Biomedical subjects

S U Kim

Publications and source records attributed to S U Kim.

At least 127 records · Page 7Linked to original sources

PubMed1994

Co-expression of mRNA for neurotrophic factors in human neurons and glial cells in culture.

Enriched populations of neurons and astrocytes of 93-99% purity were obtained from mixed cultures of four human fetal brains. Total cellular RNA was extracted from cell pellets and reverse transcribed into cDNA. Five microliters of cDNA were subjected to PCR amplification using primers specific for sequences of NGF, BDNF, NT-3 and CNTF. PCR products were separated through 5% acrylamide gel and identified by DNA sequencing. Results showed that neurons expressed detectable levels of mRNA for NGF in all four cultures; BDNF and NT-3 mRNA was seen only in two cultures; CNTF mRNA was not detected in all four cultures. Astrocytes expressed mRNA for NGF, BDNF, and NT-3 but not for CNTF in all cultures examined. Astrocytic expression of mRNA for NGF, BDNF and NT-3 was found during the active cell proliferation as well as at a phase of mitotic quiescence. This study provides evidence that dissociated cell cultures of human neurons produce NGF, BDNF and NT-3 in early stages of their development and that astrocytes are constitutively committed to synthesize neurotrophic factors, NGF, BDNF and NT-3. The active synthesis of selected neurotrophic factors by neurons and astrocytes is relevant in supporting migration, survival and differentiation of developing neurons in vivo.

Astrocytes↗

PubMed1994

Intracellular calcium concentrations during metabolic inhibition in the motoneuron cell line NSC-19.

Changes in the concentrations of intracellular free calcium ([Ca2+]i) and adenine nucleotides were determined in response to metabolic inhibitors in the motoneuron cell line NSC-19. The NADH dehydrogenase inhibitor amobarbital (Amytal) and the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) were used to alter energy metabolism. Exposure of cells to 5 mM Amytal did not significantly change ATP concentrations but produced transient elevations of [Ca2+]i of approximately 80 nM, which were reduced by 32% when cells were studied in Ca(2+)-free solutions. CCCP (10 microM) caused a transient reduction in ATP concentration of 33%. CCCP also produced sustained elevations of [Ca2+]i of about 280 nM, which were reduced by 47% when in Ca(2+)-free solutions. In spite of the sustained elevation of [Ca2+]i induced by CCCP, NSC-19 showed no reduction in cell viability after 48 h compared with controls. Ruthenium red, a blocker of Ca2+ uptake by mitochondria, had little effect on the CCCP-induced [Ca2+]i increment. KCl or glutamate did not produce significant changes in [Ca2+]i, indicating that these cells do not possess significant numbers of voltage-dependent Ca2+ channels or excitatory amino acid receptor-gated channels. [Ca2+]i values in these cells were modified by changes in extracellular Ca2+ concentrations. In Ca(2+)-containing solutions, inhibition of Na+/Ca2+ exchange by amiloride and bepridil led to increased [Ca2+]i, as did blockade of Ca2+ ATPase by vanadate, suggesting that membrane transporters are important in Ca2+ efflux in NSC-19. The present studies indicate that exposure of NSC-19 cells to Amytal and CCCP produces Ca2+ increments by release from internal stores, as well as by transmembrane influx. These results demonstrate that small increments in [Ca2+]i can be produced by metabolic inhibitors or other compounds and that such changes are not associated with immediate cell death. Changes in [Ca2+]i could potentially result in abnormal cell function secondary to altered action of Ca(2+)-dependent enzymes.

Adenine Nucleotides↗

PubMed1993

Glutamate neurotoxicity in mesencephalic dopaminergic neurons in culture.

The neurotoxic effect of glutamate in cultured mouse mesencephalic dopaminergic neurons was investigated. Neuron-rich cell cultures were prepared from 13-14-day-old fetal mouse ventral mesencephalic tissue. Cultures were exposed to glutamate for 10 min and evaluated for glutamate neurotoxicity (GNT) 18-24 hr later by tyrosine hydroxylase (TH) immunostaining, microtubule associated protein-2 (MAP2) immunostaining, and radiolabeled dopamine uptake assay. In glutamate-exposed cultures, the number of TH-positive neurons and the level of dopamine uptake were reduced to 40% (35-45%) and 50% (47-52%), respectively, of control cultures. The number of MAP2-positive neurons was also reduced to 47%, indicating that the GNT was not restricted or selective to dopaminergic neurons. It is concluded that GNT was mediated by the N-methyl-D-aspartic acid (NMDA) receptor from the following observations: 1) GNT was completely blocked by MK-801, an NMDA receptor antagonist; 2) NMDA itself was as toxic as glutamate; 3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor, did not block GNT; 4) kainate did not show neurotoxicity at a low concentration; and 5) two modulators of the NMDA receptor, 7-chlorokynurenic acid and magnesium, were effective in blocking GNT. Protective effects of phorbol myristate acetate, a tumor promoter, and gangliosides (GM1 and GT1b) on GNT were also demonstrated. Possible interactions between GNT and several protein kinase cascades were also investigated. Forskolin, an activator of adenyl cyclase and protein kinase A, showed some protective effect on GNT. But okadaic acid, an inhibitor of phosphatases, and genistein, a tyrosine kinase inhibitor, did not show any protective effect. These results suggest that 1) glutamate is capable of causing neuronal death in the substantia nigra; 2) GNT on dopaminergic neurons is mainly mediated by the NMDA receptor under the conditions of our study; 3) protein kinase C translocation is a key mechanism of GNT; and 4) there is an interplay of a signal transduction system in the pathomechanism of GNT.

Amino Acids↗

PubMed1993

Midkine that promotes survival of fetal human neurons is produced by fetal human astrocytes in culture.

Midkine (MK), a retinoic acid responsive gene product, is a novel heparin-binding growth factor with an apparent molecular weight of 14-kDa. In fetal human neuron cultures, addition of recombinant human MK (10-100 ng/ml) resulted in a 3-fold increase in the number of surviving neurons under the serum-free culture condition. Production of MK was identified in the conditioned medium of astrocyte-enriched cultures by immunoblotting but not in the medium of neuron-enriched cultures. The level of MK production was not affected by treatment with retinoic acid. These results indicate that in the developing human central nervous system, astrocytes secrete MK that exhibits potent trophic activity to neurons.

Animals↗

PubMed1993

Midkine, a novel neurotrophic factor, promotes survival of mesencephalic neurons in culture.

Midkine (MK), a retinoic acid responsive gene product, is a novel neurotrophic factor with a molecular weight of 14,000. MK at the optimal concentration of 10 ng/ml promoted the survival of mouse mesencephalic neurons in culture as demonstrated by the increased number of microtubule-associated protein-2 (MAP2) immunostaining-positive and tyrosine hydroxylase immunostaining-positive neurons and an elevated uptake of radiolabeled dopamine. These results suggest that MK has ubiquitous neurotrophic activity towards mesencephalic neurons which contain a large number of dopaminergic neurons.

Animals↗

PubMed1993

Retinoic acid responsive gene product, midkine, has neurotrophic functions for mouse spinal cord and dorsal root ganglion neurons in culture.

Midkine (MK) is the product of a retinoic acid responsive gene and is a member of a new family of heparin-binding growth factors. Neurotrophic effects of MK were examined using cultured spinal cord and dorsal root ganglion (DRG) neurons derived from fetal mouse. MK, which was added to the culture medium at concentrations of 1-100 ng/ml, promoted survival of both types of neurons approximately 5-fold after 7 days in culture. For spinal cord neurons, the increased survival was reflected in an increase of choline acetyltransferase activity. MK also promoted neurite extension in spinal cord (2-fold) and DRG (1.7-fold) neurons. The survival-promoting activity of MK to these neurons was comparable to that of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF). In spite of its significant effects on fetal neurons, MK was ineffective in sustaining survival of DRG neurons derived from postnatal mice. From these results, we conclude that MK is a neurotrophic factor to embryonic spinal cord and DRG neurons, and we propose that MK plays a significant role in embryogenesis of the nervous system.

Animals↗

PubMed1993

Midkine is a mediator of retinoic acid induced neuronal differentiation of embryonal carcinoma cells.

Midkine (MK) is a novel growth factor and is the product of a retinoic acid responsive gene. When P19 murine embryonal carcinoma (EC) cells were exposed to MK, they differentiated into neurons, and the neuronal differentiation was accompanied by expression of choline acetyltransferase activity. Synthesis and release of MK in the EC cells treated with retinoic acid were shown by Western blot analysis, and rabbit anti-MK antibody attenuated the action of retinoic acid to induce the neuronal differentiation. These results indicate that MK is one of the mediators of retinoic acid action to induce the neuronal differentiation in EC cells.

Animals↗

PubMed1993

Midkine, a novel neurotrophic factor, is present in senile plaques of Alzheimer disease.

An affinity purified antibody specific for midkine (MK) stained senile plaques in the brain of Alzheimer disease (AD) patients. After formic acid treatment, plaque staining was dramatically enhanced, and almost all beta-amyloid protein (BAP) deposits were also immunoreactive for MK. MK-immunoreactivity was not observed in normal cellular components nor in other pathological lesions including tangles in AD brain. Control brain sections were not immunoreactive for MK. The presence of MK in AD brain but not in control brain was confirmed by Western blotting. MK appears to be involved in the pathological process leading to senile plaque formation.

Aged↗

PubMed1993

Specific expression of inositol 1,4,5-trisphosphate 3-kinase in dendritic spines.

Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase (IP3K) in the rat cerebral cortex and hippocampus was studied immunohistochemically. In both regions, the major structure expressing a high level of IP3K was the dendritic spines of pyramidal neurons, where immunoreactivity was associated with the spine apparatuses and plasmalemma. The postsynaptic densities showed the most intense labelling. Taking into account the results of our previous observations, which demonstrated the restricted localization of the enzyme in the dendritic spines of Purkinje and basket cells in cerebellum, IP3K may be localized specifically in dendritic spines in various regions of the central nervous system, and involved in synaptic signal transduction at the spines.

Animals↗

PubMed1993

Human astrocytes and cytokines: tumor necrosis factor alpha and interferon gamma do not promote astrocytic proliferation.

Astrocyte cell cultures were established from human fetal brains and exposed to human recombinant tumor necrosis factor alpha (TNF alpha) and human recombinant interferon gamma (IFN gamma), and the proliferative response was assessed by bromodeoxyuridine-immunostaining technique. TNF alpha showed an inhibitory activity, while IFN gamma induced a slight increase in the number of astrocytes undergoing cell division. However, in both experiments differences were not statistically significant. Previous studies have demonstrated that TNF alpha and IFN gamma are potent mitogens for adult astrocytes in both in vivo and in vitro conditions. Our results suggest that in developing brain the immunomodulatory activity of cytokines, TNF alpha and IFN gamma, is different from that observed in adult brain.

Astrocytes↗

PubMed1993

Developmental profile of inositol 1,4,5-trisphosphate 3-kinase in rat cerebellar cortex: light and electron microscopic immunohistochemical studies.

Developmental expression and intracellular distribution of inositol 1,4,5-trisphosphate 3-kinase in the rat cerebellar cortex were studied immunohistochemically. Immunoreactivity appeared first at postnatal day 1 in the rostral region of the cerebellum and by day 15 had extended throughout the whole cerebellum, being localized in the Purkinje cell layer. Shortly after the expression of the enzyme in each Purkinje cell, the labelling showed a tendency to accumulate in the dendrites in a fine granular pattern. Electron microscopy revealed that immunoreactivity was present in the Purkinje dendritic trunks with accentuation in the distal segments during the early postnatal period, thereafter becoming concentrated in the dendritic spines at later developmental stages. Labelling was associated mainly with the plasmalemma, including the postsynaptic densities and open coated vesicles, and the subplasmalemmal vesicles of the smooth endoplasmic reticulum. Immunoreactivity was also evident in the perisomatic processes of immature Purkinje cells, which are transient projections synapsing with climbing fibers. In developing Purkinje axons, immunoreactivity was accentuated in the distal segments, associated with the plasmalemma and synaptic vesicles. These results suggest that inositol 1,4,5-trisphosphate 3-kinase is involved in the dendritic arborization and subsequent spine synaptogenesis of Purkinje cells, and that the developing presynaptic nerve endings of these cells are another functional site for the enzyme.

Aging↗

PubMed1993

Effects of tumor necrosis factor on inward potassium current and cell morphology in cultured human oligodendrocytes.

The effects of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on inward rectifier potassium [K(IR)] currents and on cell morphology have been studied in cultured human oligodendrocytes. Cell-attached patches were used to isolate and record unitary currents through an inward rectifier K+ channel with a conductance of 23 pS. In control oligodendrocytes the mean open times showed an exponential dependence on patch potential with an e-fold decrease over a patch hyperpolarization of 28 mV. Treatment of oligodendrocytes with rhTNF (at 250 ng/ml for 24-48 h) had significant actions to diminish the mean open times of K(IR) relative to control values. At cell resting potential the mean open times were reduced by 60% after rhTNF application; the amplitudes of unitary currents or the extrapolated zero-current potentials were not significantly changed by the cytokine. The rhTNF treatments were not cytotoxic to cultured human oligodendrocytes; however, in some experiments rhTNF caused evident retraction of cell processes.

Aged↗

PubMed1993

CD44 expression in human astrocytes and oligodendrocytes in culture.

CD44 is a glycoprotein present on the surface of some lymphocyte cell populations and other non-lymphoid cells, and is involved in many functions related to cell-cell and cell-matrix interactions. In this study, expression of CD44 antigen in primary neural cell cultures derived from fetal and adult human brains was investigated. In cultures processed for double immunofluorescence staining, approximately 80% of fetal astrocytes and more than 95% of adult astrocytes expressed the CD44 antigen on the cell bodies and processes; CD44 was also detected in 50-60% of adult oligodendrocytes. Neurons in fetal brain cell cultures did not express CD44 at all. Western blot analysis performed in astrocyte- and in neuron-enriched cultures confirmed the results from immunostaining and showed that the antibody against CD44 reacted with a polypeptide, of approximately 80 kD, that is present exclusively in the astrocyte-enriched cultures, but absent in neuron-enriched cultures. Our results indicate that CD44 glycoprotein is constitutively expressed in the human cells of glial cell lineage and its role is likely to be associated with normal neuroglia-mediated adhesion/recognition processes.

Aging↗

PubMed1993

Novel isoforms of mouse myelin basic protein predominantly expressed in embryonic stage.

Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well-known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.

Animals↗

PubMed1992

Tedisamil blocks a calcium-dependent potassium channel in cultured motoneurons.

A calcium-dependent potassium channel K(Ca) has been isolated in mouse motoneurons. With physiological concentrations of potassium across inside-out patches, a 100 pS K(Ca) channel was activated when the bath solution of Ca2+ was in excess of 1 microM. Introduction of the drug tedisamil, a blocker of repolarizing potassium channels in cardiac cells, at concentrations in the range 0.2-10 microM, caused a dose-dependent decrease in the mean open times for K(Ca). The drug action was consistent with open channel block of K(Ca) with an onward (blocking) rate constant of 6 x 10(7) M-1 s-1. Tedisamil, at concentrations of 1 microM and 5 microM, also blocked the K(Ca) channel when applied to outside-out patches with a similar potency as found with internal application. A large conductance K(Ca) channel in hippocampal neurons is also blocked by a number of putative Class III antiarrhythmic drugs, including tedisamil; thus, these agents may have utility in the characterization of the properties of K(Ca) channels in various cells.

Animals↗

PubMed1992

Differential distribution of cellular forms of beta-amyloid precursor protein in murine glial cell cultures.

The production and localization of cell-associated forms of beta-amyloid precursor protein (APP) of Alzheimer's disease was investigated in primary cultures of mouse glial cells. In both oligodendrocytes and astrocytes, immunofluorescence staining with an antibody against the carboxy terminus of APP revealed an intense cytoplasmic immunoreactivity. Immunoblotting of the cell extracts detected differences in the composition of APP between oligodendrocytes and astrocytes, notably the abundance of 107 kDa subtype in oligodendrocytes. Differences in immunoblot patterns were also noted between two buffer-insoluble, membrane-rich subcellular fractions of the glial cells, nuclear-mitochondrial and microsomal; the 119 kDa APP was enriched in the former, whereas the 73 and 115 kDa APPs in the latter. The results suggest that each APP subspecies may play a distinct functional role in different cell types and subcellular fractions.

Amyloid beta-Protein Precursor↗

PubMed1992

Leukemia inhibitory factor (LIF) mediated increase of choline acetyltransferase activity in mouse spinal cord neurons in culture.

The effects of leukemia inhibitory factor (LIF) on choline acetyltransferase (ChAT) enzyme activity in cultured mouse spinal cord neurons were examined. The administration of LIF to cultures at concentrations of 10 U/ml and higher enhanced ChAT activity approximately 3- to 4-fold in cultured spinal cord neurons. Among neurotrophic factors tested, basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) stimulated the development of ChAT activity but to a smaller extent than LIF, while interleukin 3 (IL-3), interleukin 6 (IL-6) and nerve growth factor (NGF) showed no apparent effect on ChAT development. Our results indicate that LIF, which has not been known to have any trophic effect on mammalian central nervous system neurons to date, acts as a potent differentiation factor for ChAT in cholinergic neurons of mouse spinal cord in culture.

Animals↗

PubMed1992

Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase in rat cerebellar cortex.

Subcellular localization of inositol 1,4,5-trisphosphate 3-kinase in the rat cerebellar cortex was studied immunohistochemically using a monoclonal antibody. Electron microscopy revealed intense immunoreactivity in the dendritic spines of Purkinje cells forming synapses with the parallel fibers, climbing fibers and recurrent collaterals of Purkinje cell axons. The labelling was associated with the hypolemmal cisternae, surrounding matrix and plasmalemma including the postsynaptic densities. Weaker immunoreactivity was present in the dendritic spines of basket cells and in certain segments of Purkinje cell recurrent collaterals. The postsynaptic regions of the dendritic trunks of Purkinje and basket cells were negative. These results indicate that inositol 1,4,5-trisphosphate 3-kinase is distributed amongst the spines of various synaptic relations with different electrophysiological properties, and that axon terminals of certain cell types are another functional site for the enzyme.

Animals↗