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1.
H K Narang 《Journal of molecular biology》1990,216(3):469-473
The nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles from normal and scrapie-infected hamster brains were examined by electron microscopy. After spreading on collodion-coated grids circular molecules of approximately 15.7 kb corresponding in size to mitochondrial DNA (mtDNA) were observed both in normal and scrapie-infected brains. In nucleic acid preparations from scrapie-infected brains multimeric mtDNA and single-stranded DNA strands of about 0.49 X 10(6) daltons were also visualized. These findings demonstrate the presence of a single-stranded DNA in scrapie-infected brains and are consistent with previous data based on enzyme digestion of nucleic acids isolated from scrapie-infected brains. 相似文献
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Two cDNA libraries were constructed from brain membrane and cytoskeletal preparations purified from scrapie-infected hamster brains. Four recombinants strongly preferential to the scrapie cytoskeletal preparation were identified by the differential hybridization of 7,000 recombinants. These clones were not, however, preferential to total nucleic acids extracted from scrapie-infected hamster brains. DNA sequence analysis revealed all four clones to have significant sequence similarities to the mouse mitochondrial genome. This correlation led us to consider a mitochondrial association with scrapie infectivity. Brain mitochondria were purified by sucrose gradient density centrifugation and found to contain high infectivity. Removal of mitochondrial outer membranes by osmotic shock or digitonin treatment resulted in no detectable loss of titer. 相似文献
5.
PrP(Sc), the only identified component of the scrapie prion, is a conformational isoform of PrPc. The physiological role of PrPc, a glycolipid-anchored glycoprotein, is still unknown. We have shown previously that neuronal nitric oxide synthase (nNOS) activity is impaired in the brains of mice sick with experimental scrapie as well as in scrapie-infected neuroblastoma cells. In this work we investigated the cell localization of nNOS in brains of wild-type and scrapie-infected mice as well as in mice in which the PrP gene was ablated. We now report that whereas in wild-type mice, nNOS, like PrPc, is associated with detergent-insoluble cholesterol-rich membranous microdomains (rafts), this is not the case in brains of scrapie-infected or in those of adult PrP(0/0) mice. Also, adult PrP(0/0), like scrapie-infected mice, show reduced nNOS activity. We suggest that PrPc may play a role in the targeting of nNOS to its proper subcellular localization. The similarities of nNOS properties in PrP(0/0) as compared with scrapie-infected mice suggest that at least this role of PrPc may be impaired in scrapie-infected brains. 相似文献
6.
Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain. 总被引:2,自引:0,他引:2
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This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed. 相似文献
7.
PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27-30 kDa (PrP27-30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27-30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive. 相似文献
8.
Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. 总被引:47,自引:12,他引:47
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R J Kascsak R Rubenstein P A Merz M Tonna-DeMasi R Fersko R I Carp H M Wisniewski H Diringer 《Journal of virology》1987,61(12):3688-3693
Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent. 相似文献
9.
Lee KH Jeong BH Jin JK Meeker HC Kim JI Carp RI Kim YS 《Biochemical and biophysical research communications》2006,349(1):122-130
Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrPSc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210+/-5 days) compared to sR1 (235+/-10 days) after intraperitoneal injection. The levels of PrPSc in sP8 were significantly greater than sR1 at 210+/-5 days, but levels of PrPSc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV. 相似文献
10.
Purification of scrapie agent from infected animal brains and raising of antibodies to the purified fraction 总被引:2,自引:0,他引:2
K Takahashi M Shinagawa S Doi S Sasaki H Goto G Sato 《Microbiology and immunology》1986,30(2):123-131
The fraction (P4) containing scrapie infectivity was obtained by treatment of scrapie-infected mouse brains with the detergent sarcosyl, differential centrifugation, and proteolytic enzyme digestion. Scrapie infectivity in the P4 fraction was purified 239-2,390 times with respect to protein. Similar fractions were also prepared from the brain of a sheep naturally infected with scrapie. Morphological observation of the P4 fractions revealed that the main components were unique rods of 3-5 X 60-200 nm, which resembled scrapie-associated fibrils (SAF) or prion rods. The P4 fractions formed three major broad bands of polypeptides with molecular weights (MWs) of about 24.5K, 21K, and 17K dalton (Kd) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some low MW polypeptides were also present in the fraction. Rabbits immunized with this fraction prepared from mouse brains raised antibodies against the three major polypeptides. 相似文献
11.
Localization of a human gene homologous to the PrP gene on the p arm of chromosome 20 and detection of PrP-related antigens in normal human brain 总被引:10,自引:0,他引:10
N K Robakis E A Devine-Gage E C Jenkins R J Kascsak W T Brown M S Krawczun W P Silverman 《Biochemical and biophysical research communications》1986,140(2):758-765
Infectious fractions prepared from scrapie-infected hamster brains contain a protein, PrP 27-30, which shares antigenic determinants with polypeptides found in similarly prepared fractions from patients with Creutzfeldt-Jakob disease. cDNA sequences encoding the hamster PrP 27-30 identified homologous sequences in the human genome as well as in normal human brain mRNA preparations. Antibodies raised against the mouse PrP's identified antigenically related peptides in both normal hamster and human brain as well as in scrapie-infected hamster brain and CJD-affected human brain. By using in situ hybridization we localized the homologous human genomic sequences on the short arm of chromosome 20. Our results indicate that the reportedly unique proteins detected in human CJD preparations derive from normal human gene products. 相似文献
12.
Jae-Kwang Jin Byungki Jang Hyoung Tae Jin Eun-Kyoung Choi Cha-Gyun Jung Hiroyasu Akatsu Jae-Il Kim Richard I. Carp Yong-Sun Kim 《PloS one》2015,10(4)
PrPSc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrPC) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrPSc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer’s disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases. 相似文献
13.
Quantitative immunogold procedure was used to study the distribution of metallothionein I/II (MT-I/II) at the ultrastructural level in the perivascular areas, including microvascular endothelial cells (ECs) and astrocytes with their perivascular end-feet, in brains of scrapie-infected hyperglycemic (diabetic) and normoglycemic (non-diabetic) mice. Samples of the fronto-parietal cortex obtained from diabetic and non-diabetic scrapie-infected, as well as from non-infected (control) SJL/J mice, were processed for immunocytochemical examination. In control mice, the labelling of the ECs was of low intensity, restricted to few immunogold particles in the cytoplasm. More intense labelling was present in the cytoplasm of astrocytic perivascular processes and perikarya, where it was associated with endoplasmic reticulum and fibrils. A few immunosignals were also present inside the nuclei of astrocytes. In diabetic mice the labelling of the EC cytoplasm was slightly increased, whereas in the cytoplasm of perivascular processes and pericarya of astrocytes, including their nuclei, there was significant enhancement of labelling. In these cells the density of immunosignals was highest in the areas of cytoplasm containing bundles of fibrils. In non-diabetic, scrapie-infected mice the intensity of immunolabelling was higher than in control mice but slightly lower than in diabetic mice. These results are similar to those in Alzheimer’s disease reported by other authors, and suggest that neurodegenerative diseases as well as metabolic stress enhance the metallothionein expression in perivascular regions of brain cerebral cortex, predominantly in astrocytes. 相似文献
14.
K Basler B Oesch M Scott D Westaway M W?lchli D F Groth M P McKinley S B Prusiner C Weissmann 《Cell》1986,46(3):417-428
15.
Jin JK Kim NH Min DS Kim JI Choi JK Jeong BH Choi SI Choi EK Carp RI Kim YS 《Journal of neurochemistry》2005,92(3):452-461
Mitochondrial dysfunction and free radical-induced oxidative damage are critical factors in the pathogenesis of neurodegenerative diseases. Recently, phospholipid breakdown by phospholipase D (PLD) has been recognized as an important signalling pathway in the nervous system. Here, we examined the expression of PLD and alteration of membrane phospholipid in scrapie brain. We have found that protein expression and enzyme activity of PLD1 were increased in scrapie brains compared with controls; in particular, there was an increase in the mitochondrial fraction. PLD1 in mitochondrial membranes from scrapie brains, but not from control brains, was tyrosine phosphorylated. Furthermore, the concentration of mitochondrial phospholipids such as phosphatidylcholine and phosphatidylethanolamine was increased and the content of phosphatidic acid, a product of PLD activity, was up-regulated in the mitochondrial membrane fractions. Immunohistochemically, PLD1 immunoreactivity was significantly increased in activated astrocytes in both cerebral cortex and hippocampus of scrapie brains. Taken together, these results suggest that PLD activation might induce alterations in mitochondrial lipids and, in turn, mediate mitochondrial dysfunction in the brains of scrapie-infected mice. 相似文献
16.
Alessandro Di Martino Jiri Safar Lanfranco Callegaro Norman Salem Jr. Clarence J. Gibbs Jr. 《Neurochemical research》1993,18(8):907-913
Ganglioside composition in brains of terminally ill LVG/LAK golden Syrian hamsters infected with the 263K strain of the scrapie agent was analyzed. Results were compared to those obtained from noninfected animals matched by age, sex, and strain. Gangliosides extracted from scrapie-infected animals showed little change in major components, while an increased number of new alkali-labile species appeared. Additionally, the animal strain employed demonstrated a significant polymorphism in brain ganglioside composition. No significant changes in incubation time, clinical development or pathologic features of scrapie were associated with this polymorphism. 相似文献
17.
Molecular characteristics of the major scrapie prion protein 总被引:18,自引:0,他引:18
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. 相似文献
18.
Previous studies showed that the histopathological changes found in the brains of scrapie-infected animals included amyloid plaque formation, vacuolation, gliosis and neuronal and neurite degeneration. There were differences in the histopathological findings as a function of the scrapie strain-host combination. NADPH-diaphorase (NADPH-d) has been shown to be a selective histochemical marker for neurons containing nitric oxide (NO) synthase. Neuronal cell damage caused by NOS in brain has been reported to be associated with many neurodegenerative diseases. In this study, we used NADPH-d histostaining to investigate changes in the NOS system in brains of 139H- and 263K-infected hamsters and compared the results to normal hamster brain (NHB) injected animals. We observed that some of the NADPH-d histostaining neurons in the cortex of scrapie-infected hamsters appeared to be atrophic: the neurons were smaller and had fewer neurites. The NADPH-d histostaining intensity of neurons or astrocytes in septum, thalamus, hypothalamus and amygdala of 139H- and 263K-infected hamsters was greater than in control hamsters. Astrocytes in the thalamus, hypothalamus and lower part of the cortex (layers 4 to 6) in 263K-infected hamsters were more intensely stained for NADPH-d than in either 139H-infected hamsters or controls. Our results suggest that changes in NADPH-d system might play a role in the diversity of scrapie induced neurodegenerative changes. 相似文献
19.
Ultrastructural studies of scrapie-associated fibrils and prion protein from hamster brains infected with scrapie 总被引:1,自引:0,他引:1
P P Liberski A P?ucienniczak E Hrabec A Bogucki 《Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society》1989,27(1):3-9
We report here about the purification of prion protein 27-30 (PrP 27-30) and scrapie-associated fibrils (SAF) from hamsters infected with the 263K strain of scrapie. Ultrastructural analysis of fractions from scrapie-infected brains revealed numerous fibrils measuring approximately 20 nm in diameter and 100-200 nm in length. The substructure of these fibrils consisted of protofilaments which were usually straight and rarely helically arranged. We conclude that the electron microscopic appearance of SAF depends much on the purification scheme. 相似文献
20.
Birkmann E Schäfer O Weinmann N Dumpitak C Beekes M Jackman R Thorne L Riesner D 《Biological chemistry》2006,387(1):95-102
A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage. 相似文献