Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

A physicochemical model for rationalizing SARS-CoV-2 concentration in sewage. Case study: The city of Thessaloniki in Greece.

Detection of SARS-CoV-2 in sewage has been employed by several researchers as an alternative early warning indicator of virus spreading in communities, covering both symptomatic and asymptomatic cases. A factor that can seriously mislead the quantitative measurement of viral copies in sewage is the adsorption of virus fragments onto the highly porous solids suspended in wastewater, making them inaccessible. This depends not only on the available amount of suspended solids, but also on the amount of other dissolved chemicals which may influence the capacity of adsorption. On this account, the present work develops a mathematical framework, at various degrees of spatial complexity, of a physicochemical model that rationalizes the quantitative measurements of total virus fragments in sewage as regards the adsorption of virus onto suspended solids and the effect of dissolved chemicals on it. The city of Thessaloniki in Greece is employed as a convenient case study to determine the values of model variables. The present data indicate the ratio of the specific absorption (UV254/DOC) over the dissolved oxygen (DO) as the parameter with the highest correlation with viral copies. This implies a strong effect on viral inaccessibility in sewage caused (i) by the presence of humic-like substances and (ii) by virus decay due to oxidation and metabolic activity of bacteria. The present results suggest days where many fold corrections in the measurement of viral copies should be applied. As a result, although the detected RNA load in June 2020 is similar to that in April 2020, virus shedding in the city is about 5 times lower in June than in April, in line with the very low SARS-CoV-2 incidence and hospital admissions for COVID-19 in Thessaloniki in June.

Photocatalytic degradation of prions using the photo-Fenton reagent.

Prions are proteinaceous infectious agents postulated to be the causative agents of a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). A known iatrogenic transmission route of TSEs to humans occurs via prion-contaminated surgical instruments or biological materials. Prions, unlike most common pathogens, exhibit an extraordinary resistance to conventional decontamination procedures. We have recently demonstrated that the application of TiO(2)-based heterogeneous photocatalytic oxidation is able to significantly reduce prion infectivity. The present study investigates the potential of a homogeneous photocatalytic method, based on the photo-Fenton reagent, to degrade prion proteins. We show that the photo-Fenton reagent efficiently degrades not only recombinant prion proteins, but also the total protein amount from brain preparations of naturally or experimentally infected species and PrP(Sc) (PrP scrapie) contained in sheep scrapie brain homogenates.

Cellular prion protein co-localizes with nAChR beta4 subunit in brain and gastrointestinal tract.

PrP(C), the cellular isoform of prion protein, is widely expressed in most tissues, including brain, muscle and gastrointestinal tract. Despite its involvement in several bioprocesses, PrP has still no apparent physiological role. During propagation of transmissible spongiform encephalopathies (TSE), prion protein is converted to the pathological isoform, PrP(Sc), in a process believed to be mediated by unknown host factors. The identification of proteins associated with PrP may provide information about both the biology of prions and the pathogenesis of TSE. Thus far, PrP(C) has been shown to interact with synaptic proteins, components of the cytoskeleton and intracellular proteins involved in signalling pathways. Here, we describe the association of PrP with the beta4 subunit of nicotinic acetylcholine receptor (nAChR), as indicated by co-immunoprecipitation assays and double-label immunofluorescence. The interaction between prion protein and native beta4 subunit was further studied by affinity chromatography, using immobilized and refolded recombinant PrP as a bait and brain homogenates from normal individuals. Additionally, the participation of beta4 subunit in the pathogenesis of TSE was studied by in vivo assays. beta4(-/-) and wild-type mice were challenged with the RML (Rocky Mountain Laboratories) infectious agent. Transgenic animals displayed altered incubation times but the deletion of beta4 subunit did not result in a significant change of the incubation period of the disease. Our results suggest that PrP(C) is a member of a multiprotein membrane complex participating in the formation and function of alpha3beta4 nAChR.

Atomic force microscopy to characterize the molecular size of prion protein.

The conformational transition of alpha-helix-rich cellular prion protein (PrP(C)) to an isomer with high beta-sheet content is associated with transmissible spongiform encephalopathies. With the ultimate long-term goal of using imaging techniques to study PrP aggregation, we report the results of initial experiments to determine whether PrP molecules could be visualized as single molecules, and if the observed size corresponded to the calculated size for PrP. The investigation of single molecules, and not those embedded into larger aggregates, was the key in our experimental approach. Using atomic force microscopy (AFM) as an imaging method, the immobilization of recombinant histidine (His)10-tagged PrP on mica was performed in the presence of different heavy metal ions. The addition of Cu2+ resulted in an enhanced PrP immobilization, whereas Ni2+ reduced coverage of the surface by PrP. High-resolution data from dried PrP preparations provided a first approximation to geometrical parameters of PrP precipitates, which indicated that the volume of a single PrP molecule was 30 nm3. Molecular dynamics simulations performed to complement the structural aspects of the AFM investigation yielded a calculated molecular volume of 33 nm3 for PrP. These experimentally observed and theoretically expected values provide basic knowledge for further studies on the size and composition of larger amyloidal PrP aggregates, PrP isoforms or mutants such as PrP molecules without octarepeats.

Photocatalytic degradation and drug activity reduction of Chloramphenicol.

The photocatalytic degradation of Chloramphenicol, an antibiotic drug, has been investigated in aqueous heterogeneous solutions containing n-type oxide semiconductors as photocatalysts. The disappearance of the organic molecule follows approximately a pseudo-first-order kinetics according to the Langmuir-Hinshelwood model. It was observed that, with TiO(2) P-25 as photocatalyst, quantitative degradation of the organic molecule occurs after 4h of illumination. During this time, the dechlorination of the substrate is complete, while the organic nitrogen was recovered in the form of nitrate and ammonium ions. The effect of temperature on the degradation rate of Chloramphenicol shows similar apparent activation energies for both TiO(2) P-25 and ZnO photocatalysts. The initial apparent photonic efficiency (zeta(0)) of the photo-oxidation and the mineralization under various experimental conditions have been calculated, while the Kirby-Bauer disc diffusion method showed a 100% reduction of the drug activity after 90 min of photocatalytic treatment.

CSF analysis in patients with sporadic CJD and other transmissible spongiform encephalopathies.

Patients with suspected Creutzfeldt-Jakob disease (CJD) often have routine cerebrospinal fluid (CSF) analysis performed to exclude treatable inflammatory conditions; however, little information is available about the range of results obtained for CSF tests in patients with sporadic CJD and other transmissible spongiform encephalopathies (TSE). Data from 450 patients with sporadic CJD and 47 patients with other TSEs were collected as part of an EC-supported multinational study. Raised white cell counts of >5 cells/microl were found in three of 298 patients with sporadic CJD, with two cell counts of 7 cells/microl and one of 20 cells/microl. Total protein concentrations of >0.9 g/l were found in five of 438 patients with sporadic CJD, although none had a concentration of >1 g/l. CSF oligoclonal IgG was detected in eight of 182 sporadic CJD patients. Of the patients with other TSEs, six had elevated cell counts ranging from 6 to 14 cells/microl but none had total protein concentrations of >0.9 g/l and one patient had detectable oligoclonal IgG. None of the patients with sporadic CJD or other TSEs had abnormalities in all three tests.

Non-competitive inhibition of ornithine decarboxylase by a phosphopeptide and phosphoamino acids.

In Tetrahymena pyriformis the cytosolic ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity is considerably inhibited by the presence of polyamines in the growth medium, while the nuclear ornithine decarboxylase is only slightly affected. Experimental evidence suggests that the presence of putrescine and/or spermidine elicits the appearance of non-competitive inhibitors of ornithine decarboxylase. One of the inhibitors has a molecular weight of 25,000 and properties of antizyme. In addition, two other low molecular weight inhibitors are extracted, one which is a phosphoserine oligopeptide, and the other which is phosphotyrosine. All inhibit non-competitively the homologous and heterologous (Escherichia coli and rat liver) ornithine decarboxylases. Similarly, non-competitive inhibition was obtained when the commercially available phosphoamino acids were tested against the already mentioned ornithine decarboxylases.

Purification and properties of ornithine decarboxylase from Tetrahymena pyriformis.

In Tetrahymena pyriformis, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities are present in the cytosolic and nuclear fractions and reach maximal values in the middle and late log phases of growth, respectively. The two activities have been purified to homogeneity by ammonium sulfate fractionation (20-45%), anion-exchange chromatography (DEAE-Bio-Gel A), gel-filtration (Sephadex G-150 and Sephadex G-100 superfine) and hydrophobic chromatography (Phenyl-Sepharose). Both the crude and the purified enzyme preparations are inactivated irreversibly by alpha-difluoromethylornithine, a suicide inhibitor of mammalian ornithine decarboxylase. The enzyme preparations from the nucleus and cytosol each showed a single band on polyacrylamide gel electrophoresis under native and denaturing conditions and on acrylamide gel electrofocusing. Both activities show the same pH optima (8.6) isoelectric point (5.3), molecular weight (64 000) and Kmorn (4.7 microM). The Km for L-lysine is 0.5 mM. The two activities also cross-react with acidic antizyme extracted from E. coli mutant MA 255. Based on the physicochemical properties, one can safely conclude that cytosolic and nuclear activities reside on the same protein molecule.

Antigenic profile of human recombinant PrP: generation and characterization of a versatile polyclonal antiserum.

We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antiserum was evident from the range of animal species and immunochemical techniques where it could be applied successfully. Antigen absorption studies revealed differences in the location and number of epitopes remaining after incubation with soluble or aggregated antigen.Our knowledge concerning immunoprophylaxis against prion diseases and the important role played by conformational changes of PrP is increasing rapidly. The findings reported here should add to this body of knowledge.

Analysis of Creutzfeldt-Jakob disease infectious fractions by gel permeation chromatography and sedimentation field flow fractionation.

Gel 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 M(r) of approximately 1.5 x 10(7) daltons and a mean radius of approximately 30 nm. The experimental evidence further solidifies the concept of an infectious agent that resembles a viral core rather than a simple protein.

Expression of the prion protein in the rat forebrain--an immunohistochemical study.

The cellular prion protein (PrP(C)) is crucial for the development of transmissible spongiform encephalopathies (TSEs), where the pathogenic scrapie isoform (PrP(Sc)) of the same protein, is considered to be the principal or sole infectious agent. Here, we report findings on PrP(C) expression in the rat forebrain, using immunohistochemical techniques on free floating sections of 60 microm thickness. Along with neurons and astrocytes in the gray matter, PrP(c) was detected for the first time in glial cells of the white matter and in cells of circumventricular organs. PrP(C) positive cellular processes were also found to be closely associated with intraparenchymal blood vessels, often in the form of end feet. Interestingly, PrP(C) expression was observed in areas where PrP(Sc) deposition in late stages of infection has been earlier reported in the rat and other species.

Identification of proteins co-purifying with scrapie infectivity.

PrP(C), the cellular isoform of prion protein, is widely expressed in most tissues. Despite its involvement in several bioprocesses it still has no apparent physiological role. During propagation of Transmissible Spongiform Encephalopathies, PrP(C) is converted to the pathological isoform, PrP(Sc), in a process believed to be mediated by unknown host factors. PrP(Sc) has altered biochemical properties and forms amyloid aggregates that display infectious characteristics. PrP(Sc) is also the major component in biochemically enriched infectious samples. Other molecules co-purify with it, but the protein content of these aggregates remains unknown. The goal of this project was to identify other host molecules with high affinity for PrP(Sc). Here, we present the identification of protein molecules that co-purify with PrP(Sc) isolated from naturally scrapie-infected ovine brain tissue. The infectious preparations were analyzed by two-dimensional gel electrophoresis and unknown proteins were identified by LC-MS/MS. These proteins may prove to be strategic targets for prevention and therapy of prion diseases.

Detection, quantification, and glycotyping of prion protein in specifically activated enzyme-linked immunosorbent assay plates.

The conversion of a normal glycoprotein, prion protein (PrP(C)), to its abnormal protease-resistant isoform (PrP(Sc)) seems to be one of the main factors underlying the pathogenesis of spongiform encephalopathies. There are many studies indicating that PrP interacts with glycosaminoglycans, and we exploited this interaction to develop a sensitive solid phase assay for detection of both PrP forms. Glycosaminoglycans, such as chondroitin sulfate and heparin, were immobilized by their negative charge to enzyme-linked immunosorbent assay (ELISA) plate wells activated by glutaraldehyde and spermine. PrP in the samples examined (recombinant PrP or tissue homogenate) was allowed to interact with glycans. The interaction of recombinant PrP was more efficient against immobilized chondroitin sulfate of type A, and a linear correlation with concentration was demonstrated. From this curve, the concentration of each one of the PrP isoforms in biological samples can be determined. In addition, and taking into account that glycosylation of prion protein is species specific, we used similarly activated ELISA plate wells to determine different PrP glycoforms. A monoclonal antibody against PrP was immobilized, and PrP present in the samples (brain homogenates) was bound and visualized by various lectins. The most interesting outcome of the study is the differential binding of ricinus communis agglutinin I to the normal and scrapie brain homogenates. Dattura stramonium lectin and wheat germ agglutinin seem to bind almost equally to both samples, and all three have an increased sensitivity to PrP(Sc) after proteinase K digestion.

CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease.

OBJECTIVES: To analyze the diagnostic sensitivity and specificity of various brain-derived proteins (14-3-3, Tau, neuron specific enolase [NSE], and S100b) in the CSF of patients with Creutzfeldt-Jakob disease (CJD) and to analyze biologic factors that modify these parameters. METHODS: CSF was tested for 14-3-3, Tau, NSE, and S100b in 1,859 patients with sporadic, genetic, iatrogenic, and variant CJD, and in 1,117 controls. RESULTS: The highest sensitivity was achieved for 14-3-3 and Tau in sporadic CJD (85% and 86%), and a combined determination of 14-3-3 and Tau, S100b, or NSE increased the sensitivity to over 93%. A multivariate analysis showed that the sensitivity of all tests was highest in patients with the shortest disease duration, age at onset >40 years, and homozygosity at codon 129 of the prion protein gene. In a group of patients with repeated lumbar punctures, a second test also increased the diagnostic sensitivity. CONCLUSIONS: The detection of elevated levels of brain-derived proteins in the CSF in patients with suspected Creutzfeldt-Jakob disease is a valuable diagnostic test. A second lumbar puncture may be of value in patients with atypical clinical course in whom the first test was negative.

Tissue plasminogen activator in brain tissues infected with transmissible spongiform encephalopathies.

Prion propagation involves conversion of host PrP(C) to a disease-related isoform, PrP(Sc), which accumulates during disease and is the principal component of the transmissible agent. Proteolysis seems to play an important role in PrP metabolism. Plasminogen, a serine protease precursor, has been shown to interact with PrP(Sc). Plasminogen can be proteolytically activated by tissue plasminogen activator (tPA). Recent reports imply a crosstalk between tPA-mediated plasmin activation and PrP. In our study, both tPA activity and tPA gene expression were found elevated in TSE-infected brains as compared to their normal counterparts. Furthermore, it was proved that PrP(Sc), in contrast to PrP(C), could not be degraded by plasmin. In addition, it was observed that TSE symptoms and subsequent death of plasminogen-deficient and tPA-deficient scrapie challenged mice preceded that of wild-type controls. Our data imply that enhanced tPA activity observed in prion infected brains may reflect a neuro-protective response.

Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt-Jakob disease.

A class of viruslike agents that induces Creutzfeldt-Jakob Disease (CJD) and scrapie remains undefined at the molecular level. Several investigators believe this infectious agent is constituted by a single host protein or 'prion', and have emphasized data that would seem to exclude the presence of any viral nucleic acids. However, more rigorous evaluations in scrapie have shown reasonably abundant nucleic acids. Additionally, in highly purified 120S CJD preparations that have been treated with nucleases, RNAs as long as 6,000 bases have been detected. Few nucleic acids have been characterized in either scrapie or CJD, but previous cloning experiments delineated relatively short LTR regions of the endogenous IAP retrovirus in 120S CJD preparations. We therefore used specific primers encompassing the entire IAP genome to test for the presence of long viral RNAs, and here show approximately 5,000 contiguous bases of the IAP RNA genome can be recovered from reasonable amounts of starting brain. The 3' env region of IAP is comparably truncated in CJD and normal preparations, and we find no evidence for IAP transduction of CJD-specific sequences. Because IAP cores can coencapsidate unrelated sequences, and are unusually resistant to physical and chemical treatments, it was relevant to find if cosedimenting cognate proteins of the IAP core, such as gag, could be detected. The predicted approximately 65 kd acidic gag protein, showing appropriate antigenic and nucleic acid binding features, was apparent in both one and 2-D Western blots. This data strongly indicates specific viral complexes cofractionate with the CJD agent. Interestingly, these nuclease resistant IAPs do not appear to be in morphologically recognizable 'R' particles. This cosedimenting viral assembly therefore provides a paradigm for non-particulate CJD complexes in infectious preparations. In developing strategies to identify a CJD specific sequence, cosedimenting IAPs can be used to assess the quality, length and recovery of RNAs extracted from highly resistant viral complexes.

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.