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Biomedical subjects

K Agata

Publications and source records attributed to K Agata.

At least 19 recordsLinked to original sources
PubMed2003

Intercalary regeneration in planarians.

How can a planarian regenerate its entire body from a small portion of its body? Neoblasts, the totipotent stem cells of planarian, are assumed to be able to produce all missing cell types. However, we do not know how the cell fate of these cells is controlled during regeneration. Our recent studies with molecular markers suggest that intercalary regeneration is the fundamental principle in planarian regeneration. Here, we introduce the intercalation induced by ectopic grafting along the anteroposterior (A-P), dorsoventral (D-V), and left-right (L-R) axes. Blastema formation is evoked by ectopic D-V interactions after wound closure. Intercalation between the blastema and stump induces rearrangement of the positional identities along the A-P axis. Consequently, totipotent stem cells change their differentiation patterns according to the newly rearranged positional identities along the A-P, D-V, and L-R axes. According to the classic view, the blastema is regarded as the place where undifferentiated cells accumulate and regenerative events occur. Here, we propose a new interpretation, i.e., that the blastema may work as a signaling center inducing intercalary regeneration. Also, the roles of molecules and genes involved in intercalary regeneration are discussed.

Animals↗
PubMed2001

Dorsal and ventral positional cues required for the onset of planarian regeneration may reside in differentiated cells.

We previously showed by grafting experiments that the dorsoventral (DV) interaction evokes morphogenetic events similar to those that occur in regeneration. However, it is not yet understood whether the stem cells themselves or differentiated cells have the ability to induce regeneration. Here we demonstrated by a combination of X-ray irradiation and grafting experiments that the dorsal and ventral positional cues inducing morphogenetic events are retained in X-ray-irradiated tissues, suggesting that the differentiated cells may be responsible for the positional cues. We grafted a small piece of irradiated worm, in which the stem cells were certainly eliminated, to an intact one in DV-reversed orientation. We observed that projections were developed from the host-donor boundary, as in the previous experiments. Whole-mount in situ hybridization with several markers demonstrated that the projections had a newly established DV axis and also had anterior or posterior characteristics. Furthermore, chimeric analysis with a strain-specific marker showed that the projections consisted of nonirradiated cells and that IFb-expressing cells, which normally belonged to the ventral tissue, could be generated even from the stem cells located on the dorsal side. Taken together, the findings suggest that the stem cells may simply differentiate depending on the surroundings and that differentiated cells may present positional cues that induce morphogenesis.

Animals↗
PubMed2001

Effects of brewer's yeast cell wall on constipation and defecation in experimentally constipated rats.

Brewer's yeast cell wall (BYC) was tested on constipated male Sprague-Dawley rats that had been induced by loperamide (2 mg/kg of body weight). The preventive effect of BYC on constipation was examined and compared with that of a non-fiber diet (NF) as the control. The dose-response of BYC and the effect on defecation by constipated experimental rats were also compared with the characteristics of cellulose diet (CE) group which served as a control. Defecation was observed to be greater by the rats fed with BYC than by those fed with NF or CE. The fecal water content and level of volatile fatty acids (VFA) in the cecal contents were likewise higher in the rats fed with BYC. These results indicate that the administration of BYC was effective for improving defecation and other parameters related to defecation. These favorable effects of BYC supplemented to the diet are attributed to the fermentation ability, water holding capacity and swelling force in the large intestine.

Antidiarrheals↗
PubMed2000

Planaria FoxA (HNF3) homologue is specifically expressed in the pharynx-forming cells.

We have isolated a planarian Forkhead box A (FoxA, a new name for a gene group containing HNF3 alpha,beta,gamma)-related gene, DjFoxA, and examined its spatial and temporal distribution in both intact and regenerating planarians by in situ hybridization. In intact worms, DjFoxA is specifically expressed in the cells participating in pharynx development in the region surrounding the pharynx, which is located in the central portion of the body. During regeneration, DjFoxA-positive cells appear in the pharynx-forming region and migrate to the midline to form a pharynx rudiment. These results suggest that DjFoxA is specifically expressed in the cells participating in pharynx formation and has an evolutionarily conserved function in digestive tract formation.

Amino Acid Sequence↗
PubMed1999

Neural network in planarian revealed by an antibody against planarian synaptotagmin homologue.

In order to investigate the neural connection of planarian, it is imperative to produce an antibody that specifically stains axons. To identify axon-specific genes, we constructed a cDNA library from a single eye by using a single cell PCR method, in which visual neurons are major components, and sequenced one thousand independent clones. We succeeded in the identification of a planarian homologue of synaptotagmin, Djsyt, whose specific expression in neurons was confirmed by in situ hybridization. The antibody against DjSYT specifically stained axons although its mRNA is distributed in the cell bodies. By using anti-DjSYT, we succeeded in the visualization of neural connections in planarians by whole mount staining. The anti-DjSYT antibody will become a powerful tool to analyze the molecular mechanisms underlying neural network formation in planarian.

Amino Acid Sequence↗
PubMed1999

The process of pharynx regeneration in planarians.

To understand the cellular events during planarian regeneration, we analyzed the process of pharynx regeneration in both head and tail pieces using cell-type-specific markers. Interestingly, cells expressing the pharynx-muscle-specific myosin heavy chain gene (DjMHC-A) appeared within 24 h after amputation (prior to the formation of a pharynx rudiment) in the mesenchymal space of the stump, not in the blastema region. These DjMHC-A-positive cells migrated to the midline and formed the pharynx rudiment. Even after formation of the pharynx rudiment, DjMHC-A-positive cells constantly appeared in the mesenchymal space in the region surrounding the pharynx rudiment and participated in the growth of the pharynx rudiment. These observations clearly indicated that the cells involved in pharynx-muscle formation are committed in the mesenchymal space of the stump, rather than in the blastema region or the pharynx rudiment during planarian regeneration. We also analyzed the process of regeneration of the pharynx epithelia using a monoclonal antibody and investigated the origin of the pharynx epithelia.

Animals↗
PubMed1999

The planarian HOM/HOX homeobox genes (Plox) expressed along the anteroposterior axis.

In the freshwater planarian Dugesia japonica, five cDNAs for HOM/HOX homeobox genes were cloned and sequenced. Together with sequence data on HOM/HOX homeobox genes of platyhelminthes deposited in databases, comparison of the deduced amino acid sequences revealed that planarians have at least seven HOM/HOX homeobox genes, Plox1 to Plox7 (planarian HOM/HOX homeobox genes). Whole-mount in situ hybridization and RT-PCR revealed that Plox4 and Plox5 were increasingly expressed along a spatial gradient in the posterior region of intact animals. During regeneration, Plox5 was expressed only in the posterior region of regenerating body pieces, suggesting that the gene is involved in the anteroposterior patterning in planarians. Plox5 was not found to be expressed in a blastema-specific manner, which contradicts a previous report (J. R. Bayascas, E. Castillo, A. M. Muños-Mármol, and E. Saló. Development 124, 141-148, 1997). X-ray irradiation experiments showed that Plox5 was expressed at least in some cells other than neoblasts, but that the induction of Plox5 expression during regeneration might require neoblasts.

Amino Acid Sequence↗
PubMed1999

Identification of a photoreceptor cell-specific nuclear receptor.

Nuclear receptors comprise a large and expanding family of transcription factors involved in diverse aspects of animal physiology and development, the functions of which can be modulated in a spatial and temporal manner by access to small lipophilic ligands and/or the specificity of their own localized expression. Here we report the identification of a human nuclear receptor that reveals a unique proximal box (CNGCSG) in the DNA-binding domain. The conservation of this feature in its nematode counterpart suggests the requirement for this type of P box in the genetic cascades mediated by nuclear receptors in a wide variety of animal species. The expression of this receptor, PNR (photoreceptor-specific nuclear receptor), appears strongly restricted in the retina, exclusively in photoreceptor cells. In human cell lines, PNR expression was observed in Y79 retinoblastoma along with other photoreceptor marker genes such as CRX. Among vertebrate receptors, PNR shares structural kinship with an orphan receptor TLX, and despite distinct differences in the DNA binding domain, PNR is able to recognize a subset of TLX target sequences in vitro. Analyses of the human PNR gene revealed its chromosomal position as 15q24, a site that has recently been reported as a susceptible region for retinal degeneration. These data support a role for PNR in the regulation of signalling pathways intrinsic to the photoreceptor cell function.

Amino Acid Sequence↗
PubMed1999

Expression of vasa(vas)-related genes in germline cells and totipotent somatic stem cells of planarians.

Planarians are known for their strong regenerative ability. This ability has been considered to reside in the totipotent somatic stem cell called the "neoblast." Neoblasts contain a unique cytoplasmic structure called the "chromatoid body," which has similar characteristics to the germline granules of germline cells of other animals. The chromatoid bodies decrease in number and size during cytodifferentiation and disappear in completely differentiated cells during regeneration. However, germ cells maintain the chromatoid body during their differentiation from neoblasts. These observations suggest that the chromatoid body is concerned with the totipotency of cells. To understand the molecular nature of the chromatoid body in the neoblast, we focused on vasa (vas)-related genes, since VAS and VAS-related proteins are known to be components of the germline granules in Drosophila and Caenorhabditis elegans. By PCR, two vas-related genes (Dugesia japonica vasa-like gene, DjvlgA and DjvlgB) were isolated, and they were shown to be expressed in germ cells. Interestingly, DjvlgA was also expressed in a number of somatic cells in the mesenchymal space. In regenerating planarians, accumulation of DjvlgA-expressing cells was observed in both the blastema and the blastema-proximal region. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjvlgA-expressing cells decreased drastically. These results suggest that the product of DjvlgA may be a component of the chromatoid body and may be involved in the totipotency of the neoblast.

Amino Acid Sequence↗
PubMed1999

Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes.

Homeobox genes such as orthodenticle in Drosophila and its mouse homologues, Otx1 and Otx2, are known to be essential for rostral brain development. To investigate the molecular basis of brain evolution, we searched for otd/Otx-related homeobox genes in the planarian Dugesia japonica, and identified two genes, DjotxA and B, whose expression appears to be restricted to the cephalic ganglion (brain). DjotxA was expressed more medially, in the region containing the termini of the visual axons, and in the visual cells, suggesting involvement in establishment of the visual system. DjotxB was expressed in a discrete region just lateral to the DjotxA-positive domain, but not in the more lateral branch structures, which in turn are characterized by the expression of Djotp, a planarian homeobox gene related to mouse Orthopedia (Otp). In transverse sections of planarians, DjotxA and B expression were observed only at the anterior ends of the stumps, corresponding to the regional pattern of the regenerating brain. Our findings suggest that the planarian brain is composed of structurally distinct and functionally diverse domains which are defined by the discrete expression of the three evolutionarily conserved homeobox genes.

Amino Acid Sequence↗
PubMed1999

Ectopic pharynxes arise by regional reorganization after anterior/posterior chimera in planarians.

To elucidate the mechanisms underlying pharynx regeneration in planarians, we transplanted pieces excised from various regions of the body into the prepharyngeal or postpharyngeal region, since it has been shown that such transplantation experiments can induce ectopic pharynx formation. We confirmed the ectopic formation of pharynxes by expression of the myosin heavy chain gene specific to pharynx muscles (DjMHC-A). To investigate the cellular events after grafting, we also stained such transplanted worms by in situ hybridization using neuronal cell- and mucous producing cell-type-specific marker genes which can detect formation of brain and prepharyngeal region, respectively. When the head piece was transplanted into the tail region, ectopic formation of the head, prepharyngeal and pharynx region was observed in the postpharyngeal region anterior to the graft, while these organs were formed in the reversed polarity along the anterior-posterior (A-P) axis. Furthermore, in the tail region posterior to the graft, ectopic formation of the prepharyngeal and pharynx region was observed. In the reverse combination, when a tail piece was transplanted into the prepharyngeal region, ectopic formation of prepharyngeal and pharynx region was observed in the region between the head and the graft, and an additional ectopic pharynx was also formed in reverse polarity in the region between the graft and host pharynx. These results clearly indicated that ectopic pharynxes were formed as a consequence of the regional reorganization induced by interaction between the host and graft. Furthermore, chimeric analyses demonstrated that the cells participating in ectopic pharynx formation were not exclusively derived from the host or donor cells in the worm, suggesting that the stem cells of the host and donor may change their differentiation pattern due to altered regionality. To further investigate if regional reorganization is induced after grafting, expression of a Hox gene was analyzed in the transplanted worms by whole-mount in situ hybridization. The expression of the Hox gene along the A-P axis was apparently rearranged after grafting of the head piece into the tail region. These results suggest that grafting of the head piece may rearrange the regionality of the host tail, and that stem cells in the region newly defined as pharynx-forming may start to regenerate a pharynx.

Animals↗
PubMed1999

Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH.

Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).

Animals↗
PubMed1999

Cytogenetic assignment of 29 functional genes to chicken microchromosomes by FISH.

We assigned 29 functional genes to chicken microchromosomes by fluorescence in situ hybridization (FISH). Two linkage groups in the genetic linkage map of the East Lansing breed were identified in this study by localizing the genes AGRN and H2FA to microchromosomes. The frequency of the genes mapped on 30 pairs of microchromosomes, which account for roughly 30% of the whole chicken genome, was about 40% of the 73 genes randomly mapped in our laboratory. This result confirms the important role of microchromosomes for avian genome function and supports the likelihood of a high gene density on avian microchromosomes.

Animals↗
PubMed1999

The role of dorsoventral interaction in the onset of planarian regeneration.

During planarian regeneration, dorsal and ventral tissues adhere to each other as a result of wound closure. To investigate the role of dorsoventral (DV) interaction in regeneration, dorsal and ventral portions were ectopically reoriented relative to each other by grafting. We sucked a small piece of planarian body into a Pasteur pipette, and then grafted the piece to the original position in DV-reversed orientation. 4 days after grafting, a blastema-like white region was formed on the boundary between the host and the graft. The region grew and formed a cup-shaped projection on both the dorsal and ventral sides of the worm after about 1 week. Histological analysis and in situ hybridization using dorsal and ventral markers revealed that the grafted piece maintained its original DV polarity. Analysis using DV boundary markers clearly indicated that a DV axis was newly established within the projection. Chimeric analysis using a strain-specific marker demonstrated that the projection grew on the DV boundary. Further analysis, using a marker specific for neoblasts and differentiating cells, showed that the projection was composed of neoblasts and/or neoblast-derived cells. Thus, ectopic DV interaction can cause blastema formation, outgrowth and establishment of a DV axis. In addition, staining with tissue- and region-specific markers showed that the projection appears to be identical to the structure from the grafted position to the most anterior or posterior tip. These results suggest that DV interaction evoked by wound closure has a important role in the onset of regeneration, especially for blastema formation and establishment of the DV axis during planarian regeneration.

Animals↗
PubMed1999

Increased growth of Bifidobacterium and Eubacterium by germinated barley foodstuff, accompanied by enhanced butyrate production in healthy volunteers.

Germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley mainly consists of low-lignified hemicellulose and glutamine-rich protein. GBF improves the proliferation of intestinal epithelial cells and defecation, through the bacterial production of short chain fatty acids (SCFA), especially butyrate. In this study we investigated the mechanism of production of butyrate by microflora in humans and in vitro. Daily administration of 9 g GBF for 14 successive days significantly increased fecal butyrate content. Fecal Bifidobacterium and Eubacterium were also significantly increased by GBF administration in healthy volunteers. Ten anaerobic micro-organisms selected from intestinal microflora were cultured in vitro in the medium containing GBF as a sole carbon source (GBF medium). After a 3-day incubation, 7 strains (Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Bacteroides ovatus, Clostridium butyricum, and Eubacterium limosum) lowered the medium pH producing SCFA. Eubacterium grown together with Bifidobacterium in GBF medium efficiently produced butyrate. On the other hand, GBF changed the intestinal microflora and increased probiotics such as Bifidobacterium in the intestinal tract. As a result, butyrate was produced by the mutual action of Eubacterium and Bifidobacterium. This butyrate is considered to enhance the proliferation of colonic epithelial cells.

Adult↗
PubMed1998

Identification of a receptor tyrosine kinase involved in germ cell differentiation in planarians.

To investigate external signals involved in germ cell differentiation from somatic stem cells, we have tried to identify protein kinases whose expression is regulated during the process of sexualization of asexual-state planarians. It is known that in planarians germ cells differentiate from totipotent somatic stem cells called "neoblasts" during sexualization. As a first step, we have isolated twelve protein kinase genes from cDNAs of sexual-state planarians, including three non-receptor tyrosine kinases, three receptor-tyrosine kinases and three non-receptor serine/threonine kinases, and then analyzed their expression patterns during sexualization. One of them, the DjPTK1 gene, is specifically expressed in germ cells of sexual-state planarians. DjPTK1-positive cells were also detected in the mesenchymal space during the process of sexualization, and it appears that these cells migrate to the dorsal side and then differentiate into spermatogonia/spermatocytes in testis. Sequence analysis indicated that the DjPTK1 gene encodes a receptor protein tyrosine kinase belonging to the FGFR/PDGF family. These results suggest that a receptor tyrosine kinase system may be involved both at an early stage of germ cell differentiation and in a step of germ cell maturation in planarians.

Amino Acid Sequence↗