Glycerolipid formation and PGE2 and PGF2α production of rat renal papillae in vitro, the effects of urea

The glycerolipid production by rat renal papillary slices varied inversely with the urea concentration (0a–1660 mM) whether the production was measured as labelling of the glycerol backbone from glucose or as incorporation of labelled arachidonic acid and palmitic acid. The rate of phospholipid formation was most dependent on medium urea concentrations in the range between 0 and 1100 mM. The production of prostaglandins PGE₂ and PGF_2α, measured radioimmunologically or by an isotope derivative method was in the same range inversely related to the production of glycerolipids and chain elongations. The effect of urea on prostaglandin formation is probably indirectly caused by the inhibition of the phospholipid formation and chain elongation, since the effect was abolished by 1% defatted albumin in the medium. The data suggest that the level of free arachidonic acid within the cells is controlled to an important extent by glycerolipid formation and chain elongation.     

Heat treatment results in a loss of transgene-encoded activities in several tobacco lines.

Heat treatment (37 degrees C) of transgenic tobacco (Nicotiana tabacum) plants led to a reversible reduction or complete loss of transgene-encoded activities in about 40% of 10 independent transformants carrying the luciferase-coding region fused to the 355 cauliflower mosaic virus or the soybean small subunit promoter and the nopaline synthase promoter driving the neomycin phosphotransferase gene, whereas the other lines had temperature-tolerant activities. Temperature sensitivity or tolerance of transgene-encoded activities was heritable. In some of the lines, temperature sensitivity of the transgene-encoded activities depended on the stage of development, occurring in either seedlings (40% luciferase and 50% neomycin phosphotransferase) or adult plants (both 40%). The phenomenon did not correlate with copy numbers or the homo- or hemizygous state of the transgenes. In lines harboring a temperature-sensitive luciferase activity, reduction of bioluminescence was observed after 2 to 3 h at 37 degrees C. Activity was regained after 2 h of subsequent cultivation at 25 degrees C. Irrespective of the reaction to the heat treatment, the level of luciferase RNA was slightly increased at 37 degrees C. Only in lines showing temperature sensitivity of transgene-encoded activities was the amount of luciferase and neomycin phosphotransferase strongly reduced. In sterile culture, heat treatment for 15 d did not cause visible damage or changes in plant morphology. In all plants tested a slight induction of the heat-shock response was observed at 37 degrees C.     

Fatty acid-binding to erythrocyte ghost membranes and transmembrane movement

Summary Palmitate binding to human erythrocyte ghost membranes has been investigated with ghost preparations suspended in 0.2% albumin solutions. Free unbound palmitate in the extracellular water phase was measured in equilibrium studies using albumin-filled acid loaded ghosts as small semipermeable bags. The apparent dissociation constant of binding to the membrane is 13.5 nM and the binding capacity 19 nmoles per 7.2 × 109 cells.The 0°C exchange efflux kinetics of palmitate from albumin-filled ghosts is described by a model, which provides estimates of the rate constant of membrane transfer, k₃ = 0.024 s^–1, independent of the molar ratio of palmitate to albumin () and of a mean dissociation rate constant of the palmitate-albumin complex, k₁ = 0.0015 s^–1 at 0.2, allowing for a heterogeneity of the palmitate binding to albumin.The values of a third kinetically determined dependent model constant, Q, the ratio of palmitate bound to the membrane inner surface to palmitate on intracellular albumin, are not different from the Q values obtained by equilibrium experiments.The temperature dependences of k₁ and k₃ in the interval 0°C to 15°C give activation energies of 96 and 103 kJ/mole, respectively. The 0°C exchange efflux increases about 2 fold in response to a rise of pH from 6 to 9. The results suggest a carrier mediated palmitate flux at low with a V_max about 2 pmoles min^–1 cm^–2 at 0°C pH 7.3.     

Phytochrom-induzierte Enzymbildung (Phenylalanindesaminase), ein schnell ablaufender Prozeß

Zusammenfassung Bei Erstbelichtung des Senfkeimlings mit Dauer-Dunkelrot tritt der P₇₃₀-abhängige Anstieg der Phenylalanindesaminase-Aktivität erst mit einer lag-Phase von 1,5 Std ein (Abb. 3, 4 unten). Bei einer Zweitbelichtung (Programm: Erstbelichtung — längere Dunkelperiode — Zweitbelichtung) fehlt die lag-Phase (Abb. 4, oben). Die Enzymaktivität steigt sofort linear an. Da der Anstieg der Enzymaktivität wahrscheinlich auf eine de novo Synthese von RNS und Enzymprotein zurückzuführen ist (Tabelle), so erscheint der Schluß berechtigt, daß P₇₃₀ sehr rasch eine differentielle Genaktivierung mit anschließender Enzymsynthese bewirken kann, falls die Gene der Aktivierung durch P₇₃₀ zugänglich sind. Die relativ lange lag-Phase nach Einsetzen der Erstbelichtung benötigt das P₇₃₀ offenbar dazu, die potentiell aktiven Gene (P₇₃₀) für das P₇₃₀ zugänglich zu machen. Das Problem der primären lag-Phase ist in einer vorangegangenen Arbeit zur P₇₃₀-abhängigen Anthocyansynthese ausführlich diskutiert worden (vgl. Lange, Bienger und Mohr, 1967).

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Phytochrome-mediated enzyme formation (Phenylalanine deaminase) as a rapid process

Summary In previous papers we have reported (Mohr and Durst, 1966a, b) that synthesis of phenylalanine deaminase (EC 4.3.1.5), an important enzyme of phenolic metabolism, can be stimulated by the physiologically active phytochrome (=P₇₃₀) in the mustard seedling. The data of the present paper suggest that induction of this enzyme is a rapid process if the gene in question is easily accessible for the activating action of P₇₃₀.The seedlings were irradiated with continuous standard far-red light. Longtime irradiation with far-red will maintain a low but virtually constant level of P₇₃₀ in the seedling over an extended period of time. At the moment when the far-red light is turned off the action of P₇₃₀ will virtually cease. — Fig. 3 and Fig. 4, lower part, show the kinetics of enzyme induction by P₇₃₀ in an etiolated seedling. The initial (or primary) lag-phase after the onset of far-red is 1.5 hours. If, however, a seedling which has been pre-irradiated with 12 hours of far-red is kept in darkness for 6 hours and is then re-irradiated with far-red no lag-phase for the action of the second irradiation can be found. Enzyme activity increases immediately after the onset of far-red. Since the action of the second irradiation as measured by increase of enzyme activity can be inhibited by relatively low doses of Puromycin and Cycloheximide (table) we conclude that the re-appearance of P₇₃₀ leads to de novo synthesis of enzyme protein. — Application of Actinomycin D (10 g/ml) only partially inhibits the action of the second irradiation as measured by increase of enzyme activity. This finding was to be expected. In preceding papers (e.g. Mohr and Bienger, 1967) it has been concluded that genes which have once been activated by P₇₃₀ remain less sensitive towards Actinomycin D even when P₇₃₀ has disappeared. Taking into account all available data the conclusion seems to be justified that the induction of enzyme synthesis by P₇₃₀ (i.e. differential gene activation followed by enzyme synthesis) is a rapid process if the genes are accessible for the action of P₇₃₀. The relatively long initial lag-phase (1.5 hours) is needed to make the potentially active genes (P₇₃₀) accessible for the action of P₇₃₀. The problem of how the initial lag-phase can be understood has been dealt with more in detail in a previous paper on phytochrome-mediated anthocyanin synthesis (Lange, Bienger and Mohr, 1967).

     

Blütenbildung von Pinguicula lusitanica in vitro durch Fütterung mit Pollen

Zusammenfassung Pinguicula lusitanica wurde in vitro auf stickstoff- und phorphorfreiem Mineralsalzagar kultiviert; jede Pflanze stand für sich in einem Erlenmeyerkolben.Als die Pflanzen 8 Wochen alt waren, wurden 20 Exemplare innerhalb von 5 Wochen viermal mit Pinuspollen gefüttet. 20 gleichgroße und gleichalte dienten als Kontrollen.Durch die Fütterung steigerte sich die Blattzahl und der Durchmesser der Blattrosette, die Blätter wurden intensiver grün und alterten langsamer.Vor allem wurde durch die Pollenfütterung die Blütenbildung ausgelöst. Schon nach der 2. Fütterung traten die ersten Knospen auf, eine Woche darauf blühten bereits 70% und noch vor der letzten Fütterung alle gefütterten Pflanzen, von den ungefütterten dagegen keine einzige. In dem anschließenden halben Jahr entwickelten die 20 gefütterten Pflanzen im ganzen 127 Blüten; die größte Blütenzahl einer Einzelpflanze was 14. Die nichtgefütterten Pflanzen waren auch jetzt noch rein vegetativ.

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Flowering of in vitro cultures of Pinguicula lusitanica after feeding with pinus pollen

Summary Plants of Pinguicula lusitanica were grown in individual Erlenmeyer flasks on an inorganic agar medium containing no nitrogen or phosphorus. After 8 weeks of culture, twenty of the plants were fed Pinus pollen 4 times over a period of 5 weeks.As a result of the feeding, the number of leaves as well as the diameters of the rosettes were increased. The leaves became turned a deeper green and aged more slowly.The most spectacular effect of the pollen feeding was an initiation of flowering. The first buds were already visible after the second feeding. All of the treated plants flowered before the last feeding, whereas none of the untreated plants flowered. During the following 6 months, the treated plants developed 127 flowers, the largest number on a single specimen being 14. Even after this period of time the untreated plants remained vegetative.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft     

Metabolic clearance of insulin from the cerebrospinal fluid in the anesthetized rat

Infusion of 125I-(Tyr A14)-insulin at tracer doses into the cerebrospinal fluid (CSF) resulted in a slow rate of increase in the CSF-labeled insulin during the first 2 hours with a plateau thereafter. Labeled insulin was cleared from the CSF at a higher rate than 3H-inulin, a marker of CSF bulk flow. The labeled insulin was mainly distributed in all the ventricular and periventricular brain regions. Small amounts of degraded insulin appeared in the CSF. Coinfusion with an excess of unlabeled insulin impaired the clearance and degradation of labeled insulin. It also inhibited the labeling in medial hypothalamus, olfactory bulbs and brain stem. In contrast, coinfusion of ribonuclease B (used to test the specificity of uptake) was without any effect. It was concluded that there is an active insulin intake from CSF into brain specific compartments that is presumably essential for the effects of insulin on brain function.     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP benzylaminopurine - CTAB hexadecyltrimethylammoniumbromide - Na₂EDTA ethylenediamine-tetra-acetate ferric-sodium salt - FeNaEDTA ethylenediamine-tetra-acetate ferric-sodium salt - GA₃ gibberellic acid 3 - GusA -glucuronidase - gusA -glucuronidase gene of Escherichia coli - IAA indole acetic acid - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthalene acetic acid - nptII neomycinphosphotransferase II gene - bar phosphinothricin acetyl transferase gene - PCR polymerase chain reaction - PPT phosphinothricin - STS silver thiosulphate - T-DNA transferred DNA - TDZ thidiazuron - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide - Zea trans-Zeatin