• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.191-195
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• 2003

The in vitro glycosylation of pentapeptide (Arg-Lys-Asp-Val-Tyr; RKDVY) and RNase A was carried out using PNGase F (peptide-N-glycosidase F), and the results were analyzed using MALDI-TOF-MS. Aminated N,N-diretyl chitobiose was used as the sugar in the glycosylation reaction, and the amination yield of N,N'-diacetyl chitobiose was about $60\%$. To reduce the water activity and shift the reaction equilibrium to a reverse reaction, 1,4-dioxane or ethylene glycol was used as the organic solvent in the enzymatic glycosylation. A certain extent of nonenzymatic glycosylaton, known as the Maillard reaction, was also observed, which occurs on an arginine or lysine residue when the length of tie sugar residue is one or two. However, the extent of glycosylation was much higher in the enzymatic reaction, indicating that PNGase F can be effectively used to produce glycopeptides and glycoproteins in vitro.

• Applied Biological Chemistry
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• 제24권4호
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• pp.252-259
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• 1981

Bacillus amyloliquefaciens K의 생육(生育)과 ${\alpha}-amylase$ 생산(生産)에 대한 Tunicamycin (TM)의 영향(影響)을 검토(檢討)한 결과(結果) 최소생육저지농도(最少生育沮止濃度)는 $0.25{\mu}\textrm{g}/m\ell$이고 생산(生産)된 ${\alpha}-amylase$의 내열성(耐熱性)은 $50^{\circ}C$이었다. TM 첨가량(添加量)이 $1{\mu}\textrm{g}/m\ell$인 액체배지(液體培地)에서 10시간(時間) 진탕배양(振湯培養)할 경우 균(菌)의 형태(形態)는 간형(桿形)에서 부정형(不定形)의 구형(球形)으로 변형(變形)되었으며 24시간배양(時間培養)으로 용해(溶解)되었다. TM와 자외선의 처리로 분리(分離)한 his-, $TM^{\tau}$ 변이주(變異株)의 생육(生育)은 친주(親株)와 같은 경향(傾向) 이었으나 ${\alpha}-amylase$, protease, 및 RNase의 생산량(生産量)은 떨어지는 경향(傾向)이다.

• 한국미생물·생명공학회지
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• 제30권1호
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• pp.68-72
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• 2002

RNA 함량이 증가되고, 증식속도가 더 빠른 효모 변이주를 선별하기 위해, 모균주 Saccharomyces cerevisiae MTY62 세포에 화학적 돌연변이제인 ethylmethane sulfonate를 처리하여, YPD 배지에서는 잘 자라고 KCl 함유 배지에서는 자라지 않는 변이주들을 선별하였다. 이 변이주들 중 시험관 및 플라스크 배양을 통해 균체농도와 RNA 함량이 모균주 MTY62에 비해 각각 1.5배, 1.3배 증가한 M40-10 변이주를 최종적으로 선별하였다. 변이주 M40-l0을 발효조 회분배양한 결과, 최대비증식속도는 $0.38 h^{-1}$ , RNA 농도는 3210 mg-RNA/1, RNA 함량은 183mg-RNA/g-DCW 값을 보여, 모균주에 비해 각각 23%, 15%, 12%씩 증가하였다. M40-10 변이주를 간헐적 유가배양한 결과, 최대 균체농도는 35.6 g-DCW/1을, 최대 RNA 농도는 5677mg-RNA/l을, RNA함량은 160 mg-RNA/g-DCW을 나타내어 모균주보다 우수하였다. 일정속도의 유가배양에서도 M40-10 변이주의 최대 균체농도는 46.4g-DCW/1, RNA 농도는 6270mg-RNA/1, RNA 함량은 135mg-RNA/g-DCW을 보였다. 이들 유가배양에서 배양 중반기인 20시간 전후에서는 모균주에 비해 변이주의 세포농도는 30%, RNA 농도는 10% 정도 증가되었다. 또한 유가배양 말기까지도 RNA 분해는 거의 일어나지 않아, M40-10 변이주는 산성 RNase 활성이 크게 감소한 변이주임을 알 수 있었다.

• 한국식물병리학회지
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• 제10권3호
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• pp.228-234
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• 1994

Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.

• 한국균학회지
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• 제20권3호
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• pp.234-239
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• 1992

Virus 이병(罹病) 느타리버섯 (Pleurotus spp.)으로부터 이중나선(二重螺腺) ribo 핵산(核酸 )(ds RNA)을 분리(分離)하였다. Ds RNA 는 8100 base pairs(bp)의 큰 band 와 2170, 2120, 1980 및 1840 bp의 4개 작은 band로 이루어졌다. Ds RNA 분석법(分析法)으로 느타리버섯의 Virus 이병여부(罹病與否)를 조사(調査)한 결과(結果) 균사생장(菌絲生長)이 부진(不振)하고 세균성(細菌性) 갈색(褐色) 부패병(腐敗病) 등(等)에 이병(罹病)되고 이상자실체(異常子實體)를 형성(形成)하는 느타리버섯으로부터 Virus 이병(罹病)을 확인(確因)하였으나 건전(健全)버섯으로부터는 ds RNA를 분리(分離)하지 못하였다. 이 병(病)은 해외(海外)로부터 전래(傳來)한 것으로 보인다. Ds RNA 는 저농도염류액(底濃度鹽類液) (0.1XSSC)에서 RNase A 에 용해(鎔解)되었으며 $85^{\circ}C$ 에서 특성변화(特性變化)가 발생(發生)하였다.

• 약학회지
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• 제31권2호
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• pp.116-125
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• 1987

It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of MRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5~4.0 kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4$\times$$10^5$~$10^{6}$ per $\mu\textrm{g}$ of rat liver poly (A$^+$)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.

• 약학회지
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• 제37권2호
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• pp.149-157
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• 1993

Protein methylase inhibitor which is a modulator of biological methylation has been purified and characterized from porcine liver soluble fraction by cell fractionation, Sephadex G25 chromatography, reverse phase HPLC, size exclusion HPLC. The results are summarized as follows. 1) The purified inhibitor shows apparent homogeneity, as judged by HPLC. 2) A molecular weight of the purified inhibitor which is composed of 18 amino acid residues is about 1,400 daltons. 3) A single absorption peak of ultraviolet spectrum was observed at 260nm. 4) The inhibitor was not inactivated by heating at $100^{\circ}C$ until 60min. and its activity was not influenced by treatment with digestive enzymes, such as trypsin, pepsin, pronase, chymotrypin, lysozyme, DNase, and RNase. 5) The purified inhibitor inhibited protein rnethylase I, II, III and phospholipid methyltransferase activities. 6) The purified inhibitor inhibited noncompetitively protein methylase II from porcine liver, spleen, and testis. 7) The $K_{i}$ values for protein methylase II from porcine liver, spleen, and testis were 300nM, 250nM, 297nM, respectively.

• 한국식품영양학회지
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• 제15권2호
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• pp.104-111
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• 2002

호열균 B. stearotheymophilus으로부터 단백질 고차구조 형성을 촉진하는 내열성 PPIase를 정제하기 위해, 이 균체를 대량으로 배양 집균, 파쇄하여 효소활성을 측정하였다. 효소의 활성측정은 N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(pAN)를 기질로 사용하였다. chymotrypsin은 기질 이성체(cis-trans 형)의 한쪽(trans)만을 특이적으로 분해하는 반응을 이용하여 PPIase 활성을 측정하였다. 호열균 추출시료로 부터 효소활성을 확인한 후, DEAE-sepharose CL-6B, Sephadex G-75로 정제 후, 최종적으로 Superose TM-12 (FPLC) gel-필트레이션으로 분자량 18kDa의 본 효소를 정제하였다. 정제한 효소의 화학적 특징을 조사한 결과 pH 7.5~8.0사이에 안정하였으며, 최적 pH는 8.0으로 나타났다. 그리고 $65^{\circ}C$에 30분간 열처리 후, 효소활성을 측정한 결과 50%이상의 잔존 활성을 갖는 내열성 효소임을 확인하였다. 정제 단백질의 N-말단 아미노산 분석은 Edman 분해법으로 39 아미노산 잔기를 결정하였다. 그리고 PPIase의 재구성(refolding) 반응은, 요소로 변성시킨 기질 RNase 1을 이용하여 이 단백질의 재구성 (refolding) 실험을 조사한 결과, PPIase는 변성 기질 RNase 1의 재구성(refolding)을 촉진하는데 높은 효과를 가지고 있었다. 호열균 유전자 라이브러리로부터 PPIase 유전자 약 3kb을 클로닝하였다. 재조합 플라스미드 cPI-40에서 프라이머(A-1, B-2)를 이용하여 PPIase N-말단을 코드하는 유전자를 PCR법으로 증폭하여, 염기배열을 결정한 결과 증폭된 단편은 165염기로 형성된 55 아미노산 잔기를 코드하는 open reading frame(ORF)가 연속되고 있었다. 그리고 Edman법으로 결정한 PPIase의 39아미노산 잔기가 이 배열내에 완전히 보존되어 있었다. 이 결과로부터 이 ORF는PPIase구조 유전자의 1/3에 해당하는 단편임을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제17권1호
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• pp.315-321
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• 2016

Background: Herbal medicine has becoming a potential source of treatment for different types of cancer including breast cancer. It has been shown that plants from the family Rhamnaceae possess anticancer activity. Objective: In this study, we determined the antiproliferative influence of Ziziphus spina christi- a species from this family- on the MCF-7 (human breast adenocarcinoma) cell line. Materials and Methods: The cytotoxicity of the total extract, ethanol, ethanol-aqueous (1:1) as well as aqueous fractions of Ziziphus spina christi leaves was evaluated through MTT assay against MCF-7 cell line. Cell cycle inhibition and apoptosis induction were assessed by flowcytometry cycle RNase/PI analysis and Annexin V-FLUOS, respectively. Apoptosis was also analyzed by immunoblotting assay. Results: Our results indicated that the ethanolic fraction had the lowest $IC_{50}$ value (0.02 mg/ml), induced cell cycle arrest at the G1/S phase as well as apoptosis after a 48h of treatment. Conclusions: This is the first report on anticancer effect of Ziziphus spina christi ethanolic fraction on breast cancer cells, providing a scientific basis for its utility in traditional medicine. However, further in-depth studies are needed to confirm the precise mechanisms.

• The Korean Journal of Physiology and Pharmacology
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• 제11권1호
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• pp.9-13
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• 2007

Antioxidant properties have been proposed as a mechanism for the putative anti-inflammatory effects of phenolic compounds. To reveal the relationship between antioxidant activity and anti-inflammatory effects of various antioxidants, we measured 1, 1-diphenyl-2-picryhydrazyl(DPPH)-reducing activity and examined the inhibitory effects on LPS-induced inflammation-related gene expression in the BV2 microglial cell line. Lipopolysaccharide(LPS)(0.2 ${\mu}g/ml$) was used with or without antioxidants to treat cells, and the regulation of iNOS and cytokine gene expression was monitored using an RNase protection assay(RPA). Although, all tested antioxidants had similar DPPH-reducing activity and inhibited nitrite production, but the curcuminoid antioxidants(ferulic acid, caffeic acid, and curcumin) inhibited LPS-induced gene expression(iNOS, $TNF-\alpha,\;IL-1{\beta}$, IL-6, and IL-1 Ra) in a concentration-dependent manner. Other tested antioxidants did not exhibit the same effects; N-acetylcysteine(NAC) only began to suppress $IL-1{\beta}$ gene expression just below the concentration at which cytotoxicity occurred. Moreover, the antioxidant potency of curcuminoids appeared to have no correlation with anti-inflammatory potency. Only curcumin could inhibit LPS-induced microglial activation at a micromolar level. These data suggest that curcumin may be a safe antioxidant possessing anti-inflammatory activity.