Harold M. Frost, M.D., D.Sc. (hon) -- one man's association

The author described how and when he first met Harold M. Frost, M.D., that began a journey from the Henry Ford Hospital to the University of Utah and Sun Valley Hard TissueWorkshops that sequentially developed the technology of dynamic cancellous bone histomorphometry, the ever-evolving mechanostat hypothesis and the Utah Paradigm for Bone Physiology.     

Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.     

Sphingolipid depletion increases formation of the scrapie prion protein in neuroblastoma cells infected with prions.

Sphingolipid-rich rafts play an essential role in the posttranslational (Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990) J. Cell Biol. 110, 743-752)) formation of the scrapie prion protein PrP(Sc) from its normal conformer PrP(C) (Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L., Prusiner, S. B., and Avraham, D. (1995) J. Cell Biol. 129, 121-132). We investigated the importance of sphingolipids in the metabolism of the PrP isoforms in scrapie-infected ScN2a cells. The ceramide synthase inhibitor fumonisin B(1) (FB(1)) reduced both sphingomyelin (SM) and ganglioside GM1 in cells by up to 50%, whereas PrP(Sc) increased by 3-4-fold. Whereas FB(1) profoundly altered the cell lipid composition, the raft residents PrP(C), PrP(Sc), caveolin 1, and GM1 remained insoluble in Triton X-100. Metabolic radiolabeling demonstrated that PrP(C) production was either unchanged or slightly reduced in FB(1)-treated cells, whereas PrP(Sc) formation was augmented by 3-4-fold. To identify the sphingolipid species the decrease of which correlates with increased PrP(Sc), we used two other reagents. When cells were incubated with sphingomyelinase for 3 days, SM levels decreased, GM1 was unaltered, and PrP(Sc) increased by 3-4-fold. In contrast, the glycosphingolipid inhibitor PDMP reduced PrP(Sc) while increasing SM. Thus, PrP(Sc) seems to correlate inversely with SM levels. The effects of SM depletion contrasted with those previously obtained with the cholesterol inhibitor lovastatin, which reduced PrP(Sc) and removed it from detergent-insoluble complexes.     

Spectroscopic evidence for amyloid-like interfacial self-assembly of hydrophobin Sc3

Amphipathic fungal proteins called hydrophobins are able to self-assemble into insoluble supramolecular structures at hydrophobic/hydrophilic interfaces, but the molecular mechanism and underlying protein conformation changes are not known. Secondary-structure prediction indicated that hydrophobin Sc3 is an all-beta protein. Many amyloidogenic proteins self-assemble into insoluble amyloid fibrils while undergoing a change to an all-beta conformation. In this study we show that two dyes, thioflavin T, and Congo red, which are widely used for specific detection of stacked beta sheets, interact with Sc3 assemblies in the same way as with the amyloid beta-sheet fibrils. We conclude that Sc3, and probably other hydrophobins too, self-assemble at interfaces in the same manner as amyloidogenic proteins, i.e., through beta-sheet stacking.     

Shosaku Numa, M. D., D. Med. Sc.

Shosaku Numa博士,日本人,1952年毕业于京都大学医学系,曾先后在美国哈佛大学医学院和Max-Planck细胞化学研究所接受博士后训练,现任京都大学医学院医用化学及分子遗传学教授。在利用DNA重组技术探索递质受体和离子通道的研究中,Numa博士阐明了胆碱N和M受体、钠通道、钙通道以及钙释放通道的初级结构,所获得的有关受体结构的资料还表明递质问离子通道、电压门离子通