The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these "double mutants" provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.Tick-borne encephalitis (TBE) virus is a representative of the genus Flavivirus (family Flaviviridae), which also includes several other important human pathogens, such as yellow fever virus, Japanese encephalitis virus, West Nile virus, and the dengue viruses (33). Flaviviruses are small, round, enveloped particles that contain only three structural virus proteins, i.e., the membrane-anchored surface proteins M and E and the capsid protein, C (18). The last is a relatively small protein (approximately 11 kDa) with a large content of positively charged amino acid residues that shows significantly less sequence homology among members of the genus than do the other two structural proteins (21). Nevertheless, a number of characteristics of its amino acid sequence, including the clustering of basic residues in certain sections of the sequence, the presence of two distinctive hydrophobic segments located approximately in the center of the sequence and at the carboxy-terminal end, and a predicted high propensity to adopt a predominantly alpha-helical conformation, are generally maintained among different flaviviruses, suggesting an overall conserved structural and functional organization of protein C (14,18,25).In the absence of protein C, the surface proteins M (which is first synthesized as a precursor prote...

The major birch pollen allergen Bet v 1 is one of the most extensively characterized allergens both on the molecular and the immunological level. To define conformational B cell epitopes on Bet v 1, we screened filamentous phage libraries expressing circular or linear nonapeptides to select ligands specific for anti-Bet v 1 murine monoclonal antibodies BIP1 and BIP4. The deduced amino acid sequence of the BIP1 ligand was CFPYCYPSESA, and of the BIP4-ligand, CRQTRTMPGC. Both sequences derived from the circular phage library. Alignments to the sequence of Bet v 1 showed no similarities, indicating that the antibodies most likely recognize discontinuous epitopes. Phages displaying these mimotopes were capable of inhibiting interactions of the anti-Bet v 1 monoclonals with Bet v 1 in a dose-dependent manner in ELISA. In contrast, sequence-identical synthetic peptides were ineffective in blocking the antibody-allergen interactions. This is in agreement with the conformational inhomogeneity of the peptides in solution as observed by nuclear magnetic resonance spectroscopy. Intragastric administration of phages expressing the BIP1 mimotope induced a Bet v 1-specific IgG response in Balb/c mice. We conclude that peptide mimotopes, when displayed on phages, may induce a protective IgG response preventing IgE-mediated allergic reactions, suggesting a possible clinical application.

Background: Little is known about the role of bell peppers in food allergy. We collected sera from 11 patients with food allergy to bell peppers to analyze bell pepper extracts for allergen composition. Methods: Proteins of mature fruits of eight horticultural strains of bell peppers were extracted and tested with patients’ sera for IgE binding and with monoclonal and polyclonal antibodies in immunoblot. Results: Profilin was detected in bell pepper extracts by an anti-celery profilin antibody. It showed high IgE binding activity in all extracts, which could be inhibited by recombinant birch pollen profilin. Anti-birch pollen monoclonal antibody BIP3, directed against birch pollen proteins between 30 and 69 kD, bound to bell pepper antigens of comparable molecular weights. A homologue of the major birch pollen allergen Bet v 1 was detected in four of eight horticultural strains of bell peppers, and was shown to bind IgE in 1 of the 11 patients. A 23-kD allergen of bell peppers was shown to correspond to the 23-kD major paprika allergen by IgE absorption experiments. Its N-terminal sequence showed 100% identity to P23 from tomatoes. Conclusion: The appearance of profilin in all and Bet v 1 in 50% of the tested horticultural strains indicates that bell peppers have to be considered potentially dangerous for Bet v 1- and profilin-sensitized patients. Moreover, in 4 of 8 horticultural strains of bell peppers a homologue of the osmotin-like protein P23 from tomatoes is responsible for substantial IgE binding. Contact with Bet v 1 and P23 homologues in bell peppers can therefore be minimized by avoidance of the respective horticultural strains.

Context. Recent progress in the seismic interpretation of field β Cep stars has resulted in improvements of the physics in the stellar structure and evolution models of massive stars. Further asteroseismic constraints can be obtained from studying ensembles of stars in a young open cluster, which all have similar age, distance and chemical composition. Aims. To improve our comprehension of the β Cep stars, we studied the young open cluster NGC 884 to discover new B-type pulsators, besides the two known β Cep stars, and other variable stars.Methods. An extensive multi-site campaign was set up to gather accurate CCD photometry time series in four filters (U, B, V, I) of a field of NGC 884. Fifteen different instruments collected almost 77 500 CCD images in 1286 h. The images were calibrated and reduced to transform the CCD frames into interpretable differential light curves. Various variability indicators and frequency analyses were applied to detect variable stars in the field. Absolute photometry was taken to deduce some general cluster and stellar properties. Results. We achieved an accuracy for the brightest stars of 5.7 mmag in V, 6.9 mmag in B, 5.0 mmag in I and 5.3 mmag in U. The noise level in the amplitude spectra is 50 μmag in the V band. Our campaign confirms the previously known pulsators, and we report more than one hundred new multi-and mono-periodic B-, A-and F-type stars. Their interpretation in terms of classical instability domains is not straightforward, pointing to imperfections in theoretical instability computations. In addition, we have discovered six new eclipsing binaries and four candidates as well as other irregular variable stars in the observed field.Key words. open clusters and associations: individual: NGC 884 -techniques: photometric -stars: variables: generalstars: oscillations -binaries: eclipsingThe photometric data of the variable stars are only available in electronic form at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5/micrograms per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-simulating factor (GM-CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34- MNC, the proportion of cells in S/G2M phase increased from < 0.1% to 0.75 +/- 0.4% (GM-CSF) and to 1.34 +/- 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non-cycling status of PB and progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cells sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2m phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized 'stem cells' which may require the BM micro-environment for adequate proliferation in vivo.