Bacteroides uniformis and its preferred substrate, α-cyclodextrin, enhance endurance exercise performance in mice and human males.

Although gut microbiota has been linked to exercise, whether alterations in the abundance of specific bacteria improve exercise performance remains ambiguous. In a cross-sectional study involving 25 male long-distance runners, we found a correlation between Bacteroides uniformis abundance in feces and the 3000-m race time. In addition, we administered flaxseed lignan or α-cyclodextrin as a test tablet to healthy, active males who regularly exercised in a randomized, double-blind, placebo-controlled study to increase B. uniformis in the gut (UMIN000033748). The results indicated that α-cyclodextrin supplementation improved human endurance exercise performance. Moreover, B. uniformis administration in mice increased swimming time to exhaustion, cecal short-chain fatty acid concentrations, and the gene expression of enzymes associated with gluconeogenesis in the liver while decreasing hepatic glycogen content. These findings indicate that B. uniformis enhances endurance exercise performance, which may be mediated by facilitating hepatic endogenous glucose production.

KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-ß2 suppression.

INTRODUCTION: Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-ß signalling pathway, which is involved in corneal epithelial wound healing. METHODS: HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-ß secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-ß2 treatment. RESULTS: KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-ß canonical signalling pathway, TGF-ß2 secretion by HCECs increased up to 5 folds, and several TGF-ß-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-ß signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-ß2 treatment. CONCLUSIONS: These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-ß2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-ß signalling.

Homeostasis of SLC4A11 protein is mediated by endoplasmic reticulum-associated degradation.

The cornea is an important tissue that refracts light, and the corneal endothelium prevents edema of the corneal stroma by acting as a barrier and a pump for the transport of essential molecules/ions. Sodium bicarbonate transporter-like protein 11 (SLC4A11) is a transporter present in the corneal endothelium, and its mutation causes corneal endothelial disease. Here, we aimed to investigate the degradation pathway of SLC4A11. Quantitative PCR analysis revealed that two variants of SLC4A11 transcripts, variant 2 (SLC4A11-B) and variant 3 (SLC4A11-C), were expressed in human corneal endothelial tissues. Transient overexpression of these variants in HEK293T cells revealed that SLC4A11-B abundantly localized to the cell membrane. Furthermore, SLC4A11-B-transfected HEK293T cells expressed the mature glycosylated forms and immature non-glycosylated forms of SLC4A11. Cycloheximide chase experiments revealed that mature SLC4A11 showed high degradation stability; however, degradation of immature SLC4A11-B was significantly faster than that of immature SLC4A11-C. Therefore, we performed further degradation analysis of the SLC4A11 mutants, which are classified into ER-retained and cell surface-associated mutants similar to the wild type. Compared to the wild type, ER-retained mutants S213P and W240P showed delayed degradation but the cell surface-associated mutants showed minimal degradation. Further analysis using proteasome inhibitors revealed that degradation of immature SLC4A11 was delayed after treatment with the proteasome inhibitors, MG-132 and bortezomib, and was mediated by poly-ubiquitination. Moreover, the degradation of immature SLC4A11 protein was suppressed by Eeyarestatin I, an ER-associated protein degradation (ERAD) inhibitor. Collectively, these data suggest that SLC4A11 protein is degraded via ERAD.

Identification and application of p75 neurotrophin receptor-expressing human trabecular meshwork progenitor cells.

The trabecular meshwork (TM) is a tissue that originates from the neural crest via the periocular mesenchyme and plays a role in draining water and maintaining intraocular pressure (IOP). Damage to the TM is associated with pathologically elevated IOP, and cell-based therapy is expected to restore the functions of the TM in the future. Here, we aimed to isolate and characterize TM progenitor cells (TMPs) from human TM tissues. We focused on the p75 neurotrophin receptor (p75), a stem cell marker of the neural crest. Approximately 32% of p75-expressing cells were present in the TM. P75-expressing TMPs could proliferate in serum-free culture. The colony formation efficiency of TMPs was 1.11 ±â€¯0.18%. TMPs showed a markedly lower proliferation ability for passaging. TMPs expressed neural crest markers (p75, Sry-box [SOX] 9, SOX10, transcription factor AP [TFAP] 2B); nestin; periocular mesenchymal markers (Forkhead box [FOX] C1, FOXC2, and paired-like homeodomain transcription factor 2); and CD166, but not TM differentiation markers. The TMPs differentiated into mature TM cells (dTMCs) and keratocytes. dTMCs from TMPs expressed high levels of TM markers (aquaporin 1, matrix gla protein, prostaglandin D2 synthase, and AnkG). Furthermore, the TMPs showed enhanced expression of myocilin, a glaucoma susceptibility gene, following induction of differentiation by dexamethasone. TMPs also differentiated into adipocytes, osteocytes, and chondrocytes. These data suggest that p75-expressing TMPs could be a useful cell source in cell-based therapy and pathological models of glaucoma.

STAT3 signaling maintains homeostasis through a barrier function and cell survival in corneal endothelial cells.

The cornea protects the eye from inflammation, which is one of the leading causes of blindness. Severe inflammation in the anterior chamber disrupts the barrier function of corneal endothelial cells (CECs) leading to severe visual loss. However, the mechanism by which such inflammation affects CEC function and survival is unknown. Activation of STAT3 signaling regulates various intracellular responses through inflammation and generally mediates expression of the barrier function marker zonula occludens-1 (ZO-1). In this study, we investigated the relationship between the corneal endothelial barrier function and activation of STAT3 signaling through a variety of cytokines in human CECs. Phosphorylated STAT3 (pSTAT3) was expressed in human and mouse CECs. Inhibition of pSTAT3 remarkably decreased the expression of the ZO-1 protein, reduced the trans-endothelial electric resistance, and induced cell apoptosis. The expression level of ZO-1 mRNA was correlated with that of STAT3 mRNA in the human corneal endothelium. pSTAT3 was increased with the addition of LIF, IL-6, and IFN-γ. LIF expressed in CECs suppressed pSTAT3 activation as observed experimentally using an anti-LIF antibody. Promoter regions of ZO-1 and SOCS3 were directly regulated by transcriptional activation of STAT3. These findings suggest that regulation of the STAT3 pathway is essential for corneal endothelial homeostasis via barrier function and may protect from various inflammatory factors.

Transcription factor TFAP2B up-regulates human corneal endothelial cell-specific genes during corneal development and maintenance.

The corneal endothelium, which originates from the neural crest via the periocular mesenchyme (PM), is crucial for maintaining corneal transparency. The development of corneal endothelial cells (CECs) from the neural crest is accompanied by the expression of several transcription factors, but the contribution of some of these transcriptional regulators to CEC development is incompletely understood. Here, we focused on activating enhancer-binding protein 2 (TFAP2, AP-2), a neural crest-expressed transcription factor. Using semiquantitative/quantitative RT-PCR and reporter gene and biochemical assays, we found that, within the AP-2 family, the TFAP2B gene is the only one expressed in human CECs in vivo and that its expression is strongly localized to the peripheral region of the corneal endothelium. Furthermore, the TFAP2B protein was expressed both in vivo and in cultured CECs. During mouse development, TFAP2B expression began in the PM at embryonic day 11.5 and then in CECs during adulthood. siRNA-mediated knockdown of TFAP2B in CECs decreased the expression of the corneal endothelium-specific proteins type VIII collagen α2 (COL8A2) and zona pellucida glycoprotein 4 (ZP4) and suppressed cell proliferation. Of note, we also found that TFAP2B binds to the promoter of the COL8A2 and ZP4 genes. Furthermore, CECs that highly expressed ZP4 also highly expressed both TFAP2B and COL8A2 and showed high cell proliferation. These findings suggest that TFAP2B transcriptionally regulates CEC-specific genes and therefore may be an important transcriptional regulator of corneal endothelial development and homeostasis.

Restricted Presence of POU6F2 in Human Corneal Endothelial Cells Uncovered by Extension of the Promoter-level Expression Atlas.

Corneal endothelial cells (CECs) are essential for maintaining the clarity of the cornea. Because CECs have limited proliferative ability, interest is growing in their potentially therapeutic regeneration from pluripotent stem cells. However, the molecular mechanisms of human CEC differentiation remain largely unknown. To determine the key regulators of CEC characteristics, here we generated a comprehensive promoter-level expression profile of human CECs, using cap analysis of gene expression (CAGE) with a single molecule sequencer. Integration with the FANTOM5 promoter-level expression atlas, which includes transcriptome profiles of various human tissues and cells, enabled us to identify 45 promoters at 28 gene loci that are specifically expressed in CECs. We further discovered that the expression of transcription factor POU class 6 homeobox 2 (POU6F2) is restricted to CECs, and upregulated during human CEC differentiation, suggesting that POU6F2 is pivotal to terminal differentiation of CECs. These CEC-specific promoters would be useful for the assessment of fully differentiated CECs derived from pluripotent stem cells. These findings promote the development of corneal regenerative medicine.

Spontaneous acquisition of infinite proliferative capacity by a rabbit corneal endothelial cell line with maintenance of phenotypic and physiological characteristics.

Rabbit cells and models are frequently used in pharmacological experiments and toxicity tests and are particularly useful for corneal transplant experiments. Here, we obtained a corneal endothelial cell line from normal rabbit corneal endothelium that had acquired infinite proliferative potential without requiring gene transfer. These infinitely proliferative rabbit corneal endothelial cells (iRCECs) could be cultured for > 250 generations and exhibited a greater ability to proliferate than did normal rabbit corneal endothelial cells (RCECs) cultivated by conventional methods. The results of reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining analyses revealed that the expression profiles of corneal endothelial markers, such as collagen type VIIIα1 and Na+ /K+ -ATPase, were similar to those of RCECs and in vivo corneal endothelium. Scanning electron microscopy showed that many microvilli were present on the surface of the cells and that the ultrastructure was maintained. In addition, we verified that the iRCECs had similar levels of pump function as did RCECs using an Ussing chamber system. The results of a soft agar colony-formation assay suggested that the iRCECs were not tumourigenic. Taken together, our results demonstrated that the iRCECs exhibited gene expression profiles and properties that were equivalent to those of native rabbit corneal endothelium, making these cells useful for corneal endothelial research studies. Copyright © 2015 John Wiley & Sons, Ltd.

Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

Identification and potential application of human corneal endothelial progenitor cells.

The corneal endothelium is believed to be developmentally originated from the periocular mesenchyme via the neural crest. Human corneal endothelial progenitor cells (HCEPs) have been investigated because of their potential availability for the tissue regenerative medicine. However, the existence and the properties of HCEPs have not been elucidated yet. We first established a novel serum-free culture system for HCEPs. The HCEPs highly expressed p75 neurotrophin receptor, SOX9, and FOXC2, and partially retained the properties of neural crest and periocular mesenchyme. Further, we demonstrated that HCEPs had a high proliferative potency, and the differentiated HCEP sheets had corneal endothelial function by using the Ussing chamber system and transplantation to the rabbit cornea. These findings suggest that the HCEPs can be selectively expanded from the corneal endothelium using a specific culture system and will provide cell sheets for corneal regenerative medicine.

Development of a cell sheet transportation technique for regenerative medicine.

PURPOSE: A transportation technique for cell sheets is necessary to standardize regenerative medicine. The aim of this article is to develop and evaluate a new transportation technique for cell sheets. MATERIAL AND METHODS: We developed a transportation container with three basic functions: the maintenance of interior temperature, air pressure, and sterility. The interior temperature and air pressure were monitored by a recorder. Human oral mucosal epithelial cells obtained from two healthy volunteers were cultured on temperature-responsive culture dishes. The epithelial cell sheets were transported via an airplane between the Osaka University and Tohoku University using the developed cell transportation container. Histological and immunohistochemical analyses and flow cytometric analyses for cell viability and cell purity were performed for the cell sheets before and 12 h after transportation to assess the influence of transportation on the cell sheets. Sterility tests and screening for endotoxin and mycoplasma in the cell sheets were performed before and after transportation. RESULTS: During transportation via an airplane, the temperature inside the container was maintained above 32°C, and the changes in air pressure remained within 10 hPa. The cell sheets were well stratified and successfully harvested before and after transportation. The expression patterns of keratin 3/76, p63, and MUC16 were equivalent before and after transportation. However, the expression of ZO-1 in the cell sheet after transportation was slightly weaker than that before transportation. The cell viability was 72.0% before transportation and 77.3% after transportation. The epithelial purity was 94.6% before transportation and 87.9% after transportation. Sterility tests and screening for endotoxin and mycoplasma were negative for all cell sheets. CONCLUSION: The newly developed transportation technique for air travel is essential technology for regenerative medicine and promotes the standardization and spread of regenerative therapies.

Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein-coupled receptor kinase-dependent manner.

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)-treated cells shows that G protein-coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

Authentically phosphorylated α-synuclein at Ser129 accelerates neurodegeneration in a rat model of familial Parkinson's disease.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the appearance of fibrillar aggregates of insoluble α-synuclein (α-syn) called Lewy bodies (LBs). Approximately 90% of α-syn deposited in LBs is phosphorylated at serine 129 (Ser129). In contrast, only 4% of total α-syn is phosphorylated in normal brain, suggesting that accumulation of Ser129-phosphorylated α-syn is involved in the pathogenesis of PD. However, the role of Ser129 phosphorylation in α-syn neurotoxicity remains unclear. In this study, we coexpressed familial PD-linked A53T α-syn and G-protein-coupled receptor kinase 6 (GRK6) in the rat SN pars compacta using recombinant adeno-associated virus 2. Coexpression of these proteins yielded abundant Ser129-phosphorylated α-syn and significantly exacerbated degeneration of dopaminergic neurons when compared with coexpression of A53T α-syn and GFP. Immunohistochemical analysis revealed that Ser129-phosphorylated α-syn was preferentially distributed to swollen neurites. However, biochemical analysis showed that the increased expression of Ser129-phosphorylated α-syn did not promote accumulation of detergent-insoluble α-syn. Coexpression of catalytically inactive K215R mutant GRK6 failed to accelerate A53T α-syn-induced degeneration. Furthermore, introducing a phosphorylation-incompetent mutation, S129A, into A53T α-syn did not alter the pace of degeneration, even when GRK6 was coexpressed. Our study demonstrates that authentically Ser129-phosphorylated α-syn accelerates A53T α-syn neurotoxicity without the formation of detergent-insoluble α-syn, and suggests that the degenerative process could be constrained by inhibiting the kinase that phosphorylates α-syn at Ser129.

A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation.

We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.

Phosphorylated alpha-synuclein at Ser-129 is targeted to the proteasome pathway in a ubiquitin-independent manner.

α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.

Contribution of endogenous G-protein-coupled receptor kinases to Ser129 phosphorylation of alpha-synuclein in HEK293 cells.

The majority of alpha-synuclein (alphaS) deposited in Lewy bodies, the pathological hallmark of Parkinson's disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of alphaS has been demonstrated to enhance the alphaS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of alphaS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of alphaS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of alphaS; however, knockdown of GRK2 or GRK5 did not decrease alphaS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of alphaS in HEK293 cells.

N-terminal region of alpha-synuclein is essential for the fatty acid-induced oligomerization of the molecules.

Exposure of alpha-synuclein (alphaS), a major component of Lewy bodies in Parkinson's disease, to polyunsaturated fatty acids (PUFAs) triggers the formation of soluble alphaS oligomers. Here, we demonstrate that PUFA binds recombinant alphaS protein through its N-terminal region (residues 2-60). In HEK293 cells, alphaS mutants lacking the N-terminal region failed to form oligomers in the presence of PUFA. The PUFA-induced alphaS oligomerization was accelerated by C-terminal truncation or Ser129 phosphorylation of alphaS; however, this effect was abolished by deletion of the N-terminus. The results indicate that the N-terminus of alphaS is essential for the PUFA-induced alphaS oligomerization.

An advantageous method utilizing new homogenizing device BioMasher and a sensitive ELISA to detect bovine spongiform encephalopathy accurately in brain tissue.

A new screening method was developed to detect bovine spongiform encephalopathy (BSE). This method is advantageous because it has a simpler and safer protocol than commercial kits. A new device was developed for this method; it was named the BioMasher, to homogenize brain tissue by passing it through a porous rigid polypropylene filter. In this system, a purification step was eliminated in the sample preparation. Thus, the time needed for sample pretreatment is substantially shortened, and the risk of infection during sample processing is effectively reduced. Monoclonal antibodies to prion protein were created and used to construct a sensitive sandwich enzyme-linked immunosorbent assay system. The sensitivity of this assay kit using frozen BSE-positive brain is comparable or more sensitive than commercial kits. Moreover, the detection sensitivity for deteriorated samples, which were kept at 37 degrees C for 1 day, is 10- to 30-fold more sensitive than a commercial kit.