-Bovine mammary epithelial cells contribute to the innate immune response to intramammary infections by recognizing pathogens through specialized pattern recognition receptors. Toll-like receptor 4 (TLR4) is one such receptor that binds and is activated by lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. In this study, MAC-T cells (a bovine mammary epithelial cell line) were incubated in the presence or absence of increasing concentrations of LPS for 24 h. Expression of TLR2 and TLR4 were analyzed at both mRNA and protein levels by quantitative real-time PCR (qPCR) and flow cytometry, respectively. The mRNA of both receptors were up-regulated by all concentrations of LPS used (P < 0.01). Similarly, flow cytometry with specific antibodies against TLR2 and TLR4 detected increased surface expression of these proteins. Furthermore, expression of downstream TLR4 signaling molecules was examined by qPCR following varying exposure times to 1 μg/mL of LPS. Results demonstrate that the required adaptor molecules and transcription factors were up-regulated in a time-dependent manner. Both the MyD88 dependent and independent pathways in TLR4 signaling were activated in MAC-T cells. Expression of TOLLIP increased in response to LPS as did the pro-apoptotic protease, CASP8. These results suggest that the bovine mammary epithelium possesses the necessary immune repertoires required to achieve a robust defense against E. coli. The current findings, coupled with previous findings that S. aureus ligands induce up-regulation of TLR4, may indicate a positive adaptation by mammary epithelial cells to effectively respond to different types of mastitis pathogens.lipopolysaccharide / TLR4 and TLR2 / signal transduction / bovine mammary epithelial cells / mastitis
Gram-negative and -positive bacteria elicit different response patterns by the host. The proteomic profiles of milk whey samples from cows naturally infected with Escherichia coli or Staphyloccocus aureus as compared to whey from healthy cows were determined by one-dimensional, liquid chromatography-tandem mass spectrometry (LC-MS/MS), bioinformatics processing, and pathway analyses. Since mammary epithelial cells contribute to immune responses in mammary glands, the genes of selected proteins were measured in MAC-T cells by real time quantitative PCR (qPCR) after stimulation with heat inactivated E. coli strain P4 and S. aureus strain Smith CP bacteria. A total of 173 proteins were identified including 73 proteins differentially expressed among normal, E. coli, and S. aureus treatment groups. E. coli was more effective at significantly altering the concentration of the affected proteins. The mRNA of 23 proteins out of 24 measured by qPCR was significantly altered in MAC-T cells. Pathway analyses identified top canonical pathways significantly enriched in our samples, the most significant being the acute phase response signaling pathway. Also, top networks of genes with significant associations to identified proteins were identified. Our study has demonstrated a wider proteome profile of E. coli and S. aureus mastitic milk whey, identified more low abundant defense proteins than reported before, and has linked for the first time identified proteins to several network functions and Biocarta pathways.
BackgroundCD14 is an important player in host innate immunity in that it confers lipopolysaccharide sensitivity to cell types like neutrophils, monocytes and macrophages. The study was aimed at characterizing the CD14 gene of cattle for sequence variations and to determine the effect of variations on the expression of the protein on the surfaces of monocytes and neutrophils in healthy dairy cows.ResultsFive SNPs were identified: two within the coding regions (g.A1908G and g.A2318G, numbering is according to GenBank No. EU148609), one in the 5' (g.C1291T) and two in the 3' (g.A2601G and g.G2621T) untranslated regions. SNP 1908 changes amino acid 175 of the protein (p.Asn175Asp, numbering is according to GenBank No. ABV68569), while SNP 2318 involves a synonymous codon change. Coding region SNPs characterized three gene alleles A (GenBank No. EU148609), A1 (GenBank No. EU148610) and B (GenBank No. EU148611) and two deduced protein variants A (ABV68569 and ABV68570) and B (ABV68571). Protein variant A is more common in the breeds analyzed. All SNPs gave rise to 3 haplotypes for the breeds. SNP genotype 1908AG was significantly (P < 0.01) associated with a higher percentage of neutrophils expressing more CD14 molecules on their surfaces. The promoter region contains several transcription factor binding sites, including multiple AP-1 and SP1 sites and there is a high conservation of amino acid residues between the proteins of closely related species.ConclusionThe study has provided information on sequence variations within the CD14 gene and proteins of cattle. The SNP responsible for an amino acid exchange may play an important role in the expression of CD14 on the surfaces of neutrophils. Further observations involving a larger sample size are required to validate our findings. Our SNP and association analyses have provided baseline information that may be used at defining the role of CD14 in mediating bacterial infections. The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.
The B allele of the bovine alpha (S2)-casein gene (CSN1S2) was characterized at the molecular level and the distribution of zebu-specific milk protein alleles was determined in 26 cattle breeds originating from 3 continents. The CSN1S2*B allele is characterized by a C --> T transition affecting nucleotide 17 of exon 3, which leads to a change in the eighth amino acid of the mature protein, from Ser to Phe (i.e., TCC --> TTC). DNA-based methods were developed to identify carriers of CSN1S2*B and the other alleles (CSN1S2*A, C, and D) at the same locus. CSN1S2*B and other zebu-specific milk protein alleles and casein haplotypes are widely distributed in European cattle breeds, particularly those of southeastern origin. Alleles CSN1S2*B and CSN3*H are important in searching for zebu imprints in European cattle breeds. Diversity estimates at the milk protein loci were highest in the zebus followed by southeastern European taurines. Anatolian Black had the highest number of zebu alleles among European taurines. Common, group, and intergroup haplotypes occurred in the breeds and demonstrated relationships that concurred with developmental histories, genetic makeup, and, in particular, exposed the extent of zebu influence on southeastern European cattle.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.