Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199.
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Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants. (+info)
Hydroxylation reaction catalyzed by the Burkholderia cepacia AC1100 bacterial strain. Involvement of the chlorophenol-4-monooxygenase.
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The Burkholderia cepacia AC1100 strain, known to degrade the herbicide, 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T), is able to metabolize 4-hydroxyarylaldehyde, not only into the corresponding acid, but also into a new hydroquinone, 2,5-dihydroxyarylaldehyde. When incubated with resting AC1100 cells or cell-free extracts, syringaldehyde and 3,5-dimethoxy-4-hydroxybenzaldehyde were converted into such metabolites, identified by comparison of their mass and 1H-NMR spectra with those of authentic chemically synthesized samples. With 5-bromovanillin, only one metabolite was formed, the structure of which was identified as 2, 5-dihydroxy-4-methoxy-6-bromobenzaldehyde through 1H-NMR two-dimensional NOESY experiments. All these products result formally from a para hydroxylation of the phenol followed by the cis migration of the aldehyde. This reaction is the only one to be associated with the 2,4,5-T degradation pathway, as the acid formation was retained when the AC1100 strain had lost its degradation ability. Through competitive experiments with halophenols and methimazole, an alternative substrate of flavin monooxygenase, the chlorophenol-4-monooxygenase was recognized to be the enzyme involved in the hydroxylation of 4-hydroxyarylaldehyde. The purified enzyme, previously reported to catalyze the para hydroxylation or dehalogenating hydroxylation of chlorophenols, also promotes this hydroxylation reaction in the presence of NADH and FAD. The kcat value determined for the best substrate, syringaldehyde, 0. 08 s-1, was about 20% of that obtained for 2,6-dichlorophenol hydroxylation (0.38 s-1). (+info)
Olfactory adaptation depends on the Trp Ca2+ channel in Drosophila.
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Olfactory adaptation is shown to occur in Drosophila, at both behavioral and physiological levels. In a behavioral paradigm, the extent of adaptation is shown to depend on the dose and duration of the adapting stimulus. Half-maximal adaptation occurred after 15 sec of exposure to an odor, and recovery occurred with a half-time of 1. 5 min, under a set of test conditions. Cross-adaptation was observed among all odor combinations tested, although to a lesser extent than when the same odor was used as both the adapting and the test stimulus. Mutants of the transient receptor potential (Trp) Ca2+ channel were normal in olfactory response, but defective in olfactory adaptation, when measured either behaviorally or in tests of antennal physiology. These results indicate that olfactory response and adaptation can be distinguished. Trp expression was detected in the developing antenna but, surprisingly, not in the mature antenna. These results, together with temperature-shift analysis of a temperature-sensitive trp mutant, provide evidence of a role of Trp in olfactory system development. (+info)
Odor response properties of rat olfactory receptor neurons.
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Molecular biology studies of olfaction have identified a multigene family of molecular receptors that are likely to be involved in odor transduction mechanisms. However, because previous functional data on peripheral coding were mainly collected from inferior vertebrates, it has been difficult to document the degree of specificity of odor interaction mechanisms. As a matter of fact, studies of the functional expression of olfactory receptors have not demonstrated the low or high specificity of olfactory receptors. In this study, the selectivity of olfactory receptor neurons was investigated in the rat at the cellular level under physiological conditions by unitary extracellular recordings. Individual olfactory receptor neurons were broadly responsive to qualitatively distinct odor compounds. We conclude that peripheral coding is based on activated arrays of olfactory receptor cells with overlapping tuning profiles. (+info)
cDNA cloning, expression and activity of a second human aflatoxin B1-metabolizing member of the aldo-keto reductase superfamily, AKR7A3.
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The aflatoxin B1 (AFB1) aldehyde metabolite of AFB1 may contribute to the cytotoxicity of this hepatocarcinogen via protein adduction. Aflatoxin B1 aldehyde reductases, specifically the NADPH-dependent aldo-keto reductases of rat (AKR7A1) and human (AKR7A2), are known to metabolize the AFB1 dihydrodiol by forming AFB1 dialcohol. Using a rat AKR7A1 cDNA, we isolated and characterized a distinct aldo-keto reductase (AKR7A3) from an adult human liver cDNA library. The deduced amino acid sequence of AKR7A3 shares 80 and 88% identity with rat AKR7A1 and human AKR7A2, respectively. Recombinant rat AKR7A1 and human AKR7A3 were expressed and purified from Escherichia coli as hexa-histidine tagged fusion proteins. These proteins catalyzed the reduction of several model carbonyl-containing substrates. The NADPH-dependent formation of AFB1 dialcohol by recombinant human AKR7A3 was confirmed by liquid chromatography coupled to electrospray ionization mass spectrometry. Rabbit polyclonal antibodies produced using recombinant rat AKR7A1 protein were shown to detect nanogram amounts of rat and human AKR7A protein. The amount of AKR7A-related protein in hepatic cytosols of 1, 2-dithiole-3-thione-treated rats was 18-fold greater than in cytosols from untreated animals. These antibodies detected AKR7A-related protein in normal human liver samples ranging from 0.3 to 0.8 microg/mg cytosolic protein. Northern blot analysis showed varying levels of expression of AKR7A RNA in human liver and in several extrahepatic tissues, with relatively high levels in the stomach, pancreas, kidney and liver. Based on the kinetic parameters determined using recombinant human AKR7A3 and AFB1 dihydrodiol at pH 7.4, the catalytic efficiency of this reaction (k2/K, per M/s) equals or exceeds those reported for other enzymes, for example cytochrome P450s and glutathione S-transferases, known to metabolize AFB1 in vivo. These findings indicate that, depending on the extent of AFB1 dihydrodiol formation, AKR7A may contribute to the protection against AFB1-induced hepatotoxicity. (+info)
Basic emotions evoked by eugenol odor differ according to the dental experience. A neurovegetative analysis.
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Subjective individual experiences seem to indicate that odors may form strong connections with memories, especially those charged with emotional significance. In the dental field, this could be the case with the odorant eugenol, responsible for the typical clinging odor impregnating the dental office. The odor of eugenol could evoke memories of unpleasant dental experiences and, therefore, negative feelings such as anxiety and fear, since eugenates (cements containing eugenol) are used in potentially painful restorative dentistry. This hypothesis was tested by evaluating the emotional impact of the odor of eugenol through autonomic nervous system (ANS) analysis. The simultaneous variations of six ANS parameters (two electrodermal, two thermovascular and two cardiorespiratory), induced by the inhalation of this odorant, were recorded on volunteer subjects. Vanillin (a pleasant odorant) and propionic acid (an unpleasant one) served as controls. After the experiment, subjects were asked to rate the pleasantness versus unpleasantness of each odorant on an 11-point hedonic scale. The patterns of autonomic responses, obtained for each odorant and each subject, were transcribed into one of the six basic emotions defined by Ekman et al. (happiness, surprise, sadness, fear, anger and disgust). Results were compared between two groups of subjects divided according to their dental experience (fearful and non-fearful dental care subjects) and showed significant differences only for eugenol. This odorant was rated as pleasant by non-fearful dental subjects but unpleasant by fearful dental subjects. The evoked autonomic responses were mainly associated with positive basic emotions (happiness and surprise) in non-fearful dental subjects and with negative basic emotions (fear, anger, disgust) in fearful dental subjects. These results suggest that eugenol can be responsible for different emotional states depending on the subjects' dental experience, which seems to confirm the potential role of odors as elicitors of emotional memories. This study also supports the possible influence of the ambient odor impregnating the dental office, strengthening a negative conditioning toward dental care in some anxious patients. (+info)
Study of the (S)-hydroxynitrile lyase from Hevea brasiliensis: mechanistic implications.
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Investigations of the (S)-selective hydroxynitrile lyase from Hevea brasiliensis were performed by electrospray mass spectroscopy, (1)H-NMR and with an enzyme activity assay. For the trans-cyanohydrin reaction (transcyanation) a two step reaction could be established. The results furthermore indicate a fast deactivation of the enzyme at low pH and a strong substrate dependence of its stability. They rule out an enzyme-HCN complex or a covalently bound carbonyl compound. Therefore the earlier postulated reaction intermediate as well as the proposed action of the catalytic triad have to be reevaluated. The calculated molecular mass could be confirmed by mass spectroscopy. (+info)
Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans.
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Caenorhabditis elegans uses a variety of attractive olfactory cues to detect food. We show here that the responses to olfactory cues are regulated in a dynamic way by behavioral context and the animal's previous experience. Prolonged exposure to an odorant leads to a decreased response to that odorant, a form of behavioral plasticity called olfactory adaptation. We show that starvation can increase the extent of olfactory adaptation to the odorant benzaldehyde; this effect of starvation persists for several hours after the animals have been returned to food. The effect of starvation is antagonized by exogenous serotonin, which induces many of the same behavioral responses in C. elegans as are induced by food. Starvation also inhibits recovery from adaptation to a different odorant, 2-methylpyrazine, thus enhancing olfactory memory. In addition to its effects on adaptation, starvation modulates olfactory discrimination in C. elegans; starved animals discriminate more classes of odorants than fed animals. Increased olfactory discrimination is also seen in the adaptation-defective mutant adp-1 (ky20). These various forms of behavioral plasticity enhance the ability of starved animals to respond to novel, potentially informative odorants. (+info)