• Food Science and Biotechnology
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• 제14권4호
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• pp.479-486
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• 2005

Arabinoxylan, a complex polysaccharide in cereal cell walls, has recently received research attention as a biological response modifier. The immunomodulating effect of arabinoxylans from rice bran (AXrb) was studied using a combined process of extrusion and commercial hemicellulase treatment in order to elucidate the augmentation mechanism of cell-mediated immunity in vitro. The cytotoxicity of mouse spleen lymphocytes against YAC-1 tumor cells was significantly enhanced by treatment with AXrb at $10-100\;{\mu}g/mL$. In an attempt to investigate the mechanism by which AXrb enhance NK cytotoxicity, we examined the effect of AXrb on cytokine production by spleen lymphocytes. Culture supernatants of the cells incubated with AXrb were collected and analyzed for IL-2 and IFN-${\gamma}$ synthesis by ELISA. IL-2 and IFN-${\gamma}$ production were increased significantly. These results suggest that AXrb may induce Th1 immune responses. Macrophages play an important role in host defenses against tumors by killing them and producing secretory products, which protect against bacterial, viral infection and malignant cell growth. AXrb were examined for their ability to induce secretory and cellular responses in murine peritoneal macrophages. When macrophages were treated with various concentrations ($10-100\;{\mu}g/mL$) of AXrb, AXrb induced tumoricidal activity, as well as increasing phagocytosis and the production of NO, $H_2O_2$, TNF-${\alpha}$, IL-$1{\beta}$, and IL-6. These results indicate that reactive oxygen species, reactive nitrogen species, and inflammatory cytokines are likely to be the major mediators of tumoricidal activity in AXrb-treated macrophages. Therefore, AXrb may be useful in cancer immunotherapy and it is anticipated that AXrb obtained using extrusion and subsequent enzyme treatment can be used as an ingredient in nutraceuticals and cereal-based functional food.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.631-638
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• 2008

Tandem affinity purification (TAP) method combined with LC-MS/MS is the most accurate and reliable way to study the interaction of proteins or proteomics in a genome-wide scale. For the first time, we used a TAP-tag as a mutagenic tool to disrupt protein interactions at the specific site. Although lots of commonly used mutational tools exist to study functions of a gene, such as deletional mutations and site-directed mutagenesis, each method has its own demerit. To test the usefulness of a TAP-tag as a mutagenic tool, we applied a TAP-tag to RNA polymerase II, which is the key enzyme of gene expression and is controlled by hundreds of transcription factors even to transcribe a gene. Our experiment is based on the hypothesis that there will be interrupted interactions between Pol II and transcription factors owing to the TAP-tag attached at the C-terminus of each subunit of Pol II, and the abnormality caused by interrupted protein interactions can be observed by measuring a cell-cycle of each yeast strain. From ten different TAP-tagged strains, Rpb7- and Rpb12-TAP-tagged strains show severe defects in growth rate and morphology. Without a heterodimer of Rpb4/Rpb7, only the ten subunits Pol II can conduct transcription normally, and there is no previously known function of Rpb7. The observed defect of the Rpb7-TAP-tagged strain shows that Rpb7 forms a complex with other proteins or compounds and the interruption of the interaction can interfere with the normal cell cycle and morphology of the cell and nucleus. This is a novel attempt to use a TAP-tag as a proteomic tool to study protein interactions.

• 생명과학회지
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• 제19권5호
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• pp.561-565
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• 2009

인간조직인자는 혈액응고인자 factor VII 과 복합체를 형성하며 연속적인 혈액응고 연쇄반응을 촉매하는 효소 활성체이다. 복합체 형성에 필수적인 이 조직인자의 세포 외 부분이, 기존의 융합 단백질 및 히스티딘 말단이 없는 새로운 발현 벡터에 의해 대장균 내에서 과량 발현 되었다. 봉입체 형태로 발현된 재조합 인간조직인자는 DEAE-Sephacel 크로마토그라피 기술을 적용하여 분리, 정제 및 구조적 복원이 동시에 시도 되었다. 정제된 재조합 단백질은 SDS-PAGE 분석에서 순수한 형태로 나타났으며, 생물학적 활성도 또한 기존의 조직인자와 거의 동등함을 보였다. 본 연구의 발현 및 정제 시스템은 이전의 보고에서 보여진 방법들에 비해 단백질 분해효소를 사용하지 않아 추가적인 크로마토그라피 과정이 필요 없어 좀 더 효율적이기 때문에 기존의 발현 시스템에 대해 대체할 수 있는 매우 유용한 방법으로 제공된다.

• Molecules and Cells
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• 제43권11호
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• pp.964-973
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• 2020

Recent studies have highlighted that early enhancement of the glycolytic pathway is a mode of maintaining the proinflammatory status of immune cells. Thiamine, a wellknown co-activator of pyruvate dehydrogenase complex, a gatekeeping enzyme, shifts energy utilization of glucose from glycolysis to oxidative phosphorylation. Thus, we hypothesized that thiamine may modulate inflammation by alleviating metabolic shifts during immune cell activation. First, using allithiamine, which showed the most potent anti-inflammatory capacity among thiamine derivatives, we confirmed the inhibitory effects of allithiamine on the lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and maturation process in dendritic cells. We applied the LPS-induced sepsis model to examine whether allithiamine has a protective role in hyper-inflammatory status. We observed that allithiamine attenuated tissue damage and organ dysfunction during endotoxemia, even when the treatment was given after the early cytokine release. We assessed the changes in glucose metabolites during LPS-induced dendritic cell activation and found that allithiamine significantly inhibited glucose-driven citrate accumulation. We then examined the clinical implication of regulating metabolites during sepsis by performing a tail bleeding assay upon allithiamine treatment, which expands its capacity to hamper the coagulation process. Finally, we confirmed that the role of allithiamine in metabolic regulation is critical in exerting anti-inflammatory action by demonstrating its inhibitory effect upon mitochondrial citrate transporter activity. In conclusion, thiamine could be used as an alternative approach for controlling the immune response in patients with sepsis.

• 한국미생물·생명공학회지
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• 제18권5호
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• pp.460-465
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• 1990

Asp.wentii와 Asp.nidulans의 원형질체 재생은 2-DG가 30$\mu g$/ml 첨가된 포자현탁액을 4시간 전배양 할 때 적당하였고 ergosterol, myoniositol, casamino acid, BSA가 함유된 CBE 재생용 배지에서 효과적이었으며, 30 이상 재생률을 나타내었다. 원형질체 융합은 10mM $CaCl_2$가 함유된 pH7.5의 30PEG 4000으로$37^{\circ}C$ 에서 10분간 처리했을 때 가장 양호하였으며, 융합빈도는 $8.2\times 10^{-4}$을 나타내었다. 가장 우수한 융합주인 FWN-56은 CMCase, avicelase, $\beta$-glucosidase 및 xylanase를 동시에 분비하였으며 친주에 비하여 활성이 2.3배, 1.5배, 1.8배, 2.5배 각각 증가하였고, 또한 MM에 4중 이상 보관 후의 segregant율이 1 이내였으므로 유전적 안정성은 높았으며, 분생포자 DNA함량은 1.4-1.6배였다. 또한 핵의 크기도 친주에 비하여 큰 것으로 보아 융합주임을 확인하였다.

• Applied Biological Chemistry
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• 제33권4호
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• pp.287-292
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• 1990

황금종 대두로 부터 8종의 Bowman-Birk형 proteinase inhibitor들을 DEAE-Sephadex A-50으로 전기영동상 단일 band로 순수하게 분리하였다. 이중 inhibitor VII cysteine함량이 17%로 높고 trypsin 및 chymotrypsin에 대해 각각 독립적인 결합부위를 가지고 있으며 상기 두 효소에 대한 저해활성도의 비(TIA/CIA)가 1.0으로 전형적인 Bowman-Birk trypsin inhibitor(BBTI)로 확인되었다. 본 inhibitor와 trypsin 및 chymotrypsin complex의 dissociation constant는 각각 $9.17{\times}10^{-9}M$과 $5.14{\times}10^{-8}M$로 매우 안정하였다. 또한 inhibitor Vll은 열에 매우 안정한 단백질로 $100^{\circ}C$, 6시간 처리에도 저해활성도 감소가 50%밖에 안되었다. 순수분리된 7종의 다른 isoinhibitor중 inhibitor III만이 TIA/CIA값이 1.2로 BBTI와 비슷하였으나 그외 inhibitor I, II, IV, V,및 VIII은 그 값이 $3{\sim}29$로 BBTI와는 성질이 다른 isoinhibitnr로 추정되었다.

• BMB Reports
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• 제40권2호
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• pp.239-246
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• 2007

Riboflavin synthase from Escherichia coli is a homotrimer of 23.4 kDa subunits and catalyzes the formation of one molecule each of riboflavin and 5-amino-6-ribitylamino- 2,4(1H,3H)-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrate, 6,7- dimethyl-8-ribityllumazine. Each subunit comprises two closely similar folding domains. Recombinant expression of the N-terminal domain is known to provide a $C_2$-symmetric homodimer. In this study, the binding properties of wild type as well as two mutated proteins of N-terminal domain of riboflavin synthase with various ligands were tested. The replacement of the amino acid residue A43, located in the second shell of riboflavin synthase active center, in the recombinant N-terminal domain dimer reduces the affinity for 6,7-dimethyl-8-ribityllumazine. The mutation of the amino acid residue C48 forming part of activity cavity of the enzyme causes significant $^{19}F$ NMR chemical shift modulation of trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine in complex with the protein, while substitution of A43 results in smaller chemical shift changes.

• Applied Microscopy
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• 제14권2호
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• pp.66-80
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• 1984

The organic phosphorus compounds have been widely used as an insecticide, since toxicity of these compounds is especially drastic to the insects than to men and other mammals. The organic phosphates are rapidly hydrolized and hence have little cumulative and ecologic effects. However, due to their acute toxic effects organophosphate have recorded rather high fatalities in men and domestic animals. The organic phosphorus compounds are powerful inhibitors to the carboxylic esterase enzymes such as acetylcholinesterase and pseudocholinesterase. As a result of firm binding characteristics of phosphate radicals to the active sites of enzyme, the activities of these enzymes are inhibited by the organophosphates. The organophosphates such as diazinon is easily observed from skin, gastrointestinal tract, conjunctivas and respiratory tract, and it is converted to more toxic form during metabolism in the liver The present study was carried out in order to investigate the hepatotoxicity of diazinon by observing the changes in the ultrastructure of cytoplasmic organelles of hepatic cells in albino mice. The animals were killed at 6, 12 and 24 hours after administration of 25mg/kg diazinon. The piece of hepatic tissue obtained from each animal was ultrathinly sectioned. The specimens stained by uranyl acetate and lead citrate double contrast methods were observed with JEM model 100B electron microscope. The results obtained were as follows: 1) A prominent dilatation and sacculation of the cisternae of rough endoplasmic reticulum associated with detachment of membrane bound-ribosomes, and disaggregation of the free ribosomes were recognized. 2) The hypertrophy of the smooth endoplasmic reticulum associated with depletion of the glycogen particles was observed. 3) The atrophy of cisternae of Golgi complex was observed. 4) A large number of secondary lysosomes (autophagic vacuoles and residual bodies) were formed. Consequently it is suggested that diazinon would induce disorganization of the cytoplasmic organelles of hepatocytes in albino mice.

• Journal of Microbiology and Biotechnology
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• 제6권1호
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• pp.7-11
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• 1996

MR-387Al (ARPA-Val-Pro) and A2 (AHPA-Val-Hyp) were prepared as aminopeptidase N inhibitors through the synthesis of peptide MR-387A and B analogues which contained 3-amino-2-hydroxy-4-phenyl butanoic acid (ARPA) as a zinc-chelating moiety. They are competitive inhibitors of aminopeptidase N with inhibition constants(Ki) of 4.1 $\times 10^{-7}\;and 1.1 \times 10^{-6}$ M, respectively. MR-387Al also strongly inhibited aminopeptidase B of human myelogenous leukemia K-562 cell with $IC_50$ of 0.35 $\mu$ M. Inhibitions of aminopeptidase N activity by ARPA-bearing inhibitors of various peptide chain lengths also have been studied. $IC_ 50$ values of AHPA-Val (bestatin), ARPA-Val-Pro (MR-387Al) and ARPA-Val-Pro-Leu (MR-387C) compared against porcine kidney aminopeptidase N were 20.1, 0.60 and 0.08 $\mu$ M, respectively. These results support that a multiple interaction between the $S_1\to S'_3$ sites of aminopeptidase N and the $P_1\to P'_3$ of the inhibitor plays a crucial role in stabilizing strongly the enzyme-inhibitor complex.

• Journal of Microbiology
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• 제42권3호
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• pp.188-193
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• 2004

Xanthobacter flavus strain UE15 was isolated in wastewater obtained from the Ulsan industrial complex, Korea. This strain functions as a 1,2-dichloroethane (1,2-DCA) degrader, via a mechanism of hydrolytic dechlorination, under aerobic conditions. The UE15 strain was also capable of dechlorinating other chloroaliphatics such as 2-chloroacetic acid and 2-chloropropionic acid. The dhlA gene encoding 1,2-DCA dechlorinase was cloned from the genomic DNA of the UE15 strain, and its nucleotide sequence was determined to consist of 933 base pairs. The deduced amino acid sequence of the DhlA dechlorinase exhibited 100％ homology with the corresponding enzyme from X. autotrophicus GJ10, but only 27 to 29％ homology with the corresponding enzymes from Rhodococcus rhodochrous, Pseudomonas pavonaceae, and Mycobacterium sp. strain GP1, which all dechlorinate haloalkane compounds. The UE15 strain has an ORF1 (1,356 bp) downstream from the dhlA gene. The OFR1 shows 99％ amino acid sequence homology with the transposase reported from X. autotrophicus GJ10. The transposase gene was not found in the vicinity of the dhlA in the GJ10 strain, but rather beside the dhlB gene coding for haloacid dechlorinase. The dhlA and dhlB genes were confirmed to be located at separate chromosomal loci in the Xanthobacter flavus UE15 strain as well as in X. autotrophicus GJ10. The dhlA and transposase the UE15 strain were found to be parenthesized by a pair of insertion sequences, 181247, which were also found on both sides of the transposase gene in the GJ10 strain. This unique structure of the dhlA gene organization in X. flavus strain UE15 suggested that the dechlorinase gene, dhlA, is transferred with the help of the transposase gene.