The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the hallmarks of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in ~130 days and reached the terminal stage of disease in ~150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease, confirming that these mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.

No abstract

Whether a genetic informational nucleic acid is required for the infectivity of transmissible spongiform encephalopathies is central to the debate about the infectious agent. Here we report that an infectious prion formed with bacterially expressed recombinant prion protein plus synthetic polyriboadenylic acid and synthetic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol is competent to infect cultured cells and cause prion disease in wild-type mice. Our results show that genetic informational RNA is not required for recombinant prion infectivity.

The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

Diethylnitrosamine (DEN) is a known carcinogen that can alkylate DNA molecules. In rats, DEN-induced hepatocellular carcinoma (HCC) model is well established. In this study, we used a twodimensional differential gel electrophoresis (2D-DIGE) system and liquid chromatography/mass spectrometry/mass spectrometry to identify the differential expression protein profiles between the DEN-induced HCC and healthy liver cells. Western blotting and semiquantitative RT-PCR were used to further confirm the results. Seventeen differentially expressed spots were identified in DEN-induced HCC cells. Among all, the most prominent upregulated proteins include the members of the glutathione Stransferase super family, aldo-keto reductase superfamily and proteins involved in the response to oxidative stress. Downregulation was observed in 2 proteins that were known to contribute to hepatic dysfunction. This study provides the first comprehensive protein profiling of the DEN-induced HCC in rats. This model simulates the differential protein expression of human HCC and may be useful for further understanding the mechanism of HCC tumorigenesis. ' 2008 Wiley-Liss, Inc.Key words: hepatocellular carcinoma; diethylnitrosamine; twodimensional differential gel electrophoresis; glutathione S-transferase Hepatocellular carcinoma (HCC) is a major malignancy worldwide.1 It is the fourth most common malignant tumor in the world 2 and is the second leading fetal disease in Mainland China. 3 In last 2 decades, the incidence of HCC has increased substantially all over the North America and Europe. 4 Studies have indicated HCC is associated with a major risk factor of hepatitis B and C viral infections [i.e., hepatitis B virus (HBV) and hepatitis C virus (HCV)].5 However, other factors such as aflatoxin B1 exposure, alcohol drinking and genetic defects have also aroused research interests in recent years. 6,7 To further understand the disease, animal models have been established for the study of hepatocarcinogenesis. These include the two-stage initiation-promotion 8 and the three-stage initiation, promotion and progression 9 models. The initiative period is characterized by the mutation of cells, which then undergo tumor promotion via active cell division or altered genome expression induced by chemicals.10 Thereafter, mutations continue and lead to the development of HCC cells via a number of causes such as the presence of HBV, HCV or radiation. Changes such as oxidation, chemical adducts and errors in the DNA bases and repair are also implicated in the early cellular changes.11 They are the crucial changes in the initial stage of HCC, which correspond to the histological observations including fibrosis, cirrhosis, and hepatocellular adenoma. 11In this study, diethylnitrosamine (DEN) was employed to induce HCC in rats. It is an environmental carcinogen and a hepatotoxin. It is commonly used in inducing hepatocarcinogenesis as it causes degenerative, proliferative and neoplastic lesions in the liver. DEN is a chemical agent that can alkylate DNA mo...