Earticle

The monkey 20ɑ-hydroxysteroid dehydrogenase (20ɑ-HSD) is an enzyme responsible for the catabolism in converting progesterone into biological inactive 20a-hydroxyprogesterone, thus playing a key role during the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. We have previously shown that 20ɑ-HSD in pre-ovulation and pre-parturition was highly detected in ovarian and placental tissues and localized mainly in the syncytiotrophoblast of the placenta. In this study, we focus on the analysis of molecular characterization of the monkey 20ɑ-HSD promoter region by using reporter assay systems in a Chinese hamster ovary (CHO) K1 cells. A reporter assay, using constructs (—890-Luc, —513-Luc, —306-Luc, —273-Luc and —70-Luc) of various lengths of the 5'-flanking region, revealed that the region (Ap-1) between —281 and —274 bp was essential for transcriptional activity. The absence of Ap-1 site in -273-Luc dramatically decreased to the control levels. Thus, Ap-1 site (—281→—274) (5'-TGTCTCAT-3') plays a crucial role for monkey 20α-HSD gene transcription. Next, we analyzed the monkey 20α-HSD promoter activity with treatment of prolactin, PGF2α and Forskolin in CHO-K1 cells. The 2005bp monkey promoter-pGL3 was transfected to CHO cells and added various dose-dependent treatments after incubation 24 hours. For PGF2α 0.5, 1, 5, 10 and 50 μM added and incubated during 6 hours. For Forskolin 0.1, 1, 10, 50 and 100 μM added. And finally, the luciferase activity was measured after 24 hours. Also, human prolactin (3μg/ml) was used as inhibitor. In result, PGF2a was response to a dose-dependent activation. On the other hand Forskolin was detected dose-dependent inhibition in the 2005bp 20α-HSD promoterpGL3 construct. However, prolactin was not shown any significant different. Taken together, our results indicate that monkey 20α-HSD promoter activity inhibit by forskolin. Also, PGF2a remarkedly stimulates the activity of monkey 20α-HSD promoter. We demonstrated that monkey 20a-HSD promoter regulate by AP-transcription factor, PGF2a, and Foskolin.

A형 혈우병은 혈액응고 제 8인자(FactorVIII: FVIII)가 유전적으로 결핍되어 나타나는 희귀병이며, FVIII인자는 내인성 혈액응고반응에 필수적으로 필요한 당단백질이다. 이러한 A형 혈우병 환자는 FVIII의 지속적인 주사에 의해 정상적인 혈액응고반응이 일어나고 생명연장이 가능하다. 본 연구는 인간 혈액응고 제 8인자 단백질영역 중 B-domain을 변형시킨 재조합체(FVIII-dB747)를 유즙으로 분비하는 형질전환 돼지의 후대를 계획적으로 종부시키면서 생산되는 형질전환돼지의 유즙으로부터 FVIII-dB747의 효율적인 정제를 위한 방법을 제공하기 위하여 수행되었다. 분만돈에 옥시토신을 투여하여 FVIII-dB747을 발현하는 유즙을 채취하여 원심분리를 통해 지질과 불순물을 제거하고, 유즙 내 복합적으로 존재하는 수 많은 카제인 단백질을 분획하기 위한 전처리 과정을 실시함으로써 분획의 정도를 확인하였다. 또한 전처리 과정을 통해 분획된 유즙을 이용하여 음이온 교환수지와 친화성 크로마토그래피 방법으로 FVIII-dB747의 정제를 시도하였다. 음이온 교환 크로마토그래피는 인간 혈장 유래의 FVIII을 정제하는데 사용되어지는 Q sepharose FF 컬럼을 이용하여 pH변화에 따른 분리도를 비교하였다. 또한 친화성 크로마토그래피는 B-domain이 결여된 재조합 인간 혈액응고 제 8인자(BDD rFVIII)을 정제하는데 이용 가능하다고 알려진 단일항체 리간드로 사용한 컬럼을 사용하여 수행하였다. 그 결과, 기존의 ammonium sulfate침전법을 이용한 침전방법보다는 간단하면서 효과적인 전처리 분획방법으로 개선되었으나, pH 변화에 따른 이온교환 수지를 활용한 분리법에서는 카제인 단백질이 FVIII-dB747이 포함된 분획과 함께 유출되는 현상이 발생되었다. FVIII-dB747 형질전환돼지의 유즙을 이온교환 수지로 분리할 경우 전처리과정 중 카제인 단백질을 제거하기 위한 ultra-filteration이 필요한 것을 알 수 있었다.

The present study demonstrates green approach for the production of spherical-shaped silver nanoparticles synthesized and stabilized using a bacterium, Eschericia coli. Aqueous solutions of Ag+ionsofsilverweretreatedwiththe supernatant of E. coli for the formation of Ag nanoparticles (AgNPs). The nanometallic dispersions were characterized by surface plasmon absorbance measuring at 420 nm. Transmission electron microscopy and particle analyser showed the formation of nanoparticles in the range of 15～20 nm. X-ray diffraction (XRD) analysis of the AgNPs confirmed the formation of metallic silver. Further analysis carried out by Fourier Transform Infrared Spectroscopy (FTIR), provides evidence for the presence of proteins as possible biomolecules responsible for the reduction and capping agent which helps in increasing the stability of the synthesized AgNPs. The biological activities of the synthesized particles were confirmed based on toxicity assays and lactate dehydrogenase activity (LDH) in A549 cells. The toxicity assay suggests that cell death and leakage of LDH is dose and time dependent. Further, we evaluated the impact of AgNPs on the level of phosphorylation using various kinases such as EGFR, Akt, and Erk. Further, cytotoxicity, inflammation, and developmental toxicity studies shows significant effect in mice. To understand the underlying mechanism of AgNPs on the cellular responses, we showed that AgNPs could inhibit the phosphorylation of EGFR/Akt/Erk. Together, our results indicate that AgNPs can act as an anti-proliferative molecule by targeting the inactivation of EGFR/ Akt signaling pathways.