• Journal of Radiation Protection and Research
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• v.22 no.1
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• pp.23-27
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• 1997

Radiation effects on carbohydrate moiety of chicken ovomucoid, a protease inhibitor as a typical allergenic glycoprotein of egg white, was observed. The trypsin inhibitory activity of chicken ovomucoid decreased exponentially and the inactivation was more significant irradiated in $N_2$ than in $O_2$. From the protein blotting, radiation caused protein degradation in $O_2$ and protein aggregation also in $N_2$. The patterns of carbohydrate blotting were also similar with that of protein blotting. Sugar chains in low molecular weight fraction (MW<5,000) were released by radiation and those in $O_2$ were higher than in $N_2$. From the HPLC patterns of the degradation of sugar chains, all peaks of oligosaccharides have the tendency to decrease with the increase of radiation dose and more remarkable in $O_2$ than in $N_2$. These results suggest that ionising radiation could cause the overall conformational changes of ovomucoid by the degradation and release of oligosaccharides.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.2
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• pp.113-120
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• 2000

To investigate the mechanism of smooth muscle contraction induced by emptying of intracellular $Ca^{2+}$ stores, we measured isometric contraction and $^{45}Ca^{2+}$ influx. $CaCl_2$ increased $Ca^{2+}$ store emptying- induced contraction in dose-dependent manner, but phospholipase C activity was not affected by the $Ca^{2+}$ store emptying-induced contraction. The contraction was inhibited by voltage-dependent $Ca^{2+}$ channel antagonists dose dependently, but not by TMB-8 (intracellular $Ca^{2+}$ release blocker). Both PKC inhibitors (H-7 and staurosporine) and tyrosine kinase inhibitors (genistein and methyl 2,5-dihydroxycinnamic acid) significantly inhibited the contraction, but calmodulin antagonists (W-7 and trifluoperazine) had no inhibitory effect on the contraction. The combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were greater than that of each one alone. In $Ca^{2+}$ store-emptied condition, $^{45}Ca^{2+}$ influx was significantly inhibited by verapamil, H-7 or genistein but not by trifluoperazine. However combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were not observed. Therefore, this kinase pathway may modulate the sensitivity of contractile protein. These results suggest that contraction induced by emptying of intracellular $Ca^{2+}$ stores was mediated by influx of extracellular $Ca^{2+}$ through voltage-dependent $Ca^{2+}$ channel, also protein kinase C and/or tyrosine kinase pathway modulates the $Ca^{2+}$ sensitivity of contractile protein.

• Journal of the Korean Chemical Society
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• v.41 no.4
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• pp.205-209
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• 1997

The site specificity of dephosphorylation of myelin basic protein(MBP) was studied in vitro. To assign amino acid site of dephosphorylation, MBP was phosphorylated by protein kinase C(PKC) and dephosphorylated by protein phosphatase PP2A. The phosphorylated MBP was digested by trypsine and the digested peptides were separated by a reverse phase HPLC chromatography. The radioactivity of each fraction was counted and partially sequenced. Seven radioactive peptides were observed and $Ser^{55}$ in the second peak($P_2$) shows the best susceptibility for the phosphorylation. However in the dephosphorylation, the fifth peak($P_5$) appeared to release it's phosphate group most rapidly. This result demonstrates that MBP is a suitable substrate for protein phosphatase.

• Animal cells and systems
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• v.16 no.4
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• pp.280-288
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• 2012

Targeted degradation of proteins through ubiquitin-mediated proteolysis is an important control mechanism in various cellular processes. The process of ubiquitin conjugation is achieved by three enzyme complexes, among which the ubiquitin ligase complex (E3) is in charge of substrate specificity. The SCF (SKP1-CUL1-F-box) family portrays the largest and the most characterized member of the E3 ligases. For each SCF complex, the ubiquitination target is recognized by the F-box protein subunit, which interacts with the substrate through a unique C-terminal domain. We have characterized a novel F-box protein CFL-1 that represents a single LRR-type F-box (FBXL) in the Caenorhabditis elegans genome. CFL-1 is highly homologous to FBXL20 and FBXL2 of mammals, which are known to regulate synaptic vesicle release and cell cycle, respectively. A green fluorescence protein (GFP)-reporter gene fused to the cfl-1 promoter showed restricted expression around the amphid and the anus. Modulation of CFL-1 activity by RNAi affected the time interval between defecations. RNAi-treated worms also exhibited reduced tendency to form dauer when exposed to daumone. The potential involvement of CFL-1 in the control of defecation and pheromone response adds to the ever expanding list of cellular processes controlled by ubiquitin-mediated proteolysis in C. elegans. We suggest that CFL-1, as a single LRR-type F-box protein in C. elegans, may portray a prototype gene exerting diverse functions that are allocated among multiple FBXLs in higher organisms.

• Korean Journal of Food Science and Technology
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• v.32 no.5
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• pp.1198-1205
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• 2000

Effect of inhibitions of three kinds of Ginkgo biloba extracts(Ginkgo biloba extract, Ginkgolide A, and Ginkgolide B) on induction of reactive oxygen species and release of inflammation mediator arachidonic acid were tested. Three kinds of Ginkgo biloba extracts could not inhibit the pyrogallol auto-oxidation, but they showed the hydrogen atom donating activity in DPPH assay. When 10 ${\mu}M$ hydrogen peroxide and 400 ${\mu}g/mL$ of three kinds of Ginkgo biloba extracts were added to U937 monocytic macrophage, the induction of lipid peroxidation was not observed. The Ginkgo biloba extract showed the most powerful inhibition among the extracts. And only Ginkgolide A was good for the inhibition of the protein degradation. The release of inflammation mediator arachidonic acid was induced by adding TPA and calcimycin to U937. In this assay, even 10 ${\mu}g/mL$ of three different Ginkgo biloba extracts excellently blocked the release of arachidonic acid. Particularly, the inhibition efficiency of Ginkgolide B was about 11 times higher than that of induction, and was about 4 times higher than that of the control of noninduction. This result suggests that the release of arachidonic acid is not inhibited by the antioxidant activity of Ginkgo biloba extracts, but a pre-step of the release of arachidoinc acid is inhibited by Ginkgo biloba extracts.

• BMB Reports
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• v.39 no.3
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• pp.319-328
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• 2006

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

• Toxicological Research
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• v.13 no.3
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• pp.223-228
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• 1997

Quinones have been reported to undergo nonenzymatic reaction with thiols to generate reactive oxygens. It is therefore possible that the nonenzymatic reaction of quinones with thiols in plasma could lead to potentJared cellular toxicity or disease. When 1 mM menadione was added in plasma under pH 11.2, 7.4 and 5.0, the increase in oxygen consumption rate was the order of pH 11.2 > pH 7.4 > pH 5.0. In addition, oxygen consumption rates under plasma anticoagulated with trisodium citrate solution (pH 7.85) was significantly higher than those with acid-citrate-dextrose solution (pH 6.87). SOD and catalase reduced the rate of oxygen consumption induced by menadione in plasma. Taken together, these results suggest that the menadione-induced increased oxygen consumption was due to nonenzymatic reaction of menadione with thiols in the plasma. The presence of plasma has an additive effect on the increased oxygen consumption rates induced by the menadione treatments on our model tissue, platelets, as compared between washed platelet (WP) and platelet rich plasma (PRP). Cytotoxicity, as determined by LDH release, are well correlated with the oxygen consumption rates observed in each system and strongly suggest that menadione-induced cytotoxicity can be increased with the presence of blood plasma.

• BMB Reports
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• v.37 no.6
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• pp.735-740
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• 2004

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.

• Genomics & Informatics
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• v.7 no.4
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• pp.181-186
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• 2009

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans -splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3'-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a trans-splicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

• Journal of Applied Biological Chemistry
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• v.65 no.3
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• pp.167-172
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• 2022

Pathophysiological reaction of platelets in the blood vessel is an indispensable part of thrombosis and cardiovascular disease, which is the most common cause of death in the world. In this study, we performed in vitro assays to evaluate antiplatelet activity of artemether in human platelets and attempted to identify the mechanism responsible for protein phosphorylation. Artemether is a derivative of artemisinin, known as an active ingredient of Artemisia annua, which has been reported to be effective in treating malaria, and is known to function through antioxidant and metabolic enzyme inhibition. However, the role of artemether in platelet activation and aggregation and the mechanism of action of artemether in collagen-induced human platelets are not known until now. In this study, the effect of artesunate on collagen-induced human platelet aggregation was confirmed and the mechanism of action of artemether was clarified. Artemether inhibited the phosphorylation of PI3K/Akt and Mitogen-activated protein kinases, which are phosphoproteins that are known to act in the signal transduction process when platelets are activated. In addition, artemether decreased TXA₂ production and decreased granule secretion in platelets such as ATP and serotonin release. As a result, artemether strongly inhibited platelet aggregation induced by collagen, a strong aggregation inducer secreted from vascular endothelial cells, with an IC₅₀ of 157.92 μM. These results suggest that artemether has value as an effective antithrombotic agent for inhibiting the activation and aggregation of human platelets through vascular injury.