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ABSTRACT: Mastitis, an inflammation of the mammary gland, is a major source of economic loss on dairy farms. The aim of this study was to quantify the associations between two previously identified polymorphisms in the bovine toll-like receptor 2 (TLR2) and chemokine receptor 1 (CXCR1) genes and mammary health indictor traits in (a) 246 lactating dairy cow contemporaries representing five breeds from one research farm and (b) 848 Holstein-Friesian bulls that represent a large proportion of the Irish dairy germplasm. To expand the study, a further 14 polymorphisms in immune genes were included for association studies in the bull population.
TLR4-2021 associated (P < 0.05) with both milk protein and fat percentage in late lactation (P < 0.01) within the cow cohort. No association was observed between this polymorphism and either yield or composition of milk within the bull population. CXCR1-777 significantly associated (P < 0.05) with fat yield in the bull population and tended to associate (P < 0.1) with somatic cell score (SCS) in the cows genotyped. CD14-1908 A allele was found to associate with increased (P < 0.05) milk fat and protein yield and also tended to associate with increased (P < 0.1) milk yield. A SERPINA1 haplotype with superior genetic merit for milk protein yield and milk fat percentage (P < 0.05) was also identified.
Of the sixteen polymorphisms in seven immune genes genotyped, just CXCR1-777 tended to associate with SCS, albeit only in the on-farm study. The lack of an association between the polymorphisms with SCS in the Holstein-Friesian data set would question the potential importance of these variants in selection for improved mastitis resistance in the Holstein-Friesian cow.
BMC Genetics 11/2010; 11:99. · 2.47 Impact Factor
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ABSTRACT: Mastitis is one of the most costly diseases to the dairy farming industry. Conventional antibiotic therapy is often unsatisfactory for successful treatment of mastitis and alternative treatments are continually under investigation. We have previously demonstrated, in two separate field trials, that a probiotic culture, Lactococcus lactis DPC 3147, was comparable to antibiotic therapy to treat bovine mastitis. To understand the mode of action of this therapeutic, we looked at the detailed immune response of the host to delivery of this live strain directly into the mammary gland of six healthy dairy cows. All animals elicited signs of udder inflammation 7 h post infusion. At this time, clots were visible in the milk of all animals in the investigation. The most pronounced increase in immune gene expression was observed in Interleukin (IL)-1beta and IL-8, with highest expression corresponding to peaks in somatic cell count. Infusion with a live culture of a Lc. lactis leads to a rapid and considerable innate immune response.
Journal of Dairy Research 06/2009; 76(3):340-8. · 1.34 Impact Factor
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ABSTRACT: Subsets of proteins present and the interactions between them are fundamental determinants of the properties of complex biological systems. Monoclonal antibodies (mAbs) are highly versatile tools for characterisation of such systems, being employed to analyse the structures, functions, locations and macromolecular interactions of their cognate antigens. However, production of mAbs using hybridoma technology is time-consuming, technically demanding and uses a large amount of target material. The study presented here demonstrates that a panel of synthetic single-chain fragment variable (scFv) mAbs recognising protein components of isolated terminal cisternae sarcoplasmic reticulum membranes can be rapidly selected by bacteriophage display, using small quantities of target material. The panel of scFv mAbs isolated proved useful in a wide range of immunological applications, including immunoblot, indirect immunofluorescence microscopy and for immunoprecipitation combined with identification of targets by mass spectroscopy. Such 'shotgun immunological' strategies will prove effective in characterising novel constituents of, as well as for investigating protein-protein interactions within, macromolecular structures isolated from biological systems.
International Journal of Molecular Medicine 04/2009; 23(3):399-405. · 1.98 Impact Factor
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ABSTRACT: The TEF4 gene of the non-saccharomyces yeast Yarrowia lipolytica encodes an EF1Bgamma protein with structural similarity to the glutathione transferases (GSTs). This 1203bp gene was cloned, over-expressed in Escherichia coli, and the recombinant protein characterized. DNA sequencing of the cloned gene agreed with the recently completed Y. lipolytica genome and showed 100% identity to a previously reported 30-residue N-terminal sequence for a 110kDa Y. lipolytica GST, except that it encoded two additional N-terminal residues (N-Met-Ser-). The recombinant protein (subunit M(r) 52kDa) was found not to possess GST activity with 1-chloro-2,4-dinitrobenzene. Partial tryptic digestion released two fragments of M(r) 22 and 18kDa, which we interpret as N- and C-terminal domains. Homology modeling confirmed that the N-terminal domain of Y. lipolytica TEF4 encodes a GST-like protein.
Biochemical and Biophysical Research Communications 01/2006; 337(4):1125-32. · 2.48 Impact Factor
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ABSTRACT: CD38 is a multifunctional ectoenzyme that catalyses formation of cyclic ADP ribose (cADPr), a second messenger that opens ryanodine receptor (RyR) Ca2+ channels. Despite its importance in signal transduction processes, little is known about the mechanisms regulating CD38 expression levels. In the current study, ryanodine stimulation of Ca2+ release in Namalwa cells decreased both CD38 protein abundance and cyclase activity. Reductions in cyclase activity were prevented by RyR antagonists, by lysosomal blockers, though not by calpain or proteasomal inhibitors. These findings indicate a novel negative feedback mechanism between RyR channel activity and CD38 abundance acts in cADPr signal transduction.
FEBS Letters 12/2003; 554(1-2):133-7. · 3.54 Impact Factor
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ABSTRACT: The AlkA protein from the archaebacterium Archaeglobus fulgidus was characterised with respect to release of hypoxanthine from DNA. The hypoxanthine glycosylase activity had optimal activity at 60 degrees C at pH 5.0. The enzyme released hypoxanthine from substrates with a preference for dI:dG > dI:dT > dI:dC > dI:dA. The presence of a mismatch on either side of the dIMP in the substrate reduced excision efficiency of the hypoxanthine residue at neutral pH, while a mismatch on both sides of the dIMP resulted in total loss of excision. Release of hypoxanthine from DNA required a minimum of two bases on the 5' side and four bases on the 3' side of the dIMP residue.
FEBS Letters 04/2003; 540(1-3):171-5. · 3.54 Impact Factor
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ABSTRACT: The DNA helicase UvrD (helicase II) protein plays an important role in nucleotide excision repair, mismatch repair, rolling circular plasmid replication, and in DNA replication. A homologue of the Escherichia coli uvrD gene was previously identified in Thermus thermophilus; however, to date, a UvrD helicase has not been purified and characterized from a thermophile. Here we report the purification and characterization of a UvrD protein from Thermus thermophilus HB8. The purified UvrD has a temperature range from 10 degrees to >65 degrees C, with an optimum of 50 degrees C, within the temperature limits of the assay. The enzyme had a requirement for divalent metal ions and nucleoside triphosphates which related to enzyme activity in the order ATP > dATP > dGTP > GTP > CTP > dCTP > UTP. A simple real-time helicase assay was developed that should facilitate detailed kinetic studies of the enzyme. Evaluation of helicase substrates using this assay showed that the enzyme was highly active on a double-stranded DNA with 5' recessed ends in comparison with substrates with 3' recessed or blunt ends, and supports enzyme translocation in a 3'-5' direction relative to the strand bound by the enzyme.
Extremophiles 02/2003; 7(1):35-41. · 2.94 Impact Factor
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ABSTRACT: Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.
Journal of Biological Chemistry 02/2003; 278(5):3048-54. · 4.77 Impact Factor
