• Clinical and Experimental Pediatrics
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• 제51권1호
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• pp.73-77
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• 2008

목 적 : p16 유전자는 염색체 9p21에 위치하는데 종양억제 유전자 중 하나로 cyclin-dependent kinase의 억제제로 작용하며 Rb 인산화를 억제한다. 다양한 종류의 종양에서 p16 유전자의 결실 또는 과메틸화가 발견되고 있는데 이는 급성림프구성백혈병에서도 흔히 발견되는 이상 소견으로 높은 빈도로 나타나고 있지만 급성림프구성백혈병의 예후와 p16 유전자의 연관성에 대해서는 아직 논란의 여지가 있다. 본 연구에서는 면역조직화학염색으로 확인한 p16 단백질의 소실과 급성림프구성백혈병 환아들의 임상 경과와의 연관성에 대하여 조사하고자 하였다. 방 법 : 1998년 1월부터 2006년 12월까지 급성림프구성백혈병으로 진단된 74명의 진단 시 골수 슬라이드에서 p16 단백질 면역조직화학염색을 하였다. 환아들의 임상 양상, 검사실 소견, 치료 후 경과에 대해서 후향적으로 조사하였다. 결 과 : 74명 중 12명에서 p16 단백질이 면역화학염색 결과 음성이었다. 이들 중 남아가 7명 여아가 5명이었으며 진단 시 연령의 중앙값은 5.8(1.3-18.8)세였다. 백혈구 수의 중앙값은 17,225 $(500-403,300)/{\mu}L$ 이었으며 면역표현형은 early pre-B CALLA (+)형이 7명, T 세포형은 5명이었다. 진단 시 예후 중간군이었던 두 명의 환아들에서 골수 재발 하였으며 3명의 환아들이 유방암의 가족력이 있었다. 4명이 사망하여 8년 생존율은 $53.5{\pm}18.7%$였다. 결 론 : p16 단백질의 소실은 소아 급성림프구성백혈병에서 불량한 예후와 연관된 인자로 추정되며 임상에서 진단 시 p16에 대한 유전자 검사뿐만 아니라 단백질에 대해서도 검사가 필요할 것으로 생각된다. 하지만 좀더 많은 환자들에 대한 분석이 더 정확한 연관성을 밝히는데 도움이 될 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• 한국발생생물학회지:발생과생식
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• 제4권2호
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• pp.161-173
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• 2000

컴퓨터 세포동결기를 이용하여 생쥐 배아를 동결할 때 액체질소 (L$N_2$)의 분사속도가 해빙 후 배아의 미세구조, 기능 및 발달에 미치는 영향을 알아보고자 하였다. 이를 위해 배아는 동결을 하지 않은 대조군 (control) 및 동결군에서 L$N_2$의 분사속도에 따라 고속분사군 (120 infusion/min group 1), 저속분사군 (50 infusion/min; group 2)으로 나누었다. ICR 계열의 생쥐의 2 세포기 배아를 사용하였으며, 동결 및 해빙은 저속동결-급속해빙 방법을 사용하였다. 각 군에 따라 해빙 후 배아의 생존율과 세포질이 양호하고 분절화가 없는 2세포기 배아를 대상으로 포배 발달율 및 할구수를 측정하였다. 공초점 현미경을 이용하여 배아 내에서의 $H_2O$$_2$, 활성 미토콘드리아의 분포, 막전위차 및 actin filament를 측정하였으며, TUNEL 방법을 이용하여 DNA 분절화를 확인하였다. 동결-해빙 후 건강한 2 세포기 배아의 회수율은 group 1 (50.7%)에 비해 group 2 (34.6%)에서 현저히 감소했다 (p<0.05). 포배기 배아의 발생율 (86.7%, 76.7% vs. 44.0%)과 할구수 (79.5$\pm$12.9, 71.6$\pm$8.0 vs. 62.5$\pm$4.7)는 대조군 혹은 group 1에 비해 group 2에서 유의한 차이를 보였다 (p<0.05). H$_2$0$_2$의 상대적 강도는 group 2에서 유의하게 증가하였다 (15.3$\pm$3.0, 16.6$\pm$1.6 vs. 23.4$\pm$1.8, p<0.05). 활성 미토콘드리아의 분포는 정상적인 배아에서는 균등하게 분포하는 반면 배발달이 정지된 배아에서는 원형질막 주위에 몰리고 응집된 양상을 보였다. 그러나 대조군, group 1, group 2에서는 모두 균등하게 분포하여 각 군간에 차이가 없었다. 미토콘드리아의 JC-1 염색 결과는 대조군과 group 1의 경우 590 nm의 파장으로 발산되는 미토콘드리아가 group 2에 비하여 유의하게 증가하였다 (17.2$\pm$3.8, 17.4$\pm$1.3 vs. 13.2$\pm$2.0, p<0.05). 2세포기 배아내 미세섬유 (actin filament)는 대조군 및 group 1의 경우 균일하게 분포하는 반면, group 2에서는 부분적인 결손과 응집현상이 관찰되었다. DNA 분절율 (30.8%, 36.0% vs. 65.6%; p<0.05)은 group 2에서 유의하게 증가하였다. 동결시 액체질소의 분사속도는 해빙 후 배아 발달에 매우 중요한 요인으로 작용하며, L$N_2$의 분사속도의 증가는 동결과 정에서 하강 온도의 미세한 변화를 감소시켜 세포내 골격구조와 미토콘드리아의 상해를 감소시켜 $H_2O$$_2$의 발생과 DNA 분절화를 감소시켜 배아 발생을 호전시키는 것으로 사료된다.

• 한국미생물·생명공학회지
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• 제48권2호
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• pp.158-166
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• 2020

Paenibacillus woosongensis의 xylanase 유전자를 클로닝하고 그 염기서열을 결정하였다. Xylanase 유전자는 xyn10A로 명명되었으며, 481 아미노잔기로 구성된 단백질을 코드하는 1,446개 뉴클레오티드로 구성되었다. 추론된 아미노산 배열에 따르면 Xyn10A는 glycosyl hydrolase family 10 xylanase와 상동성이 높은 활성영역과 카르복실 말단에 탄수화물을 결합하는 것으로 추정되는 영역이 포함된 다영역 효소로 확인되었다. DEAE-Sepharose와 Phenyl-Separose 컬럼 크로마토그래피 과정을 통해 P. woosongensis xyn10A 유전자를 함유한 재조합 대장균의 균체 파쇄상등액으로부터 Xyn10A를 정제하였다. 정제된 Xyn10A의 아미노 말단 배열이 GIANGSKF로 결정되었으며 이는 SignalP5.0 server로 예측된 signal peptide의 다음 아미노산 배열과 정확하게 일치하였다. 정제된 Xyn10A는 33 kDa 크기의 절단된 단백질이며 균체내 분해에 의해 카르복시 말단에서 CBM이 제거된 것으로 판단된다. 정제된 효소는 최적 pH와 온도가 6.0과 55-60℃이며 oat spelt xylan에 대한 반응 동력학적 계수 V_max와 K_m이 298.8 U/mg과 2.47 mg/ml로 각각 나타났다. 효소는 birchwood xylan이나 oat spelt xylan보다 arabinoxylan에 대한 활성이 높았으며 para-nitrophenyl-β-xylopyranoside에 대해 낮은 활성을 보였다. Xyn10A의 활성은 Cu²⁺, Mn²⁺과 SDS에 의해서 크게 저해되었으며 K⁺, Ni²⁺과 Ca²⁺에 의해는 상당하게 증진되었다. 또한 이 효소는 xylobiose 보다 중합도가 큰 자일로올리고당을 분해하였으며, 자일로올리고당의 최종 가수분해 산물은 xylose와 xylobiose로 확인되었다.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2006년도 춘계학술대회
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• pp.40-51
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• 2006

To investigate the association between Korean red ginseng (KRG) intake in HIV-1 infected patients and occurrence of grossly deleted nef genes ($g{\Delta}nef$), we characterized nef genes in 10 long-term slow progressors (LTSP) infected with HIV-1 subtype B and 34 control patients. LTSP was defined whose the annual decrease in CD4 T cells was less than $20/{\mu}l$ over 10 years in the absence of antiretroviral therapy. They were treated with KRG for a prolonged period. Nef genes were amplified from peripheral blood mononuclear cells (PBMC) using nested PCR and products were sequenced directly. Patient CD4 T cell counts decreased from $444{\pm}207/{\mu}l$ to $294{\pm}177/{\mu}l$ over $136{\pm}23$ months of KRG intake. This corresponds to an annual decrease in the level of CD4 T cells of $13.3/{\mu}l$. A total of 479 nef genes were amplified from 137 PBMC samples. Nine out of the 10 patients, 47 (34.3%) out of the 137 samples, and 92 out of the 479 genes revealed $g{\Delta}nef$. The deletion extended outside the nef gene in 25 $g{\Delta}nef$ obtained from 6 patients. The proportion of samples with $g{\Delta}nef$ (34.3%) was significantly higher than 4.8% in control patients (P<0.001). In addition, it significantly increased as the duration of KRG intake prolongs (P<0.01). These data suggest the possibility that occurrence of $g{\Delta}nef$ might be associated with long-term intake of KRG.

• Journal of Microbiology and Biotechnology
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• 제24권8호
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• pp.1081-1087
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• 2014

Aspergillus oryzae is generally recognized as safe, but it is closely related to A. flavus in morphology and genetic characteristics. In this study, we tested the aflatoxigenicity and genetic analysis of nine commercial A. oryzae strains that were used in Korean soybean fermented products. Cultural and HPLC analyses showed that none of the commercial strains produced detectable amount of aflatoxins. According to the molecular analysis of 17 genes in the aflatoxin (AF) biosynthetic pathway, the commercial strains could be classified into three groups. The group I strains contained all the 17 AF biosynthetic genes tested in this study; the group II strains deleted nine AF biosynthetic genes and possessed eight genes, including aflG, aflI, aflK, aflL, aflM, aflO, aflP, and aflQ; the group III strains only had six AF biosynthetic genes, including aflG, aflI, aflK, aflO, aflP, and aflQ. With the reverse transcription polymerase chain reaction, the group I A. oryzae strains showed no expression of aflG, aflQ and/or aflM genes, which resulted in the lack of AF-producing ability. Group II and group III strains could not produce AF owing to the deletion of more than half of the AF biosynthetic genes. In addition, the sequence data of polyketide synthase A (pksA) of group I strains of A. oryzae showed that there were three point mutations (two silent mutations and one missense mutation) compared with aflatoxigenic A. flavus used as the positive control in this study.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.768-774
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• 2007

To study the effect of dietary docosahexaenoic acid (DHA) enrichment on the expression of hepatic genes in pigs, weaned, crossbred pigs (30 d old) were fed diets supplemented with either 2% tallow or DHA oil for 18 d. Hepatic mRNA was extracted. Suppression subtractive hybridization was used to explore the hepatic genes that were specifically regulated by dietary DHA enrichment. After subtraction, we observed 288 cDNA fragments differentially expressed in livers from pigs fed either 2% DHA oil or 2% tallow for 18 d. After differential screening, 7 genes were found to be differentially expressed. Serum amyloid A protein 2 (SAA2) was further investigated because of its role in lipid metabolism. Northern analysis indicated that hepatic SAA2 was upregulated by dietary DHA enrichment (p<0.05). In a second experiment, feeding 10% DHA oil for 2d significantly increased the expression of SAA2 (compared to the 10% tallow group; p<0.05). The porcine SAA2 full length cDNA sequence was cloned and the sequence was compared to the human and mouse sequences. The homology of the SAA2 amino acid sequence between pig and human was 73% and between pig and mouse was 62%. There was a considerable difference in SAA2 sequences among these species. Of particular note was a deletion of 8 amino acids, in the pig compared to the human. This fragment is a specific characteristic for the SAA subtype that involved in acute inflammation reaction. Similar to human and mouse, porcine SAA2 was highly expressed in the liver of pigs. It was not detectable in the skeletal muscle, heart muscle, spleen, kidney, lung, and adipose tissue. These data suggest that SAA2 may be involved in mediation of the function of dietary DHA in the liver of the pig, however, the mechanism is not yet clear.

• Journal of Microbiology
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• 제38권2호
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• pp.66-73
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• 2000

To investigate the genetic relationship among 12 species belonging to the Fusarium section Martiella, Dlaminia, Gibbosum, Arthrosporiella, Liseola and Elegans, the internal transcribed spacer(ITS) regions of ribosomal DNA (rDNA) were amplified with primer pITS1 and pITS4 using the polymerase chain reaction(PCR). After the amplified products were digested with 7 restriction enzymes, restriction fragment length polymorphism (RFLP) patterns were analyzed. The partial nucleotide sequences of the ITS region were determined and compared. Little variation was observed in the size of the amplified product having sizes of 550bp or 570bp. Based on the RFLP analysis, the 12 species studied were divided into 5 RFLP types. In particular, strains belonging to the section Martiella were separated into three RFLP types. Interestingly, the RFLP type of F. solani f. sp. piperis was identical with that of isolates belonging to the section Elegans. In the dendrogram derived from RFLP analysis of the ITS region, the Fusarium spp. examined were divided into two major groups. In general, section Martiella excluding F. solani f. sp. piperis showed relatively low similarity with the other section. The dendrogram based on the sequencing analysis of the ITS2 region also gave the same results as that of the RFLP analysis. As expected, 5.8S, a coding region, was highly conserved, whereas the ITS2 region was more variable and informative. The difference in the ITS2 region between the length of F. solani and its formae speciales excluding F. solani f. sp. piperis and that of other species was caused by the insertion/deletion of nucleotides in positions 143-148 and 179-192.

• BMB Reports
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• 제43권5호
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• pp.355-362
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• 2010

The sterile alpha motif (SAM) is a putative protein interaction domain involved in a wide variety of biological processes. Here we report the identification and characterization of a novel gene, SAMD4B, which encodes a putative protein of 694 amino acids with a SAM domain. Northern blot and RT-PCR analysis showed that SAMD4B is widely expressed in human embryonic and adult tissues. Transcriptional activity assays show SAMD4B suppresses transcriptional activity of L8G5-luciferase. Over-expression of SAMD4B in mammalian cells inhibited the transcriptional activities of activator protein-1 (AP-1), p53 and p21, and the inhibitory effects can be relieved by siRNA. Deletion analysis indicates that the SAM domain is the main region for transcriptional suppression. The results suggest that SAMD4B is a widely expressed gene involved in AP-1-, p53- and p21-mediated transcriptional signaling activity.

• Journal of Microbiology and Biotechnology
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• 제34권8호
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• pp.1718-1726
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• 2024

Development of novel antibacterial agents is imperative due to the increasing threat of antibiotic-resistant pathogens. This study aimed to develop the enhanced antibacterial activity and in-vivo efficacy of a novel truncated endolysin, CHAP^SAP26-161, derived from the endolysin Lys^SAP26, against multidrug-resistant bacteria. CHAP^SAP26-161 exhibited higher protein purification efficiency in E. coli and antibacterial activity than Lys^SAP26. Moreover, CHAP^SAP26-161 showed the higher lytic activity against A. baumannii with minimal bactericidal concentrations (MBCs) of 5-10 ㎍/ml, followed by Staphylococcus aureus with MBCs of 10-25 ㎍/ml. Interestingly, CHAP^SAP26-161 could lyse anaerobic bacteria, such as Clostridioides difficile, with MBCs of 25-50 ㎍/ml. At pH 4-8 and temperatures of 4℃-45℃, CHAP^SAP26-161 maintained antibacterial activity without remarkable difference. The lytic activity of CHAP^SAP26-161 was increased with Zn²⁺. In vivo tests demonstrated the therapeutic effects of CHAP^SAP26-161 in murine systemic A. baumannii infection model. In conclusion, CHAP^SAP26-161, a truncated endolysin that retains only the CHAP domain from Lys^SAP26, demonstrated enhanced protein purification efficiency and antibacterial activity compared to Lys^SAP26. It further displayed broad-spectrum antibacterial effects against S. aureus, A. baumannii, and C. difficile. Our in vitro and in-vivo results of CHAP^SAP26-161 highlights its promise as an innovative therapeutic option against those bacteria with multiple antibiotic resistance.