Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.     

Protoplasmic streaming in the giant unicellular green algaAcetabularia mediterranea

Summary Protoplasmic Streaming inAcetabularia mediteranea has been studied by microcinematography in 1. germinating zygotes, 2. germlings before the differentiation of rhizoids and apices, 3. young cells with rhizoids and apices, 4. vegetative cells-several centimeters in length, 5. cells with a maximum sized cap, containing secondary nuclei, and 6. cells after cyst formation. Intracellular transport is found to occur at a network-system of thin filaments and at a different system of headed streaming bands. At the network of filaments chloroplasts are found to move at a velocity of 1–2 m/sec. Headed streaming bands move along the filaments and may lead without interruption from the rhizoid to the apex of the cell andvice versa. The front zone of the streaming bands is occupied by a leading cytoplasmic head-structure. Small vesicles, polyphosphate granula and secondary nuclei are the predominant moving structures in headed streaming bands. The velocity of these particles is found to be 3–11 m/sec. The filament system is found during all developmental stages. Headed streaming bands are undetectable in germinating zygotes and develop from small cytoplasmic droplets in germlings to broad heavily loaded bands in the huge vegetative cell.Transport of secondary nuclei by headed streaming bands is not observed during mitotic divisions and after cyst formation, though moving bands are still present for several weeks after cyst formation.     

The role of vanadium in green plants

Cells of Chlorella pyrenoidosa, derived from vanadium free agar slants, respond with great sensitivity to microamounts of vanadium, added as NH₄VO₃ to autotrophic liquid cultures. Between 0.01 and 1 g V per litre nutrient medium (2·10^-10-2·10^-8g-at/l), the algae respond with a continuous increase in dry weight. At higher V-concentrations, further enhancement in biomass is accompanied by a additional increase in chlorophyll content. Maximum V-effect on both parameters was found to be at 500g V/l (10^-5 g-at/l). Dry weight as well as chlorophyll content of Chlorella are decreased by concentrations above 25 mg V/l; 100 mg V/l (2·10^-3 g-at/l) stop growth and cause death of the cells. The toxic threshold for the V-content in the algae was determined to be at 150–200 g V/g (3–4·10^-6 g-at/g) dry weight.Two different pH-optima for a positive vanadium action on dry weight and chlorophyll biosynthesis were established, the first at pH 7, the other in the range pH 7.5–8. Two sites of vanadium action in green algae are discussed.Part I: Arch. Microbiol. 105, 77–82 (1975)     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Multinuclear stages of the life cycle ofAcetabularia mediterranea

Summary The reproductive stages of the life cycle ofAcetabularia mediterranea have been studied in Feulgen-stained material. Light-microscopical photographs of secondary nuclei, cyst formation, gamete formation, gamete release, zygote formation and early development of the germlings are presented. The time course of cytological events during gamete formation is described. Mitoses are found between 16 and 24 hours after the induction of cyst germination.The author is indebted to Mrs. S.Artelt who helped with the prepration of the specimens and the photographs.     

Fledermäuse im Windkanal

Zusammenfassung In einem Windkanal wurde für eine Myotis lucifugus die Abhängigkeit der Fluggeschwindigkeit (v_F) und der Geschwindigkeit über Grund (v_G) von der Windgeschwindigkeit (v_W) bestimmt. Die Fledermaus flog bei Windstille mit einer mittleren v_F von etwa 4,5 m/sec. Bei zunehmenden Gegenwinden erhöhte sie v_F und verringerte v_G, um bei v_W=7,7 m/sec für kurze Zeit stationären Flug (v_G=0) zu erreichen. Die Flügelschlagfrequenz lag bei Gegenwinden von 0–7,7 m/sec zwischen 10–11/sec. Bei zunehmenden Rückenwinden wurde der Flug immer mehr dem Rüttelflug ähnlich und die Flügelschlagfrequenz stieg bis 16/sec an. Die v_G blieb nahezu konstant in einem Bereich zwischen 4,5–5 m/sec. Bei Myotis lucifugus, Chilonycteris rubiginosa, Carollia perspillicata und Rhinolophus ferrum-equinum wurde die Flügelstellung während der Lautaussendung ermittelt. Alle Arten erzeugten entweder einen Einzellaut oder eine Gruppe von Lauten pro Flügelschlag.

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Bats in the wind tunnel

Summary In an experimental wind tunnel air speed (v_F) and ground speed (v_G) of a Myotis lucifugus were measured as a function of wind speed (v_W). The bat had a v_F of about 4,5 m/sec in still air. With head winds it increased v_F and lowered v_G to reach stationary flight (v_G=0) at a v_W of 7,7 m/sec. The rate of wing motion remained at about 10–11/sec at head winds from 0–7,7 m/sec. With tail winds the bat changed to a semi-hovering flight with wing beat frequencies rising to about 16/sec and v_F dropping to almost zero at 4–5 m/sec tailwinds. The v_G remained nearly constant between about 4,5–5 m/sec. (Figs. 2 and 3). The wing positions during which orientation sounds were emitted were determined for Myotis lucifugus (Fig. 4), Chilonycteris rubiginosa, Carollia perspillicata and Rhinolophus ferrum-equinum (Fig. 5). All bats emitted either one single sound or a group of sounds per wing beat.

     

Influence of T-2 and HT-2 Toxin on the Blood-Brain Barrier In Vitro: New Experimental Hints for Neurotoxic Effects

The trichothecene mycotoxin T-2 toxin is a common contaminant of food and feed and is also present in processed cereal derived products. Cytotoxic effects of T-2 toxin and its main metabolite HT-2 toxin are already well described with apoptosis being a major mechanism of action. However, effects on the central nervous system were until now only reported rarely. In this study we investigated the effects of T-2 and HT-2 toxin on the blood-brain barrier (BBB) in vitro. Besides strong cytotoxic effects on the BBB as determined by the CCK-8 assay, impairment of the barrier function starting at low nanomolar concentrations were observed for T-2 toxin. HT-2 toxin, however, caused barrier disruption at higher concentrations compared to T-2 toxin. Further, the influence on the tight junction protein occludin was studied and permeability of both toxins across the BBB was detected when applied from the apical (blood) or the basolateral (brain) side respectively. These results clearly indicate the ability of both toxins to enter the brain via the BBB.     

Signaling Governed by G Proteins and cAMP Is Crucial for Growth,Secondary Metabolism and Sexual Development in Fusarium fujikuroi

The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs). In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA), to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s) exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.     

Regulation of secretory activity in the albumen gland of the pulmonate snail Lymnaea stagnalis (L.)

The secretory activity of the albumen gland of the freshwater snail Lymnaea stagnalis was studied morphometrically (both at the light- and at the electron-microscope level) and biochemically under the following experimental conditions: (1) glandular tissue was implanted into acceptor snails and the glandular activity of the implants was compared to that of the glands of the donors and acceptors; (2) glandular activity was measured at various periods after oviposition; and (3) the activity was measured during a 24 h cycle (diurnal activity). The results indicate that cellular release of secretion material is regulated by a nervous mechanism, whereas synthetic activity is under hormonal control.