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At least 19 recordsLinked to original sources
PubMed2005

Biochemical evidence for the proteolytic degradation of infectious prion protein PrPsc in hamster brain homogenates by foodborne bacteria.

PrP(Sc) is a general term to describe the infectious agent causing transmissible spongiform encephalopathy (TSE), and the protease-resistant form of cellular PrP(C). In this study, we have identified several protease-secreting bacteria able to degrade PrP(Sc) under more or less native conditions (30 degrees C, pH 8), focusing on strains isolated mainly from cheese. One hundred and ninty-nine protease-secreting isolates belonging to the Actinomycetales and Bacillales were screened for the expression of PrP(Sc) degrading activity by a Western blot procedure. Only 6 strains belonging to the following species were found to exhibit such an activity: Arthrobacter nicotianae, Bacillus licheniformis, Brachybacterium conglomeratum, Brachybacterium tyrofermentans and Staphylococcus sciuri and Serratia spp. As revealed by a general protease assay based on dye-labeled Azocoll substrate, the PrP(Sc) degrading activity was not directly correlated to the total level of secreted proteolytic activity of these organisms. This indicates that specific proteases are required for the degradation of PrP(Sc). Our study also suggests the potential use of such starter bacteria or their proteases for application in PrP(Sc) degradation and decontamination under native conditions.

Actinomycetales↗
PubMed1997

Biochemical properties of protease resistant prion protein PrPsc in natural sheep scrapie.

Prions are infectious agents involved in neurodegenerative diseases, such as scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cows and Creutzfeldt-Jakob disease (CJD) in humans. These pathogens are characterized by unusual properties, and, in particular, by their strong resistance to common procedures of disinfection used against conventional microorganisms. A major component of highly infectious fractions is a proteinase K-resistant prion protein PrPsc (PrP-res), the normal host prion protein PrPc being sensitive to PK (PrP-sen). We used a biochemical approach to further characterize PrPsc protein in natural sheep scrapie. Western blot analyses using rabbit antiserum that recognized both normal and pathologic sheep prion proteins, were undertaken to study the biochemical behaviour of PrPsc extracted from brains of sheep naturally infected with scrapie after protease digestion and under denaturing conditions. Increasing concentrations of urea (1-7 M) or GdnSCN (0.25-3 M) and different pH from 2 to 11 were tested for their effects on protease resistance of PrPsc. Alkaline pH (pH = 10) and high concentrations of urea (> 3 M) and GdnSCN (> 0.75 M) greatly decreased the protease resistance of the prion protein. Identical experiments carried out on three different sheep from the same flock gave similar results. The biochemical behaviour of PrPsc under denaturing conditions and in the presence of proteinase K could thus provide a biochemical means for further characterization of different natural scrapie isolates.

Animals↗
PubMed2003

Accumulation of pathogenic prion protein (PrPSc) in nervous and lymphoid tissues of sheep with subclinical scrapie.

All sheep older than 1 year of age from a flock of the Rygja breed in which clinical scrapie was detected for the first time in two animals (4%) were examined for accumulation of pathogenic prion protein (PrPSc) by immunohistochemistry in the obex, the cerebellum, and the medial retrophayngeal lymph node. In addition, six lambs, 2-3 months old, all offspring of PrPSc-positive dams, were examined for PrPSc in the ileal Peyers' patch (IPP), the distal jejunal lymph node, the spleen, and the medial retropharyngeal lymph node (RPLN). In this flock, 35% (17/48) of the adult sheep showed accumulation of PrPSc, an eightfold increase compared with clinical disease. All positives carried susceptible PrP genotypes. Three sheep had deposits of PrPSc in the RPLN and not in the brain, suggesting that this organ, easily accessible at slaughter, is suitable for screening purposes. Two 7-year-old clinically healthy homozygous V136Q171 ewes showed sparse immunostaining in the central nervous system and may have been infected as adults. Further, two littermates, 86-days-old, showed PrPSc in the IPP. Interestingly, one of these lambs had the intermediate susceptible PrP genotype, VA136QR171. In addition to early immunolabeling in the dorsal motor nucleus of the vagal nerve, a few of the sheep had early involvement of the cerebellum. In fact, a 2-year-old sheep had sparse deposits of PrPSc in the cerebellum only. Because experimental bovine spongiform encephalopathy (BSE) in sheep seems to behave in a similar manner as natural scrapie, these results, particularly regarding spread of infectivity, may have implications for the handling of BSE should it be diagnosed in sheep.

Animals↗
PubMed2003

Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice.

Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.

Animals↗
PubMed2003

Detection of bovine spongiform encephalopathy, ovine scrapie prion-related protein (PrPSc) and normal PrPc by monoclonal antibodies raised to copper-refolded prion protein.

Prion-related protein (PrP) is a glycosylphosphatidylinositol-linked cell-surface protein expressed by a wide variety of cells, including those of the nervous system and the immune system. Several functions of normal cellular PrP (PrPc) have been proposed that may be associated with the capacity of this protein to bind copper. In the present study, we describe the generation of a panel of monoclonal antibodies raised to copper-refolded PrP, which may be used to analyse the normal and disease-associated forms of this protein. The anti-PrP monoclonal antibodies were reactive by Western blot and ELISA with recombinant murine PrPc refolded in the presence or absence of either copper or manganese, and with the disease-susceptible allelic form V136R154Q171 ('VRQ'; where single-letter amino-acid notation has been used) and disease-resistant allelic form A136R154R171 ('ARR') of recombinant ovine PrPc. FACS analysis of lymphoid cells using these monoclonal antibodies showed that wild-type non-activated mouse lymphocytes expressed little, if any, PrPc. These monoclonal antibodies were shown to react with the unglycosylated and monoglycosylated forms of PrPSc (abnormal disease-specific conformation of PrP) in prion-infected tissue samples from all of the different species tested by Western blot. In addition, this analysis allowed one to make a distinction between bovine spongiform encephalopathy ('BSE') and scrapie PrPSc) isolates from experimentally infected sheep on the basis of their different electrophoretic mobilities.

Animals↗
PubMed2004

Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie.

Recently, pathological prion protein PrP(Sc), the putative key constituent of infectious agents causing transmissible spongiform encephalopathies (TSEs), was found in muscles of rodents experimentally infected with scrapie and in patients with Creutzfeldt-Jakob disease (CJD). For the assessment of risk scenarios originating from these findings (e.g., alimentary transmission of pathogens associated with bovine spongiform encephalopathy [BSE] and chronic wasting disease [CWD] via tainted beef and game or iatrogenic dissemination of CJD agent through contaminated surgical instruments) more detailed information about the time course of PrP(Sc) accumulation in muscles at preclinical and clinical stages of incubation is needed. Here we show that PrP(Sc) in muscles of hamsters fed with scrapie can be detected prior to the onset of clinical symptoms, but that the bulk of PrP(Sc) was deposited late in clinical disease. Additionally, regarding the question of how muscles become invaded, we report on the intramuscular location of PrP(Sc) and substantial indications for centrifugal spread of infection from spinal motor neurons to myofibers. Our findings in a well-established animal model for TSEs contribute to a better assessment of the risks for public health emanating from "Prions in skeletal muscle" and provide new insights into the pathophysiological spread of TSE agents through the body.

Administration, Oral↗
PubMed2006

Use of streptomycin for precipitation and detection of proteinase K resistant prion protein (PrPsc) in biological samples.

The ability of streptomycin to form multimolecular aggregates with pathogenic prion proteins and their recovery by precipitation via a low-speed centrifugation step has been demonstrated; these novel properties of streptomycin make it a useful substance that increases the sensitivity of laboratory diagnostic techniques for prion infections in man and animals.

Animals↗
PubMed2003

Sodium hydroxide renders the prion protein PrPSc sensitive to proteinase K.

Sodium hydroxide (NaOH) solutions are widely used for the purification of contaminated equipment, as they are known to inactivate a variety of pathogens. However, information about their effect on agents causing transmissible spongiform encephalopathy (TSE) is sparse and contradictory. Scrapie hamster brain homogenate, containing the disease-associated form of the prion protein (PrP(Sc)), was exposed to NaOH. Kinetics studies showed that treatment of brain homogenate with millimolar concentrations of NaOH rapidly abolished the proteinase K-resistant form of the prion protein (PrP(res)). NaOH treatment converted PrP(Sc) into a protease-sensitive form, either in solution or when adsorbed to a metallic surface. If infectivity of TSEs is linked with PrP(res), the results imply that inactivation of TSE occurs more efficiently than currently assumed.

Animals↗
PubMed2006

Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle.

A food-borne origin of the transmission of bovine spongiform encephalopathy (BSE) to cattle is commonly assumed. However, the fate of infectious prion protein during polygastric digestion remains unclear. It is unknown at present, whether infectious prion proteins, considered to be very stable, are degraded or inactivated by microbial processes in the gastrointestinal tract of cattle. In this study, rumen and colon contents from healthy cattle, taken immediately after slaughter, were used to assess the ability of these microbial consortia to degrade PrP(Sc). Therefore, the consortia were incubated with brain homogenates of scrapie (strain 263K) infected hamsters under physiological anaerobic conditions at 37 degrees C. Within 20 h, PrP(Sc) was digested both with ruminal and colonic microbiota up to immunochemically undetectable levels. Especially polymyxin resistant (mainly gram-positive) bacteria expressed PrP(Sc) degrading activity. These data demonstrate the ability of bovine gastrointestinal microbiota to degrade PrP(Sc) during digestion.

Animals↗
PubMed2002

Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein-prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases.

Animals↗
PubMed2004

Fatal familial insomnia: the first account in a family of Chinese descent.

BACKGROUND: Fatal familial insomnia (FFI) is an autosomal dominant disease linked to a mutation in the prion protein gene. Fatal familial insomnia is characterized by sleep disturbance and loss of neurons, with gliosis in the thalamic nuclei. OBJECTIVE: To describe the clinical, neurophysiological, radiological, and neuropathological data in a Chinese family with FFI. SETTING: Tertiary referral university hospital setting. PATIENTS: Patient 1 was a 36-year-old man who presented with insomnia and myoclonus. In the subsequent 9 months, he developed ataxia and dementia, followed by death. Patient 2 was the aunt of patient 1, and presented at the age of 47 years with insomnia, myoclonus, and dementia; her condition declined during a 12-month period. Genetic analysis was performed, followed by neuropathological and biochemical analysis of the disease-associated form of the prion protein PrPSc on the postmortem brain specimen. RESULTS: Molecular analysis demonstrated an aspartic acid to asparagine mutation at codon 178 and homozygosity for methionine at codon 129. Both patients showed severe neuronal loss and prominent gliosis in the thalamus and brainstem involvement, with evidence of astrogliosis in the inferior olivary nucleus. Patient 1 also had neuronal loss and astrogliosis in the region of the superior colliculus and in the periaqueductal region. PrPSc was detected on Western blot analysis, and had a wide distribution. The strongest signals were present in the amygdala, hypothalamus, caudate, parahippocampal gyrus, periaqueductal gray matter, and mediodorsal thalamus. CONCLUSIONS: To our knowledge, this is the first report of FFI in a family of Chinese descent. This supports the worldwide distribution of FFI, and despite differences in genetic background, the clinical and pathological findings are similar to those found in white patients with FFI.

Adult↗
PubMed1990

Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein.

Permethylation of the glycan isolated from the glycoinositol phospholipid (GPI) anchor of the scrapie prion protein (PrPSc) trimethylates a free hexosamine to form a quarternary ammonium salt, substantially increasing the sensitivity for analysis by mass spectrometry. This derivatization induces specific fragmentation reactions in collision-induced dissociation spectra obtained on a four-sector tandem mass spectrometer, identifying the branching pattern of the PrPSc GPI glycan.

Glycolipids↗
PubMed1989

Unraveling prion diseases through molecular genetics.

Prions are transmissible pathogens that cause degenerative diseases in humans and animals. Unique attributes of prion diseases include infectious, sporadic and genetic manifestations, as well as progression to death, all in the absence of a detectable immune response. Prions are resistant to chemical procedures that modify or destroy nucleic acids and are composed largely of a protein, designated PrPSc. Molecular cloning of a cognate cDNA established a cellular host origin for PrPSc protein and a convergence with the genetics of host susceptibility. The murine PrP gene is linked to the Prn-i gene which determines incubation times in experimental scrapie. Mice with long incubation times have unusual PrP alleles encoding phenylalanine and valine at codons 108 and 189. Moreover, the ataxic form of Gerstmann-Sträussler syndrome (a rare human neurodegenerative disorder) has been defined as an autosomal dominant disorder with a PrP mis-sense mutation at codon 102 linked to the predisposition locus. These studies argue that amino acid substitutions in 'PrP' genes may modulate initiation and development of prion diseases.

Animals↗
PubMed2004

Detection of prion after decontamination procedures: comparative study of standard Western blot, filter retention and scrapie-cell assay.

Prion diseases such as Creutzfeldt-Jakob disease represent a unique infection control problem because the infectious agent exhibits an unusual resistance to conventional chemical and physical decontamination methods. We investigated the reliability and sensitivity of a filter retention assay and standard Western blot to detect the scrapie form of cellular prion protein (PrPSc), the most commonly used surrogate prion disease marker, on tissue infected samples, treated with five commercially available decontamination solutions currently used in hospitals. Major discrepancies were observed between the two immunoblot methods. By using Western blot, we observed that three decontamination solutions have a strong ability to reduce PrPSc levels, whereas in the filter assay, none have this effect and two even enhanced the detection of PrPSc. We used an original and rapid ex vivo approach called the scrapie-cell assay to analyse the persistence of infectivity on scrapie-treated tissues. We observed that tissues remained infectious after treatment with the decontaminant in concordance with in vivo data. This study suggests that conventional PrPSc detection methods are not adapted for the rapid study of a large number of prion decontaminants tested on infectious tissues, and that the scrapie-cell assay could be proposed as a relevant alternative method.

Animals↗
PubMed2005

Simple and specific detection of abnormal prion protein by a magnetic bead-based immunoassay coupled with laser-induced fluorescence spectrofluorometry.

Transmissible spongiform encephalopathies (TSEs), also termed prion diseases, are fatal neurodegenerative conditions that affect both humans and animals. The transmissibility and fatal nature of TSEs necessitate their rapid and accurate diagnosis. Laser-induced fluorescence (LIF) spectrofluorometry is useful for obtaining measurements on fluorescence-labeled targets with a high degree of sensitivity. In the present study, we applied this technology to the immunological detection of abnormal prion protein, PrPSc, which is a universal diagnostic marker for TSEs. The assay format consists of a magnetic bead-based sandwich immunoassay utilizing a biotin-conjugated capture antibody and a fluorophore-labeled detector antibody. By using one pair of anti-PrP monoclonal antibodies (MAbs), PrPSc in brain homogenates from various experimental and natural TSEs can be easily detected with high specificity. Furthermore, the assay proved to be applicable for the detection of PrPSc in the lymph nodes from deer with TSE. The sensitivity of the assay was shown to be comparable to standard immunoblotting, but has several advantages over conventional tests, in terms of flexibility, simplicity, specificity, and run time. These results provide an important basis for the development of an early diagnostic test with potential for multi-sample analysis.

Animals↗
PubMed1998

Capillary isoelectric focusing of the scrapie prion protein.

Prion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and humans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an abnormal isoform of a host-encoded glycoprotein that is characterized by its insolubility and partial resistance to proteases. Another characteristic of the scrapie prion protein (PrPsc) is the wide range of isoelectric points (pI values) that have been observed on conventional isoelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing (cIEF) to characterize the pI values for PrPsc isolated from sheep and hamster brain. We used a Beckman 5500 P/ACE using UV detection at 280 nm. A cIEF 3-10 Kit from Beckman Instruments was used to perform the analysis. The PrPsc was solubilized in 0.01 M Tris-HCl, pH 8.00 containing 2 mM EDTA. 5% SDS and 10% hexafluoroisopropanol at 100 degrees C for 10 min. The solubilized PrPsc was placed over a high-performance hydrophilic interaction column. After elution, the peaks were concentrated and assayed for immunoreactivity with specific antisera. The peaks that contained immunoreactivity were then placed on the cIEF capillary. The samples containing PrPsc were solubilized in 1% n-octylglucoside before isoelectric focusing. The scrapie infected sheep sample had peaks with pI values ranging from 5.2 to 3.00 with a major peak at 3.09. The normal sheep brain had pI values that were higher. The hamster adapted scrapie strain had peaks with pI values ranging from 6.47 to 3.8. These pI values were slightly higher than those obtained for the sheep samples. The use of cIEF to determine the pI values of PrPsc led to the identification of a major species of PrPsc from sheep with a very acidic pI.

Animals↗
PubMed1994

Histopathological changes in the islets of Langerhans in scrapie 139H-affected hamsters.

Previous studies showed that the 139H strain of scrapie injected into hamsters caused obesity, a marked hypertrophy of the islets of Langerhans, generalized endocrinopathy and marked hypoglycaemia-hyperinsulinaemia. In the current study, female weanling Syrian hamsters (LVG/LAK strain) were inoculated intracerebrally with scrapie strain 139H or 263K, or with normal hamster brain. Sections of the pancreas stained with haematoxylin and eosin or Gomori's one-step trichrome were examined by light microscopy. The 139H-affected hamsters showed extensive vacuolization, cellular hypertrophy, cellular atrophy, cytoplasmic vesicles and nuclear pathological changes in the islets of Langerhans. Also observed were abnormal structures, termed blood vessel cores, in the islets of 139H-affected hamsters. These structures were almost always centrally located within islets and were surrounded by B cells, some of which were abnormally elongated. None of these pathological changes were seen in the islets of Langerhans in control or 263K-affected hamsters. The level of scrapie-specific protease resistant protein (PrPSc) in pancreas was much lower than that in brain, a finding consistent with previous data showing low scrapie infectivity titres in pancreas.

Animals↗