• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2004년도 제4회 발생공학 국제심포지움 및 학술대회
• /
• pp.106-107
• /
• 2004

• 한국당과학회:학술대회논문집
• /
• 한국당과학회 2006년도 제1회 연례학술대회
• /
• pp.56-56
• /
• 2006

• Journal of Microbiology and Biotechnology
• /
• 제15권4호
• /
• pp.722-727
• /
• 2005

Cytosolic sialidase (Neu2), a member of the sialidase family that is responsible for hydrolysis of sialic acid from the terminal position of sialoglycoconjugates, is poorly expressed in skeletal muscle and not detected in any other adult tissues. Thus, we isolated Neu2 cDNA using splicing by overlap extension (SOEing). In order to further characterize this enzyme, a His-tagged derivative was expressed in the bacterial expression system and purified by $Ni^{2+}$-affinity chromatography. A recombinant product of approximately 42 kDa had sialidase activity toward 4-methyl-umbelliferyl-$\alpha$-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH and temperature of the recombinant Neu2 for 4MU-NeuAc was 6.0 and $37.5^{\circ}C$, respectively. The metal ions, such as $Cu^{2+}\;and\;Cd^{2+}$, showed strong inhibitory effect on the activity of the enzyme. The enzyme efficiently hydrolyzed the gangliosides GM3 and GD3 and had relatively low activities on ganglioside GD1a and GD1b, $\alpha$2-3 sialyllactose, and sialylated glycoproteins such as fetuin, transferrin, and orsomucoid, but had hardly any activities on $\alpha$2-6 sialyllactose and ganglioside GM1 and GM2. We concluded that the recombinant Neu2 has a sialidase activity toward glycoproteins as well as gangliosides.

• Journal of Periodontal and Implant Science
• /
• 제39권sup2호
• /
• pp.231-237
• /
• 2009

Purpose: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite $AB_2$ toxin (CdtB: the enzymatic A subunit; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for $GM_1$ ganglioside. Methods: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to $GM_1$ ganglioside was checked through ELISA. Results: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to $GM_1$ ganglioside, while CdtA alone did not. Conclusions: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.

• Journal of Microbiology and Biotechnology
• /
• 제19권1호
• /
• pp.108-112
• /
• 2009

A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-${\mu}m$ crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-${\mu}m$ permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH 7.0, containing 1.0 M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of $3.1\;EU/{\mu}g$ of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a $G_{M1}$ ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The $G_{M1}$ ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

• 한국동물학회지
• /
• 제39권4호
• /
• pp.410-418
• /
• 1996

Gangiloside는 포유동물세포에 편재하는 막성분으로서, 이들은 세포상호간의 접착, 분화 및 정보전달과정에 과여하는 것으로 알려지고 있다. Rat난소는 주요한 Gangiloside로서 GM3를 함유하고 있으며, 본 연구에서는 폐쇄난포에서 이들의 분포여부와 난포의 발달 과정에서의 변화여부를 조사하기 위하여, Rat 난소의 동결절편을 이용해 GM3를 포함 11종류의 Gangilo-series Gangiloside에 대해 특이한 단일항체로서 염색시켰다. 폐쇄난포들에서 GM3는 난포발달이 진행되는 동안 시간적, 공간적으로 서로 다른 양식으로 발현하였다. 그러나 GM1을 포함한 다른 종류의 Gangiloside들은 면역조직화학적으로 검출되지 않았다. 일차난포에서 관찰되는 폐쇄난포들에서 GM3는 모든 교막세포와 난자에 인접한 과립막세포의 일부에서 발현하였다. 이차난포의 시기에서 이들 폐쇄난포의 GM3는 모든 교막세포와 과립막세포들에서 발현하였다. 이어서 발달하고 있는 그라프난포의 시기에서 관찰되는 폐쇄난포의 GM3발현은 이차난포에서의 분포패턴과 유사함을 보여 주었다.

• BMB Reports
• /
• 제44권12호
• /
• pp.799-804
• /
• 2011

Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.

• 한국동물학회지
• /
• 제33권1호
• /
• pp.108-118
• /
• 1990

혈청을 비롯해서 extracellular matrix에 존재하는 당단백질인 fibronectin은 근세포의 융합과 밀접한 관계가 있는 것으로 알려져 있다. 본 연구실에서는 최근에 근세포가 분화하는 동안에 fibronectin의 수준이 감소되며, 이러한 감소는 fibronectin의 28 kDa fragment에 대한 수용체으 유용성이 감소하는 결과로 밝혀낸 바 있다. 본 연구에서는 근세포으 융합을 억제하는 물질로 알려진 EGTA를 이용하여 근세포의 융합과 28 kDa fragmen receptor의 관계를 검토하여 보았다.EGTA를 처리한 경우 EGTA를 처리하지 않은 근세포에 비해서 fibronectin의 수준과 28 kDa fragmen binding이 훨씬 적게 감소하였으며, 융합이 봉쇄된 근세포에서 EGTA를 제거하여 융합을 재개시키면 fibronectin의 수준과 28 kDa fragmen의 binding이 정상 근세포 수준으로 환원되었다. 이상의 실험 결과로 볼 때 28 kDa fragmen에 대한 수용체의 감소 또는 변화가 근세포의 분화과정에서 일어나는 fibronectin 수준의 감소와 연관성이 있음을 알 수 있다. 한편, 배양액 내에 trypsin을 처리한 경우에는 처리하지 않은 경우에 비해서 28 kDa fragmen의 binding이 현저하게 감소되었고, gangliosides를 처리한 상태에서는 gangliosides의 농도에 정비례해서 28 kDa fragmen의 binding이 감소되었다. 이 밖에 gel overlay technique을 이용하여 28 kDa fragmen가 SDS-PAGE gel에서 분자량이 약 43kDa인 단백질 및 gangliosides와 binding하는 사실을 알 수 있었다. 이러한 실험 결과를 종합하여 볼 때, 근세포의 융합은 28 kDa fragmen에 대한 receptor의 감소와 관계가 있으며, 그 수용체는 gangliosides와 비슷한 당을 가지고 있는 당단백질일 것으로 추정된다.

• 한국당과학회:학술대회논문집
• /
• 한국당과학회 2006년도 제1회 연례학술대회
• /
• pp.28-29
• /
• 2006

• Asian Pacific Journal of Cancer Prevention
• /
• 제17권4호
• /
• pp.1643-1647
• /
• 2016

Since their discovery in 1940, it has been well established that gangliosides are associated with a number of biological pathways and cellular processes such as growth, differentiation and toxin uptake. Gangliosides are glycosphingolipids containing neuraminic acid which are expressed on the plasma membrane of cells particularly in the nervous system. Heterogeneity and structural variation in the carbohydrate chains of gangliosides contributes to unique features of each of these molecules. Thirty five years ago it was discovered that aberrant glycosylation occurs in a variety of human cancers, including aberrant glycosylation of gangliosides. Ganglioside expression in terms of quality and quantity varies in different cancers and different roles may be played. Gangliosides, by affecting the immune system, including esxpression of cytokines and adhesion molecules, may inhibit anti-tumor mechanisms, as well as having direct impact on angiogenesis, cell movement and metastasis. It should be noted that different kinds of gangliosides do not all act by the same mechanisms.