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.     

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.     

Does Nest Defence Deter or Facilitate Cowbird Parasitism? A Test of the Nesting-cue Hypothesis

The nesting-cue hypothesis poses that avian brood parasites use nest-defence responses directed toward them by hosts as cues to locate nests to parasitize. Hosts that respond more intensely to brood parasites should provide more cues about nest location than those hosts giving lower intensity responses. Thus, the nesting-cue hypothesis predicts that within a species parasitized nest owners should respond more intensely than unparasitized nest owners to cowbirds perched near and away from nests. This assumes that hosts respond to cowbirds when they are encountered away from the nest and that host responses gradually increase in intensity as the cowbird nears the nest. The nesting-cue hypothesis, its assumptions and prediction were tested using six host species of the brown-headed cowbird (Molothrus ater, Icterinae). We presented a female cowbird model at three distances from host nests and compared the responses elicited. All species responded to the cowbird at all distances, which supports the first assumption. Some of the rarely elicited responses (e.g. contacts) and the proximity of the host to the model varied significantly with distance, which suggests that cowbirds could use nest defence by the host as cues to the location of an active nest. However, parasitized nest owners did not respond more intensely than unparasitized nest owners to the cowbird positioned at any of the distances from the nest, which does not support the nesting-cue hypothesis itself. Further considerations are discussed that suggest that nest defence is not likely to be used as a nest-location cue.     

Changes in protein composition of facultatively aerobic sulfur-dependent archaebacteria depending on growth conditions

Cell-free extracts of facultatively anaerobic, sulfur-dependent archaebacteria Acidianus infernus (DSM 3191), and Acidianus brierleyi (DSM 1651) were examine by two-dimensional gel electrophoresis. 56 out of 250 protein spots were induced in anaerobically grown cells of A. infernus compared with 57 out of 251 in aerobically grown cells. In aerobically grown cells of A. brierleyi 62 out of 160 spots were induced, compared with 84 out of 182 in anaerobically grown cells. Changes in the protein patterns of both species were not comparable.     

Effect of human chorionic gonadotropin and estradiol in vivo on estradiol binder and cytochrome P-450 concentrations in rat testis

The effects of single administration to adult male rats in vivo of various amounts of human chorionic gonadotropin (HCG) and of single or repeated injections of estradiol on testicular cytoplasmic estradiol binder concentrations and on microsomal progesterone-binding cytochrome P-450 were compared. Half-life periods of HCG-induced loss of estradiol binder and cytochrome P-450 concentrations are identical (6 h) whereas a strong dissociation of these half-life periods are evident after chronic estradiol treatment (less than 2 h for the estradiol binder, about 35 h for cytochrome P-450). Depletion of cytoplasmic estradiol binder is not a sufficient condition for mediation of effects on cytochrome P-450 content. Rate of replenishment of microsomal cytochrome P-450 is similar after HCG or estradiol treatment. Both HCG- and estradiol-induced loss of cytochrome P-450 occur not only in Leydig cells but also in microsomes prepared from seminiferous tubules. Additional information is presented contradicting the hypothesis that loss of cytochrome P-450-dependent steroidogenic enzymes caused by HCG could be mediated by estrogens.