Structural Differences between Aβ(1-40) Intermediate Oligomers and Fibrils Elucidated by Proteolytic Fragmentation and Hydrogen/Deuterium Exchange

The aggregation of amyloid-β protein (Aβ) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Aβ(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Aβ peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.     

CHARACTER DISCRIMINATORY POWER,CHARACTER-SET CONGRUENCE,AND THE CLASSIFICATION OF INDIVIDUALS FROM HYBRID ZONES: AN EXAMPLE USING STONE CRABS (MENIPPE)

Many investigators categorize individuals from hybrid zones to facilitate comparisons among genotypic classes (e.g., parental, F₁, backcross) for comparative studies in which components of fitness or geographic variation are being analyzed. Frequently, multiple character sets representing genetically independent traits are used to classify these individuals and various methodologies are employed to combine the classifications obtained from the different character sets. We adapted the principles of total evidence and taxonomic congruence (two formalized approaches used by systematists in formulating phylogenetic hypotheses) to address the problem of discriminating hybridizing species and classifying individuals from hybrid zones. As our model, we used two morphological (coloration and morphometric) and two molecular (allozyme and mitochondrial DNA restriction-fragment-length polymorphism) character sets that differentiate two stone crab species (Menippe adina and M. mercenaria). Using principal-components analysis, we determined that combining character sets and eliminating characters or character sets that did not have large eigenvector coefficients for the principal component that best separated the two species yielded the highest level of discrimination between species and allowed us to classify a broad range of morpho-genotypes as hybrids. For the stone crabs, three diagnostic allozyme loci and five diagnostic coloration characters best separated the species. The two character sets were not completely congruent, but they agreed in their classification of 50% of the individuals from the hybrid zone and rarely strongly disagreed in their classifications. Classification discrepancies between the two character sets probably represent variation between traits in interspecific gene flow rather than intraspecific, ecologically mediated variation. Our results support the assertions of previous investigators who espoused the benefits associated with using multiple character sets to classify individuals from hybrid zones and demonstrate that, if character sets are reasonably congruent and numerically balanced, combining diagnostic characters from multiple character sets (a total-evidence approach) can enhance discriminatory power between species and facilitate the assignment of hybrid-zone individuals to genotypic classes. On the contrary, classifying hybrid-zone individuals using character sets separately (a taxonomic-congruence approach) provides the opportunity to compare levels of introgression between species and to assess reasons for discordance among the data sets.     

Effect of Enzymes on the Composition and Structure of Chromobacterium violaceum Cell Envelopes

Cell envelopes of Chromobacterium violaceum were isolated and treated under controlled conditions with trypsin, Pronase, lipase, phospholipase C, lysozyme, and a mixture of enzymes produced by a bacteriolytic Pseudomonas sp. After each enzyme treatment, losses in dry weight, protein, lipid, carbohydrate, 2,6-diaminopimelic acid, and total phosphorus were determined. Electron-microscopic examination of the enzyme-treated envelopes indicated complete or partial loss of envelope rigidity or some envelope fragmentation, or both. Each enzyme hydrolyzed at least one envelope component and liberated several others into the supernatant fluid, where they appeared as nondialyzable particulate components, identified by means of electron microscopy. Unlike the other enzymes, the Pseudomonas sp. enzyme mixture partially liberated all major envelope components except phosphorus, heptose, and 2-keto-3-deoxy octonic acid. In spite of these large losses, the envelopes preserved some features of their integrity and elongated shape.     

Characterization of isolectins inTetracarpidium conophorum seeds (Nigerian walnut)

A lectin preparation obtained fromTetracarpidium conophorum (Nigerian walnut) by affinity chromatography of seed extracts on lactose-agarose has been shown to contain two components by gel filtration on Sephadex G150. The larger componentTetracarpidium conophorum agglutinin I (TCAI) is a disulphide-bonded 70 kDa homodimer whereas the second component TCAII is a 34 kDa monomeric protein. Amino terminal aminoacid sequencing shows identity in TCAI and TCAII for the first fifteen residues after which the sequences diverge. TheN-terminal sequences of TCAI and TCAII show identity with sequences in the B-chains of ricin andRicinus communis agglutinin I (RCAI) in eleven of the initial fifteen residues. Thereafter TCAI appears to be homologous to the ricin B chain whereas TCAII is more homologous with the B chain of RCAI. A limited screening of the carbohydrate-binding specificity of TCAII by affinity chromatography of defined oligosaccharides on TCAII Sepharose columns shows that the binding specificity reported earlier for affinity purifiedTetracarpidium conophorum isolectins (Sato S, Animashaun T, Hughes RC (1991)J Biol Chem 266:11485–94) reflects the binding properties of TCAII which is the major isolectin in unfractionated lectin preparations.     

Testing the success of palaeontological methods in the delimitation of clam shrimp (Crustacea,Branchiopoda) on extant species

Fossil spinicaudatan taxonomy heavily relies on carapace features (size, shape, ornamentation) and palaeontologists have greatly refined methods to study and describe carapace variability. Whether carapace features alone are sufficient for distinguishing between species of a single genus has remained untested. In our study, we tested common palaeontological methods on 481 individuals of the extant Australian genus Ozestheria that have been previously assigned to ten species based on genetic analysis. All species are morphologically distinct based on geometric morphometrics (p ≤ 0.001), but they occupy overlapping regions in Ozestheria morphospace. Linear discriminant analysis of Fourier shape coefficients reaches a mean model performance of 93.8% correctly classified individuals over all possible 45 pairwise species comparisons. This can be further increased by combining the size and shape datasets. Nine of the ten examined species are clearly sexually dimorphic but male and female morphologies strongly overlap within species with little influence on model performance. Ornamentation is commonly species-diagnostic; seven ornamentation types are distinguished of which six are species-specific while one is shared by four species. A transformation of main ornamental features (e.g. from punctate to smooth) can occur among closely related species suggesting short evolutionary timescales. Our overall results support the taxonomic value of carapace features, which should also receive greater attention in the taxonomy of extant species. The extensive variation in carapace shape and ornamentation is noteworthy and several species would probably have been assigned to different genera or families if these had been fossils, bearing implications for the systematics of fossil Spinicaudata.