Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Die Photosynthese von Meerespflanzen in ihrer Beziehung Zum Salzgehalt

Zusammenfassung Werden Blattstücke vonPosidonia oceanica oder Thallusteile vonUlva lactuca unter sonst optimalen Assimilationsbedingungen aus Meerwasser in Süßwasser, überführt, so sinkt hier die apparente Assimilation beiPosidonia bis zu Null oder gar zu negativen Werten ab, beiUlva bis zu etwa 23% der Leistung im Meerwasser. Bei Rückführung in Meerwasser wird die Assimilationsrate ebenso schnell wieder erhöht, die Ausgangswerte werden allerdings innerhalb der Versuchszeit — einige 20-min-Perioden — meist nicht in voller Höhe wieder erreicht.Folgt der Wechsel zwischen Meer- und Süßwasser im Wechsel von 20 min, so reagieren die Versuchspflanzen mit der gleichen Präzision wie auf das Aus- und Einschalten einer Lichtquelle.Wird anPosidonia die photosynthetische Rate in verschiedenen Stufen verdünnten Meerwassers (Verdünnung nicht mit dest. Wasser, sondern mit Leitungswasser von bekanntem Bicarbonatgehalt und p_H) bestimmt und in Prozenten der Leistung in Meerwasser ausgedrückt, so ergibt sich die in Abb. 3 dargestellte Kurve. Innerhalb der Versuchszeit scheint jeder Meerwasserkonzentration ein bestimmter Photosynthesewert zugeordnet zu sein, ein Verhalten, das auf das Vorhandensein von Gleichgewichtszuständen hindeutet, die zwischen dem photosynthetischen System und dem Zustand des Außenmediums bestehen.Infolge der überaus raschen Reaktion der Pflanzen auf Änderungen im Außenmedium wird als Arbeitshypothese die Vermutung ausgesprochen, daß an der Änderung der photosynthetischen Rate bei Übertragung in und Rückführung aus Süßwasser in Meerwasser nicht Zustandsveränderungen imZellinneren, sondern Zustände der Membranen verantwortlich sind, welche das Ausmaß der CO₂-Permeation bestimmen und damit einen begrenzenden Faktor der Photosynthese darstellen.Mit 4 Textabbildungen     

Das Wachstum infiltrierter Keimlinge

Zusammenfassung Werden Keimlinge vonHelianthus annuus undVicia faba mittels einer Wasserstrahlpumpe mit Wasser infiltriert, so führt dies sofort in allen Organen der Pflanze zu einer sehr starken und mitunter völligen Hemmung des Wachstums. Wirkt der Unterdruck in Luft ein, so daß es hernach zu keiner Wasserfüllung der Interzellularen kommt, so unterbleibt jede Wachstumshemmung.Die Frage nach der Kausalbeziehung zwischen Infiltration und Wachstumshemmung konnte nicht geklärt werden, da die nächstiliegende Annahme, Infiltration führe zu einer Atmungshemmung, durch das Experiment nicht bestätigt werden konnte.Es wird darauf hingewiesen, daß die Zufuhr von Wirk- oder Nährstoffen durch Infiltration eine Methode ist, die in wachsenden Organen nur mit großem Vorbehalt angewendet werden darf, da eine im Wachstum weitgehend gehemmte Pflanze sich in einem anomalen Zustand befindet.Mit 8 Textabbildungen.     

Untersuchungen über das osmotische Verhalten der GrünalgeValonia ventricosa

Zusammenfassung 1.Valonia ventricosa ist eine pantropisch verbreitete marine Grünalge (einziger Fundort im Mittelmeer: Insel Ibiza). Die hier dargestellten Untersuchungen wurden an der Ostküste Venezuelas durchgeführt.2. Die osmotischen Werte (kryoskopisch bestimmt) liegen 1 bis 3 atm über dem Wert des Meerwassers.3. Wachstum und Alter der Zellen ändern den osmotischen Wert nicht.4. Aus Meerwasser in destilliertes Wasser überführt, tritt sehr rasche Abnahme des osmotischen Wertes des Zellsaftes ein (innerhalb von 160 Minuten von 26 auf 2 atm).5. Infolge der größeren relativen Oberfläche nimmt der osmotische Wert kleinerer Zellen viel schneller als der größerer.6. In konzentriertem Meerwasser (31 atm) ändern sich die Zellsaftwerte innerhalb von 3 Stunden nicht.7. In stärker konzentriertem Meerwasser sterben die Zellen rasch ab. Ihr Zellsaft wird damit zum Spielball der Außenbedingungen.8.Valonia ventricosa ist somit ein stenohaliner Organismus ohne erkennbare Fähigkeit zur Osmoregulation.9. Zugabe von CaCl₂ zum destillierten Wasser verlangsamt zunächst die Abnahme des osmotischen Wertes.10. Die chemische Untersuchung des Zellsaftes zeigt auch bei dieser Art das Vorherrschen des Kaliums, das gegenüber dem Meerwasser 66fach konzentriert ist.11. Die Zellwand färbt sich mit Methylenblau stark an, ist jedoch für diesen Farbstoff in keiner Richtung permeable. Angefärbte Zellwände scheinen eine geringe Ionenpermeabilität zu besitzen.12. Elektronenoptische Untersuchnugen der Zellwände zeigen, daß diese aus 40 bis 50 Lamellen bestehen, wobei benachbarte Lamellen eine überkreuzte Paralleltextur (Fischgrätenmuster) besitzen. Da die Zellen — im Gegensatz zu anderenValonia-Arten — nicht in destilliertem Wasser platzen, muß die Zellwand entweder hohe Drucke auszuhalten vermögen oder eine relativ geringe Wasserpermeabilität besitzen.

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Investigations on the osmotic behaviour of the green algaValonia ventricosa

Among the marine Chlorophyta,Valonia ventricosa represents a pantropic species; one of its few extratropical localities is the island Ibiza in the Mediterranean. Our physiological investigations were carried out during several months between 1960 and 1963 in the coastal waters of Venezuela (Mochima Bay near Cumana). The following results were obtained: 1. The osmotic values (measured with the kryoskopic method) are 1 to 3 atm higher than those of the seawater (salinity 36–37 ). 2. Neither size nor age of the cells influence the osmotic characteristics of the cell sap. 3. A transfer from marine to distilled water causes a rapid decrease of the osmotic values (within 160 minutes from 26 to 2 atm). Due to the bigger relative surface, this decrease is more rapid in small cells than in the bigger ones. 4. In concentrated seawater with 31 atm the osmotic values of the cells did not change within 3 hours. 5. In more concentrated or in diluted seawater, the cells are irreversibly damaged within a short time.Valonia ventricosa can therefore be considered as a stenohaline aliga without any recognizable osmoregulation. 6. Addition of CaCl₂ delays the decrease of the osmotic value. 7. Chemical analysis of the cell sap demonstrates the well-known prevalence of potassium, which is 66 times more concentrated than in seawater. 8. The cell wall can be easily stained with methyleneblue and in this case the permeability for anorganic ions is probably reduced. 9. Photographs taken with the electron-microscope show in cross section the multilammellate nature of the cell wall and the change of the fibrillar-direction from one lamella to the other, giving the picture of a cross-fibrillar structure. Since the cells — in contradiction to those of otherValonia species — do not burst in distilled water, it must be assumed that the cell wall structure is able to resist high pressures (about 26 atm) or is characterized by a relatively low water-permeability.

     

Use of Solid-Phase Extraction To Determine Ergosterol Concentrations in Plant Tissue Colonized by Fungi

At present, the ergosterol and acetate-to-ergosterol techniques are generally considered to be the methods of choice to quantify fungal biomass, growth rate, and productivity under natural conditions. Both methods rely on the accurate isolation and quantification of ergosterol, a major membrane component of eumycotic fungi. Taking advantage of the solid-phase extraction (SPE) technique, we present a novel method to determine the ergosterol concentration in lipid extracts derived from plant tissues and dead organic matter colonized by fungi. In this method, a primary alkaline extract is acidified and passed through a reversed-phase (C(inf18)) SPE column. The column is then washed with an alkaline methanol-water solution to eliminate interfering substances and increase pH and is thoroughly dried in air. Ergosterol is eluted with alkaline isopropanol. This eluting solvent was chosen to produce a strongly basic pH of the final extract and thus confer stability on the ergosterol molecule before high-performance liquid chromatography analysis. The recovery of ergosterol from plant tissues and the O(infhf) horizon of a woodland soil ranged from 85 to 98%, and the overall extraction efficiency was similar to that obtained by a conventional procedure involving liquid-liquid extraction. Potential pitfalls of ergosterol analysis by SPE include (i) insufficient acidification before sample loading on the extraction column, resulting in a poor affinity of ergosterol for the sorbent; (ii) incomplete drying of the sorbent bed before the elution step; and (iii) chemical breakdown of ergosterol after elution, which was found to be related to a low pH of the final extract and a high concentration of matrix compounds. When these pitfalls are avoided, SPE is an attractive alternative to existing methods of ergosterol analysis of environmental samples.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Enzymatic properties of human glucuronyltransferase and a sensitive method for its assay in a stable B lymphocyte cell line

Properties of lymphocyte glucuronyltransferase were studied in homogenates of SN1006 cells. A sensitive assay procedure for lymphocyte glucuronyltransferase was developed utilizing radioactive testosterone as the acceptor substrate and TLC for separation of the metabolite. The method is capable of detecting picomolar quantities of the product. The enzyme activity exhibited a broad pH optimum, and was subject to activation by the detergent Lubrol WX and Mn++ ions. The activity conformed to the Michaelis-Menten kinetics giving apparent Km values of 0.8 mM and 11 microM, for UDPGA and testosterone, respectively. 4-Methylumbelliferone, a-naphthol and p-nitrophenol behaved as competitive inhibitors of testosterone glucuronidation. The results indicate that the method could be used for genetic studies of human lymphocyte glucuronyltransferase, and that the enzyme is of consequence in detoxication of exogenous as well as endogenous substrates.     

A field facility to simulate climate warming and increased nutrient supply in shallow aquatic ecosystems

Global warming and excess nitrogen deposition can exert strong impacts on aquatic populations, communities, and ecosystems. However, experimental data to establish clear cause-and-effect relationships in naturally complex field conditions are scarce in aquatic environments. Here, we describe the design and performance of a unique outdoor enclosure facility used to simulate warming, increased nitrogen supply, and both factors combined in a littoral freshwater wetland dominated by common reed, Phragmites australis. The experimental system effectively simulated a 2.8 °C climate warming scenario over an extended period, capturing the natural temperature variations in the wetland at diel and seasonal scales with only small deviations. Excess nitrogen supply enhanced nitrate concentrations especially in winter when it was associated with increased concentration of ammonium and dissolved organic carbon. Nitrogen also reduced dissolved oxygen concentrations, particularly in the summer. Importantly, by stimulating biological activity, warming enhanced the nitrogen uptake capacity of the wetland during the winter, emphasizing the need for multifactorial global change experiments that examine both warming and nitrogen loading in concert. Establishing similar experiments across broad environmental gradients holds great potential to provide robust assessments of the impacts of climate change on shallow aquatic ecosystems.     

Diversity patterns of leaf-associated aquatic hyphomycetes along a broad latitudinal gradient

Information about the global distribution of aquatic hyphomycetes is scarce, despite the primary importance of these fungi in stream ecosystem functioning. In particular, the relationship between their diversity and latitude remains unclear, due to a lack of coordinated surveys across broad latitudinal ranges. This study is a first report on latitudinal patterns of aquatic hyphomycete diversity associated with native leaf-litter species in five streams located along a gradient extending from the subarctic to the tropics. Exposure of leaf litter in mesh bags of three different mesh sizes facilitated assessing the effects of including or excluding different size-classes of litter-consuming invertebrates. Aquatic hyphomycete evenness was notably constant across all sites, whereas species richness and diversity, expressed as the Hill number, reached a maximum at mid-latitudes (Mediterranean and temperate streams). These latitudinal patterns were consistent across litter species, despite a notable influence of litter identity on fungal communities at the local scale. As a result, the bell-shaped distribution of species richness and Hill diversity deviated markedly from the latitudinal patterns of most other groups of organisms. Differences in the body-size distribution of invertebrate communities colonizing the leaves had no effect on aquatic hyphomycete species richness, Hill diversity or evenness, but invertebrates could still influence fungal communities by depleting litter, an effect that was not captured by the design of our experiment.     

Phylogenies and traits provide distinct insights about the historical and contemporary assembly of aquatic insect communities

The assumption that traits and phylogenies can be used as proxies of species niche has faced criticisms. Evidence suggested that phylogenic relatedness is a weak proxy of trait similarity. Moreover, different processes can select different traits, giving opposing signals in null model analyses. To circumvent these criticisms, we separated traits of stream insects based on the concept of α and β niches, which should give clues about assembling pressures expected to act independently of each other. We investigated the congruence between the phylogenetic structure and trait structure of communities using all available traits and all possible combinations of traits (4095 combinations). To account for hierarchical assembling processes, we analyzed patterns on two spatial scales with three pools of genera. Beta niche traits selected a priori – i.e., traits related to environmental variation (e.g., respiration type) – were consistently clustered on the smaller scale, suggesting environmental filtering, while α niche traits – i.e., traits related to resource use (e.g., trophic position) – did not display the expected overdispersion, suggesting a weak role of competition. Using all traits together provided random patterns and the analysis of all possible combinations of traits provided scenarios ranging from strong clustering to overdispersion. Communities were phylogenetically overdispersed, a pattern previously interpreted as phylogenetic limiting similarity. However, our results likely reflect the co‐occurrence of ancient clades due to the stability of stream habitats along the evolutionary scale. We advise ecologists to avoid using combinations of all available traits but rather carefully traits based on the objective under consideration. Both trait and phylogenetic approaches should be kept in the ecologist toolbox, but phylogenetic distances should not be used as proxies of traits differences. Although the phylogenetic structure revealed processes operating at the evolutionary scale, only specific traits explained local processes operating in our communities.