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S Kohsaka

Publications and source records attributed to S Kohsaka.

At least 91 records · Page 5Linked to original sources
PubMed1996

Functional implication of secretory proteases derived from microglia in the central nervous system.

We have demonstrated that microglia produce certain secretory proteases which have been found to be important determinants of microglial properties, in surrounding cells and regenerative processes. In recent years, it has become clear that secretory proteases, particularly PGn-PA (plasminogen-plasminogen activator) system, work not only on catalysis of proteins in the extracellular space but also on cell growth, cell function, differentiation, proliferation and remodeling. These diverse effects may be derived from the unique structures of these enzymes, including their accessary domains. In particular, kringle domains have been shown to be important for interactions with other proteins. The results of these studies indicate that microglial secretory proteases participate to a great extent in physiological processes involving the regulation of neuronal growth, neuronal function and regenerative stages in the CNS.

Central Nervous System↗
PubMed1995

Structure of rat annexin V gene and molecular diversity of its transcripts.

We report here the isolation and characterization of the entire rat annexin V gene, which is shown to span about 40 kbp, and to consist of 14 exons, 13 introns and two promoters. Previous studies found that both human and chicken annexin V genes bear 13 exons and one promoter, which possess no TATA sequence or CCAAT box, and have a high GC content. In the present study, we found that one of the promoters for rat annexin V shares these features, which are typical among promoters for housekeeping genes, and that this promoter, as expected, exerted its transcription in all of tissues we examined. In contrast, another promoter was found to possess not only a TATA box but also a glucocorticoid-responsive element, and was shown to express messages in a relatively limited number of tissues, including brain. We also demonstrated the presence of at least four species of rat annexin V transcripts that were generated by distinct transcription initiation and alternative splicing.

Alternative Splicing↗
PubMed1995

Expression, purification, and neurotrophic activity of amyloid precursor protein-secreted forms produced by yeast.

The secreted form of amyloid precursor protein (APPs) including most of the extracellular domain of APP is released from the cell surface, suggesting physiological significance of APPs in vivo. We used the methylotrophic yeast Pichia pastoris as a host system for the production of recombinant APPs (rAPPs). Two rAPPss derived from isoforms of APP (APP695 and APP770) were secreted into the culture medium from the yeast, which carried cDNA encoding the N-terminal portion of APP under the control of a P. pastoris alcohol oxidase promoter. Like APPss produced by the transfected COS-1 cells, the purified rAPPss from yeast were shown to be biologically active in terms of neurite outgrowth of embryonic rat neocortical explants. These rAPPss could be valuable tools for investigating the biological functions of APPss.

Amino Acid Sequence↗
PubMed1995

Apoptotic cell death of a temperature-sensitive central neuronal cell line.

A neuron-like cell line HS-2, derived from a primary fetal rat (E17) hippocampal cell culture using the temperature-sensitive SV 40 large T antigen, exhibits flat shape and grows well in culture medium with 5% fetal calf serum (FCS) at the permissive temperature (PT, 33.5 degrees C). At the non-permissive temperature (NPT, 38.5 degree C), many, but not all cells, have a neuronal shape with processes. The addition of dibutyryl-cAMP promotes the morphological changes in the cells to a neuron-like shape with long neurite-like processes and the cells exhibit neuron-specific enolase- and glutamic acid decarboxylase-immunoreactivity. Apoptotic cell death also occurs in these cultures at the NPT. DNA fragmentation and chromatin condensation that are characteristic of apoptosis occur within 8 h of being placed at the NPT. By 48 h after being placed at the NPT, the number of surviving cells decreases by 40% in the presence of 5% FCS. This cell line should be useful for investigating the mechanisms of 'programmed cell death' of neurons, which appears to occur during brain development and possibly in CNS degenerative diseases.

Animals↗
PubMed1995

Neurotrophic and neuroprotective effects of neuron-specific enolase on cultured neurons from embryonic rat brain.

We previously reported that the gamma gamma-isozyme of enolase, NSE), one of the glycolytic enzymes, promoted the survival of embryonic rat neocortical neurons in culture, but alpha alpha-isozyme (non-neuronal enolase) had no effect. In the present study, the neurotrophic effects of NSE on cultured mesencephalic and spinal neurons from rat embryo were examined. NSE promoted the survival of neurons not only in neocortical cultures but also in mesencephalic and spinal cord cultures. Furthermore, NSE showed neuroprotective action on cultured neocortical neurons in a low-oxygen atmosphere. By contrast, non-neuronal enolase did not show any neurotrophic or neuroprotective activities. To clarify the mechanism of the neurotrophic effect of NSE, the binding of NSE to cultured neurons was determined by radio-receptor assay using 125I-labelled NSE. The specific binding, which was dose-dependent, saturable, and calcium-dependent, could be detected. These results suggest that NSE has neurotrophic and neuroprotective effects on rather a broad spectrum of neurons in the central nervous system. The existence of specific binding of NSE to cultured neurons suggests the possibility that receptor-like or carrier-like molecules on the neuronal surface are involved in the neurotrophic activity of NSE.

Animals↗
PubMed1995

Expression and distribution of low density lipoprotein receptor-related protein mRNA in the rat central nervous system.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor that binds to the protease inhibitor alpha 2-macroglobulin (alpha 2 M). LRP has also been identified as the apolipoprotein E (apoE) receptor that mediates lipid metabolism. Recently it has been reported that apoE4, one of three common isoforms of apoE, is a main risk factor of Alzheimer's disease (AD). Moreover, all three of these proteins are reported to accumulate in the senile plaques in the brains of Alzheimer's patients. To understand the roles of LRP in the normal development of the central nervous system (CNS) and in the pathogenesis of AD, we studied the developmental expression and localization of LRP mRNA in the CNS. We used Northern blot analysis to investigate the developmental profile of LRP mRNA in the rat brain. LRP mRNA was first detected as early as in 18-day-old embryonic rat brain and was continuously expressed thereafter. A particularly high level of expression of the mRNA was observed in the perinatal stage. We also determined the cellular distribution of LRP mRNA in the CNS of 20-day-old embryonic and 6-week-old adult rat brains by in situ hybridization using a digoxigenin-labeled antisense riboprobe to LRP mRNA. In the embryonic rat brain, LRP mRNA was highly expressed in most of the cells, mainly neurons and glial cells. In the adult rat, LRP mRNA was expressed mostly in neurons in both the brain and the spinal cord. These results suggest that LRP plays crucial roles in development of the brain.

Alzheimer Disease↗
PubMed1994

Modulatory effect of plasminogen on NMDA-induced increase in intracellular free calcium concentration in rat cultured hippocampal neurons.

The effect of plasminogen on the intracellular Ca2+ concentration ([Ca]i) was examined in cultured rat hippocampal neurons. Plasminogen (100 nM) did not increase [Ca]i in the presence of tetrodotoxin (TTX, 3 microM) but increased [Ca]i in the absence of TTX. In all the cells which responded to plasminogen (100 nM), NMDA (5 microM) also increased [Ca]i in the presence of TTX. Furthermore, plasminogen (100 nM) enhanced the NMDA-evoked [Ca]i increase, and the potentiation by plasminogen was blocked by an NMDA receptor blocker, 2-amino-phosphonovalerate (APV). These data suggest that plasminogen enhances glutamate-evoked [Ca]i increase through modulation of NMDA receptor in hippocampal neurons.

2-Amino-5-phosphonovalerate↗
PubMed1994

Synthetic peptide corresponding to 30 amino acids of the C-terminal of neuron-specific enolase promotes survival of neocortical neurons in culture.

Neuron-specific enolase (NSE), one of the glycolytic enzymes, is a gamma gamma-isozyme of enolase that is specifically expressed in neurons. Our previous studies demonstrated that NSE promotes survival of rat embryonic neocortical neurons in culture but that the alpha alpha-isozyme (non-neuronal enolase; NNE) has no effect. In this study, we found that a synthetic peptide corresponding to the C-terminal portion of NSE (404-433) also promotes the survival of neocortical neurons. By contrast, a synthetic peptide of the C-terminal portion of NNE (404-433) has no effect on neuronal survival. These findings would be important for further analysis of the neurotrophic mechanism of NSE.

Amino Acid Sequence↗
PubMed1994

Neurotrophic effects of annexin V on cultured neurons from embryonic rat brain.

Recombinant human annexin V showed survival promoting activity in embryonic rat neocortical and mesencephalic neurons in vitro. The neurotrophic effect was observed from a relatively low dose and in a dose-dependent manner. The neurotrophic activity of annexin V was completely blocked by anti-annexin V antibody. Northern blot analysis demonstrated that annexin V mRNA is expressed in non-neuronal cells in the CNS. These results suggest that annexin V plays certain roles in the CNS as a paracrine-type neurotrophic factor.

Amino Acid Sequence↗
PubMed1994

Expression of the alpha 2-macroglobulin-encoding gene in rat brain and cultured astrocytes.

The alpha 2-macroglobulin (alpha 2M), a protease inhibitor, is a major acute-phase protein in rats, and is produced in the liver during acute inflammation. Recently, it has been demonstrated that alpha 2M is also produced by cultured astrocytes from newborn rat brain and has neurite-promoting activity. Here, we found that the expression of the alpha 2M gene was significantly enhanced in the brain following intraperitoneal injection of the neurotoxicant, kainic acid (KA), suggesting that alpha 2M acts as an acute-phase protein in the brain, as in the case of the liver, and may be involved in neural repair processes. Expression of alpha 2M in cultured astrocytes was shown to be stimulated by interleukin-6 (IL-6) and/or leukemia inhibitory factor (LIF) in the presence of glucocorticoid. The amount of mRNAs for IL-6 and LIF increased in the brain of KA-injected rats prior to alpha 2M induction. These results strongly suggested that IL-6 and LIF are involved in alpha 2M induction in the brain, as in the case of the liver. Analysis of the cis-acting element(s) and the trans-acting factor(s) suggested that the regulatory mechanism for alpha 2M expression in astrocytes was similar to that in inflamed liver.

Animals↗
PubMed1994

Glial conditioned medium alters the expression of amyloid precursor protein in SH-SY5Y neuroblastoma cells.

The effect of conditioned medium from astroglia or microglia cultures on the mRNA expression of amyloid precursor protein (APP) in SH-SY5Y human neuroblastoma cells was examined. When incubated with conditioned medium of glial cells, SH-SY5Y cells expressed a larger amount of APP mRNA than the control cells. Reverse transcription followed by the polymerase chain reaction showed marked induction of expression of APP isoforms containing a Kunitz-type protease inhibitor domain. Our results suggest that glial cells may contribute to the regulation of expression of APP in neurons.

Amyloid beta-Protein Precursor↗
PubMed1994

Histochemical and immunohistochemical studies on keratan sulfate in the anterior segment of the developing human eye.

Changes in the distribution of keratan sulfate in the anterior segment of developing ocular tissue were investigated histochemically and immunohistochemically using human eyes from 5 weeks gestation to 2 years after birth. Enzyme digestion methods with streptomyces hyaluronidase, chondroitinase and keratanase showed that the main component of glycosaminoglycans in the cornea was hyaluronic acid at the early stage and chondroitin sulfate from the middle stage to the neonatal stage. Keratan sulfate was found at the neonatal stage. Immunohistochemical staining, however, showed that keratan sulfate was present from 6 weeks around mesenchymal cells which migrated into the anterior portion. The stroma and endothelial cells of the cornea, trabecular tissue, iris, choroidal tissue of the ciliary body and anterior tunica vasculosa lentis were the next tissues to show positive for immunostaining. Keratan sulfate became limited to the cornea and lumbus and mostly disappeared from the other anterior segment tissues after birth. These findings suggest that tissues which seem to originate from mesenchymal cells and surround the chamber space contain keratan sulfate during the developmental stage.

Adolescent↗
PubMed1994

Plasminogen binds specifically to alpha-enolase on rat neuronal plasma membrane.

Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factor-like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat alpha-enolase. In fact, PGn was found to bind to purified rat alpha-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat alpha-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of alpha-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat alpha-enolase antibody, and pretreatment with alpha-enolase antibody markedly diminished the PGn-binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that alpha-enolase is present on the cell surface of a certain population of neurons. These results suggest that alpha-enolase may function as a PGn-binding molecule on the neuronal cell surface.

Amino Acid Sequence↗
PubMed1994

A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.

We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M. Maekawa, S. Nakamura, and S. Hattori, J. Biol. Chem. 268:22948-22952, 1993). On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP. The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa. The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene. When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1. Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene. Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues. A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue. Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype. In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase. These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Plasminogen mediates an interaction between microglia and dopaminergic neurons.

Plasminogen (PGn), which was identified as a secretory product from microglia, increased dopamine uptake and the number of tyrosine-hydroxylase-expressing neurons in cultured rat mesencephalic neurons. 125I-PGn was found to bind specifically much more to mesencephalic neurons compared to other glial cells. The binding was also recognized in plasma membrane prepared from embryonic rat brain. Furthermore, ligand blotting analysis revealed that 125I-PGn binds to a 45-kD protein in plasma membrane. These results suggest that PGn-dependent neurotrophic effects may be induced through the specific binding of PGn to the 45-kD PGn-receptor-like protein in neurons.

Animals↗