Thiacetazone (TAZ) and ethionamide (ETA) are, respectively, thiourea and thioamide-containing second line antitubercular prodrugs for which there is an extensive clinical history of cross-resistance in Mycobacterium tuberculosis. EtaA, a recently identified flavin-containing monooxygenase (FMO), is responsible for the oxidative activation of ETA in M. tuberculosis. We report here that EtaA also oxidizes TAZ and identify a sulfinic acid and a carbodiimide as the isolable metabolites. Both of these metabolites are derived from an initial sulfenic acid intermediate. Oxidation of TAZ by EtaA at basic pH favors formation of the carbodiimide, whereas neutral or acidic conditions favor formation of the sulfinic acid. The same metabolites are formed from TAZ by human FMO1 and FMO3. The sulfenic acid and carbodiimide metabolites, but not the sulfinic acid product, readily react with glutathione, the first to regenerate the parent drug and the second to give a glutathione adduct. These reactions that may contribute to the antitubercular activity and/or toxicity of TAZ.

Introns constitute most of the length of typical pre-mRNAs in vertebrate cells. Thus, the turnover rate of introns may significantly influence the availability of ribonucleotides and splicing factors for further rounds of transcription and RNA splicing, respectively. Given the importance of intron turnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1 Cb1 , the first intron from the constant region of the mouse T-cell receptor-b (TCR-b) gene. Using a tetracycline (tet)-regulated promoter, we demonstrate that spliced IVS1 Cb1 and its pre-mRNA had half-lives of 6.0 6 1.4 min and 3.7 6 1.0 min, respectively. We also examined the half-lives of these transcripts by using actinomycin D (Act.D). Act.D significantly stabilized IVS1 Cb1 and its pre-mRNA, suggesting that Act.D not only blocks transcription but exerts rapid and direct posttranscriptional effects in the nucleus. We observed that in vivo spliced IVS1 Cb1 accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1 Cb1 as a lariat did not result from an intrinsic inability to be debranched, as it could be debranched in vitro, albeit somewhat less efficiently than an adenovirus intron. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1 Cb1 lariats cofractionated with pre-mRNA, but not always with mRNA in the nucleus. Some IVS1 Cb1 also appeared to be selectively exported to the cytoplasm, whereas TCR-b pre-mRNA remained in the nucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions both with G-protein coupled seven transmembrane receptors and with the nuclear hormone receptor PPARgamma. Synthesis and biological activities of fluorinated analogues of LPA are still relatively unknown. In an effort to identify receptor-selective LPA analogues and to document in detail the structure-activity relationships of fluorinated LPA isosteres, we describe a series of monofluorinated LPA analogues in which either the sn-1 or the sn-2 hydroxy group was replaced by fluorine, or the bridging oxygen in the monophosphate was replaced by an alpha-monofluoromethylene (-CHF-) moiety. The sn-1 or sn-2 monofluorinated LPA analogues were enantiospecifically prepared from chiral protected glycerol synthons, and the alpha-monofluoromethylene-substituted LPA analogues were prepared from a racemic epoxide with use of a hydrolytic kinetic resolution. The sn-2 and sn-1 fluoro LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. The alpha-monofluoromethylene LPA analogues were unique new nonhydrolyzable ligands with surprising enantiospecific and receptor-specific biological readouts, with one compound showing a 1000-fold higher activity than native LPA for one receptor subtype.

The metabolically stabilized LPA analogue, 1-oleoyl-2-O-methyl-rac-glycerophosphothioate (OMPT), is a potent agonist for the LPA(3) G-protein-coupled receptor. A new enantiospecific synthesis of both (2R)-OMPT and (2S)-OMPT is described. Calcium release assays in both LPA(3)-transfected insect Sf9 and rat hepatoma Rh7777 cells showed that (2S)-OMPT was 5- to 20-fold more active than (2R)-OMPT. Similar results were found for calcium release, MAPK and Akt activation, and IL-6 release in human OVCAR3 ovarian cancer cells.

The vast majority of mammalian genes are interrupted by non-coding segments of DNA termed introns. Introns are spliced out of RNA transcripts as lariat structures, and then are typically debranched and rapidly degraded. Here, we described an unusual spliced intron from the constant region of the T cell receptor-beta (TCR-beta) locus that is relatively stable in mammalian cells. This intron, IVS1C beta 1, accumulates as a set of lariat RNA structures with different length tails in the nucleus of T cells. The accumulation of this spliced intron is developmentally regulated during murine thymocyte ontogeny. The property of stability appears to be evolutionarily conserved since the human version of this intron also accumulates in T cells. The stability is selective since other spliced TCR-beta introns do not detectably accumulate in T cells. The unusual stability of this intron does not depend on T cell specific factors since non-T cells transfected with TCR-beta gene constructs also accumulate spliced IVS1C beta 1. The discovery of a mammalian intron that accumulates as a lariat in vivo provides an opportunity to elucidate mechanisms that regulate intron debranching, stability, and nuclear localization.

A versatile, efficient method for the preparation of alpha-monofluoromethylene (-CHF-) phosphonates from alpha-fluorovinylphosphonate provides access to a class of lysophosphatidic acid (LPA) receptor-subtype agonists. In addition, sn-2 O-methylation of alpha-monofluoromethylene phosphonates using trimethylsilyldiazomethane generated sn-1-acyl, 2-O-methyl alpha-monofluoromethylene derivatives. Finally, a novel method for the selective etherification of 1,2-diols was developed and a new class of sn-1 O-methyl, 2-acyl alpha-monofluoromethylene LPA analogues was prepared. [reaction: see text]

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions with seven-transmembrane G-protein-coupled receptors (GPCRs) and the nuclear hormone receptor PPARgamma. To identify receptor-selective LPA analogues, we describe a series of fluorinated LPA analogues in which either the sn-1 or sn-2 hydroxyl group was replaced by a fluoro or fluoromethyl substituent. We also describe stabilized phosphonate analogues in which the bridging oxygen of the monophosphate was replaced by an alpha-monofluoromethylene (-CHF-) or alpha-difluoromethylene (-CF(2)-) moiety. The sn-2- and sn-1-fluoro-LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. We first tested these LPA analogues on insect Sf9 cells induced to express human LPA(1), LPA(2), and LPA(3) receptors. While none of the analogues were found to be more potent than 1-oleoyl-LPA at LPA(1) and LPA(2), several LPA analogues were potent LPA(3)-selective agonists. In contrast, 1-oleoyl-LPA had similar activity at all three receptors. The alpha-fluoromethylene phosphonate analogue 15 activated calcium release in LPA(3)-transfected insect Sf9 cells at a concentration 100-fold lower than that of 1-oleoyl-LPA. This activation was enantioselective, with the (2S)-enantiomer showing 1000-fold more activity than the (2R)-enantiomer. Similar results were found for calcium release in HT-29 and OVCAR8 cells. Analogue 15 was also more effective than 1-oleoyl-LPA in activating MAPK and AKT in cells expressing high levels of LPA(3). The alpha-fluoromethylene phosphonate moiety greatly increased the half-life of 15 in cell culture. Thus, alpha-fluoromethylene LPA analogues are unique new phosphatase-resistant ligands that provide enantiospecific and receptor-specific biological readouts.

The aim of the study was to determine whether or not dexmedetomidine- (DEX-) based intravenous infusion in dental implantation can provide better sedation and postoperative analgesia via suppressing postoperative inflammation and oxidative stress. Sixty patients were randomly assigned to receive either DEX (group D) or midazolam (group M). Recorded variables were vital sign (SBP/HR/RPP/SpO2/RR), visual analogue scale (VAS) pain scores, and observer's assessment of alertness/sedation scale (OAAS) scores. The plasma levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), antioxidant superoxide dismutase (SOD), and the lipid peroxidation product malondialdehyde (MDA) were detected at baseline and after 2, 4, and 24 h of drug administration. The VAS pain scores and OAAS scores were significantly lower for patients in group D compared to group M. The plasma levels of TNF-α, IL-6, and MDA were significantly lower in group D patients than those in group M at 2 h and 4 h. In group M, SOD levels decreased as compared to group D at 2 h and 4 h. The plasma levels of TNF-α, IL-6, and MDA were positively correlated with VAS pain scores while SOD negatively correlated with VAS pain scores. Therefore, DEX appears to provide better sedation during office-based artificial tooth implantation. DEX offers better postoperative analgesia via anti-inflammatory and antioxidation pathway.