• Biomolecules & Therapeutics
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• 제29권6호
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• pp.650-657
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• 2021

Metformin is an anti-diabetic drug and has anticancer effects on various cancers. Several studies have suggested that metformin reduces cell proliferation and stimulates cell-cycle arrest and apoptosis. However, the definitive molecular mechanism of metformin in the pathophysiological signaling in endometrial tumorigenesis and metastasis is not clearly understood. In this study, we examined the effects of metformin on the cell viability and apoptosis of human cervical HeLa and endometrial HEC-1-A and KLE cancer cells. Metformin suppressed cell growth in a dose-dependent manner and dramatically evoked apoptosis in HeLa cervical cancer cells, while apoptotic cell death and growth inhibition were not observed in endometrial (HEC-1-A, KLE) cell lines. Accordingly, the p27 and p21 promoter activities were enhanced while Bcl-2 and IL-6 activities were significantly reduced by metformin treatment. Metformin diminished the phosphorylation of mTOR, p70S6K and 4E-BP1 by accelerating adenosine monophosphate-activated kinase (AMPK) in HeLa cancer cells, but it did not affect other cell lines. To determine why the anti-proliferative effects are observed only in HeLa cells, we examined the expression level of liver kinase B1 (LKB1) since metformin and LKB1 share the same signalling system, and we found that the LKB1 gene is not expressed only in HeLa cancer cells. Consistently, the overexpression of LKB1 in HeLa cancer cells prevented metformin-triggered apoptosis while LKB1 knockdown significantly increased apoptosis in HEC-1-A and KLE cancer cells. Taken together, these findings indicate an underlying biological/physiological molecular function specifically for metformin-triggered apoptosis dependent on the presence of the LKB1 gene in tumorigenesis.

• 유기물자원화
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• 제16권4호
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• pp.64-73
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• 2008

glysosyl hydrolase의 하나인 CCF 유사 유전자를 지렁이 Eisenia anderi의 중장으로부터 분리하여 클로닝하였다. 이 유전자의 전체 염기서열 크기는 1,152bp로 나타났으며 개시코돈을 포함하여 384개의 아미노산을 인코딩한다. N-말단 지역의 17개 잔기들은 signal peptide이다. CCF 관련 유전자의 아미노산 서열을 분석한 결과 본 연구에서 분리한 CCF 유사 유전자는 glycosyl hydrolase family 16 (GHF16)에 속하며, 다른 종의 지렁이에서 밝혀진 CCF 및 CCF-유사 단백질과 79~99%의 높은 상동성을 보였다. 여러 지렁이 종에서 분리된 CCF 및 CCF-유사 단백질들은 가수분해활성에 중요한 polysacchride-binding motif와 glucanase motif가 100% 상동성을 나타냈다. 본 연구의 대상종과 유사종인 E. fetida에서 분리된 CCF는 이 종의 서식지가 미생물 활성이 높은 부패 유기질층이기 때문에 다른 종의 CCF에 비해 높은 기질인식 특이성을 갖고 있는 것으로 알려져 있다. 이러한 사실은 본 연구에서 분리한 CCF도 넓은 기질 특이성을 갖고 있을 가능성을 제시해 주고 있으며 이는 산업적 응용 측면에서 유용한 특징 중의 하나라고 생각된다. BLASTX를 이용한 계통수 분석 결과 GHF16 효소는 크게 후생동물군, 녹색식물군, 진정세균군 등의 세 그룹으로 나눌 수 있었으며, 후생동물군의 GHF16은 다시 촉수담륜동물형와 탈피동물형(후생동물형 포함)으로 나뉘어졌다. 본 연구에 의해 E. andrei로부터 분리된 CCF-유사 단백질의 더 많은 생물학적 특성 연구를 통해 ${\beta}$-D-글루칸의 분해 및 생산을 조절하는 산업적 활용이 가능할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제24권2호
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• pp.267-271
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• 1997

Apoptosis, programmed cell death, is posulated to occur in granulosa cells in ovarian follicular atresia. bcl-2 gene serves as protector from apoptosis and, thus, is associated with increased cell survival. TRPM-2 gene expression has been implicated as a trigger of apoptosis in rat prostate, uterus and mammary gland. Our objective was to determine if bcl-2 and TRPM-2 are expressed in luteinized human GC and, therefore, have regulatory functions for apoptosis in GC. Human GC were obtained via oocyte retrival from the infertile patients stimulated with exogeneous gonadotropins while undergoing IVF. GC were isolated from follicular fluid using Percoll gradient centrifugation. The GC were further purified with anti-CD45 magnetic beads to remove contaminating WBC's. RT-PCR were performed to analyze the mRNA expression of bcl-2 and TRPM-2 in the GC. The PCR primers were designed to amplify a 195 bp fragment of bcl-2 and a 174 bp fragment of TRPM-2. The PCR products were electrophoresed on 4% agarose gel. Three separate experiments indicated that both bcl-2 and TRPM-2 are concurrently expressed in human GC. We cultured granulosa cells with FSH (1 ng/ml) for 1 day to investigate the relative changes of TRPM-2 mRNA level with RNAse protection assay. When we cultured GC with serum free medium for 1 day TRPM-2 mRNA level increased with 1.3 fold, however it was decreased 0.64 fold with FSH. Therefore we conclude that bcl-2 and TRPM-2 are concurrently expressed and that the interaction of their products may be involved in GC apoptosis. And TRPM-2 may be regulated with FSH.

• KSBB Journal
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• 제18권6호
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• pp.522-528
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• 2003

본 연구는 식중독 환자로부터 분리한 장염비브리오균 120 균주를 세균학적 특성시험, 항균제 감수성 여부, 독소 유전자와 독소조절유전자의 검출 및 보유 여부를 PCR, RPLA 로 시험하였으며, 각 지역간의 분리 세균을 GS-PCR, PFGE 시험으로 병원체 상관 관계를 분석한 결과 다음과 같은 결론을 얻었다. 장염비브리오균은 0%의 NaCl 농도에서는 성장을 보이지 않았고, 8% NaCl이 첨가된 농도에서는 성장을 보였다. 국내 설사환자의 장염비브리오균의 O, K 혈청형은 17가지로 나타났으며, 이 중 O3:K6형이 68.3%로 가장 많았다. Ampicillin 등 18가지 항균제 시험에서 Ampicillin, Ticacillin에 높은 내성을 보였으며, Ampicillin, Ticacillin, Vancomycin에 동시 내성을 나타낸 경우가 52.5%로 나타났다. 독소조절 유전자 toxR은 PCR시험에서 시험균주 모두 368bp 크기의 유전자를 가지고 있었으며 장염 비브리오균의 조기진단에 유용한 것으로 확인되었고, 독소유전자 tdh는 120균주 중 109 균주만이 199bp 크기의 유전자를 보유하고 11균주가 음성이었다. trh 독소 유전자를 보유한 균주는 시험균주 중 3 균주만이 250bp 크기의 유전자를 보유하고 있었으며, tdh, trh 유전자를 동시에 가지고 있는 균주는 3균주로 이 균주의 TDH 독소 생성능은 x16 정도로 독소 생성능이 미약했다. 독소생성 Kanagawa 시험에서 120균주 중 107균주가 양성반응 을 나타내었으며, Kanagawa 양성반응을 보인 균주은 모두 tdh 유전자를 보유하고 있었다. Group Specific-PCR에서 최근에 유행을 일으키는 O3:K6 혈청형의 유연관계를 찾는데 유용한 것으로 나타났다. 조절유전자 toxRS 염기서열을 분석한 결과 같은 혈청형이라도 3균주는 7개의 염기서열 차이를 나타내었으며, 이는 현재 유행을 일으키는 균주와는 다른 균주임을 확인하였다. PFGE로 보다 더 세분화하여 혈청관계를 분류할 수 있었으며, 식중독 환자 유래 장염비브리오균은 3가지 type으로 나타났으며, 이들 균주 사이에는 상호 밀접한 상관관계를 나타내었다. 따라서 PFGE 기법으로 얻어진 결과는 분자 역학적으로 유용한 도구로 사용될 수 있음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1547-1556
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• 2009

A novel thermostable $\alpha$-glucosidase (TtGluA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in E. coli and characterized. The TtgluA gene contained 2,253 bp, which encodes 750 amino acids. The native TtGluA was a trimer with monomer molecular mass of 89 kDa shown by SDS-PAGE. The purified recombinant enzyme showed hydrolytic activity on maltooligosaccharides, p-nitrophenyl-$\alpha$-D-glucopyranide, and dextrin with an exotype cleavage manner. TtGluA showed preference for short-chain maltooligosaccharides and the highest specific activity for maltose of 3.26 units/mg. Maximal activity was observed at $60^{\circ}C$ and pH 5.5. The half-life was 2 h at $60^{\circ}C$. The enzyme showed good tolerance to urea and SDS but was inhibited by Tris. When maltose with the concentration over 50 mM was used as substrate, TtGluA was also capable of catalyzing transglycosylation to produce $\alpha$-1,4-linked maltotriose and $\alpha$-1,6-linked isomaltooligosaccharides. More importantly, TtGluA showed exclusive regiospecificity with high yield to produce $\alpha$-1,6-linked isomaltooligosaccharides when the reaction time extended to more than 10 h.

• Fisheries and Aquatic Sciences
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• 제16권4호
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• pp.311-318
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• 2013

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme was a serine-esterase and was active against $C_2$, $C_4$, $C_8$ and $C_{10}$ p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and $30^{\circ}C$, respectively. Relative activity remained up to 45% even at $5^{\circ}C$ with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, $Zn^{2+}$, and $Hg^{2+}$ ions.

• Journal of Microbiology and Biotechnology
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• 제33권4호
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• pp.449-462
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• 2023

Previously, we confirmed that Mychonastes sp. 246 methanolic extract (ME) markedly reduced the viability of BxPC-3 human pancreatic cancer cells. However, the underlying mechanism ME remained unclear. Hence, we attempted to elucidate the anticancer effect of ME on BxPC-3 human pancreatic cancer cells. First, we investigated the components of ME and their cytotoxicity in normal cells. Then, we confirmed the G1 phase arrest mediated growth inhibitory effect of ME using a cell counting assay and cell cycle analysis. Moreover, we found that the migration-inhibitory effect of ME using a Transwell migration assay. Through RNA sequencing, Gene Ontology-based network analysis, and western blotting, we explored the intracellular mechanisms of ME in BxPC-3 cells. ME modulated the intracellular energy metabolism-related pathway by altering the mRNA levels of IGFBP3 and PPARGC1A in BxPC-3 cells and reduced PI3K and mTOR phosphorylation by upregulating IGFBP3 and 4E-BP1 expression. Finally, we verified that ME reduced the growth of three-dimensional (3D) pancreatic cancer spheroids. Our study demonstrates that ME suppresses pancreatic cancer proliferation through the IGFBP3-PI3K-mTOR signaling pathway. This is the first study on the anticancer effect of the ME against pancreatic cancer, suggesting therapeutic possibilities and the underlying mechanism of ME action.

• Asian-Australasian Journal of Animal Sciences
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• 제26권8호
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• pp.1144-1151
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• 2013

In this study, protein domains with cellulase activity in goat rumen microbes were investigated using metagenomic and bioinformatic analyses. After the complete genome of goat rumen microbes was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered, including only those with 70% identity, 100-bp matches, and thresholds below $E^{-10}$ using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 1431 species was analyzed, among which Prevotella ruminicola 23 bacteria and Butyrivibrio proteoclasticus B316 were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4) were obtained through blast searches using the enzyme.dat file provided by the NCBI database. After translating the nucleotide sequence into a protein sequence using Interproscan, 28 protein domains with cellulase activity were identified using the HMMER package with threshold E values below $10^{-5}$. Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, cellulase, and Glyco hydro 10 were present in bacterial species with strong cellulase activities. Furthermore, correlation plots clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. This is the first metagenomic analysis of cellulase activity protein domains using bioinformatics from the goat rumen.

• International Journal of Industrial Entomology and Biomaterials
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• 제7권2호
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• pp.145-149
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• 2003

ApolipophorinIII (apoLp-III) is a protypical exchangeable apolipoprotein that is abundant in hemolymph of many insect species. Its function lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. But, recent studies with naive Galleria mellonella-apoLp-III gave first indication of an unexpected role of that protein in insect immune activation. In this research, we cloned a cDNA encoding putative apoLp-III from the silkworm, Bombyx mori injected with E. coli and characterized its role. We constructed a cDNA library using whole bodies of B. mori larvae injected with E. coli, carried out the differential screening, and selected the up-regulated clones. Among these clones, we focused on a cDNA showing a high sequence similarity to the apolipophorinIII from other insects and analyzed the nucleotide and deduced amino acid sequences. The pupative B. mori Jam123 apoLp-III cDNA contained 1,131 bp encoding 186 amino acid residues. Phylogenetic analysis revealed that the nucleotide and amino acid sequences of the B. mori apoLp-III cDNA formed a highly inclusive subgroup with Bombycidae. But, it was interesting that B. mori Jam123 is closer to B. mandarina than B. mori P50 and B. mori N4. Northern blot analysis showed a signal in the fat body, posterior silkgland and midgut.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.821-827
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• 2008

The unicellular green alga Dunaliella salina is a halotolerant eukaryotic organism. Its halophytic properties provide an important advantage for open pond mass cultivation, since D. salina can be grown selectively. D. salina was originally described by E. C. Teodoresco in 1905. Since that time, numerous isolates of D. salina have been identified from hypersaline environments on different continents. The new Dunaliella strain used for this study was isolated from the salt farm area of the west coastal side of South Korea. Cells of the new strain were approximately oval- or pear-shaped (approximately $16-24\;{\mu}m$ long and $10-15\;{\mu}m$ wide), and contained one pyrenoid, cytoplasmatic granules, and no visible eyespot. Although levels of $\beta$-carotene per cell were relatively low in cells grown at salinities between 0.5 to 2.5 M NaCl, cells grown at 4.5 M NaCl contained about a ten-fold increase in cellular levels of $\beta$-carotene, which demonstrated that cells of the new Korean strain of Dunaliella can overaccumulate $\beta$-carotene in response to salt stress. Analysis of the ITS1 and ITS2 regions of the new Korean isolate showed that it is in the same clade as D. salina. Consequently, based on comparative cell morphology, biochemistry, and molecular phylogeny, the new Dunaliella isolate from South Korea was classified as D. salina KCTC10654BP.