Immunochemical analysis of the carbohydrate moiety of yeast killer toxin K28

Killer toxin K₂₈, a 16 kd protein secreted by the wine yeast Saccharomyces cerevisiae strain 28, was reversibly bound by a column of Concanavalin A-Sepharose, confirming its glycoprotein nature. HPLC analysis of acid hydrolyzates of K₂₈ toxin as well as Western-blots of -eliminated and/or endo H-treated killer toxin preparations probed with polyclonal -toxin antibodies revealed that the carbohydrate moiety of K₂₈ consists of D-mannose only, which is O-glycosidically linked via Ser/Thr residues to the protein part. The change in gel mobility of K₂₈ after -elimination was caused by a decrease in molecular mass of about 1,800, corresponding to a carbohydrate moiety of 10 mannose residues per killer toxin molecule.     

Global analysis of cellular protein translation by pulsed SILAC

Current methods for system‐wide gene expression analysis detect changes in mRNA abundance, but neglect regulation at the level of translation. Pulse labeling with stable isotopes has been used to measure protein turnover rates, but this does not directly provide information about translation rates. Here, we developed pulsed stable isotope labeling by amino acids in cell culture (pSILAC) with two heavy isotope labels to directly quantify protein translation on a proteome‐wide scale. We applied the method to cellular iron homeostasis as a model system and demonstrate that it can confidently identify proteins that are translationally regulated by iron availability.     

Elektronenmikroskopische Untersuchungen zur Dynamik neurosekretorischer Zellen von Enchytraeus (Oligochaeta)

Zusammenfassung Von lichtmikroskopischen Befunden der Neurosekretion bei dem Oligochaeten Enchytraeus ausgehend, haben wir an zwei elektronenmikroskopisch genau bezeichneten und festgelegten Zellen bzw. Zelltypen, die bereits lichtmikroskopisch charakterisiert worden waren, Untersuchungen über die submikroskopisch faßbare Zelldynamik durchgeführt. Die beiden Arten neurosekretorischer Zellen (Q-Zelle und P-Zellen) sind elektronen-mikroskopisch schon durch ihre Lage zu erfassen. Sie können nicht nur durch die Zellgröße, sondern auch durch ihre Elementargranula, den Aufbau des endoplasmatischen Retikulums und den Golgi-Apparat eindeutig unterschieden werden. Wie schon in den lichtmikroskopischen Untersuchungen wurde die Sekretionsaktivität mit der Amputation ausgelöst. Sowohl in der Q -als auch in der P-Zelle bewirkt die Amputation eine unmittelbare Sekretentleerung. Die darauf einsetzende Phase der Sekretproduktion ist submikroskopisch durch eine erhöhte Zahl von Golgistrukturen in diesen Zellen, durch das deutlich in Erscheinung tretende granuläre endoplasmatische Retikulum und durch eine fortschreitende Vergrößerung und Verdichtung von Lysosomen in beiden Zelltypen gekennzeichnet. Für die Q-Zelle sind weiterhin die Verstärkung des diesen Zelltyp besonders noch charakterisierenden Bereiches von Membranzisternen und die dortige Ribosomenbildung typisch. Auf Grund der Feststellungen wird die Frage der Beziehung einzelner Strukturen in diesen beiden Zelltypen zur Produktion des Neuro-sekrets diskutiert. Die elektronenmikroskopische Untersuchung führte zur Entdeckung eines weiteren Zelltyps, der im Lichtmikroskop bisher nicht erkannt worden war und der sich durch besonderen Reichtum an Mitochondrien und großen Lipoid (?)-Komplexen auszeichnet (M-Zelle). Über seine Bedeutung ist jedoch noch keine Aussage möglich.

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Summary The cytophysiology of two types of neurosecretory cells (Q and P cell) in the brain of the oligochete Enchytraeus was studied at the ultrastructural level. These cell types can be identified by their location, and particularly by the size difference of their elementary granules. Amputation of the last ten segments caused a release of secretory product followed by a phase of renewed production. This was characterized by changes in the endoplasmic reticulum, the Golgi apparatus, and the lysosomes. The role of these structures in the production of neurosecretory material was discussed. Furthermore, a cell type with extraordinarily numerous mitochondria, hitherto unknown in Enchytraeus, was described. Its function has not yet been determined.

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Mit Unterstützung durch die Sächsische Akademie der Wissenschaften zu Leipzig.

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Herrn Prof. Dr. F. Seidel, Marburg, zum 70. Geburtstag gewidmet.     

Simultaneous saccharification and protein enrichment fermentation of sugar beet pulp

Summary A product with 40 % protein content was obtained from sugar beet pulp (1.25–2.0 mm) in 48 h one stage (simultaneous) saccharification/fermentation process under optimized conditions using a specific enzyme mixture andCandida tropicalis strain, also saving about 40 % enzymes in comparison to a 2-stage process.     

Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures

Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15–16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 μM) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 ± 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 ± 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD₂ ? PGF_2α > TXB₂ ? PGE₂) and astroglial cells (PGD₂ > TXB₂ ? PGF_2α ? PGE₂ ? 6-ketoPGF_1α). Preincubation with arachidonic acid (1 μg/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4–6-fold by addition of the calcium ionophore A23187 (1 μM). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Purification and some properties of an extracellular l-amino acid oxidase from Cellulomonas cellulans AM8 isolated from soil

Summary The soil isolate Cellulomonas cellulans AM8 produces an extracellular l-amino acid oxidase (L-AAO) with broad substrate specificity. The strain produced up to 0.35 unit (U)/ml of the extracellular L-AAO in a simple medium containing glycerol and yeast extract. The enzyme was easily purified up to 30 U/mg protein using Phenyl-Sepharose fast flow. The purified enzyme migrated as single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 55 kDa. On native PAGE the molecular mass was approx. 300 000 kDa, which may be due to aggregation. With the exception of glycine, proline, and threonine, all the amino acids normally constituting proteins were oxidized. The V _max values from 0.7 to 35.2 U/mg for aspartic acid and lysine, respectively, and the K _m values from 0.007 to 7.1 mm for cysteine and valine, respectively, were obtained at 25° C and pH 7.0 in oxygen-saturated solutions. The L-AAO had a pH optimum of 6.5–7.5. It was stable for several months at — 30° C and for some days at 35° C. Ferricyanide served as an electron acceptor with a V _max of 50 U/mg and K _m for 0.3 mm with phenylalanine as the substrate. Correspondence to: R. D. Schmid     

The challenges and scope of theoretical biology

Scientific theories seek to provide simple explanations for significant empirical regularities based on fundamental physical and mechanistic constraints. Biological theories have rarely reached a level of generality and predictive power comparable to physical theories. This discrepancy is explained through a combination of frozen accidents, environmental heterogeneity, and widespread non-linearities observed in adaptive processes. At the same time, model building has proven to be very successful when it comes to explaining and predicting the behavior of particular biological systems. In this respect biology resembles alternative model-rich frameworks, such as economics and engineering. In this paper we explore the prospects for general theories in biology, and suggest that these take inspiration not only from physics, but also from the information sciences. Future theoretical biology is likely to represent a hybrid of parsimonious reasoning and algorithmic or rule-based explanation. An open question is whether these new frameworks will remain transparent to human reason. In this context, we discuss the role of machine learning in the early stages of scientific discovery. We argue that evolutionary history is not only a source of uncertainty, but also provides the basis, through conserved traits, for very general explanations for biological regularities, and the prospect of unified theories of life.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.