Monitoring intracellular levels of XylR in Pseudomonas putida with a single-chain antibody specific for aromatic-responsive enhancer-binding proteins.
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We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo. (+info)
Studies on the reaction of p-hydroxybenzyl alcohol and hydroxyl radicals.
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The reaction of p-hydroxybenzyl alcohol and hydroxyl radicals generated by the Fenton reaction is studied. The products of the reaction are separated and identified by high-performance liquid chromatography (HPLC)-diode-array detection and HPLC-mass spectrometry. According to the structures of the products, a mechanism of the reaction is proposed. (+info)
Glycosides of benzyl and salicyl alcohols from Alangium chinense.
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From the water-soluble fraction of the dried leaves of Alangium chinense, three new glycosides, benzyl alcohol beta-D-glucopyranosyl-(1 --> 2)-[beta-D-xylopyranosyl-(1 --> 6)]-beta-D-glucopyranoside, 2'-O-beta-D-glucopyranosylsalicin, and 2'-O-beta-D-glucopyranosyl-6'-O-beta-D-xylopyranosylsalicin were isolated along with seven known glycosides. The structures of the new compounds were determined by spectroscopic and chemical means. (+info)
Selective adaptation to noxious foods by a herbivorous insect.
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When animals repeatedly sample a noxious food over a period of 1-4 days, they can markedly reduce their aversive behavioral response to the diet's unpleasant taste (e.g. 'bitterness') or toxic effects. This long-term adaptation process is selective, however, permitting insects to adapt physiologically to some but not all noxious foods. We hypothesized (i) that the selective nature of this adaptation process stems from the fact that some unpalatable foods are toxic while others are harmless and (ii) that insects have more difficulty adapting to foods that are both unpalatable and toxic. Our model system consisted of Manduca sexta caterpillars and two compounds that taste bitter to humans and elicit an aversive behavioral response in this insect (salicin and aristolochic acid). We found that 2 days of exposure to a salicin diet completely adapted the aversive response of the caterpillars to salicin, but that exposure to an aristolochic acid diet failed to adapt the aversive response to aristolochic acid. We determined that M. sexta could not adapt to the aristolochic acid diet because it lacked mechanisms for reducing the compound's toxicity. In contrast, the salicin diet did not produce any apparent toxic effects, and the caterpillars adapted to its aversive taste within 12 h of exposure. We also found that the salicin adaptation phenomenon (i) was mediated by the central gustatory system, (ii) generalized to salicin concentrations that were twice those in the adapting diet and (iii) offset spontaneously when the caterpillar was transferred to a salicin-free diet. We propose that toxicity is a more significant barrier to dietary adaptation than 'bitterness' in this insect. (+info)
Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.
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Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene. (+info)
Integration of global regulation of two aromatic-responsive sigma(54)-dependent systems: a common phenotype by different mechanisms.
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Pseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related sigma(54)-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding sigma(54) RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of sigma(54)-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the native Pseudomonas putida and a heterologous Escherichia coli host. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed. (+info)
Inhibitory effects of pre-ischemic and post-ischemic treatment with FR 168888, a Na+/H+ exchange inhibitor, on reperfusion-induced ventricular arrhythmias in anesthetized rat.
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Effects of pre-ischemic and post-ischemic treatment with FR 168888 (5-hydroxymethyl-3-(pyrrol-1-yl) benzoylguanidine methanesulfonate), a Na+/H+ exchange inhibitor, on reperfusion-induced ventricular arrhythmias were examined in an ischemia/reperfusion model of anesthetized rat. FR 168888 (0.3 mg/kg) significantly reduced the incidence of ventricular fibrillation (VF) and mortality induced by reperfusion following 5-min coronary occlusion, when it was intravenously administered 5 min before coronary artery occlusion. Post-ischemic treatment with FR 168888 (0.3-10 mg/kg), i.e. given 3 min after the start of occlusion, reduced the incidence of VF and mortality. In order to examine the optimal time of administration, FR 168888 (3 mg/kg) was administered 1 or 3 min after the start of occlusion or immediately before reperfusion. There was no significant difference in the reduction of VF and mortality among the three post-ischemic treatment groups. FR 168888 (3 and 10 mg/kg) significantly increased the blood pressure during ischemia without affecting the heart rate. These results indicate that FR 168888 has antiarrhythmic effects on reperfusion-induced arrhythmias even administered after coronary occlusion. (+info)
Enzymatic synthesis of a capsinoid by the acylation of vanillyl alcohol with fatty acid derivatives catalyzed by lipases.
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Capsinoids are a novel group of compounds produced by the Capsicum plant. We synthesized a capsinoid by the lipase-catalyzed esterification of vanillyl alcohol with fatty acid derivatives in an organic solvent. The use of seven out of 17 commercially available lipases, especially Novozym 435, was applicable to the synthesis of vanillyl nonanoate, a model compound of capsinoids. The yield of vanillyl nonanoate under the optimum conditions of 50 mM vanillyl alcohol and 50 mM methyl nonanoate in 500 microl of dioxane, using 20 mg of Novozym 435 and 50 mg of 4 A molecular sieves at 25 degrees C, was 86% in 20 h. Several capsinoid homologues having various acyl chain lengths (C6-C18) were synthesized at 64-86% yields from the corresponding fatty acid methyl ester. The natural capsinoids, capsiate and dihydrocapsiate, were obtained by a 400-fold-scale reaction at these optimum conditions in 60% and 59% isolated yields, respectively. (+info)