Improved purification of the membrane-bound hydrogenase–sulfur-reductase complex from thermophilic archaea using ε-aminocaproic acid-containing chromatography buffers

A hydrogenase–sulfur reductase (SR) complex was purified from membrane preparations of the extremely thermophilic, acidophilic archaeon Acidianus ambivalens using a combination of sucrose density gradient centrifugation and column chromatography (FPLC). All chromatographic steps were performed in the presence of 0.5% ε-aminocaproic acid resulting in the elution of the SR complex as a sharp peak. In contrast, chromatography using buffers without ε-aminocaproic acid, or in the presence of detergents, were not successful. The purified A. ambivalens SR complex consisted of at least four subunits with relative molecular masses of 110 000, 66 000, 39 000 and 29 000, respectively. A similar procedure was applied to purify the membrane-bound hydrogenase from Thermoproteus neutrophilus, a non-related extremely thermophilic but neutrophilic archaeon, which consisted of only two subunits with relative molecular masses of 66 000 and 39 000, respectively.     

Common CFTR mutations are not likely to predispose to chronic bronchitis in Northern Germany

The frequency of six common mutations in the cystic fibrosis transmembrane conductance regulator gene was studied in 100 patients hospitalized with chronic bronchitis. Only one patient with chronic bronchitis and diffuse bronchiectasis was heterozygous for the common F508 mutation. R553X, G542X, G551D, N1303K and 621+1GT were not detected. This result is not significantly different from the frequency of cystic fibrosis carriers in Northern Europe. Predisposition of heterozygotes for chronic bronchitis is therefore unlikely.     

Engineering of functional supramacromolecular complexes of proteins (enzymes) using reversed micelles as matrix microreactors.

The size of the inner water cavity of reversed micelles formed in a triple system 'water-surfactant-organic solvent' can be widely varied by changing the degree of surfactant hydration. This gives grounds to use reversed micelles as matrix microreactors for the design of supramolecular complexes of proteins. Using ultracentrifugation analysis, it has been demonstrated that the oligomeric composition of various enzymes (ketoglutarate dehydrogenase, alkaline phosphatase, lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) solubilized in reversed micelles of Aerosol OT [sodium bis(2-ethylehexyl)sulfosuccinate] in octane changes upon variation of the degree of hydration. An oligomeric complex forms under conditions when the radius of the micelle inner cavity is big enough to incorporate this complex as a whole. At lower degrees of hydration the micelles 'uncouple' such complexes to their components. The catalytic properties of various oligomeric complexes have been studied. Possibilities of using reversed micelles for the separation of subunits of oligomeric enzymes under non-denaturating conditions have been demonstrated. In particular, the isolated subunits of alkaline phosphatase, lactic dehydrogenase and glyceraldehyde-3-phosphate have been found to be active in Aerosol OT reversed micelles. The dependences of the catalytic activity of oligomeric enzymes represent saw-like curves. The maxima of the catalytic activity observed at these curves relate to the functioning of various oligomeric forms of an enzyme. The radii of the micelle inner cavity under conditions when these maxima are observed correlate with the linear dimensions of the enzyme oligomeric forms. Correlation of the position of a maximum with the shape of an oligomeric complex is discussed.     

Elektronenmikroskopische Untersuchungen über die Fibrillogenese in der Haut menschlicher Embryonen

Zusammenfassung Hautstücke aus der Rückengegend von zwei menschlichen Embryonen mit einer Scheitel-Steißlänge von 62 und 128 mm (Mens II und V) wurden elektronenmikroskopisch untersucht.Das subepidermale Bindegewebe des jüngeren Embryos enthält Fibroblasten mit einem oder mehreren Fortsätzen, zwischen denen einzelne Fibrillen oder kleine Fibrillenbündel liegen. Das endoplasmatische Retikulum dieser Elemente ist stark ausgeprägt. Sein Hohlraumsystem hat in den einzelnen Zellen einen verschiedenen Füllungsgrad. Die Membranen liegen entweder dicht zusammen oder sind mehr oder weniger auseinandergedrängt. Auf diese Weise können große Zisternen mit granulärem Inhalt entstehen. Den Membranen sitzen 80–100 Å und 160 Å dicke Granula auf. Außerdem werden Vesiculae von 150–400 Å Durchmesser an den Membranen beobachtet. Frei im Cytoplasma liegen zahlreiche Vesiculae mit Durchmessern bis zu 6000 Å. Die Dicke der Fibrillen variiert nur wenig; sie beträgt durchschnittlich 200 Å, die Perioden sind 300–400 Å lang.Die Fibroblasten in der Haut eines 5 Monate alten Embryos sind den Fibroblasten des jüngeren Embryos sehr ähnlich, doch ist hier die Zahl der vesikulären Strukturen geringer. Im Interzellularraum verlaufen nunmehr Fasern aus 100 und mehr Fibrillen. Die durchschnittliche Fibrillendicke beträgt 300 Å; die Perioden sind 400–500 Å lang.Das endoplasmatische Retikulum in den Fibroblasten wird für die Kollagensynthese verantwortlich gemacht, die man sich folgendermaßen vorstellen kann : Der Fibroblast liefert wahrscheinlich das Kollagen in Form des monomeren Tropokollagenmoleküls. Dieses Material sammelt sich in den Zisternen an und wird dann nach außen abgegeben. Extrazellulär bauen sich aus diesen Vorstufen Fibrillen auf. Aus diesem Grunde lassen sich Fibrillen auch nur extrazellulär elektronenmikroskopisch nachweisen. Die Zellmembran scheint eine Rolle bei der Ausrichtung der Fibrillenbündel zu spielen. Die vesikulären Strukturen der Fibroblasten werden mit der Mukopolysaccharidsynthese in Zusammenhang gebracht, deren Bedeutung für die Fibrillogenese diskutiert wird.Im Coriumbereich menschlicher Embryonen kommen noch zwei andere Zelltypen vor, die für undifferenzierte Mesenchymzellen und Histiozyten gehalten werden.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Electron microscopic study of the effect of colchicine on limb bud cartilage in organ culture (mouse embryos)]

Limb buds of 11-day-old mouse embryos were cultured. On day 5, 0.1 mM colchicine was added for 3, 6, 12 or 24 h in vitro. 3 or 6 h after application of colchicine, electron-dense granula appear to an increasing extent. These granula can be interpreted as collagen-containing secretion granula and as an indication of a secretion inhibition. The peripheral localization of the granula groups speaks for an effect on the actual discharge of the secretion after having passed the Golgi apparatus. Additional effects of colchicine are fragmentation of the Golgi apparatus and an increased occurrence of bundles of fine filaments. Since only few microtubuli occur in the periphery of the chondroblasts and recent studies indicate an influence of colchicine not only on the microtubule system, a site of action at the functional unity 'cell membrane-microfilaments' is discussed. 12 or 24 h after colchicine application, the contents of the congested secretion granula change. The occurrence of cross-striated structures indicates an activity of procollagen peptidase in the granula in the case of secretion inhibition. In addition, cytolysosomes (autophagic vacuoles) develop. The occurrence of polymorphism of the granula contents under these conditions is attributed to an effect of lysosomal enzymes.     

<Emphasis Type="Italic">Tarsius wallacei</Emphasis>: A New Tarsier Species from Central Sulawesi Occupies a Discontinuous Range

On the basis of distinguishing characteristics of various genetic markers, pelage color, tail tuft, and vocalizations, we describe a new species of the genus Tarsius Storr 1780. The new taxon Tarsius wallacei sp. nov. occupies a disjunct range in Central Sulawesi, Indonesia. The two isolated populations differ significantly in body size, but are alike in color, tail tuft dimensions, vocalizations, and genetic composition. Morphologically, the new species is similar to other Sulawesi lowland tarsiers. In the field, it can be distinguished from its congeners via a characteristic duet song and its yellow-brown pelage coloration and a copper-colored throat. Genetic analyses prove Y-chromosomal and mitochondrial DNA sequences and also microsatellite allele frequencies to be absolutely diagnostic.     

Copper related toxic effects on cellular protein metabolism in human astrocytes

INTRODUCTION: Copper overload due to a defect in the ATPase 7B mediated copper excretion within hepatocytes produces the phenotype of Wilson disease. The overload of hepatocytes with copper results in necrotic liver cells and is accompanied by a high concentration of blood copper levels. That occurs to be the reason for increasing neurological copper concentration. Although copper is linked to oxidation, there are no data on the direct copper related effects in human brain cells. AIM: To test the copper induced changes in protein oxidation in human astrocyte like cells. METHODS: We used U87 cells as model for human astrocytes. Cells were treated with increasing concentrations of copper(II)-chloride in Dulbeccos minimal essential medium. Subsequently, at different time points we investigated: cellular growth, cellular survival under copper treatment, the concentration of oxidized tryptophane in GADPH in vitro as well as the carbonyl concentration and the concentration of oxidized proteins in vivo in U87 glial cells. RESULTS: The viability of cells decreased with both increasing copper concentration and duration of treatment. The concentration of oxidized proteins was directly correlated to the increase of copper concentration and duration of exposure. CONCLUSION: These observations demonstrate the similarities between copper treatment and treatment with other commonly used oxidants, including hydrogen peroxide. Furthermore, the vulnerability of astrocytes towards copper exposure could be demonstrated. Therefore, these data give further insights into understanding of copper metabolism, which in turn is important to reveal the exact pathological mechanism in copper related diseases such as Wilson disease.     

Screening and evaluation of resistance to downy mildew (Peronospora parasitica) and clubroot (Plasmodiophora brassicae) in genetic resources of Brassica oleracea

52 entries including landraces, old cultivars and wild accessions of B. oleracea and closely related Brassica species were screened for resistance against downy mildew and clubroot. Several accessions resistant to downy mildew and a few to clubroot were found. Genetic inheritance of the resistance in downy mildew was investigated by screening F1 and BC1F1 offspring from three resistant landrace accessions crossed with both a resistant and a susceptible father. The seedling resistance against downy mildew was found to be inherited recessively. This is a bit surprising as earlier papers mostly report of inheritance controlled by a single dominant gene. Previous screenings of B. oleracea resistance against downy mildew at the cotyledon stage have been done with P. parasitica isolated from B. oleracea as the original host plant. The recessive nature of the cotyledon resistance found in this screening might be due to the fact that the P. parasitica isolate was collected from B. napus fields. The clubroot seedling resistance was found to be controlled by recessive inheritance after screening the F1 offspring, this in agreement with earlier results/reports.     

Lessons to be learned from primary renal cell carcinomas: novel tumor antigens and HLA ligands for immunotherapy

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T-lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are trying to define a larger number of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here, we present the results of a large-scale analysis of 13 RCC specimens. We were able to identify more than 700 peptides, mostly from self-proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them, we were able to characterize HLA ligands after extraction from the tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.Tobias Krüger and Oliver Schoor contributed equally to this work.