Characterization of a Pseudomonas putida allylic alcohol dehydrogenase induced by growth on 2-methyl-3-buten-2-ol.
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We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium Pseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of Acinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB. (+info)
A physiological model for tert-amyl methyl ether and tert-amyl alcohol: hypothesis testing of model structures.
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The oxygenate tert-amyl methyl ether (TAME) is a gasoline fuel additive used to reduce carbon monoxide in automobile emissions. To evaluate the relative health risk of TAME as a gasoline additive, information is needed on its pharmacokinetics and toxicity. The objective of this study was to use a physiologically-based pharmacokinetic (PBPK) model to describe the disposition of TAME and its major metabolite, tert-amyl alcohol (TAA), in male Fischer-344 rats. The model compartments for TAME and TAA were flow-limited. The TAME physiological model had 6 compartments: lung, liver, rapidly perfused tissues, slowly perfused tissues, fat, and kidney. The TAA model had 3 compartments: lung, liver, and total-body water. The 2 models were linked through metabolism of TAME to TAA in the liver. Model simulations were compared with data on blood concentrations of TAME and TAA taken from male Fischer-344 rats during and after a 6-hour inhalation exposure to 2500, 500, or 100 ppm TAME. The PBPK model predicted TAME pharmacokinetics when 2 saturable pathways for TAME oxidation were included. The TAA model, which included pathways for oxidation and glucuronide conjugation of TAA, underpredicted the experimental data collected at later times postexposure. To account for biological processes occurring during this time, three hypotheses were developed: nonspecific binding of TAA, diffusion-limited transport of TAA, and enterohepatic circulation of TAA glucuronide. These hypotheses were tested using three different model structures. Visual inspection and statistical evaluation involving maximum likelihood techniques indicated that the model incorporating nonspecific binding of TAA provided the best fit to the data. A correct model structure, based upon experimental data, statistical analyses, and biological interpretation, will allow a more accurate extrapolation to humans and, consequently, a greater understanding of human risk from exposure to TAME. (+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)
Optical imaging of odorant representations in the mammalian olfactory bulb.
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We adapted the technique of intrinsic signal imaging to visualize how odorant concentration and structure are represented spatially in the rat olfactory bulb. Most odorants activated one or more glomeruli in the imaged region of the bulb; these optically imaged responses reflected the excitation of underlying neurons. Odorant-evoked patterns were similar across animals and symmetrical in the two bulbs of the same animal. The variable sensitivity of individual glomeruli produced distinct maps for different odorant concentrations. Using a series of homologous aldehydes, we found that glomeruli were tuned to detect particular molecular features and that maps of similar molecules were highly correlated. These characteristics suggest that odorants and their concentrations can be encoded by distinct spatial patterns of glomerular activation. (+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)
Olfactory event-related potentials in young and elderly adults: evaluation of tracking task versus eyes open/closed recording.
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The purpose of the present study was to evaluate olfactory event-related potentials (OERPs) elicited by amyl acetate from subjects performing a visuomotor tracking task compared with the no-task conditions of eyes open and eyes closed. Task condition did not produce any reliable effects for any amplitude measure. Task type weakly influenced only P2 latency. Elder adults evinced smaller P2 and N1/P2 amplitudes and longer N1 and P2 latencies than young adults. The results suggest that tracking task performance is not necessary to obtain robust OERPs from normal subjects of a wide age range. (+info)
Lipid-dependent activation of protein kinase C-alpha by normal alcohols.
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Significant stimulation of protein kinase C-alpha (PKCalpha) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO). The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED(50)) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol. Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity. (31)P NMR experiments did not reveal formation of non-lamellar phases with octanol. Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PKC-bilayer binding. (+info)
Odorants presented to the rat nasal cavity increase cortical blood flow.
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Complaints about unpleasant environmental odorants, both outdoor and indoor, are increasingly being reported. The main complaints of health symptoms from environmental odorants are eye, nose and throat irritation, headache and drowsiness. Complaints may arise from the stimulation of olfactory receptors or trigeminal chemoreceptors. Stimulation of cerebrovascular nociceptors originating from a branch of the trigeminal nerve may be associated with an increase in cortical blood flow which is thought to be related to headache. Since odorants are reported to elicit headaches, the possibility that odorants may increase cortical blood flow was examined. Cortical blood flow was monitored in rats using a laser-Doppler flowmeter. The flowmeter probe was placed over the left frontal cortex while propionic acid, cyclohexanone, amyl acetate or butanol was delivered to the nasal cavity via an olfactometer. Cortical blood flow increased as the concentration increased for three of the odorants tested. The greatest increase in blood flow occurred to the presentation of propionic acid, followed by cyclohexanone and amyl acetate. There was no response to butanol. These data demonstrate that odorants can alter cerebrovascular blood flow, which may account, in part, for one of the health symptoms reported for odorants. (+info)