CRF-immunoreactive nerve fibers in the circumventricular organs of the monkey,Macaca fuscata

Summary The occurrence of CRF (corticotropin-releasing factor)-immunoreactive nerve fibers in the circumventricular organs of adult male monkeys, Macaca fuscata, was studied on serially sectioned brains, by means of the peroxidase-antiperoxidase technique in combination with a highly specific and sensitive CRF antiserum. CRF-containing nerve fibers were found in high concentrations in the infundibulum and, in addition, in small numbers in the posterior lobe, organum vasculosum laminae terminalis, subfornical organ, and area postrema; they were missing in the pineal body and the subcommissural organ. The CRF immunoreactive nerve fibers distributed in these organs were located in the proximity of the blood vessels.Supported by a grant (No. 56440022) from the Ministry of Education, Science, and Culture, Japan     

Three-dimensional distribution of the clear zone of migrating osteoclasts on dentin slices in vitro

Osteoclasts are cells that dynamically alternate resorption and migration on bone surfaces, and have the special structure called ruffled borders and clear zones by transmission electron microscopy (TEM). However, TEM features, especially the distribution of the clear zone of osteoclasts during migration, remains unclear. This study aimed to examine osteoclasts cultured on dentin slices by TEM and clarify the features of migrating osteoclasts, especially the three-dimensional distribution of clear zones. Osteoclasts obtained from mice were cultured with dentin slices for 72 h, and then cells were fixed and the tartrate-resistant acid phosphatase (TRAP) activity was detected. Specimens were embedded in Epon, then TRAP-positive cells were serially sectioned by alternating semithin and ultrathin sections. The cells were examined by TEM and the three-dimensional structures were reconstructed by computer. By TEM, most TRAP-positive cells were resorbing osteoclasts with ruffled borders and a clear zone. There were osteoclasts without ruffled borders, and these cells had clear zone-like structures and lamellipodia. The three-dimensional reconstruction showed that resorbing osteoclasts had rounded contours and ring-shaped clear zones encircling ruffled borders, and that osteoclasts without ruffled borders had irregular and flat shapes; the clear zone-like structures showed a dot or patch-like distribution. The presence of lamellipodia of the osteoclasts without ruffled borders shows that the cells are migrating osteoclasts. These results suggest that dot or patch-like distribution is the feature of the clear zone of osteoclasts during migration, and that these structures play the role of focal contacts and adhesion to the dentin surfaces during cell migration.     

Gene expression and localization of diacylglycerol kinase isozymes in the rat spinal cord and dorsal root ganglia

The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKζ and DGKι in the DRG. By immunohistochemical analysis, DGKζ immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKι immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKζ in the nucleus and DGKι in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKζ between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.The work was supported by grants-in-aid from the Ministry of Education, Science, Culture, and Sports of Japan (M.T., K.G.) and from the Ono Medical Research Foundation, Kato Memorial Bioscience Foundation, and Janssen Pharmaceutical (K.G.) and by the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Supporting cell proliferation after hair cell injury in mature guinea pig cochlea in vivo

In cold-blooded animals, lost sensory hair cells can be replaced via a process of regenerative cell proliferation of epithelial supporting cells. In contrast, in mammalian cochlea, receptor (hair) cells are believed to be produced only during embryogenesis; after maturity, sensory or supporting cell proliferation or regeneration are thought to occur neither under normal conditions nor after trauma. Using bromodeoxyuridine (BrdU) as a proliferation marker, we have assessed cell proliferation activity in the mature organ of Corti in the cochlea of young guinea pigs following severe damage to the outer hair cells induced by kanamycin sulfate and ethacrynic acid. Although limited, we have found BrdU-labeled nuclei in the regions of Deiters cells when BrdU is given for 3 days or longer. When BrdU is given for 10 days, at least one labeled nucleus can be observed in the organ of Corti in approximately half of the ears; proliferating cells typically appear as paired daughters, with one nucleus being displaced away from the basement membrane to the position expected of the hair cells. Double-staining with antibodies to cytokeratin, vimentin, and p27 have shown that the BrdU-labeled nuclei are located in cells phenotypically similar to Deiters cells. Most of the uptake of BrdU occurs 3–5 days following ototoxic insult, and the number of BrdU-labeled cells does not decrease until 30 days following insult. These findings indicate that Deiters cells in the mature mammalian cochlea maintain a limited competence to re-enter the cell cycle and proliferate after hair cell injury, and that they can survive at least for 1 month.This work was supported by the Ministry of Health, Labour, and Welfare, Japan (grants 12120101, 15110201) and by the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant 13470357) to T.Y.     

Cellular and subcellular localization of kainic acid in the marine red alga Digenea simplex

Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as “granule bodies”, which were found only in the pericentral cells of the thallus. This research was supported by Ministry of Education, Culture, Sports, Science and Technology to R.S. (13660206).     

Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-μm and 15-μm paraffin-embedded tissue sections from prostatic carcinoma

 Interphase fluorescence in situ hybridization (FISH) was performed on 15-μm-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific α-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-μm sections with signal counts obtained from 5-μm sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (χ² test), we transferred the signal frequencies into a cytogenetic classification (−7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-μm-thick sections in all 19 tumors, polysomy 7 (>3 spots) in 18/19 cases, and monosomy 7 (−7) in 13/19 cases. In 5-μm sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-μm or 15-μm sections, the following discrepancies were noted: in 15-μm sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-μm sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-μm) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-μm) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers. Accepted: 7 November 1996     

Immunohistological studies of masked indoleamine cells in the area postrema of the rat

Summary Masked indoleamine cells (MICS) in the area postrema and adjacent areas in the rat were immunohistochemically studied (the peroxidase-antiperoxidase method) using a serotonin antiserum. After pretreatment with nialamide (200–300 mg/kg), immunoreactive MICS could be observed. They were small cells (about 12 m in diameter) with several processes and were distributed in nearly all parts of the area postrema and also in the nucleus tructus solitarii. Following a single intraventricular injection of 75 g 5,6-dihydroxytryptamine, the immunoreactivity of these cells conspicuously decreased for several days. The submicroscopical structure of the cells was investigated using immunoelectron microscopy. Immunoreactive products were observed in the cytoplasm as particles with a diameter of 25–40 nm and high electron density, but these were not found in the nucleus or cell organelles.This work was supported by a grant (No. 57214028) from the Ministry of Education, Science and Culture, Japan     

Regulatory considerations on transgenic livestock in Japan in relation to the Cartagena protocol

In Japan, the development and application of living modified organisms (LMOs) are regulated by law (conservation and sustainable use of biological diversity law). Procedures are classed as type 1 for the use of LMOs where no preventive measures against their dispersal into the environment are required and type 2 for the use of LMOs where preventive measures are stipulated. Development and research on transgenic livestock falls under the responsibility of the Ministry of Education, Culture, Science, Sports and Technology. Field use of transgenic livestock is controlled by the Ministry of Agriculture, Forestry and Fisheries. The author describes risk assessment and management of transgenic livestock by both ministries.     

Immunohistochemical demonstration of serotonin nerve fibers in the cat neurohypophysis

Summary Distribution of the serotonin nerve fibers in the neurohypophysis of adult cats was demonstrated using the peroxidase-antiperoxidase method. The serotonin nerve fibers were distributed in the internal and external layers of the infundibulum, terminating on the wall of the capillary loops and the outer surface of the external layer. A small number of the fibers scattered about in the posterior lobe penetrated the intercellular spaces in the intermediate lobe.Supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, JapanTo whom offprint requests should be sent     

VAMP2 is expressed in muscle satellite cells and up-regulated during muscle regeneration

Membrane trafficking is one of the most important mechanisms involved in the establishment and maintenance of the forms and functions of the cell. However, it is poorly understood in skeletal muscle cells. In this study, we have focused on vesicle-associated membrane proteins (VAMPs), which are components of the vesicle docking and fusion complex, and have performed immunostaining to investigate the expression of VAMPs in rat skeletal muscle tissue. We have found that VAMP2, but not VAMP1 or VAMP3, is expressed in satellite cells. VAMP2 is also expressed in myofibers in the soleus muscle and nerve endings. This is consistent with previous studies in which VAMP2 has been shown to regulate GLUT4 trafficking in slow-twitch myofibers in soleus muscle and neurotransmitter release in nerve endings. As satellite cells are quiescent myogenic cells, the expression of VAMP2 has further been examined in regenerating muscles after injury by the snake venom, cardiotoxin; we have observed enhanced expression of VAMP2 in immature myotubes with a peak at 3 days after injury. Our findings suggest that VAMP2 plays roles in quiescent satellite cells and is involved in muscle regeneration. The nature of the material transported in the VAMP2-bearing vesicles in satellite cells and myotubes is still under investigation. This work was supported by a research grant (17A-10) for nervous and mental disorders from the Ministry of Health, Labor, and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the Hydra GLW-amide neuropeptide family

The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH₂. To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons. This work was supported by grants from the Ministry of Education, Sports, and Culture, Japan.     

Distributional pattern of serotonin-immunoreactive nerve fibers in the lateral geniculate nucleus of the rat,cat and monkey (Macaca fuscata)

Summary The distribution of serotonin-containing nerve fibers in the lateral geniculate nucleus (LGN) of the rat, cat, and monkey (Macaca fuscata) was studied by use of the peroxidase-antiperoxidase method and an antiserum against serotonin. In all three species, the pattern of fibers was denser in the ventral portion of the LGN (LGNv) than in the dorsal nuclear portion (LGNd). In the LGNd of rat, serotonin-immunoreactive fibers were evenly distributed in the form of a dense network, but in cat and monkey there were marked regional differences. Serotonin-immunoreactive elements were most numerous in the C complex and medial interlaminal nucleus of cat, and in the S layer and interlaminar zones of Macaca fuscata.Supported by a grant from the Ministry of Education, Science, and Culture of Japan (No. 57214028)     

Custom-shaping system for bone regeneration by seeding marrow stromal cells onto a web-like biodegradable hybrid sheet

New bone for the repair or the restoration of the function of traumatized, damaged, or lost bone is a major clinical need, and bone tissue engineering has been heralded as an alternative strategy for regenerating bone. A novel web-like structured biodegradable hybrid sheet has been developed for bone tissue engineering by preparing knitted poly(DL-lactic-co-glycolic acid) sheets (PLGA sheets) with collagen microsponges in their openings. The PLGA skeleton facilitates the formation of the hybrid sheets into desired shapes, and the collagen microsponges in the pores of the PLGA sheet promote cell adhesion and uniform cell distribution throughout the sheet. A large number of osteoblasts established from marrow stroma adhere to the scaffolds and generate the desired-shaped bone in combination with these novel sheets. These results indicate that the web-like structured novel sheet shows promise for use as a tool for custom-shaped bone regeneration in basic research on osteogenesis and for the development of therapeutic applications.This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Health and Labour Sciences Research Grants (translational research), and the Organization for Pharmaceutical Safety and Research (to A.U.)     

Vascular endothelial growth factor inhibits the function of human mature dendritic cells mediated by VEGF receptor-2

Dendritic cells (DCs) are the most potent antigen-presenting cells and play a central role in the host-antitumor immunity. Since it has been reported that vascular endothelial growth factor (VEGF) inhibits the functional maturation of immature-DCs and impairs DC differentiation, it is important to elucidate the mechanisms of VEGF-induced DC-dysfunction. To investigate the effects of VEGF against human mature DCs, we investigated how VEGF affects mature DCs with regards to phenotype, induction of apoptosis, IL-12(p70) production and the antigen-presenting function evaluated by allogeneic mixed leukocyte reaction (allo-MLR). We generated monocyte-derived DCs matured with lipopolysaccharide, OK-432 or pro-inflammatory cytokine cocktails. As a result, VEGF treatment did not alter the mature DCs with regard to phenotype, IL-12(p70) production and induction of apoptosis. As a novel and important finding, VEGF inhibited the ability of mature DCs to stimulate allogeneic T cells. Furthermore, this VEGF-induced DC dysfunction was mainly mediated by VEGF receptor-2 (VEGF R2). These observations were confirmed by the findings that the VEGF-induced DC dysfunction was recovered by anti-human VEGF neutralizing mAb or anti-human VEGF R2 blocking mAb, and that placenta growth factor (PlGF), VEGF R1-specific ligand, did not have any effect against mature DCs. Some modalities aiming at reversing mature-DC dysfunction induced by VEGF will be needed in order to induce the effective antitumor immunity. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan.     

Isomermis bipapillatus n. sp. and Gastromermis mesostoma n. sp. (Mermithidae: Nematoda), parasites of Simulium japonicum (Simuliidae: Diptera) in Japan

Summary Two new species of the Mermithidae (Nematoda) are described from the Japanese blackfly Simulium japonicum Matsumura. They are the first representatives of Gastromermis and Isomermis from Japan. Gastromermis mesostoma n. sp. is characterized by a terminal or near terminal mouth, 6 hypodermal cords and the absence of cloacal or vulvar lips. Isomermis bipapillatus n. sp. is characterized by the small size of the spicules, their curved shape and by a pair of terminal hypodermal protuberances in the tail. This work was in part supported by a grant (No. 58570188) from the Japanese Ministry of Education, Science and Culture This work was in part supported by a grant (No. 58570188) from the Japanese Ministry of Education, Science and Culture     

Immunodetection of osteopontin at sites of resorption in the pulp of rat molars.

Osteopontin (OPN) has been proposed to act as a substrate for osteoclast adhesion during bone resorption. The aim of the present study was to examine the presence and distribution of OPN at sites of resorption in traumatized radicular pulp. The upper first molars of 6-week-old male Sprague-Dawley rats were luxated and then repositioned in the original sockets. The animals were sacrificed by intracardiac perfusion at 10 and 14 days after tooth reimplantation. The teeth were decalcified in EDTA and then processed for embedding in paraffin for histochemistry or LR White resin for immunocytochemistry. Odontoclasts were identified by their multinucleated morphology and expression of tartrate-resistant acid phosphatase (TRAP). Osteopontin was immunolocalized using postembedding colloidal gold labeling with a chicken egg yolk anti-rat OPN antibody. After reimplantation of the teeth, TRAP-positive cells were present along the pulp dentin wall. Osteopontin was not consistently detected at exposed predentin/dentin surfaces. However, gold particles were often found at the margin of resorption lacunae. Labeling was also seen over the Golgi region and cytoplasmic vesicles of odontoclasts and of neutrophils and fibroblast-like cells. The results suggest that accumulation of OPN at the predentin/dentin surface is not a prerequisite for adhesion of odontoclasts to the wall substance and that recruited odontoclasts produce OPN locally to mediate cell and/or matrix events within the resorption lacuna.     

Treatment of diabetic wounds with fetal murine mesenchymal stromal cells enhances wound closure

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF ^E6-EGFP cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF ^E6-EGFP cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF ^E6-EGFP cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF ^E6-EGFP cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF ^E6-EGFP cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies. This work was supported by a Grant-in Aid for the High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the AGU High-Tech Research Center Project, the 2003-Multidisciplinary Research Project from MEXT, and grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

The diversity of nuclear cycle in microcyclic rust fungi (Uredinales) and its ecological and evolutionary implications

 Nine types with 11 variations of nuclear cycle and associated metabasidium development were distinguished in microcyclic rust fungi. An additional type was recognized in rust fungi with an expanded life cycle. A significant proportion of rust fungi with a reduced life cycle is assumed to have lost a sexual genetic recombination process, being either apomictic or asexual in reproduction. Most species that retain a sexual process in the microcyclic life cycle seem to have become homothallic. During life cycle evolution by the omission of spore stages, these traits might have had a selective advantage for those species that had less opportunity to encounter a genetically different but sexually compatible mate because of isolated patchy distribution or a short growing season. The findings that different populations of a morphologically identifiable species exhibit two or more distinct patterns of nuclear cycle and different metabasidium development indicate that microcyclic lineages might have evolved independently and repeatedly from a macrocyclic parental species. Those lineages are morphologically the same but would differ from each other in their genetics and biology. Received: July 5, 2002 / Accepted: August 5, 2002 Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research (no. 09640744) from the Ministry of Education, Science and Culture (now the Ministry of Education, Culture, Sports, Science and Technology), Japan. Correspondence to:Y. Ono     

Immunohistochemical study of nuclear changes associated with male germ cell death and spermiogenesis

In a previous study on the effects of gestational and lactational exposure of para-nonylphenol on male rats, we noted in both induced and uninduced rats, that variations in cleaved caspase-3 immunostaining patterns were associated with distinct nuclear alterations in mainly basally located germ cells (spermatogonia and preleptotene spermatocytes). These were re-analysed and compared with cleaved caspase-3-labeled germ cells in the aging human and the spermatogenically active catfish testis. In the rat testes, cytoplasmic immunostaining was progressively associated with lateral compression of the nucleus, its break up into large pieces which can contain immunostained marginated chromatin masses. The pale remnants of the nucleus continued to shrink in size concomitant with the appearance of blue-purplish stained regions in the cytoplasm similar in color to the condensed chromatin in spermatids, a condition which was TUNEL-negative. These large clumps of chromatin also eventually disappeared, giving rise to cells resembling cytoplasmic ghosts, a condition which was TUNEL-positive. By contrast, the immunolabeled nuclei of human and catfish germ cells condensed into a single mass, after which they lost immunoreactivity. To exclude the possibility that these observations could reflect alterations in Sertoli nuclei, rat testicular sections were probed with a mouse anti-human GATA-4 monoclonal (MHM) antibody. The MHM was, however, the second of two GATA-4 antibodies tested, with a goat anti-mouse polyclonal (GMP) initially used to label the rat Sertoli nuclei. GMP unexpectedly, but distinctly labeled the complete development of the acrosome in the rat testis, a fortuitous finding with utility for staging of the seminiferous epithelium.     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum

Summary The localization of serotonin (5-HT)-immunoreactive nerve fibers in the cerebellum of the rat and cat was investigated by means of the peroxidase-anti-peroxidase (PAP) method using highly specific antibodies to 5-HT.Serotonin-containing nerve fibers were distributed throughout the entire cerebellum including the deep cerebellar nuclei, while 5-HT-positive neuronal somata were not detected in the cerebellum of either species. A different pattern of 5-HT innervation was found among the three layers of the cerebellar cortex. There were also interspecific differences in the pattern of distribution of 5-HT. In the rat, the pool of 5-HT nerve fibers mainly consisted of tangential elements, which were predominant in the molecular layer, while in the cat only a few 5-HT fibers were found in the molecular layer of the cerebellar cortex; dense networks of 5-HT nerve fibers were present in the granular layer. Some differences are evident in the pattern of distribution of 5-HT fibers in cerebellar regions classified on an anatomical and functional basis.This work was supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan