We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.

The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp. CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli. The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps. The 4-chlorobenzoate:coenzyme A ligase was found to be a homodimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (Km = 100 microM), coenzyme A (Km = 80 microM), and 4-chlorobenzoate (Km = 9 microM) at a rate of 30 s-1 at pH 7.5 and 25 degrees C. Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3-dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active. The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a Km = 15 microM and kcat = 0.3 s-1 at pH 7.5 and 25 degrees C and to be catalytically inactive toward hydration of crotonyl-CoA, alpha-methylcrotonyl-CoA, and beta-methylcrotonyl-CoA. The 4-hydroxybenzoate-coenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a Km = 5 microM and kcat = 7 s-1 at pH 7.5 and 25 degrees C, and to also catalyze the hydrolyses of benzoyl-coenzyme A and 4-chlorobenzoate-coenzyme A. Acetyl-coenzyme A, hexanoyl-coenzyme A, and palmitoyl-coenzyme A were not hydrolyzed by the thioesterase.

Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultra-performance high/low collision switching (UPLC-MS E ) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3 following a simple chemical derivatization procedure with deuterated acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives that inhibit histone demethylases in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors, and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.

In this article, I argue that the ritual experience of water-baptism plays an essential role in Paul's metaphorical expression and rhetorical purpose in 1 Corinthians 12:13. To explore the role of baptism, I use conceptual blending theory from cognitive linguistics to define and demonstrate the metaphorical ways in which ritual functions in the human mind. In so doing, I emphasize the performance of a ritual itself and the contextual perception of its performance, arguing for a metaphorical relationship between the two. I apply conceptual blending analysis to interpret the complex interplay of three metaphors in 1 Corinthians 12:13. I argue that Paul forms a conceptual blend of three metaphors in this verse, and that baptism, the water-rite, plays a pivotal role in this blend by providing the physical pattern of immersion and the cultural understanding of this immersion as a new belonging. Using baptism, Paul achieves his purpose of re-picturing the reception of the Spirit and appealing for social union. This verse thus presents an excellent case of the role of ritual in the emergence of early Christianity and the explanatory power of ritual studies to the New Testament texts.

In this article, I argue that the ritual experience of water-baptism plays an essential role in Paul's metaphorical expression and rhetorical purpose in 1 Corinthians 12:13. To explore the role of baptism, I use conceptual blending theory from cognitive linguistics to define and demonstrate the metaphorical ways in which ritual functions in the human mind. In so doing, I emphasize the performance of a ritual itself and the contextual perception of its performance, arguing for a metaphorical relationship between the two. I apply conceptual blending analysis to interpret the complex interplay of three metaphors in 1 Corinthians 12:13. I argue that Paul forms a conceptual blend of three metaphors in this verse, and that baptism, the water-rite, plays a pivotal role in this blend by providing the physical pattern of immersion and the cultural understanding of this immersion as a new belonging. Using baptism, Paul achieves his purpose of re-picturing the reception of the Spirit and appealing for social union. This verse thus presents an excellent case of the role of ritual in the emergence of early Christianity and the explanatory power of ritual studies to the New Testament texts.