Figure 1.
The effect of polysaccharides on the amplification of PrPSc derived from BSE-affected cattle.
A. The effect of sodium dextran sulfate with a high molecular weight (MW) ranging from 900 to 2000 kDa (sodium dextran sulfate, DSS-H) on BSE-PrPSc amplification. The PrPSc seed (10% brain homogenate) was diluted to 10−2 to 10−3 in PrPC substrate, and the diluted samples were amplified in the presence of DSS-H at 0–1%. The samples before (left panel) and after (right panel) amplification were analyzed by WB after digestion with PK. “N” designates controls in which the PrPC substrate alone was treated in the same manner. B. The effect of dextran compounds on BSE-PrPSc amplification. The PrPSc seed was diluted to 10−4, and amplification was performed in the presence or absence (“No additive”) of dextran compounds at 0.5%. “No seed” designates the control in which the PrPC substrate alone was amplified without dextran compounds. DSS-L: sodium dextran sulfate with a low MW, ranging from 5 to 6 kDa; DSP: potassium dextran sulfate (1.5 to 1.9 kDa); DEAE-dextran hydrochloride (50 kDa); dextran I (15–20 kDa), II (35–50 kDa), III (50–70 kDa), IV (200 kDa), and V (190–230 kDa). C. The effect of glycosaminoglycans (sodium chondroitin sulfate C: CSS; sodium heparan sulfate: HSS; heparan sulfate proteoglycan: HSPG) and a sulfated polysaccharide (λ-carrageenan: λ-Cag) on BSE-PrPSc amplification. The PrPSc seed was diluted to 10−4, and amplification was performed in the presence of each reagent at the final concentration indicated in the figure.
Figure 2.
The effect of DSP and PPS on the amplification of scrapie and BSE PrPSc.
A. The PrPSc seed (10% brain homogenate from scrapie-infected sheep) was diluted to 10−3, and amplification was performed in the presence or absence (“No additive”) of sulfated compounds (potassium dextran sulfate: DSP and pentosan polysulfate: PPS). “No seed” designates the control in which PrPC substrate (10% homogenate of normal mouse brain) was amplified without sulfated compounds. B. Negative symbols indicate the results obtained for the control samples without BSE PrPSc seed, while positive symbols indicate the results obtained for the samples containing BSE PrPSc seed diluted to 10−4. Amplification was performed in the presence of PPS at the concentrations indicated.
Figure 3.
Optimal DSP concentration for BSE PrPSc amplification.
The PrPSc seed was diluted to 10−4, and amplification was performed in the presence of the potassium dextran sulfate (DSP). “N” designates the control in which only PrPC substrate was amplified.
Figure 4.
BSE-PrPSc detection sensitivity.
A. The PrPSc seed was diluted to 10−4 to 10−11 with PrPC substrate, and samples were serially amplified in the presence of 0.5% potassium dextran sulfate (DSP). The duplicate amplified samples were analyzed after each round of amplification (R1–R4) by WB after PK digestion. B. No spontaneous generation of PrPSc was observed. Samples labeled “1” to “8” contained only PrPC substrate and were amplified in the presence of 0.5% DSP.
Figure 5.
Immunohistological analysis of mice that succumbed to disease following intracerebral inoculation with PMCA product.
Results from mice inoculated with a 10% brain homogenate from BSE-infected cattle are shown for comparison. No vacuolation or PrPSc accumulation was observed in the control mice inoculated with 0.05% DSP.
Table 1.
Mean incubation time of TgBoPrP mice following intracerebral inoculation.
Figure 6.
Distribution of PrPSc in cattle inoculated with BSE.
A. Tissue distribution of PrPSc in the terminal disease stage in cow 5550. Quadruplicate samples of each tissue and bodily fluid were serially amplified, and the samples were analyzed after each round of amplification (R1–R4) by WB after digestion with PK. Horizontal lines indicate the positions of molecular weight markers corresponding to 37 kDa, 25 kDa, 20 kDa, and 15 kDa. Ns: No seed control. B. Negative control reaction for serial PMCA in each tissue. Quadruplicate samples of each tissue and bodily fluid from uninfected cow 2914 were serially amplified, and the samples were analyzed by WB following digestion with PK after each round of amplification. C: Cervical region, T: Thoracic region, L: Lumbar region, nt: Not tested.
Table 2.
Detection of PrPSc by conventional WB analysis.
Figure 7.
PrPSc in spleen and bodily fluid samples obtained in the terminal disease stage.
A total of 10 tissue pieces (0.2 g each) were cut from the central portion of the spleen (a–e) and splenic hilum (f–j) for amplification. Duplicate samples of spleen tissue from BSE-affected cows 5550 (A), 5499 (B), and 5468 (C) were examined. Blood and CSF samples from cows 5499 (B) and 5468 (C) were also examined. Horizontal lines indicate the positions of molecular weight markers corresponding to 37 kDa, 25 kDa, 20 kDa, and 15 kDa. WBC: White blood cells, CSF: Cerebrospinal fluid, Ns: No seed control.
Figure 8.
Detection of PrPSc by serial DSP-PMCA in saliva and salivary glands.
Salivary PrPSc was evaluated in samples from an uninfected control cow (5660) and samples obtained from BSE-inoculated cows (5468, 5499, and 5550) in the terminal disease stage. Salivary PrPSc was also evaluated in an asymptomatic cow (9007) that had been intracerebrally inoculated with BSE. Horizontal lines indicate the positions of molecular weight markers corresponding to 37 kDa, 25 kDa, 20 kDa, and 15 kDa. Ns: No seed control.