × 10bacteria); lane 5, cow 1490 pre-infection sample from the quarter receiving 1 × 10; lane 6, cow 1490 post-infection sample (1 × 10bacteria); lane 7, cow 1592 pre-infection control from the quarter receiving 1 × 10; lane 8, cow 1592 post-infection sample (1 × 10bacteria). Arrows denote the positions of PTX3 immunoreactive bands (monomer and dimer) detected using immunoaffinity purified antibodies raised to rPTX3.<p><b>Copyright information:</b></p><p>Taken from "Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection"</p><p>http://www.biomedcentral.com/1746-6148/4/18</p><p>BMC Veterinary Research 2008;4():18-18.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2430192.</p><p></p

Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection-9

G activities for two different preparations of rtnPTX3 (each 12.5 ng/well) are shown. The controls consisted of PBS and BSA (12.5 ng/well). Significance was measured by ANOVA relative to the PBS control (*, P < 0.05).<p><b>Copyright information:</b></p><p>Taken from "Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection"</p><p>http://www.biomedcentral.com/1746-6148/4/18</p><p>BMC Veterinary Research 2008;4():18-18.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2430192.</p><p></p

Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection-6

Taken before and 16 h after the intramammary challenge. Data from cows 1419, 1490 and 1592 were used in the analyses. Bars represent the means ± SEM for two independent samples. The asterisk denotes significant expression difference (1 way ANOVA, P < 0.01) compared with the corresponding post-infection control sample for each cow. No PTX3 expression could be detected in milk from the intra-animal control udders or in the pre-infection samples.<p><b>Copyright information:</b></p><p>Taken from "Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection"</p><p>http://www.biomedcentral.com/1746-6148/4/18</p><p>BMC Veterinary Research 2008;4():18-18.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2430192.</p><p></p

Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection-0

Mammary tissues of three Holstein Friesian cows at peak lactation 16 hours following intramammary infusion with increasing doses of into the separate udder quarters. Data for each gene are expressed as relative fold changes compared to expression in the intra-animal control tissue for each animal. The genes tested included: (a) α-S1-casein () (turquoise diamond) and β-casein () (red triangle); (b) Interleukin 8 () (blue square), Interleukin 1β () (red diamond), Tumor necrosis factor α () (yellow diamond), Interleukin 6 () (green circle) and CD14 antigen () (black triangle); (c) S100 calcium-binding protein A12 () (blue diamond) and Pentraxin-3 () (pink square). Expression data for each gene were analysed by ANOVA. The superscript letters are specific for each gene tested and signify significant differences (P < 0.01) between a specific infection dose and the corresponding intramammary control for each cow.<p><b>Copyright information:</b></p><p>Taken from "Identification of immune genes and proteins involved in the response of bovine mammary tissue to infection"</p><p>http://www.biomedcentral.com/1746-6148/4/18</p><p>BMC Veterinary Research 2008;4():18-18.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2430192.</p><p></p