Esculetin, a Coumarin Derivative, Exhibits Anti-proliferative and Pro-apoptotic Activity in G361 Human Malignant Melanoma

BACKGROUND: Although esculetin, a coumarin compound, is known to induce apoptosis in human cancer cells, the effects and molecular mechanisms on the apoptosis in human malignant melanoma (HMM) cells are not well understood yet. In this study, we investigated the anti-proliferative effects of esculetin on the G361 HMM cells. METHODS: We analyzed the anti-proliferative effects and molecular mechanisms of esculetin on G361 cells by a 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, 4\',6-diamidino-2-phenylindole staining and Western blotting. RESULTS: Esculetin exhibited significant anti-proliferative effects on the HMM cells in a dose-dependent manner. Interestingly, we found that esculetin induced nuclear shrinkage and fragmentation, typical apoptosis markers, by suppression of Sp1 transcription factor (Sp1). Notably, esculetin modulated Sp1 downstream target genes including p27, p21 and cyclin D1, resulted in activation of apoptosis signaling molecules such as caspase-3 and PARP in G361 HMM cells. CONCLUSIONS: Our results clearly demonstrated that esculetin induced apoptosis in the HMM cells by downregulating Sp1 protein levels. Thus, we suggest that esculetin may be a potential anti-proliferative agent that induces apoptotic cell death in G361 HMM cells.

Immunosuppression of xenograft rejection in porcine kidney PK15 cells by porcine IL-18

Xenotransplantation, the transplantation of cells, tissues or organs between individuals of different species, would resolve the current shortage of organs, but rejection remains the major hurdle to successful xenotransplantation. In the present study, we analyzed mixed lymphocyte reactions (MLRs) and used 51Cr release assays in order to identify the proliferation and expansion of mouse CD8+ cytotoxic T lymphocyte cells against PK15, PK15/pIL-18 or PK15/mIL-18 cells. In addition, we identified T cell populations in mouse splenocytes and lymph node cells using two-color flow cytometry. It was found that the CD8+T cells of xenograft recipients proliferated extensively and that the survival rates of populations of PK15/mIL-18 or PK15/pIL-18 cells were higher than untransfected controls. Moreover, CD3+T cells were increased in mice injected with PK15 cells or PK15/pIL-18 cells but PK15/pIL-18 cell numbers were lower in lymph nodes than untransfected controls. CD8+T cells numbers were reduced in the lymph nodes of PK15/pIL-18 injected mice. These results suggest that porcine IL-18 regulates anti-pig cellular rejection in C57BL/6 mice.

Preparation and Characterization of SDF-1alpha Recombinant Adenovirus for ex vivo Expansion of Hematopoietic Stem Cells

Stromal cell-derived factor 1 (SDF-1/CXCL12) is a multifunctional cytokine implicated in normal hematopoiesis. We previously reported that SDF-1alpha enhanced the survival of hematopoietic stem and progenitor cells in synergy with other cytokine such as GM-CSF, steel factor, or thrombopoietin. As adult stem cells are very rare, many investigators are trying to expand hematopoietic stem/progenitor cells in vitro. In this study, we constructed an adenoviral vector and produced high titer of recombinant adenoviruses directing robust expression of SDF-1alpha determined by ELISA. We also produced control empty adenoviruses and recombinant LacZ adenoviruses. In order to check the feasibility of SDF-1alpha in ex vivo expansion system, we compared HUVEC cells tranduced by a SDF-1alpha recombinant virus with HUVEC cells transduced by a LacZ recombinant virus in supporting activity of hematopoietic cells, and found that expression of SDF-1alpha in HUVEC cells increased viable blood cell population obtained from the same number of CD34+ cells. The SDF-1alpha recombinant adenovirus seems to be useful for future application in hematopoiesis studies.

The Emerging Role of Natural Killer Cells in Innate and Adaptive Immunity

In the early host defense system, effector function of natural killer (NK) cells results in natural killing against target cells such as microbe-infected, malignant, and certain allogenic cells without prior stimulation. NK cell cytotoxicity is selectively regulated by homeostatic prevalence between a repertoire of both activating and inhibitory receptors, and the discrimination of untransformed cells is achieved by recognition of major histocompatibility complex (MHC) class I alleles through inhibitory signals. Although it is well known that the bipotential T/NK progenitors are derived from the common precusor, functional mechanisms in terms of the development of NK cells remain to be further investigated. NK cells are mainly involved in innate immunity, but recent studies have been reported that they also play a critical role in adaptive immune responses through interaction with dendritic cells (DC). This interaction will provide effector functions and development of NK cells, and elucidation of its precise mechanism may lead to therapeutic strategies for effective treatment of several immune diseases.

Development of PCR-ELISA for Detection of HIV-1 in Biomedical Products / 대한진단검사의학회지

BACKGROUND: Biomedical products such as viral vaccines can be contaminated with hazardous viruses during manufacturing processes and storage, thus causing harmful side effects. To assure the safety of biomedical products, highly effective and sensitive methods should be available to detect contaminating viruses. In this study, we performed recovery tests to determine the limit of detection of HIV-1. METHODS: An HIV-1 plasmid preparation was serially diluted and spiked into various culture media (DMEM, RPMI-1640, IMDM, GICM, and SDM) containing 10% fetal bovine serum (FBS). The HIV-1 plasmid was detected by PCR alone or a combination of PCR and ELISA (PCR/ELISA). RESULTS: When spiked into DMEM, RPMI, and IMDM, less than 4x10(-2) ng of HIV-1 plasmid was not detectable as HIV-1 PCR products in agarose gel. Intra- and inter-assays (n=6) showed that the PCR-ELISA system could detect PCR products diluted as much as 1, 875 times from HIV-1 plasmid serially spiked in various media. CONCLUSIONS: The PCR/ELISA system can be useful for the detection of trace amounts of hazardous viruses which may be present as contaminants in biological products.

Germinal Center Formation and Integrins Expression in the Rat Thymus After 5-Fluorouracil Treatment / 대한해부학회지

Thymus is a lymphoid organ forming T-cells from hematogenous stem cells. There is no report on the germinal centers in the thymus except the Myasthenia gravis in human. In this study, new germinal centers were experimentally induced from 6 days after 5-FU treatment in the Sprague-Dawley rats. With germinal center formation, proteins of molecular weight 123 kDa on 6 days after 5-FU treatment, and 162 kDa and 205 kDa on 9 days after 5-FU treatment were increased in 5-FU treated group. These proteins revealed alpha 1 -and beta 1 -integrins on integrin Western Blot. In this experiment, it was cosidered that alpha 1-integrins and beta 1 -integrins were the key proteins for proliferation of B cells within the newly formed thymic germinal centers and the massive apoptotic disposal of B-cells from the germinal centers, and the new formation of T cells within the cortex.

Histological and Biochemical Study on the Effect of Cadmium Chloride on the Rat Testis / 대한해부학회지

Although cadmium is a well known heavy metal which has an influence testis and brings about male infertility, the mechanism of action in the testis is still fully unknown. In these experiment, cadmium chloride 4 mg/kg of body weight administered intraperitoneally to the rat (Sprague-Dawley) and sacrificed after 1 week, and morphological changes were observed by LM and TEM. In addition, electrophoresis, immunoprecipitation and Western blotting and N-terminal analysis performed to reveal the protein changes. 1. Major findings under light microscope were hemorrhagic necrosis and death of all the spermatogenic cells and supporting cells within the seminiferous tubules, and decreased volume of ECM, many apoptotic bodies, and death of interstitial cells and fibroblasts within interstitium. 2. The EM findings were disruption of nuclear membrane and disappearance of cell organelles of spermatogenic cells and supporting cells within seminiferous tubules, and decreased filopodia, increased inclusion bodies, vacuolation and apoptotic changes of the interstitial cells and fibroblastic cells, many short electron-dense collagen fibers in the extracellular matrix of interstitium. 3. Two proteins of molecular weight 42 kDa and 21 kDa which disappeared after cadmium treatment were rat collagen type I alpha 2. According to the above results, it is considered that cadmium degrades the collagen of the wall of small blood vessels within seminiferous tubules and interstitium and disrupts vascular walls, which results hemorrhagic necrosis, death of all the spermatogenic cells, and the death of interstitial cells and fibroblastic cells.