• 한국막학회:학술대회논문집
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• 한국막학회 2004년도 Proceedings of the second conference of aseanian membrane society
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• pp.82-85
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• 2004

Microinterface of W/Omicroemulsion prepared by phosphatidylcholine was used as reaction media of hydrolysis of phosphatidylcholine by phospholipaseA$_2$. Phosphatidylcholine was used as an amphiphile and was acted as a substrate. Organic phase of W/Omicroemulsion in this study was prepared by mixed organic solvents i.e. 2,2,4-trimethylpentane (isooctane) as a main solvent and 1-butanol as a co-solvent. The effect of added 1-butanol was remarkable not only on reaction beginning but also on high reaction rate. The hydrolysis reaction was dramatically initiated when 1-butanol was injected into the running isooctane/PC system. The enhancement by 1-butanol addition into single organic solvent was our original finding compare with previous conventional organic solvent. The reaction rate was elevated by the added amount of 1-butanol. The enhanced reaction rate was about 150-folds. This enhancement was speculated as 1-butanol adsorption on the microinterface. The adsorbed 1-butanol improved the properties of microinterface, especially its mobility was increased by difference of the chain length between phosphatidylcholine and 1-butanol. PhospholipaseA$_2$ molecules were located on the microinterface due to modified mobility of microinterface. Located phospholipaseA$_2$ on the microinterface reacted easily with phosphatidylcholine molecule. As a result high reaction rate was obtained. Microinterfacial properties were successfully improved by adsorbed 1-butanol molecule, and were favorable to appear higher reactivity of phospholipaseA$_2$.

• Archives of Pharmacal Research
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• 제18권3호
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• pp.189-194
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• 1995

The focus of this study was to investigate the influences of enzymatic scavengers of active oxygen metabolites and phospholipase $A_2$ inhibitor on hepatic secretory and microsomal function during hepatic ischemia/reperfusion. Rats were pretreated with free radical scavengers such as superoxide dismutase (SOD), catalase, deferoxamine and phospholipase $A_2$ inhibitor such as quinacrine and then subjected to 60 min. no-flow hepatic ischemia in vivo. After 1, 5 hr of reperfusion, bile was collected, blood was obtained from the abdominal aorta, and liver microsomes were isolated. Serum aminotransferase (ALT) level was increased at 1 hr and peaked at 5 hr. The increase in ALT was significantly attenuated by SOD plus catalase, deferoxamine and quinacrine especially at 5 hr of reperfusion. The wet weight-to-dry weight ratio of the liver was significantly increased by ischemia/reperfusion. SOD and catalase treatment minimized the increase in this ratio. Hepatic lipid peroxidiltion was elevated by ischemia/reperfusion, and this elevation was inhibited by free radical scavengers and quina crine. Bile flow and cholate output, but not bilirubin output, were markedly decreased by ischemia/reperfusion and quinacrine restored the secretion. Cytochrome $P_{450}$ content was decreased by ischemia/reperfusion and restored by free radical scavengers and quinacrine to the level of that of the sham operated group. Aminopyrine N-demethylase activity was decreased and aniline p-hydroxylase was increased by ischemia/reperfusion. The changes in the activities of the two enzymes were prevented by free radical scavengers and quinacrine. Our findings suggest that ischemia/reperfusion diminishes hepatic secretory functions as well as microsomal drug metabolizing systems by increasing lipid peroxidation, and in addition to free radicals, other factors such as phospholipase $A_2$ are involved in pathogenes of hepatic dysfunction after ischemia/reperfusion.

• The Korean Journal of Physiology and Pharmacology
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• 제1권1호
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• pp.19-25
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• 1997

The C-terminus ends of the second putative transmembrane domains of both $M_1$ and $M_2$ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in $M_1$ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposedfashion (LYT-LYT) in the sequence of $M_2$ receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of $M_1$ receptors to the corresponding $M_2$ receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT $M_1$ receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant $M_1$ receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant $M_1$ receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in $M_1$ muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of $M_1$ receptors to the stimulation of phospholipase C.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1998년도 학술발표회
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• pp.31-31
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• 1998

The role of a phosphoinositide-specific phospholipase C, PLC-deltal, in the bradykinin receptor-mediated signaling pathway was investigated using a clone of stably overexpressed PLC-deltal in rat pheochromocytoma (PC12) cells. Stimulation with bradykinin induced significantly higher [Ca$\^$2＋/]i rise in PLC-deltal-overexpressed cells (PC12-D1) than in the wild type (PC12-W) and the vector-transfected (PC12-V) cells.(omitted)

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2000년도 춘계학술대회
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• pp.15-16
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• 2000

Transient forebrain ischemia results in delayed neuronal death in the CA1 region of the hippocampus after injury, which is, at least in part, a consequence of excessive generation of reactive oxygen species. Previous in vitro studies using cell cultures or brain slices have demonstrated that phospholipase D (PLD) in the nervous system is involved in the signaling mechanism in response to a variety of agonists. Several recent studies have shown that reactive oxygen species stimulate phospholipase D (PLD) activity in several kinds of cells. Therefore, this raises the possibility that PLD activity is enhanced in the ischemic brain. Meanwhile, osteopontin (OPN) was initially identified as a sialoglycoprotein in bone, but has since been found in various tissues. Although not much is known about its function, OPN seems to play an important role in inflammation and tissue repair. Recently, it was reported that OPN was upregulated in the activated microglia after focal brain ischemia, suggesting that OPN might play a role in wound healing after a focal stroke.

• Asian-Australasian Journal of Animal Sciences
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• 제15권8호
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• pp.1091-1097
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• 2002

Capacitated goat spermatozoa generated diacyl glycerol (DAG) when suspended in Krebs-Ringer bicarbonate medium and induced by progesterone or $Ca^{2+}$ ionophore A23187. We have added Sn-1-oleoyl-2-acetyl glycerol externally, to study the effect of DAG in goat sperm acrosomal exocytosis. Addition of neomycin abolished the DAG generating capacity of progesterone in a dose dependent manner, suggesting the involvement of a phosphoinositidase C activated phospholipase C system in the process. The level of increase in phosphatidic acid was considerably low and was produced well after the DAG generation thereby suggesting the involvement of a DAG kinase which phosphorylates DAG to produce PA. The inhibition of progesterone mediated effect by inhibitors of $GABA_A/Cl^{-}$ channel and $Ca^{2+}$ channels further supports the evidence that the events of binding of agonist to the receptor(s), opening of $Ca^{2+}$ channels and the activation of phospholipase C are reconciled to perform the function of acrosome reaction in capacitated goat spermatozoa.

• 식물조직배양학회지
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• 제27권5호
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• pp.375-377
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• 2000

It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the hydrolysis of phosphatidyl-4,5-bisphosphate catalysed by phosphatidylinositol - specific phospholipase C yields to D-myo-inositol - 1,4,5-trisphosphate and diacylglycerol, which are well known second messengers. The binding of InsP$_3$to a receptor located on the endoplasmic reticulum triggers a calcium release from the endoplasmic reticulum. We have detected and partially characterised key components of phosphoinositide signaling. First, tobacco microsomal fraction and plasma membrane PI-PLC. Consecutively, using a radioligand binding assay we have identified a $Ca^{2+}$ -dependent high affinity InsP$_3$binding site in microsomal membrane fraction vesicle preparation and then we have measured inositol-1,4,5-trisphosphate induced calcium release from tobacco microsomal fraction. These findings suggest that phosphoinositide signaling system is present and operates in the tobacco suspension culture.e.

• Archives of Pharmacal Research
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• 제21권4호
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• pp.423-428
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• 1998

The ligand binding signals to a wide variety of seven transmembrane cell surface receptors are transduced into intracellular signals through heterotrimeric G-proteins. Recently, there have been reports which show diverse coupling patterns of ligand-activated receptors to the members of Gq family $\alpha$ subunits. In order to shed some light on these complex signal processing networks, interactions between G$\alpha$q family of G protein and neurokinin-2 receptor as well as muscarinic M$_{1}$ receptor, which are considered to be new thearpeutic targets in asthma, were studied. Using washed membranes from Cos-7 cells co-transfected with different G.alpha.q and receptor cDNAs, the receptors were stimulated with various concentrations of carbachol and neurokinin A and the agonist-dependent release of [$^3H$]inositol phosphates through phospholipase C beta-1 activation was measured. Differential coupling of Gaq family of G-protein to muscarinic M$_{1}$ receptor and neurokinin-2 receptor was observed. The neurokinin-2 receptor shows a ligand-mediated response in membranes co-transfected with G$\alpha$q, G$\alpha$11 and G$\alpha$14 but not G$\alpha$16 and the ability of the muscarinic $M_1$ receptor to activate phospholipase C through G$\alpha$/11 but not G$\alpha$14 and G$\alpha$16 was demonstrated. Clearly G$\alpha$/11 can couple $\M_1$ and neurokinin-2 receptor to activate phospholipase C. But, there are differences in the relative coupling of the G$\alpha$14 and G$\alpha$16 subunits to these receptors.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.19-26
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• 1996

A phospholipase C (PLC) inhibitor (MT267-2B) was isolated from the culture broth of actinomycetes isolate MT2617-2 by the extraction with n-butanol and column chromatographic techniques. The molecular weight of the inhibitor was 1057, by the spectroscopic analyses of IR, $^{13}C$-and $^{1}H$-NMR and ESI-MS. The chemical structure of MT2617-2B was found to be a macrolide compound consisted of a hemiketal ring, polyhydroxyl and polymethyl groups, which had a malonate and guanidine group as its side chain. MT2617-2B produced its two isomers having the same molecular weight by standing in methanol solution at room temperature. Therefore, MT2617-2B was identified as copiamycin and niphithricin A, macrolide antibiotics. The values of $IC_{50}$ against PLC-${\gamma}$1 and PLC-${\beta}$1 were 25 and 50${\mu}$g/ml, respectively. MT2617-2B had antimicrobial activities against Staphylococcus aureus and Candida albicans, but not against Escherichia coli.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.71-76
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• 2002

Soil samples were screened for actinomycete strains capable of producing phospholipase D, and a strain, Streptomyces sp. YU100, showing a high transphosphatidylation activity was isolated. This strain secreted phospholipase D in a culture broth after 12 h of cultivation, and its productivity continued to increase for 36 h of fermentation. In addition, its transphosphatidylation rate of phosphatidylcholine to phosphatidylserine was almost $68\%$ within 1 h. The morphological and chemotaxonomical characteristics showed that this strain could be classified as a number of the Streptomycetaceae family, particularly due to the spiral form of its spore chain consisting of 60-70 smooth spores $(0.75{\times}1.0{\mu}m$) on an aerial mycelium, FA-2c type of fatty acid profile in the cell wall, and LL-DAP component in the cell wall peptidoglycan. A phylogenetic analysis of the 16S rDNA provided a clue that the strain YU100 was actually a member of the genus Streptomyces, because the determined sequence exhibited a higher homology with Streptomyes sp. ASB27, S. peucetius JCM9920, and S. griseus ATCC10137. A dendrogram based on the 16S rDNA sequences also showed a phylogenetic relationship between the strain YU100 and these strains. However, the strain YU100 has not yet been assigned to a particular species, because of absence of any other classified species with a high matching score.