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Synthesis and characterization of stereospecific 1-propargyl-2-(dimethoxymethyl)-1-cyclohexanols.

Stereochemical isomers with hydroxy groups were synthesized by reacting 2-(dimethoxymethyl)cyclohexanone with propargylmagnesium bromide. The stereo chemical structures were identified by NMR spectral interpretation and the geometry optimization. To assist the NMR interpretation, geometry optimization based on semi-empirical AM1 and PM3 methods was applied. Throughout this study, the structures of the two isomers were all determined and 1H and 13C NMR spectra were fully assigned. It was proven that the less polar isomer is an axial alcohol and the more polar one is an equatorial alcohol.

Alkynes↗

Synthesis and biological activity of conformationally controlled 2-PAM derivatives.

A series of conformationally controlled 2-PAM derivatives were prepared from 2-acetylpyridine and 2,3-pyrido[b]cycloalkenones in two steps and their reactivities towards parathion poisoned AChE were evaluated. The most planar 2,3-pyrido[b]cyclohexanone oxime methiodide showed an activity comparable to 2-PAM implying E-syn is that the most active conformation of 2-PAM in the biological system.

Acetylcholinesterase↗

[Toxic anosmia and rhinitis due to occupational solvent exposure].

A patient's case report documents the development of anosmia and rhinitis caused by occupational exposure to organic solvents, including tetrahydrofuran, cyclohexanone, acetone and other ketones. Physicians should inquire about possible mucosal irritation or prenarcotic symptoms during exposure. If an occupational disease is assumed, notification must be sent to the Institution for Statutory Accident Insurance and Prevention (Berufsgenossenschaft). Some diseases, for example olfactory disorders due to chemical mixtures, are not included in the German list of occupational diseases. In this case, a special law (Sozialgesetzbuch VII) regulates compensation.

Adult↗

Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106.

Sequencing of a genomic library prepared from Pseudomonas fluorescens DSM 50106 identified an orf showing 29% identity to a C alpha-dehydrogenase of Pseudomonas paucimobilis and high homology to several sequences with unknown functions derived from genome projects. The corresponding gene adhF1 encodes a dehydrogenase of 296 amino acids with a calculated molecular mass of 31.997 kDa. The gene was functionally expressed in E. coli using a rhamnose inducible expression system. The resulting recombinant enzyme was active in the pH range 6-10 (best pH 8) and at 5-25 degrees C. This dehydrogenase converts cyclic ketones to the corresponding alcohols utilizing the cofactor NADH. The highest activity was found for cyclohexanone. The enzyme also exhibits high stereoselectivity in the desymmetrization of the prochiral ketone acetophenone, producing optically pure ( R)-alpha-phenyl ethanol (>99%ee) at high conversion (95%).

Alcohol Dehydrogenase↗

Colorimetric estimation of inorganic phosphate in colored and/or turbid biological samples: assay of phosphohydrolases.

A simple method of inorganic phosphate determination for colored and/or turbid biological samples is described. The procedure is mild, and so is suitable for routine phosphohydrolase assays. Following deproteinization by ice-cold trichloroacetic (or silicotungstic) acid, the sample was treated with acid-washed charcoal to remove interference due to color. The phosphate in the colorless supernatant was assayed either by measuring the phosphomolybdate spectrophotometrically at 310 nm, following its extraction in organic solvents or by a modified Fiske and Subbarow method. The turbidity interference in the latter case was eliminated either by centrifugation, by sodium dodecyl sulfate treatment, or by extraction of reduced phosphomolybdate blue color by cyclohexanone. Though deproteinization by silicotungstic acid eliminated the turbidity problem, its use in conjunction with charcoal treatment was not convenient.

Animals↗

Analysis of aldehydic lipid peroxidation products in rat liver and hepatocytes by gas chromatography and mass spectrometry of the oxime-tert-butyldimethylsilyl derivatives.

A method for analysis of aliphatic aldehydes in biological samples is described. Cyclohexanone is added as internal standard and the samples are treated with hydroxylamine and perchloric acid. The oximes are extracted and converted to the oxime-tert-butyldimethylsilyl derivatives, which are quantitated by capillary gas chromatography and identified by mass spectrometry. The characteristic M-57 fragment ions in the mass spectra enabled a rapid identification of the derivatives of the aldehydes, alkanals, alk-2-enals, alka-2,4-dienals, and 4-hydroxyalk-2-enals, which in addition gave rise to characteristic double peaks in the gas chromatographic analysis. The method was applied to analysis of autoxidized arachidonic acid, ADP-Fe3(+)-treated rat hepatocytes, and rat liver given a single dose of ethanol, 5 g/kg. The amounts of hexanal and 4-hydroxynon-2-enal were not increased 6 h after the administration of ethanol.

Adenosine Diphosphate↗

Ethanol metabolism in isolated hepatocytes. Effects of methylene blue, cyanamide and penicillamine on the redox state of the bound coenzyme and on the substrate exchange at alcohol dehydrogenase.

Ethanol metabolism in hepatocytes increases the NADH/NAD+ ratio. The mechanism was investigated by measurements of the redox state of the coenzyme bound to alcohol dehydrogenase and of ethanol-acetaldehyde exchange and concomitant hydrogen transfer between ethanol molecules. Isolated hepatocytes from fed rats were incubated with cyclohexanone and cyclohexanol or with [1,1-2H2]-and [2,2,2-2H3]ethanol, followed by gas chromatographic determination of the redox state and isotope analysis of the ethanol by gas chromatography-mass spectrometry, respectively. Cyanamide and methylene blue decreased the redox shift caused by ethanol and increased the rates of acetaldehyde reduction during the exchange. Both drugs increased the extent of hydrogen transfer between ethanol molecules during oxidoreduction. Penicillamine had no significant effect on the ethanol-induced change in redox state of the bound coenzyme although it decreased the rate of acetaldehyde reduction. The results indicate that methylene blue inhibits aldehyde dehydrogenase and that accumulation of acetaldehyde decreases the redox effects of ethanol. The redox effect appears to result primarily from rapid elimination of acetaldehyde and equilibration with the NAD system on the alcohol dehydrogenase, but is not enhanced by further decreases in acetaldehyde concentration. Thus, penicillamine could probably be used to decrease the concentration of acetaldehyde without increasing the redox effects.

Acetaldehyde↗

Receptor cell responses to odorants: similarities and differences among odorants.

An extensive exploration of the discriminating properties of olfactory receptors cells has been conducted in our laboratory through the statistical processing of single cell responses recorded in the frog's olfactory epithelium. Similarities between odorant stimulating properties could be demonstrated by comparing the complex response profiles of receptor cells, resulting in the concept of odorant groups which depicts consistent relationships found between some odorous compounds. The recording technique limits the number of odorants which can be tested and compared within the same series of experiments. Thus, based upon previous studies we have chosen a specific set of 20 odorants whose characteristics are known but, up to this time, have not been compared in the same experimental system. These odorants were: acetophenone, anisole, n-butanol, DL-camphor, cyclodecanone, 1,8-cineole, p-cymene, D-citronellol, n-heptanol, isoamyl acetate, isovaleric acid, D-limonene, methyl amylketone, L-menthol, phenol, thiophenol, pyridine, thymol, cyclohexanol, cyclohexanone. The pattern of similarities between these odorants, as delineated with the aid of correlation coefficient computation, factor analysis and non-hierarchical taxonomy, confirms the reality of several odorant groups previously suggested and describes their cross-relationships. The receptor mechanisms underlying these odorant groups are discussed. Because reliable prominent features of the olfactory stimulus space can be established on an objective basis, the findings are proposed as a reference for future studies on other aspects of olfactory discrimination.

Animals↗

Olfactory discrimination of structurally related molecules: receptor cell responses to camphoraceous odorants.

Electrophysiological investigations of the selective sensitivity of receptor cells have shown that odorants could be distributed into groups according to the profiles of responses that they elicited from a population of receptor cells. As the concept of odorant group plays an important role in the understanding of olfactory coding, an attempt was made to investigate in detail the group properties of a number of structurally and qualitatively related molecules: 1,8 cineole, DL-camphor, borneol, iso-borneol, adamantane, fenchone, fenchyl alcohol, 3,3,5-tri-methyl cyclohexanone, camphene, 1,1,2,2-tetra-bromoethane. These odorants, along with several other representatives of other odorant groups, were utilized to stimulate a set of 71 individually recorded receptor cells. Factor analysis of electrophysiological data demonstrated that the grouping of odorants with camphor could be predicted on the basis of the shape and size of the molecules. The analysis also showed that even a clearly defined odorant group such as the camphor group is not likely to be determined by a single type of receptor site.

Animals↗

The binding of an aminoazo dye carcinogen to a specific methionine residue in rat liver alcohol dehydrogenase in vivo.

On the administration of 3'-methyl-N,N-dimethyl-4-aminoazobenzene to rats pure aminoazo dye-bound alcohol dehydrogenase accounting for 45% of the total soluble protein bound aminoazo dye is isolated from the liver soluble supernatant. Tryptic digestion of that purified aminoazo dye-bound enzyme yields an aminoazo dye-bound nonapeptide which has a sequence identical to amino acids 301-309 in the known sequence of alcohol dehydrogenase (H. Jornvall and O. Markovic, Eur. J. Biochem., 29 (1972) 167-174) with the exception of methionine 306 which is replaced by an aminoazo dye modified amino acid. The nature of the aminoazo dye adduct was determined by studying the structure of the related tetrapeptide obtained by Pronase B digestion and shown by proton NMR spectroscopy and fast atom bombardment mass spectroscopy to have the structure 3-(Val. Asn. Pro. Homocystein-S-yl)-4-methylamino-3'-methylazobenzene. This carcinogen-protein adduct is assumed to arise from attack of the ultimate carcinogenic metabolite, N-sulphonyloxy-4-methylamino-3'-methylazobenzene (FF. Kadlubar, J.A. Miller and E.C. Miller, Cancer Res., 36 (1976) 2350-2359) at the sulphur of methionine 306 followed by spontaneous S-demethylation. This highly specific reaction of carcinogen with alcohol dehydrogenase lowers its Vmax and increases its Km with cyclohexanone thereby reducing its catalytic efficiency for this substrate. This highly specific reaction of the carcinogen with alcohol dehydrogenase may be regarded as a major detoxication reaction.

Alcohol Dehydrogenase↗

A study of muscarinic receptor heterogeneity with weak antagonists.

A study of heterogeneity among muscarinic receptors was carried out with new rigid molecules, comprising structures in the fused quinuclidine-valerolactone, quinuclidine-cyclohexenone, quinuclidine-cyclohexanone and quinuclidine-cyclohexane derivatives. These are structurally related to the potent muscarinic agent, 3-acetoxyquinuclidine but substantially different from in it conformation. All proved to antagonize acetylcholine-like activity, but to a different extent in different systems. The equipotent molar ratio with respect to atropine (as 1) was: isolated guinea pig ileum, 10,000-1,000; salivary gland (mouse), 1,000-100; superior cervical ganglion (cat), 100-10; CNS (mouse), approximately 10. It is suggested that the rigid structure induces a three-point constrained fit in the receptor (onium group, hydrophobic moiety and carbonyl group), but that not all muscarinic receptors are capable of responding equally. In this case, receptor specificity of the drug is a direct consequence of its graded departure from the preferred conformation of acetycholine and, therefore, is necessarily associated with partial loss of potency.

Animals↗

Mechanisms of epidermal growth factor-induced contraction of guinea pig airways.

We investigated the functional effects of epidermal growth factor (EGF) on guinea pig airways in vitro. EGF (3 ng/ml to 1 microgram/ml) induced a concentration-dependent contraction in epithelium-denuded strips. The average maximal contraction was 0.64 +/- 0.1 g (mean +/- S.E., for n = 27), which was 72.0 +/- 9.5% of the 100 mM KCl-induced contraction. The EC50 was 12.3 +/- 1.6 ng/ml. The presence of the epithelium significantly suppressed the EGF-induced contraction (P < 0.01). EGF-induced contraction was abolished by cyclooxygenase inhibitors (indomethacin and ibuprofen) and a 5-lipoxygenase inhibitor, 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benz oqu inone (AA-861). It was also inhibited by a leukotriene-receptor antagonist, 8-[p-(4-phenylbutyloxy)benzoyl]amino-2-(tetrazol-5-yl)-4-oxo -4H-1-benzopyran hemihydrate (ONO-1078) but not affected by a thromboxane A2-synthetase inhibitor, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid (OKY-046) or a thromboxane A2-receptor antagonist, 9,11-epithio-11,12-methano-thromboxane A2 (ONO-3708). A phospholipase A2 inhibitor (mepacrine) inhibited the EGF-induced contraction but a diacylglycerol-lipase inhibitor, 1,6-di-(O-(carbamoyl)cyclohexanone oxime)hexane (U-57908) and a phospholipase D inhibitor (wortmannin) did not affect it. A tyrosine kinase inhibitor (genistein) abolished it. Measurement of prostanoids showed that EGF (300 ng/ml) did not increase the prostaglandin F2 alpha level in either epithelium-intact or epithelium-denuded strips. In epithelium-intact strips, EGF significantly increased the prostaglandin E2 concentration (P < 0.01). These results suggest that EGF causes contraction of guinea pig airway smooth muscle by activating tyrosine kinase followed by phospholipase A2 activation, and that arachidonic acid metabolites, especially leukotrienes, may have important roles in this contraction.

Animals↗

A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase.

The use of enzymes requiring a cofactor as substrate in organic synthesis is still a problem since the cofactors are expensive. This study deals with a new approach consisting of using fragments of NAD+. Three fragments of NAD(H) are examined. The activities of NMN+ and NMNH are greatly improved by the addition of adenosine in ethanol oxidation and in cyclohexanone reduction, respectively. Nicotinamide mononucleoside is not active in the ethanol oxidation but the addition of AMP promotes this reaction.

Adenosine↗

Decrease in oligodendrocyte carbonic anhydrase activity preceding myelin degeneration in cuprizone induced demyelination.

Both immunohistochemical and biochemical evidence is presented to show for the first time that carbonic anhydrase II (CA II) activity falls in the brain of mice in cuprizone (bis(cyclohexanone)oxalyldihydrazone) induced demyelination well before demyelination develops. This fall began during the first week, whereas the first signs of myelin degeneration induced by cuprizone did not appear until 3 weeks and demyelination in the superior cerebellar peduncle in the mouse took 6-8 weeks to develop. The findings suggest that oligodendrocyte CA II activity is essential either for the survival of oligodendrocytes or for the maintenance of central myelin.

Animals↗

Hyperthermic and anorectic effects of oxazolidines derived from L-ephedrine in rats.

Oxazolidines synthesized from (-) ephedrine have been proposed as potential pro-drugs, but no pharmacological data on these compounds has been yet reported. In this study, four such compounds are tested in rats for ephedrine-like activity using the hyperthermia and anorexia models. The compounds were synthesized by reaction of (-) ephedrine with salicylaldehyde, acetone, cyclohexanone, and benzaldehyde, respectively. The results showed that all of the compounds decreased food intake significantly, but only the acetone and the salicylaldehyde derivatives caused a significant elevation of body temperature. All of the compounds were less effective than (-) ephedrine in the anorexia model. The acetone and salicylaldehyde derivatives showed similar potency to (-) ephedrine in the hyperthermia model.

Animals↗

Effect of odor quality and intensity on conditioned odor aversion learning in the rat.

Odor quality and intensity were varied to test the ability of rats to associate odor with an induced illness. Rats were allowed 10 minutes access to water on each of nine days; deodorized air was directed towards each rat's nose while drinking at familiarization and recovery sessions (days 1-5 and 7-8, respectively) and odorized air at treatment and test sessions (days 6 and 9, respectively). Each rat was injected with LiCl following its drinking period on day 6. The difference between the amount of water consumed on day 6 and day 9 gave a measure of the conditioned aversion. Only mild or no aversion occurred with odors of n-butyric acid, benzylamine, cyclohexanone, and n-butanol. Strong conditioned aversions were obtained to odors of triethylamine, 1,4-cineole, and isoamyl acetate, and the degree of aversion increased linearly with the log of odor concentration. The effect of odor quality, intensity and presentation method, and the role of the different chemoreceptor systems in the acquisition of odor aversions are discussed.

Animals↗

Rat fetuses respond to chemical stimuli in gas phase.

Fetuses exhibit behavioral responses to intraoral infusions of chemical solutions presented in liquid phase, including an increase in overall activity and the production of a species-typical action pattern, facial wiping. A series of experiments were conducted in which chemical compounds with strong olfactory characteristics--citral and cyclohexanone--were presented in gas phase to rat fetuses on day 20 of gestation. Fetuses exhibited similar behavioral responses to stimuli presented in liquid and gas phases, suggesting a common basis for the processing of prenatal chemosensory information. Chemosensory stimuli in gas phase are sufficient to elicit both behavioral activation and a stereotypic action pattern of the rat fetus, patterns that anticipate postnatal olfactory-directed behavior. These results suggest that prenatal chemosensory experience, which normally occurs in an aqueous medium, may be relevant to the development of postnatal behavior that is directed by airborne olfactory cues.

Animals↗

Concentration-dependent behavioral changes in mice following short-term inhalation exposure to various industrial solvents.

Mice were exposed during a 4-hr period to various concentrations of 13 aliphatic or aromatic solvents which affect primarily the central nervous system (CNS). The test compounds were benzyl chloride, butyl alcohol, chlorobenzene, cyclohexanone, 1,2-dichloroethylene, diisobutyl ketone, isopropyl acetate, methyl ethyl ketone, styrene, tetrachloroethylene, 1,1,1-trichloroethane, toluene, and ortho-xylene. After exposure, measurements were made to see whether these neurotoxicants would decrease the immobility developed in a "behavioral despair" swimming test. Each chemical was shown to reduce the total duration of immobility measured over a 3-min period in a concentration-related manner. The systematic determination of the atmospheric concentrations responsible for a 50% decrease in immobility (ID50) permitted classification of the solvents in terms of their relative potencies. The possibility of using such experimental data as tentative guidelines for setting safe levels of work exposure to the neurotoxicants was suggested, considering the existence of quantitative relationships between the ID50 values and the current occupational standards.

Animals↗