• The Korean Journal of Physiology and Pharmacology
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• v.13 no.3
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• pp.189-194
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• 2009

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic $Ca^{2+}$ level $([Ca^{2+}]_i)$, and $Ca^{2+}$ sensitivity in guinea pig ileum. Forskolin (0.1 nM ${\sim}$ 10 ${\mu}M$) inhibited high $K^+$ (25 mM and 40 mM)- or histamine (3 ${\mu}M$)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high $K^+$-evoked contractions. Spontaneous changes in $[Ca^{2+}]_i$ and contractions were inhibited by forskolin (1 ${\mu}M$) without changing the resting $[Ca^{2+}]_i$. Forskoln (10 ${\mu}M$ ) inhibited muscle tension more strongly than $[Ca^{2+}]_i$ stimulated by high $K^+$, and thus shifted the $[Ca^{2+}]_i$-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 ${\mu}M$) inhibited both $[Ca^{2+}]_i$ and muscle tension without changing the $[Ca^{2+}]_i$-tension relationship. In ${\alpha}$-toxin-permeabilized tissues, forskolin (10 ${\mu}M$) inhibited the 0.3 ${\mu}M$ $Ca^{2+}$-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the $Ca^{2+}$-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in $Ca^{2+}$ sensitivity of contractile elements in high $K^+$-stimulated muscle and a decrease in $[Ca^{2+}]_i$ in histamine-stimulated muscle.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.333-339
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• 2003

It has been demonstrated that an unidentified cytosolic factor(s) reduces $K_{ACh}$ channel function. Therefore, this study attempted to elucidate the cytosolic factor. Fresh cytosol isolated from normal heart (FC) depressed the $K_{ACh}$ channel activity, but cytosol isolated from the ischemic hearts (IC) did not modulate the channel function. Electrophorectic analysis revealed that a protein of ${\sim}80 kDa was markedly reduced or even lost in IC. By using peptide sequencing analysis and Western blot, this 80 kDa protein was identified as transferrin (receptor-mediated $Fe^{3+}$ transporter, 76 kDa). Direct application of transferrin (100 nM) to the cytoplasmic side of inside-out patches decreased the open probability ($P_o$, 12.7${\pm}6.4%, n=4) without change in mean open time (${\tau}_o$, $98.5{\pm}1.3$%, n=4). However, the equimolar apotransferrin, which is free of $Fe^{3+}$, had no effect on the channel activity (N*$P_o$, $129.1{\pm}13.5$%, n=3). Directly applied $Fe^{3+}$ (100 nM) showed results similar to those of transferrin (N*$P_o$: $21.1{\pm}3.9$%, n=5). However $Fe^{2+}$ failed to reduce the channel function (N*$P_o$, $106.3{\pm}26.8$%, n=5). Interestingly, trivalent cation La3+ inhibited N*$P_o$ of the channel ($6.1{\pm}3.0$%, n=3). Taken together, these results suggest that $Fe^{3+}$ bound to transferrin can modulate the $K_{ACh}$ channel function by its electrical property as a polyvalent cation.

• Journal of Nutrition and Health
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• v.25 no.1
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• pp.3-11
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• 1992

This paper examines the effects of dietary fats on the fatty acid composition and market enzyme activites during liver damage in 2-acetylaminofluorene treated rats. Weaning Sprague-Dawley male rats were fed the diet of beef tallow(BT source of sturated fatty acid) corn oil(CO source of n-6 fatty acid) and perilla oil(PO source of n-3 fatty acid) at the level of 15% fat. Ten days after feeding 2-acetylaminofluorene(2-AAF) was injected intraperitoneally twice every week at the level of 50mg/kg body weight for 7 weeks. Liver microsomal and cytosolic fractions were collected to determine the microsomal fatty acid composition lipid peroxide(malondialdehyde MDA) contents glucose 6-phosphatase(G6 Pase) activity cytochrome(Cyt) P-450 contents and cytosolic glutathione S-transferase(G6 Pase) activity cytochrome(Cyt) P-450 contents and cytosolic glutathione S-transferase(GST) activity. The fatty acid composition in microsomal fraction was reflected by different dietary fats. By 2-AAF treatment linoleic acids were increased regardless of the diet MDA contents were higher in CO group than it was in BT group. However 2-AAF treatment decreased MDA contents in all dietary groups. G6Pase activity of BT group was higher than those of the other gropus. CO group had the highest Cyt P-450 contents and 2-AAF treatment lowered Cyt P-450 contents only in CO gropu GST activites were higher in CO than in BT group whereas the enzyme activites were increased by 20AAF treatment in all dietary groups. These results suggest that dietary fats and 2-AAF treatment in all dietary groups,. These results suggest that dietary fats and 2-AAF treatment affect microsomal fatty acid composition The enzyme activities concerned with liver damage were influenced differently by dietary fats and 2-AFF treatment Although PO diet contains much more polyunsaturated fatty acids than CO diet PO diet doesn't cause more oxidant stress compared with CO diet in these data.

• Journal of Life Science
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• v.21 no.8
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• pp.1100-1108
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• 2011

The immunomodulating effects of moxifloxacin seem to be effective in downregulating inflammatory reactions. This presumed effect was tested in endotoxin (ETX)-induced acute lung injury (ALI) in rats. After moxifloxacin treatment (10 mg/kg) of ETX-given rats, lung myeloperoxidase (MPO) activity, bronchoalveolar-lavage (BAL) protein, and the number of neutrophils in the BAL cells were measured. Light and electron microscopic structures were also examined. Electron microscopic $CeCl_3$ histochemistry for the detection of hydrogen peroxide in the lungs and immunohistochemistry of cytosolic phospholipase A2 (cPLA2) in the lung tissues and BAL cells were performed. To examine the expression of TNF${\alpha}$ in the lungs, western blotting was carried out with the lung tissues. ETX had accumulated neutrophils in the lungs, which was followed by lung leak. Oxidative stress occurred, and increased expression of cPLA2 in the lung tissues and BAL cells was observed in the ETX-given rats. Simultaneously, the expression of TNF${\alpha}$ was enhanced by ETX. Moxifloxacin, however, decreased all these parameters, indicating that ALI may have been ameliorated. Moxifloxacin appears to ameliorate ETX-induced ALI partially through the suppression of cPLA2 in the lungs of rats.

• BMB Reports
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• v.39 no.6
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• pp.749-758
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• 2006

The cytosolic members of the HSP70 family of proteins play key roles in the molecular chaperone machinery of the cell. In the study we cloned and sequenced the full-length cDNA of Delia antiqua HSP70 gene, which is 2461 bp long and encodes 643 a.a. with a calculated molecular mass of 70,787 Da. We investigated gene copies of cytosolic HSP70 members of 4 insect species with complete genome available, and found that they are quite variable with species. In order to characterize this protein we carried out an alignment and a phylogenetic analysis with 41 complete protein sequences from insects. The analysis divided the cytosolic members of the family into two classes, HSP70 and HSC70, distinguishable on the basis of 15 residues. HSP70 class members were slightly shorter in length and smaller in molecular mass relative to the HSC70 class members, and the conservative and functional regions in these sequences were documented. Mainly, we investigated the expression of Delia antiqua HSP70 gene, in response to diapauses and thermal stresses. Both summer and winter diapauses elevated HSP70 transcript levels. Cold-stress led to increased HSP70 expression levels in summer- and winter-diapausing pupae, but heat-stress elevated the levels only in the winter-diapausing pupae. In all cases, the expression levels, after being elevated, gradually decreased with time. HSP70 expression was low in non-diapausing pupae but was up-regulated following cold- and heat-stresses. Heat-stress gradually increased the mRNA level with time whereas cold-stress gradually decreased levels after an initial increase.

• Molecules and Cells
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• v.38 no.5
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• pp.441-451
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• 2015

Recognition of cytosolic DNA initiates a series of innate immune responses by inducing IFN-I production and subsequent triggering JAK1-STAT1 signaling which plays critical roles in the pathogenesis of infection, inflammation and autoimmune diseases through promoting B cell activation and antibody responses. The stimulator of interferon genes protein (STING) has been demonstrated to be a critical hub of type I IFN induction in cytosolic DNA-sensing pathways. However, it still remains unknown whether cytosolic DNA can directly activate the JAK1-STAT1 signaling or not. And the role of STING is also unclear in this response. In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase. Moreover, this response is not dependent on type I IFN receptors. Interestingly, STING could inhibit dsDNA-triggered activation of JAK1-STAT1 signaling by inducing SHP-1 and SHP-2 phosphorylation. In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases. Our data provide a molecular insight into the novel role of STING in dsDNA-mediated inflammatory disorders.

• Molecules and Cells
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• v.24 no.2
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• pp.261-267
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• 2007

Apoptotic signals are typically accompanied by activation of aspartate-specific cysteine proteases called caspases, and caspase-3 and -7 play crucial roles in the execution of apoptosis. Previously, using the proteomic approach, protein disulfide isomerase (PDI) was found to be a candidate substrate of caspase-7. This abundant 55 kDa protein introduces disulfide bonds into proteins (via its oxidase activity) and catalyzes the rearrangement of incorrect disulfide bonds (via its isomerase activity). PDI is abundant in the ER but is also found in non-ER locations. In this study we demonstrated that PDI is cleaved by caspase-3 and -7 in vitro. In addition, in vivo experiment showed that it is cleaved during etoposide-induced apoptosis in HL-60 cells. Subcellular fractionation showed that PDI was also present in the cytosol. Furthermore, only cytosolic PDI was clearly digested by caspase-3 and -7. It was also confirmed by confocal image analysis that PDI and caspase-7 partially co-localize in both resting and apoptotic MCF-7 cells. Overexpression of cytosolic PDI (ER retention sequence deleted) inhibited cell death after an apoptotic stimulus. These data indicate that cytosolic PDI is a substrate of caspase-3 and -7, and that it has an anti-apoptotic action.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.43-43
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• 2003

Large-conductance $Ca^{2+}$-actived $K^{+}$ channels ($BK_{Ca}$ channels) play a key role in setting the pace of contractile activity in muscle and are involved in the regulation of neurotransmitter release in neuron. $BK_{Ca}$ channels are activated by depolarizing membrane potential and the elevated level of intracellular calcium. Using yeast-two hybrid assay, we have identified a novel protein interacting with the cytosolic carboxyl terminus of rSlo, the brain isoform of rat large-conductance $Ca^{2+}$-activated $K^{+}$ channel $\alpha$-subunit. The novel gene encodes 51 kDa protein and is named as SIRK(rSlo-interacting RGS-like protein). SIRK is expressed in various tissues and localized in the cytosolic and the membrane fraction. Biochemical and immunological studies indicated that SIRK physically interacted with the cytosolic region of rSlo. To investigate whether SIRK can modulate the activity of rSlo, GFP-fused SIRK and rSlo were transiently transfected into COS-7 cells and the effects of SIRK was studied using electrophysiological means. We concluded that the overexpression of SIRK alters the surface expression of rSlo channel with only a limited effect on the biophysical characteristics of the channel.the channel.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.2
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• pp.158-161
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• 2005

We have isolated fractions from Cordyceps militaris and Paecilomyces japonica and investigated their effects on the activity of rat liver cytosolic glucokinase, a key metabolic enzyme involved in carbohydrate metabolism. The dried powder of the C. militaris and P. japonica were successively extracted with ethanol and with 70% ethanol. The residue was exhaustively extracted with hot water. The extract was dialyzed against water, and to the non-dialyzable solution was added 2 volumes of ethanol. The precipitate was collected by centrifugation dispered in water, and lyophilized to afford fraction A. The residue after hot-water extraction was suspended in 5% sodium carbonate. The final residue was suspended in 5% NaOH. The alkaline suspension was purified in a similar manner as described above to afford fraction B. Hepatic glucokinase activities of the fraction A extracted from C. militaris and P. japonica were 371.4 and 379%, respectively. The fraction B was 314.2 and 147.4%. The activity of fraction B of C. militaris extracts was higher than that of P. japonica. Liver cytosolic glucokinase activity of rats fed normal diet supplemented with 0.1% C. militaris was 1316%. In conclusion, the present study has demonstrated that C. militaris extracts were able to prevent sudden postprandial peaks in blood glucose as a result of a marked increase in the liver cytosolic glucokinase activities.

• BMB Reports
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• v.37 no.2
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• pp.239-245
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• 2004

We have previously demonstrated that the redox reactant pyruvate prevents apoptosis in the oxidant model of bovine pulmonary artery endothelial cells (BPAEC), and that the anti-apoptotic mechanism of pyruvate is mediated in part via the mitochondrial matrix compartment. However, cytosolic mechanisms for the cytoprotective feature of pyruvate remain to be elucidated. This study investigated the pyruvate protection against endothelial cytotoxicity when the glycolysis inhibitor 2-deoxy-D-glucose (2DG) was applied to BPAEC. Millimolar 2DG blocked the cellular glucose uptake in a concentration- and time-dependent manner with >85% inhibition at $\geq$5 mM within 24 h. The addition of 2DG evoked BPAEC cytotoxicity with a substantial increase in lipid peroxidation and a marked decrease in intracellular total glutathione. Exogenous pyruvate partially prevented the 2DG-induced cell damage with increasing viability of BPAEC by 25-30%, and the total glutathione was also modestly increased. In contrast, 10 mM L-lactate, as a cytosolic reductant, had no effect on the cytotoxicity and lipid peroxidation that are evoked by 2DG. These results suggest that 2DG toxicity may be a consequence of the diminished potential of glutathione antioxidant, which was partially restored by exogenous pyruvate but not L-lactate. Therefore, pyruvate qualifies as a cytoprotective agent for strategies that attenuate the metabolic dysfunction of the endothelium, and cellular glucose oxidation is required for the functioning of the cytosolic glutathione/NADPH redox system.