• Parasites, Hosts and Diseases
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• v.27 no.3
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• pp.161-170
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• 1989

The proteases of Toxoplasma gcndii were purified partially and characterisrd for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteasfs working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion rxchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotorf. The protease working at pH 6.0 was inactivated by iodoacetamide with LDso of 10-5 M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD50 of 10-5 M and was Promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.4
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• pp.149-154
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• 2007

Kainic acid (KA) causes neurodegeneration, but no consensus has been reached concerning its mechanism. Nitric oxide may be a regulator of the mechanism. We identified differentially expressed genes in the hippocampus of mice treated with kainic acid, together with or without L-NAME, a nonselective nitric oxide synthase inhibitor, using a new differential display PCR method based on annealing control primers. Eight genes were identified, including clathrin light polypeptide, TATA element modulatory factor 1, neurexin III, ND4, ATPase, $H^+$ transporting, V1 subunit E isoform 1, and N-myc downstream regulated gene 2. Although the functions of these genes and their products remain to be determined, their identification provides insight into the molecular mechanism(s) involved in KA-induced neuronal cell death in the hippocampal CA3 area.

• The Korean Journal of Mycology
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• v.26 no.1 s.84
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• pp.119-126
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• 1998

Mitochondria were isolated from Lentinus edodes and properties of the mitochondrial NADH dehydrogenase were studied. Optimal pH, temperature, and thermal stability of the enzyme were estimated to be 7.6, $33^{\circ}C$, and stable for one hour at $50^{\circ}C$. The apparent $K_m$ for the NADH was 0.33 mM. This enzyme catalyzed to transfer electrons from NADH to ferricyanide, decylubiquinone, and 2,6-dichloro-phenol-indophenol. 0.5 mM antimycin A and 0.01 mM dibromothymoquinone strongly inhibited 87.8% and 76.5% of the enzyme activities. 0.01 mM oligomycin known as an inhibitor of ATPase also strongly inhibited 79.2% of activities. 0.5 mM 5,5'-dithiobis-(2-nitrobenzoic acid) and 1.0 mM N-ethylmaleimide known as a modifier of SH group inhibited 50.4% and 36.7% of activities. 1 mM ethyl 2,4-dihydroxy-6-methyl benzoate and 10 mM orcinol, which had been known as an antibiotics isolated from Umbilicaria vellea according to our previous work, stimulated 68.4% and 48.1% of the enzyme activities.

• Journal of Acupuncture Research
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• v.19 no.2
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• pp.211-220
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• 2002

Objective : This study was undertaken to determine whether Scutellaria baicalensis Georgi extract (SbG) exerts the protective effect against oxidant-induced alterations in organic cation transport in the renal proximal tubule. Methods : Organic cation transport was estimated by examining alterations in tetraethylammonium (TEA) uptake in rabbit renal cortical slices. The slices were treated with 0.2 mM tBHP for 60 min at $37^{\circ}C$. tBl-IP caused an inhibition in TEA uptake by renal cortical slices. Such an effect was accompanied by depressed Na+-K+-ATPase activity and ATP depletion. Result : SbG prevented tBHP-induced inhibition of TEA uptake in a dose-dependent manner at the concentration ranges of 0.05-0.1%. SbG also prevented H2O2-induced reduction in TEA uptake. tBHP-induced inhibition of Na+-K+-ATPase activity and ATP depletion were significantly prevented by 0.05% SbG. Oxidants increased LDH release, which was blocked by SbG. Oxidants caused a significant increase in lipid peroxidation and its effect was prevented by SbG. Conclusion : These results suggest that SbG prevents oxidant-induced alterations in organic cation transport in rabbit renal cortical slices. Such protective effects of SbG may be attributed to inhibition of peroxidation of membrane lipid.

• Journal of Microbiology and Biotechnology
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• v.19 no.1
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• pp.37-41
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• 2009

Previous studies have demonstrated that Shewanella decolorationis S12 can grow on the azo compound amaranth as the sole electron acceptor. Thus, to explore the mechanism of energy generation in this metabolism, membranous vesicles (MVs) were prepared and the mechanism of energy generation was investigated. The membrane, which was fragmentized during preparation, automatically formed vesicles ranging from 37.5-112.5 nm in diameter under electron micrograph observation. Energy was conserved when coupling the azoreduction by the MVs of an azo compound or Fe(III) as the sole electron acceptor with $H_2$, formate, or lactate as the electron donor. The amaranth reduction by the vesicles was found to be inhibited by specific respiratory inhibitors, including $Cu^{2+}$ ions, dicumarol, stigmatellin, and metyrapone, indicating that the azoreduction was indeed a respiration reaction. This finding was further confirmed by the fact that the ATP synthesis was repressed by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). Therefore, this study offers solid evidence of a mechanism of microbial dissimilatory azoreduction on a subcell level.

• The Korean Journal of Pharmacology
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• v.18 no.2
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• pp.79-87
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• 1982

Higenamine(dl-demethylcoclaurine, dl-1-(4-hydroxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrah-ydroisoquinoline hydrochloride), which has recently been isolated from Aconite root by Drs. Kosuge and Yokota, has known to be the main cardiotonic component of the Aconite root. The present study was undertaken to investigate the effects of Higenamine on the calcium binding and release and ATPase activity of fragmented cardiac sarcoplasmic reticulum under in vitro condition. The calcium binding and release of sarcoplasmic reticulum were measured by using the double-beam spectrophotometer and the calcium sensitive dye, murexide. In the presence of $10^{-4}{\sim}5{\times}10^{-3}M$ of Higenamine, the maximal calcium binding and the initial binding rate of porcine cardiac sarcoplasmic reticulum were inhibited dose dependently by up to 43%. However, the calcium release from cardiac sarcoplasmic reticulum, which was loaded with $Ca^{++}(50{\mu}M)$, was stimulated in dose dependent manner. When incubated in the medium of 20 mM Tris-maleate(pH 7.0), 100 mM KCl, 10 mM $MgCl_2,\;0.05mM\;CaCl_2\;and\;0.014{\sim}1\;mM\;Tris-ATP\;at\;30^{\circ}C$ in the presence of Higenamine $(10^{-4}{\sim}5{\times}10^{-3}M)$, both $Ca^{++}-and\;Mg^{++}-ATPase$ of sarcoplasmic reticulum were inhibited non-competitively by Higenamine and values of $K_i$ were 4.896 mM and 6.875 mM respectively. It is suggested from the above findings that the cardiotonic effects of Higenamine might be partially explained by the inhibition of calcium binding and the stimulation of calcium release from the sarcoplasimic reticulum which may increase the free intracellular calcium that is available in the contraction of the cardiac muscle fiber.

• Biomedical Science Letters
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• v.13 no.3
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• pp.197-206
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• 2007

P2X receptors are membrane-bound ion channels that conduct $Na^+,\;K^+$, and $Ca^{2+}$ in response to ATP and its analogs. There are seven subunits identified so far ($P2X_1-P2X_7$). $P2X_2$ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that $P2X_2$ receptor activation in PC12 cells couples to $Ca^{2+}-dependent$ release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at $P2X_2$ receptors. This leads to a hypothesis that $P2X_2$ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional $P2X_2$ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS ($100\;{\mu}M)$ each), and exogenous exprerssion of ATP-binding mutant $P2X_2$ receptor subunit ($P2X_2[K69A]$). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride ($10\;{\mu}M$), did not give any effects to neurite extension. The vesicular proton pump ($H^+-ATPase$) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from $P2X_2$ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and $P2X_2$ receptors in hormone-induced cell differentiation or neuronal synaptogenesis in local acidic environments.

• The Journal of Korean Medicine
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• v.19 no.1
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• pp.251-270
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• 1998

The effects of Gamigunshimtang on the isolated perfused ischemic heart in rats, heart rates, left ventricular pressure, cardiac blood flow and cardiotoxicity were stu.died in H9C2 myoblast cell, myocardial slice culture The results were as follows: 1. The administration of Gamigunshimtang to the rat recovered effectively heart rate, left ventricular pressure and flow rate from the experimental ischemia in perfused rat heart. The release of lactic dehydrogenase after the ischemia also decreased compared to the control group. 2. The administration of Gamigunshimtang to H9C2 myoblast culture enhanced the cell proliferation and protected against doxorubicin and allylamine induced release of the lactic dehydrogenase into the culture medium. It also protected effectively against doxorubicin and allylamine induced decrease of Ca ATPase activity and the increase of NADPH-cytochrome C reductase activity in the microsome. 3. The administration of Gamigunshimtang to the rat myocardial slice culture protected effectively against doxorubicin and allylamine induced decreases of protein synthesis and ATP content, and increases of cvtosolic enzyme, creatin kinase into the medium and lipid peroxidation.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.5
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• pp.197-206
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• 2007

The aim of the present study was to investigate the effects of 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine(SKF81297), a selective agonist of dopaminergic $D_1$ receptor, on the secretion of catecholamines(CA) evoked by cholinergic stimulation and membrane-depolarization in the isolated perfused rat adrenal gland, and also to elucidate the mechanism involved. SKF81297($10{\sim}100{\mu}M$) perfused into an adrenal vein for 60 min produced dose- and time-dependent inhibition of CA secretory responses evoked by ACh(5.32 mM), high $K^+$(56 mM), DMPP($100{\mu}M$) and McN-A-343($100{\mu}M$). Also, in adrenal glands loaded with SKF81297($30{\mu}M$), the CA secretory responses evoked by Bay-K-8644($10{\mu}M$), an activator of L-type $Ca^{2+}$ channels and cyclopiazonic acid($10{\mu}M$), an inhibitor of cytoplasmic $Ca^{2+}$-ATPase were also inhibited. However, in the presence of the dopamine $D_1$ receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol(SCH23390, $3{\mu}M$), which is a selective antagonist of dopaminergic $D_1$ receptor, the inhibitory responses of SKF81297($30{\mu}M$) on the CA secretion evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644, and cyclopiazonic acid were significantly reduced. Collectively, these experimental results suggest that SKF81297 inhibits the CA secretion from the rat adrenal medulla evoked by cholinergic stimulation(both nicotininc and muscarinic receptors) and membrane depolarization. This inhibitory of SKF81297 seems to be mediated by stimulation of dopaminergic $D_1$ receptors located on the rat adrenomedullary chromaffin cells, which are relevant to extra- and intracellular calcium mobilization. Therefore, it is thought that the presence of the dopaminergic $D_1$ receptors may be involved in regulation of CA release in the rat adrenal medulla.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.187-191
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• 2005

This study was designed to investigate the changes of quantity and quality of proteins in medium and cytoplasm during in vitro maturation of bovine oocytes. The total quantity of proteins in medium decreased from 0 to 4.5 hr, but increased from 13.5 to 18 hr after the onset of in vitro maturation. The total quantity of protein in cytoplasm increased from 0 to 4.5 hr, decreased from 4.5 to 9 hr, and increased after 18 hr after the onset of in vitro maturation. A total of 298 protein spots was detected on a gel of 2D SDS-PAGE form maturation medium. Among 28 protein spots expressed significant differences in their quantity, 8 proteins were identified by peptide mass fingerprinting (aldose reductase, alpha enolase, apolipoprotein A-1 precursor, 43kDa collectin precursor, heat shock 27kDa protein, plasminogen activator inhibitor-1 precursor, thrombospondin 1, transitional endoplasmic reticulum ATPase). Among total of 35 protein spots detected on gel of 2D SDS-PACE from oorytes cytoplasm, $\beta$-tubulin was identified by peptide mass fingerprinting.