Gene expression studies using reverse transcriptase (RT)-PCR are widely used and powerful, but the results obtained from such studies are dependent on the specificity of the assay. Here we describe an assay designed to detect ovine TLR6 in blood and tissues from sheep Discrimination between TLR1 and TLR6 at the level of gene expression was challenging due to extensive tracts of homology and identity within the two sequences. Both TLR1 and 6 can form heterodimers with TLR2 in order to bind the ligands GSK2879552 inhibitor of microbial pathogens The expression of TLR6 was increased in the ileum and jejunum of sheep infected
with MAP, with a trend towards TLR6 upregulation in peripheral blood cells in response to exposure to MAP A likely role for TLR6/TLR2 heterodimers in the pathogenesis
of JD was identified. TLR6 may be a potential marker of exposure and could aid in the development of a gene signature for sheep SU5402 nmr resistant to MAP infection (C) 2010 Elsevier B V All rights reserved”
“Methyl-coenzyme M reductase (MCR) is the key enzyme in methane formation by methanogenic Archaea. It converts the thioether methyl-coenzyme M and the thiol coenzyme B into methane and the heterodisulfide of coenzyme M and coenzyme B. The catalytic mechanism of MCR and the role of its prosthetic group, the nickel hydrocorphin coenzyme URMC-099 in vivo F(430), is still disputed, and no intermediates have, been observed so far by fast spectroscopic techniques when the enzyme was incubated with the natural substrates. In the presence of the competitive inhibitor coenzyme M instead of methyl-coenzyme M, addition of coenzyme B to the active Ni(I) state MCR(red1) induces two new species called MCR(red2a) and MCR(red2r) which have been characterized by pulse EPR spectroscopy. Here we show that the two MCR(red2) signals can also be induced
by the S-methyl- and the S-trifluoromethyl analogs of coenzyme B. (19)F-ENDOR data for MCR(red2a) and MCR(red2r) induced by S-CF(3)-coenzyme B show that, upon binding of the coenzyme B analog, the end of the 7-thioheptanoyl chain of coenzyme B moves closer to the nickel center of F(430) by more than 2 angstrom as compared to its position in both, the Ni(l) MCR(red1) form and the X-ray structure of the inactive Ni(II) MCR(ox1-silent) form. The finding that the protein is able to undergo a conformational change upon binding of the second substrate helps to explain the dramatic change in the coordination environment induced in the transition from MCR(red1) to MCR(red2) forms and opens the possibility that nickel coordination geometries other than square planar, tetragonal pyramidal, or elongated octahedral might occur in intermediates of the catalytic cycle.