The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Protoplast isolation and callus production from leaves of tissue-cultured Vitis spp.

Protoplasts were isolated from mesophyll of axenic cultures of grape, Vitis rotundifolia cv. Summit and V. vinifera cv. Cabernet Sauvignon. Enzymes effective for protoplast isolation were Macerozyme R-10 (0.5% and 0.1%) and Cellulase Onozuka R-10 (1.0% and 0.5%) for V. rotundifolia and V. vinifera, respectively. Polyvinylpyrrolidone and 2-(N-morpholino)ethanesulfonic acid were essential in the isolation media. Protoplasts were purified using flotation/centrifugation. The protoplasts of V. rotundifolia cultured in Gamborg's B5 basal medium with 2.2 M 6-benzyladenine, 4.5 M 2,4-dichlorophenoxyacetic acid and 0.4% agarose gave the best plating efficiency of conditions tried in this study. Cell division occurred within 5 to 6 days and visible microcalli developed within one month. After 6 weeks in culture, microcalli transferred to liquid medium exhibited active callus growth. Protoplasts of V. vinifera cultured under these conditions had similar results.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid monohydrate - PVP polyvinylpyrrolidone - PDS potassium dextran sulfate - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate     

Respiration-dependent proton translocation and the mechanism of protonmotive force generation in Nitrobacter winogradskyi

Abstract Proton translocation associated with electron flow to oxygen has been observed with cells of Nitrobacter winogradskyi in the presence of either potassium ferrocyanide or isoascorbate plus N , N , N ', N ' tetramethyl- p -phenylenediamine. The data are consistent with a proton pumping function for the terminal oxidase, cytochrome aa ₃, in this organism as the mechanism for generating a protonmotive force. The failure of previous work with Nitrobacter [4] to detect proton translocation linked to oxidation of nitrite, the physiological substrate, is discussed.     

Elektronenmikroskopische Untersuchungen am Subcommissuralorgan des Meerschweinchens

Zusammenfassung Im Subcommissuralorgan des Meerschweinchens wird das mehrreihig hochprismatische Ependym von einem wechselnd breiten, gefäßführenden Hypendym unterlagert, das neben Astrocyten, Oligodendrocyten und Ependymfortsätzen Zellen mit den feinstrukturellen Merkmalen der Ependymzellen enthält.Die subcommissuralen Zellen in Ependym und Hypendym bilden verschiedene Arten von Sekret: Von den Cisternen des endoplasmatischen Reticulum schnüren sich helle Sekretsäckchen ab, die das Cytoplasma durchsetzen. Im apikalen Zellbereich konfluieren sie mitunter zu großen, unregelmäßig begrenzten Sekretarealen. Helle Sekretsäckchen liefern auch den Rohstoff für dichte Sekretgranula, die in manchen Zellen vom Golgi-Apparat gebildet werden; die in verschiedenen Varianten vorkommenden Granula sind apikal angehäuft. Vereinzelt anzutreffende Zellen sind mit Sekretvakuolen so dicht angefüllt, daß das Cytoplasma zu schmalen, dichten Stegen reduziert ist; der Zellkern ist pyknotisch. Die Sekretvakuolen enthalten sehr wenig flockiges Material. Im Hypendym konfluieren die Sekretvakuolen zu großen intracellulären Hohlräumen, in die gelegentlich Mikrovilli und Cilien hineinragen. Schließlich ist eine Art von apokriner Sekretion zu beobachten: Manche Ependymzellen besitzen nahezu homogene Protrusionen, die weit in den Ventrikel reichen; isoliert im Ventrikel werden organellenfreie Cytoplasmabereiche gefunden.Die Kapillaren besitzen ein unterschiedlich breites Endothel. Die Basalmembran ist an vielen Stellen aufgeweitet und umschließt kleine, helle Bezirke. Häufig ist ein echter perivasculärer Raum vorhanden; er ist mit ungeordnet liegenden Filamenten oder Kollagenfibrillen angefüllt und enthält gelegentlich Adventitiazellen. In schmalen perivasculären Spalträumen beobachtet man öfters ein Streifenmuster (Periode ca. 50 m); es handelt sich dabei um ausgedehnte, nicht fibrillär gegliederte Kollagenbereiche.Der entlang dem Recessus pinealis dünn ausgezogene supracommissurale Teil des Organs ist nur von Randbündeln der Commissura posterior unterlagert, die ein dünner Gliafilz von der Hirnoberfläche trennt.

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Electron microscope studies on the subcommissural organ of the guinea pig

Summary In the subcommissural organ of the guinea pig the ependyma is built up of several rows of prismatic cells. The hypendyma of varying width contains capillaries plus astrocytes, oligodendrocytes and ependymal cell processes as well as elements showing the structural characteristics of ependymal cells.The subcommissural cells in the ependyma and in the hypendyma form various types of secretory products: Light secretory sacs originating from the cisternae of the endoplasmic reticulum pile up in the cytoplasm. Sometimes they confluate to irregularly lined areas in the apical zone. In several cells the light secretory sacs deliver material for dense secretory granules which are produced by the Golgi apparatus; dense granules of varying shape are accumulated apically. Some cells are tightly filled with secretory vacuoles. The cytoplasm between the vacuoles is condensed and reduced to narrow rims; the nucleus is pyknotic. The secretory vacuoles contain very little fluffy material. In the hypendyma the secretory vacuoles confluate forming giant vacuoles, occasionally containing microvilli and cilia. Finally a kind of apocrine secretion is observed: Some ependymal cells have protrusions which possess a nearly homogeneous cytoplasm and extend far into the ventricular lumen. Isolated cytoplasmic areas lacking organelles are to be found within the ventricle.The endothelium of the capillaries varies in width. At some places the basement membrane is widened and encloses small areas of lower density. Often a true perivascular space is found, filled with disordered filaments or collagen fibrils; occasionally it contains adventitial cells. Sometimes a substance exhibiting a periodic pattern (period ca. 50 m) occurs in narrow perivascular spaces; this material consists of extended areas of non-fibrillar collagen.The thin supracommissural part of the organ extends along the recessus pinealis. The adjoining commissura posterior is flattened to only a few axon bundles which are separated from the cerebral surface by a thin felt of glial processes.

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Herrn Dozenten Dr. phil. Ernst Kinder zur Vollendung seines 60. Lebensjahres gewidmet.

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Die Arbeit wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft ausgeführt. — Frau H. Asam danken wir für ausgezeichnete Mitarbeit bei der Präparation; ihr und Frl. C. Degen, Frl. I. Dürr und Frau B. Rottmann für die Ausführung der photographischen Arbeiten. Herrn Dr. med. A. Meinel gebührt unser Dank für wertvolle Diskussionen und Mithilfe bei der Fixierung der Objekte.     

Degradation of the fluoroquinolone enrofloxacin by wood-rotting fungi.

The veterinary fluoroquinolone enrofloxacin was degraded in vitro by four species of wood-rotting fungi growing on wetted wheat straw containing carbonyl-14C-labeled drug. A maximum 14CO2 production of 17% per week was observed with the brown rot fungus Gloeophyllum striatum, resulting in up to 53% after 8 weeks. However, rates reached at most 0.2 and 0.9% per week, if enrofloxacin was preadsorbed to native or gamma ray-sterilized soil, respectively.     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants

Summary Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either -naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with -naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somatic embryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - WPM Woody Plant Medium (Lloyd & McCown 1980)     

Influence of photoperiod and medium formulation on peanut somatic embryogenesis

Somatic embryos were produced from peanut (Arachis hypogaea L.) immature zygotic cotyledons. Comparisons were made of the level of -naphthaleneacetic acid during induction, nitrogen formulation of the medium, and photoperiod. Over 70% embryogenesis was obtained regardless of NAA level used. Percent embryogenesis and number of embryos were markedly lower in explants induced on NAA compared to 2,4-D. Embryo production was not greatly affected by either the use of Murashige & Skoog versus Finer & Nagasawa salts or light versus dark culture conditions. However, embryo morphology was noticeably affected by photoperiod. Embryos produced under a 16 h photoperiod were tough, woody and difficult to separate for subsequent germination and conversion. Those produced under a 0-h photoperiod were succulent and pliable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) - B5 Gamborg et al. (1968) - picloram 4-amino-3,5,6-trichloropicolinic acid - FN Finer & Nagasawa (1988) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)