Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.

Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.     

Molecular characteristics of the major scrapie prion protein

A major protein was identified that purifies with the scrapie agent extracted from infected hamster brains. The protein, designated PrP 27-30, was differentiated from other proteins in purified fractions containing the scrapie agent by its microheterogeneity (Mr 27000-30000) and its unusual resistance to protease digestion. PrP 27-30 was found in all fractions enriched for scrapie prions by discontinuous sucrose gradient sedimentation or sodium dodecyl sarcosinate-agarose gel electrophoresis. It is unlikely that PrP 27-30 is a pathologic product because it was found in fractions isolated from the brains of hamsters sacrificed prior to the appearance of histopathology. If PrP 27-30 is present in normal brain, its concentration must be 100-fold lower than that found in equivalent fractions from scrapie-infected hamsters. Three protease-resistant proteins similar to PrP 27-30 were found in fractions obtained by discontinuous sucrose gradient sedimentation of scrapie-infected mouse brain. These proteins were not evident in corresponding fractions prepared from normal mouse brain. One-dimensional peptide maps comparing PrP 27-30 and normal hamster brain proteins of similar molecular weight demonstrated that PrP 27-30 has a primary structure which is distinct from these normal proteins. Heating substantially purified scrapie fractions to 100 degrees C in sodium dodecyl sulfate inactivated the prion and rendered PrP 27-30 susceptible to protease digestion. Though the scrapie agent appears to be hydrophobic, PrP 27-30 remained in the aqueous phase after extraction with organic solvents, indicating that it is probably not a proteolipid. PrP 27-30 is the first structural component of the scrapie prion to be identified.     

Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease

The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease.     

Physical and Chemical Properties of the Transmissible Mink Encephalopathy Agent

The size of the transmissible mink encephalopathy (TME) agent is estimated to be less than 50 nm on the basis of its passage through membrane filters. The agent is sensitive to ether, relatively resistant to 10% Formalin, resistant to ultraviolet irradiation, and susceptible to proteolytic digestion with Pronase. Attempts to extract an infectious nucleic acid fraction with hot phenol were unsuccessful. The results of these studies indicate that the TME agent has biochemical properties which are similar to those described for the transmissible agent of scrapie.     

Isolation and structural studies of the intact scrapie agent protein

Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had greater than 10(11) LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-428; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N alpha-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.     

Processing and degradation of exogenous prion protein by CD11c(+) myeloid dendritic cells in vitro

The immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. CD11c(+) myeloid dendritic cells (DC) could, due to their subepithelial location and their migratory capacity, be early targets for prion infection and contribute to the spread of infection. In order to analyze mechanisms by which these cells may affect prion propagation, we studied in vitro the effect of exposing such DC to scrapie-infected GT1-1 cells, which produce the scrapie prion protein PrP(Sc). In this system, the DC efficiently engulfed the infected GT1-1 cells. Unexpectedly, PrP(Sc), which is generally resistant to protease digestion, was processed and rapidly degraded. Based on this observation we speculate that CD11c(+) DC may play a dual role in prion infections: on one hand they may facilitate neuroinvasion by transfer of the infectious agent as suggested from in vivo studies, but on the other hand they may protect against the infection by causing an efficient degradation of PrP(Sc). Thus, the migrating and highly proteolytic CD11c(+) myeloid DC may affect the balance between propagation and clearance of PrP(Sc) in the organism.     

The estrogen-receptor complex is bound at unusual chromatin regions

The estrogen receptor of MCF-7 cells labeled with high specific activity estradiol was used to mark the chromatin binding sites for this regulatory molecule. Many of these sites are especially sensitive to nuclease, and produce on digestion a series of uniquely sedimenting products. Several of these have been examined in some detail in this paper. These include a form of receptor that sediments in trace digests at 9S but in more extensive digests at 7S, fast mononucleosomes of about 12.5S, and a species at 15S. Two components of digests, fast mononucleosomes and dinucleosomes were isolated and subjected to further digestion. Much of the hormone on these isolated particles was found to be sensitive to additional hydrolysis, although some was nuclease resistant. It appears that a major fraction of the hormone receptor complexes bound to MCF-7 cell chromatin occurs at nucleosome-free regions which can be detected as transient hydrolysis intermediates.     

Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis.

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.     

免疫印迹法检测牛海绵状脑病和羊瘙痒病

用大肠杆菌表达的牛朊病毒正常成熟蛋白 (BoPrPC)免疫新西兰白兔 ,获得了与朊病毒蛋白 (PrP)反应的抗体T1。根据致病型朊病毒 (PrP^SC)能抵抗蛋白酶消化的特性 ,用蛋白酶K消化脑组织提取物 ,以抗体T1进行免疫印迹反应 ,结果表明从接种羊瘙痒病朊病毒 2 6 3K的金黄地鼠脑组织提取物内检测到抗蛋白酶K消化的致病型PrPSC ,而正常金黄地鼠脑组织中没有抗蛋白酶消化的蛋白。以我国正常牛羊为材料 ,制备其脑组织提取物 ,用上述方法和抗体T1进行检测 ,结果没有发现抗蛋白酶K的任何蛋白存在 ,说明没有牛海绵状脑病和羊瘙痒病存在。用 1A8抗体也获得了同样的结果。这些结果表明可以用自制的抗血清检疫牛海绵状脑病和羊瘙痒病 ,防止其传入我国     

Scrapie strain infection in vitro induces changes in neuronal cells

PC12 cells, in the presence of nerve growth factor (NGF), support replication of the mouse-derived scrapie strains 139A and ME7, with the former yielding 100–1000-fold higher levels of infectivity. Infectivity remained cell-associated and cells did not show any gross morphological alterations, although changes were observed by electron microscopy in the form of an increased number of lipid droplets in 139A-infected cultures. Analysis of phospholipid metabolism in 139A infected cells indicated that scrapie replication did not change the inositol phosphate levels, but did stimulate phosphoinositide synthesis. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Since scrapie-infected cultures did not exhibit cell death or any gross changes, any scrapie-induced effects would probably be manifested in nonvital cellular functions. When compared to controls, infection with the 139A scrapie strain resulted in decreased activity of the cholinergic pathway-related enzymes, as well as the GABA synthetic pathway; however, the adrenergic pathway was unaffected by scrapie infection. The effects of the 139A scrapie strain on the cholinergic system appeared to be dose-dependent and were first detected prior to the detection of scrapie agent replication in these cells. No neurotransmitter-related enzymatic changes were detected in 263K- or 139R-infected PC12 cells. The enzymatic changes observed in ME7-infected PC12 cells and in Chandler agent-infected mouse neuroblastoma cells suggest that the significant changes in neurotransmitter levels in cultures exhibiting low infectivity titers must involve factors other than, but not excluding, replication of the agent. The role of additional factors is also suggested in studies of protein kinase C activity in 139A- and 139R-infected PC12 cells. These studies emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and, in addition, support the concept of variation among scrapie strains.     

Evidence for a subunit structure of chromatin in mouse myeloma cells

If micrococcal nuclease is allowed to digest chromatin as it exists inside intact nuclei isolated from mouse myeloma tissue culture cells, more than 60% of the DNA can be isolated as a homogeneous fragment on a sucrose gradient. Analytical ultracentrifugation indicates that the protected DNA is native, unnicked, and about 140 +/- 10 base pairs long. After less extensive nuclease digestion, the protected DNA migrates in gels in lengths which are integral multiples of this 140 base pair "monomer" band. A submonomer band, 105 "/- 10 base pairs long, can also be detected. Similar digestion patterns were obtained by two different nuclear isolation procedures and even when intact cells were gently lysed directly in the digestion medium. These results confirm and extend the chromatin digestion studies of previous investigators and provide support for a subunit model for eukaryotic chromatin. The single strand specific S1 nuclease did not digest intranuclear chromatin under the conditions used.     

Comparative molecular analysis of the abnormal prion protein in field scrapie cases and experimental bovine spongiform encephalopathy in sheep by use of Western blotting and immunohistochemical methods

Since the appearance of bovine spongiform encephalopathy (BSE) in cattle and its linkage with the human variant of Creutzfeldt-Jakob disease, the possible spread of this agent to sheep flocks has been of concern as a potential new source of contamination. Molecular analysis of the protease cleavage of the abnormal prion protein (PrP), by Western blotting (PrP(res)) or by immunohistochemical methods (PrP(d)), has shown some potential to distinguish BSE and scrapie in sheep. Using a newly developed enzyme-linked immunosorbent assay, we identified 18 infected sheep in which PrP(res) showed an increased sensitivity to proteinase K digestion. When analyzed by Western blotting, two of them showed a low molecular mass of unglycosylated PrP(res) as found in BSE-infected sheep, in contrast to other naturally infected sheep. A decrease of the labeling by P4 monoclonal antibody, which recognizes an epitope close to the protease cleavage site, was also found by Western blotting in the former two samples, but this was less marked than in BSE-infected sheep. These two samples, and all of the other natural scrapie cases studied, were clearly distinguishable from those from sheep inoculated with the BSE agent from either French or British cattle by immunohistochemical analysis of PrP(d) labeling in the brain and lymphoid tissues. Final characterization of the strain involved in these samples will require analysis of the features of the disease following infection of mice, but our data already emphasize the need to use the different available methods to define the molecular properties of abnormal PrP and its possible similarities with the BSE agent.     

A protease-resistant protein is a structural component of the scrapie prion

Fractions purified from scrapie-infected hamster brain contain a unique protein, designated PrP. It was labeled with N-succinimidyl 3-(4-hydroxy-5-[125I]-iodophenyl) propionate, which did not alter the titer of the scrapie prion. The concentration of PrP was found to be directly proportional to the titer of the infectious prion. Both PrP and prion infectivity were resistant for 2 hr at 37 degrees C to hydrolysis by proteinase K under nondenaturing conditions. Prolonging the digestion resulted in a concomitant decrease in both PrP and the scrapie prion. When the amino-acid-specific proteases trypsin or SV-8 protease were used instead of proteinase K, no change in either PrP or the prion was detected. The parallel changes between PrP and the prion provide evidence that PrP is a structural component of the infectious prion. Our findings also suggest that the prion contains only one major protein, namely PrP.     

Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells.

Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.     

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes

Summary The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2–3 mm³) and with volumes tissue ratios ≥2 ml/g tissue. For incubations ≤10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions ≤10 h (collagenase 2700–3900 Mandl U/ml, neutral protease 5100–10,000 caseinase U/ml, and clostripain 35–48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350–3000 U/ml, neutral protease 2550–7700 U/ml, and clostripain 18–36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1×10⁴ cells/cm². These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.     

Organization within the mammalian kinetochore

The organization within the mammalian kinetochore was examined using whole-mount electron microscopic techniques on chromosomes digested with restriction enzymes or micrococcal nuclease. These preparations revealed that a portion of the kinetochore is highly resistant to nuclease digestion and can be visualized as a discrete structure. The relationship of this structure to the remainder of the chromosome suggests that it represents the outer kinetochore plate. The plate is composed of a series of fibrillar loops that are arranged in a parallel array along the plane of the plate. These fibers are 25–30 nm in diameter. The morphology, particulate substructure, and ultimate susceptibility to nuclease digestion suggest that these fibers contain DNA. A model is presented that suggests that the outer plate contains the apexes of chromatin loops that originate within the body of the primary constriction.     

Analyses of frequency of infection, specific infectivity, and prion protein biosynthesis in scrapie-infected neuroblastoma cell clones.

Scrapie, a spongiform encephalopathy of sheep and goats, is caused by a poorly understood transmissible agent in which no nucleic acid has been conclusively identified. Biochemical characterization of agent derived from animal tissues has not been precise because of the tenacious association of the agent with tissue components. As an approach toward obtaining homogeneous preparations of agent generated in vitro, we cloned scrapie-infected neuroblastoma cells. By frequency analysis, nearly every cell in expanded cultures contained scrapie agent. We also analyzed cell-dose infectivity relationships and developed a standard curve which allowed various cultures to be compared. Since a proteinase K (PK)-resistant form of a protein designated prion protein (PrP) has been found in partially purified preparations of scrapie agent from infected animal spleens and brains, we sought to identify this protein in cell cultures. No PK-resistant PrP was found in infected or uninfected cultures, although the PK-sensitive PrP was readily detected. These results suggested that PK-resistant PrP may not be an essential component of the infectious scrapie agent.     

Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease.

It has been suggested that the infectious agents of scrapie and Creutzfeldt-Jakob disease (CJD) are 'prions' constituted by a protease resistant glycopeptide, PrP. To analyze the role of PrP in CJD infectivity we re-evaluated the biochemical characteristics of infectivity. First, when the infectious agent is not aggregated, infectivity is exquisitely sensitive to proteinase K treatment, and therefore a proteinase-K-resistant molecule (e.g. PrP) is unlikely to contain information essential for agent replication. Second, removal of sugar residues from Gp34 (the major precursor of the proteolyzed PrP band) failed to reduce infectivity. Third, one-half of the PrP peptides could be separated from significant infectivity using nondenaturing conditions with practical quantitative recovery of infectivity. These studies suggest that PrP in itself is unlikely to be the replicating component of the infectious agent. We suggest that these as yet undefined agents may consist of core protein and nucleic acid that are incompletely assembled in, and protected by, cell membranes. This hypothesis would explain the absence of conventional viral particles in these diseases, account for observed membrane pathology including altered behavior of endogenous membrane proteins, and would be consistent with the replication and transforming properties of CJD that indicate there is an agent specific nucleic acid.     

Encystment-Inducing Factor from Cultures of Acanthamoeba palestinensis

Acanthamoeba palestinensis grown in monolayer cultures encysted almost synchronously at a stationary phase, with a yield of about 80% cysts. Under these growth conditions an encystmentinducing factor was released into the medium by transforming amoebae. Cell-free supernatants induced encystment of amoebae from early-log phase cultures. The not dialyzable encystment factor was resistant to nuclease, protease and trypsin digestion, as well as to boiling, but the activity was almost completely destroyed by autoclaving. Isolation and further characterization of the factor will enable clarification of the mode of its action as a regulator of amoeba-cyst transformation.