Direct evidence of generation and accumulation of ß-sheet-rich prion protein in scrapie-infected neuroblastoma cells with human IgG1 antibody specific for ß-form prion protein.

We prepared ß-sheet-rich recombinant full-length prion protein (ß-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this ß-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to ß-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of ß-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of ß-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing ß-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to ß-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

Arterial thrombosis after intravenous infusion of oral bacterium in a rat model.

Oral bacteria have been detected at atherosclerotic plaque, aneurysms, and thrombosed arteries in Buerger disease. We explored a possible relationship between the oral bacterium Porphyromonas gingivalis and arterial thrombosis at proximal and distal sites in rats. Eighteen rats underwent subcutaneous placement of an infusion pump connected to the jugular vein. The Pg infusion group received a continuous infusion of P. gingivalis for 2 weeks, and the controls received normal saline. At 2 and 4 weeks, specimens were obtained from the iliac, superficial, and below-knee arteries, which were studied pathologically and by polymerase chain reaction (PCR) analysis to detect P. gingivalis-specific DNA. The Pg infusion group had thrombosis in 33.3% at 2 weeks and in 55.6% at 4 weeks, but normal arterial wall structure was preserved without any features of infection. Positive PCR findings were recognized in 73.3% and 22.2% at 2 and 4 weeks, respectively. At 4 weeks, thrombosis was observed in a higher proportion, with the below-knee specimens having an especially high thrombus rate (83.3%). No control specimen had thrombosis or positive PCR results. Bacteremia due to the oral pathogen P. gingivalis may lead to thrombus formation in the peripheral arteries, especially in small-sized arteries.

Reversible monomer-oligomer transition in human prion protein.

The structure and the dissociation reaction of oligomers Pr(Poligo) from reduced human prion huPrP(C)(23-231) have been studied by (1)H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The 1H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105 approximately 210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra- and/ or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrP(C*), a rare metastable form of PrP(C) stabilized at high pressure (Kachel et al., BMC Struct Biol 6:16). The results strongly suggest that the oligomeric form PrP(oligo) is in dynamic equilibrium with the monomeric forms via PrP(C*), namely huPrP(C)[left arrow over right arrow]huPrP(C*)[left arrow over right arrow]huPrP(oligo).

Vascular closure system type of nonpenetrating arcuate-legged titanium clips for graft-artery and graft-graft anastomoses: review of our clinical experience.

PURPOSE: To assess the durability of Vascular Closure System (VCS) clips for graft-artery and graft-graft anastomoses. METHODS: The subjects were 100 consecutive patients, who had undergone vascular procedures in which VCS clip application was attempted for anastomoses. The operative indications were arteriosclerosis obliterans in 69 patients, aortic aneurysm in 26, and other disorders in 5. Large clips were used for both graft-artery and graft-graft anastomoses, and medium-sized clips were used for the smaller caliber femoral or popliteal arteries. RESULTS: Vascular Closure System clips could not be applied to anastomose the graft to the artery in 13 patients because the arterial wall was too thick or stiff. Anastomosis was accomplished without any problems in 80 patients, although suture-line bleeding occurred in 7 patients. This was ameliorated by an additional clip in four patients, but interrupted sutures were needed to seal the anastomosis in the other three patients. The 1-, 3-, and 5-year cumulative primary patency rates were 98.7%, 97.4%, and 87.7%, respectively. There were two graft failures and two anastomotic aneurysms. CONCLUSION: Vascular Closure System clips were useful to coapt a prosthetic graft to an artery unless the arterial wall was thicker than 2 mm or calcified. Thus, VCS clips could be durable enough for graft-artery anastomoses in the iliac or popliteal region.