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Tadaaki Moritomo

Publications and source records attributed to Tadaaki Moritomo.

At least 19 recordsLinked to original sources
PubMed2006

Expression analysis of two Eomesodermin homologues in zebrafish lymphoid tissues and cells.

Eomesodermin (Eomes) is a T-box transcription factor that is involved in mesoderm formation in most vertebrates. Eomes is also expressed in CD8+ T cells and NK cells. No information is available on the role of Eomes in the immune system of lower vertebrates to date, although developmental studies on Eomes (Eomes1) have been performed in zebrafish. Here we report the identification of a second Eomes (Eomes2) in zebrafish and compare expression of the two Eomes genes in the immune system. Zebrafish Eomes1 and Eomes2, composed of 661 and 534 amino acids, respectively, share 49.3% amino acid identity in their coding regions and 88.7% amino acid identity in their T-box regions. Conserved synteny between regions of the human and zebrafish genomes, gene organization and phylogenetic analysis all indicate that the zebrafish Eomes2 gene is a homologue of mammalian Eomes, as previously found for zebrafish Eomes1. Eomes1 mRNA was found to be expressed in the gonad, body kidney, spleen and gill, while Eomes2 mRNA was not detected in any of these tissues. However, strong expression of both Eomes mRNAs was detected in the leukocytes from the spleen, followed by those from body kidney and peripheral blood, with expression of Eomes1 always stronger than that of Eomes2. RT-PCR analysis of body kidney cells sorted by FACS revealed that Eomes1 was expressed strongly in lymphocytes, weakly in blast cells, and was not expressed in granulocytes, while Eomes2 was expressed weakly in lymphocytes. These results suggest that both Eomes genes are involved in the zebrafish immune response, particularly in lymphocyte function as has been found in mammals.

Amino Acid Sequence↗
PubMed2006

Isolation of side population cells from ginbuna carp (Carassius auratus langsdorfii) kidney hematopoietic tissues.

Side population (SP) cells, characterized by a specific Hoechst dye efflux pattern by flow cytometry were isolated from kidney hematopoietic tissues of ginbuna carp (Carassius auratus langsdorfii). The hematopoietic activity of SP cells was evaluated by the repopulation and multilineage potential using an in vivo transplant system of ginbuna carp (donor) and ginbuna-goldfish hybrids (recipient). In a flow cytometric (FCM) analysis, a small and distinct population of ginbuna SP cells displayed efficient effluxes of Hoechst 33342 was virtually identical to the efflux observed in mammalian SP cells. The frequency of the ginbuna SP cells was 0.17+/-0.08% in the kidney hematopoietic cells. Morphologically, SP cells were composed of small lymphocyte-like cells having a thin-layered cytoplasm and a round nucleus. These characteristics of ginbuna SP cells were very similar to those of mammalian SP cells. Since cyprinid fish have two hematopoietic sites, the head (anterior) and body (posterior, trunk) kidney, the distribution of SP cells were examined in head and body kidney. The proportion of SP cells were 0.33+/-0.15% in the body kidney, but near 0% in the head kidney. After the ginbuna SP cells were injected into ginbuna-goldfish hybrids, the major types of donor-derived cells (erythrocytes, neutrophils, basophils, monocytes, thrombocytes, T and B lymphocytes) were detected in the recipient blood over a long period (9-16 months post-transplantation). In ginbuna carp, SP cells reside in the body kidney and contain primitive populations of hematopoietic stem cells (HSCs).

Acridine Orange↗
PubMed2006

Detection of DNA closely related to 'Candidatus Rickettsia principis' in Haemaphysalis danieli recovered from cattle in Xinjiang Uygur Autonomous Region Area, China.

Tick DNA samples from cattle in Xinjiang Uygur Autonomous Region Area, China, were examined for Rickettsia infection by citrate synthase gene-based PCR and sequencing. Four positive samples were detected from Haemaphysalis danieli and high levels of similarity were found with recently detected 'Candidatus Rickettsia principis.'

Animals↗
PubMed2006

Demonstration of hematopoietic stem cells in ginbuna carp (Carassius auratus langsdorfii) kidney.

A model system of ginbuna carp (clonal triploid fish, Carassius auratus langsdorfii) and ginbuna-goldfish hybrids (tetraploid fish, having three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish) was employed to demonstrate the presence of hematopoietic stem cells (HSCs) in kidney hematopoietic tissues. Kidney hematopoietic cells were obtained from ginbuna carp (S3n, donor) and injected into a ginbuna-gold fish hybrid (S4n, recipient) via the caudal sinus. The percentage of S3n cells in the S4n blood was measured by flow cytometry by the difference of DNA content from 3 to 18 months after the injection. Donor-derived major types of blood cells including erythrocytes, thrombocytes, neutrophils, basophils, monocytes, T and B lymphocytes were detected in the recipient blood over a long period (9-18 month posttransplantation). Moreover, recipient lympho-hematopoietic organs such as head kidney, body kidney, spleen, and thymus were reconstituted by donor cells. These results indicate that long-term multipotent repopulating HSCs reside in the donor kidney.

Acridine Orange↗
PubMed2005

Molecular cloning and tissue expression of canine Toll-like receptor 2 (TLR2).

Toll-like receptor 2 (TLR2) is involved in an important role on inflammatory reactions against various pathogenic microorganisms. Although some investigations suggested a relation between human and murine TLR2 gene polymorphisms and infections or inflammatory reactions, canine TLR2 gene has not been investigated. In the present study, therefore, the canine full-length of cDNA of Toll-like receptor 2 gene was sequenced and the expression of canine TLR2 mRNA was investigated in various tissues. Canine TLR2 clone was 2552bp encoding 785 amino acids. The similarities of canine TLR2 with human, mouse, rat, cattle, horse and pig TLR2 were 87.6, 72.9, 73.2, 83.1, 85.1 and 80.7% at the nucleotide sequence level, and 84.6, 68.7, 68.5, 78.8, 82.9 and 79.0% at amino acid sequence level, respectively. By a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis, mRNA of canine TLR2 was detected in blood mononuclear cells (BMCs), lymph node, lung, liver, spleen, bladder, pancreas, small intestine, large intestine and skin of the dogs.

Amino Acid Sequence↗
PubMed2005

Characteristics of NADPH oxidase genes (Nox2, p22, p47, and p67) and Nox4 gene expressed in blood cells of juvenile Ciona intestinalis.

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.

Amino Acid Sequence↗
PubMed2005

In vitro effect of recombinant human granulocyte colony-stimulating factor on canine neutrophil apoptosis.

Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.

Animals↗
PubMed2005

Cloning of canine toll-like receptor 9 and its expression in dog tissues.

Toll-like receptor 9 (TLR9) is activated by bacterial DNA and induces production of inflammatory cytokines. In this study, canine TLR9 cDNA was cloned and sequenced. Further, the expression of TLR9 mRNA was investigated in canine tissues. The full-length cDNA for the canine TLR9 gene was 3419bp encoding 1032 amino acids. The similarity of canine TLR9 with those of cat, cattle, human, mouse and pig was 90, 84.3, 83.6, 76.3 and 85.2% at the nucleotide level, and 90.5, 82.7, 81.4, 74.8 and 85.7% at the amino acid level. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, canine TLR9 mRNA was detected in venous blood mononuclear cells and lymph node and weakly in spleen, whereas it was not detected in kidney, liver, pancreas, lung, small intestine, large intestine, heart, skin, muscle, stomach and bladder.

Amino Acid Sequence↗
PubMed2005

Comparison of respiratory burst activity of inflammatory neutrophils in ayu (Plecoglossus altivelis) and carp (Cyprinus carpio).

Neutrophils of ayu (Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp (Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

Animals↗
PubMed2005

Characterisation of T cell antigen receptor alpha chain isotypes in the common carp.

T cell receptor alpha (TCRalpha) chain has been characterised in several teleost species to date. Here, a reverse transcription-polymerase chain reaction (RT-PCR) strategy was used to isolate cDNA clones encoding TCRalpha chain from an individual of the common carp (Cyprinus carpio.L.). The Valpha sequences identified were most similar to Valpha of other teleosts, and could be classified into as many as 14 Valpha families. For the Jalpha sequences, diversity comparable to that seen in other teleosts could be identified, and the J-region motif was well conserved. The Calpha sequences demonstrated the highest similarity to zebrafish Calpha and possessed a well-conserved transmembrane (TM) region. Two Calpha isotypes with a complete C region were obtained, designated Calpha1 and Calpha2, with approximately 70% similarity at the amino acid level ( approximately 85% identity at the nucleotide level), and, in addition, Calpha2 contained two unique sequences, designated Calpha2a and Calpha2b, with 93% similarity (96% identity). Therefore, the results obtained using an individual clearly showed that carp possesses at least two Calpha loci, possibly as a result of tetraploidisation.

Amino Acid Sequence↗
PubMed2005

Counting absolute number of lymphocytes in quail whole blood by flow cytometry.

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.

Animals↗
PubMed2004

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization.

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP3alpha1 and MIP3alpha2 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP3alpha1 and MIP3alpha2 contain four exons and three introns, and MIP3alpha1 and MIP3alpha2 shared the same intron/exon organization with that of human. The MIP3alpha1 and MIP3alpha2 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP3alpha2 gene was much lower than the Trsc-SCYA107 and MIP3alpha1 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP3alpha1 and MIP3alpha2 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP3alpha2 genes in PWBCs was observed at 1-12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP3alpha1 expression was observed at 3-12 h poststimulation only with PMA.

Amino Acid Sequence↗
PubMed2004

Cell culture of clonal ginbuna crucian carp hematopoietic cells: differentiation of cultured cells into erythrocytes in vivo.

We cultivated kidney hematopoietic cells from clonal triploid ginbuna crucian carp, Carassius auratus langsdorfii. Proliferating cells from hematopoietic cell cultures were harvested and injected into tetraploid hybrids (clonal triploid ginbuna and goldfish hybrid) which possess three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish (Carassius auratus). After injection of cultured triploid cells (donor cells), blood samples were obtained from tetraploid hybrids (recipients) every other week. Blood cells stained with acridine orange were measured by flow cytometry to trace the injected donor cells by means of differences in DNA content. Although erythrocytes were not produced in donor cell cultures, such cultures maintained precursor cells capable of differentiation into erythrocytes in vivo. After 4-12 weeks of transplantation, mature erythrocytes derived from the donors were observed in the blood circulation of the recipient fish. These results indicated that the ginbuna hematopoietic cell culture system is an in vivo situation suitable for the study of hematopoietic control mechanisms.

Animals↗
PubMed2004

Molecular cloning and sequencing of Japanese pufferfish (Takifugu rubripes) NADPH oxidase cDNAs.

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91phox and p22phox, and cytosolic proteins, p40phox, p47phox and p67phox. In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox clones contained open reading frames encoding 565, 186, 348, 423 and 495 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the pufferfish gp91phox, p22phox, p40phox, p47phox and p67phox sequences shared 68.0, 61.8, 53.8, 54.7 and 41.9% identity with those of human components, respectively. gp91phox has three potential N-linked glycosylation sites. gp91phox and p22phox have six and three hydrophobic regions, respectively, that are predicted to be transmembrane regions. p47phox and p67phox have two potential Src homology 3 domains and p40phox has one. The functional domains are highly conserved in many animals, though the sequence of the components of the pufferfish showed low homology with that of mammals. The Fugu NADPH oxidase genes were expressed in various tissues of unstimulated fish. The level of gp91phox, p47phox and p67phox expression were high only in the blood and kidney, while p22phox and p40phox were constitutively expressed in a wide range of tissues. These results suggest that Japanese pufferfish NADPH oxidase components possess functional activities similar to those of human.

Amino Acid Sequence↗
PubMed2004

Characterisation of a fourth immunoglobulin light chain isotype in the common carp.

Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp (Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription-polymerase chain reaction (RT-PCR) method including 5'- and 3'-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5'- and 3'-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences.

Amino Acid Sequence↗
PubMed2004

Serodiagnosis of Babesia gibsoni infection in dogs by an improved enzyme-linked immunosorbent assay with recombinant truncated P50.

The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-length), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliable reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.

Animals↗
PubMed2003

Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats.

In the present study, full length of canine and feline Toll-like receptor 4 (TLR4) cDNAs were sequenced, and the expression of canine and feline TLR4 mRNAs in dog and cat tissues were investigated. The full-length cDNA of TLR4 of dog and cat was 2709 bp encoding 637 amino acids and 3113 bp encoding 833 amino acids, respectively. The similarity of canine and feline TLR4 were 83.6% at the nucleotide sequence level and 77.6% at the amino acid sequence level. At the amino acid sequence level, canine and feline TLR4 showed sequence similarities of approximately 62-78% with those of Homo sapiens, Mus musculus, Bos taurus and Equus caballus, respectively. Southern hybridization analyses with TLR4 cDNA probes gave one distinct band in BamHI, EcoRI and HindIII digests of genomic DNA from dogs and cats, respectively, indicating the likely presence of a single TLR4 gene in each species. By RT-PCR analysis, mRNA of canine TLR4 was expressed highly in peripheral blood leukocytes (PBL), moderately in spleen, stomach and small intestine, at low levels in liver, with no expression in kidney, large intestine and skin. On the other hand, mRNA of feline TLR4 was expressed highly in lung, bladder and PBL, moderately in kidney, liver, spleen and large intestine and at low levels in pancreas and small intestine.

Amino Acid Sequence↗