• Asian Pacific Journal of Cancer Prevention
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• 제13권11호
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• pp.5563-5568
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• 2012

Based on our previous report on gastric cancer which documented ATP4A and ATP4B mRNA down-regulation in gastric tumors relative to normal gastric tissues, we hypothesized that epigenetic mechanisms could be responsible. ATP4A and ATP4B mRNA expression in gastric cancer cell lines AGS, SNU638 and NUGC-3 was examined using reverse transcriptase PCR (RT-PCR). AGS cells were treated with TSA or 5'-AzaDC and methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis were performed. MSP analysis was on DNA from paraffin embedded tissues sections and plasma. Expression analysis revealed downregulation of ATP4A and ATP4B genes in gastric cancer cell lines relative to normal gastric tissue, while treatment with 5'-AzaDC re-activated expression of both. Search for CpG islands in their putative promoter regions did not indicate CpG islands (CGI) but only further downstream in the bodies of the genes. Methylation specific PCR (MSP) in the exon1 of the ATP4B gene and exon7 in ATP4A indicated methylation in all the gastric cancer cell lines tested. MSP analysis in tumor tissue samples revealed methylation in the majority of tumor samples, 15/19, for ATP4B and 8/8 for ATP4A. There was concordance between ATP4B and ATP4A down-regulation and methylation status in the tumour samples tested. ATP4B methylation was detectable in cell free DNA from gastric cancer patient's plasma samples. Thus ATP4A and ATP4B down-regulation involves DNA methylation and methylated ATP4B DNA in plasma is a potential biomarker for gastric cancer.

• Journal of Korean Academy of Oral Health
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• 제42권4호
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• pp.224-228
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• 2018

Objectives: As a first step to study the anticaries effect of ethanol alone, we investigated the effects of ethanol on the expression levels of the atpB gene and proton permeability of Streptococcus mutans in suspension cultures. Methods: S. mutans UA159 was grown in brain heart infusion medium at either pH 4.8 or 6.8. The total extracted RNA was reverse-transcribed into cDNA using a $Superscript^{TM}$ First-Strand Synthesis System. The resulting cDNA and negative controls were amplified by ABI PRISM 7700 real-time PCR system with SYBR Green PCR Master Mix. For proton flux assay, bacterial suspensions were titrated to pH 4.6 with 0.5 M HCl, and then additional 0.5 M HCl was added to decrease the pH values by approximately 0.4 units. The subsequent increase in pH was monitored using a glass electrode. Ten percent (v/v) butanol was added to the suspensions at 80 min to disrupt the cell membrane. Results: In a concentration-dependent manner, ethanol alone not only decreased the growth rate of S. mutans and the expression of the atpB gene but also increased the proton permeability at both pH 4.8 and 6.8. Conclusions: These findings suggest that ethanol has the potential for an anticaries ingredient. We believe that ethanol may be used together with fluoride and/or other cariostatic agents in order to develop better anticaries toothpastes and/or mouthrinses.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권2호
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• pp.115-122
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• 2007

As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identification of novel genes of B.mori is conceivable by data-mining techniques. We used the in silico cloning method to get the vacuolar-type $H^+-ATP$ synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and sequencing. The V-ATPase c subunit cDNA contains a 468 bp ORF. The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-spanning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Malpighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.

• Clinical and Experimental Reproductive Medicine
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• 제34권1호
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• pp.19-31
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• 2007

목 적: 자궁내막증은 자궁내부에 존재하여야 할 자금내막조직이 자궁 외에 존재하는 질환으로 그 발생기전은 아직 명확하게 밝혀져 있지 않다. 이에 저자들은 자궁내막증 환자와 정상 대조군의 자궁내막조직 간의 유전자 발현의 차이가 자궁내막증의 발병과 관련이 있을 것이라는 가정 하에 DNA microarray 기술을 도입하여 연구를 시행하였다. 연구방법: 2002년 1월부터 2002년 12월까지의 기간 동안 본원 산부인과에서 자궁내막증 환자와 자궁내막증 이외의 다른 부인과적 질환으로 수술을 시행한 환자들을 대상으로 채취한 자궁내막 조직으로 KNU 4.8K cDNA chip을 이용하여 유전자 발현을 비교 연구하였다. 유전자칩으로 자궁내막증 조직에서 발현의 증감을 보였던 유전자 중에서 8종의 유전자를 대상으르 RT-PCR이나 real time RT-PCR 법을 통하여 그 발현 양상을 검증하였다. 결 과: 자궁내막증에 이환된 여성의 자궁내막조직에서 대조군에 비하여 높게 발현되고 있는 것으로 나타난 유전자들은 ATP synthase H transporting F1 (ATP5B), eukaryotic translation elongation factor 1, isocitrate dehydrogenase 1 (NADP+), mitochondrial ribosomal protein L3, ATP synthase H+ trarsporting (ATP5C1), LPS induced TNF-$\alpha$ factor 등으로 세포의 에너지 생성과 대사과정 및 신호전달에 관여하는 유전자들이었다. 한편 자궁내막중 환자의 자궁내막조직에서 대조군에 비하여 낮게 발현된 유전자들은 insulin like growth factor II associated protein, EGF-containing fibulin-like EMP1, matrix Gla protein, TGF beta-induced, TGF beta receptor 1(activin A receptor type II-like kinase), cystallin alpha B, fibulin 5, tissue inhibitor of metalloproteinase 3, collage type XII, alpha 1, tissue inhibitor of metalloproteinase 1, decorin 등으로 세포외기질의 구성 및 기능에 관련이 있었다. 결 론: 이상의 DNA mirroarry 및 RT-PCR을 통해 얻어진 결과에서 자궁내막증의 자궁내막조직에서 대조군에 비하여 유전자들의 발현에 차이가 있음을 확인하였다.

• 대한의생명과학회지
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• 제18권1호
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• pp.49-55
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• 2012

Numerous biochemical molecules have been implicated in the development of muscular atrophy. However, control mechanisms associated with muscular disease are not clear. The present study was conducted to investigate gene expression profiles of rat muscle during the denervation to atrophy transition processes. We isolated total RNA from rats suffering from partial muscle atrophy (P) and electromyostimulated atrophy (PE) and synthesized cDNA using annealing control primers. Using 20 ACPs for PCR, we cloned 18 DEGs using TOPO TA cloning vector, sequenced, and analyzed their identities using BLAST search. Sequences of 14 clones significantly matched database entries, while one clone was ESTs, and 3 clones were unidentified. Different expression profiles of selected DEGs between P and PE were confirmed. The troponin T, Fkbp1a, RGD1307554, Phtf1, Atp1a1 and Commd3 were highly expressed genes in the P and PE groups, while Krox-25 and TCOX2 were only expressed genes in the P group, the Sv2b and Marcks were only expressed genes in PE group. also, Cox8h was highly expressed genes in PE groups. The ASPH, ND1, and ARPL1 were highly expressed genes in the P and PE groups. List of genes obtained from the present study might provide an insight for the study of mechanism regulating muscle atrophy and electrostimulated muscle atrophy transitions. These data suggest that troponin T, Fkbp1a, RGD1307554, Phtf1, Atp1a1, and Commd3 are potentially useful as clinical biomarkers of age-related muscle atrophy and dysfunction.

• Bulletin of the Korean Chemical Society
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• 제31권4호
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• pp.887-890
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• 2010

The family of ClpB protein is a molecular chaperone which protects cellular proteins from being aggregated upon exposure to severe environmental stresses in association with DnaK/DanJ/GrpE in the ATP-dependent manner. In a psychrophilic bacterium which survives at a subzero temperature, any functional role of cold-active ClpB protein can be rather crucial. In order to identify a ClpB encoding gene from a cold-adapted bacterium whose genome sequence has not been fully discovered, we have employed a series of PCR technologies, including a gradient PCR with homologous primers, an inverse PCR and a cassette PCR. The full sequence of PaclpB gene was successfully identified and compared with those of other psychrophilic species. We have further cloned the gene in E.coli expression systems and were able to induce PaClpB protein expression by IPTG, which help us understand a molecular mechanism for survival against extremely cold environments.

• The Korean Journal of Physiology and Pharmacology
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• 제14권6호
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• pp.353-358
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• 2010

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-${\kappa}B$/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-${\kappa}B$/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-${\kappa}B$/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-${\kappa}B$/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-${\kappa}B$/Rel and p38 kinase signaling.

• 대한한방내과학회지
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• 제38권6호
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• pp.1035-1048
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• 2017

Objectives: This study was performed to investigate the effects of Gardenia jasminoides extract (GJ) on osteoclast differentiation and bone resorption in vitro. Methods: To investigate the effect of GJ on osteoclast differentiation, the mouse leukemic myeloid cell line RAW 264.7 was stimulated by RANKL (receptor activator of nuclear factor kB ligand). Osteoclast differentiation was measured by counting TRAP (+) MNC in the presence of RANKL. To elucidate the mechanism of the inhibitory effect of GJ on osteoclast differentiation, gene expression of TRAP, Cathepsin K, MMP-9, NFATc1, c-Fos, MITF, DC-STAMP, CTR, OC-STAMP and Atp6v0d2 was measured using reverse transcription-PCR (RT-PCR). Bone resorption was measured using the bone pit formation assay. Results: GJ decreased the number of TRAP (+) MNCs in the presence of RANKL. GJ inhibited the expression of cathepsin K, MMP-9, TRAP, MITF, NFATc1, c-Fos, iNON, OC-STAMP, Atp6v0d2, and DC-STAMP in the osteoclast, and inhibited bone pit formation in vitro. Conclusions: The results suggest that GJ has inhibitory effects on bone resorption resulting from inhibition of osteoclast differentiation and gene expression.

• The Plant Pathology Journal
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• 제30권4호
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• pp.375-383
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• 2014

Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5'-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum.

• 한국자원식물학회지
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• 제22권3호
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• pp.273-280
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• 2009

A cDNA clone encoding a MDR-like ABC transporter protein was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (named as Brmdr 1; GenBank accession no.: DQ296184 ) had a total length of 4222 bp with an open reading frame of 3900 bp, and encoded a predicted polypeptide of 1300 amino acids with a molecular weight of 143.1 kDa. The BrMDR1 protein shared 71.0, 62.5, 60.0 and 58.2% identity with other MDR proteins isolated from Arabidopsis thaliana (AAN28720), Coptis japonica (CjMDR), Gossypium hirsutum (GhMDR) and Triticum aestivum (TaMDR) at amino acid level, respectively. Southern blot analysis showed that Brmdr1 was a low-copy gene. Expression pattern analysis revealed that Brmdr1 constitutively expressed in the root, stem petals and stamens, but with lower expression in leaves and open flowers. The domains analysis showed that BrMDR1 protein possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranging in "TMD1-NBD1-TMD2-NBD2" direction, which is consistent with other MDR transporters. Within NBDs three characteristic motifs common to all ABC transporters, "Walker A", "Walker B" and C motif, were found. These results indicate that BrMDR1 is a MDR-like ABC transporter protein that may be involved in the transport and accumulation of secondary metabolites.