We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrP
Sc) or recombinant prion ...protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56–90, 119–127, 137–143, 143–149, 147–151, 163–169, and 219–229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrP
Sc. However, no mAbs reacted with protease-treated PrP
Sc purified from scrapie-affected mice, even when PrP
Sc was dispersed into a detergent–lipid protein complex, to reduce the size of PrP
Sc aggregates. In contrast, denaturation of PrP
Sc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrP
Sc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrP
Sc, the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrP
Sc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases.
The cell tropism of Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, in the placenta is thought to be a key event of infectious abortion, although the ...molecular mechanism for this is largely unknown. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant (TG) cells in the placenta. In the present study, we investigated mechanism of B. abortus invasion into TG cells.
We observed internalization and intracellular growth of B. abortus in cultured TG cells. A monoclonal antibody that inhibits bacterial internalization was isolated and this reacted with heat shock cognate protein 70 (Hsc70). Depletion and over expression of Hsc70 in TG cells inhibited and promoted bacterial internalization, respectively. IFN-gamma receptor was expressed in TG cells and IFN-gamma treatment enhanced the uptake of bacteria by TG cells. Administering the anti-Hsc70 antibody to pregnant mice served to prevent infectious abortion.
B. abortus infection of TG cells in placenta is mediated by Hsc70, and that such infection leads to infectious abortion.
The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrP(Sc)), is essential for prion propagation. ...Antibodies to the C-terminal portion of PrP are known to inhibit PrP(Sc) accumulation in cells persistently infected with prions. Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrP(Sc) formation reduced PrP(Sc) accumulation in cells persistently infected with prions. The 50% effective dose was as low as approximately 1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrP(C)) expressed on the cell surface. Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb-PrP(C) complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggested that anti-PrP mAb antagonizes PrP(Sc) formation by interfering with the regular PrP(C) degradation pathway.
ABSTRACT
It has been reported that macrophages degrade infectious forms of prion protein (PrPSc). In order to investigate the mechanisms underlying PrPSc degradation in macrophages, the effects of ...lysosomal and proteasomal inhibitors on macrophage cell lines which were incubated with scrapie‐affected brain homogenate were studied. PrPSc degradation was inhibited in the presence of both proteasomal and lysosomal inhibitors. Indirect fluorescence assays to determine the cellular localization of PrPSc were undertaken. PrPSc colocalized with the lysosomal membrane protein Lamp‐1 and ubiquitin, a protein that is related to the proteasome. The present data indicate that macrophages might degrade PrPSc via the lysosomal and proteasomal pathways.
A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal ...protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5-1 micromolar in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies
In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected ...with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrPSc) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrPC) at levels similar to the parental N2a cells. In contrast, there was a variation in PrPC expression in unsusceptible N2a subclones. For example, subclone N2a‐1 expressed PrPC at the same level as the parental N2a cells and prion‐susceptible subclones, whereas subclone N2a‐24 expressed much lower levels of PrP mRNA and PrPC than the parental N2a cells. There was no difference in the binding of PrPSc to prion‐susceptible and unsusceptible N2a subclones regardless of their PrPC expression level, suggesting that the binding of PrPSc to cells is not a major determinant for prion susceptibility. Stable expression of PrPC did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion‐unsusceptible N2a subclones that expressed PrPC at levels similar to prion‐susceptible subclones, indicated that a host factor(s) other than PrPC and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion‐susceptible and ‐unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation.
In prion diseases, an abnormal isoform of prion protein (PrPSc) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrPSc-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 PrPSc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrPSc-specific staining with monoclonal antibody (MAb) 132. The combination of PrPSc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrPSc from those that were PrPSc negative. The flow cytometric analysis of PrPSc revealed the appearance of PrPSc-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrPSc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrPSc revealed a continuous increase in the proportion of PrPSc-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrPSc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrPSc-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrPSc-associated pathogenesis.IMPORTANCE Although formation of PrPSc 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 PrPSc-associated pathogenesis, such as the intracerebral spread of PrPSc and clearance of PrPSc from the brain. Despite the great need for detailed analyses of PrPSc-positive neurons and glial cells, methods available for cell type-specific analysis of PrPSc have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrPSc in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrPSc-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrPSc-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.
The conversion of prion protein (PrP) from the monomeric cellular isoform to the oligomeric pathological isoform is a crucial event in the pathogenesis of prion diseases. To investigate oligomer ...formation of PrP, enhanced green fluorescent protein (EGFP)-tagged PrP (EGFP-PrP) without the glycosylphosphatidylinositol (GPI) anchor was prepared and the oligomerization of EGFP-PrP induced by sodium dodecyl sulphate (SDS) was monitored by fluorescence correlation spectroscopy (FCS). The FCS analysis indicated that soluble oligomers were formed at 0.011% SDS. Furthermore, the combination of fluorescence cross-correlation spectroscopy (FCCS) and a panel of anti-PrP monoclonal antibodies (mAbs) revealed the conformational changes in PrP. Our studies provide a method to analyze conformational changes of proteins in solution.
Peptides can be converted to highly active compounds by introducing appropriate substituents on the suitable amino acid residue. Although modifiable residues in peptides can be systematically ...identified by peptide scanning methodologies, there is no practical method for optimization at the “scanned” position. With the purpose of using derivatives not only for scanning but also as a starting point for further chemical functionalization, we herein report the “scanning and direct derivatization” strategy through chemoselective acylation of embedded threonine residues by a serine/threonine ligation (STL) with the help of in situ screening chemistry. We have applied this strategy to the optimization of the polymyxin antibiotics, which were selected as a model system to highlight the power of the rapid derivatization of active scanning derivatives. Using this approach, we explored the structure–activity relationships of the polymyxins and successfully prepared derivatives with activity against polymyxin-resistant bacteria and those with Pseudomonas aeruginosa selective antibacterial activity. This strategy opens up efficient structural exploration and further optimization of peptide sequences.
The Food Safety Commission (FSC) of Japan, established in July 2003, has its own initiative to conduct risk assessments on food stuffs known as “self-tasking assessment”. Within this framework, the ...FSC decided to conduct a risk assessment of beef and beef offal imported into Japan from countries with no previous BSE reports; thus, a methodology was formed to suit to this purpose. This methodology was partly based on the previous assessments of Japanese domestic beef and beef imported from U.S.A./Canada, but some modifications were made. Other organizations’ assessment methods, such as those used for BSE status assessment in live cattle by the OIE and EFSA’s GBR, were also consulted. In this review, the authors introduce this alternative methodology, which reflects (1) the risk of live cattle in the assessed country including temporal risks of BSE invasion and domestic propagation, with the assessment results verified by surveillance data, and (2) the risk of beef and beef offal consisting of cumulative BSE risk by types of slaughtering and meat production processes implemented and the status of mechanically recovered meat production. Other possible influencing factors such as atypical BSE cases were also reviewed. The key characteristic of the current assessment is a combination of the time-sequential risk level of live cattle and qualitative risk level of meat production at present in an assessed country.
PDF
