• Journal of Microbiology and Biotechnology
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• v.30 no.8
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• pp.1244-1251
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• 2020

Phospholipase A₂ (PLA₂) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA₂ in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA₂ was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA₂ (P-PLA₂), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA₂. Finally, we observed that the extracellular PLA₂ from the recombinant E. coli P-PLA₂ culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA₂ expression system led to extracellular production of PLA₂ to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA₂.

• Environmental Analysis Health and Toxicology
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• v.15 no.3
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• pp.69-80
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• 2000

ADP-rubosylation may be involved in the process of macrophage activation. Nitric oxide (NO) has emerged as an important intracellular and interacellular regulatory molecule with function as diverse as vasodilation, neural communication or host defense. NO is derived from the oxidation of the terminal guanidino nitrogen atom of L-arginine by the NADPH -dependent enzyme, nitric oxide synthase (NOS) which is one of the three different isomers in mammalian tissues. Since NO can exert protective or regulatory functions in the cell at a low concentration while toxic effects at higher concentrations, its role may be tightly regulated in the cell. Therefore, this paper was focused on signal transduction pathway of NO synthesis, role of endogenous TGF-$\beta$ in NO production. effect of NO on superoxide formation. Costimulation of murine peritoneal macrophages with interferon-gamma (IFN-γ) and phorbol 12-myristate 13-acetate (PMA) increased both NO secretion and mRNA expression of inducible nitric oxide synthase (iNOS) when PMA abolished costimulation. Pretreatmnet of the cells with PMA abolished costimuation effects due to the depletion of protein kinase C (PKC) activities . The involvement of PKC in NO secretion could be further confirmed by PKC inhibitor, stauroprine, and phorbol ester derivative, phorbol 12,13-didecanoate. Addition of actinomycine D in IFN-γ plus PMA stimulated cells inhibited both NO secretion and mRNA expression of iNOS indication that PMA stabilizes mRNA of iNOS . Exogenous TGF-$\beta$ reduced NO secretion in IFN -γ stimulated murine macrophages. However addition of antisense oligodeoxynucleotide (ODN) to TGF-$\beta$ to this system recovered the ability of NO production and inhibited mRNA expression of TGF-$\beta$. ACAS interactive laser cytometry analysis showed that transportation of FITC -labeled antisense ODN complementary to TGF-$\beta$ mRNA could be observed within 5 min and reached maximal intensity in 30 min in the murine macrophage cells. NO released by activated macrophages inhibits superoxide formation in the same cells . This inhibition nay be related on NO-induced auto -adenosine diphosphate (ADP) -ribosylation . In addition, ADP-ribosylation may be involved in the process of macrophage activation .

• Microbiology and Biotechnology Letters
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• v.15 no.5
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• pp.361-367
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• 1987

A DL-seleno-methionine resistant mutant, Cephalosporium acremonium MS-92 showed increased activities of sulfate and L-methionine uptake than the parent strain, and accumulated excess methionine and S-adenosylmethionine (SAM) intracellularly. And the sulfate uptake system was severely inhibited by L-cysteine. In crude enzyme extracts, the mutant MS-92 showed lower L-serine sulfhydrylase (identical with cystathionine $\beta$-synthase) activity than the parent. Also, cysteine desulfhydrylase activity, an index of intracellular L-cysteine concentration, of the mutant MS-92 was decreased by about 50% as com-pared with that of the parent. Thus, it was supposed that the mutant MS-92 should have n lower level of L-cysteine than the parent. In C. acremonium like A. nidulans, the enzymes related to the biosynthesis of methionine might be regulated by L-cysteine, but not by methionine or SAM.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.511-518
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• 1992

We subcloned a 58 KD chitinase gene of Serratia marcescens into Escherichia coli and investigated the expression and secretion of the chitinase. Chitinase was produced in E. coli by using its own promoter but the levels of enzyme were very low, less than 5 mU/m$\ell$. However, by the combined action of the chitinase and lac promoters, the chitinase activity increased up to about 80 mU/m$\ell$. The most of the chitinase produced in E. coli was localized in periplasm and the small amounts were observed in cytosol and culture medium. Intracellular chitinase activities increased in proportion to the growth of E. coli up to the early stationary phase but rapidly decreased thereafter, which was assumed to be degradation of the chitinase by E. coli proteolytic enzymes.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1744-1754
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• 2014

The hepatitis C virus (HCV) RNA genome is replicated by an RNA replicase complex (RC) consisting of cellular proteins and viral nonstructural (NS) proteins, including NS5B, an RNA-dependent RNA polymerase (RdRp) and key enzyme for viral RNA genome replication. The HCV RC is known to be associated with an intracellular membrane structure, but the cellular components of the RC and their roles in the formation of the HCV RC have not been well characterized. In this study, we took a proteomic approach to identify stomatin, a member of the integral proteins of lipid rafts, as a cellular protein interacting with HCV NS5B. Co-immunoprecipitation and co-localization studies confirmed the interaction between stomatin and NS5B. We demonstrated that the subcellular fraction containing viral NS proteins and stomatin displays RdRp activity. Membrane flotation assays with the HCV genome replication-competent subcellular fraction revealed that the HCV RdRp and stomatin are associated with the lipid raft-like domain of membranous structures. Stomatin silencing by RNA interference led to the release of NS5B from the detergent-resistant membrane, thereby inhibiting HCV replication in both HCV subgenomic replicon-harboring cells and HCV-infected cells. Our results identify stomatin as a cellular protein that plays a role in the formation of an enzymatically active HCV RC on a detergent-resistant membrane structure.

• Journal of Nutrition and Health
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• v.39 no.4
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• pp.323-330
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• 2006

Peripheral insulin resistance in obese/type II diabetes animals results from an impairment of insulin-stimulated glucose uptake into skeletal muscle. Insulin stimulate the translocation of GLUT4 from intracellular location to the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) is implicated in mediation of fusion of GLUT4-containing vesicle with the plasma membrane. Present study investigated regulatory effects of Rhodiola sachalinensis administration and exercise training on the expression of GLUT4 protein and SNAREs protein in skeletal muscles of obese Zucker rats. Experimental animals were randomly assigned into one of five groups ; lean control(LN), obese control(OB), exercise-treated(EXE), Rhodiola sachalinensis-treated(Rho), combine of Rho & EXE (Rho-EXE). All animals of exercise training (EXE, Rho-EXE) performed treadmill running for 8 weeks, and animals of Rho groups (Rho, Rho-EXE) were dosed daily by gastric gavage during the same period. After experiment, blood were taken for analyses of glucose, insulin, and lipids levels. Mitochondrial oxidative enzyme (citrate synthase, CS ; $\beta$-hydroxyacyl-CoA dehydrogenase, $\beta$-HAD) activity were analysed. Skeletal muscles were dissected out for analyses of proteins (GLUT4, VAMP2, syntaxin4, SNAP23). Results are as follows. Exercise and/or Rhodiola sachalinensis administration significantly reduced body weight and improved blood lipids (TG, FFA), and increased insulin sensitivity. Endurance exercise significantly increased the activity of mitochondrial enzymes and the expression of GLUT4 protein, however, administration of Rhodiola sachalinensis did not affect them. The effect of exercise and/or Rhodiola sachalinensis administration on the expression of SNARE proteins was unclear. Our study suggested that improvement insulin sensitivity by exercise and/or Rhodiola sachalinensis administration in obese Zucker rats is independent of expression of SNARE proteins.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.11
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• pp.4727-4732
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• 2015

Background: Folylpolyglutamate synthetase (FPGS), an important enzyme in the folate metabolic pathway, plays a central role in intracellular accumulation of folate and antifolate in several mammalian cell types. Loss of FPGS activity results in decreased cellular levels of antifolates and consequently to polyglutamatable antifolates in acute lymphoblastic leukemia (ALL). Materials and Methods: During May 1997 and December 2003, 134 children diagnosed with ALL were recruited from one hospital in Thailand. We performed a mutation analysis in the coding regions of the FPGS gene and the association between single nucleotide polymorphisms (SNPs) within FPGS in a case-control sample of childhood ALL patients. Mutation screening was conducted by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and subsequently with direct sequencing (n=72). Association analysis between common FPGS variants and ALL risk was done in 98 childhood ALL cases and 95 healthy volunteers recruited as controls. Results: Seven SNPs in the FPGS coding region were identified by mutation analysis, 3 of which (IVS13+55C>T, g.1297T>G, and g.1508C>T) were recognized as novel SNPs. Association analysis revealed 3 of 6 SNPs to confer significant increase in ALL risk these being rs7039798 (p=0.014, OR=2.14), rs1544105 (p=0.010, OR= 2.24), and rs10106 (p=0.026, OR=1.99). Conclusions: These findings suggested that common genetic polymorphisms in the FPGS coding region including rs7039789, rs1544105, and rs10106 are significantly associated with increased ALL risk in Thai children.

• International Journal of Oral Biology
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• v.41 no.4
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• pp.163-173
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• 2016

Flavonoid myricetin, usually found in tea and medicinal plants, has antioxidant and anti-inflammatory effects. Our objectives in this study were to verify the effects of myricetin on periodontal ligament fibroblasts (PDLFs) under inflammatory conditions and to observe its effects on osteoclast generation and on cytokine expression in RAW264.7 cells. To determine the effects of myricetin on PDLFs, we examined the expression and activity of proteolytic enzymes, including MMP-1, MMP-2, and MMP-8, which all play an important role in chronic periodontitis. We observed the effects of myricetin on intracellular signal transduction to verify the molecular mechanism involved. By measuring the formation of TRAP-positive multinucleated cells and the expression and activity of MMP-8, we were able to assess the effects of myricetin on osteoclast generation. In addition, by measuring the secretion of IL-6 and NO, we could evaluate the effects of myricetin on inflammatory mediators. We found that Myricetin had no effect on the viability of the PDLFs in the presence of inflammation, but it did decrease both the expression of MMP-1 and MMP-8 and the enzyme activity of MMP-2 and MMP-8 in these fibroblasts. Myricetin also decreased the lipopolysaccharide-stimulated phosphorylation of JNK, p38 signaling, IKKB, AKT, and p65RelA in the PDLFs. In the RAW264.7 cells, myricetin inhibited both the expression and the activity of MMP-8. Furthermore, Myricetin not only suppressed the generation of LPS-stimulated osteoclasts, but it also slightly inhibited LPS-stimulated degradation of IkB and decreased the release of LPS-induced IL-6 and NO. These findings suggest that myricetin alleviates the tissue-destructive processes that occur during periodontal inflammation.

• Journal of Life Science
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• v.22 no.1
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• pp.1-6
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• 2012

The present study examined the effects of consumption of Jeju ground water containing vanadium components on oxidative stress in obese (ob/ob) mice. Intake of Jeju ground water decreased the generation of oxidative stress induced-lipid peroxidation in the liver of ob/ob mice It also enhanced the enzymatic antioxidant defense system by increasing the protein expression and activity of superoxide dismutase, catalase, and glutathione peroxidase in liver tissues. Jeju ground water intake also upregulated the intracellular content of reduced glutathione. The induction of antioxidant enzyme expression by consumption of Jeju ground water was mediated by the erythroid transcription factor NF-E2 (Nrf2). Increased nuclear expression of phospho Nrf2 was observed in ob/ob mouse liver cells following ntake of Jeju ground water. These results suggest that consumption of Jeju ground water stimulated the antioxidant defense system in the livers of ob/ob mice via induction of Nrf2.

• YAKHAK HOEJI
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• v.54 no.6
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• pp.529-534
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• 2010

Alzheimer's disease (AD) is characterized pathologically by the presence of intracellular neurofibrillary tangles and deposition of ${\beta}$-amyloid ($A{\beta}$) peptides, which are generated by processing of amyloid precursor protein (APP). It is urgent to develop effective therapies for the treatment of AD, since our society rapidly accelerate aging. $A{\beta}$ peptides have been believed to be neurotoxic and now are also considered to have effects on the mechanism of memory formation. Recently, we investigated that a quinoline compound from natural product reduced the secretion of $A{\beta}$ from the neuroblastoma N2a cells (NL/N cell line) overexpressing APPswe. In this study, 3-phenyl-1-isoquinolinamine, a synthetic isoquinoline compound was analyzed to determine its effects on the metabolism of APP. It inhibited the secretion of $A{\beta}$ peptides from the N2a NL/N cell line. Beta-site APP cleaving enzyme (BACE) fluorescence resonance energy transfer (FRET) assay revealed that it inhibited BACE activity in a dose dependent manner. Immunoblotting study showed that it inhibited APP stabilization and expression and it slightly increased the stablization and the expression of ${\gamma}$-secreatase component from the N2a NL/N cell line. We suggest that 3-phenyl-1-isoquinolinamine inhibits APP metabolism and $A{\beta}$ generation by the means of BACE inhibitory mechanism. This is the first report that 3-phenyl-1-isoquinolinamine inhibits the secretion of $A{\beta}$ peptides from neuroblastoma cells.