• Asian-Australasian Journal of Animal Sciences
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• 제13권9호
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• pp.1201-1204
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• 2000

We examined expression patterns of imc-415 gene in mammary gland and in HC11 mammary epithelial cells in culture. mRNA levels of imc-415 gene were higher at pregnancy and involution stages of mouse mammary gland compared with lactation period. Expression of imc-415 gene was induced with serum starvation or treatment with Fas monoclonal antibody in HC11 mammary epithelial cells in culture.

• Fisheries and Aquatic Sciences
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• 제7권3호
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• pp.99-108
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• 2004

Vitellogenin (VTG) was purified from serum of $estradiol-l7{\beta}-treated rockfish$(Sebastes schlegeli) by precipitation with $EDTA-Mg^{2+}$ and ammonium sulfate and two step chromatography (anion exchange chromatography and gel permeation chromatography) was performed on FPLC system. Rockfish VTG (rfVTG) was characterized and its properties were determined. The monomers have apparent, molecular mass of about 188 kDa as indicated by SDS-PAGE. Amino acid composition analysis of rfVTG was similar to VTG from other oviparous teleosts. Cysteine and lysine were present at relatively high level. Leucine was present at relatively lower level than in other species. The N-terminal amino acid sequence was evaluated to identify rfVTG. Western blot analysis using an antibody against the purified VTG showed that the antibody reacted with both plasma of $estradiol-l7{\beta}-treated rockfish$ treated male and purified VTG, whereas there was no reaction with male serum of the control. An ELISA was developed using monoclonal and polyclonal antibodies against rfVTG. The assay range was 3.2 ng/mL and 1,000 ng/mL and the value of the intra and inter assay variations were within $9.7{\%}$ and $11.2{\%}$, respectively. Recovery rate was $96.8{\%}$. The sandwich ELISA could be useful for the detection of VTG and could be good for screening of estrogenic compounds.

• Fisheries and Aquatic Sciences
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• 제8권4호
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• pp.213-219
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• 2005

Vitellogenin (VTG) was purified from the blood plasma of estradiol-17$\beta$ ($E_2$)-treated male marbled sole (Limanda yokohamae) using gel filtration and anion exchange chromatography. The purity of the marbled sole VTG (msVTG) was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing. The purified msVTG was used to produce monoclonal and polyclonal antibodies in mice and rabbits, respectively, and the specificity of the polyclonal antisera for msVTG was confirmed by Western blot analysis. The antibodies cross­reacted with a protein of molecular mass approximately 160 kDa in the plasma samples of mature female marbled sole. No cross-reactivity was observed with the plasma of male fish. A direct non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed using the monoclonal anti-msVTG and polyclonal anti-msVTG antibodies, with purified msVTG as the standard protein. The values of the intra- and inter-assay variations were within the ranges of $8.l-9.8\%$ and $8.5-12.2\%$, respectively. The sensitivity was about 0.3 ng/mL. Serial dilutions of plasma from mature female sole reacted with the msVTG-antibodies in the sandwich ELISA, whereas the plasma from male fish did not. The results indicate that the maturation status of female marbled sole can be identified using a sandwich ELISA for msVTG.

• 한국발생생물학회지:발생과생식
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• 제20권4호
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• pp.315-329
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• 2016

Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-${\gamma}$) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-${\gamma}$ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-${\gamma}$ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-${\gamma}$ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-${\gamma}$ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (${\geq}1,000U/mL$) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-${\gamma}$ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-${\gamma}$ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

• IMMUNE NETWORK
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• 제2권1호
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• pp.53-59
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• 2002

Background: Clinical observations and laboratory studies have supported an immune basis for most acquired aplastic anemias, with the majority of patients responding to immunosuppressive therapy. Fas, a member of the tumor necrosis factor (TNF) receptor superfamily is a critical downregulator of cellular immune responses. Proinflammatory cytokines like interferon gamma (IFN-${\gamma}$) and TNF-${\alpha}$ can induce Fas expression and render hematopoietic progenitor cells susceptible to Fas-induced growth suppression and apoptosis. Methods: In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anemia (AA), we measured the expression of Fas antigen and caspase-3 on bone marrow (BM) mononuclear cells (MNCs) of AA in the presence or absence of IFN-${\gamma}$, TNF-${\alpha}$, or macrophage inflammatory protein 1-${\alpha}$ (MIP-$1{\alpha}$). Results: We confirmed that AA BM MNCs were more apoptotic and highly expressed Fas antigen than normal donors. Stimulation by IFN-${\gamma}$, TNF-${\alpha}$, or MIP-$1{\alpha}$ increased Fas antigen and caspase-3 expression in AA BM MNCs than BM MNCs of normal donors. Anti-Fas monoclonal antibody enhanced IFN-${\gamma}$, TNF-${\alpha}$, or MIP$1{\alpha}$ mediated caspase-3 expression in BM MNCs of normal donors. Among these three cytokines, IFN-${\gamma}$ enhanced apoptosis most strongly via Fas-caspase-3 pathway. Conclusion: These results suggest that Fas signal pathway may play a role in the pathophysiology of aplastic anemia and negative hematopoietic regulators like IFN-${\gamma}$ can induce apoptosis of bone marrow progenitors in part by Fas induction.

• Clinical and Experimental Pediatrics
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• 제46권2호
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• pp.128-136
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• 2003

목 적 : Hyper IgM syndrome(HIGM)은 크게 세 부류로 나누는데, CD40L 분자의 돌연변이로 초래되며 X-linked로 유전되는 형태를 HIGM1이라고 하고, 상염색체성 열성 형태로 유전되면서 CD40L는 정상적으로 표현되는 형태로 activation-induced cytidine deaminase(AID) 유전자에 이상 때문에 오는 경우를 HIGM2로 분류하고 있다. 다른 한 부류는 X-linked HIGM 증후군의 매우 드문 한 형태로서 발한 저하성 외배엽 이형성증이 동반된 경우로, 이 질환은 전사인자인 nuclear factor ${\kappa}B$의 활성화에 관여하는 nuclear factor ${\kappa}B$ essential modulator를 coding하는 유전자의 돌연변이 때문에 오는 것으로 알려져 있다. 연구자들은 HIGM2와 유사하지만 AID 유전자에 변이는 없는 새로운 형태의 HIGM 환자의 말초 B세포를 이용하여 병인을 조사하고자 본 연구를 시도하였다. 방 법 : 환자의 말초혈액 단핵구를 분리하고, EBV로 immortalization을 시켜 Cycle TEST PLUS DNA Reagent kit를 이용하여 cell cycle을 분석하였다. 환자의 말초혈액 T 세포에서 CD40L의 표현을 immunostaining으로 알아보고, RNA를 추출하여 RT-PCR을 하고 direct sequencing을 통하여 CD40L 유전자의 돌연변이 부위를 찾아보았다. 아울러 AID 유전자의 돌연변이를 찾기 위하여 같은 방법으로 sequencing하고 조사하였다. 환자의 림프절을 병리학적인 검사를 시행하고 CD3, CD23, CD40, Fas-L, bcl-2, BAX의 표현을 알아보기 위하여 immunostaining을 실시하였다. 결 과: 림프절의 광학적 소견은 반응성 여포증식의 소견을 보였으며, 여포와 단구양 증식은 B-세포 표시인자인 L26에 양성을 보였고, 대부분의 형질세포는 IgM에 양성을 보였다. 여포는 CD40, Fas, BAX에 양성반응을 보이고, bcl-2와 Fas-L에 음성반응을 보였다. 말초혈액 B 림프구를 이용하여 cell cycle을 분석한 결과 정상(17.9%)에 비하여 G2/mitosis phase(M3 in figure)가 현저하게 감소(8.5%)되어 있는 양상을 보여주고 있으며, IL-4로 자극한 경우에는 정상인에서 보여주는 양상으로 회복되는 양상을 볼 수 있었다. 단핵수에서 CD40L의 표현은 정상이었고 CD40L 유전자에도 돌연변이는 발견할 수 없었으며, HuAID 유전자에도 돌연변이를 발견할 수 없었다. 결 론 : 환자의 말초혈액 B림프구를 통한 연구 결과, 기존에 보고 되어진 HIGM2 형태와 임상적으로는 비슷하지만, 정상적인 AID유전자를 보이고, G2/mitosis phase가 정상에 비하여 감소된, 새로운 형태의 HIGM이라고 여겨진다.