Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)  

Gold nanoparticles (AuNPs) have been widely applied to molecular sensors due to their optical properties. We previously reported a molecular detection by observing the scattered light of AuNPs at a single nanoparticle level using dark field microscopy (DFM). Recently, a molecular detection method using digital immunoassay has been reported, taking advantage of the characteristics of DFM. However, the digital immunoassays reported so far have been performed by a conventional sandwich immunoassay, which is difficult to apply to the detection of small molecules. In this study, with the aim of small molecule detection, we developed a digital immunoassay method using an anti-immunocomplex antibody that specifically recognizes immunocomplexes of small molecules with antibodies. The number of AuNPs modified with anti-immunocomplex antibody bound to immunocomplex of estradiol and anti-estradiol antibody was counted at a single nanoparticle level using DFM. We demonstrated for the first time that estradiol molecule can be detected by digital immunoassay using DFM and an anti-immunocomplex antibody with a detection sensitivity of 1 pg/mL. Graphical abstract: (Figure presented.).

DOI： 10.1007/s44211-024-00518-6

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Gold nanoparticles (AuNPs) have been utilized as colorimetric biosensors, where target molecule-induced AuNP aggregation can be recognized by a colour change from red to blue. Particularly, single-stranded DNA (ssDNA)-immobilized AuNPs (ssDNA-AuNPs) have been applied to genetic diagnosis due to their rapid and sequence-specific aggregation properties. However, the effect of the density of immobilized ssDNA have not been investigated yet. In this study, we developed a method to control the amount of immobilized ssDNA by use of ethylene glycol, which is expected to control the ice crystal spacing in a freezing-thawing ssDNA-AuNP synthesis method. We also investigated the effect of the DNA density on the sensitivity of the target ssDNA detection, and found that the detection sensitivity was improved at lower DNA densities. To discuss the reason for the improved detection sensitivity, we modified the ssDNA-AuNPs with alkane thiol for better dispersion stability against salt. The results suggest that the DNA density, rather than the dispersion stability, has a significant impact on detection sensitivity.

DOI： 10.1039/d3ra06528f

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2023.03.060

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Language：Japanese   Publisher：(公社)日本薬理学会  

インスリン療法の皮膚合併症は,インスリン療法を阻害する要因として以前から問題となってきた.従来の皮膚合併症の中で,リポアトロフィーやインスリンアレルギーはインスリン製剤の発展とともに著明に減少したが,リポハイパートロフィーはいまだにインスリン治療患者での頻度が高い.最近,インスリン由来アミロイドーシスあるいはインスリンボールと呼ばれる皮膚合併症の報告が増えてきた.インスリン由来アミロイドーシスは,注射されたインスリンがアミロイドタンパク質となり注射部位に沈着するものであり,腫瘤形成性と非形成性の病変があり,腫瘤形成性の病変をインスリンボールと呼んでいる.インスリン由来アミロイドーシスは血糖コントロール悪化やインスリン注射量増加を生じるが,その原因はインスリン吸収を低下させることである.リポハイパートロフィーもインスリン吸収を低下させるが,インスリン由来アミロイドーシスの方がインスリン吸収の低下が大きく,臨床的影響も大きい.このため両者を鑑別診断することが重要であるが,時に鑑別が困難で,画像検査を要することがある.また通常診療ではインスリン由来アミロイドーシスの診断が難しいことが多く,そのため病態や有病率が完全には明らかにされていない.最近,インスリン由来アミロイドーシスが毒性を持つ場合があることが報告され,ミノサイクリン使用との関連が示唆された.インスリン由来アミロイドーシスおよびリポハイパートロフィーの対処法はその部位を避けてインスリン注射をすることであるが,その際にインスリン注射量を減量することが必要である.インスリン由来アミロイドーシスもリポハイパートロフィーも予防が重要であり,そのためにはインスリン注射部位の確認や適切なインスリン注射手技の指導が継続的に必要であり,多職種連携が極めて重要である.(著者抄録)

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2023&ichushi_jid=J01200&link_issn=&doc_id=20230314120012&doc_link_id=%2Fcf4yakur%2F2023%2F015802%2F015%2F0173-0177%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fcf4yakur%2F2023%2F015802%2F015%2F0173-0177%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

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DOI： 10.1007/s44211-022-00229-w

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Insulin balls, localized insulin amyloids formed at the site of repeated insulin injections in patients with diabetes, cause poor glycemic control and cytotoxicity. Our previous study has shown that insulin forms two types of amyloids; toxic amyloid formed from the intact insulin ((i)-amyloid) and less-toxic amyloid formed in the presence of the reducing reagent TCEP ((r)-amyloid), suggesting insulin amyloid polymorphism. However, the differences in the formation mechanism and cytotoxicity expression are still unclear. Herein, we demonstrate that the liquid droplets, which are stabilized by electrostatic interactions, appear only in the process of toxic (i)-amyloid formation, but not in the less-toxic (r)-amyloid formation process. The effect of various additives such as arginine, 1,6-hexanediol, and salts on amyloid formation was also examined to investigate interactions that are important for amyloid formation. Our results indicate that the maturation processes of these two amyloids were significantly different, whereas the nucleation by hydrophobic interactions was similar. These results also suggest the difference in the formation mechanism of two different insulin amyloids is attributed to the difference in the intermolecular interactions and could be correlated with the cytotoxicity.

DOI： 10.1038/s41598-022-12212-6

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Publishing type：Research paper (scientific journal)   Publisher：SAGE Publications  

Introduction

Dialysis-related amyloidosis (DRA) caused by β2-microgloblin (B2M) fibrils is a serious complication for patients with kidney failure on long-term dialysis. Deposition of B2M amyloid fibrils is thought to be due not only to serum extracellular B2M but also to infiltrating inflammatory cells, which may have an important role in B2M amyloid deposition in osteoarticular tissues in patients with DRA. Here, we asked whether B2M amyloid fibrils activate the inflammasome and contribute to formation and deposition of amyloid fibrils in cells.

Methods

Amyloid formation was confirmed by a thioflavin T (ThT) spectroscopic assay and scanning electron microscopy (SEM). Activation of inflammasomes was assessed by detecting interleukin (IL)-1β in culture supernatants from human embryonic kidney (HEK) 293T cells ectopically expressing inflammasome components. IL-1β secretion was measured by enzyme-linked immunosorbent assay. Expression and co-localization were analyzed by immunohistochemistry and dual immunofluorescence microscopy.

Results

B2M amyloid fibrils interacted directly with NLRP3/Pyrin and to activate the NLRP3/Pyrin inflammasomes, resulting in IL-1β secretion. When HEK293T cells were transfected with inflammasome components NLRP3 or Pyrin, along with ASC, pro-caspase-1, pro-IL-1β, and B2M, ThT fluorescence intensity increased. This was accompanied by IL-1β secretion, which increased in line with the amount of transfected B2M. In this case, morphological glowing of amyloid fibrils was observed by SEM. In the absence of ASC, there was no increase in ThT fluorescence intensity or IL-1β secretion, or any morphological glowing of amyloid fibrils. NLRP3 or Pyrin and B2M were co-localized in a “speck” in HEK293T cells, and co-expressed in infiltrated monocytes/macrophages in the osteoarticular synovial tissues in a patient with DRA.

Conclusion

Taken together, these data suggest that inflammasome assembly is required for the subsequent triggering of intracellular formation of B2M amyloid fibrils, which may contribute to osteoarticular deposition of B2M amyloid fibrils and inflammation in patients with DRA.

DOI： 10.1177/03946320221104554

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Other Link： http://journals.sagepub.com/doi/full-xml/10.1177/03946320221104554

Biointerfaces are regions where biomolecules, cells, and organic materials are exposed to environmental media or come in contact with other biomaterials, cells, and inorganic/organic materials. In this review article, six research topics on biointerfaces are described to show examples of state-of-art research approaches. First, biointerface design of nanoparticles for molecular detection is described. Functionalized gold nanoparticles can be used for sensitive detection of various target molecules, including chemical compounds and biomolecules, such as DNA, proteins, cells, and viruses. Second, the interaction between bacterial cell surfaces and material surfaces, including the introduction of advances in analytical methods and theoretical calculations, are explained as well as their applications to bioprocesses. Third, bioconjugation technologies for localizing functional proteins at biointerfaces are introduced, in particular, by focusing the potential of enzymes as a catalytic tool for designing different types of bioconjugates that function at biointerfaces. Forth topics is focusing on lipid–protein interaction in cell membranes as natural biointerfaces. Examples of membrane lipid engineering are introduced, and it is mentioned how their compositional profiles affect membrane protein functions. Fifth topic is the physical method for molecular delivery across the biointerface being developed currently, such as highly efficient nanoinjection, electroporation, and nanoneedle devices, in which the key is how to perforate the cell membrane. Final topic is the chemical design of lipid- or polymer-based RNA delivery carriers and their behavior on the cell interface, which are currently attracting attention as RNA vaccine technologies targeting COVID-19. Finally, future directions of biointerface studies are presented.

DOI： 10.1016/j.jbiosc.2021.12.004

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Gold nanoparticles (AuNPs) have been used as colorimetric biosensors by utilizing the difference in color between the dispersed (red) and aggregated (blue) states. We previously developed a biosensor that converts sandwich-type thrombin recognition to RNA amplification and color difference of AuNPs. But the sensitivity was insufficient because of the linear signal amplification mechanism. In this study, we designed an exponential signal amplification biosensor based on transcription-reverse transcription concerted (TRC) reaction.

DOI： 10.1007/s44211-022-00050-5

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Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bmc.2021.116391

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Publishing type：Research paper (scientific journal)   Publisher：SAGE Publications  

<sec><title>Introduction</title> Nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), an intracellular pattern recognition receptor, recognizes various pathogen-associated molecular pattern and/or damage-associated molecular pattern molecules to constitute inflammasome that act as an interleukin (IL)-1β processing platform. Injected insulin is reported to induce focal amyloidosis and the formation of subcutaneous lumps called insulin balls, but the formation of subcutaneous lumps and the underlying cytotoxic mechanism has not been elucidated.

</sec><sec><title>Methods</title> Amyloid formation was evaluated by thioflavin T spectroscopic assay and scanning electron microscopy. Binding between insulin amyloid fibrils and NLRP3 was evaluated by immunoprecipitation followed by native polyacrylamide gel electrophoresis. Inflammasome activation was evaluated by immunofluorescence speck formation called “ASC speck” and Western blotting. IL-1β secretion in culture supernatants of peripheral blood mononuclear cells was evaluated by enzyme-linked immunosorbent assay. Cytotoxicity was measured by lactate dehydrogenase release assay.

</sec><sec><title>Results</title> Insulin amyloid fibrils interact directly with NLRP3, resulting in NLRP3 inflammasome activation and pyroptotic cell death.

</sec><sec><title>Conclusion</title> Insulin ball formation and cytotoxicity may be associated with NLRP3 inflammasome activation followed by pyroptotic cell death.

</sec>

DOI： 10.1177/20587384211038357

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Other Link： http://journals.sagepub.com/doi/full-xml/10.1177/20587384211038357

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Gold nanoparticles (AuNPs) have been employed as colorimetric biosensors due to the color difference between their dispersed (red) and aggregated (blue) states. Although signal amplification reactions triggered by structural changes of the ligands on AuNPs have been widely used to improve measurement sensitivity, the use of ligands is limited. In this study, we designed a AuNP-based signal-amplifying sandwich biosensor, which does not require a conformational change in the ligands. Thrombin was used as a model target, which is recognized by two different probes. In the presence of the target, an extension reaction occurs as a result of hybridization of the two probes. Then RNA synthesis is started by RNA polymerase activation due to RNA promoter duplex formation. The amplified RNA drives aggregation or dispersion of the AuNPs, and a difference of the color if the AuNP solution is observed. As this detection system does not require a con-formational change in the ligand, it can be generically applied to a wide range ligands.

DOI： 10.3390/s21134318

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Gold nanoparticles (AuNPs) are often used for biosensing. In particular, aptamer-modified AuNPs are often used for colorimetric molecular detection, where target molecule-induced AuNP aggregates can be recognized by a color change from red to blue. However, non-specific aggregation could be induced by various compounds, leading to false-positive results. In this work we employed high-density ssDNA modification on the AuNP surface to prevent non-specific aggregation. The covalently immobilized DNA brush was used as an anchor for an aptamer specific for the target molecule. Herein, as a proof-of-concept study, we demonstrated detection of estradiol (E2), one of the endocrine-disrupting estrogen molecules as a model target, in the presence of antibiotic kanamycin (KN) as a model of co-contaminating compounds that induce non-specific aggregation of AuNPs. We also developed a smartphone dark field microscope (DFM) to visualize AuNP aggregation. Our previous study demonstrated that the observation of light scattering by AuNP aggregates with DFM can be applied for versatile molecular detection. In this work, we could successfully detect E2 with the smartphone DFM, and the results were verified by the results from a conventional benchtop DFM. This study would contribute to the future field applicability of AuNP-based sensors.

DOI： 10.1039/d0ra05149g

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Insulin balls, localized insulin amyloids formed at subcutaneous insulin-injection sites in patients with diabetes, cause poor glycemic control owing to impairments in insulin absorption. Our previous study has shown that some insulin balls are cytotoxic, but others are not, implying amyloid polymorphism. Interestingly, the patient with toxic insulin balls had been treated with antibiotic minocycline, suggesting a possible relationship between toxicity of insulin balls and minocycline. However, the direct effect of minocycline on the structure and cytotoxicity of the insulin amyloid is still unclear. Herein, we demonstrated that that minocycline at physiological concentrations induced degradation of insulin amyloids formed from human insulin and insulin drug preparations used for diabetes patients. Interestingly, the process involved the initial appearance of the toxic species, which subsequently changed into less-toxic species. It is also shown that the structure of the toxic species was similar to that of sonicated fragments of human insulin amyloids. Our study shed new light on the clarification of the revelation of insulin balls and the development of the insulin analogs for diabetes therapy.

DOI： 10.1038/s41598-021-86001-y

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Gold nanoparticles (AuNPs) are commonly used in biosensing applications. In this study, AuNPs were synthesized by using reduced bovine serum albumin (rBSA) as the reducing agent. The rBSA conjugated with AuNPs via Au-Sulfur interactions to form rBSA-functionalized AuNPs (rBSA-AuNPs). The interaction of the rBSA moieties on the rBSAAuNP surface with an anti-BSA antibody (anti-BSA) led to AuNP aggregation, which enabled the successful detection of anti-BSA at a concentration as low as 20 nM through darkfield microscopy (DFM). This study demonstrates the potential applications of protein-functionalized AuNPs in the bioanalysis of substances through DFM.

DOI： 10.2116/analsci.20SCP12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Analytical Chemistry  

DOI： 10.2116/analsci.GE2103

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© The Royal Society of Chemistry. Amyloid specific fluorescent probes are becoming an important tool for studies of disease progression and conformational polymorphisms in diseases related to protein misfolding and aggregation such as localized and systemic amyloidosis. Herein, it is demonstrated that using the amyloid specific fluorescent probes pFTAA and benzostyryl capped benzothiadiazole BTD21, structural polymorphisms of insulin amyloids are imaged in localized insulin-derived amyloid aggregates formed at subcutaneous insulin-injection sites in patients with diabetes. It is also found that pFTAA and BTD21 could discriminate structural polymorphisms of insulin amyloids, so called fibrils and filaments, formed in vitro. In addition, it is shown that insulin drug preparations used for treating diabetes formed various types of amyloid aggregates that can be assessed and quantified using pFTAA and BTD21. Interestingly, incubated pFTAA-positive insulin preparation aggregates show cytotoxicity while BTD21-positive aggregates are less toxic. From these observations, a variety of amyloid polymorphic structures with different cytotoxicities formed both in vivo and in vitro by various insulin preparations are proposed. This journal is

DOI： 10.1039/d0ra07742a

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To date, almost all case reports of insulin-derived amyloidosis described the presence of a subcutaneous mass that was observable on physical examination. This report presents two cases of insulin-derived amyloidosis without palpable masses at insulin injection sites. In both cases, blood glucose concentrations improved, and the insulin dose could be reduced by an average of 45% after changing the insulin injection sites. The insulin absorption at the site was reduced to at most 40% of that at a normal site in one case. Magnetic resonance imaging and ultrasonography were useful to screen and differentiate insulin-derived amyloidosis without a palpable mass. This report showed that insulin-derived amyloidosis without a palpable mass can be present at the insulin injection site, and has similar clinical effects to insulin-derived amyloidosis with palpable masses.

DOI： 10.1111/jdi.13199

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/sscp.201900049

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Language：English   Publishing type：Research paper (international conference proceedings)  

DOI： 10.1080/13506129.2019.1582517

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BACKGROUND: Insulin-derived amyloidosis is a skin-related complication of insulin therapy that interferes with insulin therapy. Although toxicities of in vitro-formed insulin amyloid fibrils have been well studied, the toxicity of insulin-derived amyloidosis remains to be clarified. CASE PRESENTATION: A 58-year-old man with type 2 diabetes mellitus underwent a lower limb amputation due to diabetic gangrene. Several antibiotics including minocycline were administered for infection and sepsis. A hard mass at the insulin injection sites in the lower abdomen was discovered by chance four months later. Although no abnormal findings in the surface skin of the mass were observed, necrotic tissue was seen around the mass when a biopsy was performed. Histological and toxicity studies were performed for this patient and four other patients with abdominal masses at insulin injection sites. Histological and immunohistochemical studies showed that the masses had typical characteristics of amyloid deposits in all cases, whereas necrotic findings were seen adjacent to the amyloid deposit only in the case presented. Toxicity studies indicated that the amyloid tissue from the present case had significant cell toxicity compared to the control skin tissue or the amyloid tissues from the other four cases. CONCLUSIONS: This report showed that toxic insulin-derived amyloidosis can occur. In addition, this report suggested that toxic insulin-derived amyloidosis may cause necrosis in the surrounding tissue. Although the toxic amyloid deposit of insulin-derived amyloidosis was found in only one patient, no structural differences between toxic and non-toxic deposits were seen on histological and immunohistochemical studies.

DOI： 10.1186/s12902-019-0385-0

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DOI： 10.2116/analsci.18P571

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Here we report a novel aspect of molecular chaperone prefoldin (PFD) as a biomaterial in the biocatalytic synthesis of gold nanoparticles (AuNPs) using glycerol dehydrogenase (GLD). We found that PFD could inhibit the aggregation of AuNPs during the biosynthesis, leading to the formation of AuNPs with controlled size distribution.

DOI： 10.1039/c8bm01026a

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DOI： 10.1088/1742-6596/1220/1/012051

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DOI： 10.1051/epjconf/201819003009

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DOI： 10.1177/2058738418788749

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

A quenchbody (Q-body) is an antibody-based biosensor that employs fluorescence quenching of the dye(s) attached to the antibody fragment, which are de-quenched upon antigen binding. In this study, we aimed to develop Fab type Q-bodies (UQ-bodies) to aid the diagnosis of Alzheimer's disease (AD). Characteristic senile plaques in AD consist of amyloid-β peptide (Aβ) generated from the amyloid precursor protein. Aβ42, one of the major peptide forms, aggregates fast and manifests higher neurotoxicity. Recent studies showed that Aβ oligomers, such as Aβ-derived diffusible ligand (ADDL), are more toxic than fibrils. Thus, detection of Aβ and its oligomers in body fluid might help detect deterioration caused by the disease. To this end, the Fab fragment of the anti-Aβ antibody h12A11, which binds preferentially to ADDL, was expressed in Escherichia coli, and labeled with a fluorescent dye at the N terminus of either the heavy chain, or the heavy and light chains, via Cys-containing tag(s) to prepare UQ-bodies. As a result, the double-labeled UQ-bodies detected ADDL with higher sensitivity than that for the Aβ peptide. In addition, the UQ-body could be used to image aggregated Aβ with a low background, which suggested the potential of UQ-bodies as a fast bioimaging tool.

DOI： 10.1016/j.ab.2018.04.016

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Language：English   Publisher：Springer Verlag  

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits and has the appearance of a jellyfish: its body consists of a double beta-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. The prefoldin-group II chaperonin system is thought to be important for the folding of newly synthesized proteins and for their maintenance, or proteostasis, in the cytosol. Based on structural information of archaeal prefoldins, the mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the role and mechanism of eukaryotic PFDs remain unknown. Recent studies have shown that prefoldin plays an important role in proteostasis and is involved in various diseases. In this paper, we review a series of studies on the molecular mechanisms of archaeal prefoldins and introduce recent findings about eukaryotic prefoldin.

DOI： 10.1007/s12551-018-0400-0

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Background: Alzheimer's disease is a neurodegenerative disease characterized by the interstitial deposition of amyloid β (Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβ is known to make fibrils via oligomers from monomers. Aβ has been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear. Methods: We prepared monomers, oligomers, and fibrils of Aβ, which promoted the interaction between NLRP3 and each form of Aβ and analyzed the interaction between NLRP3 and ASC induced by each form of Aβ in a cell-free system with the amplified luminescent proximity homogeneous assay. We also confirmed the physiological relevance in a cell-based assay using human embryonic kidney 293T cells and human peripheral mononuclear cells. Results: Monomers, oligomers, and fibrils of Aβ were successfully prepared. Aβ oligomers and fibrils interacted with NLRP3. Aβ oligomers and fibrils induced the interaction between NLRP3 and ASC. However, Aβ monomers did not interact with NLRP3 or induce interaction between NLRP3 and ASC in the cell-free system, and IL-1β was not secreted according to the cell-based assay. Conclusion: Oligomerized Aβ originating from non-toxic Aβ monomers directly interacted with NLRP3, leading to the activation of the NLRP3 inflammasome. This may be an attractive target for the treatment of Alzheimer's disease.

DOI： 10.1186/s41232-018-0085-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Due to the potential of gold nanoparticle (AuNP)-based trace analysis, the discrimination of small AuNP clusters with different assembling stoichiometry is a subject of fundamental and technological importance. Here we prepare oligomerized AuNPs with controlled stoichiometry through DNA-directed assembly, and demonstrate that AuNP monomers, dimers and trimers can be clearly distinguished using dark field microscopy (DFM). The scattering intensity for of AuNP structures with stoichiometry ranging from 1 to 3 agrees well with our theoretical calculations. This study demonstrates the potential of utilizing the DFM approach in ultra-sensitive detection as well as the use of DNA-directed assembly for plasmonic nano-architectures. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cplett.2017.07.012

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In this study, we firstly report inhibition of amyloid beta(42) (A beta(42)) fibrillation, which is related to the progression of Alzheimer's disease, by digestion using proteases, combined with A beta(42) isolation using an immunoaffinity membrane. Especially, digestion of A beta(42) with carboxypeptidase Y (CPY) is effective for the inhibition of A beta(42) fibrillation, indicating that carboxy-terminal digestion of A beta(42) is efficient for the prevention of its fibrillation. The combinational method of the immunoaffinity membrane with CPY digestion is applicable to suppress A beta(42) fibrillation.

DOI： 10.1246/cl.160613

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

DOI： 10.1002/advs.201600082

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Biophysical understanding of amorphous protein aggregation can significantly impact diverse area of biotechnology. Here, we report the time dependent salt-induced formation of amorphous aggregation as monitored by fluorescence self-quenching and compare the results with conventional methods for detecting protein aggregation [static light scattering (LS) and dynamic light scattering (DLS)]. As a model protein, we used a bovine pancreatic trypsin inhibitor (BPTI) variant extended by two glycines (C2G) at its C terminus, and three variants where three types of Solubility Controlling Peptide tags (SCP tags) made of five serines (C5S), alanines (C5A) or aspartic acids (C5D) were added to the C terminus of C2G. All variants have a native-like BPTI structure and trypsin inhibitory activity, but different solubilities controlled by the SCP tags. The BPTIs were labeled using NHS-Fluorescein (FAM) conjugated to BPTI's lysines, and we measured the changes in fluorescence intensity occurring upon the addition of NaCl. The fluorescence of all FAM-BPTIs decreased almost immediately, albeit to a different extent, upon addition of salt and became constant after 10 min for 24 h or more. On the other hand, LS and DLS signal changes were dependent on the type of tags. Namely, C2G's LS and DLS signals changed immediately, the signals of C5S and C5A tagged FAM-BPTIs increased slowly from 10 min to 24 h, and those of C5D remained constant. These observations indicated the presence of at least one intermediate step, with increased protein-protein interaction yielding a 'molecular condensation' phase. According to this model, C2G would rapidly turn from 'condensates' to aggregates, whereas C5S and C5A tagged FAM-BPTIs would do so slowly, and the soluble C5D tagged variant would remain in the molecular condensation state.

DOI： 10.1002/1873-3468.12439

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Localization of colorectal tumors during laparoscopic surgery is generally performed by tattooing into the submucosal layer of the colon. However, faint and diffuse tattoos may lead to difficulties in recognizing cancer sites, resulting in inappropriate resection of the colon. We previously demonstrated that yttrium oxide nanoparticles doped with the rare earth ions (ytterbium and erbium) (YNP) showed strong near-infrared (NIR) emission under NIR excitation (1550 nm emission with 980 nm excitation). NIR light can penetrate deep tissues. In this study, we developed an NIR laparoscopy imaging system and demonstrated its use for accurate resection of the colon in swine.
The NIR laparoscopy system consisted of an NIR laparoscope, NIR excitation laser diode, and an NIR camera. Endo-clips coated with YNP (NIR clip), silicon rubber including YNP (NIR silicon mass), and YNP solution (NIR ink) were prepared as test NIR markers. We used a swine model to detect an assumed colon cancer site using NIR laparoscopy, followed by laparoscopic resection. The NIR markers were fixed at an assumed cancer site within the colon by endoscopy. An NIR laparoscope was then introduced into the abdominal cavity through a laparoscopy port.
NIR emission from the markers in the swine colon was successfully recognized using the NIR laparoscopy imaging system. The position of the markers in the colon could be identified. Accurate resection of the colon was performed successfully by laparoscopic surgery under NIR fluorescence guidance. The presence of the NIR markers within the extirpated colon was confirmed, indicating resection of the appropriate site.
NIR laparoscopic surgery is useful for colorectal cancer site recognition and accurate resection using laparoscopic surgery.

DOI： 10.1007/s00464-015-4669-9

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Prefoldin is a molecular chaperone that captures an unfolded protein substrate and transfers it to a group II chaperonin. Previous studies have shown that the interaction sites for prefoldin are located in the helical protrusions of group II chaperonins. However, it does not exclude the possibility of the existence of other interaction sites. In this study, we constructed C-terminal truncation mutants of a group II chaperonin and examined the effects of these mutations on the chaperone's function and interaction with prefoldin. Whereas the mutants with up to 6 aa truncation from the C-terminus retained more than 90% chaperone activities for protecting citrate synthase from thermal aggregation and refolding of green fluorescent protein and isopropylmalate dehydrogenase, the truncation mutants showed decreased affinities for prefoldin. Consequently, the truncation mutants showed reduced transfer efficiency of the denatured substrate protein from prefoldin and subsequent chaperonin-dependent refolding. The results clearly show that the C-terminal region of group II chaperonins contributes to their interactions with prefoldin, the transfer of the substrate protein from prefoldin and its refolding. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2016.04.006

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Dark field microscopy (DFM) was employed to detect amyloid beta (A beta) fibrils-induced gold nanoparticle (AuNP) aggregation at the single-particle level, with a detection limit of 40 pM fibrils. The sensitivity of this method is higher than that of the current fibril-specific detection method using probe dye, such as thioflavin T, for which sub -mu M level of fibrils are necessary. This study further proved the potential application of DFM in the analytical methods based on AuNP aggregation.

DOI： 10.2116/analsci.32.307

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Amyloid beta (Ab) protein aggregates, which include fibrils and oligomers, are neurotoxic and are considered to cause Alzheimer's disease. Thus, separation of these Ab aggregates from biological samples is important. Herein, we report the use of strongly ferromagnetic few-layer graphene-coated magnetic nanoparticles (C/Co), which were functionalized with a cationic polymer, poly[3-(methacryloyl amino) propyl] trimethylammonium chloride (polyMAPTAC), C/Co@polyMAPTAC, for the adsorption and magnetic separation of Ab aggregates. Fast adsorption (similar to 1 min) of Ab fibrils and oligomers onto the particles was observed. Interestingly, the Ab monomer was not captured by the particles, suggesting that binding to Ab molecules is toxic species-selective. Selective adsorption was also observed in the presence of serum albumin protein. We also showed that C/Co@polyMAPTAC could reduce the cytotoxicity of the Ab aggregate solutions. This study should be useful for further elucidation of the application of nanoparticle adsorption in mediating Ab toxicity.

DOI： 10.1039/c4tb02029d

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The use of near-infrared (NIR) light over 1000 nm (OTN-NIR or second NIR) is advantageous for bioimaging because it enables deep tissue penetration due to low scattering and autofluorescence. In this report, we describe the application of rare earth ion-doped ceramic nanoparticles to cancer-targeted NIR imaging using erbium and ytterbium ion-doped yttrium oxide nanoparticles (YNP) functionalized with streptavidin via bi-functional PEG (SA-YNP). YNP has NIR emission at 1550 nm, with NIR excitation at 980 nm (NIR-NIR imaging). Cancer-specific NIR-NIR imaging was demonstrated using SA-YNP and biotinylated antibodies on cancer cells and human colon cancer tissues. NIR-NIR imaging through porcine meat of 1 cm thickness was also demonstrated, supporting the possible application of deep tissue NIR-NIR bioimaging using YNP as a probe. Our results suggest that non-invasive imaging using YNP has great potential for general application in cancer imaging in living subjects.

DOI： 10.1039/c4bm00232f

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The NADH oxidase-peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from beta-NADH passed through the secondary disulfide, Cys128-Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license

DOI： 10.1016/j.fob.2015.01.005

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DOI： 10.1021/bi5005415

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In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l-cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l-arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm-like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non-covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470-478, 2014

DOI： 10.1002/btpr.1866

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Low-temperature atmospheric-pressure plasma was applied to degenerate amyloid-beta (A beta) fibrils, which are a major component of neuritic plaque associated with Alzheimer's disease (AD). We showed that an A beta fibril exposed to a low-frequency (LF) plasma jet in aqueous solution retained its morphology, molecular weight, and cytotoxicity, but, intriguingly, decreased in protease resistance and beta-sheet content. These results suggested that an LF plasma jet could be utilized for the treatment of AD to eliminate neuritic plaque by accelerating the proteolysis of A beta fibrils. (C) 2014 AIP Publishing LLC.

DOI： 10.1063/1.4861842

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Amyloid aggregates, which include oligomers and fibrils, are considered to cause various diseases, and their detection is important. In this minireview, recent developments on biomaterials, such as proteins, nanoparticles, and chemical reagents, for detecting amyloid aggregates are discussed. In particular, the molecular chaperone prefoldin (PFD) exhibits interesting properties for the interaction and detection of amyloid oligomers. In fact, any molecule that can bind amyloid aggregates could be useful for their detection.

DOI： 10.1039/c4bm00026a

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Inhibitors of amyloid fibril formation have been at the centre of intense research efforts for the prevention of amyloidosis. Here, we hypothesise that a specific non-covalent interaction, the thiophilic interaction between the side chain of an aromatic residue in a polypeptide and a sulphur atom of the compound, effectively inhibits amyloid fibril formation. Fluorescence spectroscopy and transmission electron microscopy revealed that sulphur compounds, particularly Cys, inhibit the fibrillisation of amyloid-beta 1-40 (A beta 40) and 1-42 (A beta 42). Interestingly, aggregates of A beta 40 and A beta 42 induced by Cys were less cytotoxic than those induced by catechin, which is the most typical inhibitor of amyloid fibril formation. Because the essential amino acid, Cys, is an abundant molecule in the blood and cytosol, our data provide a new basis for the prevention of amyloid-related diseases and the elucidation of the mechanism of these diseases.

DOI： 10.1039/c3cp54245a

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Springer Verlag  

Recently, a near-infrared (NIR) Fluorescence Biomedical Imaging (FBI) attracts increasing attention from many researchers. The light wavelength region in 1000 nm to 1700 nm has been known to be a “biological window” where the both tails of scattering and infrared absorption decrease to form a valley in optical loss spectra of biological objects. The inside of biological objects which cannot be usually seen in a normal endoscopy can be seen by using NIR, because of NIR light can penetrate in biological objects, and the further development of medical equipment is expected by using NIR. We are developing towards the realization of a NIR laparoscope which composes of a NIR camera and a visible light camera. The visible light camera can observe the surface in biological objects, and the NIR camera can observe the inside of biological objects. The NIR laparoscope has a potential of the helpful in the early detection of tumor. In our current system, the images captured by a NIR laparoscope and a normal laparoscope are shown on the separate screens simultaneously. The operator need to more experience to use the system. It was difficult to figure out the clinical usefulness of the NIR laparoscope with a visible light camera. We proposed the image composite method using a projection transform and a distance correction. The proposed method, compared to existing methods, is simple and fast. The proposed composite method of the two video images from a NIR camera and a visible light camera can facilitate the diagnosis and the surgery. The proposed system can reduce not only operator’s burden but also patient one’s. We are applying the proposed method to a laparoscope and conducting the evaluation experiment of the proposed method.

DOI： 10.1007/978-3-319-02913-9_33

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Amyloid beta (A beta) peptides produced by APP cleavage are central to the pathology of Alzheimer's disease. Despite widespread interest in this issue, the relationship between the auto-assembly and toxicity of these peptides remains controversial. One intriguing feature stems from their capacity to form anti-parallel beta-sheet oligomeric intermediates that can be converted into a parallel topology to allow the formation of protofibrillar and fibrillar A beta. Here, we present a novel approach to determining the molecular aspects of A beta assembly that is responsible for its in vivo toxicity. We selected A beta mutants with varying intracellular toxicities. In vitro, only toxic A beta (including wild-type A beta(42)) formed urea-resistant oligomers. These oligomers were able to assemble into fibrils that are rich in anti-parallel beta-sheet structures. Our results support the existence of a new pathway that depends on the folding capacity of A beta.

DOI： 10.1371/journal.pone.0080262

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Nanomaterials are increasingly suggested for the selective adsorption and extraction of complex compounds in biomedicine. Binding of the latter requires specific surface modifications of the nanostructures. However, even complicated macromolecules such as proteins can afford affinities toward basic surface characteristics such as hydrophobicity, topology, and electrostatic charge. In this study, we address these more basic physical interactions. In a model system, the interaction of bovine serum albumin and amyloid beta 42 fibrillar aggregates with carbon-coated cobalt nanoparticles, functionalized with various polymers differing in character, was studied. The possibility of rapid magnetic separation upon binding to the surface represents a valuable tool for studying surface interactions and selectivities. We find that the surface interaction of A beta 42 fibrillar aggregates is mostly hydrophobic in nature. Because bovine serum albumin (BSA) is conformationally adaptive, it is known to bind surfaces with widely differing properties (charge, topology, and hydrophobicity). However, the rate of tight binding (no desorption upon washing) can vary largely depending on the extent of necessary conformational changes for a specific surface. We found that BSA can only bind slowly to polyethylenimine-coated nanomagnets. Under competitive conditions (high excess BSA compared to that for beta 42 fibrilar aggregates), this effect is beneficial for targeting the fibrillar species. These findings highlight the possibility of selective extractions from complex media when advantageous basic physical surface properties are chosen.

DOI： 10.1021/la4026427

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than similar to 40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells.

DOI： 10.1074/jbc.M113.477984

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Amyloid-beta (A beta) peptides represent key players in the pathogenesis of Alzheimer's disease (AD), and mounting evidence indicates that soluble A beta oligomers mediate the toxicity. Prefoldin (PFD) is a molecular chaperone that prevents aggregation of misfolded proteins. Here we investigated the role of PFD in M aggregation. First, we demonstrated that PFD is expressed in mouse brain by Western blotting and immunohistochemistry and found that PFD is upregulated in AD model APP23 transgenic mice. Then we investigated the effect of recombinant human PFD (hPFD) on A beta(1-42) aggregation in vitro and found that hPFD inhibited Afi fibrillation and induced formation of soluble Afi oligomers. Interestingly, cell viability measurements using the 3-(4,5-dirnethyldnazol-2-y1)-2,5-diphenyltetrazoliurn bromide assay showed that A beta oligomers formed by hPFD were 30-40% less toxic to cultured rat pheochromocytoma (PC 12) cells or primary cortical neurons from embryonic CS7BL/6CrSlc mice than previously reported A beta oligomers (formed by archaeal PFD) and A beta fibrils (p &lt; 0.001). Thioflavin T measurements and immunoblotting indicated different structural properties for the different A beta oligomers. Our findings show a relation between cytotoxicity of A beta ofigomers and structure and suggest a possible protective role of PFD in AD.

DOI： 10.1021/bi301705c

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Photon counting histogram (PCH) is a fluorescence fluctuation method that can quantify the brightness and concentration of different fluorescent molecules in solution at a single molecule level using confocal optics. The current method assumes that the point spread function (PSF) of confocal optics is Gaussian function. However, this could be problematic when the actual PSF profile is not Gaussian. Here, we propose an improved PCH analysis method using numerical PSF data. This method does not require any analytical equation for the PSF profile, and can handle any arbitrary shape of PSF. We report a proof-of-concept estimation of this method using a model PSF. (c) 2013 The Japan Society of Applied Physics

DOI： 10.7567/JJAP.52.038001

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Small heat shock protein (sHsp) is a molecular chaperone with a conserved alpha-crystallin domain that can prevent protein aggregation. It has been shown that sHsps exist as oligomers (12-40 mer) and their dissociation into small dimers or oligomers is functionally important. Since several sHsps are upregulated and co-localized with amyloid-beta (A beta) in senile plaques of patients with Alzheimer's disease (AD), sHsps are thought to be involved in AD. Previous studies have also shown that sHsp can prevent A beta aggregation in vitro. However, it remains unclear how the quaternary structure of sHsp influences A beta aggregation. In this study, we report for the first time the effect of the quaternary structure of sHsp on A beta aggregation using sHsp from the fission yeast Schizosaccharomyces pombe (SpHsp16.0) showing a clear temperature-dependent structural transition between an oligomer (30 degrees C) and dimer (50 degrees C) state. A beta aggregation was inhibited by the oligomeric form of SpHsp16.0. In contrast, amyloid fibrils were formed in the presence of dimeric SpHsp16.0. Interestingly, these amyloid fibrils consisted of both A beta and SpHsp16.0 and showed a low ThT intensity and low cytotoxicity due to their low binding affinity to the cell surface. These results suggest the formation of novel fibrillar A beta amyloid with different characteristics from that of the authentic A beta amyloid fibrils formed in the absence of sHsp. Our results also suggest the potential protective role of sHsp in AD under stress conditions. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.12.059

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Dark field imaging was employed to visualize and quantify complementary DNA-induced gold nanoparticle aggregation at the single-particle level, with a detection limit of 100 fM DNA, which allows the highly sensitive detection of single nucleotide polymorphisms (SNPs). Results suggested that the aggregation process is consistent with the cluster-cluster model.

DOI： 10.1039/c3cc44182b

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Mass production of trimer rings consisting of 20 nm gold nanoparticles by using DNA template is demonstrated. Three kinds of DNA-monoconjugated gold nanoparticles are programmed to self-assemble into a trimer ring structure with nanoscale gaps through simple hybridization process. Self-assembled gold trimer rings are immobilized on a centimeter-scale quartz substrate to investigate their optical properties by the far-field transmission spectroscopy. Quantitative characterization of the gold trimer rings by atomic force microscope measurements reveals that 43% of gold nanoparticles keep forming the ring configuration on the substrate after the immobilizing process. In the far-field transmission spectroscopy, the trimer ring sample clearly exhibits two absorption dips in the visible spectrum, while monomer one has a single dip. The experimental results are in good agreement with the corresponding numerical simulations, proving that the unique spectral feature for the trimer ring arises from the hybridization of plasmon resonances of gold nanoparticles. The plasmonic responses of gold nanoparticle assemblies can be entirely controlled by designing DNA templates and thus may open up a novel approach for the realization of large-scale optical metamaterials.

DOI： 10.1021/jp302363v

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Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states. © 2012 by the authors
licensee MDPI, Basel, Switzerland.

DOI： 10.3390/ijms13021461

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Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.

DOI： 10.3390/ijms13021461

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Amyloid beta (A beta) oligomer is a known causative agent of Alzheimer's disease (AD). For early recognition of AD, development of an A beta oligomer detection system is vital. We developed a novel detection system using the molecular chaperone, Prefoldin (PFD), which can specifically interact with amyloid oligomers. A beta oligomer detection was achieved with a PFD-coated rnicroplate, following the enzyme-linked immunosorbent assay (ELISA) technique. The results suggest that this system specifically detects A beta oligomers at their physiological concentrations. The problematic nonspecific adsorption of A beta to the microplate can be effectively blocked with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer. This novel A beta oligomer detection method may be applied for AD early recognition. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2011.12.005

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DOI： 10.1002/cbic.201100467

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DOI： 10.2116/analsci.28.73

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Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (Vpr(N17C74)) contained a bona fide nuclear localization signal and it is targeted Vpr(N17C74) to the nuclear envelope and then imported into the nucleus by importin alpha (Imp alpha) alone. The interaction between Imp alpha and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to Vpr(N17C74). This study investigated the nuclear import of full-length Vpr using the three typical Imp alpha isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Imp alpha isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM) region (which is also essential for the binding of CAS, the export receptor for Imp alpha) in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Imp alpha isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1-mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Imp alpha complex.

DOI： 10.1371/journal.pone.0027815

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In this study, we investigated the possibility of employing an insulin amyloid without the cell binding motif sequence as a novel cell adhesion material. Immobilized insulin amyloid on cell culture plates shows higher cell adhesion properties compared with untreated plates. Interestingly, cells adhered to the insulin amyloid displayed marked cell proliferation. It was also demonstrated that the insulin amyloid interacted with fibronectin present within the serum.

DOI： 10.1246/cl.2011.315

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Synthetic biomaterials, such as polymers and self-assembling polypeptides have been developed for cell culture. The self-assembly of misfolded proteins, which is so-called &apos;amyloid&apos;, has also been demonstrated to work as a cell adhesive biomaterial. In this study, we demonstrated that two morphologically different insulin amyloids, fibrils and filaments, can be used as biomaterials for cell culture. We previously showed that the cytotoxicity of insulin filaments is markedly lower than that of fibrils. Both types of insulin amyloid-coated dishes showed higher cell adhesion and cell proliferation ability compared to non-coated dishes. Interestingly, insulin filaments showed higher cell adhesion and cell proliferation ability compared to insulin fibrils. These results strongly suggest that insulin amyloids, and insulin filaments in particular, can be used as a biomaterial for cell culture surfaces. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.reactfunctpolym.2010.10.012

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DOI： 10.2142/biophys.51.S79_1

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Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group ll chaperonin remains unclear. As prefoldin binds the chaperonin beta subunit more strongly than the alpha subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group ll chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpn beta subunits. Interestingly, chaperonin complexes containing two beta subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of beta subunits. The result suggests that all four beta tentacles of prefoldin interact with the helical protrusions of CPN in the PFD CPN complex as the previously proposed model that two adjacent PFD 0 subunits seem to interact with two CPN adjacent subunits. (C) 2010 Elsevier B.V. All rights reserved.

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DOI： 10.1016/j.bbapap.2010.04.013

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Prefoldin (PFD) is a hexameric chaperone that captures a protein substrate and transfers it to a group II chaperonin (CPN) to complete protein folding. We have studied the interaction between PFD and CPN using those from a hyperthermophilic archaeon, Thermococcus strain KS-1 (T. KS-1). In this study, we determined the crystal structure of the T. KS-1 PFD beta 2 subunit and characterized the interactions between T. KS-1 CPNs (CPN alpha and CPN beta) and T. KS-1 PFDs (PFD alpha 1-beta 1 and PFD alpha 2-beta 2). As predicted from its amino acid sequence, the PFD beta 2 subunit conforms to a structure similar to those of the PFD beta 1 subunit and the Pyrococcus horikoshii OT3 PFD beta subunit, with the exception of the tip of its coiled-coil domain, which is thought to be the CPN interaction site. The interactions between T. KS-1 CPNs and PFDs (CPN alpha and PFD alpha 1-beta 1; CPNa and PFD alpha 2-beta 2; CPN beta and PFD alpha 1-beta 1; and CPN beta and PFD alpha 2-beta 2) were analyzed using the Biacore T100 system at various temperatures ranging from 20 to 45 degrees C. The affinities between PFDs and CPNs increased with an increase in temperature. The thermodynamicparameters calculated from association constants showed that the interaction between PFD and CPN is entropy driven. Among the four combinations of PFD CPN interactions, the entropy difference in binding between CPN beta and PFD alpha 2-beta 2 was the largest, and affinity significantly increased at higher temperatures. Considering that expression of PFD alpha 2-beta 2 and CPN beta subunit is induced upon heat shock, our results suggest that PFD alpha 1-beta 1 is a general PFD for T. KS-1 CPNs, whereas PFD alpha 2-beta 2 is specific for CPN beta. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2010.04.046

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DOI： 10.1111/j.1742-4658.2010.07567.x

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Alzheimer&apos;s disease (AD) is an age-related, progressive degenerative disorder that is characterized by synapse and neuron loss in the brain and the accumulation of protein-containing deposits (referred to as &apos;senile plaques&apos;) and neurofibrillary tangles. Insoluble amyloid beta-peptide (A beta) fibrillar aggregates found in extracellular plaques have long been thought to cause the neurodegenerative cascades of AD. However, accumulating evidence suggests that prefibrillar soluble A beta oligomers induce AD-related synaptic dysfunction. The size of A beta oligomers is distributed over a wide molecular weight range (from &lt; 10 kDa to &gt; 100 kDa), with structural polymorphism in A beta oligomers of similar sizes. Recent studies have demonstrated that A beta can accumulate in living cells, as well as in extracellular spaces. This review summarizes current research on A beta oligomers, focusing on their structures and toxicity mechanism. We also discuss possible formation mechanisms of intracellular and extracellular A beta oligomers.

DOI： 10.1111/j.1742-4658.2010.07568.x

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Language：English   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2009.12.3569

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group 11 chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshu OT3 (PhPFD). exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity Out results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak. (C) 2009 Elsevier Inc All rights reserved.

DOI： 10.1016/j.bbrc.2009.11.081

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Photocyclodimerization of 2-anthracenecarboxylate mediated by molecular chaperone protein was performed for the first time to afford chiral syn-head-to-tail and anti-head-to-head dimers (2 and 3) in 10% and 16% enantiomeric excess, respectively, with enhanced yields of sterically and electrostatically less-favored head-to-head dimers (3 and 4).

DOI： 10.1039/b9pp00186g

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We report on a novel facile method towards a synthesis of cyclic assembled gold nanoparticles (AuNPs) by mixing each of the DNA-monoconjugated AuNPs, i.e., "DNA building blocks&apos;&apos; for cyclic assembly, and UV-induced interstrand crosslinking. By using our designed template DNA for cyclic assembly which possesses complementary sequences for cyclic assembly of the AuNPs, we demonstrated the formation of the cyclic assembly of AuNPs, i.e., triangular and square assemblies of AuNPs, which were confirmed by TEM analysis.

DOI： 10.1039/c0cc00305k

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S34_2

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Language：English   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.1377

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HINDAWI PUBLISHING CORPORATION  

The use of near infrared (NIR) light for biomedical photonics in the wavelength region between 800 and 2000 nm, which is called "biological window", has received particular attention since water and biological tissues have minimal optical loss due to scattering and absorption as well as autofluorescence in this region. Recent development of InGaAs CCD enables observations in this wavelength region. In the present paper, we report development of Yb and Er-doped yttrium oxide nanoparticles (Y(2)O(3):YbEr-NP) which show strong NIR emission under NIR excitation (NIR-NIR emission). We also demonstrate that NIR emission can be observed through swine colon wall. Based on these results, we propose a possible application of Y(2)O(3):YbEr-NP for cancer diagnosis and therapy using NIR-NIR imaging system. Our results also suggest potential applications of Y(2)O(3):YbEr-NP for noninvasive detection of various diseases.

DOI： 10.1155/2010/491471

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Language：English   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

DOI： 10.1016/j.jbiosc.2009.08.305

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Language：English   Publisher：AMER CHEMICAL SOC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Amyloid fibrils are associated with more than 20 diseases, including Alzheimer&apos;s disease and type 11 diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel beta-sheet structure, whereas the insulin fibrils have a parallel beta-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.

DOI： 10.1016/j.bpj.2008.12.3957

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

One of the great challenges of oncology is to improve methods for early tumor detection. Thus tumor cell-targeted optical imaging has been intensively studied. Bioimaging with upconversion (UC) phosphors (UCPs) is of considerable interest due to a variety of possible applications taking advantage of infrared-to-visible luminescence. Here we report for the first time tumor cell-targeted UC imaging using UCPs modified with cyclic RGD peptide (RGD-Y(2)O(3)). Cyclic RGD peptide binds specifically to integrin alpha(nu)beta(3) which is highly expressed in a tumor cell surface of certain cancer types but not in normal tissues. Since UC emission from RGD-Y(2)O(3) was observed for U87MG cancer cell (high integrin alpha(nu)beta(3) expression), but not for MCF-7 cancer cell (low integrin alpha(nu)beta(3) expression), this UC imaging is considered to be integrin alpha(nu)beta(3) specific. The non-invasive imaging of integrin alpha(nu)beta(3) expression using UCP-based probes will have great potential in cancer imaging in general in living subjects. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.02.004

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S72_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S160_6

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Language：English   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2009.09.1406

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The conformational changes of GroEL during the ATPase cycle in the presence of GroES were studied by measuring the fluorescence intensity time course of intrinsic tyrosine Y506, which is located near the nucleotide-binding site. A GroEL solution containing GroES was mixed with an ATP solution to initiate the reaction cycle. The tyrosine fluorescence intensity relative to that without the nucleotide reached 112% within the dead time of the apparatus (&gt;15 s(-1)) and further increased to 123% at 0.57 s(-1) followed by a decrease to 102% at 0.32 s(-1). An initial conformational change and a second intermediate state were expected to occur in ATP-bound GroEL because similar changes were observed for the ATPase-deficient D398A mutant. The conformational change to the third intermediate state corresponded to a process during or after ATP hydrolysis because D398A had no decreasing phase. The second intermediate state before ATP hydrolysis was characterized for the first time.

DOI： 10.1007/s10930-008-9157-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Alzheimer&apos;s disease (AD) is a neurological disorder characterized by the presence of amyloid beta (A beta) peptide fibrils and oligomers in the brain. It has been suggested that soluble A beta oligomers, rather than A beta fibrils, contribute to neurodegeneration and dementia due to their higher level of toxicity. Recent studies have shown that A beta is also generated intracellularly, where it can subsequently accumulate. The observed inhibition of cytosolic proteasome by A beta suggests that A beta is located within the cytosolic compartment. To date, although several proteins have been identified that are involved in the formation of soluble A beta oligomers, none of these have been shown to induce in vitro formation of the high-molecular-mass (&gt; 50 kDa) oligomers found in AD brains. Here, we examine the effects of the jellyfish-shaped molecular chaperone prefoldin (PFD) on A beta(1-42) peptide aggregation in vitro. PFD is thought to play a general role in de novo protein folding in archaea, and in the biogenesis of actin, tubulin and possibly other proteins in the cytosol of eukaryotes. We found that recombinant Pyrococcus PFD produced high-molecular-mass (50-250 kDa) soluble A beta oligomers, as opposed to A beta fibrils. We also demonstrated that the soluble A beta oligomers were more toxic than A beta fibrils, and were capable of inducing apoptosis. As Pyrococcus PFD shares high sequence identity to human PFD and the PFD-homolog protein found in human brains, these results suggest that PFD may be involved in the formation of toxic soluble A beta oligomers in the cytosolic compartment in vivo.

DOI： 10.1111/j.1742-4658.2008.06727.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Upconverting (UC) phosphors (UCPs) are ceramic materials doped with rare earth ions. These materials can absorb and upconvert infrared (IR) radiation to emit visible light by the stepwise excitation among discrete energy levels of the rare earth ions. UCPs are potentially useful reagents for use in bioimaging since the use of low energy photons avoids photo-toxicity. The use of UCP nanoparticles as bioimaging probes requires surface modifications in an effort to improve dispersion stability in aqueous milieu. In this study, we covalently attached poly(ethylene glycol) (PEG) to the surface of Er-doped Y(2)O(3) nanoparticles and firstly demonstrated that PEG covalently bound to the Y(2)O(3) surface markedly improved dispersion stability in water. UC emission of PEG-modified Er-Y(2)O(3) nanoparticles excited with IR light was successfully observed. We also showed that PEG-modified Er-Y(2)O(3) nanoparticles exhibit no cell-toxicity. These observations lend strong support to the potential use of PEG-modified UCP nanoparticles as bioimaging tools.

DOI： 10.1007/s10853-008-2776-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by similar to 2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of similar to 3- and similar to 5-s duration (Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an similar to 5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state.

DOI： 10.1074/jbc.M804090200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Group II chaperonin (CPN) cooperates with prefoldin (PFD), which forms a jellyfish-shaped heterohexameric complex with a molecular mass of 87 kDa. PFD captures an unfolded protein with the tentacles and transfers it to the cavity of CPN. Although X-ray crystal structures Of CPN and PFD have been reported, no structural information has been so far available for the terminal regions of the PFD tentacles nor for the C-terminal segments of CPNs, which were regarded to be functionally significant in the previous studies. Here we report C-13 NMR analyses on archaeal PFD, CPAT, and their complex, focusing on those structurally uncharacterized regions. The PFD and CPN complexes selectively labeled with 13C at methionyl carbonyl carbons were separately and jointly subjected to NMR measurements. 13 C NMR spectral data demonstrated that the N-terminal segment of the alpha and beta subunits of PFD as well as the C-terminal segments of the CPN hexadecamer retain significant degrees of freedom in internal motion even in the complex with a molecular mass of 1.1 MDa.

DOI： 10.1002/prot.21606

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Language：English   Publishing type：Research paper (international conference proceedings)  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S132_6

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S32_6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Feasibilities to stabilize CdSe/ZnS/trioctylphosphineoxide (TOPO) nanocrystals (quantum dots, QDs) in aqueous solutions with prefoldin macromolecules in their bioactive states are reported. Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group 11 chaperonins. As a protein folding intermediate is captured within its central cavity, so CdSe/ZnS/TOPO QDs would also be included within this cavity. It is also found the QDs can be much more dispersed in aqueous solutions and suspended for certain period of time by adding trace amount of t-butanol in the buffer prior to the mixing of the QDs mother solution. While biochemical procedures are evaluated with ordinary fluorescence measurements, possible complex formations are also evaluated with TIRFM single-molecule detection techniques. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jlumin.2007.02.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOP PUBLISHING LTD  

Protein nanopatterning techniques to fulfil the requirement for reducing nonspecific adsorption are demonstrated. A target protein is selectively immobilized on a hydroxy-terminated self-assembled monolayer template, the pattern of which is modified with streptavidin used as the intermediating molecule. 3-aminopropyltrimethoxysilane (APTES) used as the intermediating molecule induces nonspecific adsorption of proteins due to nonspecific adsorption of APTES itself. The data indicate that reducing nonspecific adsorption of both the target protein and the intermediating molecule is important for selectivity improvement in protein patterning. As a result of the refinement, a protein nanopattern of 250 nm in diameter has been fabricated.

DOI： 10.1088/0957-4484/18/30/305304

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S281_3

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S32_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group 11 chaperonin for correct folding. The manner by which prefoldin interacts with a group IT chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group IT chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus a. chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2006.08.088

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Chaperonin is a double ring-shaped oligomeric protein complex, which captures a protein in the folding intermediate state and assists its folding in an ATP-dependent manner. The chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, is a group II chaperonin and is composed of two distinct subunits, alpha and beta. Although these subunits are highly homologous in sequence, the homo-oligomer of the beta-subunit is more thermostable than that of the alpha-subunit. To identify the region responsible for this difference in thermostability, we constructed domain-exchange mutants. The mutants containing the equatorial domain of the beta-subunit were more resistant to thermal dissociation than the mutants with that of the alpha-subunit. Thermostability of a beta-subunit mutant whose C-terminal 22 residues were replaced with those of the alpha-subunit decreased to the comparable level of that of the alpha-subunit homo-oligomer. These results indicate that the difference in thermostability between alpha and beta-subunits mainly originates in the C-terminal residues in the equatorial domain, only where they exhibit substantial sequence difference.

DOI： 10.1007/s00792-006-0519-y

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S320_1

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Other Link： http://orcid.org/0000-0001-8849-4217

Language：English   Publishing type：Research paper (scientific journal)   Publisher：48  

Group II chaperonins, found in Archaea and in the eukaryotic cytosol, act independently of a cofactor corresponding to GroES of group I chaperonins. Instead, the helical protrusion at the tip of the apical domain forms a built-in lid of the central cavity. Although many studies on the lid's conformation have been carried out, the conformation in each step of the ATPase cycle remains obscure. To clarify this issue, we examined the effects of ADP-aluminum fluoride (AlFx) and ADP-beryllium fluoride (BeFx) complexes on alpha-chaperonin from the hyperthermophilic archaeum, Thermococcus sp. strain KS-1. Biochemical assays, electron microscopic observations, and small angle x-ray scattering measurements demonstrate that alpha-chaperonin incubated with ADP and BeFx exists in an asymmetric conformation; one ring is open, and the other is closed. The result indicates that alpha-chaperonin also shares the inherent functional asymmetry of bacterial and eukaryotic cytosolic chaperonins. Most interestingly, addition of ADP and BeFx induced alpha-chaperonin to encapsulate unfolded proteins in the closed ring but did not trigger their folding. Moreover, alpha-chaperonin incubated with ATP and AlFx or BeFx adopted a symmetric closed conformation, and its functional turnover was inhibited. These forms are supposed to be intermediates during the reaction cycle of group II chaperonins.

DOI： 10.1074/jbc.M506785200

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We have expressed, purified, and characterized one small heat shock protein of the fission yeast Schizosaccharomyces pombe, SpHsp16.0. SpHsp16.0 was able to protect citrate synthase from thermal aggregation at 45 degrees C with high efficiency. It existed as a hexadecameric globular oligomer near the physiological growth temperature. At elevated temperatures, the oligomer dissociated into small species, probably dimers. The dissociation was completely reversible, and the original oligomer reformed immediately after the temperature dropped. Large complexes of SpHsp16.0 and denatured citrate synthase were observed by size exclusion chromatography and electron microscopy following incubation at 45 degrees C and then cooling. However, such large complexes did not elute from the size exclusion column incubated at 45 degrees C. The denatured citrate synthase protected from aggregation was trapped by a GroEL trap mutant at 45 degrees C. These results suggest that the complex of SpHsp16.0 and denatured citrate synthase at elevated temperatures is in the transient state and has a hydrophobic nature. Analyses of the interaction between SpHsp16.0 and denatured citrate synthase by fluorescence cross-correlation spectrometry have also shown that the characteristics of SpHsp16.0-denatured citrate synthase complex at the elevated temperature are different from those of the large complex obtained after the shift to lowered temperatures.

DOI： 10.1074/jbc.M504121200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/smll.200500091

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The N-terminal domain (N-domain) of the firefly luciferase from Photinus pyraris has weak luminescence activity, and shows a unique light emitting profile with very long rise time of more than several minutes. Through a sensitive assay of the reaction intermediate luciferyl-adenylate (LH2-AMP), we found that the slow increase in the N-domain luminescence faithfully reflected the concentration of dissociated LH2-AMP. No such correlation was observed for wild-type or mutant enzymes with short rise time, except one with longer rise time. The results suggest that the C-terminal domain plays an indispensable role in efficiently coupling adenylation and oxidative steps. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.07.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Prefoldin is a chaperone that captures a protein-folding intermediate and transfers it to the group 11 chaperonin for correct folding. However, kinetics of interactions between prefoldin and substrate proteins have not been investigated. In this study, dissociation constants and dissociation rate constants of unfolded proteins with prefoldin were firstly measured using fluorescence microscopy. Our results suggest that binding and release of prefoldin from hyperthermophilic archaea with substrate proteins were in a dynamic equilibrium. Interestingly, the release of substrate proteins from prefoldin was facilitated when chaperonin was present, supporting a handoff mechanism of substrate proteins from prefoldin to the chaperonin. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.05.061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INST PURE APPLIED PHYSICS  

DNA micropatterns have been for the first time fabricated on a single-crystal diamond surface in conjunction with the photolithography technique. A new chemical modification process for producing amine groups inside patterned regions and a passivation layer terminated with fluorine outside patterned regions is demonstrated. The resulting amine groups within patterned areas and fluorine termination outside patterned areas on the single-crystal diamond surface were characterized by spatially resolved X-ray photoelectron spectroscopy. Amine-terminated oligonucleotides were then linked to the amine patterned regions using a crosslinker. It was revealed that hybridization on DNA-patterned diamond is specific and selective, with a low background outside the patterns and strong binding to complementary probe DNA immobilized inside the patterns but no binding to noncomplementary probe DNA similarly immobilized inside the patterns. These results suggest that DNA micropatteming on a single-crystal diamond may serve as an ideal platform for future biochips and biosensors.

DOI： 10.1143/JJAP.44.L295

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S165_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S39_1

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：WORLD SCIENTIFIC PUBL CO PTE LTD  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioimaging Society  

Subcellular microenvironments within a living cell are expected to intimately correlate with cellular events including cell division, phagocytosis, locomotion, and intracellular transport. Thus, understanding diffusion processes of macromolecules in subcellular compartments is important to elucidate functions of these compartments. Some biologically active molecules are reported to move by Brownian motion at low mobility due to association and dissociation of cellular components. Measurement of diffusion of inert macromolecules including green fluorescent protein (GFP) is also important to evaluate anomalous diffusion of biologically active macromolecules. Here, we analyzed mobility of GFP in the nucleoplasm, nucleolus, and cytoplasm using fluorescence correlation spectroscopy (FCS). GFP molecules were transiently expressed in HeLa cells, and distributed widely throughout the nucleus. FCS analysis indicated that GFP molecules move by Brownian motion with two different diffusion coefficients. About 97% of GFP in the nucleoplasm and 87% of GFP in the nucleolus moved with diffusion coefficients of 68 μm⊃2;⁄s and 45 μm⊃2;⁄s at 37°C, respectively. These diffusion coefficients were 2 to 3-fold smaller than that in solution (126 μm⊃2;⁄s). The remaining GFP molecules moved slower at 3.8 μm⊃2;⁄s in the nucleoplasm, and 4.5 μm⊃2;⁄s in the nucleolus. Diffusion coefficients decreased to 28 μm⊃2;⁄s in the nucleoplasm, and to 12 μm⊃2;⁄s in the nucleolus at 23°C. Amount of slow components decreased to 3% in the nucleolus, and was undetectable in the nucleoplasm at 23°C. These results indicated that diffusion of protein molecules was different in different nuclear microstructures of a living cell, and was temperature-dependent.

DOI： 10.11169/bioimages.13.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A novel fabrication process of silicon microstructure array for preferential immobilization of biomolecules is proposed. We perform electron beam lithography on a self-assembled monolayer (SAM), and achieve high-density silicon patterns terminated with both 3-aminopropyltriethoxysilane (APTES) and octadecyltrimethoxysilane (ODS). The amino-terminated surface produces the site-directed covalent immobilization of DNA inside the pattern, while the hydrophobic surface of the ODS-SAM prevents the adsorption. As a result, we have succeeded in immobilizing the DNA within the amino-modified area. By using this methodology, we demonstrate the miniaturization of deoxyribonucleic acid (DNA) chip. After the covalent attachment of the amino-modified oligonucleotides to the microstructures, we hybridize the immobilized DNA with the target DNA labeled with a fluorescent dye. The signals from the DNA chip exhibit the specific binding due to the DNA-DNA interaction. These results show the feasibility of this technique for high-density information storage and biochip miniaturization. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.apsusc.2004.05.033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N- and C-terminal regions of the prefoldin beta subunit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.

DOI： 10.1074/jbc.M402889200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

To elucidate the exact role of the helical protrusion of a group II chaperonin in its molecular chaperone function, three deletion mutants of the chaperonin from a hyperthermophilic archaeum (Thermococcus sp. strain KS-1) lacking one-third, two-thirds, and the whole of the helical protrusion were constructed. The helical protrusion is thought to be substituted for the co-chaperonin GroES of a group I chaperonin and to be important for binding to unfolded proteins. Protease sensitivity assays and small angle x-ray scattering experiments were performed to demonstrate the conformation change of the wild type protein and the deletion mutants by adenine nucleotides. Whereas the binding of ATP to the wild type protein induced a structural transition corresponding to the closure of the built-in lid, it did not cause significant structural changes in deletion mutants. Although the mutants effectively protected proteins from thermal aggregation, ATP-dependent protein folding ability was remarkably diminished. We conclude that the helical protrusion is not necessarily important for binding to unfolded proteins, but its ATP-dependent conformational change mediates folding of captured unfolded proteins.

DOI： 10.1074/jbc.M400839200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

The site-directed covalent immobilization of amino-terminated DNA oligonucleotides on microstructured patterns at silicon surfaces generated by the methodology of electron beam (EB) lithography was investigated. The microstructured patterns characterized by scanning electron microscopy (SEM) revealed remarkably regular in size and shape. After treatment with different time of activation (10 s and 30 min), self-assembled layers of 3-aminopropyltriethoxysilane (APTES) on silicon surfaces characterized by X-ray photoelectron spectroscopy (XPS) were demonstrated to obtain similar N 1s peaks. The immobilization specificity was evaluated by means of 5' amino-modified oligonucleotides labeled with Cy 5 at its 3' end attached onto microstructured patterns. The high-density DNA array (40,000 spots per cm(2)) was achieved, and the resulting array exhibited the specific binding due to DNA-DNA interaction. Additional studies indicated hardly visible signals when non-complementary probes were immobilized on the microstructured patterns. The deposition of DNA in a microstructure array using this technique is precise and homogeneous, showing the potential for high-density information storage and the miniaturization for biosensors and biochips. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2003.08.023

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S32_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S154_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S33_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S46_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S32_4

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Publisher：2  

DOI： 10.1016/S1570-9639(03)00179-1

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, a-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.

DOI： 10.1021/bp034015n

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Firefly luciferase catalyzes highly efficient emission of light from the substrates luciferin, Mg-ATP, and oxygen. A number of amino acid residues are identified to be important for the luminescent activity, and almost all the key residues are thought to be located in the N-terminal 529 domain (1-437), except one in the C-terminal domain, Lys(529), which is thought to be critical for efficient substrate orientation. Here we show that the purified N-terminal domain still binds to the substrates luciferin and ATP with reduced affinity, and retains luminescent activity of up to 0.03% of the wild-type enzyme (WT), indicating that all the essential residues for the activity are located in the N-terminal domain. Also found is low luminescence enhancement by coenzyme A (CoA), which implies a lower product inhibition than in the WT enzyme. These findings have interesting implications for the light emission reaction mechanism of the enzyme, such as reaction intermediates, product inhibition, and the role of the C-tenninal domain. (C) 2003 Elsevier B.V All rights reserved.

DOI： 10.1016/S1570-9639(03)00179-1

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Scopus

PubMed

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S60_4

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S63_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Qbeta replicase functioning in Escherichia coli is an RNA-dependent RNA polymerase composed of one phage-coded subunit and three host-coded proteins: ribosomal protein S1, and protein elongation factors EF-Tu and EF-Ts. Qbeta replicase lacking ribosomal protein S1 (alpha-less replicase) is capable of replicating some small RNAs. We attempted to create functional alpha-less replicase by co-expression of the mRNAs that code for the subunits of alpha-less replicase in a rabbit reticulocyte cell-free translation system. Replicase activity, however, could not be detected when both EF-Tu and EF-Ts were co-expressed with the phage-coded subunit. On the other hand, active alpha-less replicase was obtained when an EF-Ts-EF-Tu fusion protein was co-expressed with the phage-coded subunit. Consequently, we succeeded in generating genetically engineered active alpha-less Qbeta replicase which functions in a eukaryotic cell-free system.

DOI： 10.1263/jbb.93.20

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S71_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S72_1

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：SOC CHEMICAL ENG JAPAN  

The structural information of protein molecules on solid phase, such as conformation and orientation , has attracted much attention. In this report, we try to evaluate structural change of proteins via measurement of the adsorption amount of non-ionic detergent (TritonX-100) to proteins immobilized covalently on the matrix surface by a surface plasmon resonance (SPR) sensor. The adsorption amount of detergent to alpha -glucosidase and apomyoglobin is dependent on the amount of immobilized proteins and their surface net hydrophobicity. Moreover, it is observed that the adsorption amount of detergent to proteins increases in proportion to the denaturation degree of proteins induced by 6M guanidinium chloride solution. Consequently, it is revealed that measurement of the adsorption amount of detergent to proteins is applicable to detecting conformational changes of proteins on solid phase.

DOI： 10.1252/kakoronbunshu.27.191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.

DOI： 10.1093/oxfordjournals.jbchem.a002818

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S106_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S94_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation, In this study, firefly luciferase was immobilized on agarose beads at a high concentration, Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution, Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase, These novel observations are described in detail in this paper.

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.40.S174_3

CiNii Books

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Other Link： http://orcid.org/0000-0002-1719-0643

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.39.S181_3

CiNii Books

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Language：English   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

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Language：English   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER DIABETES ASSOC  

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J-GLOBAL

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Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

CiNii Books

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2016232388

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2016231972

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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CiNii Books

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J-GLOBAL

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Language：Japanese   Publisher：南江堂  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2013167601

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：化学工業社  

CiNii Books

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Molecular chaperone prefoldin (PFD) assists protein folding by inhibition of protein aggregation in the cell. In this study, we investigated an interaction between gold nanoparticles (Au NPs) and the molecular chaperone PFD. We demonstrated that addition of PFD inhibits salt-induced aggregation of Au NPs. Interestingly, the interaction between PFD and Au NPs was observed to be Au-size dependent. As PFD has no thiol groups in the molecule, hydrophobic interaction is considered to be the primary interaction. This is the first demonstration of size-specific stabilization of Au NPs with non-covalent bonding. (C) 2010 Elsevier B. V. All rights reserved.

DOI： 10.1016/j.cplett.2010.10.035

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BLACKWELL PUBLISHING  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BLACKWELL PUBLISHING  

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DOI： 10.11491/scej.2008.0.644.0

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DOI： 10.11491/scej.2007.0.393.0

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S321_3

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOPHYSICAL SOCIETY  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOPHYSICAL SOCIETY  

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CiNii Books

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S32_2

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Language：Japanese   Publisher：The Japanese Association for Crystal Growth (JACG)  

We have performed optical observation of the cooperation between the group II chaperon and the prefoldin from the hyperthermophilic archaea and kinetic analyses. Our results clearly demonstrate physical interaction and functional cooperation of them in protein folding.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOPHYSICAL SOCIETY  

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Language：Japanese   Publisher：The Spectroscopical Society of Japan  

DOI： 10.5111/bunkou.51.3

CiNii Books

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CiNii Books

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CiNii Books

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

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