• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.559-559
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• 2005

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.7
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• pp.936-940
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• 2008

The PCR-amplified prion protein (PrP) gene was sequenced to determine the frequency of scrapie-associated as well as novel PrP genotypes in 72 healthy goats representing five breeds. A total of six genotypes were detected, resulting from the three reported 143 (H/R), 154 (R/H) and 240 (S/P) and the two novel 39 (S/R) and 185 (I/F) amino acid polymorphisms. Of the four silent mutations 42 (a$\rightarrow$g), 138 (c$\rightarrow$t), 231 (c$\rightarrow$a) and 237 (g$\rightarrow$c) detected in this study, 237 (g$\rightarrow$c) is novel. A genotype (SIP/RFP) harboring three amino acid polymorphisms 39 (S/R), 185 (I/F) and 240 (S/P) was found in few goats. Although both scrapie-associated genotypes with 143 (H/R) and 154 (R/H) polymorphisms and others with 39 (S/R), 185 (I/F) and 240 (S/P) polymorphisms were present in the studied Pakistani goats, their frequency was lower than that of the wild-type genotype SHRIS/SHRIS (34.7%). These results emphasize the need for further sequencing of the PrP gene in a large number of goats representing the five studied breeds, so that overall PrP variability can be assessed in these breeds in research addressing future concerns about scrapie.

• Journal of Microbiology and Biotechnology
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• v.28 no.10
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• pp.1749-1759
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• 2018

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with >95% purity. The purified recPrPs predominantly adopted an ${\alpha}$-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.

• Toxicological Research
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• v.28 no.1
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• pp.5-9
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• 2012

It has been shown that the accumulation of prion in the cytoplasm can result in neurodegenerative disorders. Synthetic prion peptide 106-126 (PrP) is a glycoprotein that is expressed predominantly by neurons and other cells, including glial cells. Prion-induced chronic neurodegeneration has a substantial inflammatory component, and an increase in the levels of matrix metalloproteinases (MMPs) may play an important role in neurodegenerative development and progression. However, the expression of MMPs in PrP induced rat astrocytes and microglia has not yet been compared. Thus, in this study, we examined the fluorescence intensity of CD11b positive microglia and Glial Fibrillary Acidic Protein (GFAP) positive astrocytes and found that the fluorescent intensity was increased following incubation with PrP at 24 hours in a dose-dependent manner. We also observed an increase in interleukin-1 beta (IL-$1{\beta}$) and tumor necrosis factor alpha (TNF-${\alpha}$) protein expression, which are initial inflammatory cytokines, in both PrP induced astrocytes and microglia. Furthermore, an increase MMP-1, 3 and 11 expressions in PrP induced astrocytes and microglia was observed by real time PCR. Our results demonstrated PrP induced activation of astrocytes and microglia respectively, which resulted in an increase in inflammatory cytokines and MMPs expression. These results provide the insight into the different sensitivities of glial cells to PrP.

• BMB Reports
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• v.42 no.7
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• pp.444-449
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• 2009

Different neurodegenerative disorders like prion disease, is caused by protein misfolding conformers. Reverse-transfected cytosolic prion protein (PrP) and PrP expressed in the cytosol have been shown to be neurotoxic. To investigate the possible mechanism of neurotoxicity due to accumulation of PrP in cytosol, a PrP mutant lacking the signal and GPI (CytoPrP) was introduced into the SH-SY5Y cell. MTT and trypan blue assays indicated that the viability of cells expressing CytoPrP was remarkably reduced after treatment of MG-132. Obvious apoptosis phenomena were detected in the cells accumulated with CytoPrP, including loss of mitochondrial transmembrane potential, increase of caspase-3 activity, more annexin V/PI-double positive-stained cells and reduced Bcl-2 level. Moreover, DNA fragmentation and TUNEL assays also revealed clear evidences of late apoptosis in the cells accumulated CytoPrP. These data suggest that the accumulation of CytoPrP in cytoplasm may trigger cell apoptosis, in which mitochondrial relative apoptosis pathway seems to play critical role.

• 한국환경농학회:학술대회논문집
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• 2009.07a
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• pp.273-282
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• 2009

The transmissible spongiform encephalopathies (TSEs) disease group are fatal neurodegenerative disorders affecting a wide range of hosts. The group includes kuru and Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats and Bovine spongiform encephalopathy (BSE) in cattle. The exact nature of the infectious agent involved in the transmission of these diseases remains controversial. However, a central event in their pathogenesis is the accumulation in infected tissues of an abnormal form of a host-encoded protein, the prion protein (PrP). Whereas the normal cellular protein is fully sensitive to protease ($PrP^{sen}$), the disease-associated prion protein ($PrP^d$) is only partly degraded ($PrP^{res}$), its amino-terminal end being removed. BSE was first reported in the mid-80s in the UK. Ten years later, a new form of human prion disease, variant CJD (vCJD) developed in the wake of the BSE epidemic, and there is now strong scientific evidence that vCJD was initiated by the exposure of humans to BSE-infected tissues, thus indicating a zoonotic disease. However, the ban on the feeding of animal-derived proteins to ruminants, and the apparent lack of vertical transmission of BSE, have led to a decline in the incidence of the disease within cattle herd and therefore, an assumed decreased risk for human contacting vCJD. The origin of the original case(s) of BSE still remains an enigma even though three hypotheses have been raised. Hypotheses are i) sheep- or goat-derived scrapie-infected tissues included in meat and bone meal fed to cattle, ii) a previously undetected sporadic or genetic bovine TSE contaminating cattle feed or iii) originating from a human TSE through animal feed contaminated with human remains. A host cellular membrane protein ($PrP^C$), which is abundant in central nervous system tissue, appear to be conformationally altered in the diseased host into a prion protein ($PrP^{Sc}$). This $PrP^{Sc}$ is detergent insoluble and partially protease-resistant ($PrP^{res}$). The term $PrP^{res}$ is normally used to describe the protein detected after protease treatment, in techniques such as Western immunoblotting, and enzyme-linked immunosorbant assay using fresh/frozen tissue. Immunohistochemistry may performed with formalin-fixed tissues. Also, clinical signs of the BSE are one of the major diagnostic indicators. Recently, atypical forms (known as H- and L-type) of BSE have appeared in several European countries, Japan, Canada and the United States. An unusual case was also reported in a miniature zebu. The atypical BSE fall into two groups based on the relative molecular mass (Mm) of the unglycosylated $PrP^{res}$ band relative to that of classical BSE, one of the higher Mm (H-type) and the other lower (L-type). Both types have been detected worldwide as rare cases in older animals, at a low prevalence consistent with the possibility of sporadic forms of prion diseases in cattle. This raises the unwelcome possibility that vCJD could increase in the human population. Now, active surveillance program against BSE is going on in Korea. In regional veterinary service lab, ELISA is applied to screen the BSE in slaughter and confirmatory tests by Western immunoblotting and immunohistochemisty are carried out if there are positive or suspect in the screening test. Also, the ruminant feed ban is rigorously enforced. Removal of specified risk materials such as brain and spinal cord from cattle is mandatory process at slaughter to prevent the infected material from entering the human food chain.

• BMB Reports
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• v.42 no.6
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• pp.331-337
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• 2009

We investigate the correlation between the glycosylation modified prion proteins and apoptosis. The wild-type PRNP gene and four PRNP gene glycosylated mutants were transiently expressed in HeLa cells. The effect of apoptosis induced by PrP mutants was confirmed by MTT assay, Hochest staining, Annexin-V staining and PI staining. ROS test detected ROS generation within the cells. The mitochondrial membrane potential was analyzed by the flow cytometry. The expression levels of Bcl-xL, Bax, cleaved Caspase-9 proteins were analyzed by Western Blot. The results indicated that the expressed non-glycosylated PrP in HeLa cells obviously induced apoptosis, inhibited the growth of cells and reduced the mitochondrial membrane potential, and more ROS generation and low levels of the apoptosis-related proteins Bcl-xL, the activated the cleaved Caspase-9 proteins were found. The apoptosis induced by non-glycosylated PrP demonstrates that its underlying mechanism correlates with the mitochondria-mediated signal transduction pathway.

• Journal of the Korean Society of Tobacco Science
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• v.31 no.1
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• pp.29-38
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• 2009

Although prion diseases, a group of fatal neurodegenerative diseases of human and animals, are presumed to be caused by several mechanisms including abnormal change of prion protein, oxidative stress is still believed to play a central role in development of the diseases. Cigarette smoking has a few beneficial effects on neuronal diseases such as Alzheimer's disease and Parkinson's disease despite of many detrimental effects. In this study, we investigated how chronic cigarette smoking could exert such beneficial effect against oxidative damage. For this study, homogenates of 87V scrapie-infected brain was inoculated on intracerebral system of IM mice through stereotaxic microinjection and biochemical properties concerning with oxidative stress were examined. The scrapie infection decreased the activity of mitochondrial Mn-containing superoxide dismutase by 50% of the control, meanwhile the effects on other antioxidant enzymes including Cu or Zn-containing superoxide dismutase were not significant. Additionally, the infection elevated superoxide level as well as monoamine oxide-B (MAO-B) in the infected brain. Interestingly, many of the detrimental effects were improved in partial or significantly by long-term cigarette smoke exposure (CSE). CSE not only completely prevented the generation of mitochondrial superoxide but also significantly (p<0.05) decreased the elevated mitochondrial MAO-B activity in the infected brain. Concomitantly, CSE prevented subsequent protein oxidation and lipid peroxidation caused by scrapie infection; however, it did not affect the activities of antioxidant enzymes. These results suggest that chronic exposure of cigarette smoke contribute to in part preventing the progress of neurodegeneration caused by scrapie infection.

• Proceedings of the Korean Magnetic Resonance Society Conference
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• 2002.08a
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• pp.82-82
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• 2002

• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.1023-1031
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• 2017

The conformational change of cellular prion protein ($PrP^C$) to its misfolded counterpart, termed $PrP^{Sc}$, is mediated by a hypothesized cellular cofactor. This cofactor is believed to interact directly with certain amino acid residues of $PrP^C$. When these are mutated into cationic amino acid residues, $PrP^{Sc}$ formation and prion replication halt in a dominant negative (DN) manner, presumably due to strong binding of the cofactor to mutated $PrP^C$, designated as DN PrP mutants. Previous studies demonstrated that plasminogen and its kringle domains bind to PrP and accelerate $PrP^{Sc}$ generation. In this study, in vitro binding analysis of kringle domains of plasminogen to Q167R DN mutant PrP (PrPQ167R) was performed in parallel with the wild type (WT) and Q218K DN mutant PrP (PrPQ218K). The binding affinity of PrPQ167R was higher than that of WT PrP, but lower than that of PrPQ218K. Scatchard analysis further indicated that, like PrPQ218K and WT PrP, PrPQ167R interaction with plasminogen occurred at multiple sites, suggesting cooperativity in this interaction. Competitive binding analysis using $\small{L}$-lysine or $\small{L}$-arginine confirmed the increase of the specificity and binding affinity of the interaction as PrP acquired DN mutations. Circular dichroism spectroscopy demonstrated that the recombinant PrPs used in this study retained the ${\alpha}$-helix-rich structure. The ${\alpha}$-helix unfolding study revealed similar conformational stability for WT and DN-mutated PrPs. This study provides an additional piece of biochemical evidence concerning the interaction of plasminogen with DN mutant PrPs.