We report the proteomic characterization of the venom of the medically important North American western diamondback rattlesnake, Crotalus atrox, using two complementary approaches: snake venomics (to gain an insight of the overall venom proteome), and two solid-phase combinatorial peptide ligand libraries (CPLL), followed by 2D electrophoresis and mass spectrometric characterization of in-gel digested protein bands (to capture and "amplify" low-abundance proteins). The venomics approach revealed approximately 24 distinct proteins belonging to 2 major protein families (snake venom metalloproteinases, SVMP, and serine proteinases), which represent 69.5% of the total venom proteins, 4 medium abundance families (medium-size disintegrin, PLA(2), cysteine-rich secretory protein, and l-amino acid oxidase) amounting to 25.8% of the venom proteins, and 3 minor protein families (vasoactive peptides, endogenous inhibitor of SVMP, and C-type lectin-like). This toxin profile potentially explains the cytotoxic, myotoxic, hemotoxic, and hemorrhagic effects evoked by C. atrox envenomation. Further, our results showing that C. atrox exhibits a similar level of venom variation as Sistrurus miliarius points to a "diversity gain" scenario in the lineage leading to the Sistrurus catenatus taxa. On the other hand, the two combinatorial hexapeptide libraries captured distinct sets of proteins. Although the CPLL-treated samples did not retain a representative venom proteome, protein spots barely, or not at all, detectable in the whole venom were enriched in the two CPLL-treated samples. The amplified low copy number C. atrox venom proteins comprised a C-type lectin-like protein, several PLA(2) molecules, PIII-SVMP isoforms, glutaminyl cyclase isoforms, and a 2-cys peroxiredoxin highly conserved across the animal kingdom. Peroxiredoxin and glutaminyl cyclase may participate, respectively, in redox processes leading to the structural/functional diversification of toxins, and in the N-terminal pyrrolidone carboxylic acid formation required in the maturation of bioactive peptides such as bradykinin-potentiating peptides and endogenous inhibitors of metalloproteases. Our findings underscore the usefulness of combinatorial peptide libraries as powerful tools for mining below the tip of the iceberg, complementing thereby the data gained using the snake venomics protocol toward a complete visualization of the venom proteome.

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrPTSE), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrPTSE type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.

The use of combinatorial peptide ligand libraries, containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, allowed discovering and identifying a large number of previously unreported proteins in cow's whey. Whereas comprehensive whey protein lists progressively increased in the last 6 years from 17 unique gene products to more than 100, our findings have considerably expanded this list to a total of 149 unique protein species, of which 100 were not described in previous proteomics studies. As an additional interesting result, a polymorphic alkaline protein was observed with a strong positive signal when blotted from an isoelectric focusing separation in gel and tested with sera of allergic patients. This polymorphic protein, found only after treatment with the peptide library, was identified as an immunoglobulin (Ig), a minor allergen that had been largely amplified. The list of cow's whey components here reported is by far the most comprehensive at present and could serve as a starting point for the functional characterization of low-abundance proteins possibly having novel pharmaceutical, diagnostic, and biomedical applications.

The beer proteome has been evaluated via prior capture with combinatorial peptide ligand libraries (ProteoMiner as well as a homemade library of reduced polydispersity) at three different pH (4.0, 7.0, and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could categorize such species in 20 different barley protein families and 2 maize proteins, the only ones that had survived the brewing process (the most abundant ones being Z-serpins and lipid transfer proteins). In addition to those, we could identify 40 unique gene products from Saccharomyces cerevisiae, one from S. bayanus and one from S. pastorianus as routinely used in the malting process for lager beer. These latter species must represent trace components, as in previous proteome investigations barely two such yeast proteins could be detected. Our protocol permits handling of very large beer volumes (liters, if needed) in a very simple and user-friendly manner and in a much reduced sample handling time. The knowledge of the residual proteome in beers might help brewers in selecting proper proteinaceous components that might enrich beer flavor and texture.

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The oxidation of free DHA, DHA mixed with PC, and DHA incorporated into PC, PE, or TG was evaluated to determine which lipid provided DHA with the best protection against oxidation. DHA was either situated at the sn-1 position, sn-2 position, or both positions of the phospholipid, whereas the TG contained DHA at all positions. All lipids were incubated as bulk lipids, in chloroform, or as an emulsion in contact with air at 25-30 degrees C for 28 d. Since DHA, which is highly sensitive to oxidation, has a great impact on our health and is desired as a food additive, the stability of this FA is of great importance. This study was mainly focused on the primary oxidation products, which were monitored as eight monohydroperoxy-DHA isomer groups, the total amount of polyhydroperoxides, and the PV. However, a measure of secondary oxidation products, the carbonyl value, was also monitored. We found that DHA was most protected against hydroperoxide formation when it was incorporated at one position of either PC or PE. In these lipids, hydroperoxide formation at carbon atoms 4, 7, 8, and 11 was completely prevented. DHA mixed with PC was also protected, although to a lesser extent, and all hydroperoxide isomers were detected. In contrast, PC and TG containing DHA at all positions should be avoided, since they were highly oxidized.

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To describe a novel molecular and pathological phenotype of Creutzfeldt-Jakob disease. Patient: A 69-year-old woman with behavioral and personality changes followed by rapidly evolving dementia. Results: Postmortem examination of the brain showed intracellular prion protein deposition and axonal swellings filled with amyloid fibrils. Biochemical analysis of the pathological prion protein disclosed a previously unrecognized PrP Sc tertiary structure lacking diglycosylated species. Genetic analysis revealed a wild-type prion protein gene. The prion agent responsible for this atypical phenotype was successfully passaged to bank voles. Conclusion: To our knowledge, our results define a new human prion disorder characterized by intracellular accumulation of a novel type of pathological prion protein.