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S Kohsaka

Publications and source records attributed to S Kohsaka.

At least 109 records · Page 6Linked to original sources
PubMed1993

Plasminogen-binding protein associated with the plasma membrane of cultured embryonic rat neocortical neurons.

To investigate the receptor-like molecule(s) for plasminogen (PGn) on the neuronal surface, the properties of binding of PGn to the plasma membrane of cultured embryonic rat neocortical neurons were investigated. [125I]PGn was found to specifically bind to the plasma membrane depending on the incubation temperature and time. The binding was also affected strongly by ionic strength and slightly by Ca2+. Furthermore, ligand blotting analysis revealed that [125I]PGn binds to a major protein with an apparent molecular weight of 45 kDa among plasma membrane proteins. These results suggest that the 45-kDa protein is a PGn receptor-like molecule on the neuronal surface.

Animals↗
PubMed1993

Plasminogen promotes the development of rat mesencephalic dopaminergic neurons in vitro.

In order to investigate the physiological significance of plasminogen, which is produced by and secreted from microglia, we determined the effects of plasminogen on the development of primary cultured mesencephalic neurons from 16-day-old embryonic rats. Plasminogen markedly increased dopamine uptake and the dopamine content of cultured neurons. Furthermore, the number of mesencephalic dopaminergic neurons, which was determined by immunostaining with tyrosine hydroxylase antibody, was significantly increased in the presence of plasminogen. However, plasminogen had only a slight effect on gamma-aminobutyric acid (GABA) uptake and glutamic acid decarboxylase activity in mesencephalic neurons. The effect on dopaminergic neurons was also observed in the culture in the presence of cytosine arabinoside. In addition, plasminogen had no effect on the number of glial fibrillary acidic protein-positive cells. These results suggest that plasminogen acts directly on neurons and enhances the development of dopaminergic neurons in a rather selective manner.

Animals↗
PubMed1993

Interleukin-2 enhances the viability of primary cultured rat neocortical neurons.

We determined the neurotrophic activity of interleukin-2 (IL-2) on primary cultured neocortical neurons from embryonic rat brain. IL-2 clearly enhanced the viability of cultured neurons in a dose-dependent manner. The neurotrophic effect of IL-2 was completely neutralized by IL-2 antibody. Furthermore, expression of IL-2 receptor mRNA was more pronounced in neurons than in other cultured cells such as astroglia and microglia. These results strongly suggest that IL-2 plays certain roles in the central nervous system as a neurotrophic factor.

Animals↗
PubMed1993

Microglial conditioned medium promotes survival and development of cultured mesencephalic neurons from embryonic rat brain.

We previously reported that microglial conditioned medium (Mic-CM) has a neurotrophic effect on cultured rat neocortical neurons [Nakajima et al. (1989): Biomed Res 10:411-423]. In order to investigate the interaction between microglia and neurons in more detail, we determined the effects of Mic-CM on the primary cultured mesencephalic neurons from 16-day embryonic rats. The addition of Mic-CM to the culture medium significantly enhanced the survivability of neurons and promoted neurite extension in a low cell-density culture condition. In a high cell-density culture condition, Mic-CM markedly increased dopamine uptake, which was quantified by assessing the specific [3H]dopamine uptake, and also increased the dopamine content of cultured cells. Furthermore, the number of mesencephalic dopaminergic neurons, which was determined by quantitative analysis of tyrosine hydroxylase (TH)-immunoreactive cells, increased significantly in the presence of Mic-CM. These results suggest that Mic-CM enhances survival or maturation of TH-positive neurons present in cultures of the embryonic mesencephalon and that these neurotrophic effects may be due to a diffusible factor(s) from microglia.

Animals↗
PubMed1993

Functional roles of microglia in the brain.

It has been suggested that microglia, a type of glial cells in the central nervous system, play various important roles in normal and pathologic brains. In this article, we discussed the association or roles of microglia in injury and in brain diseases such as Alzheimer's disease, AIDS dementia complex, multiple sclerosis and ischemia. Furthermore, microglia-derived cytotoxic products and other secretory factors were summarized. In addition to the pathological aspects, secretory factors that showed neurotrophic effects were described with special reference to their physiological significance in the neuronal growth, neuronal function and regeneration processes. Accumulated evidence suggests that microglia are associated with not only brain pathology but also normal physiology in the brain.

Animals↗
PubMed1993

Molecular cloning of rat growth inhibitory factor cDNA and the expression in the central nervous system.

Human growth inhibitory factor (GIF) is a new metallothionein-related molecule whose expression is markedly reduced in Alzheimer's disease (AD) brain. We have recently isolated a full-length cDNA for human GIF that has a striking homology to metallothioneins (MTs). As an initial step to better understanding of the biological functions of GIF, we have isolated a full-length cDNA for rat GIF. Comparison of the predicted amino acid sequence with that of human GIF revealed a strikingly high homology (84% identity). Rat GIF cDNA also showed a striking homology to rat MT-1 and MT-II (66% and 62% identities on amino acid sequences, respectively). All cysteine residues were conserved among rat GIF, human GIF and MTs, indicating that cysteine residues play an important function in these molecules. Compared to mammalian MTs, there were 3 bp and 12 bp insertions near the amino-terminal and carboxy-terminal regions in the rat GIF. The rat GIF mRNA was found to be expressed exclusively in the central nervous system, being expressed predominantly in rat cortical astrocytes in primary culture. Although the rat GIF mRNA expression was low during the fetal stage, a dramatic increase of the rat GIF mRNA expression was demonstrated during the postnatal period of 10-17 days. These results indicate that transcriptional regulation of the rat GIF gene is quite different from that of MTs despite its strikingly high homology with MTs.

Amino Acid Sequence↗
PubMed1993

Microglia-derived plasminogen enhances neurite outgrowth from explant cultures of rat brain.

Recently, we reported the production and secretion of plasminogen (Pg) in cultured rat brain microglia [Nakajima et al., (1992) Fedn. Eur. Biochem. Socs Lett. 308, 179-182]. To investigate the physiological significance of Pg, we determined the effect of Pg on neurite outgrowth of cultured neocortical explants of an embryonic rat brain in serum-free chemically defined medium. Pg markedly enhanced the neurite outgrowth. Although plasmin, which is derived from Pg by activation by urokinase (UK), had a similar effect in this explant culture system, UK itself did not show any effect. Furthermore, we studied the characteristics of Pg binding to cultured neocortical neurons dissociated from an embryonic 16-day-old rat brain by using 125I-Pg. Specific binding of Pg to neocortical neurons was detected and Scatchard plot analysis revealed high- and low-affinity binding sites on the neurons. The estimated dissociation constants of high- and low-affinity binding sites were approximately 16.1 and 124.2 nM, respectively. These results suggest that microglia-derived Pg plays certain roles in the regulation of neurite extension through binding to the surface of neocortical neurons.

Animals↗
PubMed1993

Microglia-derived elastase produces a low-molecular-weight plasminogen that enhances neurite outgrowth in rat neocortical explant cultures.

In the course of analysis of plasminogen in microglial conditioned medium (Mic-CM), novel low-molecular-weight (LMW) zymogen with a molecular mass of approximately 36 kDa was detected by casein-urokinase zymography. Because this form was produced when rat native plasminogen was incubated with Mic-CM, a specific protease in the Mic-CM was thought to be responsible for the production of LMW plasminogen. The production of LMW plasminogen was strongly inhibited by elastase inhibitors. Furthermore, elastase (pancreatic or leukocyte) was also found to produce LMW zymogen from native plasminogen. These results indicate that LMW plasminogen is produced through limited proteolysis by an elastase-like protease in Mic-CM. To determine the biochemical characteristics of LMW plasminogen, rat native plasminogen was cleaved by pancreatic elastase, and the fragments (LMW plasminogen and nonzymogen fragments) were purified by several kinds of column chromatography. Amino acid sequence analysis revealed that LMW plasminogen is a carboxy-terminal region that contains the fifth kringle domain and a protease active site, and the amino acid sequence is identical to that of LMW plasminogen produced by Mic-CM. On the other hand, the nonzymogen fragment was the amino-terminal region containing four kringle domains. The effects of native plasminogen and the fragments on neurite outgrowth of rat brain explant were examined. LMW plasminogen promoted neurite outgrowth as well as did native plasminogen, whereas nonzymogen fragments did not. These results suggest that LMW plasminogen, which is produced from native plasminogen by elastase, may be a physiologically active molecule that mediates the intercellular interaction between microglia and neurons.

Amino Acid Sequence↗
PubMed1992

Retinoic acid enhances the secretion of plasminogen from cultured rat microglia.

To determine the amount of plasminogen in microglial conditioned medium, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for rat plasminogen was established. Weak cross-reactivity with human serum plasminogen was observed, while no reactivity was detected with frog and carp plasminogen. The specificity of the immunosorbent assay was confirmed by Western blotting. The secretion of plasminogen into the microglial culture medium was quantified by using the established ELISA and was found to be increased depending on the culture time and number of microglia. The secretion was increased about 5-fold by stimulation with retinoic acid, while interleukin-1, and basic fibroblast growth factor showed no significant effect.

Animals↗
PubMed1992

Production and secretion of plasminogen in cultured rat brain microglia.

The production and secretion of plasminogen in cultured rat brain microglia was investigated. Urokinase-dependent caseinolytic activity was detected by zymography in microglial conditioned medium with a molecular weight of about 90 kDa. The 90-kDa protein was also detected by Western blotting with anti-rat plasminogen antiserum in the non-reducing condition. Immunoprecipitation with plasminogen antiserum following [35S]methionine labelling revealed that the plasminogen detected in microglial conditioned medium is synthesized in microglia. The amount of plasminogen in the conditioned medium was increased by stimulation with lipopolysaccharide. These results show that cultured microglia produce plasminogen and secrete it into the culture medium.

Animals↗
PubMed1992

Generation of microglial cell lines by transfection with simian virus 40 large T gene.

Microglial cells, which were isolated from a primary culture of neonatal rat brain, were transfected with temperature-sensitive simian virus 40 (SV40) large T gene by the calcium phosphate precipitation method. Four weeks after transfection, several colonies were generated, and cloned cells were characterized. One of the cloned cells (RBM129) proliferated actively at 37 degrees C and the dividing rate was significantly decreased at 40.5 degrees C. The expression of large T antigen was detected by western blotting in cells incubated at both 37 degrees C and 40.5 degrees C. The cell line showed high activity of non-specific esterase, isolectin B4 binding and phagocytosis. Also the cells were stained by ED 1 monoclonal antibody. These results indicate that these cells were derived from rat brain microglia, and immortalized by large T gene.

Animals↗
PubMed1992

Microglia isolated from rat brain secrete a urokinase-type plasminogen activator.

In a previous study, we found particular proteases which degrade myelin basic protein (MBP) in a conditioned medium of cultured rat brain microglia. The MBP degrading activity in microglial-conditioned medium (Mic-CM) increased markedly in the presence of plasminogen. By Sephadex G-150 column chromatography, plasminogen-dependent MBP degrading activity was eluted at the position of about 47 kDa and 28 kDa. Furthermore slight plasminogen-dependent protease activity in the presence of fibrin (tissue plasminogen activator activity) was detected at a molecular weight of about 68 kDa. The two molecular forms (47 kDa and 28 kDa) of plasminogen-dependent protease were demonstrated by casein-zymography, and it was suggested that they were urokinase type-plasminogen activators (uPA). This suggestion was confirmed by immunoblotting using anti-uPA antiserum. The unique 28 kDa type was considered to be produced from the 47 kDa form by limited proteolysis. Secretion of PA from microglia was demonstrated by cell zymography. In contrast, significant secretion of plasminogen activator inhibitor could not be detected in the Mic-CM. In addition, lipopolysaccharide significantly decreased the secretion of PA from microglia, while interleukin-1 and basic fibroblast growth factor enhanced the secretion.

Animals↗
PubMed1992

De novo production of alpha 2-macroglobulin in cultured astroglia from rat brain.

Previous studies from our laboratory demonstrated that alpha 2-macroglobulin (alpha 2M) is one of the neurite-promoting factors in the conditioned medium of astroglia. In the present study, we further examined the de novo production of alpha 2M in cultured astroglia by determining the expression of alpha 2M mRNA, and the biosynthesis of [35S]methionine-labeled alpha 2M protein. We analyzed the mRNA of cultured astroglia by differential hybridization using specific probes to alpha 2M and its homologous protein, alpha 1-inhibitor 3 (alpha 1I3), after amplification of reverse-transcribed cDNA with the polymerase chain reaction. The result clearly showed that only alpha 2M mRNA is expressed in cultured astroglia. Northern blotting analysis revealed that alpha 2M mRNA is expressed mainly in the astroglia and is not detected in neurons, microglia and meningeal fibroblasts. Furthermore, the biosynthesis of alpha 2M protein in the astroglia was confirmed by an immunoprecipitation experiment after labeling of each type of cell with [35S]methionine. It was concluded that alpha 2M is produced in the cultured astroglia which is the major source of alpha 2M production among various types of cells in rat brain.

Animals↗
PubMed1992

Cortical reflex myoclonus associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS): a case report.

A 9-year-old female MELAS patient with myoclonus is reported, with emphasis on the results of electrophysiological studies of the myoclonus. At age 5 years she experienced a stroke-like episode, and a diagnosis of MELAS was made at age 6 years on the basis of muscle biopsy findings. At age 9 years spontaneous and segmental myoclonus, predominantly affecting the upper extremities, developed because of complications. Electrophysiological examination, including of somatosensory-evoked potentials (SEPs) and averaged EMG for long loop reflexes, revealed so-called "giant SEP" and enhanced long loop reflexes reflecting cortical hyperexicitability. Jerk-locked averaging yielded no myoclonus related spikes, but myoclonus-contingent 4-5 Hz theta bursts appeared. These findings suggest that some types of MELAS may be associated with cortical types of myoclonus.

Child↗
PubMed1992

Identification of elastase as a secretory protease from cultured rat microglia.

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.

Animals↗
PubMed1992

Tetrahydrobiopterin-dependent functional recovery in 6-hydroxydopamine-treated rats by intracerebral grafting of fibroblasts transfected with tyrosine hydroxylase cDNA.

Fibroblasts (NRK-49F) were transfected with human type 2 tyrosine hydroxylase (TH; EC 1.14.16.29) cDNA, to clarify the mechanism involved in amelioration of parkinsonism by intracerebral grafting of catecholaminergic neurons and to investigate its possible use as a donor material. These genetically manipulated fibroblasts did not develop into a mass of tissue, and survived well in the host striatum. Expression of the TH minigene in the cells was successful even when they were transplanted into the host brain. Intracerebral microdialysis revealed that a measurable amount of L-3,4-dihydroxyphenylalanine (L-DOPA) was not spontaneously released from the implanted cells into the host striatum. However, release of a large amount of L-DOPA from the cells was observed when (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) was perfused through a dialysis probe. Finally, we investigated whether these BH4-dependent L-DOPA-secreting fibroblasts are able to ameliorate the abnormal behavior of 6-hydroxydopamine-treated rats. Apomorphine-induced rotating behavior was not reversed by the grafting alone, whereas a marked reduction in drug-induced circling was observed temporarily after BH4 was perfused around the implanted cells. These findings indicate that TH cDNA-transfected non-neuronal cells might be able to be used as donor material for intracerebral grafting and ameliorate the abnormal behavior of rats with experimental Parkinson's disease.

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