• Microbiology and Biotechnology Letters
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• v.44 no.1
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• pp.55-61
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• 2016

Sepiapterin, a precursor for tetrahydrobiopterin, is produced in higher mammals using guanosine triphosphate (GTP) as a biosynthetic intermediate. Four genes involved in GTP biosynthesis, namely those of guanosine monophosphate kinase (gmk), nucleoside diphosphate kinase (ndk), guanosine phosphate synthetase (guaA), and inosine-5'-monophosphate dehydrogenase (guaB), were expressed in sepiapterin-producing recombinant Escherichia coli BL21(DE3) to increase intracellular GTP concentration and to improve sepiapterin production concomitantly. Coexpression of gmk, ndk, guaA, and guaB, doubled the intracellular GTP concentration and increased the maximum sepiapterin concentration up to $126.1{\pm}19.3mg/l$ (an increase of 43% compared with control cells) in batch-cultivated recombinant E. coli.

• Korean Journal of Microbiology
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• v.20 no.4
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• pp.201-209
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• 1982

An enzyme, named GTP cyclohydorlase, that catalizes the hydrolytic removal of carbon No.S of GTP has been partially purified from extracts of Pseudomonas putida (IAM 1506). The enzyme exists in two molecuar weight forms : a high molecular weight form (150,000) and a low molecular weight from (40,000). The high molecular weight form has been purified 25-fold. Some of the properties of the enzyme are as follows : It functions optimally at pH8.0, and at $52^{\circ}C$. The Km value for GTP is $20{\mu}M$. Divalent cations $(Cd^{2+}\;and\;Hg^{2+})$ 2+/) at a concentration of 5mM inhibit completely the enzyme activity. No metal ion including $Mg^{2+}$ is needed for the catalysis. The enzyme is heat labile ; its half at $57^{\circ}C$ is 1.5 min. Of a number of nucleotides tested, only GDP was used to any extent as substrbte in place of GTP. One of the products of the enzyme is determined to be a dihydro-neopterin compound.

• BMB Reports
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• v.39 no.5
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• pp.492-501
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• 2006

Since differentiation therapy is one of the promising strategies for treatment of leukemia, universal efforts have been focused on finding new differentiating agents. In that respect, we used guanosine 5'-triphosphate (GTP) to study its effects on K562 cell line. GTP, at concentrations between 25-200 ${\mu}M$, inhibited proliferation (3-90%) and induced 5-78% increase in benzidine-positive cells after 6-days of treatments of K562 cells. Flow cytometric analyses of glycophorine A (GPA) showed that GTP can induce expression of this marker in more mature erythroid cells in a time- and dose-dependent manner. These effects of GTP were also accompanied with inhibition of DNA synthesis (measured by [$^3H$]-thymidine incorporation) and early S-phase cell cycle arrest by 96 h of exposure. In contrast, no detectable effects were observed when GTP administered to unstimulated human peripheral blood lymphocytes (PBL). However, GTP induced an increase in proliferation, DNA synthesis and viability of mitogen-stimulated PBL cells. In addition, growth inhibition and differentiating effects of GTP were also induced by its corresponding nucleotides GDP, GMP and guanosine (Guo). In heat-inactivated medium, where rapid degradation of GTP via extracellular nucleotidases is slow, the anti-proliferative and differentiating effects of all type of guanine nucleotides (except Guo) were significantly decreased. Moreover, adenosine, as an inhibitor of Guo transporter system, markedly reduced the GTP effects in K562 cells, suggesting that the extracellulr degradation of GTP or its final conversion to Guo may account for the mechanism of GTP effects. This view is further supported by the fact that GTP and Guo are both capable of impeding the effects of mycophenolic acid. In conclusion, our data will hopefully have important impact on pharmaceutical evaluation of guanine nucleotides for leukemia treatments.

• BMB Reports
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• v.28 no.1
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• pp.72-78
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• 1995

Time-dependent inactivation of E. coli GTP cyclohydrolase I with a 2',3'-dialdehyde derivative of GTP (oGTP) was directed to the active site of the enzyme, and was dependent on the concentration of oGTP. The kinetics of inactivation were biphasic with a rapid reaction occurring immediately upon exposure of the enzyme to oGTP followed by a slow rate of inactivation. The $K_i$ value of oGTP for the enzyme was 0.25 mM. Inactivation was prevented by preincubation of the enzyme with GTP, the substrate of the enzyme. At 100% inactivation, 2.3 mol of [8.5'-$^3H$]oGTP were bound per each enzyme subunit, which consists of two identical polypeptides. The active site residue which reacted with the affinity label was lysine. oGTP interacted selectively with the ${\varepsilon}$-amino group of lysine in the GTP-binding site to form a morpholine-like structure which was stable without sodium borohydride treatment. However, triphosphate group was eliminated during the hydrolysis step. To identify the active site of the enzyme, [8.5'-$^3H$]oGTP-labeled enzyme was cleaved by endoproteinase Lys-C, and the $^3H$-labeled peptide was purified by HPLC. The amino acid sequence of the active site peptide was Pro-Ser-Leu-Ser-Lys, which corresponds to the aminoterminal sequence of the enzyme.

• Journal of Life Science
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• v.29 no.2
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• pp.256-264
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• 2019

Lymphotoxin ${\beta}$ receptor ($LT{\beta}R$), a member of the tumor necrosis factor receptor family, plays an important role in lymphoid tissue's architecture and organogenesis. In contrast, MLCK and ROCK play critical roles in the regulation of stress fiber (SF) formation in cells. To determine whether $LT{\beta}R$ stimulation in fibroblastic reticular cells (FRCs) is involved in these signaling pathways, myosin light chain kinase inhibitor-7 (ML-7) was used to inhibit them. ML7-treated FRCs completely blocked SFs and showed retraction and shrinkage processes comparable to those observed in agonistic anti-$LT{\beta}R$ antibody-treated cells. The inhibition of ROCK activity with Y27632-induced changes in actin cytoskeleton organization and cell morphology in FRCs. Actin bundles rearranged into SFs, and phospho-myosin light chain (p-MLC) co-localized in FRCs. We checked the level of Rho-guanosine diphosphate (RhoGDP)/guanosine triphosphate (GTP) exchange activity using FRC lysate. When $LT{\beta}R$ was stimulated with agonistic anti-$LT{\beta}R$ antibodies, Rho-GDP/GTP exchange activity was markedly reduced. Regarding $LT{\beta}R$ signaling with a focus on MLCK inhibition, we showed that the phosphorylation of MLCs was reduced by $LT{\beta}R$ stimulation in FRCs. Cytoskeleton components, such as tubulin, b-actin, and phospho-ezrin proteins acting as membrane-cytoskeleton linkers, were produced in de-phosphorylation, and they reduced expression in agonistic anti-$LT{\beta}R$ antibody-treated FRCs. Collectively, the results suggested that MLCK and ROCK were simultaneously responsible for SF regulation triggered by $LT{\beta}R$ signaling in FRCs.

• Biomedical Science Letters
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• v.20 no.2
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• pp.70-76
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• 2014

In this study, we investigated the effect of DMSO, a highly dipolar organic liquid, in collagen ($5{\mu}g/ml$)-stimulated platelet aggregation. DMSO inhibited platelet aggregation at 0.5% by inhibiting production of thromboxane $A_2$ ($TXA_2$) which was associated with blocking cyclooxygenase (COX)-1 activity and $TXA_2$ synthase. In addition, DMSO significantly increased the formation of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) and cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). On the other hand, DMSO (0.1~0.5% concentration) did not affect the LDH release which indicates the cytotoxicity. Based on these results, DMSO has anti-platelet effect by regulation of several platelet signaling pathways, therefore we suggest that DMSO could be a novel strategy on many thrombotic disorders.

• Journal of Life Science
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• v.28 no.9
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• pp.1100-1117
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• 2018

The Ras superfamily of small G-proteins acts as a molecular switch on the intracellular signaling pathway. Upon ligand stimulation, inactive GTPases (Ras-GDP) are activated (Ras-GTP) using guanine nucleotide exchange factor (GEF) and transmit signals to their downstream effectors. Following signal transmission, active Ras-GTP become inactive Ras-GDP and cease signaling. However, the intrinsic GTPase activity of Ras proteins is weak, requiring Ras GTPase-activating protein (RasGAP) to efficiently convert RAS-GTP to Ras-GDP. Since deregulation of the Ras pathway is found in nearly 30% of all human cancers, it might be useful to clarify the structural and physiological roles of Ras GTPases. Recently, RasGAP has emerged as a new class of tumor-suppressor protein and a potential therapeutic target for cancer. Therefore, it is important to clarify the physiological roles of the individual GAPs in human diseases. The first RasGAP discovered was RASA1, also known as p120 RasGAP. RASA1 is widely expressed, independent of cell type and tissue distribution. Subsequently, neurofibromatosis type 1 (NF1) was discovered. The remaining GAPs are affiliated with the GAP1 and synaptic GAP (SynGAP) families. There are more than 170 Ras GTPases and 14 Ras GAP members in the human genome. This review focused on the current understanding of Ras GTPase and RasGAP in human diseases, including cancers.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.314-321
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• 2015

Background: Ginseng belongs to the genus Panax. Its main active ingredients are the ginsenosides. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the gastrointestinal (GI) tract. To understand the effects of ginsenoside Re (GRe) on GI motility, the authors investigated its effects on the pacemaker activity of ICCs of the murine small intestine. Methods: Interstitial cells of Cajal were dissociated from mouse small intestines by enzymatic digestion. The whole-cell patch clamp configuration was used to record pacemaker potentials in cultured ICCs. Changes in cyclic guanosine monophosphate (cGMP) content induced by GRe were investigated. Results: Ginsenoside Re ($20-40{\mu}M$) decreased the amplitude and frequency of ICC pacemaker activity in a concentration-dependent manner. This action was blocked by guanosine 50-[${\beta}-thio$]diphosphate [a guanosine-5'-triphosphate (GTP)-binding protein inhibitor] and by glibenclamide [an adenosine triphosphate (ATP)-sensitive $K^{+}$ channel blocker]. To study the GRe-induced signaling pathway in ICCs, the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) and RP-8-CPT-cGMPS (a protein kinase G inhibitor) were examined. Both inhibitors blocked the inhibitory effect of GRe on ICC pacemaker activity. L-NG-nitroarginine methyl ester ($100{\mu}M$), which is a nonselective nitric oxide synthase (NOS) inhibitor, blocked the effects of GRe on ICC pacemaker activity and GRe-stimulated cGMP production in ICCs. Conclusion: In cultured murine ICCs, GRe inhibits the pacemaker activity of ICCs via the ATP-sensitive potassium ($K^{+}$) channel and the cGMP/NO-dependent pathway. Ginsenoside Re may be a basis for developing novel spasmolytic agents to prevent or alleviate GI motility dysfunction.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.51 no.2
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• pp.163-169
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• 2018

This study examined the expression of heat shock protein 70 (Hsp70) in red seabream Pagrus major infected by the, acanthocephalan parasites Longicollum pagrosomi. We cloned the full-length Hsp70 cDNA from the liver of the red seabream. The full-length cDNA had a 1,950 bp open reading frame (ORF) that encoded a protein of 650 amino acids. The deduced amino acid sequence of Hsp70 contained all of the conserved Hsp70 family signature sequences and an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding motif, including the EEVD (consensus sequence that terminates in Hsp70 family) consensus sequence. The expression of Hsp70 mRNA was upregulated int the fish head-kidney and liver, as determined by quantitative real-time PCR. We quantified the Hsp70 mRNA expression in normal red seabream and fish infected fish by L. pagrosomi. The expression of Hsp70 mRNA was significantly higher in the infected red seabream. These results suggest that Hsp70 play a role of protection against stress and inflammation caused by the parasite and may help maintain homeostasis.

• Journal of Life Science
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• v.27 no.10
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• pp.1199-1206
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• 2017

The lymphotoxin ${\beta}$ receptor ($LT{\beta}R$), a member of the tumor necrosis factor receptor family, plays an important role in lymphoid tissue's architecture and organogenesis. We found that $LT{\beta}R$ stimulation induced changes in stress fibers (SFs) in fibroblastic reticular cells (FRCs). MLCK and ROCK play critical roles in the regulation of SF formation in cells. The present study was performed to investigate the antifibrotic effects on SF regulation of $LT{\beta}R$ signaling, with a focus on MLCK inhibition. The effect of $LT{\beta}R$ on the SF change was analyzed using immunoblot and fluorescence assays and agonistic $anti-LT{\beta}R$ antibody-treated FRCs. In addition, we checked the level of Rho-guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange activity with FRC lysate. Phospho-ezrin proteins acting as membrane-cytoskeleton linkers completely de-phosphorylated in agonistic $anti-LT{\beta}R$ antibody-treated FRCs. The actin bundles rearranged into SFs, where phospho-myosin light chain (p-MLC) co-localized in FRCs. ML7-treated FRCs completely blocked SFs and showed retraction and shrinkage processes comparable to those observed in agonistic $anti-LT{\beta}R$ antibody-treated cells. Inhibition of ROCK activity induced changes in the actin cytoskeleton organization; however, some SFs remained in the cells, while they were completely disrupted by MLCK inhibition with ML7. We showed that the phosphorylation of MLC was completely abolished with $LT{\beta}R$ stimulation in FRCs. When $LT{\beta}R$ was stimulated with the agonistic $anti-LT{\beta}R$ antibody, the Rho-GDP/GTP exchange activity was reduced, however, the activity was not completely abolished. Collectively, the results illustrated that MLCK was potently responsible for the SF regulation triggered via $LT{\beta}R$ signaling in FRCs.