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.     

Cation-distribution in developing follicles of the fruit fly Drosophila melanogaster

Abstract. Cations were precipitated with potassium antimonate in ovarian follicles of Drosophila and the distribution of the formed precipitates was studied. The precipitates were analyzed with a laser microprobe mass analyzer (LAMMA) and found to contain a high concentration of calcium; potassium and sodium were also detected. On counting the antimon precipitates in stage 10B follicles with the electron microscope, few precipitates per unit area were found in anterior nurse cells, but more in posterior nurse cells; the highest precipitate density occurred consistently in the oocyte. When follicles of different stages were compared, the precipitate density was found to increase in the ooplasm and in the posterior nurse cells during vitellogenesis, whereas it remained nearly constant in the anterior nurse cells. Thus, the ratio of precipitates between the posterior and anterior end of the follicle increases during vitellogenesis. It begins to decrease at the time when the nurse cells collapse. These results suggest that the electrical polarity observed in polytrophic ovarioles may be based on differences in the cation distribution along the antero-posterior axis of the follicle.     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphotyrosine.

A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.     

Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.

A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli.     

Secretogranin II: Relative Amounts and Processing to Secretoneurin in Various Rat Tissues

Abstract: Secretoneurin is a 33-amino-acid peptide produced in vivo from secretogranin II. An antiserum raised against this peptide recognizes both the free peptide and its precursors. By HPLC and radioimmunoassay we characterized the immunoreactive molecules and determined the levels of immunoreactivity in various rat organs. In adrenal medulla and to a lesser degree in the anterior pituitary processing of secretogranin II to secretoneurin was very limited, whereas in all other organs studied (brain, intestine, endocrine pancreas, thyroid gland, and posterior pituitary) a high degree of processing was apparent. Thus, practically all of the immunoreactivity was present as free secretoneurin. This was also true for serum. When the total amount of secretoneurin immunoreactivity was calculated for the various organs, the largest pools in descending order were in the intestine, CNS, anterior pituitary, pancreas, and adrenal gland. This makes it likely that secretoneurin in serum is mainly derived from the intestine. The high degree of processing of secretogranin II in most organs is consistent with the concept that this protein acts as a precursor of a functional peptide, i.e., secretoneurin.     

Formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines in human urine

Human urine samples were examined for the occurrence of formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines generated by condensation of the methanol oxidation product with biogenic amines. Positive results were obtained for the tryptamine condensation product 1,2,3,4-tetrahydro-β-carboline and the serotonine condensation product 6-hydroxy-1,2,3,4-tetrahydro-β-carboline as well as for the condensation products with tyramine, dopamine, adrenaline and noradrenaline 1,2,3,4-tetrahydroisoquinoline, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, 4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and the metabolite 6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline. Negative results were obtained for N-methyl-1,2,3,4-tetrahydroisoquinoline and 6,7-di-methoxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,2,3,4-tetrahydro-β-carboline, 6-methyl-1,2,3,4-tetrahydro-β-carboline, and 6-methoxy-1,2,3,4-tetrahydro-β-carboline in samples of chronic alcoholics as well as in the urine of healthy volunteers. No correlation between alcohol ingestion or state of alcoholization could be demonstrated.     

Cell-contact mediated modulation of the sialylation of contactinhibin

Contactinhibin was found to be involved in contact-dependent inhibition of growth. The growth inhibitory activity of contactinhibin is mediated by N-linked oligosaccharides with desialylated -glycosidically linked, terminal galactose residues. Here we show that in sparse human fibroblasts contactinhibin was expressed in a biologically inactive, highly sialylated form both on the plasma membrane and intracellularily, while in confluent cells plasma membrane localized contactinhibin was present in a biologically active, low sialylated form. Plasma membranes were shown to contain a glycoprotein sialidase which is suggested to be engaged in the activation of contactinhibin in a cell contact-dependent manner.