An experimental examination of the scrapie agent in cell membrane mixtures: III. Studies of the operational size
Abstract
Membrane preparations from scrapie-affected mouse brain were ultrasonicated, passed through filters of 50 nm and 20 to 35 nm pore size and the filtrates analysed using exclusion chromatography, isopycnic sedimentation and electron microscopy.
A high recovery of scrapie activity was found in the 50 nm filtrates and all evidence indicated association of this activity with membrane fragments. Although a significant amount of lipoprotein material was recovered in 20 to 35 nm filtrates, membrane fragments were absent and no scrapie activity was found.
These observations are consistent with the membrane hypothesis of scrapie: it is suggested that the operational size of the agent is much larger than the radiation target size.
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Scrapie infectivity is independent of amyloid staining properties of the N-Terminally truncated prion protein
2000, Journal of Structural BiologyThe rion rotein undergoes a profound conformational change when the ellular isoform (PrPC) is converted into the disease-causing form (PrPSc). Limited proteolysis of PrPSc produces PrP 27-30, which readily polymerizes into amyloid. To study the relationship between PrP amyloid and infectivity, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes α-helix formation, modified the ultrastructure of PrP amyloid and decreased the β-sheet content as well as prion infectivity. HFIP reversibly decreased the binding of Congo red dye to the PrP amyloid rods while inactivation of prion infectivity was irreversible. In contrast, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolished Congo red binding. Solubilization using various solvents and detergents produced monomeric and dimeric PrP that lacked infectivity. Proteinase K resistance of detergent-treated PrP 27-30 showed no correlation with scrapie infectivity. Our results separate prion infectivity from the amyloid properties of PrP 27-30 and underscore the dependence of prion infectivity on PrPSc conformation. These findings also demonstrate that the specific β-sheet-rich structures required for prion infectivity can be differentiated from those required for amyloid formation.
Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions
1999, Biophysical JournalThe structural transition from the cellular prion protein (PrPC) that is rich in α-helices to the pathological form (PrPSc) that has a high β-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation.
The key must fit: Macrophages transport prion infection to the central nervous system and may determine the sites of infection within it
1997, Medical HypothesesIt is suggested that the agent for transmissible spongiform encephalopathies is trasferred from an original peripheral site of infection into the brain by recruited and selected circulating macrophages/monocytes. It is because of this selection that strains of disease appear to be different when infecting separate species, but retain characteristics when infecting a single species.
Transmission of spongiform encephalopathy agents or prions
1996, Medecine et Maladies InfectieusesLes agents transmissibles non conventionnels (ATNC) ou prions induisent des maladies neurologiques lentes toujours mortelles chez l'homme et chez l'animal. Leur nature n'est toujours pas connue avec précision. Sur le plan neuropathologique, l'atteinte du système nerveux central se caractérise par une spongiose neuronale et une gliose avec hyperastrocytose, en l'absence de tout signe inflammatoire central ou périphérique. Sur le plan biochimique, les prions induisent l'accumulation, proportionnellement au titre infectieux, d'une protéine de l'hôte, la PrP, sous une isoforme résistante partiellement à la protéinase K. Pour certains auteurs, cette protéine modifiée serait l'agent lui-même, alors que d'autres associent à ces agents une information génétique dont la nature reste à préciser. Les ATNC sont particulièrement résistants aux procédés de décontamination physiques et chimiques. Ils sont transmissibles expérimentalement et au travers d'actes chirurgicaux ou médicaux impliquant le système nerveux central : utilisation d'instruments de neurochirurgie insuffisamment décontaminés, d'électrodes profondes, greffes de tympan, de cornée ou de dure mère, traitement par des hormones d'origine hypophysaire. Ces accidents dramatiques justifient amplement la mise en place de measures de santé publique visant à minimiser le risque lié aux ATNC.
Transmissible spongiform encephalopathy agents (TSA) or prions are the aetiologic agents of the transmissible spongiform subacute encephalopathies (TSSE) which can be described in humans and animals. TSSE are transmissible among individuals of the same species and some of different species. These diseases stem from a very specific category of agents that have specific physicochemical and biological properties. Their nature remains still unknown. Transmissibility has been proven experimentally and in natural situations such as the outbreak of CJD among children treated with extractive growth hormone and the recent surge of a new disease decimating British cattle, the bovine spongiform subacute encephalopathy, or disease of the mad cow. This double causality has a number of consequences, one of them is the requirement of a strict compliance to safety regulations in the preparation of biological products used in human therapy, in grafting and blood donation.
Assessment of risk of transmission of unconventional agents through human albumin
1996, Annales Francaises d'Anesthesie et de ReanimationLes agents transmissibles non conventionnels (ATNC) ou prions sont à l'origine de maladies lentes dégénératives du système nerveux central, les encéphalopathies subaiguës spongiformes transmissibles (ESST). Leur nature exacte n'est pas encore connue avec précision, même s'il semble que leur composante principale voire exclusive soit une isoforme pathologique d'une protéine de l'hôte, la protéine du prion (PrP). À ce jour, il n'existe aucun test de dépistage des sujets infectés. Les ATNC résistent à de nombreux procédés d'inactivation, habituellement efficaces en virologie. Des accidents de contamination iatrogène ont été rapportés: ils ont toujours concerné l'utilisation des substances, de greffons, ou d'instruments ayant été en contact avec le système nerveux central. Chez un individu atteint, l'infectiosité est détectable majoritairement dans le système nerveux central. Cependant, de faibles niveaux de ≪ virémie ≫ peuvent être mis en évidence: cette infectiosité est toujours associée aux cellules blanches du sang, et jamais au plasma, au sérum, ou aux cellules rouges. Par ailleurs, les études épidémiologiques les plus récentes montrent que la transfusion sanguine n'est pas vectrice de contamination par les ATNC. Le risque de transmission d'un ATNC au travers de l'administration d'albumine n'est donc que théorique.
Transmissible spongiform encephalopathy agents (TSA) or prions induce neurodegenerative diseases in humans and animals. Their nature is still unknown, even if the main component of infectivity is identified as an abnormal isoform of a host-encoded protein, the prion protein (PrP). Today, no diagnostic test is available routinely for the detection of infected patients. TSA are resistant to most of the physical and chemical procedures that are efficient against other micro-organisms, latrogenic transmissions of TSA have been reported in the past: they always involved either brain derivatives or instruments that have been in contact with infected central nervous system. In an infected individual, infectivity is mostly detectable in brain. However, a persistent low-level viremia can be demonstrated in association with the white blood cells; infectivity is never found in plasma, serum or in red blood cells. Epidemiological data do not evidence any relationship between spongiform encephalopathies and blood transfusion. Therefore, in 1995, TSA transmission trough albumin is only a theoretical risk.
Analysis of Creutzfeldt-Jakob disease infectious fractions by gel permeation chromatography and sedimentation field flow fractionation
1992, Virus ResearchGel permeation chromatography and sedimentation field flow fractionation (SF3) were used to further analyze highly infectious fractions from Creutzfeldt-Jakob disease (CJD) infected hamster brain. These analyses defined the relative molecular mass and physical size of the Creutzfeldt-Jakob disease (CJD) agent with greater precision than previously possible. Highly purified disaggregated fractions yielded single, homogeneous Gaussian peaks with both methods. The relevant analytical peaks contained protein-nucleic acid complexes with an Mrr of ~ 1.5 × 107 daltons and a mean radius of ~ 30 nm. The experimental evidence further solidifies the concept of an infectious agent that resembles a viral core rather than a simple protein.