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.     

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.     

Biotization of Arabidopsis thaliana with Pseudomonas putida and assessment of its positive effect on in vitro growth

Determination of the optimal inoculation method and concentration to use for plant-bacteria interaction studies is important in many cases, such as the phytoremediation of heavy metals and other toxic compounds in contaminated areas. The aim of this study was to compare different concentrations and times of inoculation of Pseudomonas putida with various growth stages of Arabidopsis thaliana in 14-d in vitro cultures. A significant beneficial impact of the bacterium was detected in the shoot length and root weight of seedlings. The highest shoot length and root fresh and dry weights were detected in 14-d and 2 × 10³ cfu mL⁻¹ inoculated samples. In addition, the increase in root weight could be visualized with crystal violet staining, as relatively more root hair and lateral root formation occurred in seedlings inoculated with moderate concentrations of bacteria, possibly due to the ability of P. putida to produce indole-acetic acid. Moreover, the highest photosynthetic pigment accumulation was obtained with the highest bacterial inoculum (2 × 10⁶ cfu mL⁻¹), which was tested in 0- or 3-d-old seedlings. Rhizospheric bacterial colonization was also visualized with GFP-labeled bacteria by confocal microscopy. These results showed that biotization of A. thaliana with P. putida KT2440 did not cause severe oxidative stress in seedlings, because H₂O₂ accumulation levels together with CAT and POX activities were not significantly induced. Therefore, this strain could be used for several applications based on plant-bacteria interactions.