• Molecules and Cells
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• v.23 no.1
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• pp.11-16
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• 2007

N,N-dimethyl-D-erythro-sphingosine (DMS) is an N-methyl derivative of sphingosine and an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In the present study, we examined the effects of DMS on intracellular $Ca^{2+}$ concentration, pH, and glutamate uptake in human 1321N1 astrocytes. DMS increased intracellular $Ca^{2+}$ concentration and cytosolic pH in a concentration-dependent manner. Pretreatment of the cells with the $G_{i/o}$ protein inhibitor PTX and the PLC inhibitor U73122 had no obvious effect. However, removal of extracellular $Ca^{2+}$ with the $Ca^{2+}$ chelator EGTA or depletion of intracellular $Ca^{2+}$ stores with thapsigargin impeded the DMS-induced increase of intracellular $Ca^{2+}$ concentration. Pretreatment of cells with $NH_4Cl$ or monensin reduced the DMS-induced $Ca^{2+}$ increase. However, inhibition of the DMS-induced $Ca^{2+}$ increase with BAPTA did not influence the DMS-induced pH increase. DMS also inhibited glutamate uptake by the 1321N1 astrocytes in a concentration-dependent manner. It also increased intracellular $Ca^{2+}$ and pH in PC12 neuronal cells. Our observations on the effects of DMS on 1321N1 astrocytes and PC12 neuronal cells point to a physiological role of DMS in the brain.

• Journal of Nutrition and Health
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• v.32 no.4
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• pp.369-375
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• 1999

Hyperthroidism in known to alter the activity of a number of enzymes involved in the catabolism of histidine to CO2. 10-Formyltetrahydrofolate dehydrogenase(EC 1.5, 1.6, 10-formyl-THE dehydrogenase) catalyzes the NADP-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In previous studies, 10-formyl-THF dehydrogenase purified from rat and pig liver was coidentified with the cytosolic folate-binding protein. In this study, we investigated the effects of feeding thyroid powder (TP) and thiouracil(TU) on the folate-binding properties of 10-formyl-THE dehydrogenase, the uptake of an injected dose of [3H] folate, and the metabolism of labeled folate to pteroylopoly-${\gamma}$-glutamate in rat liver. The initial hepatic uptake(24hr) of the labeled folate dose was higher in TU-rats and slightly higher in TP-rats in controls. With longer time periods, decreased hepatic uptake of labeled folate was observed in TP-animals compared to euthroid animals, and high levels of hepatic uptake of labeled folate were maintained in TU-animals. This data shows that high levels of thyroid hormone decreased the retention of folate in rat liver. Folate polygutamate chain length was shorter in TU-rats than controls, which suggests that thyroid states do not affect the ability to synthesize pteroylpolyglutamates and that folate polyglutamate might be modulated by altered folate pool size. The ability of 10-formyl-THE dehydrogenase to bind folate in rat liver was similar in both TP-and TU-rats although dehydrogenase activity was changed by thyroid sates.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.6
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• pp.363-369
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• 2005

It is imperative to analyse brain injuries directly in real time, so as to find effective therapeutic compounds to protect brain injuries by stress. We established a system which could elucidate the real time $Ca^{2+}$ dynamics in an organotypic cultured hippocampal slice by the insults of artificial stress hormone, dexamethasone. The real time $Ca^{2+}$ dynamics could continuously be detected in cornus ammonis 3 (CA3) of the organotypic hippocampus for 8 hours under confocal microscopy. When dexamethasone concentration was increased, the $Ca^{2+}$ was also increased in a dose dependent manner at $1{\sim}100{\mu}M$ concentrations. Moreover, when the organotypic cultured hippocampal slice was treated with a glutamate receptor antagonist together with dexamethasone, the real time $Ca^{2+}$ dynamics were decreased. Furthermore, we confirmed by PI uptake study that glutamate receptor antagonist reduced the hippocampal tissue damage caused by dexamethasone treatment. Therefore, our new calcium ion dynamics system in organotypic cultured hippocampal slice after dexamethasone treatment could provide real time analysis method for investigation of brain injuries by stress.

• Microbiology and Biotechnology Letters
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• v.21 no.6
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• pp.527-533
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• 1993

Aminolevulinic acid(ALA) was shown to be synthesized via active pathways of either C4 or C5 ALA biosynthesis in cells of a photosynthetic bacterium, Rhodocyclus gelatinosus KUP-74, where the C5 pathway was appeared to be preferntially expressed in the cells. It was strongly suggested that L-glutamine might be utilized more effectively than L-glutamate to synthesize ALA via C5 pathway in this bacterium from the fact of relationship between the cellular uptake rates of glutamate and its Gamma-derivaties and corresponded ALA productivities in vitro and in vivo.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.13-21
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• 1996

Intracellular calcium concentration ($[Ca^{2+}]_i$) may play a crucial role in a variety of neuronal functions. Here we report that in primary culture of mouse cerebellar granule cells nicotinic acetylcholine receptors (nAChRs) are expressed in a specific developmental stage and involved in the regulation of intracellular calcium homeostasis. Nicotine-mediated calcium responses were measured using $^{45}Ca^{2+}$ or fluorometrically using the calcium-sensitive fluorescent dye fura-2. Maximal uptake of $^{45}Ca^{2+}$ evoked by nicotine in mouse cerebellar granule cells were revealed $8{\sim}12$ days in culture. In contrast, nicotine did not alter the basal $^{45}Ca^{2+}$ uptake in cultured glial cells. In cerebellar granule cells nicotine-evoked $^{45}Ca^{2+}$ uptake was largely blocked by the NMDA receptor antagonists. Glutamate pyruvate transaminase (GPT). which removes endogenous glutamate, also prevented nicotine effects, implying the indirect involvement of glutamate in nicotine-mediated calcium responses. Fluorometric studies using fura-2 showed two phases of nicotine-evoked $[Ca^{2+}]_i$ rises: the initial rising phase and the later plateau phase. Interestingly, the NMDA receptor antagonists and GPT appeared to inhibit only the later plateau phase of nicotine-evoked $[Ca^{2+}]_i$ rises. The present results imply that nicotine mediated $^{45}Ca^{2+}$ uptake and $[Ca^{2+}]_i$ rises are attributed to the calcium fluxes through both nAchRs and NMDA receptors in a time-dependent manner. Consequently, nAChRs may play an important role in neuronal development by being expressed in a specific developmental stage and regulating the intracellular calcium homeostasis.

• Macromolecular Research
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• v.14 no.3
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• pp.387-393
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• 2006

Nanoparticles of Poly(L-glutamic acid) (PG) conjugated to the anticancer drug paclitaxel and targeted moiety folic acid (FA) were synthesized and characterized in vitro. The nanoparticles were designed to take advantage of FA targeting to folate receptor (FR) positive cancer cells. The chemical composition of the conjugate was characterized by $^1H-NMR$, FTIR and UV/vis spectroscopy. The selective cytotoxicity of the FA-PG-paclitaxel conjugates was evaluated in FR positive cancer cells. The interaction of the conjugate was visualized by fluorescence microscopy with results confirming the successful preparation of the conjugate and the production of nanoparticles of about 200-300 nm in diameter. The amount of paclitaxel conjugated to FA-PG was 25% by weight. Cellular uptake of the conjugate was FA dependent, and the conjugate uptake was mediated specifically by the folate receptor. These results demonstrate the improved selective toxicity and effective delivery of an anticancer drug into FR bearing cells in vitro.

• Biomolecules & Therapeutics
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• v.17 no.2
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• pp.175-180
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• 2009

Taurine is the most abundant free amino acid in the retina and transported into retina via taurine transporter (TauT) at the inner blood-retinal barrier (iBRB). In the present study, we investigated whether the taurine transport at the iBRB is regulated by oxidative stress or disease-like state in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) used as an in vitro model of iBRB. First, [$^3H$]taurine uptake and efflux by TR-iBRB were regulated in the presence of extracellular $Ca^{2+}$. [$^3H$]Taurine uptake was inhibited and efflux was enhanced under $Ca^{2+}$ free condition in the cells. In addition, oxidative stress inducing agents such as tumor necrosis factor-$\alpha$ (TNF-$\alpha$), lipopolysaccharide (LPS), diethyl maleate (DEM) and glutamate increased [$^3H$]taurine uptake and decreased [$^3H$]taurine efflux in TR-iBRB cells. Whereas, 3-morpholinosydnonimine (SIN-1), which is known to NO donor decreased [$^3H$]taurine uptake. Lastly, TR-iBRB cells exposed to high glucose (25 mM) medium and the [$^3H$]taurine uptake was reduced about 20% at the condition. Also, [$^3H$]taurine uptake was decreased by cytochalasin B, which is known to glucose transport inhibitor. In conclusion, taurine transport in TR-iBRB cells is regulated diversely at extracellular $Ca^{2+}$, oxidative stress and hyperglycemic condition. It suggested that taurine would play a role as a retinal protector in diverse disease states.

• YAKHAK HOEJI
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• v.43 no.6
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• pp.851-860
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• 1999

The effects of antimycin A(AA), dodium azide ($NaN_3$) and 2,4-dinitrophenol (DNP), which inhibit mitochondrial ATP production, on cellular functions of cultured astrocytes were studied. High concentrations of AA $(50{\;}\mu\textrm{g}/ml),{\;}NaN_3$ (100mM) and DNP (20mM) significantly decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, which was known to be related to mitochondrial function and then cel viability. AA ($50{\;}\mu\textrm{g}/ml$) increased lactate dehydrogenase (LDH) release and decreased [$^3H$] glutamate uptake, suggesting severe damage of cellular function by the concentrations of the compounds. Meanwhile, low concentrations of AA $(\leq{;\}10{\;}\mu\textrm{g}/ml),{\;}NaN_3{;\}(\leq{\;}50mM)$ and DNP ($\leq{\;}5mM$) significantly increased MTT reduction, the effect of which was specific to astrocytes. AA (5 and $10{\;}\mu\textrm{g}/ml$) did not affect LDH release and [$^3H$] glutamate uptake, indicating that these compounds increased MTT reduction at the low concentrations without cellular membrane damage. However, the low concentrations of AA produced significant decrease of MTT reduction in a glucose-free medium. Low concentrations of AA (1 and $5{\;}\mu\textrm{g}/ml$) did not change ATP production of astrocytes in the medium containing 10 mM glucose, but completely inhibited in a glucose-free medium, suggesting marked increase of cytosolic ATP production by the blockade of mitochondrial ATP production with low concentrations of AA. These results suggest that astrocytes have ability to enhance neuronal function or survival under conditions of incomplete ischemia or early by enhancement of glycolysis, and that cellular reduction of MTT occurs not only mitochondrially but also extramitchondrially.

• The Korean Journal of Pharmacology
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• v.24 no.1
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• pp.17-29
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• 1988

The author examined the effects of lidocaine on the veratrine-or potassium-induced release of neurotransmitters to determine the possible role of amino acid neurotransmitters in lidocaine-induced convulsion. The examined transmitters were gamma-aminobutyric acid (GABA), aspartic acid, glutamic acid and norepinephrine which are released from the synaptosomes. Furthermore, the effects of lidocaine on the binding to receptors and synaptosomal uptake of the two transmitters, GABA and glutamic acid, were determined in crude synaptic membranes and synaptosomes. In addition, the effects of propranolol, norepinephrine and serotonin on the release of amino acid neurotransmitters were also examined. The veratrine-induced release of GABA was most severely inhibited by lidocaine and propranolol, while norepinephrine and serotonin reduced the release of aspartic acid and glutamic acid more than the GABA release. Generally the potassium-induced release was much more resistant to the lidocaine action than the veratrine-induced release. Among the neurotransmitters examined, the aspartic acid release was most prone to the lidocaine action, while the GABA release was most resistant. Concentrations of lidocaine below 1 mM did not significantly change the GABA and glutamic acid receptor binding and uptake. These results indicate that the blocking of sodium channels by lidocaine can result in the selective depression of the GABA release. This may result in unlimited excitation of the central nervous system.

• Biomolecules & Therapeutics
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• v.20 no.3
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• pp.245-255
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• 2012

Amyloid-${\beta}$ peptide ($A{\beta}$) is still best known as a molecule to cause Alzheimer's disease (AD) through accumulation and deposition within the frontal cortex and hippocampus in the brain. Thus, strategies on developing AD drugs have been focused on the reduction of $A{\beta}$ in the brain. Since accumulation of $A{\beta}$ depends on the rate of its synthesis and clearance, the metabolic pathway of $A{\beta}$ in the brain and the whole body should be carefully explored for AD research. Although the synthetic pathway of $A{\beta}$ is equally important, we summarize primarily the clearance pathway in this paper because the former has been extensively reviewed in previous studies. The clearance of $A{\beta}$ from the brain is accomplished by several mechanisms which include non-enzymatic and enzymatic pathways. Nonenzymatic pathway includes interstitial fluid drainage, uptake by microglial phagocytosis, and transport across the blood vessel walls into the circulation. Multiple $A{\beta}$-degrading enzymes (ADE) implicated in the clearance process have been identified, which include neprilysin, insulin-degrading enzyme, matrix metalloproteinase-9, glutamate carboxypeptidase II and others. A series of studies on $A{\beta}$ clearance mechanism provide new insight into the pathogenesis of AD at the molecular level and suggest a new target for the development of novel therapeutics.