Mass spectral characterization of two onion constituents with prostaglandin E-like activity as lipoxygenase metabolites of linoleic acid

Onions ($\text{Allium cepa}$ have been used in folk medicine for the treatment of hypertension and gastrointestinal ulcers, which are cases where the administration of prostaglandins is considered. Using bioassay guided fractionation and gas chromatography/mass spectrometry (GC/MS) we have isolated two biologically active fractions with PGE-like properties and have characterized these products as lipoxygenase metabolites of linoleic acid.For the isolation of the active principles, onion bulbs were homogenized in phosphate buffer and the PGE-like products were isolated using Amberlite XAD-2 absorption, silicic acid column chromatography and silicagel thin layer chromatography. The PGE-like activity was estimated in a cascade superfusion system in which the isolated rabbit coeliac artery, the rabbit mesenteric artery and the rat fundus were used as assay organs.For the GC/MS characterization, various types of volatile derivatives were prepared in order to facilitate the structure elucidation. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation. The active fractions yielded identical electron-impact mass spectra, indicating that they are stereoisomeric, and each fraction was identified as a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic and of 9,10,13-trihydroxy-11-octadecenoic acid. With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butyl boronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol function.The isomeric trihydroxylated octadecenoic acids have been described for the first time as metabolites of linoleic acid in wheat flour incubates. In this system, the trihydroxylated octadecenoic acids were shown to be formed through a sequence of three reactions, including an initial 9- or 13-lipoxygenation yielding hydroperoxy octadecadienoic acids, followed by rearrangement into unstable allylic epoxy hydroxy octadecenoic acids, which in turn are hydrolyzed to trihydroxy octadecenoic acids. Furthermore, structurally related trihydroxy octadecadienoic acids have also recently been isolated from a plant source, more specifically from the roots of $\text{Bryonia Alba}$, which are used for the same medicinal purposes as onion bulbs.     

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.     

Flavonoids from Haplophyllum pedicellatum, H. robustum AND H. glabrinum

Haplophyllum pedicellatum, H. robustum and H. glabrinum all yielded the known compound gossypetin 8,3′-dimethyl ether 3-rutinoside. In addition the first two species afforded isorhamnetin and its 3-rutinoside. A new glycoside, gossypetin 8,3′-dimethyl ether 3-glucoside was obtained from H. pedicellatum together with the 3-malonylrutinoside, 3-malonylglucoside and 3-galactoside of isorhamnetin plus kaempferol 3-malonylglucoside. H. robustum yielded isorhamnetin 7-glucoside and 3-glucoside and quercetin 3-galactoside, while H. glabrinum was found to contain gossypetin 8-methyl ether 3-malonylrutinoside in addition to kaempferol and isorhamnetin 3-glucoside.     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Changes in macromolecule syntheses under special consideration of UNA and the energy providing system in imbibing embryos of different agedagrostemma githago L. Seeds

Changes in macromolecule syntheses, especially RNA synthesis, and the energy providing system were investigated in seeds ofAgrostemma githago aged for different periods. In embryos of aged seeds all macromolecule syntheses start later and reach a lower level than young ones. It was found that the synthesis of rRNA in embryos of aged seeds is reduced whereas the synthesis of poly (A⁺) RNA in relation to the total RNA synthesis is highly increased as well as the amount of this RNA species with long poly (A) chains. The results are discussed in connection with the decreased protein synthesis and the reduced ATP content and ATP formation ability in embryos during the long time storage of seeds.     

Parasitic fungi from the province of Manisa

This paper is a study of the parasitic fungi of Manisa. 32 species of parasitic fungi have been discovered of which 2 species are new for the Turkish parasitic fungal flora. Also, new hosts for 13 of these species are reported in Turkey for the first time.     

Alpha-fetoprotein synthesis in clonogenic cells of rat hepatoma McA-RH 7777

Production of alpha-fetoprotein (AFP) was determined in single cells of hepatoma McA-RH7777 and in the clones of their progeny. To elucidate the heritability of this trait in a series of cell generations, a variety of local hemolysis in gel was devised. According to the method the cells and red cells conjugated with protein A were placed on the polylysine covered surface and layered with agarose gel containing antibodies. AFP production by single cells was determined from the formation of plaques--areas of red cell hemolysis. The cells forming the plaques (+AFP) and not forming them (-AFP) were distinguished and their reproduction was followed up. After 7-14 days the cells were fixed and stained by the immunoperoxidase technique with antibodies to AFP. High efficacy of the cloning has been demonstrated for both +AFP- and -AFP-cells (69 and 71%). Negative cells preserved their phenotype more frequently, producing homogenous negative clones, whereas +AFP cells gave "negative" clones in 1/3 of the cases. Both cells gave mixed clones in a small percentage of the cases. At present the AFP trait in these cells is being studied by recloning.     

Cell surface component(s) involved in rat hepatocyte intercellular adhesion

Aggregation of rat hepatocytes was effectively inhibited by monovalent antibodies (Fab fragments) directed against hepatocyte plasma membranes, but monovalent antibodies against some distinct, known hepatocyte surface antigens had no effect. Surface antigens, which neutralized the Fab inhibiting effect on cell aggregation, could be solubilized from plasma membranes by limited proteolytic digestion. Thus, hepatocyte intercellular adhesion seems to involve specific cell surface components, which may be proteins or protein derivatives.