• IMMUNE NETWORK
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• 제13권2호
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• pp.63-69
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• 2013

IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membranebound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-${\alpha}$. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the $CD8^+$ T cell-depleted mice than in $CD4^+$ T cell-depleted or normal mice. These results suggest that $CD8^+$ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and $CD4^+$ helper T cells.

• Applied Biological Chemistry
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• 제20권1호
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• pp.26-32
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• 1977

대두(大豆)(Glycine max cultivar Gwang-gyo)의 각 성숙시기(成熟時期)에 나타나는 7S globulin을 분리하여 Davis 방법(方法)에 의한 전기영동과 PAWU용매에 의해서 유리(遊離)되는 그들의 subunit를 전기영동한 결과 7S globulin중의 복합단백질간(複合蛋白質間)에는 그 구성(構成) subunit에 유사성(類似性)이 있음을 시사하였다. 7S globulin의 복합단백질(複合蛋白質)을 DEAE-Sephadex A-50으로 크로마토그라피하여 분리하였다. 이때 pH 7.6의 인산완충액(燐酸緩衝液)에서 NaCl의 농도구배(濃度句配)가 0.28M부터 0.40M 사이에서 두 개의 분획(分劃)으로 분리되었다. 이들 명(名) 단백질(蛋白質)의 subunit를 5M urea와 1% SDS로 유리(遊離)시켜 7.5% acrylamide-PAWU gel과 5.6% acrylamide-SDS gel에서 전기영동하였다. 그 결과 subunit의 하전량(荷電量)에 의해서 분리되는 PAWU gel전기영동에서 7S globulin이 5개의 주 분리대로 분리되고 그중 2개의 분리대가 7S-A globulin과 7S-B globulin에 공유(共有)되어 있었다. 또 subunit의 분자량(分子量)에 따라서 분리되는 SDS gel 전기영동에서는 7S globulin이 7개의 주 분리대를 나타내는데 그 중에서 3개의 분리대가 7S-A와 7S-B 분획에 공유(共有)되어 있었다. 따라서 7S globulin의 복합단백질간(複合蛋白質間)에는 구성(構成) subunit간(間)에 유사성(類似性)이 있는 것으로 나타났다.

• 한국식품과학회지
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• 제22권2호
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• pp.134-139
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• 1990

누룩으로부터 ${\beta}-galactosidase$ 활성을 가지는 균주를 순수분리하여 분리된 균주(NY-15)의 ${\beta}-galactosidase$를 정제하였다. 다량배양하여 모은 균체를 8%(v/v) toluene 처리에 의한 추출액을 정제의 출발 물질로 하여 acetone 침전에 의해서 탈지처리를 한 후 40-60%포화의 ammonium sulfate 분획을 하고 DEAE-cellulose, DEAE-Sephadex A-50의 이온교환 chromatography를 한 후 Agarose-PAPT를 이용한 affinity chromatography에 의해서 정제하였다. 이 정제법에 의해서 ${\beta}-galactosidase$는 조효소 추출액으로부터 40배 정제되었으며 회수율은 7.7%였나. 누룩 yeast의 ${\beta}-galactosidase$는 2개의 subunit로 되어 있으며, 각 subunit 분자량은 각각 96,000과 130,000이었다. 효소활성에의 최적 pH는 7.5이었고, 최적온도는 $40^{\circ}C$이었다.

• Journal of Plant Biology
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• 제33권1호
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• pp.59-64
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• 1990

In order to study the effect of GA3 on the phosphorylation of ribosomal proteins during germination in Zea mays, ribosomal proteins were labelled with 32P, extracted, electrophoresed and autoradiographed. There are five phosphorylated ribosomal proteins. One of these is in 40S subunit and has molecular weight of 33,000 daltons. Others are in 60S subunit and have molecular weights of 37,000, 16,000, 15,200 and 13,500, respectively. Phosphorylation of ribosomal proteins was increased maximum 47.7% in shoots of Zea mays treated with GA3.

• ALGAE
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• 제33권1호
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• pp.49-54
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• 2018

Red algal mitochondrial genomes (mtDNAs) can provide useful information on species identification. mtDNAs of Pyropia / Porphyra (Bangiales, Rhodophyta) have shown diverse variation in their size and gene structure. In particular, the introns and intronic open reading frames found in the ribosomal RNA large subunit gene (rnl) and cytochrome c oxidase subunit 1 gene (cox1) significantly vary the mitochondrial genome size in Pyropia / Porphyra species. In this study, we examined the exon / intron structure of rnl and cox1 genes of Pyropia yezoensis at the intraspecific level. The combined data of rnl and cox1 genes exhibited 12 genotypes for 40 P. yezoensis strains, based on the existence of introns. These genotypes were more effective to identify P. yezoensis strains in comparison to the traditional DNA barcode cox1 marker (5 haplotypes). Therefore, the variation in gene structure of rnl and cox1 can be a novel molecular marker to discriminate the strains of Pyropia species.

• Journal of Animal Science and Technology
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• 제50권2호
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• pp.177-184
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• 2008

Acid-labile subunit(ALS)는 85-kDa 크기의 당단백질로서 7.5-kDa의 insulin-like growth factor(IGF) 및 40~45-kDa IGF-binding protein-3와 결합하여 150-kDa ternary complex를 형성하는 혈장단백질이다. 선행연구에서 본 연구진은 reverse transcription-polymerase chain reaction(RT-PCR) 방법으로 돼지(porcine) ALS(pALS)의 coding sequence를 합성하여 plasmid vector에 삽입시켜 ‘expression construct’를 제작한 바 있다. 그러나 본 expression construct의 pALS coding sequence에는 PCR error로 추정되는 원인으로 말미암아 2개의 bases에서 mis-sense mutation이 일어난 것이 발견되었다. 본 연구에서는 ‘site-directed mutagenesis’ 방법으로 pALS의 올바른 coding sequence를 합성하여 ‘insert DNA’의 마지막 codon 다음에 ‘His-tag’ sequence가 위치한 pET- 28a(+) plasmid expression vector에 삽입하였다. 본 expression construct는 E. coli BL21(DE3) 세포에서 ‘induction’ 시켰고, 발현된 유전자재조합(recombinant) peptide는 Ni-affinity chromato- graphy로 정제하였다. 이렇게 affinity chro- matography로 정제된 peptide는 SDS-PAGE에서 66kDa 위치에 single band를 나타냄으로써 recombinant pALS의 예상된 질량과 일치하였다. 이상의 결과는 본 연구에서 recombinant pALS peptide가 성공적으로 발현정제되었음을 시사한다.

• Journal of Plant Biology
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• 제32권1호
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• pp.33-40
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• 1989

Polysomal polyadenylated mRNAs which were purified from pea leaves were fractionated by sucrose grandient sedimentation. Fractions corresponding to the peak at 11.5S were found to contain mostly mRNA encoding the precursor polypeptide to the small subunit of ribulose bisphosphate carboxylase (rbcS) by in vitro translation in wheat germ extract. Double-stranded cDNA which was synthesized from the 11.5S mRNA was cloned into Hind III site of plasmid pBR 325. A cDNA clone, H24, was identified to code for rbcS. In vitro translation product of the hybridization-selected mRNA was molecular weight 20,000, presumably the precursor of rbcS. The nucleotide sequences of the H24 showed almost complete homology with the sequences encoding the transit peptide of the rbcS-3A gene which was reported by Fluhr et al.(1986).

• International Journal of Oral Biology
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• 제40권4호
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• pp.205-210
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• 2015

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase ${\beta}$-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC $25611^T$. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

• Asian Pacific Journal of Cancer Prevention
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• 제15권16호
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• pp.6911-6917
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• 2014

Background: Hepatocellular carcinoma is a complex and heterogeneous tumor with poor prognosis due to frequent intrahepatic spread and extrahepatic metastasis. The molecular mechanisms underlying HCC pathogenesis still remain obscure. Objectives: We aimed to investigate the abundance and the DNA binding activity of nuclear factor kappa B/p65 subunit in peripheral blood mononuclear cells from patients with HCC and to assess its prognostic significance and association with hypoxia inducible factor one alpha (HIF-$1{\alpha}$) in blood. Subjects and methods: This study was carried out on 40 patients classified equally into liver cirrhosis (group I) and HCC (group II), in addition to 20 healthy volunteers (group III). All groups were subjected to measurement of NF-${\kappa}B$/P65 subunit expression levels by real time-PCR, and DNA binding activity was evaluated by transcription factor binding immunoassay. Serum HIF-$1{\alpha}$ levels were estimated by enzyme-linked immunosorbent assay (ELISA). Significant increase of both the expression level and DNA binding activity of NF-${\kappa}B$/P65 subunit together with serum HIF-1 alpha levels was noted in HCC patients compared to liver cirrhosis and control subjects, with significant positive correlation with parameters for bad prognosis of HCC. In conclusion, NF-${\kappa}B$ signaling is activated in HCC and associated with disease prognosis and with high circulating levels of HIF-1 alpha.

• Molecules and Cells
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• 제20권2호
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• pp.288-296
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• 2005

Cytokines interleukin (IL) 12 and 23 play critical roles in linking innate and adaptive immune responses. They are members of heterodimeric cytokines, sharing a subunit p40. Although IL12/23 p40 is mainly induced in macrophages and dendritic cells (DCs) after stimulation with microbial Toll-like receptor ligands, methods to monitor the cells that produce IL12/23 p40 in vivo are limited. Recently, the mouse model to track p40-expressing cells with fluorescent reporter, yellow fluorescent protein, has been developed. Macrophages and DCs from these mice faithfully reported p40 induction using the fluorescent marker. Here we took advantage of these reporter mice to screen bio-compounds for p40-inducing activity. After screening hundreds of compounds, we found several extracts inducing IL12/23 p40 gene expression. Treatment of DCs with these extracts induced the expression of MHC class II and co-stimulatory molecules, which implies that these might be useful as adjuvants. Next, the in vivo target immune cells of candidate compounds were examined. The reporter system can be useful to identify cells producing IL12 or IL23 in vivo as well as in vitro. Thus, our cytokine reporter system proved to be a valuable reagent for screening for immunostimulatory molecules and identification of target cells in vivo.