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Molecular Cloning and Tissue Distribution of Troponin I from the Japanese Pearl Oyster, Pinctada fucata

American Journal of Molecular Biology 2019年第2期9卷 29-40页

作者： Daisuke Funabara yoshinori urakawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ and is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of troponin I (TnI) in vertebrates has been extensively characterized, but its role in molluscan muscles has not yet been elucidated. Our previous work suggested that the troponin C subunit has a role in adductor phasic muscle but not in catch muscle. Here, we investigated the molecular characteristics of TnI from the bivalve Japanese pearl oyster, Pinctada fucata to aid the elucidation of the function of molluscan muscle troponin. We determined the primary structure of the full-length TnI protein from the P. fucata adductor muscle (Pifuc-TnI) and found that it is composed of 286 amino acid residues with a predicted molecular weight of 33,737. Motif structure predictions and multiple sequence alignments revealed that Pifuc-TnI has a 138 residue extension at its N-terminus compared with rabbit TnI. This is analogous to characterized TnIs from other mollusks. However, unlike scallop TnI, Pifuc-TnI is predicted to contain two cAMP-dependent protein kinase phosphorylation sites, at residues 39 - 45 (RRGTEDD) and 145 - 151 (KKKSKRK). Phylogenetic analysis indicated that Pifuc-TnI and molluscan TnIs were grouped into the same clade. Pifuc-TnI gene structure predictions using Splign alignment of our obtained cDNA and genome sequences indicated that Pifuc-TnI consists of fifteen exons, with the start and stop codons located in exon 2 and exon 11, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnI gene is predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, and is not expressed in the gill, mantle or foot. These findings suggest that TnI, as a component of the troponin complex, plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction.

关键词： Adductor Muscle Catch Contraction Pinctada fucata Troponin Troponin I

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Molecular Cloning and Tissue Distribution of Troponin C from the Japanese Pearl Oyster, Pinctada fucata

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American Journal of Molecular Biology 2018年第3期8卷 166-177页

作者： Daisuke Funabara yoshinori urakawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin C (TnC) is one of the subunits of troponin. Troponin, which is activated by Ca2+ binding, is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of TnC in vertebrates has been characterized in detail, but the role of TnC in molluscan muscles is still unclear. In this work, we investigated whether TnC plays a role in the catch contraction of molluscan smooth muscle in the bivalve Japanese pearl oyster Pinctada fucata. We determined the full-length primary structure of the TnC protein from the P. fucata adductor muscle (Pifuc-TnC), and found it is composed of 150 amino acid residues with a predicted molecular weight of 17,400. Multiple sequence alignments indicated that it had four EF-hand motifs, but only one (site IV) was predicted to have Ca2+-binding ability. This is analogous to characterized TnCs from other mollusks. Three-dimensional modeling of Pifuc-TnC using SWISS-MODEL indicated the presence of a short loop within the α-helix connecting the site II and III EF-hand motifs. We predicted the gene structure of Pifuc-TnC using Splign alignment of our obtained cDNA and genome sequences and elucidated that Pifuc-TnC consists of five exons, with the start and stop codons located in exon 1 and exon 5, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnC gene is predominantly expressed in adductor phasic muscle and rarely in adductor catch muscle, gill, mantle and foot. These findings suggest that TnC may not have a role in catch muscle contraction.

关键词： Adductor Muscle Catch Contraction EF-Hand Troponin Troponin C

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Troponin T from the Japanese Pearl Oyster Pinctada fucata: Molecular Cloning, Tissue Distribution, Gene Structure, and Interaction Analysis with Tropomyosin

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American Journal of Molecular Biology 2020年第1期10卷 61-73页

作者： Daisuke Funabara yoshinori urakawa Daisuke Ishikawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.

关键词： Adductor Muscle Catch Contraction Pinctada fucata Troponin T Tropomyosin

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Ca2+-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster, Pinctada fucata

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American Journal of Molecular Biology 2018年第4期8卷 205-214页

作者： Daisuke Funabara Daisuke Ishikawa yoshinori urakawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca2+ binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca2+. To confirm the number of Ca2+-binding sites in Pifuc-TnC and whether Ca2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca2+, confirming that site IV is the only Ca2+-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca2+ compared with the condition when Ca2+ was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca2+, suggesting that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca2+. In combination, these findings suggest that Ca2+ binding to site IV induces a conformational

关键词： Ca2+-Binding Catch Muscle Conformational Change EF-Hand Troponin C

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Experimental study on boundary lubricity of superficial area of articular cartilage and synovial fluid

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Friction 2024年第5期12卷 981-996页

作者： Wenxiao LI Takehiro MORITA yoshinori SAWAE Departure of Mechanical Engineering Kyushu UniversityFukuoka8190395Japan International Institute for Carton-Neutral Energy Research Kyushu UniversityFukuoka8190395Japan Advanced Research Center for Biomechanics Kyushu UniversityFukuoka8190395Japan

The boundary lubrication mechanism at the articulating surface of natural synovial joints has been the subject of much discussion in *** this study,to elucidate the lubricating function of the superficial area of articular cartilage and synovial fluid(SF),cartilage specimens were processed with four different treatments:gentle and severe washing with detergent,incubation in NaCl solution,and trypsin digestion to selectively remove certain constituents from the cartilage ***,the frictional characteristics were examined in phosphate-buffered saline(PBS)and SF against *** reciprocating sliding tests with a spherical glass probe and square articular cartilage specimens were performed at low contact loads in the mN range to extract the frictional behavior in the superficial area of the cartilage ***,the cartilage surface was observed to confirm the effects of treatments on the morphology of the cartilage surface using a fluorescence microscope and water-immersion *** coefficient of friction(COF)of the prepared cartilage specimens was varied from 0.05 to over 0.3 in ***,a certain group of cartilage specimens exhibited a low COF of less than 0.1 with limited *** the low COF group of specimens,all four treatments increased the COF in PBS to different extents,and fluorescence microscopy revealed that the integrity of the cartilage surface was deteriorated by *** means that the intact cartilage surface had lubricating constituents to maintain low friction,and the removal of such constituents resulted in the loss of the intrinsic boundary lubricity of the cartilage *** variation in the COF of the cartilage specimens was suppressed in SF because it had a clear boundary lubrication effect on the cartilage *** lubricating effect of SF could be confirmed even after degenerative treatment.

关键词： articular cartilage synovial fluid the uppermost superficial layer boundary lubrication

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Regeneration mechanisms and therapeutic strategies for neuromuscular junctions in aging and diseases

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Neural Regeneration Research 2025年第1期20卷 193-194页

作者： Masashi Fujitani Abu Md Mamun Tarif yoshinori Otani Department of Anatomy and Neuroscience Faculty of MedicineShimane UniversityShimaneJapan

The neuromuscular junction(NMJ)is an essential synaptic structure composed of motor neurons,skeletal muscles,and glial cells that orchestrate the critical process of muscle contraction(Li et al.,2018).The typical NMJ structure is classically described as having a“pretzel-like”shape in mice(Figure 1),whereas human NMJs have a smaller,fragmented structure throughout *** NMJs exhibit smaller or fragmented endplates,partial denervation,reduced numbers of synaptic vesicles,abnormal presynaptic mitochondria,and dysfunctional perisynaptic Schwann cells(Alhindi et al.,2022).

关键词： aging structure Figure

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Development of a scintillating-fiber-based beam monitor for the coherent muon-to-electron transition experiment

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Nuclear Science and Techniques 2024年第4期35卷 156-164页

作者： Yu Xu Yun-Song Ning Zhi-Zhen Qin Yao Teng Chang-Qing Feng Jian Tang Yu Chen yoshinori Fukao Satoshi Mihara Kou Oishi School of Physics Sun Yat-sen UniversityGuangzhou 510275China State Key Laboratory of Particle Detection and Electronics University of Science and Technology of ChinaHefei 230026China Department of Modern Physics University of Science and Technology of ChinaHefei 230026China High Energy Accelerator Research Organization(KEK) Ibaraki 305‑0801Japan Department of Physics Imperial College LondonLondon SW72AZUK

The coherent muon-to-electron transition(COMET)experiment is a leading experiment for the coherent conversion of μ^(-)N→e^(-)N using a high-intensity pulsed muon beamline,produced using innovative slow-extraction ***,it is critical to measure the muon beam *** set up a muon beam monitor(MBM),where scintillating fibers woven in a cross shape were coupled to silicon photomultipliers to measure the spatial profile and timing structure of the extracted muon beam for the *** MBM detector was tested successfully with a proton beamline at the China Spallation Neutron Source and took data with good performance in the commissioning *** development of the MBM,including its mechanical structure,electronic readout,and beam measurement results,are discussed.

关键词： Beam instrumentation Profile monitor Scintillating fiber Silicon photomultipliers

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Ssu72磷酸酶缺失导致减数第二次分裂过程中纺锤体交叉

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遗传 2024年第6期46卷 502-508页

作者：闫静亮马玲玲渡边嘉典江南大学未来食品科学中心无锡214122 江南大学生物工程学院无锡214122

Ssu72磷酸酶是酵母裂解/多聚腺苷化因子(cleavage/polyadenylation factor,CPF)复合物的组成成分,可以催化RNA聚合酶II的C末端结构域(C-terminal domain,CTD)S5-P、S7-P的去磷酸化。后又有研究指出Ssu72磷酸酶参与有丝分裂过程中染色体... 详细信息

Ssu72磷酸酶是酵母裂解/多聚腺苷化因子(cleavage/polyadenylation factor,CPF)复合物的组成成分,可以催化RNA聚合酶II的C末端结构域(C-terminal domain,CTD)S5-P、S7-P的去磷酸化。后又有研究指出Ssu72磷酸酶参与有丝分裂过程中染色体凝聚力的调控。为进一步明确Ssu72磷酸酶是否会影响裂殖酵母减数分裂过程中染色体的分离,本研究利用绿色荧光蛋白(green fluorescent protein,GFP)标记着丝粒、红色荧光蛋白标记微管蛋白Atb2,并在荧光显微镜下实时观察ssu72Δ细胞的整个减数分裂染色体分离过程。结果表明,ssu72Δ细胞在减数第二次分裂中后期发生纺锤体交叉,并且这种纺锤体的交叉产生了一种新型的孢子排布缺陷模式。本研究为高等生物中磷酸酶Ssu72的研究提供重要的借鉴意义。

关键词：裂殖酵母减数分裂 Ssu72 纺锤体交叉

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Recovery of rare-earth metal neodymium from aqueous solutions by poly-γ-glutamic acid and its sodium salt as biosorbents:Effects of solution pH on neodymium recovery mechanisms

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Journal of Rare Earths 2018年第5期36卷 528-536页

作者： Misaki Hisada yoshinori Kawase Research Center for Biochemical and Environmental Engineering Department of Applied Chemistry Toyo University 2100 Kujirai kawagoe Saitama 350-8585 Japan

For recovery of metals from low-concentration sources, biosorption is one of promising technologies and poly-γ-glutamic acid（γ-PGA） has been known as a potential biosorbent for recovery of heavy metals from aqueous solutions. Effects of solution pH on recovery of rare-earth metal Nd are systematically examined to clarify mechanisms of Nd recovery by y-PGA and its sodium salt（y-PGANa）. The recovery efficiency of Nd by y-PGA increases from 2.4 to 19.6% as pH increases from 2 to 4. Subsequently the Nd recovery efficiencies for y-PGA and y-PGANa remain almost constant in the range of pH from 4 to 7. For pH 〉 7 the increase in Nd recovery is significant and 100% recovery of Nd is achieved at pH 9. The pH dependency on Nd recovery by y-PGANa is similar to that by y-PGA. Contributions of adsorption and precipitation/coagulation to Nd recovery process are quantified. Whereas the adsorption dominates Nd recovery at lower pH（〈～4）, the precipitation/coagulation controls Nd recovery process for pH 〉 7. At higher pH, purple gel precipitates are observed. The maximum adsorption capacities for γ-PGA and yPGANa are 215 mg-Nd/（g-γ-PGA） at pH 4 and 305 mg-Nd/（g-y-PGANa） at pH 3, respectively. From the spectra of FT-IR and XPS, the biosorption of Nd onto y-PGA and y-PGANa via electrostatic interaction with carboxylate anions at pH 3 is verified. The Nd complexation with amide and carboxylate anion groups on γ-PGA and γ-PGANa may also contribute to the Nd recovery. The biosorption isotherms for Nd recovery by γ-PGA and γ-PGANa can be satisfactorily fitted by the Langmuir model. The thermodynamic studies suggest that the biosorptions of Nd by γ-PGA and γ-PGANa are endothermic. The utilization of γ-PGA and γ-PGANa as potential and eco-friendly biosorbents for the highly effective recovery of Nd from aqueous solution is confirmed.

关键词： Rare-earth metal Nd recovery Poly-y-glutamic acid Biosorption Precipitation Coexisting cations

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Estimating camera parameters from starry night photographs

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Computational Visual Media 2020年第4期6卷 445-454页

作者： Naoto Ishikawa yoshinori Dobashi Graduate School of Information Science and Technology Hokkaido UniversitySapporoJapan

We propose an efficient, specific method for estimating camera parameters from a single starry night image. Such an image consists of a collection of disks representing stars, so traditional estimation methods for common pictures do not work. Our method uses a database, a star catalog, that stores the positions of stars on the celestial sphere. Our method computes magnitudes(i.e., brightnesses) of stars in the input image and uses them to find the corresponding stars in the star catalog. Camera parameters can then be estimated by a simple geometric calculation. Our method is over ten times faster and more accurate than a previous method.

关键词： astrophotography constellation star identification pattern matching

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