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Biomedical subjects

S Kohsaka

Publications and source records attributed to S Kohsaka.

At least 127 records · Page 7Linked to original sources
PubMed1991

Dissociation of neurite-promoting activity and protease-inhibiting function of alpha 2-macroglobulin in culture.

The relationship between the neurite-promoting effect and the protease-inhibiting function of alpha 2-macroglobulin (alpha 2M) was examined in a culture of dissociated neurons from embryonic rat neocortical tissue. The neurite-promoting effect of various protease inhibitors (soybean trypsin inhibitor, alpha 1-antitrypsin, aprotinin, phenylmethanesulfonyl fluoride, leupeptin, antipain, human alpha 2M and purified rat alpha 2M) was investigated. At lower concentrations, only alpha 2M significantly promoted neurite outgrowth. Furthermore, alpha 2M-trypsin complexes, which had lost their protease-inhibiting function and exposed their receptor-recognition site, promoted neurite outgrowth more efficiently than native alpha 2M. These results indicate the possibility that the neurite-promoting effect of alpha 2M is mediated by its receptor-binding activity but is not directly related to its protease-inhibiting function.

Amyloid beta-Protein Precursor↗
PubMed1991

Production of basic fibroblast growth factor in cultured rat brain microglia.

The production of basic fibroblast growth factor (bFGF) in cultured rat brain microglia was investigated. Rat brain microglia were found to express mRNA of bFGF in analysis by polymerase chain reaction (PCR) technique. Basic FGF was also detected in microglial cell lysate by Western blot analysis. These results indicate that microglia produce bFGF and possibly contribute to the regulation of neuronal development and regeneration.

Animals↗
PubMed1991

Core protein of chondroitin sulfate proteoglycan promotes neurite outgrowth from cultured neocortical neurons.

Chondroitin sulfate proteoglycan (CS-PG) was purified from rat brain and examined for its effect on neurite outgrowth in primary cultures of embryonic rat neocortical neurons. Neurite outgrowth was increased in culture wells coated with CS-PG. The core protein and glycosaminoglycan (GAG) prepared from the CS-PG were also examined for neurite-promoting activity. The activity was observed in culture wells coated with the core protein but not with GAG. These results suggest that CS-PG stimulates neurite outgrowth from the cultured neurons via its core protein.

Aggrecans↗
PubMed1991

Neuronal survival factor from bovine brain is identical to neuron-specific enolase.

Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high-speed centrifugation was used as the starting material. At the step of DE-52 ion-exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti-F14 antiserum recognized mainly a 30-kDa protein in F14 and anti-F23 antiserum recognized mainly a 44-kDa protein in F23. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of F23, the 44-kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron-specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti-NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose-dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.

Animals↗
PubMed1991

Effects of astroglia on the development of cultured neurons from embryonic rat cerebral cortex.

1. Previous studies from our laboratory demonstrated that subcultured astroglia enhance neurite outgrowth and survival of cultured neurons from embryonic rat cerebral cortex, but suppress proliferation of neuroblasts. 2. In the present study, the mechanisms of these three effects were further investigated. 3. Dissociated neurons were seeded on poly-L-lysine-coated coverslips which were plated on subcultured astroglia, and the survival, proliferation and neurite outgrowth of the neurons were investigated. Under these conditions, survival and antimitotic effects were also observed, while neurite extension was not stimulated. 4. The results clearly indicate that neuronal survival and proliferation are regulated by soluble factors produced by astroglia. 5. We also postulated that the neurite-promoting effect of astroglia is mediated by cell-cell contact. 6. This idea was confirmed by the finding that neurite extension was enhanced when the neurons were cultured directly on heat-treated astroglia. 7. The neurite-promoting effect was found to be specific to astroglia. 8. We preliminarily characterized the astroglial surface neurite-promoting factors (ASNPFs). 9. The relationship of laminin to ASNPFs was examined by using antibody to laminin. Laminin antibody did not inhibit the ASNPF activity. 10. The effect of digestion of heat-treated astroglia with enzymes (sialidase and endo-beta-galactosidase) on the ASNPF activity was also examined. 11. These enzyme treatments did not inhibit the ASNPF activity. 12. These results suggest that enhancement of the neurite-promoting activity is not associated with the sugar moiety of ASNPFs.

Animals↗
PubMed1990

Alpha 2-macroglobulin is an astroglia-derived neurite-promoting factor for cultured neurons from rat central nervous system.

The neurite promoting factors in the astroglial conditioned medium (As-CM) were characterized by using primary cultures of embryonic rat neocortical neurons. The factors in the As-CM bind to lectins such as wheat germ agglutinin (WGA), suggesting that they contain sugar moieties. When the WGA-bound fractions were applied on a Superose 6 column, the activity was recovered mainly in two fractions, peak I and peak II. The peak II fraction was further purified by Mono Q anion exchange chromatography. A single protein band of 180 kDa was detected in the final Mono Q fraction by sodium dodecylsulfate polyacrylamide gel electrophoresis. The molecular weight coincided with that of alpha 2-macroglobulin (alpha 2M). Western blotting showed that the single protein band was reacted with anti-alpha 2M antibody but not with anti-fibronectin and anti-laminin antisera. The neurite-promoting activity of the Mono Q fraction was inhibited by anti-alpha 2M antibody. Furthermore, commercially available alpha 2M also promotes neurite outgrowth in our assay system. These results strongly suggested that alpha 2M is one of the neurite-promoting factors in the As-CM.

Animals↗
PubMed1990

Tetrahydrobiopterin-dependent production of L-dopa in NRK fibroblasts transfected with tyrosine hydroxylase cDNA: future use for intracerebral grafting.

In the present study, tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was transfected into cultured fibroblasts and the production of L-3,4-dihydroxyphenylalanine (L-DOPA) was determined. Type 2 TH cDNA was transfected into fibroblasts (NRK-49F) derived from the normal rat kidney, and the expression of the TH minigene was screened by immunocytochemical staining and immunoblotting analysis with TH antiserum. Several clones of the NRK transfectants that produce TH molecules were obtained. The expressed TH molecules showed high enzyme activity in a complete assay system in vitro. However, L-DOPA was not detected in the cultured cells due to the possible absence of de novo synthesis of (6R)-L-erythro-tetrahydrobiopterin (BH4) in these cells. When BH4 was added to the medium, a large amount of L-DOPA was detected not only in the cells but also in the medium. These findings may aid in regulating the amount of L-DOPA secretion from cells after they are transplanted into the brain.

Animals↗
PubMed1990

Histochemical studies on hyaluronic acid in the developing human retina.

Changes in the distribution of hyaluronic acid in the developing human retina were investigated histochemically with alcian blue staining and the Streptomyces hyaluronidase digestion method using 56 human embryos and fetuses ranging from 5 to 41 weeks of gestational age. Hyaluronic acid was first detected in the inner layer of the retina at 12 weeks. The site of accumulation extended towards the outer layer by 20 weeks. At the neonatal stage, longitudinal fibers, possibly the processes of Müller cells, were proved to contain hyaluronic acid. These findings suggest that Müller cells produce hyaluronic acid transiently from 12 weeks' gestation to the neonatal stage.

Aged↗
PubMed1990

Xenogeneic neural transplantation: role of vasculature and MHC antigen in immunological rejection.

The reconstruction of blood vessels in a graft is one of the important events inducing immunological rejection. First, we investigated the reconstruction of blood vessels in a graft by using a monoclonal antibody against mouse endothelial surface antigen 1. Secondly, the lymphocyte proliferative response to Ia antigen were examined in mixed lymphocyte cultures by using monoclonal antibodies against Ia antigen. The results show that the blood vessels originating in the host tissue inoculate with those originating in the donor graft tissue and that the expression of Ia antigen on the vascular endothelial cells plays an important role in the immunological rejection through sensitization of peripheral lymphocytes.

Animals↗
PubMed1990

[An analysis of ictal EEGs by a newly developed ambulatory cassette EEG system].

We evaluated the efficiency of a newly developed cassette EEG recording device and our system for detecting and analyzing ictal EEGs in epileptic children. We got 13 ictal records from 16 epileptic children. We could precisely differentiate and diagnose their classifications of seizures by comparative analyses of ictal records and clinical symptoms. We discussed the characteristics of our system, and described actual analysis methods in 3 cases as examples. It was concluded that our system offered valuable information about ictal events in epileptic children.

Adolescent↗
PubMed1990

[The new cassette EEG recording system: its application to epileptic children].

We designed a new cassette EEG recording system which consists of a recording block (an ambulatory-type data recorder and a newly developed pre-amplifier) and a reproduction block (a fixed-type data recorder, a personal computer equipped with A/D converting board and an intervening EEG device for a bandpass filter). This system was applied to 36 epileptic children, and 33 ictal records were obtained. The seizure type could be precisely classified by a comparative analysis of the data computed from this system and from the ictal symptoms. Three representative cases were presented with actual analysis procedures.

Child↗
PubMed1989

Mosaic reconstruction of blood vessels in mouse neocortical tissue transplanted into the third ventricle of rat brain.

Newborn mouse neocortical tissue was transplanted into the third ventricle of rat brain and the reconstruction and the origin of the blood vessels were investigated by using a monoclonal antibody against mouse endothelial surface antigen-1 (MESA-1). It was clearly demonstrated that some of the blood vessels in the graft originated in the donor mouse neocortical tissue. An India ink perfusion experiment revealed that the blood was supplied to the MESA-1-positive blood vessels. Furthermore, electron microscopic immunohistochemical studies demonstrated the existence of a mosaic reconstruction of blood vessels which consisted of mouse- and rat-derived vascular endothelial cells. It was concluded that the blood vessels originating in the donor tissue and those originating in the host tissue inoculate with each other in the grafted tissue.

Animals↗
PubMed1989

Expression of Ia antigen on vascular endothelial cells in mouse cerebral tissue grafted into the third ventricle of rat brain.

The mechanisms of the immunological rejection after xenogeneic neural transplantation were investigated with special reference to the expression of class II major histocompatibility complex (MHC) antigen (Ia antigen) on the grafted tissue. Tissue from a newborn mouse cerebral cortex was transplanted into the third ventricle of a 4-week-old rat brain. Infiltration of cytotoxic T-cells into the grafted tissue was investigated immunohistochemically by using a monoclonal antibody (OX-8). The infiltration began 8 days after transplantation and continued until about 4 weeks when the tissue was completely rejected. The expression of Ia antigen was also investigated immunohistochemically. The Ia antigen was first detected in the grafted tissue at 6 days after transplantation. The Ia antigen was considered to be expressed on the vascular endothelial cells judging from the staining patterns and the location of India ink which was perfused from the host's left cardiac ventricle. The perfusion experiments with India ink also revealed that blood was supplied to the grafted tissue from 5 days after transplantation. These results suggest that the expression of Ia antigen on the vascular endothelial cells renders the grafted tissues competent to initiate and participate in the immune reaction. The results also raise a possibility that the expression of Ia antigen is triggered by blood supplied from the host brain. In addition, the results indicate that the Ia-positive blood vessels do not originate in the host brain but are intrinsic to the grafted tissue.

Animals↗
PubMed1989

Changes in ganglioside composition and morphological features during the development of cultured astrocytes from rat brain.

Changes in ganglioside content over a period of days were examined in astrocytes obtained via cell passage from rat cerebral cortex. Thin-layer chromatography revealed that, in the astrocytes, ganglioside GM1 was absent, the predominant ganglioside being GM3. Also, an increased GD3 content in long-term astrocyte cultures was detected. The morphological features of astrocytes were also studied using immunoperoxidase staining. Astroglial features were characterized by high levels of glial fibrillary acidic protein (GFAP) and vimentin, which are the major intermediate-filament proteins present in astrocytes at an early culture stage. In long-term-cultured (greater than 7 months) astrocytes, vimentin and GFAP were increased in process-bearing cells. Ganglioside GD3 recognized by R24 monoclonal antibody was also expressed in these cells. These results suggest that the increase of ganglioside GD3 in long-term-cultured astrocytes may be related to the appearance of multistellate cells showing strong reactivity against GFAP and vimentin during development over a specified period in culture.

Animals↗
PubMed1989

Synthesis of L-3,4-dihydroxyphenylalanine by tyrosine hydroxylase cDNA-transfected C6 cells: application for intracerebral grafting.

In the present study, we obtained genetically manipulated nonneuronal cells which synthesize a catecholamine precursor for future use in intracerebral grafting. Human type 1 tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was inserted into eukaryotic expression vector pKCRH2 and was co-transfected into C6 cells with plasmid pSV2neo. Expression of the TH minigene was screened by immunohistochemical staining with TH antibody and immunoblotting analysis. Several clones of the C6 transfectants that produce TH molecules were obtained. These cells showed TH activity, and the product, L-3,4-dihydroxyphenylalanine (L-DOPA), was detected intracellularly due to the absence of L-amino acid decarboxylase (EC 4.1.1.28) activity. It was found that a large amount of L-DOPA was released from the cells into the culture medium. These transfectants were transplanted into rat brain, and the expression of TH was examined immunohistochemically. On the 10th day following transplantation, a mass of C6 cells which was heavily stained with TH antibody was observed in the brain. These findings may provide us with an opportunity to investigate the effects of intracerebral transplantation of nonneuronal cells that produce catecholamine or its precursor.

Animals↗
PubMed1989

[Regulation of neuronal development by astroglia].

Accumulating lines of evidence indicated that glial cells play important roles in regulating the neuronal development. It has been reported by a number of authors that astroglia promote the survival of neurons and the neurite outgrowth by several diffusible factors and membrane-associated factors. In the present article, we have reviewed the astroglia-derived bioactive substances which possibly affect the neuronal development in the central nervous system.

Animals↗