Anti-prion protein (PrP) monoclonal antibody 132, which recognizes mouse PrP amino acids 119-127, enables us to reliably detect abnormal isoform prion protein (PrP.sup.Sc) in cells or frozen tissue ...sections by immunofluorescence assay, although treatment with guanidinium salts is a prerequisite. Despite the benefit of this mAb, the mechanism of PrP.sup.Sc -specific detection remains unclear. Therefore, to address this mechanism, we analyzed the reactivities of mono- and bivalent mAb 132 to recombinant mouse PrP (rMoPrP) by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In ELISA, binding of the monovalent form was significantly weaker than that of the bivalent form, indicating that bivalent binding confers a higher binding stability to mAb 132. Compared with other anti-PrP mAbs tested, the reactivity of bivalent mAb 132 was easily affected by a decrease in antigen concentration. The binding kinetics of mAb 132 assessed by SPR were consistent with the results of ELISA. The dissociation constant of the monovalent form was approximately 260 times higher than that of the bivalent form, suggesting that monovalent binding is less stable than bivalent binding. Furthermore, the amount of mAb 132 that bound to rMoPrP decreased if the antigen density was too low to allow bivalent binding. If two cellular PrP (PrP.sup.C) are close enough to allow bivalent binding, mAb 132 binds to PrP.sup.C . These results indicate that weak monovalent binding to monomeric PrP.sup.C diminishes PrP.sup.C signals to background level, whereas after exposure of the epitope, mAb 132 binds stably to oligomeric PrP.sup.Sc in a bivalent manner.
The formation of protease-resistant prion protein (PrP-res or PrPSc) involves selective interactions between PrP-res and its normal protease-sensitive counterpart, PrP-sen or PrPC. Previous studies ...have shown that synthetic peptide fragments of the PrP sequence corresponding to residues 119–136 of hamster PrP (Ha119–136) can selectively block PrP-res formation in cell-free systems and scrapie-infected tissue culture cells. Here we show that two other peptides corresponding to residues 166–179 (Ha166–179) and 200–223 (Ha200–223) also potently inhibit the PrP-res induced cell-free conversion of PrP-sen to the protease-resistant state. In contrast, Ha121–141, Ha180–199, and Ha218–232 were much less effective as inhibitors. Mechanistic analyses indicated that Ha166–179, Ha200–223, and peptides containing residues 119–136 inhibit primarily by binding to PrP-sen and blocking its binding to PrP-res. Circular dichroism analyses indicated that Ha117–141 and Ha200–223, but not non-inhibitory peptides, readily formed high β-sheet structures when placed under the conditions of the conversion reaction. We conclude that these inhibitory peptides may mimic contact surfaces between PrP-res and PrP-sen and thereby serve as models of potential therapeutic agents for transmissible spongiform encephalopathies.
In prion diseases, an abnormal isoform of prion protein (PrP
) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrP
-positive ...neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrP
in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrP
-specific staining with monoclonal antibody (MAb) 132. The combination of PrP
staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrP
from those that were PrP
negative. The flow cytometric analysis of PrP
revealed the appearance of PrP
-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrP
in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrP
revealed a continuous increase in the proportion of PrP
-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrP
from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrP
-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrP
-associated pathogenesis.
Although formation of PrP
in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrP
-associated pathogenesis, such as the intracerebral spread of PrP
and clearance of PrP
from the brain. Despite the great need for detailed analyses of PrP
-positive neurons and glial cells, methods available for cell type-specific analysis of PrP
have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrP
in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrP
-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrP
-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.
Abstract
Intracellular dynamics of an abnormal isoform of prion protein (PrP
Sc
) are tightly associated with prion propagation. However, the machineries involved in the intracellular trafficking of ...PrP
Sc
are not fully understood. Our previous study suggested that PrP
Sc
in persistently prion-infected cells dynamically circulates between endocytic-recycling compartments (ERCs) and peripheral regions of the cells. To investigate these machineries, we focused on retrograde transport from endosomes to the trans-Golgi network, which is one of the pathways involved in recycling of molecules. PrP
Sc
was co-localized with components of clathrin-coated vesicles (CCVs) as well as those of the retromer complex, which are known as machineries for retrograde transport. Fractionation of intracellular compartments by density gradient centrifugation showed the presence of PrP
Sc
and the components of CCVs in the same fractions. Furthermore, PrP
Sc
was detected in CCVs isolated from intracellular compartments of prion-infected cells. Knockdown of clathrin interactor 1, which is one of the clathrin adaptor proteins involved in retrograde transport, did not change the amount of PrP
Sc
, but it altered the distribution of PrP
Sc
from ERCs to peripheral regions, including late endosomes/lysosomes. These data demonstrated that some PrP
Sc
is transported from endosomes to ERCs by CCVs, which might be involved in the recycling of PrP
Sc
.
Since high levels of prions, the causative agent of bovine spongiform encephalopathy (BSE), accumulate in the brain and spinal cord, contamination of beef carcasses with central nervous system tissue ...(CNST) may occur at post-slaughter process. In this study, we investigated CNST contamination on the surface of beef carcasses using glial fibrillary acidic protein as a marker after splitting and evaluated the effects of washing procedures on contamination removal. High levels of CNST contamination was detected immediately after splitting, especially in the area close to the spinal column. This suggests that spinal cord fragments are attached to carcasses at the time of splitting even though the spinal cords have been removed by vacuum before splitting. Steam cleaning or manually washing with normal pressure water around the spinal column, performed prior to washing with high-pressure water, was found to be effective for reducing the level of CNST contamination. Furthermore, manually washing with high-pressure water could reduce CNST contamination to almost negligible levels. These results are useful for preparation of appropriate sanitation standard operating procedures to reduce the risk of CNST contamination of carcasses for prevention of exposure to BSE prion via the food chain.
Infection prevention and control (IPC) in veterinary medicine is crucial to protect patients, owners, staff, and the public. An IPC programme is recommended for every animal hospital. The objective ...of this retrospective longitudinal study was to describe the changes in bacterial and multidrug-resistant (MDR) bacterial isolates and self-reported hand hygiene awareness and practices after an IPC programme to assess the long-term effect of this programme in small animal veterinary medicine. The IPC programme was implemented at our veterinary teaching hospital in April 2018, which included the establishment of an infection control task force, regular IPC lectures and poster campaigns, infrastructure improvement, and manual refinement. Laboratory-based surveillance was retrospectively conducted before and after the programme (January 2016–December 2022). Level and slope changes in bacterial isolates were evaluated using interrupted time-series analysis. Self-reported hand hygiene awareness and practices were assessed using an annual questionnaire. Additionally, hygiene product purchases during the study period were investigated.
The monthly number of total and MDR bacterial isolates decreased significantly after the programme (MDR level change: −0.426; 95% confidence interval: −0.744, −0.109; P = 0.009; and MDR slope change: −0.035; 95% confidence interval: −0.058, −0.011; P = 0.003). Additionally, awareness of hand hygiene before touching animals improved after the programme. Overall self-reported hand hygiene practices improved, and hygiene product purchases significantly increased. These results suggested that the IPC programme may have long-term effects regarding reducing total and MDR bacterial isolates and improving hand hygiene awareness in veterinary medicine.
•Infection prevention and control (IPC) programmes promote IPC like hand hygiene.•The number of bacterial isolates decreased after the IPC programme.•The number of multidrug-resistant bacterial isolates also decreased.•The changes of isolates were minimal after the coronavirus disease 2019 pandemic.•Overall hand hygiene awareness was improved after the IPC programme.
The AT-tailing method is a labelling technique that utilises oligo(dA–dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. ...This method uses an oligo(dA–dT)-conjugated primary antibody (direct immunoAT method) or an oligo(dA–dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base oligo(dA–dT)-conjugated antibodies (IgG–ATs) were prepared in advance by conjugating maleimide-activated oligo(dA–dT) to IgG via free sulfhydryl residues that had been introduced on the surface of IgG using Traut's reagent. Following the reaction with the target antigen and the IgG–AT, oligo(dA–dT) was elongated by Δ
Tth DNA polymerase in the presence of dATP, dTTP and biotinylated dUTP, consequently labelling the antigen–antibody complex with a large amount of biotin. To initially evaluate the immunoAT method, the presence or absence of prion protein (PrP
sc) was determined in formalin-fixed and paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing IgG–AT and AT-tailing methods. PrP
sc detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method.
We developed prototype CdTe detectors with segmented electrodes using inkjet drawing in ink containing Gold nanoparticles by exploiting the sintering process for metallization. The resulting ...electrode patterns demonstrated that the inkjet technology is capable of forming fine detection elements with submillimeter alignment and gaps of several tens of micrometers. Moreover, it was shown that they are suitable for the construction of medical imaging scanners. In addition, it was also demonstrated that the proposed approach is compatible with conventional processes with respect to detector performance. The developed prototype detectors achieved good energy resolution that was comparable to other detectors based on conventional methods. In particular, an energy resolution of 1.7 % was obtained at 662 keV for a detector with pixel sizes of 2×2 mm2. It was also determined that gold nanoparticle inks can be metallized at a relatively low cure temperature of approximately 200 °C and this feature facilitates the fabrication of detectors by employing CdTe, which is a heat-sensitive material.
The total synthesis of the depsipeptide natural product plusbacin A
(
) utilizing solid-phase peptide synthesis (SPPS) was disclosed. A 3-hydroxy-proline derivative compatible with Fmoc SPPS was ...prepared by a diastereoselective Joullié-Ugi three-component reaction (JU-3CR)/hydrolysis sequence. After peptide elongation on the solid support, cleavage of the peptide from the resin, followed by macrolactamization and global deprotection, gave plusbacin A
(
).
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