Stem cell therapy for ischemic stroke holds great promise for the treatment of neurological impairment and has moved from the laboratory into early clinical trials. The mechanism of action of stem cell therapy includes the bystander... more
Stem cell therapy for ischemic stroke holds great promise for the treatment of neurological impairment and has moved from the laboratory into early clinical trials. The mechanism of action of stem cell therapy includes the bystander effect and cell replacement. The bystander effect plays an important role in the acute to subacute phase, and cell replacement plays an important role in the subacute to chronic phase. Intraarterial (IA) transplantation is less invasive than intraparenchymal transplantation and can provide more cells in the affected brain region than intravenous transplantation. However, transplanted cell migration was reported to be insufficient, and few transplanted cells were retained in the brain for an extended period. Therefore, the bystander effect was considered the main mechanism of action of IA stem cell transplantation. In most clinical trials, IA transplantation was performed during the acute and subacute phases. Although clinical trials of IA transplantation...
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Prions are unconventional pathogens that encode the pathogenic information in conformations of the constituent abnormal isoform of prion protein (PrPSc), independently of the nucleotide genome. Therefore, conformational diversity of PrPSc... more
Prions are unconventional pathogens that encode the pathogenic information in conformations of the constituent abnormal isoform of prion protein (PrPSc), independently of the nucleotide genome. Therefore, conformational diversity of PrPSc underlies the existence of many prion strains and species barriers of prions, although the conformational information is extremely limited. Interestingly, differences between polymorphic or species-specific residues responsible for the species/strain barriers are often caused by conservative replacements between hydrophobic amino acids. This implies that subtle differences among hydrophobic amino acids are significant for PrPSc structures. Here, we analyzed the influence of different hydrophobic residues on the structures of an in-register parallel β-sheet amyloid of α-synuclein (αSyn) using molecular dynamics (MD) simulation, and applied the knowledge from the αSyn amyloid to modeling a local structure of human PrPSc encompassing residues 107–143....
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Background and Purpose— Intra-arterial cell transplantation offers a novel therapeutic strategy for stroke; however, it remains unclear how the timing of cell administration affects cell distribution, brain repair processes and functional... more
Background and Purpose— Intra-arterial cell transplantation offers a novel therapeutic strategy for stroke; however, it remains unclear how the timing of cell administration affects cell distribution, brain repair processes and functional recovery. Here, we investigate the hypothesis that the timing of cell transplantation changes the behavior of the cell graft and the host environment in a way that affects functional recovery. Methods— Rats received human mesenchymal stem cells (hMSCs) via the internal carotid artery at 1, 4 or 7 days (D1, D4 or D7) after middle cerebral artery occlusion and reperfusion. Animals were sacrificed at various time points to assess cell distribution in correlation with the host cerebral hemodynamics, serum levels of matrix metallopeptidase-9 (MMP-9), infiltration of activated microglia, expression of brain derived neurotrophic factor (BDNF), angiogenesis, presence of reactive astrocytes, and neurological recovery. Results— hMSCs were widely distributed ...
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Diversity of prion strains is one of the most mysterious traits of prions because they are mere aggregates of abnormally-folded forms of single protein species, prion protein (PrPSc), without genome. Although the strain-specific... more
Diversity of prion strains is one of the most mysterious traits of prions because they are mere aggregates of abnormally-folded forms of single protein species, prion protein (PrPSc), without genome. Although the strain-specific properties are hypothesized to be enciphered in the strain-specific structures of PrPSc instead of nucleotide genome, specifically what structure can code the information remains an enigma due to the incompatibility of PrPSc with structural analyses. Although the strain diversity was regarded as unique to prions, recently other disease-associated amyloids of α-synuclein (αSyn) or tau are also reported to have “strains”. As detailed structures of αSyn amyloid are already identified and the properties of mutant αSyn associated with familial Parkinson’s diseases, e.g. A53T, H50Q, and G51D, have been characterized, structure-phenotype relations of this type of amyloid could be investigated by using the αSyn amyloid as a model. Here we intensively investigated th...
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Background and purpose: Cell transplantation therapy holds great potential to improve impairments after stroke. However, the importance of donor age on therapeutic efficacy is uncertain. We investigate regenerative capacity of... more
Background and purpose: Cell transplantation therapy holds great potential to improve impairments after stroke. However, the importance of donor age on therapeutic efficacy is uncertain. We investigate regenerative capacity of transplanted cells focusing on donor age (young vs. old) for ischemic stroke. Methods: The value of platelet-derived growth factor (PDGF)-BB secreted from human mesenchymal stem cells (hMSC) was analyzed regarding in two age groups; young (20-30 years) and old (57-65 years) in vitro. Male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion, and received young or old hMSC trans-arterially at 24 h after stroke. Functional recovery was assessed with modified neurological severity score (mNSS). Structural recovery was assessed on neovascularization and endogenous cell migration as well as trophic factor secretion. Results: The value of PDGF-BB was significantly higher in young hMSC (40.47±4.29 pg/ml/10 4 cells) than that in old hMSC (2...
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We report the general autopsy findings of abnormal prion protein (PrP) deposits with their seeding activities, as assessed by the real‐time quaking‐induced conversion (RT‐QuIC) method, in a 72‐year‐old female patient with sporadic... more
We report the general autopsy findings of abnormal prion protein (PrP) deposits with their seeding activities, as assessed by the real‐time quaking‐induced conversion (RT‐QuIC) method, in a 72‐year‐old female patient with sporadic Creutzfeldt–Jakob disease (sCJD). At 68 years of age, she presented with gait disturbance and visual disorders. Electroencephalography showed periodic synchronous discharge. Myoclonus was also observed. A genetic test revealed that PRNP codon 129 was methionine/methionine (MM). She died of pneumonia three years and four months after disease onset, and a general autopsy was performed. The brain weighed 650 g and appeared markedly atrophic. Immunohistochemistry for PrP revealed synaptic PrP deposits and coarse PrP deposits in the cerebral cortices, basal ganglia, cerebellum, and brainstem. Western blot analysis identified type 1 proteinase‐K‐resistant PrP in frontal cortex samples. PrP deposits were also observed in systemic organs, including the femoral nerve, psoas major muscle, abdominal skin, adrenal medulla, zona reticularis of the adrenal gland, islet cells of the pancreas, and thyroid gland. The RT‐QuIC method revealed positive seeding activities in all examined organs, including the frontal cortex, femoral nerve, psoas major muscle, scalp, abdominal skin, adrenal gland, pancreas, and thyroid gland. The following 50% seeding dose (SD50) values were 9.5 (frontal cortex); 8 ± 0.53 (femoral nerve); 7 ± 0.53 (psoas major muscle); and 7.88 ± 0.17 (scalp). The SD50 values for the adrenal gland, dermis, pancreas, and thyroid gland were 6.12 ± 0.53, 5.25, 4.75, and 4.5, respectively. PrP deposits in general organs may be associated with long‐term disease duration. This case indicated the necessity for general autopsies in sCJD cases to establish strict infection control procedures for surgical treatment and to examine certain organs.
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BACKGROUND The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of... more
BACKGROUND The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other than immunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid. METHODS We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed according to Parchi's classification. We assessed SD50 in brains after incubation in formalin solution for over 1 month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings. RESULTS The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combined formalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathological findings in most brain regions examined. CONCLUSION RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously been amenable to analysis.
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Background: Neuropathology in sporadic Creutzfeldt–Jakob disease (sCJD) brains is usually investigated using formalin-fixed brains and formic acid-treated brains. However, brain tissues prepared in these ways are not amenable to... more
Background: Neuropathology in sporadic Creutzfeldt–Jakob disease (sCJD) brains is usually investigated using formalin-fixed brains and formic acid-treated brains. However, brain tissues prepared in these ways are not amenable to biochemical analyses. We therefore developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brains. Methods: We used endpoint RT-QuIC assays to analyse SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2T cases and 2 MM2C cases) according to Parchi’s classification. We estimated SD50 in brains after incubation in formalin solution for over 1 month, and checked SD50 after treating formalin-fixed brain samples with formic acid. We also compared the neuropathological findings with SD50 from formalin-fixed brain samples from MM1, MM2T, and MM2C patients. Findings: RT-QuIC enables the study of formalin-fixed brains in sCJD patients that have not previously been amenable to analysis. Interpretation: The SD50 values of formalin...
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The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the... more
The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant varia...
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Prion diseases are characterized by accumulation of amyloid fibrils. The causative agent is an infectious amyloid that is comprised solely of misfolded prion protein (PrPSc). Prions can convert PrPC to proteinase-resistant PrP (PrP-res)... more
Prion diseases are characterized by accumulation of amyloid fibrils. The causative agent is an infectious amyloid that is comprised solely of misfolded prion protein (PrPSc). Prions can convert PrPC to proteinase-resistant PrP (PrP-res) in vitro; however, the intermediate steps involved in the spontaneous conversion remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation in the absence of PrPSc. We found that rPrP underwent liquid-liquid phase separation at the interface of the aqueous two-phase system (ATPS) of polyethylene glycol (PEG) and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired proteinase-resistant amyloid accompanied by β-sheet conversion, as confirmed by western blotting, Fourier transform infrared spectroscopy, and Congo...
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Nicaraven, a chemically synthesized hydroxyl radical-specific scavenger, has been demonstrated to protect against ischemia-reperfusion injury in various organs. We investigated whether nicaraven can attenuate radiation-induced injury in... more
Nicaraven, a chemically synthesized hydroxyl radical-specific scavenger, has been demonstrated to protect against ischemia-reperfusion injury in various organs. We investigated whether nicaraven can attenuate radiation-induced injury in hematopoietic stem/progenitor cells, which is the conmen complication of radiotherapy and one of the major causes of death in sub-acute phase after accidental exposure to high dose radiation. C57BL/6 mice were exposed to 1 Gy c-ray radiation daily for 5 days in succession (a total of 5 Gy), and given nicaraven or a placebo after each exposure. The mice were sacrificed 2 days after the last radiation treatment, and the protective effects and relevant mechanisms of nicaraven in hematopoietic stem/progenitor cells with radiation-induced damage were investigated by ex vivo examination. We found that post-radiation administration of nicaraven significantly increased the number, improved the colony-forming capacity, and decreased the DNA damage of hematopo...
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Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy are characterized by aggregation of abnormal α-synuclein (α-syn) and collectively referred to as α-synucleinopathy. Because these diseases have different... more
Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy are characterized by aggregation of abnormal α-synuclein (α-syn) and collectively referred to as α-synucleinopathy. Because these diseases have different prognoses and treatments, it is desirable to diagnose them early and accurately. However, it is difficult to accurately diagnose these diseases by clinical symptoms because symptoms such as muscle rigidity, postural dysreflexia, and dementia sometimes overlap among these diseases. The process of conformational conversion and aggregation of α-syn has been thought similar to that of abnormal prion proteins that cause prion diseases. In recent years, in vitro conversion methods, such as real-time quaking-induced conversion (RT-QuIC), have been developed. This method has succeeded in amplifying and detecting trace amounts of abnormal prion proteins in tissues and central spinal fluid of patients by inducing conversion of recombinant prion proteins via shaking. ...
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Human prion diseases are neurodegenerative disorders caused by prion protein. Although infectivity was historically detected only in the central nervous system and lymphoreticular tissues of patients with sporadic Creutzfeldt-Jakob... more
Human prion diseases are neurodegenerative disorders caused by prion protein. Although infectivity was historically detected only in the central nervous system and lymphoreticular tissues of patients with sporadic Creutzfeldt-Jakob disease, recent reports suggest that the seeding activity of Creutzfeldt-Jakob disease prions accumulates in various non-neuronal organs including the liver, kidney, and skin. Therefore, we reanalyzed autopsy samples collected from patients with sporadic and genetic human prion diseases and found that seeding activity exists in almost all digestive organs. Unexpectedly, activity in the esophagus reached a level of prion seeding activity close to that in the central nervous system in some CJD patients, indicating that the safety of endoscopic examinations should be reconsidered.
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Prion is a unique infectious agent which consists solely of abnormally-folded prion protein (PrPSc) but possesses virus-like features, e.g. existence of strain diversity, adaptation to new hosts and evolutionary changes. These biological... more
Prion is a unique infectious agent which consists solely of abnormally-folded prion protein (PrPSc) but possesses virus-like features, e.g. existence of strain diversity, adaptation to new hosts and evolutionary changes. These biological phenomena were attributed to the structural properties of PrPSc due to lack of genetic material of prion. Therefore, regardless of incompatibility with high-resolution structural analysis, many structural models of PrPSc have been hypothesized based on limited structural information and, recently, models consisting solely of β-sheets and intervening loops/kinks have been suggested, i.e. parallel in-register β-sheet models and β-solenoid model. Given the relatively simple structural models of PrPSc, we utilized values of theoretical β-sheet or random-coil propensity (Pβ or Pc, respectively) calculated by secondary structure prediction with a neural network to analyze interspecies transmissions of prion, because numerical conversion of the primary str...
Prions are composed solely of the pathological isoform (PrP) of the normal cellular prion protein (PrP). Identification of different PrP structures is crucially important for understanding prion biology because the pathogenic properties... more
Prions are composed solely of the pathological isoform (PrP) of the normal cellular prion protein (PrP). Identification of different PrP structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrP However, these structures remain yet to be identified, because of the incompatibility of PrP with conventional high-resolution structural analysis methods. Previously, we reported that the region between the first and the second alpha-helix (H1~H2) of PrP might cooperate with the more C-terminal side region for efficient interactions with PrP From this starting point, we created a series of PrP variants with two cysteine substitutions (C;C-PrP) forming a disulfide crosslink between H1~H2 and the distal region of the third helix (Ctrm). We then assessed the conversion capabilities of the C;C-PrP variants in N2a cells infected with mouse-adapted scrapie prions (22L-ScN2a). Specifically, ...
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Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrP) in the central nervous system. Although several small compounds that bind to... more
Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrP) in the central nervous system. Although several small compounds that bind to normal PrP (PrP) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrP in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a β-adrenergic blocker for hypertension, also reduced the accumulation of PrP in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their ...
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Immune complexes (ICs) may clearly reflect immunological abnormalities caused by disease, especially for autoimmune diseases. Although ICs have been detected in cerebrospinal fluid (CSF) from patients with CNS autoimmune diseases,... more
Immune complexes (ICs) may clearly reflect immunological abnormalities caused by disease, especially for autoimmune diseases. Although ICs have been detected in cerebrospinal fluid (CSF) from patients with CNS autoimmune diseases, identities of antigens in such ICs have not been comprehensively determined. We used immune complexome analysis, in which nano-liquid chromatography-tandem mass spectrometry is employed to comprehensively identify antigens incorporated into ICs in biological fluids, to characterize ICs in CSF samples from patients with CNS autoimmune diseases, and to find disease-specific IC antigen to a certain CNS autoimmune disease. Also, we compared the IC antigens we identified with the reported CSF proteome or with the published plasma proteome to examine if the method is distinguished from the conventional CSF proteome analysis. We identified 176 antigens in 78 CSF samples. We then assessed the overlaps among these antigens, the CSF proteome, and the plasma proteome...
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Sporadic human prion diseases are defined on the basis of clinical features, with periodic sharp discharge (PSD) on electroencephalograms (EEG), a positive 14-3-3 protein assay of CSF samples, and abnormal signals on cerebral cortex on... more
Sporadic human prion diseases are defined on the basis of clinical features, with periodic sharp discharge (PSD) on electroencephalograms (EEG), a positive 14-3-3 protein assay of CSF samples, and abnormal signals on cerebral cortex on diffusion-weighted (DWI) MR images. It is essential to detect the abnormal prion protein in neuropathological or immunochemical detection of brain tissues when we diagnose definite cases for human prion disease. We performed definite diagnosis of sporadic human prion disease in alive patients. Recently, testing of CSF with a new in vitro abnormal prion protein amplification technology, designated real-time quaking-induced conversion (RT-QUIC), has shown considerable promise as a highly specific diagnostic test for human prion disease.
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Cell transplantation therapy offers great potential to improve impairments after stroke. However, the importance of donor age on therapeutic efficacy is unclear. We investigated the regenerative capacity of transplanted cells focusing on... more
Cell transplantation therapy offers great potential to improve impairments after stroke. However, the importance of donor age on therapeutic efficacy is unclear. We investigated the regenerative capacity of transplanted cells focusing on donor age (young vs. old) for ischaemic stroke. The quantities of human mesenchymal stem cell (hMSC) secreted brain-derived neurotrophic factor in vitro and of monocyte chemotactic protein-1 at day 7 in vivo were both significantly higher for young hMSC compared with old hMSC. Male Sprague-Dawley rats subjected to transient middle cerebral artery occlusion that received young hMSC (trans-arterially at 24 h after stroke) showed better behavioural recovery with prevention of brain atrophy compared with rats that received old hMSC. Histological analysis of the peri-infarct cortex showed that rats treated with young hMSC had significantly fewer microglia and more vessels covered with pericytes. Interestingly, migration of neural stem/progenitor cells ex...
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Influenza virus infections are serious public health concerns throughout the world. The development of compounds with novel mechanisms of action is urgently required due to the emergence of viruses with resistance to the... more
Influenza virus infections are serious public health concerns throughout the world. The development of compounds with novel mechanisms of action is urgently required due to the emergence of viruses with resistance to the currently-approved anti-influenza viral drugs. We performed in silico screening using a structure-based drug discovery algorithm called Nagasaki University Docking Engine (NUDE), which is optimised for a GPU-based supercomputer (DEstination for Gpu Intensive MAchine; DEGIMA), by targeting influenza viral PA protein. The compounds selected by NUDE were tested for anti-influenza virus activity using a cell-based assay. The most potent compound, designated as PA-49, is a medium-sized quinolinone derivative bearing a tetrazole moiety, and it inhibited the replication of influenza virus A/WSN/33 at a half maximal inhibitory concentration of 0.47 μM. PA-49 has the ability to bind PA and its anti-influenza activity was promising against various influenza strains, including...
Prion diseases are caused by deposition of abnormal prion protein aggregates (PrP(Sc)) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrP(Sc) deposits. We synthesized several... more
Prion diseases are caused by deposition of abnormal prion protein aggregates (PrP(Sc)) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrP(Sc) deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [(125)I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (Kd) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and i...
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Prions are unique infectious agents, consisting solely of abnormally-folded prion protein (PrPSc). However, they possess virus-like features, including strain diversity, the ability to adapt to new hosts and to be altered evolutionarily.... more
Prions are unique infectious agents, consisting solely of abnormally-folded prion protein (PrPSc). However, they possess virus-like features, including strain diversity, the ability to adapt to new hosts and to be altered evolutionarily. Because prions lack genetic material (DNA and RNA), these biological phenomena have been attributed to the structural properties of PrPSc. Therefore, many structural models of the structure of PrPSc have been proposed based on the limited structural information available, regardless of the incompatibility with high-resolution structural analysis. Recently hypothesized models consist solely of β-sheets and intervening loops/kinks; i.e. parallel in-register β-sheet and β-solenoid models. Owing to the relative simplicity of these structural models of PrPSc, we hypothesized that numerical conversion of the primary structures with a relevant algorithm would enable quantitative comparison between PrPs of distinct primary structures. We therefore used the ...
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The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we... more
The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we investigated whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, can convert Escherichia coli-derived recombinant αSyn (r-αSyn) to fibrils. Diffuse neocortical DLB yielded 50% seeding dose (SD50) values of 10(7)~10(10)/g brain. Limbic DLB was estimated to have an SD50 value of ~10(5)/g brain. Furthermore, RT-QUIC assay discriminated DLB from other neurological and neurodegenerative disorders. Unexpectedly, the prion-like seeding was reconstructed in reactions seeded with oligomer-like species, but not with insoluble aggregates of r-αSyn, regardless of Ser129 phosphorylation status. Our findings suggest that RT-QUIC using r-αSyn can be applied to detect see...
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Accidental transmission of prions during neurosurgery has been reported as a consequence of re-using contaminated surgical instruments. Several decontamination methods have been studied using the 263K-hamster prion; however, no studies... more
Accidental transmission of prions during neurosurgery has been reported as a consequence of re-using contaminated surgical instruments. Several decontamination methods have been studied using the 263K-hamster prion; however, no studies have directly evaluated human prions. A newly developed in vitro amplification system, designated real-time quaking-induced conversion (RT-QuIC), has allowed the activity of abnormal prion proteins to be assessed within a few days. RT-QuIC using human recombinant prion protein (PrP) showed high sensitivity for prions as the detection limit of our assay was estimated as 0.12 fg of active prions. We applied this method to detect human prion activity on stainless steel wire. When we put wires contaminated with human Creutzfeldt-Jakob disease brain tissue directly into the test tube, typical PrP-amyloid formation was observed within 48 hours, and we could detect the activity of prions at 50% seeding dose on the wire from 10(2.8) to 10(5.8) SD50. Using thi...
Removal of pathogenic organisms from reprocessed surgical instruments is essential to prevent iatrogenic infections. Some bacteria can make persistent biofilms on medical devices. Contamination of non-disposable equipment with prions also... more
Removal of pathogenic organisms from reprocessed surgical instruments is essential to prevent iatrogenic infections. Some bacteria can make persistent biofilms on medical devices. Contamination of non-disposable equipment with prions also represents a serious risk to surgical patients. Efficient disinfection of prions from endoscopes and other instruments such as high-resolution cameras remains problematic because these instruments do not tolerate aggressive chemical or heat treatments. Herein, we develop a new washing system that uses both the alkaline and acidic water produced by electrolysis. Electrolyzed acidic water, containing HCl and HOCl as active substances, has been reported to be an effective disinfectant. A 0.15% NaCl solution was electrolyzed and used immediately to wash bio-contaminated stainless steel model systems with alkaline water (pH 11.9) with sonication, and then with acidic water (pH 2.7) without sonication. Two bacterial species (Staphylococcus aureus and Pse...
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The accumulation of abnormal prion protein (PrP(Sc)) converted from the normal cellular isoform of PrP (PrP(C)) is assumed to induce pathogenesis in prion diseases. Therefore, drug discovery studies for these diseases have focused on the... more
The accumulation of abnormal prion protein (PrP(Sc)) converted from the normal cellular isoform of PrP (PrP(C)) is assumed to induce pathogenesis in prion diseases. Therefore, drug discovery studies for these diseases have focused on the protein conversion process. We used a structure-based drug discovery algorithm (termed Nagasaki University Docking Engine: NUDE) that ran on an intensive supercomputer with a graphic-processing unit to identify several compounds with anti-prion effects. Among the candidates showing a high-binding score, the compounds exhibited direct interaction with recombinant PrP in vitro, and drastically reduced PrP(Sc) and protein-aggresomes in the prion-infected cells. The fragment molecular orbital calculation showed that the van der Waals interaction played a key role in PrP(C) binding as the intermolecular interaction mode. Furthermore, PrP(Sc) accumulation and microgliosis were significantly reduced in the brains of treated mice, suggesting that the drug c...
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Research Interests: Kinetics, Brain, Mice, Cellular and Molecular Neurobiology, Animals, and 10 morePrions, Prion disease, Analytical Ultracentrifugation, Neurosciences, Conformational Change, Biochemistry and cell biology, Immunoblotting, Prion Protein, Creutzfeldt-Jakob disease, and Pharmacology and pharmaceutical sciences
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A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP(Sc)).... more
A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP(Sc)). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP(Sc) results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP(Sc). However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the majo...