Biomedical subjects

S U Kim

Publications and source records attributed to S U Kim.

At least 109 records · Page 6Linked to original sources

PubMed1995

Complement and cytokine gene expression in cultured microglial derived from postmortem human brains.

Microglia were successfully cultured from human brain tissue from normal and neurologically diseased cases obtained 3.5-10 hours postmortem. Final cell preparations were more than 99% pure as judged by latex bead phagocytosis, expression of microglial phenotypic markers, and absence of astrocytic markers. The expression of complement genes C1qB, C3, and C4 as well as genes for interleukin-(IL-)1 alpha, IL-1 beta, IL-6, tumor necrosis factor (TNF)alpha, IL-1 receptor antagonist, and transforming growth factor beta, but not inducible nitric oxide synthase, by these cells was detected by polymerase chain reaction (PCR) analysis. The pattern of gene expression was evaluated following stimulation of the cells with lipopolysaccharide, phorbol myristate acetate, gamma interferon, and beta amyloid peptide. There was considerable variation in gene response to these activating agents. However, it was of interest that beta-amyloid peptide (1-40) increased the expression of IL-1 beta mRNA in these cells. The number of cases in this study was too small to permit evaluation of microglial response according to the disease state, but the results demonstrate the potential for such studies in the future.

Aged↗

PubMed1995

Low-affinity nerve growth factor receptor immunoreactivity in the human urinary bladder.

The localization of low-affinity nerve growth factor receptor (LNGFR) in the human urinary bladder was examined immunohistochemically using the mouse monoclonal antibody (ME20-4) against human LNGFR. LNGFR immunoreactivity was present in the human urinary bladder. The distribution of LNGFR-positive fibers was more abundant in the mucosa than in the muscle layer. Results also showed that some LNGFR-positive fiber bundles contained tyrosine hydroxylase immunoreactivity. Electron microscopic examination revealed that LNGFR immunoreactivity was located on the surface of Schwann cells, and frequently on the interface of axons and Schwann cells.

Adolescent↗

PubMed1995

Myelin basic protein does not have a mitogenic effect on adult oligodendrocytes.

Increased numbers of oligodendrocytes and remyelination are frequently observed in multiple sclerosis plaques. It is presumed the increased numbers of oligodendrocytes are due to cell division, but this has not been proven. The mitogens within the lesion which might be responsible for this are unknown. Since oligodendrocyte proliferation occurs in areas in which there is myelin breakdown, we undertook the present study to determine if myelin basic protein (MBP) or its breakdown products could induce oligodendrocyte proliferation. MBP, or MBP digested by the neutral proteinase plasmin, was added in three concentrations to the media of adult bovine oligodendrocytes in culture. Oligodendrocytes were identified by staining for galactocerebroside. Bromodeoxyuridine incorporation was used as a measure of cell division. Oligodendrocytes were found to divide only rarely in regular culture media, in the presence of MBP, plasmin, or MBP digested by plasmin. The results indicate that MBP is not a significant mitogen for the mature oligodendrocyte.

Animals↗

PubMed1995

Properties of inward and outward potassium currents in cultured mouse motoneurons.

Inward rectifier potassium currents and calcium-dependent potassium currents have been studied in cultured embryonic mouse motoneurons. Sustained unitary inward rectifier potassium currents were recorded from cell-attached patches and the channel conductance was dependent on external K+ concentration with a value of 25 pS when external K+ was 140 mM. The channel open probability exhibited a sigmoidal dependence on potential with the largest values (near 0.7) at depolarizing patch potentials. Inactivating inward rectifier potassium currents were also recorded in some cell-attached patches following voltage steps to hyperpolarizing potentials with the rate of inactivation faster with larger hyperpolarizing steps. Whole-cell inward rectifier potassium currents increased from an initial level to a steady-state level with hyperpolarizing steps to -120 mV from a holding potential of -60 mV; with larger hyperpolarizing commands the peak currents decayed to the steady-state. The steady-state current-voltage relation exhibited a region of negative slope resistance. External Cs+ (0.5-1 mM) reduced the amplitudes of macroscopic currents and diminished the open times of unitary currents consistent with block of open rectifying channels with an estimated KD for channel block of 1 mM. A large conductance calcium-dependent potassium channel was isolated in inside-out patches with a conductance of 240 pS with symmetrical 140 mM K+ across the patches and a conductance of 110 pS when the external K+ was reduced to 5 mM. With symmetrical K+ the channel open probability exhibited a sigmoid dependence on potential with the largest values, in excess of 0.8, associated with patch depolarization. The dependence of open probability on potential was dependent on the concentrations of internal Ca2+ and external K+. Properties of inward rectifier and calcium-dependent K+ channels, such as the voltage dependence of open probability, are involved in the establishment of cellular excitability in motoneurons. Future studies will be useful to investigate whether channel properties of motoneurons are altered after cell treatment with neurotoxic agents including oxygen radicals or excitotoxic amino acids.

Animals↗

PubMed1995

Expression of proteolipid protein gene is directly associated with secretion of a factor influencing oligodendrocyte development.

Oligodendroglial cell death in the myelin proteolipid protein (PLP) mutants can be partially rescued by the environment factor(s) supplied by the wild-type cells in vivo and in vitro. It is possible that the presence of PLP or DM-20 results in secretion of a factor or factors in the CNS influencing oligodendrocyte development. We previously showed that DM-20 mRNA is produced in G26 mouse oligodendroglioma, B104 rat neuroblastoma, and B16 mouse melanoma but not in NIH3T3 mouse fibroblast cell lines. Culture supernatants from these cell lines were added to primary glial cell cultures from embryonic day 17 mouse brain. After 4 days, the number of oligodendrocytes present in cultures with supernatants from DM-20-producing cells (G26, B104, and B16) was significantly higher than that of control cultures but not with the NIH3T3 supernatant. To investigate more directly whether the PLP gene expression is involved in this process, NIH3T3 cells (nonneural cells) were forced to produce PLP or DM-20. By addition of the supernatants from the PLP/DM-20 transformants, the number of oligodendrocytes in the mixed glial cell cultures increased. This clearly demonstrates that the expression of the PLP gene is sufficient for and directly associated with secretion of a factor, which influences the oligodendrocyte development.

3T3 Cells↗

PubMed1995

Cytokines and growth factors induce HSP27 phosphorylation in human astrocytes.

In previous studies, an enhanced expression with abnormal phosphorylation of a low molecular weight heat shock protein, HSP27, was identified in reactive astrocytes and Rosenthal fibers in Alexander disease brain. To investigate the relevance of HSP27 to the pathological change of astrocytes, phosphorylation of HSP27 was examined in astrocyte cultures isolated from fetal human brains. In unstimulated human astrocytes, HSP27 was identified in the cytoplasm and was comprised of a major unphosphorylated "a" isoform and a minor monophosphorylated "b" isoform. The level of HSP27 phosphorylation was elevated greatly after a 30 minute exposure to heat shock, sodium arsenite, interleukin-1 (IL-1 alpha and IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) with an increased expression of a diphosphorylated "c" isoform. Treatment with interferon-beta (IFN-beta), platelet-derived growth factor-AA, leukemia inhibitory factor, phorbol 12-myristate 13-acetate, and dibutyryl cyclic AMP stimulated phosphorylation of HSP27 moderately, while IFN-gamma, TNF-beta, basic fibroblast growth factor, epidermal growth factor, or fetal bovine serum did not significantly alter the level of HSP27 phosphorylation. Total amount of the HSP27 protein and its cytoplasmic localization were unaffected by any of these reagents. These results indicate that HSP27 is a constitutive protein in human astrocytes. The induction of HSP27 phosphorylation by a specific set of cytokines and growth factors suggests that HSP27 is a key cellular substrate by which signaling events are mediated in human astrocytes under normal and pathological conditions.

Astrocytes↗

PubMed1995

Expression and distribution of GAP-43 in human astrocytes in culture.

By combining mRNA analysis and immunocytochemistry, we investigated the expression of the growth associated protein 43 (GAP-43) in enriched populations of astrocytes, obtained from mixed cultures of human fetal brains. Total cellular RNA was extracted from cell pellets and reverse transcribed into cDNA; cDNA was subjected to PCR amplification using primers specific for GAP-43 and PCR products were separated through polyacrylamide gels. Double immunofluorescence staining was performed on dissociated cell cultures using antibodies to glial fibrillary acidic protein (GFAP) and to GAP-43. Results showed that both transcription and translation for GAP-43 occur in cultured astrocytes. GAP-43 immunoreacting material was detected in the cell processes and diffusely in the cytoplasm of GFAP-positive astrocytes, during early stages of maintenance in vitro. In older cultures, GAP-43 immunoreactivity persisted in a large percentage of cells, with a tendency to accumulate in perinuclear areas. These observations provide evidence that GAP-43 is not restricted to neuronal cells. The close spatial association with cytoskeletal constituents, as observed in astrocytes, suggests a role for this protein in the control of cell shape, motility and adhesion processes.

Astrocytes↗

PubMed1995

Ganglioside markers GD3, GD2, and A2B5 in fetal human neurons and glial cells in culture.

Expression of ganglioside markers GD3, GD2, and A2B5 was investigated in primary cell cultures isolated from fetal human brains of 12-15 weeks' gestation by immunocytochemistry. None of neuron-specific enolase (NSE)+ neurons expressed GD3, while large numbers of NSE+ neurons expressed GD2 (72%) or A2B5 (48%). In GFAP+ astrocytes, GD3 was expressed in a small population (3%) with a high proliferative capacity. GD2 expression was observed in 20% of GFAP+ astrocytes, while A2B5 was identified in a very small number (2%) of GFAP+ astrocytes. GD2 was coexpressed in a small population (11%) of GD3+ astrocytes, while A2B5 was colocalized in more than 50% of GD3+ astrocytes. In galactocerebroside+ oligodendrocytes, GD3 expression was not observed but a small population (8-9%) expressed GD2 and A2B5. In Ricinus communis agglutinin (RCA)+ microglia, neither GD3, GD2, nor A2B5 were identified. Our results indicate that in fetal human brain cell cultures, GD3 is expressed in a small population of astrocytes, while both GD2 and A2B5 are expressed in a large population of neurons and smaller populations of astrocytes and oligodendrocytes. Our results suggest that both GD3 and A2B5, cell type-specific markers for oligodendrocyte-type 2 astrocyte progenitor cells in the rat central nervous system, could not be utilized as valid markers for glial precursor cells in fetal human brain cell cultures.

Bromodeoxyuridine↗

PubMed1995

Differential effects of beta and gamma interferons on expression of major histocompatibility complex antigens and intercellular adhesion molecule-1 in cultured fetal human astrocytes.

We investigated the modulatory effects of human interferon beta (IFN-beta) and gamma (IFN-gamma) on expression of class I and II major histocompatibility complex (MHC) antigens and intercellular adhesion molecule-1 (ICAM-1) in fetal human astrocytes in culture using flow cytometry and immunocytochemistry. Under the baseline condition, class I MHC antigen and ICAM-1 were expressed in a moderate number (23 to 76%) of astrocytes, whereas class II MHC antigen was expressed in only a small number (0.3 to 8%) of astrocytes. Following a 72-hour treatment with IFN-gamma (10 to 100 U/ml), expression of all three antigens increased greatly. Expression of class I MHC antigen was also elevated by exposure to IFN-beta (10 and 100 IU/ml). However, IFN-beta did not significantly induce expression of class II MHC antigen or ICAM-1. Furthermore, IFN-beta significantly reduced IFN-gamma-induced expression of class II MHC antigen but not of class I MHC antigen or of ICAM-1. The differential effects of IFN-beta and IFN-gamma on expression of class I and II MHC antigens and ICAM-1 in fetal human astrocytes suggest that interferons serve as modulators of astrocyte function at sites of inflammation in the human CNS.

Abortion, Legal↗

PubMed1995

GERI-BP001 compounds, new inhibitors of acyl-CoA: cholesterol acyltransferase from Aspergillus fumigatus F37. I. Production, isolation, and physico-chemical and biological properties.

GERI-BP001 compounds, new inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT), were isolated from a culture broth of Aspergillus fumigatus F37 by acetone extraction, EtOAc extraction, SiO2 column chromatography, and reverse phase HPLC. GERI-BP001 M, A, and B inhibit ACAT activity in an enzyme assay system using rat liver microsomes by 50% at concentrations of 42, 94, and 40 microM, respectively.

Animals↗

PubMed1995

Down-regulation of transforming growth factor-beta gene expression by antisense oligodeoxynucleotides increases recombinant interferon-gamma-induced nitric oxide synthesis in murine peritoneal macrophages.

Increasing evidence indicates that the production of nitric oxide (NO) by inducible NO synthase (iNOS) is tightly regulated. Transforming growth factor-beta (TGF-beta) is a family of multifunctional peptides secreted during macrophage activation, but several lines of evidence suggest that TGF-beta is selectively suppressive for macrophage NO production. We therefore reasoned that a strategy employing oligodeoxynucleotides (ODN) complementary to TGF-beta mRNA (antisense ODN) might increase NO production in interferon-gamma (IFN-gamma) treated murine peritoneal macrophages. To evaluate this concept, we tested the effects of antisense ODN targeted to TGF-beta mRNA (25-mer ODN complementary to TGF-beta mRNA sequences) by introducing them into the medium of cultured macrophages. Phosphorothioation of ODN was employed to retard their degradation. Antisense ODN had no effect on NO production by itself, whereas IFN-gamma alone had a modest effect. When antisense ODN were used in combination with IFN-gamma, there was a marked cooperative induction of NO production. These effects of antisense ODN were associated with decreased TGF-beta expression in activated macrophages. However, sense ODN had no effect. Adding anti-TGF-beta antibodies to the IFN-gamma-treated macrophages mimicked the positive effect of antisense ODN on NO production. In addition, the effects of either antisense ODN or anti-TGF-beta antibodies were blocked by adding exogenous TGF-beta in cultured macrophages. These results indicate that the generation of TGF-beta by activated macrophages provides a self-regulating mechanism by which the temporal and perhaps spatial production of NO, a reactive and potentially toxic mediator, can be finely regulated.

Animals↗

PubMed1994

Proliferation and differentiation of fetal human oligodendrocytes in culture.

Phenotypic expression and proliferative capacity of the cells of oligodendrocyte lineage were investigated in primary cultures isolated from fetal human brains of 12-15 weeks' gestation using double immunolabeling with Ranscht-monoclonal antibody (R-mAb) or O4 and antibromodeoxyuridine (BrdU) antibody. Cultured cells of oligodendrocyte lineage consisted of a major population of R-mAb+O4- cells and minor populations of R-mAb-O4+ and R-mAb+O4+ cells. Most of the R-mAb+O4- cells exhibited a uni-, bi-, or tripolar immature morphology, while the majority of the R-mAb+O4+ cells exhibited a multipolar mature morphology. R-mAb-O4+ cells contained a mixture of immature and mature cell types. When incubated in serum-free culture medium containing BrdU for 4 days, 42% of total oligodendrocytes expressed nuclear BrdU immunolabeling. R-mAb+ cells exhibited a higher degree of BrdU immunolabeling, indicating that they have greater capacities for proliferation than O4+ cells. The large majority of BrdU+ cells exhibited an immature morphology. Inclusion of insulin, insulin-like growth factor (IGF)-I, basic fibroblast growth factor (bFGF), or fetal bovine serum in culture medium did not stimulate proliferation of oligodendrocytes, while platelet-derived growth factor (PDGF) or PDGF plus bFGF increased the number of R-mAb+BrdU+ and O4+BrdU+ cells over control, even though the results were not statistically significant. In addition, insulin and IGF-I induced a 3-fold increase in the number of R-mAb+O4+ cells, indicating that they promoted differentiation of oligodendrocytes. The present study indicates that fetal human oligodendrocytes in culture exhibit a considerable degree of proliferative capacity without requirement of exogenous growth factors and that both insulin and IGF-I promote their differentiation.

Animals↗

PubMed1994

HSP72 induction by heat stress in human neurons and glial cells in culture.

Expression of 72-kDa heat shock protein (HSP72) induced by heat stress was investigated in cultured neurons and glial cells isolated from fetal human brains using immunoblotting and immunocytochemistry. Under the unstressed condition, a low level of HSP72 expression was observed in astrocytes, microglia, oligodendrocytes, and neurons. Under the heat-stressed condition, an increased expression of HSP72 was observed in all cell types with specific location in the nuclear and cytoplasmic regions. Following heat stress, HSP72 was expressed intensely in more than 50% of astrocytes and microglia during 8-24 h post-recovery, while it was detectable in only 9% of oligodendrocytes and 3% of neurons at 48 h post-recovery. These results indicate that heat stress induces a predominant expression of HSP72 in astrocytes and microglia, and more limited HSP72 expression in oligodendrocytes and neurons in fetal human neural cells in culture. The differential patterns of HSP72 induction in human neural cells by heat stress suggest that cellular mechanisms by which the heat shock response is regulated are different among various cell types in the human central nervous system.

Astrocytes↗

PubMed1994

Differential expression of Lewis(x) and sialyl-Lewis(x) antigens in fetal human neural cells in culture.

Lewis(x) is a cell-surface carbohydrate antigen defined by the trisaccharide structure, Gal beta 1-->4 (Fuc alpha 1-->3) GlcNAc. Expression of Lewis(x) and sialyl-Lewis(x) antigens in primary cell cultures isolated from fetal human brains of 12-15 weeks gestation was investigated by double immunolabelling with antibodies against monomeric Lewis(x) (4C9), oligomeric Lewis(x) (FH4), and sialylated oligomeric Lewis(x) (FH6) antigens and cell type-specific markers. The monomeric Lewis(x) antigen was expressed in more than 15% of astrocytes and 100% of oligodendrocytes, whereas it was not identified in neurons or in microglia. The oligomeric Lewis(x) antigen was undetectable in any cell types, while the sialylated oligomeric Lewis(x) antigen was expressed in more than 95% of microglia but not in any other cell types. The cell type-specific expression of Lewis(x) and sialyl-Lewis(x) antigens in fetal human glial cells suggests that these fucose-containing carbohydrate molecules play roles in intercellular recognition between distinct cell types during the development of the human central nervous system.

Antibodies, Monoclonal↗

PubMed1994

HSP72 induction by heat stress is not universal in mammalian neural cell lines.

Heat-induced expression of 72-kDa heat shock protein (HSP72) was investigated in a panel of neuronal and non-neuronal cell lines by immunoblotting and immunocytochemistry using monoclonal antibodies directed to HSP72. By immunoblotting, HSP72 expression was observed in most cell lines of mouse (SN6.1b, CL8c4.7, NSC34.6, B2A, C2C12), rat (PC12, C-6, L3), and human (NB-1, GOTO, IMR-32, HeLa) origin under the heat-stressed condition. The mouse neuroblastoma cell line N18TG2, however, did not express HSP72 under the heat-stressed condition. By immunocytochemistry, HSP72 was undetectable in the heat-stressed N18TG2 cells, while it was identified in the heat-stressed SN6.1b cells, a clonal hybrid neuron between N18TG2 and mouse septal cholinergic neuron. By exposure to a priming sublethal heat shock, SN6.1b cells but not N18TG2 cells acquired a significant level of tolerance to a subsequent lethal heat shock. These results suggest that heat-induced expression of HSP72 may contribute to acquisition of the thermotolerant state in SN6.1b cells.

Animals↗

PubMed1994

Oxygen radical-induced neurotoxicity in spinal cord neuron cultures.

The neurotoxic effects of oxygen radicals on spinal cord neuron cultures derived from fetal mouse have been studied. Oxygen radicals, superoxide radical and hydrogen peroxide, were generated by adding xanthine oxidase and hypoxanthine in the culture medium. Exposure of neurons to this oxygen radical-generating system resulted in a significant cell death and decrease of choline acetyltransferase (ChAT) activity in a time-dependent manner in spinal cord neuron cultures. The decrease in cell viability and ChAT enzyme activity induced by the oxygen radicals was blocked by scavengers such as superoxide dismutase (SOD), catalase, and tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a metal chelator. Antagonists of the N-methyl-D-aspartate (NMDA) receptor, including MK801 (a noncompetitive NMDA antagonist), D-2-amino-5-phosphonovaleric acid (APV) (a competitive NMDA antagonist), and 7-chlorokynurenic acid (an antagonist at the glycine site associated with the NMDA receptor), similarly blocked oxygen radical-induced decrease in cell viability and ChAT activity in spinal cord neuron cultures. These results indicate that both oxygen radicals and excitotoxic amino acids were involved in the oxidant-initiated neurotoxicity of spinal cord neurons.

Animals↗

PubMed1994

The role of intracellular free calcium in motor neuron disease.

The intracellular calcium (Ca2+) concentrations of motoneurons can be altered by the influx of Ca2+ into the cell by the opening of voltage-dependent Ca2+ channels and ligand-gated channels linked to Ca2+ influx, especially by the N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. Intracellular Ca2+ concentration is also affected by the release of Ca2+ buffered in mitochondria and endoplasmic reticulum. Evidence that motoneurons may be selectively vulnerable to Ca(2+)-induced cell death include the following observations: (i) the presence of excitatory amino acid receptors on the cell membranes of motoneurons, some of which would permit Ca2+ influx (e.g. NMDA receptors); (ii) the availability of the presynaptic terminal for antibody-mediated effects leading to changes in cell permeability and Ca2+ influx; and (iii) the limited amounts of intracellular Ca(2+)-binding proteins such as calbindin D28K and parvalbumin in motoneurons. Elevation of intracellular free Ca2+ may also be a common event in a number of independent mechanisms leading to motoneuron death in motor neuron disease.

Animals↗

PubMed1994

Rapidly proliferating glial cells isolated from adult mouse brain have a differentiative capacity in response to cyclic AMP.

A glial cell line designated as B2 was generated from primary cultures of oligodendrocytes/astrocytes isolated from an adult BALB/c mouse brain and maintained for over 1 year. Phenotypic characteristics of B2 cells were investigated by immunolabeling with cell type-specific markers for oligodendrocytes (O4 and galactocerebroside (GalC)), astrocytes (glial fibrillary acidic protein (GFAP)), and immature neuroectodermal cells (vimentin). When cultured in a serum-containing medium, B2 cells exhibited a bipolar or a tripolar process-bearing morphology and proliferated with a 24-28 h doubling time, without requirement of exogenous growth factors. Under this culture condition, vimentin was identified in all of the B2 cells, GFAP in 7%, and O4 and GalC in less than 1% of the cells. When cultured in a serum-free medium containing 1 mM dibutyryl cyclic AMP (dbcAMP), B2 cells extended longer processes and 45% of the cells expressed cell type-specific markers for oligodendrocytes or astrocytes. GFAP was identified in 29% of B2 cells, O4 in 16%, and GalC in 6% of the cells, although, neither O4+GFAP+ nor GalC+GFAP+ cells were observed. B2 cells proliferated in response to phorbol 12-myristate 13-acetate (PMA), basic fibroblast growth factor (bFGF), insulin, insulin-like growth factor (IGF-I) and platelet-derived growth factor (PDGF), but not to dbcAMP, forskolin (FK), or retinoic acid (RA). These results indicate that B2 cells are distinct from typical oligodendrocytes and astrocytes with respect to their great proliferative potential, and suggest that B2 cells, with a capacity to differentiate into oligodendrocytes and astrocytes in response to cyclic AMP, may represent a population of glial precursor cells in the adult mouse central nervous system (CNS).

Animals↗