Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc. The PrPC-peptide complex formed fibrous aggregates and up to 65% of complexed PrPC sedimented at 100,000 x g for 1 h, whereas PrPC alone did not. These complexes were resistant to proteolytic digestion and displayed a high beta-sheet content. Unexpectedly, the peptide in a beta-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPC sensitive to protease digestion. While the pathogenic A117V mutation increased the efficacy of complex formation, anti-PrP monoclonal antibody prevented interaction between PrPC and peptides. Our findings in concert with transgenetic investigations argue that PrPC interacts with PrPSc through a domain that contains the first two putative alpha-helices. Whether PrPC-peptide complexes possess prion infectivity as determined by bioassays remains to be established.

The secondary structure of a 55-residue fragment of the mouse prion protein, MoPrP(89 -143), was studied in randomly aggregated (dried from water) and fibrillar (precipitated from water͞ acetonitrile) forms by 13

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Metaphase-II chromosome transfer (M-II transfer) is considered to be a useful technique for studying nucleus–cytoplasm relationships, or for generating oocytes with good developmental ability after transfer of the nucleus to the cytoplasm. The reconstructed oocytes carry the original genomic information within the metaphase chromosomes from the donor oocytes. The objective of the present study was to evaluate the parthenogenetic developmental ability of porcine M-II transferred oocytes. In vitro maturation was carried out as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). After culture for 44 h, cumulus cells were removed by hyaluronidase treatment and gentle pipetting. Oocytes that had extruded the first polar body were selected and centrifuged at 13 000g for 9 min to stratify the cytoplasm. The zonae pellucidae were removed after exposure to pronase, and zona-free oocytes were layered on a 300 �L discontinuous gradient (100 �L each of 45%, 30%, and 7.5%) of Percoll in TCM-HEPES supplemented with 5 �g mL-1 cytochalasin B. After centrifugation of the oocytes on the gradient in microcentrifuge tubes at 6000g for 20 s, fragmented cytoplasm with an equal volume was obtained, stained with Hoechst 33342, and classified as cytoplasm with or without chromosomes by observation with a fluorescence microscope. One fragmented cytoplasm with chromosomes and 2 fragmented cytoplasms without chromosomes were fused by electric stimulation with a single DC pulse (1.5 kV cm-1, 20 �s) and cultured temporarily for 1 h. The reconstructed oocytes were then stimulated again to induce parthenogenetic activation (0.8 kV cm-1, 30 �s, 2 DC pulses) (treatment group). Zona-free mature oocytes that had not been subjected to reconstruction were activated as a control group. The oocytes in both groups were treated with 5 �g mL-1 cytochalasin B for 2 h, and then cultured for 6 days in media (Kikuchi et al. 2002) using the WOW system (Gabor et al. 2000 Mol. Reprod. Dev.). The blastocyst formation rate in the control group (22.9 � 5.5%) was significantly higher (P < 0.05; ANOVA and PLSD-test) than that in the treatment group (7.6 � 1.8%). The total cell number per blastocyst in the control group (28.7 � 4.6) was significantly higher (P < 0.05) than that in the treatment group (16.7 � 1.0). These results suggest that reconstructed porcine oocytes following M-II transfer by centrifugation and electrofusion can develop to the blastocyst stage in vitro. This technique enables transfer of the nucleus to cytoplasm with good developmental ability without the use of a micro-manipulation system.