NADPH‐dependent acetoacetyl‐CoA (AcAc‐CoA) reductase (EC 1.1.1.36) was purified to electrophoretic homogeneity from the methylotroph Methylobacterium extorquens– a producer of poly‐3‐hydroxybutyrate. The enzyme has an Mr of 141 000 and consists of four identical subunits (Mr 31 000) and demonstrates absolute specificity for NADPH as cofactor. NADPH‐AcAc‐CoA reductase is inhibited by NADPH, AcAc‐CoA, NADP and NAD but is activated by isocitrate and ATP. The calculated Km values were 11.6 and 41 μM for AcAc‐CoA and NADPH, respectively. The results suggest that this enzyme plays a more important role in the coordinated operation of the tricarboxylic acid cycle and poly‐3‐hydroxybutyrate synthesis in M. extorquens than in the taxonomically related M. rhodesianum MB 126.
Mediated by a family of Toll-like Receptors (TLRs) responses lead to transcriptional activation and synthesis of many inflammatory genes. As an example, thousands of genes are up-regulated in response to LPS (lipopolysaccharide), with many of them undergoing specific program of activation and silencing. To investigate the breadth and specificity of LPS-induced gene expression in macrophages, we compared expressional activation in response to LPS in genetically diverse mice of subspecies M.m.musculus and M.m.domesticus using next generation (NGS) RNA-sequencing. To characterize the genetic basis of LPS-response in detail, we applied a systems genetics approach, using mapping of gene expression traits with on a panel of a second-generation F2 intercross mice. Specifically, RNA from LPS-activated F2 macrophages was RNA-sequenced and hundreds of activated genes were used as phenotypic read-outs for mapping the traits. We identified several hundreds cis- and trans-acting QTL contributing to expression variation (eQTL). Many trans eQTL cluster into major groups, some of which are confined to genomic loci that do not contain known regulators of innate immune responses. We identified 227 cis-expression quantitative trait loci (cis-eQTL) that control expression. Our data provide the first genome-wide map of major regulators of the inflammatory responses in macrophages.
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