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Biomedical subjects

Akira Kudo

Publications and source records attributed to Akira Kudo.

At least 37 records · Page 2Linked to original sources
PubMed2004

Twist functions in vertebral column formation in medaka, Oryzias latipes.

Medaka twist, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the sclerotome during embryogenesis. We previously established a line of twist-EGFP transgenic medaka, whose EGFP expression is regulated by the twist promoter; therefore, we could observe the behavior of sclerotomal cells in vivo. In the transgenic medaka embryos, EGFP-positive sclerotomal cells migrated dorsally around the notochord and the neural tube, where at a later stage the vertebral column would be formed. This finding strongly suggests that twist-expressing sclerotomal cells participate in vertebral column formation in medaka. To clarify the function of twist gene in the sclerotome, we performed knockdown analysis of twist by using two kinds of morpholino antisense oligonucleotides targeted against twist (MO1 and MO2). Both the MO1 and MO2 morphants exhibited absence of neural arches, which are bilaterally paired, dorsomedially oriented bones on the dorsal aspect of the centrum. In addition, MO2, which blocks translation of only endogenous twist mRNA in the twist-EGFP transgenic medaka, did not affect the migration pattern of EGFP-positive cells, revealing that the migration of sclerotome-derived cells were normal in the absence of twist gene function. These results demonstrate that medaka twist functions in vertebral column formation by regulating the sclerotomal cell differentiation.

Amino Acid Sequence↗
PubMed2004

Large-scale analysis of the genes involved in fin regeneration and blastema formation in the medaka, Oryzias latipes.

Medaka is an attractive model to study epimorphic regeneration. The fins have remarkable regenerative capacity and are replaced about 14 days after amputation. The formation of blastema, a mass of undifferentiated cells, is essential for regeneration; however, the molecular mechanisms are incompletely defined. To identify the genes required for fin regeneration, especially for blastema formation, we constructed cDNA libraries from fin regenerates at 3 days postamputation and 10 days postamputation. A total of 16,866 expression sequence tags (ESTs) were sequenced and subjected to BLASTX analysis. The result revealed that about 60% of them showed strong matches to previously identified proteins, and major signaling molecules related to development, including FGF, BMP, Wnt, Notch/Delta, and Ephrin/Eph signaling pathways were isolated. To identify novel genes that showed specific expression during fin regeneration, cDNA microarray was generated based on 2900 independent ESTs from each library which had no sequence similarity to known proteins. We obtained 6 candidate genes associated with blastema formation by gene expression pattern screening in competitive hybridization analyses and in situ hybridization. Olrfe16d23 and olrfe14k04 were expressed only in early regenerating stages when blastema formation was induced. The expression of olrf5n23, which encodes a novel signal peptide, was detected in wound epidermis throughout regeneration. Olrfe23l22, olrfe20n22, and olrfe24i02 were expressed notably in the blastema region. Our study has thus identified the gene expression profiles and some novel candidate genes to facilitate elucidation of the molecular mechanisms of fin regeneration.

Animals↗
PubMed2004

A mutation in the gene for delta-aminolevulinic acid dehydratase (ALAD) causes hypochromic anemia in the medaka, Oryzias latipes.

A genetic screen for mutations affecting embryogenesis in the medaka, Oryzias latipes, identified a mutant, whiteout (who), that exhibited hypochromic anemia. The who mutant initially had the normal number of blood cells, but it then gradually decreased during the embryonic and larval stages. The blood cells in the who mutants show an elongated morphology and little hemoglobin activity. Genetic mapping localized who to the vicinity of a LG12 marker, olgc1. By utilizing the highly conserved synteny between medaka and pufferfish, we identified a gene for delta-aminolevulinic acid dehydratase (ALAD), which is the second enzyme in the heme synthetic pathway, as a candidate for who. We found a missense mutation in the alad gene that was tightly linked to the who phenotype, strongly suggesting that the hypochromic anemia phenotype in the who mutant is caused by a loss of the alad function. Thus, who mutants represent a model for the human disease ALAD-deficiency porphyria.

Anemia, Hypochromic↗
PubMed2004

Characterization of mutations affecting embryonic hematopoiesis in the medaka, Oryzias latipes.

In a genetic screen for mutations affecting organogenesis in the medaka, Oryzias latipes, we identified eight mutants with defects in embryonic hematopoiesis. These mutations were classified into seven complementation groups. In this paper, we characterize the five mutants that were confirmed in the next generation. The beni fuji mutant was defective in the generation of blood cells, exhibiting reduced blood cells at the initiation of circulation. Mutations in two genes, lady finger and ryogyoku, caused abnormal morphology of blood cells, i.e., deformation, along with a progressive decrease in the number of blood cells. The sekirei mutant exhibited photosensitivity with autofluorescent blood cells. Mutations in kyoho resulted in huge blood cells that were approximately three times longer than the wild-type blood cells. The spectrum of phenotypes identified in this study is similar to that of the zebrafish hematopoietic mutants except for the huge blood cells in kyoho. Our results demonstrate that medaka, as well as zebrafish, is a useful model to study hematopoiesis.

Animals↗
PubMed2004

The novel medaka transglutaminase gene is expressed in developing yolk veins.

The vascular system is highly conserved in all vertebrates in the aspects of anatomy as well as in the genetic mechanism governing it. The embryo of the medaka, Oryzias latipes is small and transparent, providing many advantages for the experimental analysis of the vertebrate vascular system. We isolated a novel medaka transglutaminase gene, termed embryonic transglutaminase, and found that it showed the highest homology to the coagulation factor XIII A subunit of mammals. This gene is expressed in the anterior lateral plate mesoderm, and then expressed specifically in yolk veins consisting two ducts of Cuvier and the vitellocaudal vein. Our data is the first finding that a coagulation factor XIII-like gene is expressed in the early vascular development of vertebrates.

Amino Acid Sequence↗
PubMed2004

The mechanism of sperm-oocyte fusion in mammals.

Sperm-oocyte fusion is one of the most impressive events in sexual reproduction, and the elucidation of its molecular mechanism has fascinated researchers for a long time. Because of the limitation of materials and difficulties in analyzing membrane protein-protein interactions, many attempts have failed to reach this goal. Recent studies involving gene targeting have clearly demonstrated the various molecules that are involved in sperm-oocyte binding and fusion. Sperm ADAMs (family of proteins with a disintegrin and metalloprotease domain), including fertilin alpha, fertilin beta and cyritestin, have been investigated and found to be important for binding rather than for fusion and painstaking studies have raised suspicions that their putative receptors, oocyte integrins, are necessary for the sperm-oocyte interaction. Recently, several studies have focused the spotlight on CD9 and glycosylphosphatidylinositol (GPI)-anchored proteins on oocytes, and epididymal protein DE on sperm, as candidate molecules involved in sperm-oocyte fusion. Lack of, or interference with the function of, these proteins can disrupt the sperm-oocyte fusion without changing the binding. In this review we highlight the candidate molecules involved in the sperm-oocyte interaction suggested from the recent progress in this research field.

Animals↗
PubMed2003

A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport.

Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2003

The divergent expression of periostin mRNA in the periodontal ligament during experimental tooth movement.

A novel 90-kDa protein named periostin, which is preferentially expressed in the periosteum and the periodontal ligament (PDL), may play a role in bone metabolism and remodeling. However, the precise role of periostin in the PDL remains unclear. Therefore, we examined the expression of periostin mRNA during experimental tooth movement. Experimental tooth movement was achieved in 7-week-old male Sprague-Dawley rats. In control specimens without tooth movement, the expression of periostin mRNA was uniformly observed in the PDL surrounding the mesial and distal roots of the upper molars and was weak in the PDL of the root furcation area. The periostin mRNA-expressing cells were mainly fibroblastic cells in the PDL and osteoblastic cells on the alveolar bone surfaces. The divergent expression of periostin mRNA in the PDL began to be observed at 3 h and continued up to 96 h after tooth movement. The maximum changes, which showed stronger staining in the pressure sites than in the tension sites, were observed at 24 h. The expression of periostin mRNA in the PDL 168 h after tooth movement exhibited a similar distribution to that of the control specimens. These results suggest that periostin is one of the local contributing factors in bone and periodontal tissue remodeling following mechanical stress during experimental tooth movement.

Animals↗
PubMed2003

Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo.

Osteosarcoma by nature shows aggressive pulmonary metastasis; however, the underlying molecular mechanisms remain unclear. We previously showed that N-cadherin and cadherin-11 (OB-cadherin), which are highly expressed in normal osteoblasts, are anomalously expressed in human osteosarcoma (Kashima et al., Am J Pathol 1999;155:1549-55). In the present study, we examined the role of cadherins in osteosarcoma metastasis using the mouse osteosarcoma cell line Dunn and its highly metastatic subline LM8. Oligonucleotide array and RT-PCR analyses demonstrated that Dunn and LM8 cells did not express appreciable levels of several members of the cadherin family, and Western blot analysis confirmed that Dunn and LM8 cells did not express P-cadherin, E-cadherin, N-cadherin or cadherin-11 protein. We therefore investigated the functional consequences of cadherin overexpression on cell migration and in vivo metastatic potential of LM8 cells. Several LM8 clones were isolated which expressed exogenous N-cadherin and cadherin-11 localized to the cell membrane and able to bind to beta-catenin. Overexpression of N-cadherin or cadherin-11 in LM8 cells did not affect cell proliferation but caused an inhibitory effect on cell migration in vitro. In vivo analysis showed that N-cadherin- and cadherin-11-overexpressing cells exhibited a marked reduction in their ability to form pulmonary metastases, with significant decreases in lung weight and the number and weight of metastatic lesions, as well as the size and weight of primary lesions at the s.c.-inoculated site. These observations demonstrate that disruption of N-cadherin- and cadherin-11-mediated cell-cell adhesion is critical in the pulmonary metastasis of osteosarcoma.

Animals↗
PubMed2003

EBF-regulating Pax5 transcription is enhanced by STAT5 in the early stage of B cells.

Pax5 is an essential transcription factor for B cell development, and it is reported that Pax5 expression was reduced in the IL-7 receptor (IL-7R) knockout mouse. To investigate whether signals from the IL-7R regulate Pax5 transcription, we searched the consensus sequence of signal transducers and activators of transcription (STAT) in the Pax5 promoter region, since STAT is one of the components of cytokine signal transduction. A STAT-binding motif, termed SBM, was identified at 1,118 bp upstream of the transcriptional start site, and SBM completely overlapped with the binding site for early B cell factor (EBF). STAT5 was phosphorylated in the presence of IL-7 in the IL-7-dependent preB cell line, PreBR1, and phosphorylated-STAT5 as well as EBF was found to bind to the SBM. Moreover, we also revealed STAT5 binding to SBM in PreBR1 cells by chromatin immunoprecipitation assay. Transient co-transfection of reporter genes together with expression vectors of a constitutive active form of STAT5 and EBF into NIH3T3 cells demonstrated that STAT5 enhanced EBF-regulating transcription. Our results suggest that STAT5 phosphorylated by IL-7 can directly up-regulate Pax5 transcription in early B cells.

Animals↗
PubMed2003

TRANCE together with IL-7 induces pre-B cells to proliferate.

TRANCE (TNF-related activation-induced cytokine)-deficient mice completely lack osteoclasts, and develop severe osteopetrosis. These mice also show a defect in their pre-B cell differentiation. In the present study, the expression of TRANCE was examined in pre-B cell lines using flow cytometry and reverse transcription-PCR. Three pre-B cell lines, 18-81, B3P816-1, and 38B9, expressed TRANCE on their surface, and two pre-B cell lines, 7OZ/3 and NFS5, at the late pre-B cell stage, expressed it at low levels, although their mRNA expression was normal. Another pre-B cell line, 38-C-13, at the intermediate stage between pre-B and immature B cells, did not express TRANCE. The IL-7-dependent pre-B cell line PreBR, which expresses the pre-B cell receptor on the cell surface, also expressed TRANCE. When differentiation of PreBR cells was induced in vitro by removing IL-7 from cultures, TRANCE expression dropped; it was restored by the addition of IL-7, suggesting that TRANCE functions in cooperation with IL-7. To examine the function of TRANCE, we introduced the TRANCE gene into PreBR cells and established two transfectants that constitutively expressed TRANCE, even in the absence of IL-7. In these transfectants, after removal of IL-7, the number of cells that succeeded in kappa chain rearrangement was decreased to one third; and CD40 expression decreased to less than one tenth. Moreover, the percentage of cells in the S/G2/M phase was increased by 50% over the mock transfectant. These findings indicate that, before kappa chain rearrangement occurs, TRANCE together with IL-7 induces pre-B cells to proliferate and makes this rearrangement more efficient.

Animals↗
PubMed2003

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8.

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.

Amino Acid Sequence↗
PubMed2003

Osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in DAP12-deficient mice.

Deletions in the DAP12 gene in humans result in Nasu-Hakola disease, characterized by a combination of bone fractures and psychotic symptoms similar to schizophrenia, rapidly progressing to presenile dementia. However, it is not known why these disorders develop upon deficiency in DAP12, an immunoreceptor signal activator protein initially identified in the immune system. Here we show that DAP12-deficient (DAP12(-/-)) mice develop an increased bone mass (osteopetrosis) and a reduction of myelin (hypomyelinosis) accentuated in the thalamus. In vitro osteoclast induction from DAP12(-/-) bone marrow cells yielded immature cells with attenuated bone resorption activity. Moreover, immature oligodendrocytes were arrested in the vicinity of the thalamus, suggesting that the primary defects in DAP12(-/-) mice are the developmental arrest of osteoclasts and oligodendrocytes. In addition, the mutant mice also showed synaptic degeneration, impaired prepulse inhibition, which is commonly observed in several neuropsychiatric diseases in humans including schizophrenia, and aberrant electrophysiological profiles in the thalami. These results provide a molecular basis for a unique combination of skeletal and psychotic characteristics of Nasu-Hakola disease as well as for schizophrenia and presenile dementia.

Alleles↗
PubMed2003

TRAF5 functions in both RANKL- and TNFalpha-induced osteoclastogenesis.

Although TRAF6 is essential for both RANKL- and TNFalpha-induced osteoclastogenesis, it has remained unclear whether other members of the TRAF family are involved in osteoclastogenesis. We examined TRAF5 function in both RANKL- and TNFalpha-induced osteoclastogenesis by using osteoclast progenitor cells from TRAF5-deficient mice. The results demonstrated that RANKL or TNFalpha did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c-jun N-terminal kinase (JNK) and NF-kappaB activation was apparently observed in osteoclast progenitor cells. In the parathyroid hormone (PTH)-induced hypercalcemia model, calcium concentration peaked at day 3 after administration. However, in TRAF5-deficient mice, this peak was delayed and found at day 5, showing less effective osteoclast differentiation. Thus, we have provided the first evidence showing that TRAF5 is involved in osteoclastogenesis.

Animals↗
PubMed2003

Osteoclastogenesis-related antigen, a novel molecule on mouse stromal cells, regulates osteoclastogenesis.

Osteoclastogenesis is regulated by RANKL expressed on stromal cells. In this study, we sought to isolate a new surface molecule regulating osteoclastogenesis on stromal cells by generating monoclonal antibodies. A rat was immunized with the mouse stromal cell line, TSB13, which can support osteoclastogenesis, and a monoclonal antibody, A15-1, was obtained. A15-1 bound to a surface antigen on TSB13 cells, termed osteoclastogenesis-related antigen (OCRA), and immunoprecipitation with this antibody revealed that OCRA was a 220-kDa molecule. By means of flow cytometry, the A15-1 antigen (OCRA) was found to be expressed on various mesenchymal cell lines but not on hematopoietic cell lines, and the expression level of OCRA on the TSB13 cells was slightly increased by treatment with 1alpha,25(OH)2D3. When osteoclast progenitors and TSB13 cells were co-cultured in the presence of 1alpha,25(OH)2D3, the addition of A15-1 inhibited osteoclast differentiation in a dose-dependent manner; however, no significant inhibition of soluble RANKL-induced osteoclastogenesis was observed, suggesting that A15-1 inhibited only stromal cell-dependent osteoclastogenesis. The same inhibitory effect of A15-1 was also observed when primary bone marrow-derived stromal cells were used. The osteoclastogenesis-promoting effects of other osteotropic factors, such as parathyroid hormone (PTH) and interleukin (IL)-1beta, were also inhibited by A15-1. Time-course analysis of osteoclast differentiation in vitro indicated that the initial 2 days of treatment with A15-1 was sufficient for inhibition, suggesting that A15-1 inhibits the early stages of osteoclast differentiation. Finally, we investigated the in vivo effects of A15-1 on PTH-induced hypercalcemia in mice. Treatment with A15-1 significantly decreased the osteoclast surface in the PTH-administered mice. Taken together, our data indicate that OCRA, a novel A15-1-detected antigen, regulates stromal cell-dependent osteoclastogenesis.

Animals↗
PubMed2002

An alternatively spliced cadherin-11 enhances human breast cancer cell invasion.

Although reduced levels of the epithelial cell adhesion molecule E-cadherin are often associated with poorly differentiated breast cancers, recent studies show that expression of other cadherins such as N-cadherin, P-cadherin, and the mesenchymal cadherin-11 is actually elevated in invasive breast cancers and cell lines. Cadherin-11 is unique among cadherins in that it exists as two alternatively spliced forms that are expressed together in the same cell. We now show that expression of wild-type cadherin-11, with or without coexpression of the COOH-terminal truncated splice variant, promotes epithelial differentiation of the cadherin-negative SKBR3 cell line. Exogenous wild-type cadherin-11 association with and membrane recruitment of beta-catenin and p120 are unaffected by coexpression of the truncated variant. Cadherin-11-expressing cells exhibit modest changes in cell proliferation and no change in anchorage-independent growth. However, coexpression of wild-type cadherin-11 and the splice variant promotes a dramatic increase in the ability of SKBR3 cells and E-cadherin-positive MCF7 cells to traverse Matrigel-coated filters. Biochemical studies indicate that the truncated variant may be secreted from the cell and/or enter a detergent-insoluble compartment. These data suggest that the presence of the cadherin-11 splice variant promotes invasion of cadherin-11-positive breast cancer cells.

Adherens Junctions↗
PubMed2002

Infertility of CD9-deficient mouse eggs is reversed by mouse CD9, human CD9, or mouse CD81; polyadenylated mRNA injection developed for molecular analysis of sperm-egg fusion.

CD9 is a membrane protein belonging to the tetraspanin family. Despite CD9's broad tissue distribution, the only abnormality observed in CD9-deficient mice was infertility of females, which was responsible for a defect in the sperm-egg fusion process. However, the function of CD9 in sperm-egg fusion is not clear at all because the technique to analyze the activity of molecules in sperm-egg fusion has not been established. We demonstrated that the exogenous mouse CD9, expressed by polyadenylated mRNA injection at the germinal-vesicle stage oocytes, was precisely localized to the egg plasma membrane, and the expression reversed the infertility of CD9(-/-) eggs. Then, two other tetraspanins, human CD9 and mouse CD81, overexpressed with this technique on CD9(-/-) eggs restored the fertilization rate up to approximately 90 and approximately 50% against that of wild type eggs, respectively. Moreover, in the presence of an anti-mouse CD9 mAb, which blocks sperm-egg fusion, expression of human CD9 or mouse CD81 on eggs also rescued the fusibility. These results suggested that human CD9 plays a crucial role in human fertilization, and mouse CD81 has the potential to compensate for CD9 function in sperm-egg fusion. In addition, the polyadenylated mRNA injection is effective for molecular analysis of sperm-egg fusion.

Animals↗