• Molecules and Cells
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• 제26권1호
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• pp.12-17
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• 2008

The TrkA tyrosine kinase is activated by autophosphorylation in response to NGF, and plays an important role in cell survival, differentiation, and apoptosis. To investigate its role in cell fate determination, we produced stable TrkA-inducible SK-N-MC and U2OS cell lines using the Tet-On system. Interestingly, TrkA overexpression induced substantial cell death even in the absence of NGF, by stimulating ERK phosphorylation and caspase-7 activation leading to PARP cleavage. TrkA-mediated cell death was shown by the annexin-V binding assay to be, at least in part, apoptotic in both SK-N-MC and U2OS cells. Furthermore, the truncated form (p18) of Bax accumulated in the TrkA-induced cells, suggesting that TrkA induces mitochondria-mediated apoptosis. NGF treatment augmented the cell death induced by TrkA overexpression. This TrkA-induced cell death was blocked by the tyrosine kinase inhibitors, K-252a and GW441756. Moreover, TrkA overexpression inhibited long-term proliferation of both the neuronal SK-N-MC cells and the non-neuronal U2OS cells, suggesting a potential role of TrkA as a tumor suppressor.

• Reproductive and Developmental Biology
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• 제29권1호
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• pp.57-62
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• 2005

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.

• Biomolecules & Therapeutics
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• 제6권4호
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• pp.351-357
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• 1998

In order to understand the mechanism of the regulation of CYPIAI gene expression and ethoxy-resorufin deethylase (EROD) activity in ex vivo system, we have studied the action of TCDD and 3MC in theisolated perfused male rat liver. CYPIAI myNA level and EROD activity were measured in rat liver that wasisolated and perfused with va.ious chemicals such as 2,3,7,8-tet.achlorodibenzo-p-dioxin (TCDD), 3-methyl-cholanthrene (3MC), $17{\beta}$-est.adios ($E_2$), morin. TCDD or 3MC alone perfusion into male rat liver resulted in increase of CYPIAI mRNA level and the magnitude of stimulation was one and half times higher with TCDD treatment than 3MC treatment. However $E_2$ perfusion into male rat liver showed slight stimulation of CYPIAI mRNA level. When $10_{-8}$ M $E_2$ was perfused concomitantly with either $10_{-9}$ M TCDD or $10_{-9}$ M 3MC, stimulated CYPIAI mRNA by either TCDD or 3MC was inhibited. Morin was examined for its effects on CYPIAI mRNA level and result was similar to that was observed with estrogen except that morin alone did not change the level of CYPIAI mRNA. EROD activity was also stimulated with either TCDD or 3MC perfusion, and the magnitude of EROD stiumlation was similar to that of CYPIAI mRNA stimulation in response to TCDD or 3MC perfusion. This data is different from the data that we have obtained with female rat liver. Concomitant perfusion either $E_2$ or morin with TCDD or 3MC inhibited 3MC perFusion or TCDD perfusion stimulated EROD activity. These data confirm the hypothesis that TCDD and 3MC might act through the same mechanism of action on the regulation of CYPIAI gene expression in male rat liver.

• Journal of Microbiology
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• 제40권3호
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• pp.211-218
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• 2002

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

• Reproductive and Developmental Biology
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• 제30권3호
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• pp.157-162
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• 2006

Endogenous 84 amino acid parathyroid hormone(PTH) is synthesized as a pre-pro hormone by the chief cells of the parathyroid glands. Physiological actions of PTH include regulation of bone metabolism, renal tubular reabsorption of calcium and phosphate, and intestinal calcium absorption. In addition, PTH stimulates new bone formation by extraordinary stimulation of osteoblastic activity and decreasing calcium excretion by the kidney. In this study, we constructed and tested retrovirus vectors designed to express the human parathyroid hormone(hPTH) gene under the control of the tetracycline-inducible promoters. To increase the hPTH gene expression at turn-on state, woodchuck hepatitis virus posttranscriptional regulatory element(WPRE) sequence was also introduced into retrovirus vector at downstream region of either the hPTH gene or the sequence encoding reverse tetracycline-controlled transactivator(rtTA). Transformed primary culture cells(porcine fetal fibroblast, PFF, chicken embryonic fibroblast, CEF) were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the hPTH gene expression level using two step RT-PCR and ELISA Higher hPTH expression($3{\tims}10^4\;pg/ml,\;5.3{\times}10^4\;pg/ml$) and tighter expression control(up to 8 fold) were observed from the vector in which the WPRE sequence was placed at downstream of the hPTH gene. The resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2745-2748
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• 2012

Aims: To study KIT (CD117) expression in thymic epithelial tumors in China, and investigate diagnostic and clinical significance. Material and Methods: Thymic epithelial tumors (TETs) from 102 patients (3 type A, 29 type AB, 5 type B1, 22 type B2, 29 typeB3 and 16 thymic carcinomas) were examined. Immunohistochemical staining with an antic-kit monoclonal antibody was performed on a tissue microarray. Relationships between KIT positive expression and the TET clinical characteristics (WHO histologic classification and Masaoka stage system) were analysed. Results: The KIT positive expression rate was significantly higher in thymic carcinoma (60%, 9/16) than in thymoma (8%, 7/86), a strong correlation being found with the WHO classification, but not the Masaoka tumor stage. The overall survival for patients with KIT positive lesions was significantly worse. Conclusions: KIT is a good molecule marker to differentially diagnose thymic carcinoma from thymoma, while also serving as a predictor of prognosis for TETs. Further research into KIT mutations in Chinese TETs should be conducted to assess the efficacy of targeted therapy.

• Korean Journal of Organic Agriculture
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• 제31권4호
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• pp.381-394
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• 2023

This study was conducted to monitor the antibiotics resistance of human-harmful bacteria isolated in the agricultural environment for hot peppers (Capsicum annuum) and tomato (Lycopersicon esculentum). As a result, we isolated 120 bacterial species (34 on fruits, 48 in soil, 21 in water, and 17 in manure), identified them with the 16S rRNA sequence, analyzed minimum inhibitory concentration (MIC) for 26 antibiotics using Sensititre ARIS Hi-Q system and then evaluated whether each bacterial genus acquired resistance for the tested antibiotics or not, according to the CLSI criteria. From difference in MIC between eco-friendly (EFM) and practical (PFM) cultivation farms, Klebsiella spp. isolated from EFM was resistant to ampicillin (AMP) and nalidixic acid (NAL), and that isolated from PFM was resistant to streptomycin (STR) and tetracycline (TET). Enterobacter spp. isolated from EFM was resistant to AMP and azithromycin (AZI), and that isolated from PFM was resistant to AMP, AZI, and STR. Meanwhile, Pseudomonas spp. isolated from EFM and PFM were all resistant to AMP, AZI, cefotaxime (FOT), cefoxitin (FOX), ceftriaxone (AXO), CHL, NAL, and STR. Staphylococcus spp. isolated from EFM and PFM were resistant to gentamycin (GEN), STR, and kanamycin (KAN), and in particular, that from EFM showed resistance for erythromycin (ERY). In conclusion, our study suggested that EFM lead STR antibiotics resistance for human-harmful bacteria to decrease, because only the bacteria isolated from hot pepper and tomato crop with PFM have showed resistance against STR antibiotics, regardless of bacterial genus.