Search PubMed⌕ Search

Biomedical subjects

H Okano

Publications and source records attributed to H Okano.

At least 73 records · Page 4Linked to original sources
PubMed2000

Targeted expression of baculovirus p35 caspase inhibitor in oligodendrocytes protects mice against autoimmune-mediated demyelination.

The mechanisms underlying oligodendrocyte (OLG) loss and the precise roles played by OLG death in human demyelinating diseases such as multiple sclerosis (MS), and in the rodent model of MS, experimental autoimmune encephalomyelitis (EAE), remain to be elucidated. To clarify the involvement of OLG death in EAE, we have generated transgenic mice that express the baculovirus anti-apoptotic protein p35 in OLGs through the Cre-loxP system. OLGs from cre/p35 transgenic mice were resistant to tumor necrosis factor-alpha-, anti-Fas antibody- and interferon-gamma-induced cell death. cre/p35 transgenic mice were resistant to EAE induction by immunization with the myelin oligodendrocyte glycoprotein. The numbers of infiltrating T cells and macrophages/microglia in the EAE lesions were significantly reduced, as were the numbers of apoptotic OLGs expressing the activated form of caspase-3. Thus, inhibition of apoptosis in OLGs by p35 expression alleviated the severity of the neurological manifestations observed in autoimmune demyelinating diseases.

Animals↗
PubMed2000

Drob-1, a Drosophila member of the Bcl-2/CED-9 family that promotes cell death.

The Bcl-2/CED-9 family of proteins, which includes both antiapoptotic and proapoptotic members, plays key regulating roles in programmed cell death. We report here the identification and characterization of Drob-1, the first Drosophila member of the Bcl-2/CED-9 family to be isolated. Drob-1 contains four conserved Bcl-2 homology domains (BH1, BH2, BH3, and BH4) and a C-terminal hydrophobic domain. Ectopic expression of Drob-1 in the developing Drosophila eye resulted in a rough-eye phenotype. Furthermore, when overexpressed in Drosophila S2 cells, Drob-1 induced apoptosis accompanied by elevated caspase activity. This Drob-1-induced cell death, however, could not be antagonized by baculovirus p35, a broad-spectrum caspase inhibitor. Drob-1 was localized to the intracytoplasmic membranes, predominantly to the mitochondrial membranes, and a mutant Drob-1 lacking the hydrophobic C terminus lost both its mitochondrial localization and its proapoptotic activity. These results suggest that Drob-1 promotes cell death by inducing both caspase-dependent and -independent pathways at the mitochondria. Our identification of Drob-1 and further genetic analysis should provide increased understanding of the universal mechanisms by which the Bcl-2/CED-9 family members and other related proteins regulate apoptosis.

Amino Acid Sequence↗
PubMed2000

The bHLH gene hes1 as a repressor of the neuronal commitment of CNS stem cells.

Hes1 is one of the basic helix-loop-helix transcription factors that regulate mammalian CNS development, and its loss- and gain-of-function phenotypes indicate that it negatively regulates neuronal differentiation. Here we report that Hes1(-/-) mice expressed both early (TuJ1 and Hu) and late (MAP2 and Neurofilament) neuronal markers prematurely, and that there were approximately twice the normal number of neurons in the Hes1(-/-) brain during early neural development. However, immunochemical analyses of sections and dissociated cells using neural progenitor markers, including nestin, failed to detect any changes in Hes1(-/-) progenitor population. Therefore, further characterization of neural progenitor cells that discriminated between multipotent and monopotent cells was performed using two culture methods, low-density culture, and a neurosphere assay. We demonstrate that the self-renewal activity of multipotent progenitor cells was reduced in the Hes1(-/-) brain, and that their subsequent commitment to the neuronal lineage was accelerated. The Hes1(-/-) neuronal progenitor cells were functionally abnormal, in that they divided, on average, only once, and then generated two neurons, (instead of one progenitor cell and one neuron), whereas wild-type progenitor cells divided more. In addition, some Hes1(-/-) progenitors followed an apoptotic fate. The overproduction of neurons in the early Hes1(-/-) brains may reflect this premature and immediate generation of neurons as well as a net increase in the number of neuronal progenitor cells. Taken together, we conclude that Hes1 is important for maintaining the self-renewing ability of progenitors and for repressing the commitment of multipotent progenitor cells to a neuronal fate, which is critical for the correct number of neurons to be produced and for the establishment of normal neuronal function.

Animals↗
PubMed2000

GAL4/UAS-WGA system as a powerful tool for tracing Drosophila transsynaptic neural pathways.

Visualization of specific transsynaptic neural pathways is an indispensable technique for understanding the relationship between structure and function in the nervous system. Here, we demonstrate the application of the wheat germ agglutinin (WGA) transgene technique for tracing transsynaptic neural pathways in Drosophila. The intracellular localization of WGA was examined by immunoelectron microscopy. WGA signals were detected in granule-like structures in both the outer photoreceptor cells expressing WGA and the second-order laminar neurons. Misexpression of tetanus toxin (TNT), which inactivates N-synaptobrevin, in the outer photoreceptor cells resulted in the elimination of on/off transients in electroretinogram (ERG) recordings and in a great reduction in WGA transfer into laminar neurons, suggesting that anterograde WGA transsynaptic transfer is dependent mainly on synaptic transmission. Retrograde WGA transfer was also detected upon its forced expression in muscle cells. WGA primarily expressed in muscle cells was taken up by motoneuron axons and transported to their cell bodies in the ventral nerve cord, suggesting that WGA can trace motoneuronal pathways in combination with the muscle-specific GAL4 driver. Thus, the GAL4/UAS-WGA system should facilitate the dissection of the Drosophila neural circuit formation and/or synaptic activity in various regions and at various developmental stages.

Animals↗
PubMed2000

Role of caspase-1 subfamily in cytotoxic cytokine-induced oligodendrocyte cell death.

Oligodendrocytes are myelin forming cells in mammalian central nervous system. About 50% of oligodendrocytes (OLGs) undergo cell death in normal development. In addition, OLG cell deaths have been observed in demyelinating diseases including multiple sclerosis (MS). Clinical observations and in vitro cell culture studies have suggested that cytokines mediate OLG cell damage in multiple sclerosis (MS). Among the cytokines, tumor necrosis factor (TNF) is thought to be one of the mediators responsible for the damage of OLGs in MS. The administration of TNF-alpha to primary cultures of OLGs induced DNA fragmentation, and significantly decreased the number of live OLGs. Chemical inhibitors Ac-YVAD-CHO (a specific inhibitor of caspase-1 (ICE)-like proteases) enhanced the survival of TNF-alpha treated OLGs better than Ac-DEVD-CHO (a specific inhibitor of caspase-3 (CPP32)-like proteases). These results indicate that caspase-1-mediated cell-death pathway are activated in TNF-induced OLG cell death. Caspase-11 is involved in activation of caspase-1. Oligodendrocytes from caspase-11-deficient mice are partially resistant to TNF-induced OLG cell death. Our results suggest that the inhibition of caspase-1 sufamily may be a novel therapeutic approach to treat MS.

Animals↗
PubMed2000

Expression patterns of musashi homologs of the ascidians, Halocynthia roretzi and Ciona intestinalis.

The gene family encoding RNA-binding proteins includes important regulators involved in the neurogenesis in both protostomes and deuterostomes. We isolated cDNAs of the ascidian homolog of one of the RNA-binding proteins, MUSASHI, from Halocynthia roretzi and Ciona intestinalis. The predicted amino acid sequences contained two RNA-recognition and RNA-binding motifs in the N-terminus and an ascidian-specific YG-rich domain in the C-terminus. Maternal transcripts of musashi were ubiquitous in early cleavage-stage embryos. Ascidian musashi had three domains of zygotic expression: the brain, nerve cord, and mesenchyma. The temporal order of the onset in these domains was highly divergent between the two species of ascidian examined.

Amino Acid Sequence↗
PubMed2000

Dissection of signaling cascades through gp130 in vivo: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses.

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Acute-Phase Reaction↗
PubMed2000

Components of the SWI/SNF complex are required for asymmetric cell division in C. elegans.

Asymmetric cell division is a fundamental process that produces cellular diversity during development. We have identified two mutants in C. elegans (psa-1 and psa-4) in which the asymmetry of T cell division is disrupted. psa-1 and psa-4 encode homologs of yeast SWI3 and SWI2/SNF2, respectively, which are components of the SWI/SNF complex. We show by RNA interference assay that homologs of other components of SWI/SNF are also involved in T cell division. psa-1 and psa-4 are likely to be required in the T cell during mitosis to cause asymmetric cell division. Because the SWI/SNF complex is required for asymmetric division in S. cerevisiae, these results demonstrate that at least some aspects of the mechanism of asymmetric cell division are conserved between yeast and a multicellular organism.

Adenosine Triphosphatases↗
PubMed2000

In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Adult↗
PubMed2000

Conditional expression of anti-apoptotic protein p35 by Cre-mediated DNA recombination in cardiomyocytes from loxP-p35-transgenic mice.

p35, a viral inhibitor of caspase, prevents cell death induced by various stimuli. We established an experimental system to study the involvement of caspases in cell death, using primary cultured cells from p35 transgenic mice in which the p35 open reading frame (ORF) had been disrupted by the insertion of a DNA segment flanked by loxP sites, the Cre recognition sites. In this system, p35 expression can be initiated by Cre recombinase. Cardiomyocytes, which are highly sensitive to hypoxic stress, were infected with an adenovirus carrying the cre gene (AxCANCre). Expression of p35 by infection with AxCANCre resulted in inhibition of caspase-3 activation and resistance to hypoxia-induced cell death. Hypoxia-induced cytochrome c release was also attenuated in p35-expressing cardiomyocytes. Our transgenic mice can be used as an experimental model for studying the involvement of caspases in various degenerative diseases as well as programmed cell death both in vitro and in vivo.

Animals↗
PubMed2000

MSI-1, a neural RNA-binding protein, is involved in male mating behaviour in Caenorhabditis elegans.

BACKGROUND: Neural RNA-binding proteins are thought to play important roles in neural development and the functional regulation of postmitotic neurones by mediating post-transcriptional gene regulation. RNA-binding proteins belonging to the Musashi family are highly expressed in the nervous system; however, their roles are poorly understood. RESULTS: We identified a Caenorhabditis elegans Musashi homologue, MSI-1, whose RNA-recognition motifs show extensive similarity to those of Drosophila and vertebrate Musashi proteins. We isolated a msi-1 mutant and found males with this mutation to have a mating defect. C. elegans male mating behaviour includes a distinct series of steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. msi-1 is required for the turning and vulva location steps. Like other Musashi family members, MSI-1 is expressed specifically in neural cells, including male-specific neurones required for turning and vulva location. However, msi-1 was not expressed in proliferating neural progenitors in C. elegans, unlike the Musashi family genes in other systems. CONCLUSIONS: Our results suggest that msi-1 is expressed specifically in postmitotic neurones in C. elegans. msi-1 is required for full development of male mating behaviour, possibly through regulation of msi-1 expressing neurones.

Alleles↗
PubMed2000

Mutations modulating the Argos-regulated signaling pathway in Drosophila eye development.

Argos is a secreted protein that contains an EGF-like domain and acts as an inhibitor of Drosophila EGF receptor activation. To identify genes that function in the Argos-regulated signaling pathway, we performed a genetic screen for enhancers and suppressors of the eye phenotype caused by the overexpression of argos. As a result, new alleles of known genes encoding components of the EGF receptor pathway, such as Star, sprouty, bulge, and clown, were isolated. To study the role of clown in development, we examined the eye and wing phenotypes of the clown mutants in detail. In the eye discs of clown mutants, the pattern of neuronal differentiation was impaired, showing a phenotype similar to those caused by a gain-of-function EGF receptor mutation and overexpression of secreted Spitz, an activating ligand for the EGF receptor. There was also an increased number of pigment cells in the clown eyes. Epistatic analysis placed clown between argos and Ras1. In addition, we found that clown negatively regulated the development of wing veins. These results suggest that the clown gene product is important for the Argos-mediated inhibition of EGF receptor activation during the development of various tissues. In addition to the known genes, we identified six mutations of novel genes. Genetic characterization of these mutants suggested that they have distinct roles in cell differentiation and/or survival regulated by the EGF receptor pathway.

Animals↗
PubMed2000

The interaction between the Drosophila secreted protein argos and the epidermal growth factor receptor inhibits dimerization of the receptor and binding of secreted spitz to the receptor.

Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor alpha-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.

Animals↗
PubMed2000

Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells.

In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopus proteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in the CNS of all the vertebrates tested. Double or triple immunostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form 'neurospheres' in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(-) cells, which included Talpha1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(-). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ.

Amino Acid Sequence↗
PubMed2000

Caspases determine the vulnerability of oligodendrocytes in the ischemic brain.

Although oligodendrocytes (OLGs) are thought to be vulnerable to hypoxia and ischemia, little is known about the detailed mechanism by which these insults induce OLG death. From the clinical viewpoint, it is imperative to protect OLGs as well as neurons against ischemic injury (stroke), because they are the only myelin-forming cells of the central nervous system. Using the Cre/loxP system, we have established a transgenic mouse line that selectively expresses p35, a broad-spectrum caspase inhibitor, in OLGs. After hypoxia, cultured OLGs derived from wild-type mice exhibited significant upregulation of caspase-11 and substantial activation of caspase-3, which led to cell loss. Expression of p35 or elimination of caspase-11 suppressed the caspase-3 activation and conferred significant protection against hypoxic injury. Expression of p35 in OLGs in vivo resulted in significant protection from ischemia-induced cell injury, thus indicating that caspases are involved in the ischemia-induced cell death of OLGs. Furthermore, the induction of caspase-11 was evident in the ischemic brains of wild-type mice, and OLGs exhibited resistance to brain ischemia in mice deficient in caspase-11, suggesting that caspase-11 is critically implicated in the mechanism(s) underlying ischemia-induced OLG death. Caspases may therefore offer a good therapeutic target for reducing ischemia-induced damage to OLGs.

Animals↗
PubMed2000

The C. elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis.

Septins have been shown to play important roles in cytokinesis in diverse organisms ranging from yeast to mammals. In this study, we show that both the unc-59 and unc-61 loci encode Caenorhabditis elegans septins. Genomic database searches indicate that unc-59 and unc-61 are probably the only septin genes in the C. elegans genome. UNC-59 and UNC-61 localize to the leading edge of cleavage furrows and eventually reside at the midbody. Analysis of unc-59 and unc-61 mutants revealed that each septin requires the presence of the other for localization to the cytokinetic furrow. Surprisingly, unc-59 and unc-61 mutants generally have normal embryonic development; however, defects were observed in post-embryonic development affecting the morphogenesis of the vulva, male tail, gonad, and sensory neurons. These defects can be at least partially attributed to failures in post-embryonic cytokineses although our data also suggest other possible roles for septins. unc-59 and unc-61 double mutants show similar defects to each of the single mutants.

Amino Acid Sequence↗