We have generated transgenic mice that expressed mouse extracellular superoxide dismutase (EC-SOD) in their skin. In particular, the expression plasmid DNA containing human keratin K14 promoter was used to direct the keratinocyte-specific transcription of the transgene. To compare intron-dependent and intron-independent gene expression, we constructed two vectors. The vector B, which contains the rabbit -globin intron 2, was not effective for mouse EC-SOD overexpression. The EC-SOD transcript was detected in the skin, as determined by Northern blot analysis. Furthermore, EC-SOD protein was detected in the skin tissue, as demonstrated by Western blot analysis. To evaluate the expression levels of EC-SOD in various tissues, we purified EC-SOD from the skin, lungs, brain, kidneys, livers, and spleen of transgenic mice and measured its activities. EC-SOD activities in the transgenic mice skin were approximately 7 fold higher than in wild-type mice. These results suggest that the mouse overexpressing vector not only induces keratinocyte-specific expression of EC-SOD, but also expresses successfully functional EC-SOD. Thus, these transgenic mice appeared to be useful for the expression of the EC-SOD gene and subsequent analysis of various skin changes, such as erythema, inflamation, photoaging, and skin tumors.

Interlellkin • 8(IL-8) is an important cytokine involved in tllmor growth and angiogenesis in a variety of malig nancies. bllt the regll lation of IL-8 in 01 외 cancer cells are llnderstood . We invesLigated whether mi togen-activated protein kinases pathway is involved in iron chelator-mediated lL-8 produdion in inunortalized and malignant oral keratinocytes. In this study we examined the role of p38 and extracellular signal- reglllated kinase• 1/2 in the expression of [L-8 by DFO. Incllbation of IHOK and HN12 cel ls with DF'O increased the expression of 11-8 mRNA. as well as the release of IL-8 protein. The signal transdllction study revealed that both p38 and ERK1/2 were significantly activated in response to DFO. Accord ingly. the selective inhibitors for both kinases‘ eit her a lone or combination. abolished DFO- induced lL-8 secretion. indicating an importance of MAP kinase pathway. Interestingly. however‘ inhibition of the p38 and ERK pathway more attenuated IL-8 secretion in IHOK than in HN12 cells. DFO induced NF-kB activation , suggesting a NF-kB- dependent mechanism in DFO- induced IL-8 production. In addition, p38 and ERK inhi bition resulted in the accelerated degradation of lL-8 mRNA, suggesting that in IHOK and HN12 cells, p38 and ERK cunLr iullLe Lo DFO imluced IL-8 secretion by IHOK and HN12 cells via a posttranscriptional mechanism that involves stabilization 01' the IL-8 transcript. Finally. we investigatecl llsing specific inhibitors : 8NP and G8NO for NO c1onor. PDTC for potent inhibitor of NF-kB. Cycloheximide for inhibition of de novo protein synthesis. We observecl 8NP ancl PD1'C clepenclent IL-8 gene incluction in IHOK cell s. but not in HN12 cells used specific inhibitors‘ Collectively. these results demonstrate that‘ targeting MAP kinase ancl NF-kB pathway may be a potentiaI approacb to controlling the angiogenes is ancl growth 이 human oral cancers

Studies to evaluate distribution of markers in normal keratinocyte and their immortalized keratinocyte are appropriate to evaluate the normal and preneoplastic lesion of oral cancers as biochemical and cytochemical changes associate with tumorigenesis being not completely understood. Complementary DNA microarray containing 6000 sequence -verified cDNA elements was used to systematically characterize the variation in gene expression patterns of NHOK cells vs. immortalized keratinocyte by HPV16 E6-E7(IHOK). Examination of gene expression that is 85 clones cDNAs exhibits greater than 2 fold overexpression in NHOK probes relative to IHOK probe, 147 cDNAs reveal greater 2 fold overexpression in IHOK relative to NHOK probe.The high similarity in gene expression (96.5%) between IHOK and NHOK cells suggests that only an additional 232/6720 (3.5%) of the genome is differentially gene activated during HPV16 immoratlized keratinocyte growth and differentiation. Examination of gene expression that differs between NHOK and IHOK cellsapprear to be related to : cell adhesion & recognition, cell cycle regulator, apoptosis, transciption factors, growth factors and therir receptors, cytoskeletal and extracellular matrix proteins, signal transduction modulators and effectors, and miscellaneous. The gene expression of cell recognition factor such as endothelin 1, collagen IV, fibronectin, and SPR1 in IHOK were upregulated. Distinct or duplicated cDNA clones representing the same gene were typically clustered in adjacent rows in the clustered gene map. Therefore the differentially expressed and identified genes should be informative in studying oral epithelial cell carcinogenesis and such studies should foster the research of molecular markers allowing to assess the phenotypeof malignant epithelial tumor.

Nitric oxide (NO) has been known to inf1uence cell fate through apoptotic or necrotic cell death. Here, we investigated the role of nitric oxide on the growth and viability of immortalized human salivary gland (HSG) cells 띠 vitro. Treatrnent of HSG with a NO donor, S-nitroso-N-acetyl-DL-penκi1lamine (SNAP), significantly diminished the growth rate of HSG in a concentration dependent manner. However, this retardation of cell비ar growth rate was not corresponded to the apoptotic cell death of HSG cells, because there were no characteristic apopto디c features such as condensation of nuclear chromatin, nuclear fragmentation, and the apoptotic peak of propidium iodide (PI)-stained nuclei by flow cytome띠. 까ùs implies that HSG cells are resistant to NO-mediated 다π。to:잉city. 1n SNAP treated HSG cells, cell cycle analysis revealed that the number of G2/M phase increased markedly, according to while the percentage of cells in GO/Gl and S phases was not significantly affected. Otherwise, high concentrations of SNAP increased both P1 and annexin V positive cells. 1nterestingly, preincubation of HSG cells with iron chelator, deferoxamine (DFO), significantly diminished NO cytotoxicity more than when HSG cells are only incubated with SNAP which su잃.ests the role of iron homeostasis in NO-mediated cell death of HSG cells. 1n addi디。n ， treatrnent of HSG cells with SNAP specifically cleaved iron regulatory protein-2 (IRP2) while not affecting 1RP1. Collectively, the mπent results s멍gest that NO has a potential to control HSG cell growth through cell cycle arresting at G2/M phase. 1n addi디on ， intracellular iron homeostasis nùght play an important role in regulating cell survival of HSG cells

Immunoregulative action of 70% ethyl alcohol fraction of Cervus nippon was investigated in vitro. The fraction enhanced the proliferation of thymocytes and the population of CD4^+CD8^- single-positive. cells in thymocytes. CN-E enhanced [Ca^2+]_i, the production of TNF-α and IL-1β, and phagocytosis in peritoneal macrophages. The fraction did not induce the proliferation of splenocytes and DNA fragmentation in thymocytes. These results indicate that 70% ethyl alcohol fraction of Cervus nippon regulates immunological action by stimulating murine thymocytes and macrophages.

당화효소와 단백질분해효소의 생산능력이 강한 Aspergillus usamii mut. shirousamii S1을 접종하여 밀가루누룩을 제조할 때에 당화효소와 단백질분해효소의 생산을 위한 배양조건을 검토하였다. 밀가루를 가열처리했을 때에는 날밀가루에 비하여 단백질분해효소와 유기산의 생산은 증가되었으나 당화효소와 호정화효소의 생산은 감소되었다. 밀가루에 염산 0.5%를 함유하는 물을 급수했을 때에는 당화효소, 호정화효소 및 단백질분해효소의 생산이 감소되었다. 당화효소 생산의 최적급수비율은 32%이고, 단백질분해효소 생산의 최적급수비율은 28%이었다. 당화효소 생산의 최적온도는 36℃이고, 단백질분해효소 생산의 최적온도는 28℃이었다. 당화효소력은 120시간 배양시에 최고치에 도달하였고, 단백질분해효소력은 96시간 배양시에 최고치에 도달하였다. 당화효소의 생산은 곰팡이 접종 후 즉시 성형하는 것보다 24시간 전배양을 한 후에 성형하는 것이 좋았고, 단백질분해효소의 생산은 성형을 하지 않는 것이 좋았다.