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

Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00401-023-02553-5

researchmap

Other Link： https://link.springer.com/article/10.1007/s00401-023-02553-5/fulltext.html

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

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

Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

The low patency of synthetic vascular grafts hinders their practical applicability. Polyvinyl alcohol (PVA) is a non-toxic, highly hydrophilic polymer; thus, we created a PVA-coated polycaprolactone (PCL) nanofiber vascular graft (PVA–PCL graft). In this study, we examine whether PVA could improve the hydrophilicity of PCL grafts and evaluate its in vivo performance using a rat aorta implantation model. A PCL graft with an inner diameter of 1 mm is created using electrospinning (control). The PCL nanofibers are coated with PVA, resulting in a PVA–PCL graft. Mechanical property tests demonstrate that the PVA coating significantly increases the stiffness and resilience of the PCL graft. The PVA–PCL surface exhibits a much smaller sessile drop contact angle when compared with that of the control, indicating that the PVA coating has hydrophilic properties. Additionally, the PVA–PCL graft shows significantly less platelet adsorption than the control. The proposed PVA–PCL graft is implanted into the rat’s abdominal aorta, and its in vivo performance is tested at 8 weeks. The patency rate is 83.3% (10/12). The histological analysis demonstrates autologous cell engraftment on and inside the scaffold, as well as CD31/α-smooth muscle positive neointima regeneration on the graft lumen. Thus, the PVA–PCL grafts exhibit biocompatibility in the rat model, which suggests that the PVA coating is a promising approach for functionalizing PCL.

DOI： 10.3389/fcvm.2022.946899

researchmap

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

DOI： 10.1007/s00535-021-01846-4.

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

BACKGROUND: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. METHODS: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. RESULTS: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. CONCLUSIONS: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

DOI： 10.1007/s00535-021-01846-4

PubMed

researchmap

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

Background:

Extracellular vesicles (EV) released from neurons into the blood can reflect the state of nervous tissue. Measurement of neuron derived EV (NDE) may serve as an indicator of brain injury.

Methods:

A sandwich immunoassay was established to measure plasma NDE using anti-neuron CD171 and anti-EV CD9 ([CD171 ⁺ CD9⁺]). Plasma samples were obtained from commercial sources, cross-country (n = 9), football (n = 22), soccer (n = 19), and rugby (n = 18) athletes over time. Plasma was also collected from patients undergoing total aortic arch replacement (TAR) with selective cerebral perfusion during cardiopulmonary bypass before and after surgery (n = 36).

Results:

The specificity, linearity, and reproducibility of NDE assay (measurement of [CD171 ⁺ CD9⁺]) were confirmed. By scanning electron microscopy and nanoparticle tracking, spherical vesicles ranging in size from 150 to 300 nm were confirmed. Plasma levels of NDE were widely spread over 2 to 3 logs in different individuals with a significant age-dependent decrease. However, NDE were very stable in each individual within a ± 50% change over time (cross-country, football, soccer), whereas rugby players were more variable over 4 years. In patients undergoing TAR, NDE increased rapidly in days post-surgery and were significantly ( P = .0004) higher in those developing postoperative delirium (POD) (n = 13) than non-delirium patients (n = 23).

Conclusions:

The blood test to determine plasma levels of NDE was established by a sandwich immunoassay using 2 antibodies against neuron (CD171) and exosomes (CD9). NDE levels varied widely in different individuals and decreased with age, indicating that NDE levels should be considered as a normalizer of NDE biomarker studies. However, NDE levels were stable over time in each individual, and increased rapidly after TAR with greater increases associated with patients developing POD. This assay may serve as a surrogate for evaluating and monitoring brain injuries.

DOI： 10.1177/11772719221128145

researchmap

Other Link： https://journals.sagepub.com/doi/full-xml/10.1177/11772719221128145

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fnana.2021.759804. eCollection 2021.

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography (i.e., serial section SEM). This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods.

DOI： 10.3389/fnana.2021.759804

researchmap

Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Ghrelin, a circulating orexigenic hormone secreted from the stomach, stimulates appetite and food intake by activating the hypothalamic arcuate nucleus. Administration of exogenous ghrelin exerts anabolic effects, causing weight gain, increased adiposity, and decreased metabolism. Body temperature (BT), which is determined by the balance of heat production and heat loss, must be strictly regulated to maintain proper cellular function and metabolism. However, the role of ghrelin in thermoregulation remains unclear. In this study, we found that ghrelin was essential for decreasing BT when mice are placed under calorie restriction. Elevated ghrelin concentrations induced by fasting correlated with significant decreases in BT, a hibernation-like state called torpor. Ghrelin-deficient (Ghrl^−/−) animals could not enter torpor. The BT of Ghrl^−/− mice also remained high under restricted feeding, but the animals gradually entered precipitous hypothermia, indicating thermoregulatory impairment. These effects of ghrelin on thermoregulation were the result of suppression of sympathetic nervous system activity input to brown adipose tissue; in the absence of ghrelin, it was not possible to suppress uncoupling protein 1 (ucp1) expression and decrease BT in low-energy states. Together, these findings demonstrate that ghrelin is an essential circulating hormone involved in lowering BT.

DOI： 10.1038/s41598-021-97440-y

researchmap

Other Link： https://www.nature.com/articles/s41598-021-97440-y

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

Trunk muscles in vertebrates are classified as either dorsal epaxial or ventral hypaxial muscles. Epaxial and hypaxial muscles are defined as muscles innervated by the dorsal and ventral rami of spinal nerves, respectively. Each cluster of spinal motor neurons passing through dorsal rami innervates epaxial muscles, whereas clusters traveling on the ventral rami innervate hypaxial muscles. Herein, we show that some motor neurons exhibiting molecular profiles for epaxial muscles follow a path in the ventral rami. Dorsal deep-shoulder muscles and some body wall muscles are defined as hypaxial due to innervation via the ventral rami, but a part of these ventral rami has the molecular profile of motor neurons that innervate epaxial muscles. Thus, the epaxial and hypaxial boundary cannot be determined simply by the ramification pattern of spinal nerves. We propose that, although muscle innervation occurs via the ventral rami, dorsal deep-shoulder muscles and some body wall muscles represent an intermediate group that lies between epaxial and hypaxial muscles.

DOI： 10.1111/joa.13219

PubMed

researchmap

Language：Japanese   Publisher：(公社)日本顕微鏡学会  

切片SEM法は、新たな走査電子顕微鏡(SEM)イメージング技法であり、スライドガラスに貼り付けた樹脂包埋組織切片から反射電子(BSE)像を観察する手法である。この手法では、高度なミクロトーム技術を必要とすることなく、透過電子顕微鏡のような超薄切片像を取得することができる。そこで本稿では、私たちが独自に開発した3つの切片SEM法、1)準超薄切片から超薄像をイメージする手法(準超薄切片SEM法)、2)蛍光像と準超薄切片BSE像の相関顕微鏡観察法(CLEM法)、3)凍結薄切法(徳安法)との組み合わせ法、について紹介する。さらに、新たな3Dイメージング技法である「連続切片SEM法」についても紹介し、切片のSEMイメージング技術の可能性について考察する。(著者抄録)

researchmap

Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2020&ichushi_jid=J04085&link_issn=&doc_id=20200528350003&doc_link_id=10.11410%2Fkenbikyo.55.1_18&url=https%3A%2F%2Fdoi.org%2F10.11410%2Fkenbikyo.55.1_18&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_2.gif

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

The β3-adrenergic receptor (β3AR) is related to myocardial fatty acid metabolism and its expression has been implicated in heart failure. In this study, we investigated the role of β3AR in sepsis-related myocardial dysfunction using lipopolysaccharide (LPS)-induced endotoxemia as a model of cardiac dysfunction. We placed mice into three treatment groups and treated each with intraperitoneal injections of the β3AR agonist CL316243 (CL group), the β3AR antagonist SR59230A (SR group), or normal saline (NS group). Survival rates were significantly improved in the SR group compared with the other treatment groups. Echocardiography analyses revealed cardiac dysfunction within 6-12 h of LPS injections, but the outcome was significantly better for the SR group. Myocardial ATP was preserved in the SR group but was decreased in the CL-treated mice. Additionally, quantitative PCR analysis revealed that expression levels of genes associated with fatty acid oxidation and glucose metabolism were significantly higher in the SR group. Furthermore, the expression levels of mitochondrial membrane protein complexes were preserved in the SR group. Electron microscope studies showed significant accumulation of lipid droplets in the CL group. Moreover, inducible nitric oxide synthase (iNOS) protein expression and nitric oxide were significantly reduced in the SR group. The in vitro study demonstrated that β3AR has an independent iNOS pathway that does not go through the nuclear factor-κB pathway. These results suggest that blockading β3AR improves impaired energy metabolism in myocardial tissues by suppressing iNOS expression and recovers cardiac function in animals with endotoxin-induced heart failure.NEW & NOTEWORTHY Nitric oxide production through stimulation of β3-adrenergic receptor (β3AR) may improve cardiac function in cases of chronic heart failure. We demonstrated that the blockade of β3AR improved mortality and cardiac function in endotoxin-induced heart failure. We also determined that LPS-induced inducible nitric oxide synthase has a pathway that is independent of nuclear factor-κB, which worsened cardiac metabolism and mortality in the acute phase of sepsis. Treatment with the β3AR antagonist had a favorable effect. Thus, the blockade of β3AR could offer a novel treatment for sepsis-related heart failure.

DOI： 10.1152/ajpheart.00108.2019

PubMed

researchmap

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

The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20°C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20°C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60°C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40°C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should find wide appeal for the 3D analysis of cellular organelles in various cell types.

DOI： 10.2220/biomedres.41.161

PubMed

researchmap

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

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

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

OBJECTIVES: Continuously growing rodent incisors have an apically located epithelial stem cell compartment, known as an "apical bud" (AB). Few studies have described the morphological features of ABs and stem cell niches in continuously growing premolars/molars. We attempted to clarify the relationship between the three-dimensional configuration of ABs and the stem cell niches in guinea pig cheek teeth. METHODS: We perfusion-fixed four-week-old guinea pigs, then decalcified their premolars/molars to produce serial paraffin sections, which we immunostained for Sox2. We reconstructed the serial sections using image processing and analysis software. We processed undecalcified samples for scanning electron microscopy by KOH digestion. RESULTS: Two types of epithelia with M and Δ shapes surrounded the S-shaped dental papilla in the apical region of the premolars/molars, and there were three Sox2-positive epithelial bulges above the M- and Δ-shaped epithelia. Sox2-positive epithelial stem cell niches were restricted to the apical side, and cell proliferation and differentiation immediately proceeded in the crown-analogue dentin. The Sox2-positive epithelial stem cell niches were sparsely distributed and extended to the occlusal side. We also detected continuously proliferating cells in the cervical loop and Hertwig's epithelial root sheath of the root-analogue dentin. CONCLUSIONS: Our findings suggest that guinea pig cheek teeth have three ABs, and the complex configuration of these types of teeth may be attributed to the prompt formation of crown-analogue dentin followed by the long-term formation of root-analogue dentin.

DOI： 10.1016/j.job.2019.01.002

PubMed

researchmap

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

Authorship：Lead author,　Corresponding author   Language：English  

DOI： 10.21820/23987073.2019.4.46

researchmap

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

Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.

DOI： 10.1242/jcs.228007

PubMed

researchmap

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

DOI： 10.1042/BCJ20180832

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Secretogranin III (SgIII), a member of the granin family, binds bothtoanother granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knock down of Sg III in adrenocorticotropic hormone (ACTH)-producingAtT-20cellslargelyimpairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretionofactive insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.

DOI： 10.1210/en.2017-00636

Scopus

PubMed

researchmap

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

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

Scanning electron microscopes have longer focal depths than transmission electron microscopes and enable visualization of the three-dimensional (3D) surface structures of specimens. While scanning electron microscopy (SEM) in biological research was generally used for the analysis of bulk specimens until around the year 2000, more recent instrumental advances have broadened the application of SEM; for example, backscattered electron (BSE) signals under low accelerating voltages allow block-face and section-face images of tissues embedded in resin to be acquired. This technical breakthrough has led to the development of novel 3D imaging techniques including focused ion beam SEM, serial-block face SEM and serial section SEM. Using these new techniques, the 3D shapes of cells and cell organelles have been revealed clearly through reconstruction of serial tomographic images. In this review, we address two modern SEM techniques: section-face imaging of resin-embedded tissue samples based on BSE observations, and serial section SEM for reconstruction of the 3D structures of cells and organelles from BSE-mode SEM images of consecutive ultrathin sections on solid substrates.

DOI： 10.1093/jmicro/dfy028

PubMed

researchmap

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

We describe a novel immuno-scanning electron microscopy (SEM) technique that combines both Tokuyasu’s cryosectioning and section SEM methods. In this technique, semithin cryosections, cut according to the Tokuyasu method, were adhered to glass microscope slides, immunostained for bio-molecules of interest and observed by confocal laser scanning microscopy. The same sections were subsequently embedded in epoxy resin and ultrathin sections were cut on an ultramicrotome. These were then observed by SEM using a backscattered electron detector. Correlation between immunofluorescence and SEM images was performed in the same area of the cryosection. Immuno-SEM was also performed using a FluoroNanogold-labeled secondary antibody. This novel immuno-SEM method can provide ultrastructural information of cell organelles in relation to associated molecules, such as Golgi- and ER-associated proteins. This novel immuno-SEM technique has the potential to be widely used.

DOI： 10.2220/biomedres.39.21

Scopus

PubMed

researchmap

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

DOI： 10.2220/biomedres.39.197

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The effects of warm machine perfusion preservation of liver grafts donated after cardiac death on the intracellular three-dimensional ultrastructure of the organelles in hepatocytes remain unclear. Here we analyzed comparatively the ultrastructure of the endomembrane systems in porcine hepatocytes under warm ischemia and successive hypothermic and midthermic machine perfusion preservation, a type of the warm machine perfusion. Porcine liver grafts which had a warm ischemia time of 60 minutes were perfused for 4 hours with modified University of Wisconsin gluconate solution. Group A grafts were preserved with hypothermic machine perfusion preservation at 8 degrees C constantly for 4 hours. Group B grafts were preserved with rewarming up to 22 degrees C by warm machine perfusion preservation for 4 hours. An analysis of hepatocytes after 60 minutes of warm ischemia by scanning electron microscope revealed the appearance of abnormal vacuoles and invagination of mitochondria. In the hepatocytes preserved by subsequent hypothermic machine perfusion preservation, strongly swollen mitochondria were observed. In contrast, the warm machine perfusion preservation could preserve the functional appearance of mitochondria in hepatocytes. Furthermore, abundant vacuoles and membranous structures sequestrating cellular organelles like autophagic vacuoles were frequently observed in hepatocytes after warm machine perfusion preservation. In conclusion, the ultrastructure of the endomembrane systems in the hepatocytes of liver grafts changed in accordance with the temperature conditions of machine perfusion preservation. In addition, temperature condition of the machine perfusion preservation may also affect the condition of the hepatic graft attributed to autophagy systems, and consequently alleviate the damage of the hepatocytes.

DOI： 10.1371/journal.pone.0186352

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

In the Japanese Archipelago, Ezohelix gainesi, a member of bradybaenid land snails, is endemic mainly to the island of Hokkaido. During July to August of 2016, a survey to detect trematode infections from E. gainesi was carried out at a forest city park in Asahikawa, Hokkaido. Systemic infections of the snails with sporocysts containing short-tailed cercariae were found in 5.3% of 94 individuals examined. Furthermore, most of them (90.4%) harbored non-encysted metacercariae within their kidneys. A DNA sequence identification revealed that both of the sporocyst and the metacercaria belong to an unknown species of the family Brachylaimidae. The metacercariae showed a genetic diversity with 6 haplotypes of mitochondrial DNA (mtDNA) even in the limited sampling area. A definitive host of the unknown species could not be determined, although 34 field mice (Apodemus speciosus) and 21 voles (Myodes rufocanus) from the city park were examined for intestinal parasites. To examine the adult stage, the metacercariae were perorally administrated to mice, together with anti-inflammatory treatment with methylprednisolone. Fully matured adult worms were recovered from the intestinal ileum 8 and 14 days postinfection. The gravid adults showed typical features of the genus Brachylaima. A morphological and biogeographical evaluation prompted us to propose Brachylaima ezohelicis sp. nov. for the parasite from E. gainesi. The autochthony of the first intermediate host and the spatial heterogeneity of mtDNA suggest that the new species found in the city park is not a recently expanded population of immigrant origin. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.parint.2017.01.015

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The structure of the Golgi apparatus has been extensively examined by light and electron microscopy, but details of its three-dimensional (3D) structure have remained unclear because of the technical limitations of conventional microscopy techniques. To overcome this problem, we have developed several novel scanning electron microscopy (SEM) methods for observing the 3D structure of subcellular organelles including the Golgi apparatus: (1) an osmium maceration method that facilitates SEM observation of membranous organelles, including the Golgi apparatus, by selectively removing soluble cytoplasmic proteins, (2) an osmium impregnation/maceration method that combines an osmium impregnation method with the osmium maceration method to determine the polarity of the Golgi apparatus by SEM, (3) a correlative light and SEM method that combines a cryosectioning technique with the osmium maceration method to enable correlation of the immunocytochemical distribution of molecules with the 3D ultrastructure of the Golgi apparatus, and (4) array tomography based on the systematic collection and integration of SEM images of serial ultrathin sections on glass slides for revealing the 3D ultrastructure of the entire Golgi apparatus. Together, the novel SEM techniques listed above can reveal the complete 3D structure of the Golgi apparatus in different cell types.

DOI： 10.1007/s12565-016-0380-8

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

The increased discharge of gonadotropin releasing hormone (GnRH) from hypothalamic neurons after castration specifically stimulates pituitary gonadotropes. To elucidate the putative effects of GnRH on the three-dimensional ultrastructure of gonadotropes, we examined osmium-macerated pituitary tissues of male rats at various time points after castration by high resolution scanning electron microscopy (SEM) combined with immunocytochemistry. Two days after castration, the Golgi apparatus was disassembled into small stacks; patch-like, tubuloreticular clusters of endoplasmic reticulum (ER) membranes were present; and spherically enlarged mitochondria were accumulated in the central area of the stimulated gonadotropes. These acute changes were indiscernible by 1 week after castration, and then the pituitary gonadotropes of castrated animals gradually became hypertrophic, finally exhibiting the characteristic "signet-ring" appearance, with markedly dilated cisterns of the rough ER. Upon SEM observation, the inner surface of the cavity was mostly flat, and openings connecting adjacent lumens of the ER were sparse. Proliferation of the osmiophilic tubular network of the ER-Golgi intermediate compartment was observed in the persistently stimulated gonadotropes, indicating a marked increase in trafficking of secretory proteins between the Golgi and ER. The acute and chronic changes in the gonadotropes after castration revealed in the present study by SEM provide evidence for a putative link between the intracellular signaling events evoked by GnRH and the ultrastructural dynamics of the organelles of the secretory pathway.

DOI： 10.2220/biomedres.38.1

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

In the cochlea, a high K+ environment in the endolymph is essential for the maintenance of normal hearing function, and the transport of K+ ions through gap junctions of the cochlear epithelium is thought to play an important role in endolymphatic homeostasis. The aim of the present study was to demonstrate the three-dimensional (3D) ultrastructure of spiral ligament root cells and interdental cells, which are located at both ends of the gap junction system of the cochlea epithelium. Serial semi-thin sections of plastic-embedded rat cochlea were mounted on glass slides, stained with uranyl acetate and lead citrate, and observed by scanning electron microscopy (SEM) using the backscattered electron (BSE) mode. 3D reconstruction of BSE images of serial sections revealed that the root cells were linked together to form a branched structure like an elaborate "tree root" in the spiral ligament. The interdental cells were also connected to each other, forming a comb-shaped cellular network with a number of cellular strands in the spiral limbus. Furthermore, TEM studies of ultra-thin sections revealed the rich presence of gap junctions in both root cells and interdental cells. These findings suggest the possibility that both root cells and interdental cells contribute to K+ circulation as the end portion of the epithelial cell gap junction system of the cochlea.

DOI： 10.2220/biomedres.38.239

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

Thiamine pyrophosphatase (TPPase) cytochemistry is an established method for specific labeling of the trans-Golgi cisterns in tissue sections. Herein, we combined this enzyme cytochemical method with array tomography using scanning electron microscopy (SEM), a new imaging technique based on collection of backscattered electron (BSE) images of consecutive resin-embedded sections on glass slides, to detect the entire three-dimensional (3D) organization of the Golgi apparatus with sufficient spatial resolution. As the signal intensity of BSE depends on the atomic number of the materials, lead precipitates confined to the trans-Golgi cisterns after TPPase cytochemistry were clearly observed by BSE-mode SEM. The mild fixative used for TPPase cytochemistry also enabled accurate identification of target gonadotropes in the composite pituitary tissue by immunocytochemical staining. By 3D reconstruction of the entire trans-Golgi cisterns based on serial ultrathin section images of tissues after TPPase cytochemistry, we detected ultrastructural differences in the 3D configuration of the Golgi apparatus between cerebellar Purkinje cells and pituitary gonadotropes. The appropriate combination of enzyme cytochemistry and/or immunostaining with array tomography will further clarify the relationship between the organization and functional states of the Golgi apparatus.

DOI： 10.2220/biomedres.38.285

Web of Science

researchmap

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

Lymphocyte recruitment into the portal tract is crucial not only for homeostatic immune surveillance but also for many liver diseases. However, the exact route of entry for lymphocytes into portal tract is still obscure. We investigated this question using a rat hepatic allograft rejection model.
A migration route was analyzed by immunohistological methods including a recently developed scanning electron microscopy method. Transmigration-associated molecules such as selectins, integrins, and chemokines and their receptors expressed by hepatic vessels and recruited T-cells were analyzed by immunohistochemistry and flow cytometry.
The immunoelectron microscopic analysis clearly showed CD8 beta(+) cells passing through the portal vein (PV) endothelia. Furthermore, the migrating pathway seemed to pass through the endothelial cell body. Local vascular cell adhesion molecule-1 (VCAM-1) expression was induced in PV endothelial cells from day 2 after liver transplantation. Although intercellular adhesion molecule-1 (ICAM-1) expression was also upregulated, it was restricted to sinusoidal endothelia. Recipient T-cells in the graft perfusate were CD25(+)CD44(+)ICAM-1(+)CXCR3(+)CCR5(-) and upregulated alpha 4 beta 1 or alpha L beta 2 integrins. Immunohistochemistry showed the expression of CXCL10 in donor MHCIIhigh cells in the portal tract as well as endothelial walls of PV.
We show for the first time direct evidence of T-cell transmigration across PV endothelial cells during hepatic allograft rejection. Interactions between VCAM-1 on endothelia and alpha 4 beta 1 integrin on recipient effector T-cells putatively play critical roles in adhesion and transmigration through endothelia. A chemokine axis of CXCL10 and CXCR3 also may be involved.

DOI： 10.1007/s00535-016-1169-1

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three-dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis-trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis- or trans-faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis-Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis-elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron-mode SEM. Prolonged osmication by osmium impregnation (2% OsO4 solution at 40 degrees C for 40 h) and osmium maceration (0.1% OsO4 solution at 20 degrees C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.
Lay Description The Golgi apparatus consists of several layers of flattened cisternae with distinct polarity. Its entry and exit sides are termed the cis-faces (or forming) and trans-faces (or maturing), respectively. The 3D fine structure of the Golgi apparatus, as well as the rough endoplasmic reticulum and mitochondria, can be observed by scanning electron microscopy (SEM), after the soluble cytoplasmic proteins have been selectively removed from the cells by immersing the tissue samples in diluted OsO4 for several days (osmium maceration method). We have analysed the 3D organisation of the Golgi apparatus in various differentiated cells by high-resolution SEM of osmium-macerated tissues, and have demonstrated the diversity in the organisation of the Golgi apparatus. However, we have often faced difficulties in specifying the polarity of the Golgi apparatus, because there were no appropriate ways to label the cis- or trans-faces of the Golgi apparatus by SEM. To overcome this problem, in the present study, we used osmium impregnation for preparation of the specimens for SEM. Impregnation of OsO4 into tissue samples at a relatively high temperature (40 degrees C) for several tens of hours resulted in accumulation of osmium deposits exclusively in the cis-Golgi elements. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically labelled the cis-elements of the Golgi apparatus with osmium deposits. The intensity of backscattered electron (BSE)-mode signals depends on the atomic number of the specimen composition (osmium, atomic number 76). Osmium accumulated in the cis-Golgi elements could be clearly visualised owing to the intense signals of BSE-mode SEM, compared with the low background signals of the surrounding organic compounds. Prolonged exposure to OsO4 during the impregnation and maceration processes hardly damaged the 3D fine structure of the cell organelles, including the Golgi apparatus. This novel preparation method enabled us to analyse the polarity of the Golgi apparatus by SEM, with enough information about the surrounding 3D ultrastructure. The method will contribute to our understanding of the organisation of the entire Golgi apparatus in various differentiated cells that has rarely been achieved by transmission electron microscopy.

DOI： 10.1111/jmi.12379

Web of Science

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/jmicro/dfv360

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Although many studies of the Golgi apparatus structure have been performed by light and electron microscopy, the full shape of the Golgi apparatus remained unclear due to the technical limitations of the previously applied microscopy techniques. In this study, we used serial section scanning electron microscopy (SEM) for the morphological study of the Golgi apparatus. This method is useful for three-dimensional (3D) reconstruction of cellular structures without requiring specialized instruments, unlike focused ion beam SEM (FIB-SEM) and serial block face SEM (SBF-SEM). Using the serial section SEM method developed by our laboratory, we investigate the 3D shape of the osmium-impregnated Golgi apparatus in rat epididymal cells, pancreatic acinar cells and gonadotropes. The combination of serial section SEM and a 3D reconstruction technique enabled us to elucidate the entire shape of the Golgi apparatus in these cells. The full shape of the Golgi apparatus in epididymal cells formed a basket-like structure with oval-shaped cisterns, while the Golgi apparatus in an acinar cell from the pancreas was composed of elongated ribbon-like structures that were connected to each other, making a coarse network. The overall image of the Golgi apparatus cisterns from a gonadotrope looked like a spherical cage. This study has clearly shown that entire 3D shape of the Golgi apparatus varies depending on the cell type and that the Golgi cisterns network appears as a single mass located in the large region of the cytoplasm.

DOI： 10.1093/jmicro/dfv360

Web of Science

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/jmi.12379

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.

DOI： 10.1369/0022155415609099

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM.

DOI： 10.1093/jmicro/dfv042

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

Although a number of papers have given useful information on splenic microcirculation by light and/or scanning electron microscopy, controversies remain as to the vascular arrangement, especially in the human spleen. The present study re-examined the microvasculature of the human spleen using a three-dimensional reconstruction of immunohistochemically stained tissue sections, and showed that the central artery does not directly issue follicular arteries in the human spleen; follicular arteries are derived from penicillar arteries outside the follicle and end in the white pulp. We found that the splenic follicle is surrounded by an elaborate system of anastomosed capillaries in both the marginal zone and the superficial layer of the white pulp. Most of these capillaries are also branches of the penicillar arterioles that are issued from the central artery in the same, or a different, white pulp system. Because the endothelia of these capillaries are widely open in the marginal zone, this vascular network may play a major role in supplying blood to the marginal zone. The accumulation of sialoadhesin-positive macrophages was also observed around the vascular network, suggesting an important role for this structure as the front line of immune response.

DOI： 10.2220/biomedres.36.195

Web of Science

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1369/0022155415609099

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

In contrast to the widely accepted images of the Golgi apparatus as a cup-like shape, the Golgi in pituitary gonadotropes is organized as a spherical shape in which the outer and inner faces are cis- and trans-Golgi elements, respectively. At the center of the spherical Golgi, a pair of centrioles is situated as a microtubule-organizing center from which radiating microtubules isotropically extend toward the cell periphery. This review focuses on the significance of the characteristic organization of the Golgi and microtubule network in gonadotropes, considering the roles of microtubule-dependent membrane transport in the formation and maintenance of the Golgi structure. Because the highly symmetrical organization of the Golgi is possibly perturbed in response to experimental treatments of gonadotropes, monitoring of the Golgi structure in gonadotropes under various experimental conditions will be a novel in vivo approach to elucidate the biogenesis of the Golgi apparatus. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mce.2013.10.003

Web of Science

researchmap

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

DOI： 10.1016/j.mce.2013.10.003

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

DOI： 10.11410/kenbikyo.49.3_171

researchmap

Language：English   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/jid.2013.60

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histological Documentation  

Gonadotropin-releasing hormone (GnRH) agonists exert acutely stimulatory action on gonadotropes, but thereafter suppress paradoxically gonadotropin synthesis and release by receptor desensitization. To examine whether GnRH signaling affects the morphological characteristics in membranous organelles related to the synthesis of gonadotropin, we have analyzed the ultrastructural changes in the ER and Golgi apparatus of male rat pituitary gonadotropes during sustained treatment with a GnRH agonist, leuprorelin. In pituitary gonadotropes at 1 day after the onset of treatment, clusters of the tubuloreticular ER appeared, and the globular Golgi apparatus was transiently disassembled into isolated small-sized stacks. However, 1 week after the onset of the treatment, the tubuloreticular ER was seemingly converted to rough ER with regularly stacked sheets and the scattered Golgi stacks converged to form globular structures. In the following chronic phase of the treatment, the ER cisterns remained flattened and the trans-Golgi compartment appeared to be collapsed. Sustained treatment with leuprorelin could also restore the enlarged Golgi apparatus and expand the cisterns of the rough ER
a feature that was seen in hypertrophic gonadotropes of castrated rats. These findings indicated that the ultrastructure of the membranous organelles changed because of the chronic suppressive effects of leuprorelin on gonadotropes both in the physiological and stimulated states. The acute and chronic ultrastructural changes in the ER and Golgi apparatus during sustained leuprorelin treatment also suggests that GnRH signaling cross-talks with the regulation of the morphological characteristics in membranous organelles related to gonadotropin synthesis.

DOI： 10.1679/aohc.74.41

Scopus

researchmap

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

Axonal injury and demyelination are observed in demyelinating diseases such as multiple sclerosis. However, pathological changes that underlie these morphologies are not fully understood. We examined in vivo morphological changes using a new histological technique, scanning electron microscopy (SEM) with osmium maceration method to observe three-dimensional structures such as myelin and axons in the spinal cord. Myelin basic protein-deficient shiverer mice and mice with experimental autoimmune encephalomyelitis (EAE) were used to visualize how morphological changes in myelin and axons are induced by dysmyelination and demyelination.SEM revealed following morphological changes during dysmyelination of shiverer mice. First, enriched mitochondria and well-developed sER in axons were observed in shiverer, but not in wild-type mice. Second, the processes from some perinodal glial cells ran parallel to internodes of axons in addition to the process that covered the nodal region of the axon in shiverer mice. Last, this technique left myelin and axonal structures undisturbed. Moreover, SEM images showed clear variations in the ultrastructural abnormalities of myelin and axons in the white matter of the EAE spinal cord. This technique will be a powerful tool for identifying the mechanisms underlying the pathogenesis in demyelination. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society.

DOI： 10.1016/j.neures.2013.01.009

Scopus

PubMed

researchmap

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

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

DOI： 10.1679/aohc.74.41

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

DOI： 10.1016/j.neures.2013.01.009

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

Histological changes were observed in peripheral nerves following end-to-side nerve coaptation to determine the effects of perineurial opening and deliberate donor nerve injury during surgery. Twenty rats were randomised into four groups as follows: group 1, end-to-side nerve coaptation without perineurial opening; group 2, end-to-side nerve coaptation with simple perineurial opening; group 3, end-to-side nerve coaptation with partial crush injury after perineurial opening; group 4, end-to-side nerve coaptation with partial neurotomy after perineurial opening. Seven days after coaptation of the musculocutaneous (recipient) nerve to the ulnar (donor) nerve, the nerves were immunohistochemically analysed using antibodies against neurofilament-H (RT97) and phosphorylated GAP-43 (p-GAP-43). The former labels all axons, including regenerating axons and degenerated axonal debris, while the latter only labels regenerating axons. Results demonstrated no regenerating nerves in the recipient nerve of group 1. In group 2, because nerve herniation from the perineurial opening partially injured donor nerve fibres, some regenerating axons extended proximally and distally along the partially injured fibres in the donor nerve; some of these regenerating axons also extended into the recipient nerve via the perineurial opening. In groups 3 and 4, thin regenerating axons were more prominent in recipient and donor nerves compared with group 2. Statistical evaluation revealed increased efficacy of perineurial opening and deliberate donor nerve injury in end-to-side nerve coaptation, suggesting that partial nerve fibre herniation with partial axonotmesis or neurotomesis was important for effective axonal regeneration in end-to-side nerve coaptation.

DOI： 10.3109/2000656X.2012.696264

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Reactive oxygen species (ROS) are toxic but essential molecules responsible for host defense and cellular signaling. Conserved NADPH oxidase (NOX) family enzymes direct the regulated production of ROS. Hydrogen peroxide (H2O2) generated by dual oxidases (DUOXs), a member of the NOX family, is crucial for innate mucosal immunity. In addition, H2O2 is required for cellular signaling mediated by protein modifications, such as the thyroid hormone biosynthetic pathway in mammals. In contrast to other NOX isozymes, the regulatory mechanisms of DUOX activity are less understood. Using Caenorhabditis elegans as a model, we demonstrate that the tetraspanin protein is required for induction of the DUOX signaling pathway in conjunction with the dual oxidase maturation factor (DUOXA). In the current study, we show that genetic mutation of DUOX (bli-3), DUOXA (doxa-1), and peroxidase (mlt-7) in C. elegans causes the same defects as a tetraspanin tsp-15 mutant, represented by exoskeletal deficiencies due to the failure of tyrosine cross-linking of collagen. The deficiency in the tsp-15 mutant was restored by co-expression of bli-3 and doxa-1, indicating the involvement of tsp-15 in the generation of ROS. H2O2 generation by BLI-3 was completely dependent on TSP-15 when reconstituted in mammalian cells. We also demonstrated that TSP-15, BLI-3, and DOXA-1 form complexes in vitro and in vivo. Cell-fusion-based analysis suggested that association with TSP-15 at the cell surface is crucial for BLI-3 activation to release H2O2. This study provides the first evidence for an essential role of tetraspanin in ROS generation.

DOI： 10.1371/journal.pgen.1002957

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

In polarized exocrine cells, the Golgi apparatus is cup-shaped and its convex and concave surfaces are designated as cis and trans faces, functionally confronting the rough endoplasmic reticulum and the cell surface, respectively. To clarify the morphological characteristics of the Golgi apparatus in non-polarized endocrine cells, the investigators immunocytochemically examined its precise architecture in pituitary gonadotropes, especially in relation to the arrangement of the intracellular microtubule network. The Golgi apparatus in the gonadotropes was not cup-shaped but ball-shaped or spherical, and its outer and inner surfaces were the cis and trans faces, respectively. Centrioles were situated at the center of the Golgi apparatus, from which radiating microtubules isotropically extended to the cell periphery through the gaps in the spherical wall of the Golgi stack. The shape of the Golgi apparatus and the arrangement of microtubules demonstrated in the present study could explain the microtubule-dependent movements of tubulovesicular carriers and granules within the gonadotropes. Furthermore, the spherical shape of the Golgi apparatus possibly reflects the highly symmetrical arrangement of microtubule arrays, as well as the poor polarity in the cell surface of pituitary gonadotropes. (J Histochem Cytochem 60:588-602, 2012)

DOI： 10.1369/0022155412448791

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.

DOI： 10.1093/jmicro/dfr098

Web of Science

researchmap

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

researchmap

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

Background: We have investigated how recirculating lymphocytes patrol the liver in a normal steady state. Methods: Thoracic duct lymphocytes of congeneic rats were intravenously transferred to host rats and donor cell trafficking in the liver and hepatic lymph was examined. Host hepatic lymph nodes (HLNs) were selectively removed, which allowed liver-derived donor cells to collect in the thoracic duct without transit in the intervening HLNs. Results: The number of donor cells in the thoracic duct lymph significantly increased over the baseline 3, 5 and 11 h after transfer in the HLN-removed, non-pretreated, and HLN-ligated ( in which a lymph efflux was blocked) groups, respectively. Histologically, donor cells appeared in the portal area from 0.5 h after transfer and frequently attached to the basal lamina of portal vein both externally and internally. Three hours after transfer, a few donor cells appeared in the subcapsular sinus of HLNs. Conclusion: The minimal transit time of rat recirculating lymphocytes is 3 - 4 h in the liver and 5 - 8 h in the hepatic LNs, in a normal steady state. Recirculating lymphocytes might transmigrate through the portal vein as well as the sinusoid in the periportal zone. This rapid transit might enable an efficient surveillance of the liver portal area by the recirculating lymphocytes.

DOI： 10.1111/j.1478-3231.2008.01671.x

Web of Science

researchmap

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

researchmap

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

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The three-dimensional ultrastructure of the Golgi apparatus in different cells of the rat - epithelial principal cells in the epididymal duct, goblet cells in the jejunum, gonadotrophs in the pituitary grand and dorsal root ganglion cells - was studied by scanning electron microscopy (SEM) of osmium-macerated tissues. The Golgi apparatus in the epididymal principal cells took the shape of a candle flame with irregular-shaped cisterns, while those in the goblet cells of the jejunum were cup-shaped or cylindrical with flat cisterns. Gonadotrophs had a large spherical Golgi apparatus; this apparatus was composed of several concentric cisterns with large round windows through which the rough endoplasmic reticulum (rER) and mitochondria extended into the center of the globular Golgi apparatus. Dorsal root ganglion cells had several small Golgi stacks scattered in the cytoplasm. In all Golgi apparatuses of the different cells examined in the present study, the cis-most cistern was generally composed of a flattened sheet with numerous small fenestrations on its wall. On the other hand, the shape of the trans-most cistern varied by cell type; it was generally composed of tubules and/or small sheets which were sometimes connected with each other to form a rather complicated structure. The cismost cistern and the trans-most cistern were often closely associated with the rER although no direct communication was found between them. These findings indicate that the structure of the Golgi apparatus, especially its overall shape and the ultrastructure of the trans-most cistern, varies by cell type, a point to be considered in relation to the function of the individual cells.

DOI： 10.1679/aohc.69.357

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The ptilinum of the fly and the compound eye are among the most fragile organs encountered during conventional procedures of morphological sample fixation. in order to identify a fixative suitable for preparing such samples for scanning electron microscopy, we examined various fixation conditions using microwave irradiation (MWI). The conditions examined were: (i) fixatives; (ii) temperature; (iii) concentration; (iv) duration; (v) dehydration; and (vi) substitution. The identified optimal conditions were 5% glutaraldehydc with MWI (350 W, 5 min). The MWI was continued until the maximal temperature of 75degreesC was attained, followed by intermittent irradiation to maintain a temperature of 75degreesC. After irradiation, the sample was left at room temperature for 24 h in the fixative and then dehydrated in increasing concentrations of ethanol. Each step in the ethanol series lasted for 24 h. The final absolute ethanol step included three solution changes, with each incubation lasting 1 h. A subsequent stepwise substitution of t-butyl alcohol for ethanol was conducted by reducing the ratio of 100% ethanol to t-butyl alcohol from 2:1 to 1: 1 and then 1:2 (24 h each). The substitution was completed by three solution changes using 100% t-butyl alcohol, 30 min each. The best results were obtained by freeze-drying samples using t-butyl alcohol. The use of MWI improved fixative permeation, which occurred at a uniform rate throughout the sample. Comparison with temperature in a water bath at 75degreesC indicated that the fixation effect of MWI was due to its heat generation in addition to some unknown mechanism.

DOI： 10.1093/jmicro/52.5.477

Web of Science

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：公益社団法人　日本顕微鏡学会  

Language：Japanese   Publisher：(一社)日本内分泌学会  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(一社)日本解剖学会  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：English   Publisher：日本神経化学会  

researchmap

Language：Japanese   Publisher：(一社)日本解剖学会  

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(一社)日本解剖学会  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(一社)日本解剖学会  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(一社)日本解剖学会  

J-GLOBAL

researchmap

Language：Japanese   Publisher：ニュートンプレス  

CiNii Books

researchmap

Event date： 2022.3

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

Event date： 2021.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Event date： 2023.3

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

Venue：東北大学  

Event date： 2019.3

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

Event date： 2019.3

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

Venue：順天堂大学本郷キャンパス  

Event date： 2019.3

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

Venue：品川インターシティ  

Event date： 2018.11

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

Venue：パシフィコ横浜  

Event date： 2018.8

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

Event date： 2018.8

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

Venue：国民宿舎「桂浜荘」  

Event date： 2018.5

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

Venue：久留米シティプラザ  

Event date： 2018.5

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

Venue：久留米シティプラザ  

Event date： 2018.5

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

Event date： 2016.3

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

Venue：ビックパレふくしま  

Event date： 2015.12

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Event date： 2015.9

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

Event date： 2015.8

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.5

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

Presentation type：Symposium, workshop panel (nominated)  

researchmap

Presentation type：Symposium, workshop panel (public)  

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：English   Presentation type：Oral presentation (general)  

researchmap

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

researchmap

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

researchmap

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

researchmap

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

researchmap

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

researchmap

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

researchmap

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

researchmap

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

researchmap

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

researchmap

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

researchmap

researchmap

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

researchmap

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

researchmap

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

researchmap

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

researchmap

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

researchmap

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

researchmap

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

researchmap

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

researchmap

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

researchmap

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

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

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

researchmap

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

researchmap

Language：English   Presentation type：Poster presentation  

researchmap

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

researchmap

Language：English  

researchmap

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

researchmap

Presentation type：Symposium, workshop panel (public)  

researchmap

Presentation type：Oral presentation (general)  

researchmap

Presentation type：Oral presentation (invited, special)  

researchmap

Award type：Honored in official journal of a scientific society, scientific journal  Country：Japan

Award type：Honored in official journal of a scientific society, scientific journal 

researchmap

Award type：International academic award (Japan or overseas)  Country：Japan

researchmap

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

researchmap

Award type：Award from Japanese society, conference, symposium, etc. 

researchmap

Award type：Award from Japanese society, conference, symposium, etc. 

researchmap

Award type：Award from international society, conference, symposium, etc. 

researchmap

Award type：Award from international society, conference, symposium, etc. 

researchmap

Award type：Award from Japanese society, conference, symposium, etc. 

researchmap

Award type：Award from Japanese society, conference, symposium, etc. 

researchmap

旭川医大で行われたアウトリーチ活動。
電子顕微鏡の実機を使った組織・細胞のミクロの世界を紹介した。

秋葉原UDXで行われたアウトリーチ活動。
電子顕微鏡の実機を持ち運び、ミクロの世界を紹介した。

NHK科学番組に電子顕微鏡の画像を提供した。

電子顕微鏡の実機をサイパルに持ち運び、旭川市民へ電子顕微鏡の世界を紹介した。アウトリーチ活動

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

NHKスペシャル「人体」に提供した画像についてや、「ミクロの世界の魅力」について、トークショーというスタイルで子供達や一般の方々に紹介した。

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：Lecture

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：Lecture

researchmap

魔法の虫メガネ　走査電子顕微鏡の魅力～NHKスペシャル人体収録エピソード～、と題して、小学生から大人まで幅広い年代層に、電子顕微鏡の魅力について講演した。

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：Other

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：Other

researchmap

Type：Internet

researchmap

Type：Other

researchmap

Type：Seminar, workshop

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：TV or radio program

researchmap

Type：Lecture

researchmap

Audience： Schoolchildren,　Junior students

Type：Lecture

researchmap

Type：Visiting lecture

researchmap

Type：Visiting lecture

researchmap

Type：Visiting lecture

researchmap

Type：Visiting lecture

researchmap

Audience： High school students

Type：Visiting lecture

researchmap

Type：Visiting lecture

researchmap

Type：Visiting lecture

researchmap

Type：Peer review 

researchmap

第128回日本解剖学会総会・全国学術集会　
「オルガネラ・イメージングの電顕マルチモダリティ」
シンポジウム企画・座長