Biomedical subjects

Yasuo Kitagawa

Publications and source records attributed to Yasuo Kitagawa.

At least 19 recordsLinked to original sources

PubMed2007

Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells.

Matrin 3, a nuclear matrix protein has potential (1) to withhold promiscuously edited RNAs within the nucleus in cooperation with p54(nrb) and PSF, (2) to mediate NMDA-induced neuronal death, and (3) to modulate promoter activity of genes proximal to matrix/scaffold attachment region (MAR/SAR). We identified a bipartite nuclear localization signal (NLS) of chicken matrin 3 (cmatr3) at residues 583-602. By expressing green fluorescent protein (GFP) fused to the NLS mutant in chicken DT40 cells, we showed an essential role of the NLS for cell proliferation. Furthermore, we showed that both clusters of basic amino acids and a linker of the bipartite NLS were essential and sufficient for the nuclear import of GFP. Exogenous cmatr3 rescued the HeLa cells where human matrin 3 was suppressed by RNA interference, but cmatr3 containing deletions at either of the basic amino acid clusters or the linker could not.

Active Transport, Cell Nucleus↗

PubMed2006

The Sp family of transcription factors regulates the human laminin alpha1 gene in JAR choriocarcinoma cells.

Laminin-111 (alpha1beta1gamma1) is the major component of the embryonic and extra-embryonic basement membrane. The laminin alpha1 chain shows a restricted and developmentally regulated expression in basement membranes of distinct epithelial tissues while beta1 and gamma1 chains have a wide tissue distribution. To understand how human laminin alpha1 chain expression is controlled, we cloned and characterized the 5'-flanking region of the human laminin alpha1 (LAMA1) gene. Transfection studies using serially deleted promoter constructs and JAR choriocarcinoma cells revealed that the minimal promoter fragment resided in the +31 to -206 region, which contains a number of GC- and GT/A-rich motifs for the binding of the Sp family of transcription factors. Electrophoretic mobility shift assays and mutational analyses revealed that Sp1 and Sp3 bound specifically to these elements and are important for the promoter activity. Furthermore, we showed that Krüppel-like factors KLF4 and KLF6 also activate transcription of the human LAMA1 gene. Chromatin immunoprecipitation analysis demonstrated recruitment of these transcription factors to the promoter region. These results indicate that transcription of the human LAMA1 gene is controlled by a combination of the actions of Sp1/Sp3 and Krüppel-like factors, KLF4 and KLF6.

Animals↗

PubMed2006

Identification and expression of alternative splice variants of the mouse Ppp1r3b gene in lung epithelial cells.

Hepatic glycogen-targeting subunit GL of protein phosphatase-1 (PP1), which is encoded by the gene Ppp1r3b, is one of a family of proteins that target PP1 to glycogen and regulate its activity on enzymes of glycogen metabolism. GL is primarily expressed in the liver where it promotes hepatic glycogen synthesis in response to insulin. Here, we show that GL is highly expressed in embryonic mouse lungs. GL utilizes two alternative promoters and 5' non-coding exons, which produce at least three alternatively spliced transcripts that encode identical proteins. Expression of GL in the lung is detectable as early as embryonic day (E) 12.5 and increases by E16.5, however, it declines before birth. In situ hybridization of mouse embryonic lung revealed that GL is expressed in bronchial epithelial cells. Thus, the temporal and spatial expression of GL in the lung suggests that it has a potential role in glycogen metabolism during lung development.

Alternative Splicing↗

PubMed2005

Conservation of novel Mahya genes shows the existence of neural functions common between Hymenoptera and Deuterostome.

Honeybees have been shown to exhibit cognitive performances that were thought to be specific to some vertebrates. However, the molecular and cellular mechanisms of such cognitive abilities of the bees have not been understood. We have identified a novel gene, Mahya, expressed in the brain of the honeybee, Apis mellifera, and other Hymenoptera. Mahya orthologues are present in Deuterostomes but are absent or highly diverged in nematodes and, intriguingly, in two dipteran insects (fruit fly and mosquito) and Lepidoptera (silk moth). Mahya genes encode novel secretory proteins with a follistatin-like domain (Kazal-type serine/threonine protease inhibitor domain and EF-hand calcium-binding domain), two immunoglobulin domains, and a C-terminal novel domain. Honeybee Mahya is expressed in the mushroom bodies and antennal lobes of the brain. Zebra fish Mahya orthologues are expressed in the olfactory bulb, telencephalon, habenula, optic tectum, and cerebellum of the brain. Mouse Mahya orthologues are expressed in the olfactory bulb, hippocampus, and cerebellum of the brain. These results suggest that Mahya may be involved in learning and memory and in processing of sensory information in Hymenoptera and vertebrates. Furthermore, the limited existence of Mahya in the genomes of Hymenoptera and Deuterostomes supports the hypothesis that the genes typically represented by Mahya were lost or highly diverged during the evolution of the central nervous system of specific Bilaterian branches under the specific selection and subsequent adaptation associated with different ecologies and life histories.

Animals↗

PubMed2005

Isolation of two human fibroblastic cell populations with multiple but distinct potential of mesenchymal differentiation by ceiling culture of mature fat cells from subcutaneous adipose tissue.

Adipose tissue is a source of adult multipotent stem cells that can differentiate along mesenchymal lineage. When mature fat cells obtained from human subcutaneous adipose tissue were maintained with attachment to the ceiling surface of culture flasks filled with medium, two fibroblastic cell populations appeared at the ceiling and the bottom surface. Both populations were positive to CD13, CD90, and CD105, moderately positive to CD9, CD166, and CD54, negative to CD31. CD34, CD66b, CD106, and CD117, exhibited potential of unlimited proliferation, and differentiated along mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes. The population that appeared at the ceiling surface showed higher potential of adipogenic differentiation. These observations showed that the cells tightly attached to mature fat cells can generate two fibroblastic cell populations with multiple but distinct potential of differentiation. Since enough number of both populations for clinical transplantation can be easily obtained by maintaining fat cells from a small amount of subcutaneous adipose tissue, this method has an advantage in preparing autologous cells for patients needing repair of damaged tissues by reconstructive therapy.

Adipose Tissue↗

PubMed2004

Syndecan-dependent binding of Drosophila hemocytes to laminin alpha3/5 chain LG4-5 modules: potential role in sessile hemocyte islets formation.

Heparin-column chromatography and elastase-digestion of medium from hemocyte Kc167 gave Drosophila laminin alpha3/5betagamma trimer, alpha3/5LG2-3 and alpha3/5LG4-5 modules with eluting NaCl concentrations of 450, 280 and 450 mM, respectively. Kc167 cells bound dish surface with alpha3/5betagamma trimer or alpha3/5LG4-5, but not with alpha3/5LG2-3 modules. Cell binding was counteracted by treating with heparin or heparan sulfate. RNA interference of syndecan in Kc167 cells impaired the binding, but that of dally or dally-like did not. Green fluorescent protein-expressing hemocytes also bound surface with alpha3/5betagamma trimer or alpha3/5LG4-5 module. Thus, syndecan-dependent binding of hemocytes to laminin may have a potential role in sessile hemocytes islets formation in T2-A8 segments of Drosophila.

Amino Acid Sequence↗

PubMed2004

Application of RNA interference to chicken embryos using small interfering RNA.

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.

Animals↗

PubMed2004

Mammalian and Drosophila cells adhere to the laminin alpha4 LG4 domain through syndecans, but not glypicans.

We have previously shown that the LG4 (laminin G-like) domain of the laminin alpha4 chain is responsible for the significantly higher affinity of the alpha4 chain to heparin than found for other alpha chains [Yamaguchi, Yamashita, Mori, Okazaki, Nomizu, Beck and Kitagawa (2000) J. Biol. Chem. 275, 29458-29465]; four basic residues were identified to be essential for this activity [Yamashita, Beck and Kitagawa (2004) J. Mol. Biol. 335, 1145-1149]. By creating GST (glutathione S-transferase)-fused LG1, LG2, LG4 and LG5 proteins, we found that only LG4 is active for the adhesion of human HT1080 cells, human umbilical vein endothelial cells and Drosophila haemocytes Kc167 with a half-saturating concentration of 20 microg/ml. Adhesion was counteracted by treatment of the cells with heparin, heparan sulphate and heparitinase I. Upon mutating the four basic residues essential for heparin binding within LG4, the adhesion activity was abolished. Pull-down experiments using glutathione beads/GST-fusion proteins indicate a direct interaction of LG4 with syndecan-4, which might be the major receptor for cell adhesion. Neither the release of glypican-1 by treating human cells with phosphatidylinositol-specific phospholipase C nor targeted knockdown of dally or dally-like protein impaired the cell-adhesion activity. As the LG4-LG5 domain of the alpha4 chain is cleaved in vivo from the main body of laminin-8 (alpha4beta1gamma1), we suggest that the heparan sulphate proteoglycan-binding activity of LG4 is significant in modulating the signalling of Wnt, Decapentaplegic and fibroblast growth factors.

Amino Acid Sequence↗

PubMed2004

Characterization of the two distinct subtypes of metabotropic glutamate receptors from honeybee, Apis mellifera.

L-Glutamate is a major neurotransmitter at the excitatory synapses in the vertebrate brain. It is also the excitatory neurotransmitter at neuromuscular junctions in insects, however its functions in their brains remain to be established. We identified and characterized two different subtypes (AmGluRA and AmGluRB) of metabotropic glutamate receptors (mGluRs) from an eusocial insect, honeybee. Both AmGluRA and AmGluRB form homodimers independently on disulfide bonds, and bind [3H]glutamate with K(D) values of 156.7 and 80.7 nM, respectively. AmGluRB is specifically expressed in the brain, while AmGluRA is expressed in the brain and other body parts, suggesting that AmGluRA is also present at the neuromuscular junctions. Both mGluRs are expressed in the mushroom bodies and the brain regions of honeybees, where motor neurons are clustered. Their expression in the brain apparently overlaps, suggesting that they may interact with each other to modulate the glutamatergic neurotransmission.

Amino Acid Sequence↗

PubMed2004

Heparin binds to the laminin alpha4 chain LG4 domain at a site different from that found for other laminins.

We previously reported that the LG4 domain of the laminin alpha4 chain is responsible for high-affinity heparin binding. To specify the amino acid residues involved in this activity, we produced a series of alpha4 LG4-fusion proteins in which each of the 27 basic residues (arginine, R; histidine; lysine, K) were replaced one by one with alanine (A). When the effective residues R1520A, K1531A, K1533A, and K1539A are mapped on a structural model, they form a track on the concave surface of the beta-sandwich, suggesting that they interact with adjacent sulfate groups along the heparin chain. Whereas low-affinity heparin-binding sites of other LG domains have been located at the top of the beta-sheet sandwich opposite the N and C termini, the residues for high-affinity heparin binding of alpha4 LG4 reveal a new topological area of the LG module.

Amino Acid Sequence↗

PubMed2004

The changes of gene expression in honeybee (Apis mellifera) brains associated with ages.

Honeybee (Apis mellifera) worker bees (workers) are known to perform wide variety of tasks depending on their ages. The worker's brains also show the activity and behavior-dependent chemical and structural plasticity. To test if there are any changes of gene expression associated with different ages in the worker brains, we compared the gene expression patterns between the brains of newly emerged bees and old foraging workers (foragers) by macroarray analysis. The expression of genes encoding signal transduction pathway components, ion channels, and neurotransmitter transporters is elevated in the old forager brains, suggesting that the neuronal activities would be enhanced. The mRNA levels of cell adhesion protein, transcription related factors, and plasma membrane associated proteins are also increased in the old forager brains. Meanwhile, the mRNA level of one putative cell adhesion protein is decreased in the old forager brains. These results thus suggest that the dramatic changes of gene expression occur in honeybee brains associated with ages.

Age Factors↗

PubMed2003

Drosophila hemolectin gene is expressed in embryonic and larval hemocytes and its knock down causes bleeding defects.

We have previously identified and characterized Drosophila hemolectin (Hml) in the conditioned medium of a Drosophila cell line. The deduced amino acids sequence contains a number of domains conserved in human von Willebrand factor, coagulation factor V/VIII, and complement factors. To characterize Hml localization and function, we have established two transgenic lines (hml-GAL4 and UAS-hmlRNAi). By crossing hml-GAL4 with UAS-eGFP, we observed that Hml is specifically expressed in embryonic and in larval hemocytes. We determined that Hml is expressed in a subpopulation of plasmatocytes and crystal cells but not in lamellocytes. hml RNAi larvae are viable and do not display obvious developmental defects under normal conditions. However, they show a bleeding defect upon injury. We confirmed that the failure to seal a wound is not due to a defect in melanin production or in phenoloxidase activity. The expression of antimicrobial peptides was not significantly affected on hml RNAi adults. Altogether, our data strongly suggest that Hml is involved in hemostasis and/or coagulation in Drosophila larvae.

Animals↗

PubMed2003

Syndecan binding sites in the laminin alpha1 chain G domain.

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.

Amino Acid Sequence↗

PubMed2003

Functional domains of chicken mitochondrial transcription factor A for the maintenance of mitochondrial DNA copy number in lymphoma cell line DT40.

Nuclear and mitochondrial (mt) forms of chicken mt transcription factor A (c-TFAM) generated by alternative splicing of a gene (c-tfam) were cloned. c-tfam mapped at 6q1.1-q1.2 has similar exon/intron organization as mouse tfam except that the first exons encoding the nuclear and the mt form-specific sequences were positioned oppositely. When cDNA encoding the nuclear form was transiently expressed in chicken lymphoma DT40 cells after tagging at the C terminus with c-Myc, the product was localized into nucleus, whereas the only endogenous mt form of DT40 cells was immunostained exclusively within mitochondria. c-TFAM is most similar to Xenopus (xl-) TFAM in having extended C-terminal regions in addition to two high mobility group (HMG) boxes, a linker region between them, and a C-terminal tail, also found in human and mouse TFAM. Similarities between c- and xl-TFAM are higher in linker and C-terminal regions than in HMG boxes. Disruption of both tfam alleles in DT40 cells prevented proliferation. The tfam+/tfam- cells showed a 50 and 40-60% reduction of mtDNA and its transcripts, respectively. Expression of exogenous wild type c-tfam cDNA in the tfam+/tfam- cells increased mtDNA up to 4-fold in a dose-dependent manner, whereas its transcripts increased only marginally. A deletion mutant lacking the first HMG box lost this activity, whereas only marginal reduction of the activity was observed in a deletion mutant at the second HMG box. Despite the essential role of the C-terminal tail in mtDNA transcription demonstrated in vitro, deletion of c-TFAM at this region reduced the activity of maintenance of the mtDNA level only by 50%. A series of deletion mutant at the tail region suggested stimulatory and suppressive sequences in this region for the maintenance of mtDNA level.

Amino Acid Sequence↗

PubMed2003

Misexpression of mouse porcupine isoforms modulates the differentiation of P19 embryonic carcinoma cells.

Drosophila porcupine (porc) encodes an ER membrane protein that is required for the processing of the Drosophila Wnt family. Homologs of porc have been identified in various multicellular organisms and have been implicated in the biosynthesis of Wnt proteins. In contrast to Drosophila, vertebrates generate four different porc mRNAs (A-D) by alternative splicing. Murine porcD (MporcD) mRNA levels transiently increase during the neuroectodermal differentiation of P19 cells, but diminish during mesodermal differentiation. P19 cells constitutively expressing mouse porcA (MporcA), but not MporcD, undergo apoptosis by the induction of neuroectodermal differentiation. Meanwhile, P19 cells constitutively expressing MporcD, but not MporcA, do not adopt mesodermal cell morphology and fail to express myf-5 when induced to mesodermal differentiation. These results therefore demonstrate that the alternative splicing of Mporc is regulated in a cell-type specific manner, and the resulting Mporc isoforms have different functions in the neuroectodermal and mesodermal differentiation of P19 cells.

Acyltransferases↗

PubMed2003

The expression of genes encoding visual components is regulated by a circadian clock, light environment and age in the honeybee (Apis mellifera).

The honeybee, Apis mellifera, has been used as a model to study the development of the visual system and adult bee behaviour. However, the regulation of the levels of visual component genes has never been addressed in this organism. We isolated honeybee cDNAs encoding green-sensitive opsin and visual arrestin and then measured their mRNA levels in honeybee workers. Both mRNAs fluctuate on a daily cycle that depends on a pacemaker that functions separately from the pacemaker which controls rhythmic locomotor activity. The cycling-patterns of opsin and arrestin mRNAs are different from each other and are modified by light. Furthermore, light exposure can increase the absolute levels of both mRNAs and the arrestin mRNA level is also dependent on age. Consistent with these results, both mRNA levels are higher in foragers than in in-hive bees under natural conditions. This study thus shows that the expression of genes encoding visual components is regulated by multiple factors and is adjusted to the honeybees' need for vision during the day, and throughout their lives. Comparison of data obtained with honeybees and other organisms indicates that there is a link between the regulation of phototransduction components and vision-related animal behaviour.

Aging↗

PubMed2002

Identification of biologically active sequences in the laminin alpha 4 chain G domain.

Laminins are a family of trimeric extracellular matrix proteins consisting of alpha, beta, and gamma chains. So far five different laminin alpha chains have been identified. The laminin alpha 4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin alpha 4 chain. Here we have focused on the biological functions of the laminin alpha 4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-alpha 4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-alpha 4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-alpha 4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-alpha 4G. These results suggest that the four active sites are involved in the biological functions of the laminin alpha 4 chain G domain. Furthermore, rec-alpha 4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.

Amino Acid Sequence↗

PubMed2002

Drosophila segment polarity gene product porcupine stimulates the posttranslational N-glycosylation of wingless in the endoplasmic reticulum.

Wnt is a family of cysteine-rich secreted glycoproteins, which controls the fate and behavior of the cells in multicellular organisms. In the absence of Drosophila segment polarity gene porcupine (porc), which encodes an endoplasmic reticulum (ER) multispanning transmembrane protein, the N-glycosylation of Wingless (Wg), one of Drosophila Wnt family, is impaired. In contrast, the ectopic expression of porc stimulates the N-glycosylation of both endogenously and exogenously expressed Wg. The N-glycosylation of Wg in the ER occurs posttranslationally, while in the presence of dithiothreitol, it efficiently occurs cotranslationally. Thus, the cotranslational disulfide bond formation of Wg competes with the N-glycosylation by an oligosaccharyl transferase complex. Porc binds the N-terminal 24-amino acid domain (residues 83-106) of Wg, which is highly conserved in the Wnt family and stimulates the N-glycosylation at surrounding sites. Porc is also necessary for the processing of Drosophila Wnt-3/5 in both embryos and cultured cells. Thus, Porc binds the N-terminal specific domain of the Wnt family and stimulates its posttranslational N-glycosylation by anchoring them at the ER membrane possibly through acylation.

Acylation↗