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The green alga Chlorella pyrenoidosa was examined for its ability to metabolize 13-hydroperoxylinoleic and 13-hydroperoxylinolenic acids. The study showed that Chlorella extracts possessed hydroperoxide dehydrase and other enzymes of the jasmonic acid pathway. However, under normal laboratory conditions for culture growth, neither jasmonic acid nor metabolites of the jasmonic acid pathway were present in Chlorella. In vitro enzyme studies also revealed the presence of hydroperoxide lyase activity that cleaved 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acid into two products, 13-oxo-cis-9,trans-11-tridecadienoic acid and pentane (from linoleic acid) or pentene (from linolenic acid). The lyase was heat-labile, insensitive to 50 millimolar KCN, and had an approximate molecular weight of 48,000 as estimated by gel filtration. Two other products, 13-hydroxy-cis-9,trans-11,cis-15-octadecatrienoic acid and 12, 13-trans-epoxy-9-oxo-trans-10,cis-15-octadecadienoic acid, were also observed. Because these compounds are also products of nonenzymic, Fe(II)-catalyzed hydroperoxide decomposition reactions, their presence suggested that the observed lyase activity may occur via a homolytic decomposition mechanism.

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The metabolism of 13-hydroperoxylinolenic acid was examined in protoplasts and homogenates prepared from mature leaves of spinach (Spinacia oleracea L.). Chloroplast membranes were the principal site for metabolism of the compound by at least two highly hydrophobic enzyme systems, hydroperoxide lyase and hydroperoxide dehydrase, the new name for an enzyme system formerly known as hydroperoxide isomerase and hydroperoxide cyclase. Hydroperoxide lyase was most active above pH 7 and could be separated from hydroperoxide dehydrase by anion exchange chromatography. Hydroperoxide dehydrase, measured by the formation of both alpha-ketol product and 12-oxo-phytodienoic acid, had its optimum activity in the range of pH 5 to 7. Lyase was more active than dehydrase activity when the enzymes were extracted by homogenization. The reverse was true when the enzyme activities were measured in protoplasts, which are isolated by gentle extraction methods. The variation in enzyme activity ratios with extraction methods suggests that hydroperoxide lyase is activated by plant injury and thus may function in a wound response. In the absence of injury, the normal pathway of fatty acid hydroperoxide metabolism is probably by hydroperoxide dehydrase activity. The molecular weights of both the lyase and dehydrase were approximately 220,000, as estimated by gel filtration.

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12-Oxo-phytodienoic acid reductase, an enzyme of the biosynthetic pathway that converts linolenic acid to jasmonic acid, has been characterized from the kernel and seedlings of corn (Zea mays L.). The molecular weight of the enzyme, estimated by gel filtration, was 54,000. Optimum enzyme activity was observed over a broad pH range, from pH 6.8 to 9.0. The enzyme had a K(m) of 190 micromolar for its substrate, 12-oxo-phytodienoic acid. The preferred reductant was NADPH, for which the enzyme exhibited a K(m) of 13 micromolar, compared with 4.2 millimolar for NADH. Reductase activity was low in the corn kernel but increased five-fold by the fifth day after germination and then gradually declined.

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To determine whether an animal model could be used to study the susceptibility of women to antacid-induced phosphate deficiency, 6-wk-old male and female rats were given basic aluminum carbonate gel (Basaljel) (1 ml/100 g body wt) or distilled water by gastric intubation daily for 3 wk. Rats were fed either ad libitum (group 1) or by pair-feeding (group 2) with pelleted rat food containing 0.74% phosphorus. In group 1, baseline, 1-wk, and 3-wk values for serum phosphorus in Basaljel-treated females were 7.7 +/- 0.2, 6.3 +/- 0.2, and 6.2 +/- 0.2 mg/dl, respectively. Corresponding values for control females were 7.8 +/- 0.3, 7.0 +/- 0.2, and 7.3 +/- 0.2 mg/dl. Values for treated females were significantly lower (p less than 0.02) than values for control females by 1 wk of treatment. Basaljel-treated males did not differ from controls. The pattern for group 2 was similar. Intestinal absorption and intramuscular stores of phosphate were assessed in group 1. After 3 wk of treatment, [32P]phosphate assimilation from the duodenum into the body was lower in Basaljel-treated females than in controls (33.8% +/- 1.9% vs. 49.8% +/- 6.2% of the luminally administered dose, p less than 0.05). This was due to increased retention of [32P]phosphate in the intestine of treated females (19.9% +/- 2.0% vs. 11.9% +/- 2.4% for control females, p less than 0.02). Results in jejunum were similar. Total intramuscular phosphate in females was significantly lower (p less than 0.005) than in males both before and after antacid treatment. Thus hypophosphatemia in the female rat during antacid administration is probably secondary to the additive effects of decreased assimilation and decreased soft tissue stores of phosphate.

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Six plant species metabolized (18)O-labeled 12-oxo-cis,cis-10,15-phytodienoic acid (12-oxo-PDA) to short chain cyclic fatty acids. The plant species were corn (Zea mays L.), eggplant (Solanum melongena L.), flax (Linum usitatissimum L.), oat (Avena sativa L.), sunflower (Helianthus annuus L.), and wheat (Triticum aestivum L.). Among the products was jasmonic acid, a natural plant constituent with growth-regulating properties. The pathway is the same as the one recently reported by us for jasmonic acid synthesis in Vicia faba L. pericarp. First, the ring double bond of 12-oxo-PDA is saturated; then beta-oxidation enzymes remove six carbons from the carboxyl side chain of the ring. Substrate specificity studies indicated that neither the stereochemistry of the side chain at carbon 13 of 12-oxo-PDA nor the presence of the double bond at carbon 15 was crucial for either enzyme step. The presence of enzymes which convert 12-oxo-PDA to jasmonic acid in several plant species indicates that this may be a general metabolic pathway in plants.

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Linolenic acid was converted to a cyclic product, 12-oxo-phytodienoic acid, by lipoxygenase and hydroperoxide cyclase enzymes present in Vicia faba pericarp. Isotope labeling studies in which [U-14C] 12-[180] oxo-phytodienoic acid was incubated with thin sections of pericarp tissue showed that 12-oxo-phytodienoic acid is a biosynthetic precursor to jasmonic acid, a plant growth regulator which promotes senescence. Key enzymes proposed for this pathway are a reductase enzyme which reduces a double bond in the cyclopentenone ring, and beta-oxidation enzymes which remove six carbons from the carboxyl end of the molecule.

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Three oxygenated unsaturated fatty acids were investigated to determine whether they were present in seedlings of corn (Zea mays L. cv. NK PX443) and sunflower (Helianthus annuus L. cv. Sundak). The three compounds, 13-hydroxy-12-oxo-cis-9-octadecenoic acid (I), 13-hydroxy-12-oxo-cis,cis-9, 15-octadecadienoic acid (II), and 12-oxo-cis,cis-10, 15-phy-todienoic acid (III), were detected and estimated by gas chromatography-mass spectrometry selected ion monitoring of their trimethylsilyloxy, methyloxime derivatives with 20-carbon analogs added as internal standards. In corn, the concentration of III increased between 5 and 10 days, while I and II remained relatively constant. A higher concentration of II was observed in corn seedlings grown in the light than those grown in the dark. Wounding increased the levels of all three compounds. In sunflower seedlings, the concentrations of I, II, and III increased between 6 and 10 days. The intracellular concentration of III in 10-day-old light-grown seedlings was estimated to be 200 nm in corn and 40 nm in sunflower.

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The oxidation of linoleic acid in incubation mixtures containing extracts of barley lipoxygenase and hydroperoxide isomerase, and the production of these enzymes in quiescent and germinated barley, were investigated. The ratio of 9-hydroperoxylinoleic acid to 13-hydroperoxylinoleic acid was higher for incubation mixtures containing extracts of quiescent barley than for mixtures containing extracts of germinated barley; production of 13-hydroperoxylinoleic acid from germinated barley exceeded that of quiescent barley. Hydroperoxy metabolites of linoleic acid were converted to 9-hydroxy-10-oxo-cis-12-octadecenoic acid, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, and small amounts of 11-hydroxy-12,13-epoxy-cis-9-octadecenoic acid and 11-hydroxy-9,10-epoxy-cis-13-octadecenoic acid whether quiescent or germinated barley was the enzyme source; a fifth product, 13-hydroxy-12-oxo-cis-9-octadecenoic acid was formed only when germinated barley was the enzyme source.Lipoxygenase was readily extracted by buffer, but hydroperoxide isomerase was bound in a catalytically active state to the insoluble barley grist and was efficiently extracted only when Triton X-100 was included in the extraction buffer. Hydroperoxide isomerase was localized in the embryo of quiescent barley, but it was present in the embryo, acrospire, and in small but concentrated amounts in the rootlet of germinating barley. The levels of both lipoxygenase and hydroperoxide isomerase increased through the thirteenth day of germination.

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Lipoxygenase was demonstrated in young cotton seedlings. It catalyzed the oxygenation of linoleic or linolenic acid, predominantly at carbon 13, and its molecular weight was estimated by gel filtration to be 100,000. Hydroperoxide isomerase was also present and converted hydroperoxylinoleic or hydroperoxylinolenic acid to alpha- or gamma-ketols. The enzyme utilized the 13-hydroperoxy isomer in preference to the 9 isomer and its molecular weight was estimated at 250,000 by gel filtration. In addition, hydroperoxide cyclase, which catalyzes the conversion of 13-hydroperoxylinolenic acid to 12-oxo-phytodienoic acid, was present. Hydroperoxide isomerase and hydroperoxide cyclase activities could not be separated by gel filtration and ion-exchange chromatography experiments, indicating the two enzyme activities may be associated with the same protein. The activities of all three enzymes were very low in the seed but increased immediately after germination, reached a maximum after 3 to 4 days, and then declined. The results suggest a role, as yet unknown, for these enzymes during early plant development.

RESUMEN

13-[18-O]Hydroperoxylinolenic acid was permitted to react with an extract of flaxseed acetone powder containing hydroperoxide cyclase activity. The resulting product, 12-oxo-cis-10,cis-15-phytodienoic acid (12-oxo-PDA), contained 18O in the carbonyl oxygen at carbon 12, suggesting that an epoxide was an intermediate in the hyderoperoxide cyclase reaction. A substrate specificity study showed that a cis double bond beta, gamma to the conjugated hydroperoxide group was essential for the substrate to be converted to a cyclic product by hydroperoxide cyclase.

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A direct data link between a CT scanner and a radiation treatment planning system has been developed. The link transmits the data serially over a coaxial cable using the pseudo-paper-tape punch and reader (serial PIO) interfaces. The data transmission rate with error-check is approximately 25,000 8-bit bytes/s. This translates to about 7 s for transferring a CT scan with a 320-pixel diameter.

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12-Oxo-cis-10,15-phytodienoic acid is an enzymic product obtained from incubations of (9, 12, 15)-linolenic acid with extracts of flaxseed (Linum usitatissimum L.). 13-l-Hydroperoxy-cis-9, 15-trans-11-octadecatrienoic acid, a product of lipoxygenase catalysis, was an intermediate in the enzymic synthesis of 12-oxo-cis-10, 15-phytodienoic acid from (9, 12, 15)-linolenic acid. Substrate specificity studies showed that n-3,6,9 unsaturation was an absolute requirement for conversion of polyunsaturated fatty acids into analogous products containing a cyclopentenone ring. Fatty acids with 18, 20, or 22 carbons that satisfied this requirement were effective substrates. The optimum activity of the enzyme from flaxseed was at pH 7.2.

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12-Oxo-trans-10-dodecenoic acid (trans-10-ODA) is an oxidation product of polyunsaturated fatty acids in plant tissues. The structural similarity of trans-10-ODA and traumatic acid, a compound considered to be a wound hormone, suggested that trans-10-ODA might be a precursor of traumatic acid. Both trans-10-ODA and traumatic acid were active in the Wehnelt bean assay. The results were more consistent with trans-10-ODA than with traumatic acid. Cucumber (Cucumis sativus L. var. National Pickling) hypocotyls also showed a growth increase following treatment with trans-10-ODA, which suggested that trans-10-ODA has a more general influence on plant development than previously ascribed to traumatic acid.Runner beans (Phaseolus vulgaris L. var. Kentucky Wonder) were analyzed for the presence of endogenous trans-10-ODA and traumatic acid. These are the beans from which traumatic acid was originally isolated in 1939. They contained trans-10-ODA but no traumatic acid. Young beans were a better source of trans-10-ODA than older beans and an increase in the esterified form of trans-10-ODA with age may have been due to a conversion of the free acid to the esterified form. The amount of endogenous trans-10-ODA increased when bean pod tissue was sliced and wounded. Rapid stirring and the presence of oxygen increased autooxidation of trans-10-ODA to traumatic acid in runner beans, which indicated that the compound identified as traumatic acid is formed by autooxidation of trans-10-ODA and that trans-10-ODA is a natural compound with growth-regulating properties.Enzyme extracts of runner beans synthesized trans-10-ODA from linoleic acid. No enzymic synthesis of traumatic acid was observed even when cofactors were added to the reaction mixture. This confirmed the conclusion that traumatic acid is formed by autooxidation of trans-10-ODA.

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Occupational exposure to inhalation anesthetics can be substantially reduced by control measures that have been recently developed. The incentive for their use should be the suspected relationship between chronic exposure to trace concentrations of agents, such as nitrous oxide, and health hazards. Control measures include the use of a newly developed scavenging nasal mask, relatively gastight anesthetic equipment, and vented suction machine, supported by an air monitoring program. With these measures, the concentration of nitrous oxide inhaled by the oral surgeons studied was reduced 97%.

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Methods were developed for controlling the dental team's occupational exposure to nitrous oxide. The most applicable and effective use of these methods included the use of properly maintained gas delivery equipment, a double-walled scavenging nosepiece and vented suction machine, and minimizing speech by the patients. These methods were evaluated by measuring concentrations of nitrous oxide present in the air inspired by dental personnel. Before their use, the dentist inhaled 900 ppm nitrous oxide; their application reduced his inhaled concentration to 31 ppm, representing a 97% reduction. These methods were well accepted during 157 procedures completed by a group of eight dentists engaged in private practice (four general practitioners, two pedodontists, and two oral surgeons).

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Lipoxygenase (EC 1.13.1.13) was found in seedlings of Citrullus lanatus (Thunb.) Matsum. and Nakai (watermelon). The enzyme has pH optima of 4.4 and 5.5 and is inhibited by 0.2 mM nordihydroguaiaretic acid. It is present in two functional units with estimated molecular weights of 120,000 and 240,000, respectively.A new enzyme, tentatively termed hydroperoxide lyase, has been partially purified from watermelon seedlings. The enzyme, located principally in the region of the hypocotyl-root junction, catalyzes the conversion of 13-l-hydroperoxy-cis-9-trans-11-octadecadienoic acid to 12-oxo-trans-10-dodecenoic acid and hexanal. The hydroperoxide lyase enzyme from watermelon has a molecular weight in excess of 250,000, a pH optimum in the range of 6 to 6.5, and is inhibited by p-chloromercuribenzoic acid. Its presence has also been demonstrated in other cucurbits.The maximum activity of both enzymes occurs on the 6th day of germination. The identification of the products of the hydroperoxide lyase reaction suggests that lipoxygenase and hydroperoxide lyase may be involved in the conversion of certain polyunsaturated fatty acids to traumatic acid (trans-2-dodecenedioic acid).

RESUMEN

Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies.

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