Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded α2(VI) and α3(VI) chains but not on the α1(VI) chain. The interactions with the chains were inhibited by low concentrations (10–100 μM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data incidate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

High yield enzymatic conversion of intravascular leukotriene A4 in blood-free perfused lungs

Experiments to investigate the fate of intravascularly administered leukotriene (LT) A4, an unstable intermediate of LT generation, were performed in isolated, ventilated, and blood-free perfused rabbit lungs. LT extracted from the lung effluent were separated by different reverse phase and straight phase HPLC procedures as methylated and nonmethylated compounds. Identity of eluting LT was confirmed by UV spectrum analysis and immunoreactivity. Pulmonary artery injection of 75 to 300 nmol of LTA4 resulted in the rapid appearance of cysteinyl-LT as well as LTB4 in the recirculating perfusate. The yield of these enzymatically generated LTA4 metabolites vs non-enzymatic hydrolysis products (6-trans-LTB4, 5-trans-epi-LTB4, 5,6-dihydroxyeicosatetraenoic acids) ranged above 90%. Experiments with application of tritiated LTA4 showed exclusive origin of the detected LT from the exogenously applied precursor. The time course of cysteinyl-LT appearance in the perfusate suggested metabolism of LTC4 via LTD4 to LTE4, whereas there was no evidence for LTB4 omega-oxidation. In the dose range of LTA4 used, the enzymatic conversion of this LT precursor did not approach saturation. Collectively, these data indicate that the intact pulmonary vasculature contains a hitherto not described capacity for enzymatic conversion of intravascularly offered LTA4 to both cysteinyl-LT and LTB4. This may be of biological significance for a putative transcellular biosynthesis of LT in the pulmonary microcirculation upon contact with LTA4 feeder cells, such as activated granulocytes.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

Closed circular viral DNA and asymmetrical heterogeneous forms in livers from animals infected with ground squirrel hepatitis virus

To identify possible intermediates in the replication of ground squirrel hepatitis virus, we characterized the major forms of intracellular virus-specific DNA in the livers of infected ground squirrels. A variety of DNA species were found: covalently closed circular molecules, relaxed circular molecules, and a heterogeneous collection of molecules that migrated ahead of closed circular DNA during agarose gel electrophoresis. The heterogeneous DNA was at least partly single stranded, consisted of minus strands in a greater than eight-fold mass excess of plus strands, and was tightly associated with protein.     

Synthese der Hämolymphproteine und die Aufnahme der Dotterfraktion in die Oocyte unter Actinomycin-Einflu\ (Untersuchungen anMusca domestica)

Zusammenfassung Mit Hilfe von autoradiographischen und elektrophoretischen Methoden wurde die Dottereinlagerung in den wachsenden Oocyten vonMusca domestica untersucht. Sie beginnt nach 30 min im Autoradiogramm sichtbar zu werden. Durch ihre Färbbarkeit und Markierung konnte die Dotterfraktion im Pherogramm von Ovar und Hämolymphe eines mittleren Wachstumsstadiums (Stadium 3) nachgewiesen werden. Nach Abschlu\ der Vitellogenese tritt sie in der Hämolymphe nicht mehr auf. Die Einlagerung der Dotterproteine wird durch Actinomycin gestört, dagegen läuft ihre Synthese nahezu unbeeinflu\t weiter. Die Transporthemmung kann als bisher unbekannter Nebeneffekt des Actinomycins gedeutet werden.

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Synthesis of haemolymph proteine and the uptake of the yolk fraction in the oocyte during Actinomycin-treatment. (Studies onMusca domestica)

Summary By means of radioautographic and electrophoretic techniques yolk protein uptake in the growing oocytes ofMusca domestica was investigated. After 30 min yolk protein becomes visible in the radioautograms. By stainability and labeling the yolk fraction could be demonstrated in the pherogram of ovary and haemolymph in an intermediate developmental stage (stage 3). After the end of vitellogenesis it does not appear in the haemolymph. The yolk protein uptake is inhibited by Actinomycin, but the synthesis goes on nearly as normal. This inhibition can be interpretated as a new accessory effect of Actinomycin.

     

Über den Transport zelleigener Makromoleküle durch die Kernmembran

Summary The influence of O₂-deprivation and reduction of temperature on the incorporation of the RNA-precursors ³H-uridine und ³H-cytidine is investigated in various tissues of the larvae and in the ovaries of adults of the housefly Musca domestica L. While RNA-synthesis in most of the tissues is strongly reduced under anaerobic conditions, synthesis continues in a moderate extent in muscle cell nuclei and nurse cell nuclei. The RNA-macromolecules (mRNA and rRNA), however, do not migrate into the cytoplasma. RNA-synthesis within the cell nucleus is less affected by a sudden reduction of temperature than the passage of RNA through the nuclear membrane which is reduced to a very low rate. The macromolecular RNA, therefore, does not diffuse into the cytoplasma but is transported actively through the nuclear envelope. The malformations caused by anaerobiosis during embryogenesis are brought in connexion with the active RNA-transport through the nuclear envelope and the separation of transport and synthesis.

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Herrn Professor Dr. Hans Bauer zum 60. Geburtstag gewidmet.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Das Verhalten von Ameisenarbeiterinnen gegenüber der Königin nach vorangegangener weisellosigkeit

Zusammenfassung Arbeiterinnen vonMyrmica ruginodis Nyl. werden im weisellosen Zustand fertil. Sie legen aber keine Eier, wenn sie mehr als 3 Larven je Arbeiterin zu versorgen haben (Mamsch, 1965). Arbeiterinnengruppen wurden mit und ohne Larven von der Königin abgetrennt. In den eierlegenden Gruppen wurde die erneut zugesetzte eigene Königin entweder nicht beachtet oder angegriffen. In den Gruppen, in denen die Arbeiterinnen durch Larvenpflege unfruchtbar blieben, wurde die Königin intensiv beleckt und in das engere Nest eingetragen. Isolierte eierlegende Arbeitergruppen zeigten im Gegensatz zu Arbeiterinnen mit inaktiven Ovarien keine Tendenz, sich mit der Königingruppe zu vereinigen. Das veränderte Verhalten der Arbeiterinnen gegenüber der Königin ist demnach nicht vom Zeitraum der Weisellosigkeit abhängig. Es wurde nur bei eierlegenden Arbeiterinnen beobachtet.

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Summary Workers ofMyrmica ruginodis Nyl. start laying eggs after being separated from their queen. No deposition of eggs occurs in groups in which each worker has to feed 3 or more larvae (Mamsch, 1965). Worker groups rearing larvae and those without brood were separated from the queen.When the queen was put again into an egg-laying worker group, either no attention was paid to her or she was attacked. In groups of workers sterile due to rearing larvae the queen was licked intensively and carried into the nest cavity. Fertile worker groups showed no tendency to join the queen group. The change in the workers' behaviour does not depend on the time of the separation from the queen. It only occurs in groups of fertile workers.

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Résumé Des ouvrières deMyrmica ruginodis Nyl. deviennent fertiles après avoir été séparées de la reine. Mais elles ne pondent pas d'ufs, si elles ont chacune trois larves ou plus à nourrir (Mamsch, 1965). Des groupes d'ouvrières, d'une part avec larves et d'autre part sans, ont été séparés de la reine. Les groupes d'ouvrières pondeuses n'attachaient pas d'attention à la reine replacée dans le nid ou encore l'attaquaient. Dans les groupes où les ouvrières étaient restées infertiles à cause du nourrissement du couvain, la reine était léchée d'une façon intensive et portée à l'intérieur du nid. A l'opposé des ouvrières stériles, les groupes d'ouvrières pondeuses isolées ne montraient aucune tendance à joindre le groupe de la reine. Le changement du comportement des ouvrières ne dépend donc pas de la durée de l'état orphelin; il n'est observé que chez les ouvrières pondeuses.

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Herrn Prof. Dr.K. Gösswald, zum 60. Geburtstag gewidmet.