• Asian Pacific Journal of Cancer Prevention
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• 제16권1호
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• pp.367-372
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• 2015

Background: Human adiponectin (ApN), a 30 kDa glycoprotein of 244-amino acids which is predominantly produced by adipocytes, exerts its effects via two receptors, namely adiponectin receptor-1 (adipo-R1) and adiponectin receptor-2 (adipo-R2) with differential binding affinity to globular adiponectin. Adiponectin receptor expression has been studied in several cancer tissues. However, there are no studies of colorectal adenomas which are considered to be precursors for colorectal carcinoma (CRC). Objectives: In the present study, the expression of adipo-R1 and adipo-R2 was investigated immunohistochemically in colorectal adenomas and colorectal carcinoma tissues in an attempt to determine associations with these tumors. Materials and Methods: The study enrolled 50 CRC patients with tumor resection and 82 patients who were diagnosed with adenomatous polyps, classified as negative for neoplasia, low-grade dysplasia (L-GD) or high- grade dysplasia (H-GD). Results: Expression of both adipo-R1 and adipo-R2 was found to be significantly lower in the CRCs than in colorectal adenomas (tubular and tubulovillous, p=0.009 and p<0.001, respectively). Adipo-R1 and adipo-R2 expression was also significantly lower in the CRC group when compared with the groups of patients with low grade dysplasia, high-grade dysplasia or no neoplasia (p=0.012 and p<0.001, respectively). In addition, it was observed that adipo-R2 expression was generally positive in the non-neoplastic group irrespective of the adipo-R2 expression. In the L-GD, H-GD and CRC groups, the adipo-R2 result was positive whenever adipo-R1 result was positive but some patients with negative adipo-R1 had positive adipo-R2 (p<0.001, p=0.004, p<0.001, respectively). Conclusions: This study indicated that ApN may play a role in the progression of colorectal adenomatous polyps to carcinoma through actions on adipo-R1 and adipo-R2 receptors.

• Clinical and Experimental Reproductive Medicine
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• 제10권2호
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• pp.1-11
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• 1983

황소의 정액에서 베타 굴룩유로니다아제를 부분적으로 정제하였다. 이 정제과정에는 $(NH_4)_2SO_4$ 의 분획분리법, 두개의 연속적인 DEAE-셀루로오즈 콜럼 및 등전초점화법(pH4-6) 및 세파덱스 G-200의 절여과 방법이 쓰여졌다. 등전초점화(lsoelectric focusing)법을 사용했을때 pH5.13에서 베타 굴룩유로니다아제는 한 형태의 단백질로 존재하였다. 고도로 정제된 베타 굴룩유로니다아제는 전기영동법에 의해 한개의 주된 띠와 약간의 불순물의 띠로 나타났고 특수활동도는 34Units/mg 단백질로 나타났다. 이 효소는 pH 5.2 와 $48^{\circ}C$ 에서 가장 높은 활동도를 나타냈다. 알부민이나 0.15M 소금용액에서 베타 굴룩유로니다아제는 활동도가 상승됐다. 페놀프타레인-모노-베타-굴룩 유로닉산을 기질로 사용했을때 km값은 2.9mM 이었고 Vmax값은 $0.8{\mu}$mole/min 이었다. 대두의 콘카나발린 A에 흡착하는 것으로 보아 이 효소는 당단백질임이 확인됐다. 토끼, 사람의 정자 아크롬좀 추출물 및 정액에서 이 효소는 높은 활성도를 나타냈다.

• BMB Reports
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• 제39권4호
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• pp.432-440
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• 2006

A tuber lectin from Arisaema jacquemontii Blume belonging to family Araceae was purified by employing a single step affinity chromatography using column of asialofetuin-linked amino activated silica beads and the bound lectin was eluted with 100 mM glycine-HCl buffer pH 2.5. The purified A. jacquemontii lectin (AJL) showed a single protein band with an apparent molecular mass of 13.4 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing as well as non-reducing conditions. The native molecular mass of AJL determined by gel filtration on a Biogel P-200 column was 52 kDa and its carbohydrate content was estimated to be 3.40%. Thus AJL is a tetrameric glycoprotein. The purified lectin agglutinated erythrocytes from rabbit but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested except N-acetyl-D-lactosamine having minimal inhibitory sugar concentration (MIC) 25 mM. Among the glycoproteins tested only asialofetuin was found to be inhibitory (MIC $125\;{\mu}g/mL$). A single band was obtained in native PAGE at pH 4.5 while PAGE at pH 8.3 showed two bands. Isoelectric focusing of AJL gave multiple bands in the pI range of 4.6-5.5. When incorporated in artificial diet AJL significantly affected the development of Bactrocera cucurbitae (Coquillett) larvae indicating the possibility of using this lectin in a biotechnological strategy for insect management of cucurbits. Larvae fed on artificial diet containing sub-lethal dose of AJL showed a significant decrease in acid phosphatase and alkaline phosphatase activity while esterase activity markedly increased as compared to larvae fed on diet without lectin. Out of various human cancer cell lines employed in sulphorhodamine B (SRB) assay, this lectin was found to have appreciable inhibitory effect on the in vitro proliferation of HCT-15, HOP-62, SW-620, HT-29, IMR-32, SKOV-3, Colo-205, PC-3, HEP-2 and A-549 cancer cell lines by 82, 77, 73, 70, 41, 41, 37, 29, 21 and 21% respectively.

• Applied Biological Chemistry
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• 제42권4호
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• pp.304-309
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• 1999

식용버섯인 팽이버섯(Flammulina velutipes)으로부터 적혈구 응집능력을 갖는 렉틴(flammulina velutipes lectin, FVL) FVL-1과 FVL-2을 정제하였다. FVL-1과 FVL-2의 분자량은 각각 10.6 kDa과 37 kDa으로 추정되었다. FVL-2는 사람적혈구의 경우 모든 혈액형에서 응집현상을 나타내었으나, FVL-1은 O형의 적혈구에 대하여 높은 상대활성도를 나타내었다. 렉틴의 hemagglutination inhibition test 결과 FVL-1과 FVL-2는 fetuin, bovine submaxillary mucin, asialofetuin, porcine stomach mucin에 의해 적혈구 응집력이 저해되었다. FVL-1은 1.56 mM $Cu^{2+}$에서 적혈구 응집력이 저해되었으며, $Mg^{2+}$, $Mn^{2+}$, $Ca^{2+}$, $Fe^{2+}$ 등에서도 저해되었다. 반면 FVL-2는 금속 이온에 대한 저해가 나타나지 않았다. pH 안정성에 있어서 FVL-1은 pH $5{\sim}11$에서 안정하였으며, FVL-2는 pH 4까지의 범위에서 안정하였다. 온도 안정성에 있어서는 FVL-1과 FVL-2 각각 $55^{\circ}C$, $45^{\circ}C$까지 안정하였다. FVL-1은 단백질이었으며 FVL-2는 당 단백질이었다.

• KSBB Journal
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• 제14권5호
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• pp.578-584
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• 1999

겨우살이가 가지고 있는 lectin의 약리작용의 가능성에 대한 연구의 일환으로, lectin을 분리 . 정제하였으며, 분자량, subunit수, 탄수화물의 조성, 단백질의 아미노산 조성, 적혈구 응집력, 탄수화물 특이성, pH 및 열 안정성등의 생화학적 특성을 연구하였다. Lectin의 분리는 0.15 M NaCl에 의한 추출, $(NH_4)_2SO_4$ 침전, sepharose 4B affinity chromatography 및 sephadex G-150 gel filtration에 의해 11.64배 정제에 0.14%의 수율을 획득하였으며, HPLC와 전기영동을 이용하여 순도를 검정하였다. Gel filtration에 의해 분석된 lectin의 분자량은 124,000 Da이였고, SDS-PAGE에 의해 분자량이 30,000과 32,000 Da인 2개의 band로 나타나서 각각 2개의 subunit를 갖는 tetramer로 확인되었다. 겨우살이 lectin의 탄수화물은 glucose, fructose, arabinose 및 xylose를 함유하고 있다. 또한 phenlylalanine, lysine, tyrosine, aspartic acid가 비교적 풍부하였고 methionine, alanine, arginine은 비교적 적었다. Lectin은 적혈구에 종 특이성을 나타내는데 겨우살이의 혈구응집반응에서는 사람혈액형간의 차이는 없었으며, 쥐에서 가장 높았고 소에서 가장 낮았으므로 동물 종간에 대한 특이성은 뚜렸하였다. Lectin은 당 특이성을 가지는데, D-galactose, D-mannose, D-lactose 및 D-raffinose에 대해 특이성을 나타내었다. 겨우살이 lectin은 pH4~7에서 안정하였으며, 5$0^{\circ}C$까지는 10~30분 열처리해도 활성이 유지되는 열 안정성 단백질이었다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.5929-5934
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• 2013

Background: Primary brain tumors constitute a small percent of all malignant cancers, but their etiology remains poorly understood. ${\beta}3$ integrin (ITGB3) has been recognized to play influential roles in angiogenesis, tumor growth and metastasis. Intercellular adhesion molecule-1 (ICAM-1) is a surface glycoprotein important for tumor invasion and angiogenesis. The aim of this study was to investigate whether specific genetic polymorphisms of ICAM-1 and ITGB3 could be associated with brain cancer development and progression in a Turkish population. Our study is the first to our knowledge to investigate the relationship between brain tumor risk and ICAM-1 and ${\beta}3$ integrin gene polymorphisms. Materials and Methods: The study covered 92 patients with primary brain tumors and 92 age-matched healthy control subjects. Evaluation of ${\beta}3$ integrin (Leu33Pro (rs5918)) and ICAM-1 (R241G (rs1799969) and K469E (rs5498)) gene polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: According to results of our research, the A allele of the ICAM-1 R241G gene polymorphism appeared to be a risk factor for primary brain tumors (p<0.001). Similarly, the frequency of the A mutant allele of ICAM-1 R241G was statistically significant in patients with brain tumors classified as glioma (p<0.001). When allele and genotype distributions of ICAM-1 K469E, ICAM-1 R241G and ${\beta}3$ integrin Leu33Pro gene polymorphisms were evaluated with age, sex, and smoking, there were no statistically significant differences. Haplotype analysis revealed that the frequencies of GAC (rs1799969-rs5498-rs5918) and GAT (rs1799969-rs5498-rs5918) haplotypes were significantly lower in patients as compared with controls (p=0.001; p=0.036 respectively). Conclusions: This study provides the first evidence that ICAM-1 R241G SNP significantly contributes to the risk of primary brain tumors in a Turkish population. In addition, our results suggest that ICAM-1 R241G in combination ICAM-1 K469E may have protective effects against the development of brain cancer.

• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1326-1334
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• 2008

The prime-boost vaccination with DNA vaccine and recombinant viral vector has emerged as an effective prophylactic strategy to control infectious diseases. Here, we compared the protective immunities induced by multiple alternating immunizations with DNA vaccine (pCIgB) and replication-incompetent adenovirus (Ad-gB) expressing glycoprotein gB of pseudorabies virus (PrV). The platform of pCIgB-prime and Ad-gB-boost induced the most effective immune responses and provided protection against virulent PrV infection. However, priming with pCIgB prior to vaccinating animals by the DNA vaccine-prime and Ad-boost protocol provided neither effective immune responses nor protection against PrV. Similarly, boosting with Ad-gB following immunization with DNA vaccine-prime and Ad-boost showed no significant responses. Moreover, whereas the administration of Ad-gB for primary immunization induced Th2-type-biased immunity, priming with pCIgB induced Th1-type-biased immunity, as judged by the production of PrV-specific IgG isotypes and cytokine IFN-$\gamma$. These results indicate that the order and injection frequency of vaccine vehicles used for heterologous prime-boost vaccination affect the magnitude and nature of the immunity. Therefore, our demonstration implies that the prime-boost protocol should be carefully considered and selected to induce the desired immune responses.

• 한국잠사곤충학회지
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• 제49권2호
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• pp.37-42
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• 2007

락토페린 cDNA 유전자를 도입시킨 누에 형질전환 실험을 수행한 결과, 다음과 같은 결과를 얻을 수 있었다. 1. 사람 GI-101 세포주의 mRNA로부터 클로닝 된 락토페린 cDNA 유전자의 개시코돈 ATG와 종결코돈 TAA를 포함하는 open reading frame(2,136 bp) 영역을 확인하였다. 2. Sf9 배양세포의 조추출물 시료에 의한 Western blot 분석 결과, 락토페린으로 추정되는 약 80kDa의 단백질 발현을 확인하였다. 3. 누에 형질전환에 높은 전이효율과 활성을 나타내는 트랜스포존을 이용한 전이벡터 pPIGA3GFP를 개조하여 락토페린 cDNA를 삽입시킨 전이벡터 pPT-HLf를 구축하였다. 4. DNA 미량 주사법에 의한 누에 형질전환 개체의 발현 비율은 약 6.7% 정도를 나타냈다. 5. 형질전환 누에(G0) 동일한 세대간 교배 및 처리하지 않은 성충간의 역교배에 의한 차세대(G1) 개체로부터 락토페린 유전자와 동일한 크기의 2.1 kb DNA 단편을 확인 할 수 있었으며, 형질전환 G1 세대의 조추출물 시료에 의한 Western blot 분석 결과, 표준 락토페린 항체와 반응하는 약 80 kDa의 단백질 발현을 확인할 수 있었다.

• 생명과학회지
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• 제28권4호
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• pp.389-397
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• 2018

당 단백질에 붙어 있는 당사슬 구조는 형질전환 돼지 유즙으로 분비되는 의약용 단백질의 생물학적 활성, 안정성 그리고 안전성에 영향을 줄 수 있다. 형질전환 동물을 이용한 치료용 당 단백질 생산은 유선 세포에서 이루어지는 당사슬 부가능력에 의해 제한되며, 균일한 당사슬 형태를 가지는 당 단백질 생산은 도전 과제로 남아있다. ${\beta}$-1,3-N-acetylglucosaminylatransferase1 (B3GNT1) 유전자는 N-아세틸글루코사민에 갈락토오스 잔기를 부착시키는 단백질 당화기작에 중요한 효소이지만, 돼지 당 전이효소에 대한 정보는 매우 제한적이다. 따라서, 돼지 B3GNT1 (pB3GNT1) 유전자를 클로닝하고 N-아세틸글루코사민에 갈락토오스 잔기를 부착시키는 기능적 특성을 조사하였다. 몇가지 다른 프라이머를 사용하여 전체 전사영역(ORF)을 함유하는 부분적인 pB3GNT1 mRNA 염기서열을 간 조직으로부터 분리하였다. 클로닝 된 pB3GNT1의 ORF는 1,248개의 뉴클레오티드를 가지며, 415개 아미노산 잔기로 구성되어 있었다. pB3GNT1 유전자의 장기별 발현특성은 성돈 및 자돈의 여러 기관에서 분석하였다. pB3GNT1 mRNA 발현 수준은 심장, 소장 보다는 근육에서 높았지만 폐에서는 낮았다. pB3GNT1의 기능적 특성 분석을 위해 돼지 신장 세포주(PK-15)에서 pB3GNT1 유전자의 안정적인 발현을 확립하였다. 그 결과, PK-15 세포에서 pB3GNT1 발현에 의한 당화 패턴은 총 시알산 증가에는 영향을 미치지 않지만, poly-N-아세틸글루코사민은 증가하는 것으로 나타났다. 본 연구는 생물반응기로 형질전환 돼지를 이용할 때 희망하는 당사슬을 부가하여 치료 가능성을 높이며 개선된 활성을 나타내는 당단백질 생산에 도움이 될 것이다.

• IMMUNE NETWORK
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• 제9권4호
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• pp.138-146
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• 2009

Background: The MHC region of the chromosome contains a lot of genes involved in immune responses. Here we have investigated the mouse NG29/Cd320 gene in the centrometrically extended MHC region of chromosome 17. Methods: We cloned the NG29 gene by RT-PCR and confirmed the tissue distribution of its gene expression by northern blot hybridization. We generated the NG29 gene expression constructs and polyclonal antibody against the NG29 protein to perform the immunofluorescence, immunoprecipitation and flow cytometric analysis. Results: The murine NG29 gene and its human homologue, the CD320/8D6 gene, were similar in the gene structure and tissue expression patterns. We cloned the NG29 gene and confirmed its expression in plasma membrane and intracellular compartments by transfecting its expresssion constructs into HEK 293T cells. The immunoprecipitation studies with rabbit polyclonal antibody raised against the NG29-NusA fusion protein indicated that NG29 protein was a glycoprotein of about 45 kDa size. A flow cytometric analysis also showed the NG29 expression on the surface of Raw 264.7 macrophage cell line. Conclusion: These findings suggested that NG29 gene in mouse extended MHC class II region was the orthologue of human CD320 gene even though human CD320/8D6 gene was located in non-MHC region, chromosome 19p13.