Concentration Of PrP Research Articles

Effect study of exosomes derived from platelet-rich plasma in the treatment of knee cartilage defects in rats

BackgroundThe repair of articular cartilage defects has always been a difficult problem. We aimed to investigate the therapeutic effect of intra-articular injection of platelet-rich plasma (RPR) and PRP-derived exosomes (PRP-Exos) on cartilage defects in rat knee joints and then provide experience for the use of PRP-exos in cartilage defect repair.MethodsRat abdominal aortic blood was collected, and PRP was extracted by two-step centrifugation. PRP-exos were obtained by kit extraction, and PRP-exos were identified by various methods. After the rats were anesthetized, a cartilage defect subchondral bone was created at the proximal end of the origin of the femoral cruciate ligament with a drill. SD rats were divided into 4 groups, including PRP group, 50 μg/ml PRP-exos group, 5 μg/ml PRP-exos group, and control group. One week after the operation, 50 μg/ml PRP, 50 μg/ml PRP-exos, 5 μg/ml PRP-exos and normal saline were injected into the knee joint cavity of rats in each group, once a week. A total of two injections were given. On the 5th and 10th week after drug injection, the serum levels of matrix metalloproteinase 3 (MMP-3) and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) were detected by each treatment method, respectively. The rats were killed at the 5th and 10th weeks, respectively, and the cartilage defect repair was observed and scored. The defect repair tissue sections were used for HE staining and type II collagen immunohistochemical staining.ResultsThe histological results showed that both PRP-exos and PRP could promote cartilage defect repair and type II collagen formation, and the promoting effect of PRP-exos was significantly better than that of PRP. In addition, enzyme-linked immunosorbent assay (ELISA) results showed that compared with PRP, PRP-exos could significantly increase serum TIMP-1 and decrease serum MMP-3 in rats. And the promoting effect of PRP-exos was concentration dependent.ConclusionIntra-articular injection of PRP-exos and PRP can promote the repair of articular cartilage defects, and the therapeutic effect of PRP-exos is better than the same concentration of PRP. PRP-exos are expected to be an effective treatment for cartilage repair and regeneration.

Carbon Microspheres Composite PbO2 Anode to Enhance Electrode Activity for Degradation of Isopropylantipyrine: Kinetics and Mechanism

An innovative CMS-PbO2 electrode was fabricated by combining hydrothermally synthesized carbon microspheres (CMS) on a PbO2 electrode by electrodeposition. Using the CMS-PbO2 electrode, the main factors affecting Isopropylantipyrine (PRP) degradation were studied. Under optimum process conditions, the concentration of PRP was 50 mg l−1, the applied current density was 30 mA cm−2, the electrolyte (Na2SO4) concentration was 0.1 mol l−1, and pH was 7. The PRP degradation rate reached 100%, and chemical oxygen demand (COD) removal rate reached 43.42% after 120 min of electrochemical oxidation. Using field-emission scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, cyclic voltammetry, linear sweep voltammetry, and electrochemical impedance spectroscopy, the electrochemical performance of the two electrodes was discussed. The composite of carbon microspheres successfully improved the electrochemical activity of the electrode and its conductivity. Furthermore, the ability of the two electrodes to generate hydroxyl radicals was compared and the possible degradation pathway of PRP was considered. In addition, electrode stability and safety were evaluated by accelerated lifetime experiments and detection of lead ions in solution after electrochemical oxidation. The CMS-electrode was more stable and safer than the PbO2 electrode. The CMS-PbO2 electrode provides a new strategy for the treatment of pharmaceutical wastewater.

Antioxidant Activity Of Platelet Rich Plasma Wistar Rats With Dpph And Abts Test

Free radicals and reactive oxygen species (ROS) are the cause of diseases such as cancer, diabetes, cardiovascular and inflammatory. The presence of an increase in the number of free radicals and excess ROS production in the body can cause an imbalance of the immune system. Therefore, additional antioxidants are needed from outside the body. During this time there are several antioxidant synthesis drugs such as (Butyl Hydroxy Anisole) BHA and (Butyl Hydroxyl Toluene) BHT. But in its use, this drug causes side effects. Therefore, it is necessary to source antioxidants that have no side effects if consumed. PRP is a product produced from fresh whole blood that contains components of Red blood cells, White blood cells, Platelets, and Plasma. Plasma contains organic and inorganic molecules and ions wherein abundant conditions serve to transport other substances. To determine the ability of PRP as an antioxidant, fractionation, and bioactivity tests for free radical capture of 1,1-diphenyl-2-picrilhydrazil (DPPH) and color decay tests at 2,2'-azino-bis-[3-ethylbenzotiazolin sulfonate] (ABTS) in vivo.. Based on the results of this study, PRP has antioxidant activity that increases along with the decrease in THE concentration of PRP with an antioxidant activity value of 10.57% with the DPPH test and 5.85% with the ABTS test at the lowest concentration (6.25 μg / mL). IC50 averages 190.29 μg/mL with DPPH test and 126.60 μg/mL with the ABTS test. The results of this study are an indication that PRP can be used as a treatment therapy related to the anti-oxidant activity

Does the Difference in Leukocyte Concentration of PRP Affect the Short-Term Follow-Up Results in Cases Diagnosed with Early Stage Knee Osteoarthritis?

Abstract This prospective study was conducted for the clinical evaluation of pain severity and knee functionality following PRP injections with different leukocyte (WBC) concentrations applied to cases diagnosed with knee osteoarthritis. A total of 109 patients were included in the study. According to the leukocyte content the PRP injections were prepared as low-leukocyte content PRP (P-PRP) and high concentration leukocyte content PRP (L-PRP). Patients were divided into 2 groups. Group 1 (n=44) received low-leukocyte content PRP and Group II (n = 65) received high-leukocyte content PRP. The patients were evaluated clinically with Visual Analog Scale (VAS) and Knee Society Score (KSS). The changes in the PLT levels of the L-PRP group after the procedure compared to the levels prior to the procedure were found to be statistically significantly greater than the changes in the PPRP group. The mean VAS score of all the cases before treatment was 9.05±0.91 and this score decreased to 3.71±1.46 within 12 months. The increases in the mean Knee Society Score (KSS) values were determined as 16.92±1.97 within 6 months and 16.89±2.97 within 12 months in the P-PRP group and 19.71±1.24 within 6 months and 19.86±0.42 within 12 months in the L-PRP group. The most important aspect of this study is that, in contrast to many other studies, the results continued after the 6th month and were reported to be good in the 12th month. It was also recorded that L-PRP was clinically superior to P-PRP in the treatment of early stage knee osteoarthritis.

TECHNOLOGY OF OBTAINING POLYRIBOSYLRIBITOL PHOSPHATE AS AN ACTIVE PHARMACEUTICAL INGREDIENT FOR THE PRODUCTION OF POLYSACCHARIDE VACCINES

Despite the world practice, in the Russian Federation immunization against hemophilic infection is carried out only for the children from risk groups, which may be due to the lack of production of this vaccine in the Russian Federation. Therefore, the development of technology for the preparation of polyribosylribitol phosphate (PRP, the active substance of the Hib vaccine) remains relevant. Earlier, the employees of the federal state unitary enterprise “Saint-Petersburg scientific research institute of vaccines and serums and the enterprise for the production of bacterial preparations” of Federal medical and biologic agency of Russia isolated and identified the strain of Haemophilus influenzae SPB type b B-7884. The conditions for cultivation of the strain B-7884 and primary isolation of PRP from the culture liquid were arranged. The aim of the work was to study the possibility of excluding the centrifugation stage for separating H. influenzae SPB type b B-7884 biomass before clarification on the cascade of filter discs, and the development of subsequent stages of PRP isolation from the obtained intermediate product. Materials and methods. PRP isolation from an inactivated culture liquid of strain B-7884 was carried out by centrifugation, filtration, precipitation, homogenization, extraction under various conditions. The concentration of PRP in the intermediate products was determined by the orcinol method, the identity was determined by the latex agglutination test. Results and discussion. The optimal scheme for isolation and purification of the active pharmaceutical ingredient PRP, including filtration through the cascade of deep filters Zeta plus LP 60 (0.3–0.6 μm) and Zeta plus LP 90 (0.1–0.3 μm), concentration and diafiltration on ultrafiltration cassettes with nominal molecular weight cut-off of 30 kDa, precipitation with 10% cetyltrimethylammonium bromide solution (5% v/v), with subsequent homogenization of the obtained precipitate (in the presence of 12.5% of ethanol from the concentrate volume) and extraction (32.5% ethanol), filtration through depth filters Zeta Plus SP 30 and carbon filters Zeta Plus Carbon R53, and precipitation by 1.0% (v/v) 4.0 M sodium chloride solution and freezing at –(20 ± 2)°C, was developed. Conclusion. The obtained results will become the basis for the further development of the polysaccharide substance and allow us to proceed to the next stage in the development of the vaccine against Haemophilus influenzae type b – conjugation with the carrier protein.

Efficacy of freeze-dried platelet-rich plasma in bone engineering

ObjectivePlatelet-rich plasma (PRP) is typically isolated and applied immediately after preparation, making it both a time- and labor-intensive addition to the operative procedure. Thus, it would be convenient if PRP could be preserved. We evaluated the efficacy of freeze-dried PRP (FD-PRP), as compared with freshly isolated PRP (f-PRP) for bone engineering. DesignFD-PRP was prepared by lyophilization of f-PRP and was subsequently preserved at −20°C for one month. It was then rehydrated with an equal or 1/3 amount of distilled water (×1FD-PRP, ×3FD-PRP, respectively), and we assessed its gelation properties and the release of growth factors (PDGF-BB, TGF-β1, and VEGF). We also examined the bone forming ability with onlay-grafting on mice calvaria using β-TCP granules as a scaffold. ResultsFD-PRP showed comparable gelation as f-PRP. In terms of growth factor release,×1FD-PRP released identical concentrations of PDGF-BB and TGF-β1 to f-PRP, while ×3FD-PRP released approximately 3-fold concentrations when compared with f-PRP. In vivo, ×1FD-PRP promoted identical levels of the bone formation as f-PRP, and ×3FD-PRP induced more abundant bone formation. ConclusionsThese results suggest that f-PRP can be stored without functional loss by freeze-drying and the concentration of PRP may improve its efficacy in bone engineering.

Treatability and fate of various phosphorus fractions in different wastewater treatment processes

The increasingly more stringent phosphorus (P) discharge limits, which are below the concentrations reliably achievable with currently available technologies, demand for better understanding of phosphorus removal mechanisms. This study investigated the compositional fractions of phosphorus (P) in various effluents as well as the efficacy of different levels of treatment processes for removing different fractions of P in wastewater. The results showed that BNR can effectively remove most fractions of P, with relatively higher efficiencies (>93%) towards bioavailable forms of P including soluble reactive P (sRP), particulate reactive P (pRP) portion and particulate acid hydrolysable P (pAHP) and, it showed relatively lower efficiency (78%) towards organic P. Soluble acid hydrolysable P (sAHP) was not effectively removed (<40%). Chemical P removal process was more effective for elimination of sRP, sAHP and particulate organic P (pOP), but was not as effective for removing pAHP and, it exhibited nearly no removal of dissolved organic P (DOP). We found that chemical P removal process led to a significant increase in the concentration of pRP by up to 255%, indicating that these pRP (presumably as chemically bounded P) are likely formed through chemical precipitation/co-adsorption. Only 22% and 64% of the pRP was removed through tertiary clarifier and filtration, respectively. This implies that chemical addition converts sRP into particulate-associated P, mostly as pRP that was not easily removed by sedimentation and filtration, therefore, the efficacy of chemical P removal highly depends on the effectiveness of solid and liquid separation process. As more sRP and particulate P were removed through the series of treatment processes, the percentage contribution from organic P increases with the level of treatment due to its recalcitrant nature. Our results indicated that in order to achieve extremely low effluent P levels, technologies and processes that can enhance pRP and DOP removal will be required.

Diagnosis of the first cases of scrapie in Poland

This is the first report of cases of scrapie in Poland. The disease was an atypical phenotype, diagnosed in two aged sheep which were found dead. Brainstem samples from both animals were positive on the applied ELISA rapid test, while the confirmatory immunoblot indicated abnormal banding patterns of protease resistant prion protein (PrP(res)). The genotypes of these sheep were ALRQ/ALHQ and ALRQ/ALRR. The absence of premonitory clinical signs, the advanced age of the affected sheep, the higher concentration of PrP(res) in the cerebellum relative to the obex, the unusual banding profile of the prion protein and its relatively low resistance to proteolytic degradation confirmed the diagnosis of atypical scrapie (Nor98-like) in both cases.

Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor

An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP C) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3′-terminal, and a fluophor modified at 5′-terminal. It was found that the guanine bases at the 3′-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5′-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP C in the range from 1.1 to 44.7 μg/mL with a limit of detection of 0.3 μg/mL (3 σ).

A sensitive and quantitative assay for normal PrP in plasma

Transmissible spongiform encephalopathies can be transmitted by blood transfusion. The risk of spreading the disease among the human population could be mitigated with the implementation of a blood screening assay. We developed a two-antibody assay for PrP detection in plasma using the ORIGEN technology with a protocol modification to improve the limit of detection and to increase the sample volume assayed. In the standard 200 μL format, the assay had a detection limit of 7–10 pg of recombinant PrP and 3 pg in 1 mL final volume implementation. PrP concentration measured in normal and scrapie-infected hamster brains was 7.5 ± 0.9 and 57.3 ± 9.6 μg/g, respectively. After a concentration step with an immuno-affinity resin, plasma PrP c was detected by Western blot and its concentration was measured at 3.5 ± 0.8 ng/mL. From these data and assuming that blood has the same specific infectivity as brain, we estimated the concentration of abnormal PrP in hamster-infected plasma to be 32 fg/mL. The assay also detected abnormal brain PrP spiked into plasma although the limit of detection was affected. This is a novel and sensitive assay for the detection of PrP in plasma that could be developed into a platform for a plasma-based TSE test.

Exposure to low dietary copper or low copper coupled with high dietary manganese for one year does not alter brain prion protein characteristics in the mature cow1,2

It is now widely accepted that abnormal prion proteins are the likely causative agent in bovine spongiform encephalopathy. Cellular prion proteins (PrP(c)) bind Cu, which appears to be required to maintain functional characteristics of the protein. The replacement of Cu on PrP(c) with Mn has resulted in loss of function and increased protease resistance. Twelve mature cows were used to determine the effects of Cu deficiency, alone and coupled with high dietary Mn, on brain Cu and Mn concentrations and on PrP(c) functional characteristics. Copper-adequate cows were randomly assigned to treatments: 1) control (adequate in Cu and Mn), 2) Cu-deficient (-Cu), and 3) Cu-deficient plus high dietary Mn (-Cu+Mn). Cows assigned to treatments -Cu and -Cu+Mn received no supplemental Cu and were supplemented with Mo to further induce Cu deficiency. After 360 d, Cu-deficient cows (-Cu and -Cu+Mn) tended to have lesser concentrations of Cu (P = 0.09) in the obex region of the brain stem. Brain Mn tended (P = 0.09) to be greater in -Cu+Mn cattle compared with -Cu cattle. Western blots revealed that PrP(c) relative optical densities, proteinase K degradability, elution profiles, molecular weights, and glycoform distributions were not different among treatments. The concentration of PrP(c), as determined by ELISA, was similar across treatment groups. Brain tissue (obex) Mn superoxide dismutase activity was greatest (P = 0.04) in cattle receiving -Cu+Mn, whereas immunopurified PrP(c) had similar superoxide dismutase-like activities among treatments. Immunopurified PrP(c) had similar Cu concentrations across treatments, whereas Mn was undetectable. We concluded that Cu deficiency, coupled with excessive Mn intake, in the bovine may decrease brain Cu and increase brain Mn. Copper deficiency, alone or coupled with high dietary Mn, did not cause detectable alterations in PrP(c) functional characteristics.

Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein

This study describes the conversion of murine PrP(C) by PrP(Sc) from three different mouse scrapie strains (ME7, 87V and 22A) and from a mouse-passaged bovine spongiform encephalopathy (BSE) strain (BSE/Bl6). This was demonstrated by a modified, non-radioactive, cell-free conversion assay using bacterial prion protein, which was converted into a proteinase K (PK)-resistant fragment designated PrP(res). Using this assay, newly formed PrP(res) could be detected by an antibody that discriminated de novo PrP(res) and the original PrP(Sc) seed. The results suggested that PrP(res) formation occurs in three phases: the first 48 h when PrP(res) formation is delayed, followed by a period of substantially accelerated PrP(res) formation and a plateau phase when a maximum concentration of PrP(res) is reached after 72 h. The conversion of prokaryotically expressed PrP(C) by ME7 and BSE prions led to unglycosylated, PK-digested, abnormal PrP(res) fragments, which differed in molecular mass by 1 kDa. Therefore, prion strain phenotypes were retained in the cell-free conversion, even when recombinant PrP(C) was used as the substrate. Moreover, co-incubation of ME7 and BSE prions resulted in equal amounts of both ME7- and BSE-derived PrP(res) fragments (as distinguished by their different molecular sizes) and also in a significantly increased total amount of de novo-generated PrP(res). This was found to be more than twice the amount of either strain when incubated separately. This result indicates a synergistic effect of both strains during cell-free conversion. It is not yet known whether such a cooperative action between BSE and scrapie prions also occurs in vivo.

Prion Disease: Exponential Growth Requires Membrane Binding

A hallmark feature of prions, whether in mammals or yeast and fungi, is exponential growth associated with fission or autocatalysis of protein aggregates. We have employed a rigorous kinetic analysis to recent data from transgenic mice lacking a glycosylphosphatidylinositol membrane anchor to the normal cellular PrP C protein, which show that toxicity requires the membrane binding. We find as well that the membrane is necessary for exponential growth of prion aggregates; without it, the kinetics is simply the quadratic-in-time growth characteristic of linear elongation as observed frequently in in vitro amyloid growth experiments with other proteins. This requires both: i), a substantial intercellular concentration of anchorless PrP C, and ii), a concentration of small scrapies seeding aggregates from the inoculum, which remains relatively constant with time and exceeds the concentration of large polymeric aggregates. We also can explain via this analysis why mice heterozygous for the anchor-full/anchor-free PrP C proteins have more rapid incubation than mice heterozygous for anchor-full/null PrP C, and contrast the mammalian membrane associated fission or autocatalysis with the membrane free fission of yeast and fungal prions.

Variation in concentration of prion protein in the peripheral blood of patients with variant and sporadic Creutzfeldt‐Jakob disease detected by dissociation enhanced lanthanide fluoroimmunoassay and flow cytometry

A highly sensitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and flow cytometry techniques have previously been developed and employed to characterize soluble cellular prion protein (PrP(c)) expression in whole blood and separated components from healthy adult blood donors. No previous studies with these techniques have evaluated the concentration and expression of PrP in the blood of patients with variant Creutzfeldt-Jakob disease (vCJD). For blood from vCJD patients, sporadic CJD (sCJD) patients, non-CJD neurological controls, and healthy adults, PrP(c) was measured by DELFIA and cell-associated PrP was measured by flow cytometry. DELFIA analysis identified a significant reduction in the concentration of PrP(c) in the whole blood of vCJD (p = 0.012) and non-CJD neurological patients (p = 0.0004) compared with healthy adults. A significant elevation was found in plasma PrP(c) in sCJD patients compared with healthy adult (p = 0.022) and neurological controls (p = 0.050). Flow cytometry found no significant differences between groups in expression of PrP on platelets and lymphocytes, nor in sensitivity of cellular PrP to proteinase K. Neurological controls show significantly less PrP on red cells than healthy adults. There are differences in free and cell-associated PrP found in blood of CJD patients and control groups, some of which might be useful with other tests in disease profiling as an aid to diagnoses.

Synaptic prion protein immuno-reactivity in the rodent cerebellum.

The cellular prion protein PrP(c) is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrP(c) is converted to infectious protease-resistant isoforms PrP(res) or prions. Prions provoque predictable and distinctive patterns of PrP(res) accumulation and neurodegeneration depending on the prion strain and on regional cell-specific properties modulating PrP(c) affinity for infectious PrP(res) in the host brain. Synaptolysis and synaptic accumulation of PrP(res) during PrP-related diseases suggests that the synapses could be primary sites able to propagate PrP(res) and neurodegeneration in the central nervous system. In the rodent cerebellum, the present light and electron microscopic immuno-cytochemical analysis shows that distinct types of synapses display differential expression of PrP(c), suggesting that synapse-specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrP(c) remain unknown, the concentration of PrP(c) almost exclusively at the Purkinje cell synapses in the cerebellum suggests its critical involvement in the synaptic relationships between cerebellar neurons in agreement with their known vulnerability to PrP deficiencies.

Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistant state

Introduction: Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPc) into a protease-resistant form (PrP Sc) by an unknown mechanism. In vitro studies have indicated that PrP Sc can induce this conversion, consistent with proposals that PrP Sc itself might be the infectious scrapie agent. Using this cell-free model of the PrP C to PrP Sc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrP Sc-derived species that has converting activity. Results: The cell-free conversion of 35S PrP C to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrP Sc (above an apparent minimum threshold concentration) and 35S PrP C. Analysis of the physical size of the converting activity indicated that detectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrP SC and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of ≈2000 kDa. Conclusions: The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrP Sc. These results are consistent with a nucleated polymerization mechanism of PrP Sc formation and inconsistent with a heterodimer mechanism.

Heparin‐like molecules bind differentially to prion‐proteins and change their intracellular metabolic fate

PrPSc is the only known component of the scrapie prion. The difference between PrPSc and its normal isoform PrPc is probably conformational, since no difference has been found in the amino acid sequence or postranslational modifications between both proteins. Heparan sulfate (HS) has been shown to be a component of amyloid plaques in a number of diseases including the prion diseases. We now present evidence that PrP can specifically bind to heparin-like compounds and that this interaction might have a physiological significance. HS can increase the concentration of PrP in normal neuroblastoma cells, whereas low molecular weight heparin (LMWH) does not. In contrast, LMWH and other heparin-like molecules, excluding HS, can inhibit the synthesis of PrPSc in scrapie infected cells and reverse their phenotype back to normal as judged by measurement of PrPSc by immunoblotting and by infectivity experiments. Whether an interaction between PrP and glycosaminoglycans plays a direct role in the conversion of PrPc into PrPSc remains to be established.

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°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.