Self-assembly of recombinant prion protein of 106 residues

The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an alpha-helical (PrP(C)) to a beta-sheet-rich isoform (PrP(Sc)). The elucidation of the mechanism of conformational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-prone PrP(Sc) isoform. In an attempt to facilitate the structural analysis of PrP(Sc), a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP106) with two deletions (Delta23-88 and Delta141-176) was expressed and purified from Escherichia coli. PrP106 retains the ability to support PrP(Sc) formation in transgenic mice, implying that it contains all regions of PrP that are necessary for the conformational transition into the pathogenic isoform [Supattapone, S., et al. (1999) Cell 96, 869-878]. Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a concentration-dependent conformational transition to a beta-sheet-rich form. Following the conformational transition, rPrP106 possesses properties similar to those of PrP(Sc)106, such as high beta-sheet content, defined tertiary structure, resistance to limited digestion by proteinase K, and high thermodynamic stability. In GdnHCl-induced denaturation studies, a single cooperative conformational transition between the unstructured monomer and the assembled beta-oligomer was observed. After proteinase K digestion, the oligomers retain an intact core with unusually high beta-sheet content (>80%). Using mass spectrometry, we discovered that the region of residues 134-215 of rPrP106 is protected from proteinase K digestion and possesses a solvent-independent propensity to adopt a beta-sheet-rich conformation. In contrast to the PrP(Sc)106 purified from the brains of neurologically impaired animals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.     

Circadian variation of the cell proliferation in the jejunal epithelium of rats at weaning phase

Circadian variation in cell proliferation of the jejunal epithelium of 18-day-old rats was studied using the 2-h arrested metaphase score and crypt isolation method. A continuous decrease in the arrested metaphases occurred from 07.00 h to 13.00 h. From 17.00 h arrested metaphase values increased and were maintained at the higher level during the dark period as showed by Cosinor analyses (P < 0.05). These results indicate that in the young rat there is already a circadian variation in jejunal epithelial cell proliferation as early as 18 days. We can even suggest that the presence of a circadian rhythm at weaning contributes to the steady state of cell proliferation in the intestinal epithelium observed in adult life.     

A major histocompatibility complex-peptide-restricted antibody and t cell receptor molecules recognize their target by distinct binding modes: crystal structure of human leukocyte antigen (HLA)-A1-MAGE-A1 in complex with FAB-HYB3

Antibodies with T cell receptor-like specificity possess a considerable diagnostic and therapeutic potential, but the structural basis of the interaction between an antibody and an histocompatibility antigen has so far not been determined. We present here the crystal structure (at 2.15 A resolution) of the recombinant, affinity-matured human antibody fragment Fab-Hyb3 bound to the tumor-associated human leukocyte antigen (HLA)/peptide complex HLA-A1.MAGE-A1. Fab-Hyb3 employs a diagonal docking mode resembling that of T cell receptors. However, other than these natural ligands, the antibody uses only four of its six complementarity-determining regions for direct interactions with the target. It recognizes the C-terminal half of the MAGE-A1 peptide, the HLA-A1 alpha1-helix, and N-terminal residues of the alpha2-helix, accompanied by a large tilting angle between the two types of molecules within the complex. Interestingly, only a single hydrogen bond between a peptide side chain and Fab-Hyb3 contributes to the interaction, but large buried surface areas with pronounced shape complementarity assure high affinity and specificity for MAGE-A1. The HLA-A1.MAGE-A1.antibody structure is discussed in comparison with those of natural ligands recognizing HLA.peptide complexes.     

Branched polyamines cure prion-infected neuroblastoma cells

Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP(Sc), the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529-14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 microg of PPI generation 4.0/ml for 4 weeks not only reduced PrP(Sc) to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the beta-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP(Sc) to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP(Sc) from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP(Sc) from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP(Sc) clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.     

Identification of discrete classes of endosome-derived small vesicles as a major cellular pool for recycling membrane proteins

Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5'-[gamma-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.     

Zur Populationsgenetik der Adenylatkinase: Genhäufigkeit in einer südwestdeutschen Stichprobe

Summary Adenylate kinase phenotypes were determined in 407 unrelated persons from Southwestern germany. The frequencies were estimated to be AK¹=0.969, AK²=0.031.

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Direktor: Prof. Dr. med. Dr. H. Baitsch

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos)

Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard—a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used β-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. β-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.     

EFFECTS OF MASSAGE UNDER HYPOXIC CONDITIONS ON EXERCISE-INDUCED MUSCLE DAMAGE AND PHYSICAL STRAIN INDICES IN PROFESSIONAL SOCCER PLAYERS

Reports based on experiences from masseurs and players, mostly without any scientific background, suggest that the combination of a classical regeneration method (i.e. massage) with exposure to hypoxia may enhance regeneration in soccer. The aim of this study was to evaluate whether this specific combination could affect blood parameters related to muscle damage and physical strain after a soccer game. Approximately 15 hours after two separate championship games, 10 professional male outfield players of the first Austrian division were exposed to normobaric hypoxia (F_iO₂ 13.5% ∼ 4000m) or normoxia for 1 hour (30 minutes rest followed by 30 min massage) (cross-over design). Creatine kinase (CK), urea and uric acid (UA) were measured 4 days before the first game, and 15 and 63 hours after the two games. Match play increased CK values independently of the intervention. No effect of the massage in combination with hypoxia was seen. A trend was found between Δ UA ([UA] 48 hours after exposure minus [UA] before exposure) in response to hypoxia and SaO₂ measured in hypoxia (r=0.612, p=0.06). Results show that massage under hypoxic conditions had no additional positive effect on the measured parameters compared to massage alone. Solely the trend of a relationship for Δ UA and SaO₂ might indicate that redox alterations are a potential consequence of hypoxic exposure.