Staurosporine Induces ROS-Mediated Process Formation in Human Gingival Fibroblasts and Rat Cortical Astrocytes

In the present study, we investigated the effect of staurosporine on the formation of cellular processes in human gingival fibroblasts and rat astrocytes. Staurosporine caused a rapid induction of process formation in human gingival fibroblasts and rat astrocytes in a concentration dependent manner. The process formation of human gingival fibroblasts and rat astrocytes was prevented by the pretreatment with N-acetylcysteine, suggesting that staurosporine-induced ROS production was responsible for the process formation. Colchicine, a microtubule depolymerizing agent, inhibited the staurosporine-induced process formation, whereas cytochalasin D, an actin filament breakdown agent, failed to suppress the formation of cellular processes. This result indicated that polymerization of microtubule, and not actin filament, was responsible for the formation of cellular processes induced by staurosporine. In support of this hypothesis, Western blot analysis was conducted using anti-tubulin antibody, and the results showed that the amount of polymerized microtubule was increased by the treatment with staurosporine while that of depolymerized beta-tubulin in soluble fraction was decreased. These results indicate that staurosporine induces ROSmediated, microtubule-dependent formation of cellular processes in human gingival fibroblasts and rat astrocytes.ope

Antibodies against non-immunizing antigens derived from a large immune scFv library

Target-specific antibodies can be rapidly enriched and identified from an antibody library using phage display. One of the key advantages of antibody phage display compared with other antibody-producing methods such as hybridoma technique is that the antibody selection process is performed in vitro. An antibody library is a collection of a large number of various antibody genes, which can be displayed on bacteriophage surface by phage display technique to yield diverse target-specific antibodies. Large, naïve antibody libraries derived from synthetic or nonimmunized sources can yield antibodies against virtually any antigen, whereas libraries from immunized sources tend to be smaller and used exclusively against the antigen of immunization. In this report, 25 scFv libraries made from the spleens of immunized rabbits, each with a size ranging from 108 to higher than 109, were combined into a single large library with > 1010 individual clones. Panning of this combined library yielded target-specific rabbit scFv clones against several immunizing or non-immunizing antigens, including proteins and peptides. These clones were tested for their ability to recognize the target antigen in ELISA and immunoblot assay. It can be concluded from this study that the immune library contained a significant number of unimmunized clones and that a sufficiently large immune library can be utilized similarly to a naïve library;Phage display방법을 이용하면 항체 라이브러리로부터 표적 특이적 항체를 빠르게 증가시키고 확인 할 수 있다. 이런 phage display의 방법이 다른 항체제조방법과 비교했을 때 가장 큰 장점은 hybridoma 기술과 달리 in vitro에서 항체를 선별하는 방법이 실행된다는 것이다. 항체 라이브러리는 무작위적으로 다양성을 가지는 항체 유전자를 제작하고 이것을 박테리오파지의 표면에 발현하는 phage display 기술로 다양한 항체를 얻어 낼 수 있다. 그리고 합성된 라이브러리나 면역되지 않은 라이브러리로부터 유래된 크고 순수한 항체 라이브러리는 어떤 항원으로부터도 항체를 얻어낼 수 있는 반면, 면역으로 얻어낸 라이브러리는 크기가 더 작고 면역된 항원에만 이용할 수 있는 경향이 있다. 우리는 면역한 토끼의 비장으로부터 작게는 108부터 크게는 109의 크기를 가진 25개의 라이브러리를 결합하여 1010정도 크기의 큰 하나의 항체 라이브러리를 만들어 이 라이브러리로 단백질들과 펩티드들을 포함하고 있는 토끼에 면역한 항원과 그렇지 않은 항원에 대해 패닝방법으로 표적특이성을 가지는 토끼의 항체클론을 얻어 내었다. 특히 이 라이브러리로부터 Actin과 인산화된 STAT1에 대한 특이성을 가지는 항체 클론을 얻어냈다. 이 클론들은 표적에 대한 특이성을 ELISA와 Immuno blot을 이용하여 확인할 수 있었다. 이러한 결과로부터 면역 라이브러리가 많은 수의 면역되지 않은 항원에 대한 상당한 수의 항체클론을 가지고 있다는 것을 알았고, 이것은 충분히 naïve 라이브러리와 마찬가지로 활용할 수 있을 것으로 생각된다.I. INTRODUCTION 1 II. MATERIALS AND METHODS 5 A. Library 5 B. Cell lysate 5 C. Phage Antibody selection by panning 6 C.1 Panning on protein antigens 6 C.2 Panning on peptide antigens 7 D. ELISA screening for protein- and -peptide- specific antibodies 8 D.1 ELISA screening on protein antigens 8 D.2 ELISA screening on peptide antigens 9 E. Purification of scFv 9 F. Immunoblot Analysis 10 G. DNA fingerprinting analysis 11 III. RESULT 12 A. Rabbit immune library transformation and pooling 12 B. Panning and ELISA screening on rabbit serum albumin, a rabbit self-antigen 14 C. Panning on protein antigens and ELISA screening 16 C.1 Selection of significant Antibodies by finger printing and sequencing 19 D. Panning and screening of a pooled rabbit immune library performed on phosphorylated peptides 21 E. Panning on pY845 phosphopeptide antigen: a comparison of the pooled rabbit immune library with a naïve synthetic scFv library 25 F. Purification of specific Antibodies and Immunoblot assay 28 IV. DISCUSSION 30 V. REFERENCES 33 VI. 국문초록 3

Neuroprotective effect of 3-morpholinosydnonimine against Zn2+-induced PC12 cell death

Excessive intracellular accumulation of zinc (Zn2+) is neurotoxic and contributes to a number of neuropathological conditions. Here, we investigated the protective effect of 3-morpholinosydnonimine (SIN-1) against Zn2+-induced neuronal cell death in differentiated PC12 cells. We found that Zn2+-induced PC12 cell death was reduced in a concentration-dependent manner by pretreatment with SIN-1. The intracellular accumulation of Zn2+ was not affected by pretreatment with SIN-1, indicating that SIN-1-induced neuroprotection was not attributable to reduced influx of Zn2+ into cells. SIN-1C, the stable decomposition product of SIN-1, failed to prevent Zn2+-induced cell death. Furthermore, the protective effect of SIN-1 against Zn2+-induced PC12 cell death was almost completely abolished by uric acid, a free radical scavenger, suggesting that reactive oxygen and nitrogen species generated by SIN-1 may contribute to the protective effect. SIN-1 prevented the inactivation of glutathione reductase (GR) and the increase in the ratio of oxidized glutathione/total glutathione (GSSG/total GSH) induced by Zn2+. Addition of membrane permeable GSH ethyl ester (GSH-EE) to PC12 cells prior to Zn2+ treatment significantly increased cell viability. We therefore conclude that SIN-1 may exert neuroprotective effect against Zn2+-induced cell death in differentiated PC12 cells by preventing inhibition of GR and increase in GSSG/total GSH ratio.ope