1.Expression of nitric oxide synthase isoforms in the porcine ovary during follicular development.
Heechul KIM ; Changjong MOON ; Meejung AHN ; Yongduk LEE ; Hwanglyong KIM ; Seungjoon KIM ; Taeyoung HA ; Youngheun JEE ; Taekyun SHIN
Journal of Veterinary Science 2005;6(2):97-101
The expression of nitric oxide synthase (NOS) isoforms in the ovaries of pigs was examined to study the involvement of nitric oxide, a product of NOS activity, in the function of the ovary. Western blot analysis detected three types of NOS in the ovary, including constitutive neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS); eNOS immunoreactivity was more intense compared with that of iNOS or nNOS. Immunohistochemical studies demonstrated the presence of nNOS and eNOS in the surface epithelium, stroma, oocytes, thecal cells, and endothelial cells of blood vessels. Positive immunoreactions for nNOS and iNOS were detected in the granulosa cells from multilaminar and antral follicles, but not in those of unilaminar follicles. iNOS was detected in the surface epithelium, oocytes, and theca of multilaminar and antral follicles. Taking all of the findings into consideration, the observed differential expression of the three NOS isoforms in the ovary suggests a role for nitric oxide in modulating reproduction in pigs.
Animals
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Blotting, Western/veterinary
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Female
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Immunohistochemistry/veterinary
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Nerve Tissue Proteins/*biosynthesis
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Nitric Oxide/metabolism
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Nitric Oxide Synthase/*biosynthesis
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Nitric Oxide Synthase Type I
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Nitric Oxide Synthase Type II
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Nitric Oxide Synthase Type III
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Ovarian Follicle/*enzymology/growth&development
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Swine/*physiology
2.Effects of insulin-like growth factor II on regulating nitric oxide synthase gene expression in mouse osteoblast-like MC3T3-E1 cells.
Wei-lian SUN ; Li-li CHEN ; Jie YAN ; Zhong-sheng YU
Chinese Journal of Stomatology 2004;39(3):201-204
OBJECTIVETo study the effects of insulin-like growth factor II (IGF-II) on regulating the levels of nitric oxide (NO) and the mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells.
METHODSMouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different intervals of time, MTT colorimetry was used for examining the cell proliferation. Nitrate reductase method was applied for detecting the NO concentrations in cell culture supernatants and RT-PCR employed for determining the levels of cellular iNOS and eNOS mRNAs.
RESULTSAfter the MC3T3-E1 cells were treated with IGF-II at the dosages of 1 microg/L for 72 h, 10 and 100 microg/L for 24, 48 and 72 h respectively, all the MTT values increased markedly (P < 0.05 or P < 0.01). After the cells were treated for 48 and 72 h at the dosage of 100 microg/L IGF-II respectively, the levels of NO in the supernatants of cell cultures and cellular iNOS mRNA decreased significantly (P < 0.01). However, the levels of eNOS mRNA in the cells treated with any of the IGF-II dosages for the different times were stable (P > 0.05).
CONCLUSIONSIGF-II at the dosages of 1 approximately 100 microg/L showed the effects on promoting proliferation, which as probably due to the maintenance of low NO levels. Inducible NOS gene expression at the level of transcription was down regulated in the MC3T3-E1 cell treated with higher dosage of IGF-II (100 microg/L) but eNOS mRNA was not, which might be one of the mechanisms for the maintenance of low NO levels.
3T3 Cells ; Animals ; Insulin-Like Growth Factor II ; pharmacology ; Mice ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; biosynthesis ; genetics ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Osteoblasts ; cytology ; drug effects ; enzymology ; RNA, Messenger ; biosynthesis
3.Effect of spinal cord injury on iNOS and Bcl-2 gene expression in spermatogenic cell of the male rat.
Yong WEI ; Xiao-Bin ZHANG ; Jie ZHANG ; Fan CHENG
National Journal of Andrology 2003;9(1):28-54
OBJECTIVESTo investigate the cause of spermatogenic cells decreasing after spinal cord injury.
METHODSImmunohistochemical S-P method was used to detect the expression of iNOS and Bcl-2 protein in the testis of spinal cord injured and sham-operated adult rats in second week and fourth week after operation.
RESULTSSecond week and fourth week after operation, the expression of iNOS of operated rats increased significantly (P < 0.05). Fourth week after operation, the number of Bcl-2 positive cells in spinal cord injured group decreased significantly (P < 0.05).
CONCLUSIONSThe expression veition of iNOS and Bcl-2 is the cause that inducing the decrease of spermatogenic cells in testis of spinal cord injured rats.
Animals ; Male ; Nitric Oxide Synthase ; biosynthesis ; genetics ; Nitric Oxide Synthase Type II ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism
4.Effects of bilirubin on alveolar macrophages in rats with emphysema and expression of iNOS and NO in them.
Jianqiang LI ; Hui ZHAO ; Manjing SONG ; Yongjian XU ; Zhenxiang ZHANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(4):322-341
To explore the effects of bilirubin on alveolar macrophages (AM) and expression of iNOS and NO in them in emphysema model, the rats were pretreated with bilirubin before exposed to smoke. AM were isolated from bronchoalveolar lavage fluid (BALF) and cultured. Pathological microscopic examination of AM and immunohistochemical analysis of iNOS were performed. Nitric oxide (NO) content in the samples was determined by nitrate reductase technique. The results showed both alveoli and alveolar septum appeared normal in size and shape in normal group. AM showed kidney-shaped nucleus and were rich in Golgi complexes and primary lysosomes in the cytoplasm. The inner membrane of mitochondrion was continuous. Most cristae of the mitochondria were intact. In model group, the alveoli were expanded, ruptured and bullaes were formed. Both the population and sizes of AM increased significantly. Secondary lysosomes were rich in the cytoplasm. Deformation and pyknosis of the nucleus, swelling of the mitochondrions and rupture of the inner mitochondrial membrane could also be seen. At high magnification, most of the mitochondrial cristae were broken, or completely lost at certain points. In bilirubin group, alveoli partly expanded and the population of AM also increased, with morphological changes being slighter than that in model group. Both NO contents and expression of iNOS in model group were higher than those in normal group (P<0.05). In bilirubin group the two indice were lower than those in model group (P<0.05). Our findings suggested that high expression of iNOS and high NO content in AM accelerate the development of emphysema associated with smoking in rats. Bilirubin may exert protective effects on AM and retards the development of emphysema in rats.
Animals
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Antioxidants
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pharmacology
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Bilirubin
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pharmacology
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Cells, Cultured
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Emphysema
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metabolism
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pathology
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Female
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Macrophages, Alveolar
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drug effects
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metabolism
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pathology
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Male
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Nitric Oxide
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biosynthesis
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Nitric Oxide Synthase
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biosynthesis
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Nitric Oxide Synthase Type II
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Rats
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Rats, Wistar
5.Activation of nuclear factor kappaB and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans.
Yan-hua ZENG ; Yi-mou WU ; Wen-bo ZHANG ; Min-jun YU ; Cui-ming ZHU ; Li-zhi TAN
Chinese Medical Journal 2004;117(7):997-1001
BACKGROUNDThis study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.
METHODSMouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor kappaB (NF-kappaB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting.
RESULTSM. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-kappaB activation, a possible mechanism for the induction of iNOS expression.
CONCLUSIONThis study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-kappaB.
Animals ; Bacterial Proteins ; pharmacology ; Cells, Cultured ; Enzyme Induction ; Lipoproteins ; pharmacology ; Membrane Proteins ; pharmacology ; Mice ; Mycoplasma penetrans ; chemistry ; NF-kappa B ; metabolism ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; biosynthesis ; Nitric Oxide Synthase Type II ; RNA, Messenger ; analysis
6.Progesterone exerts neuroprotective effect on hypoxic-ischemic encephalopathy-induced brain damage via inhibition expression of inducible nitric oxide synthase and nitric oxide production.
Xiao-yin WANG ; Xiao-juan LI ; Dong-liang LI ; Cong-rui WANG ; Xue-peng GUO
Chinese Journal of Applied Physiology 2012;28(3):253-262
7.Glucocorticoid receptor agonist dexamethasone attenuates renal ischemia/reperfusion injury by up-regulating eNOS/iNOS.
Jiong ZHANG ; Jun-hua LI ; Le WANG ; Min HAN ; Fang XIAO ; Xiao-qin LAN ; Yue-qiang LI ; Gang XU ; Ying YAO
Journal of Huazhong University of Science and Technology (Medical Sciences) 2014;34(4):516-520
The aim of this study was to determine the effect of dexamethasone (DEX) on renal ischemia/reperfusion injury (IRI). C57BL/6 mice were randomly divided into Sham group, IRI group and DEX group. The mice in IRI and DEX groups subjected to renal ischemia for 60 min, were treated with saline or DEX (4 mg/kg, i.p.) 60 min prior to I/R. After 24 h of reperfusion, the renal function, renal pathological changes, activation of extracellular signal-regulated kinase (ERK) and glucocorticoid receptor (GR), and the levels of iNOS and eNOS were detected. The results showed DEX significantly decreased the damage to renal function and pathological changes after renal IRI. Pre-treatment with DEX reduced ERK activation and down-regulated the level of iNOS, whereas up-regulated the level of eNOS after renal IRI. DEX could further promote the activation of GR. These findings indicated GR activation confers preconditioning-like protection against acute IRI partially by up-regulating the ratio of eNOS/iNOS.
Animals
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Dexamethasone
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pharmacology
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Gene Expression Regulation, Enzymologic
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drug effects
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Glucocorticoids
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pharmacology
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Male
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Mice
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Nitric Oxide Synthase Type II
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biosynthesis
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Nitric Oxide Synthase Type III
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biosynthesis
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Receptors, Glucocorticoid
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agonists
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Reperfusion Injury
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enzymology
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pathology
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Up-Regulation
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drug effects
8.Mechanism of apoptotic effects induced by 5-fluorouracil on human liver carcinoma Bel7402 cell line.
Junmei JIANG ; Jiyong LIU ; Juren ZHU ; Chongmei YANG ; Anzhong ZHANG
Chinese Medical Journal 2002;115(7):968-971
OBJECTIVETo evaluate the effect of endogenous nitric oxide (NO) on the ability of 5-fluourouracil (5-FU) to induce apoptosis in the liver carcinoma Bel7402 cell line, and to observe the anti-tumor mechanism and effective adjuvant of 5-FU.
METHODSCells were cultured under routine conditions with Dulbecco's modified Eagle's medium (DMEM) without L-Arginine (L-Arg). We observed the expression of inducible nitric oxide synthase (iNOS) and apoptosis of cells induced by 5-FU with L-Arg added to the medium. The production of nitric oxide was determined by the cell expression of iNOS detected by immunohistochemical staining, and by the concentrations of nitrite and nitrate in the supernatant.
RESULTS5-fluourouracil significantly increased the iNOS expression to 0.1687 +/- 0.01968 (P < 0.05, vs control group), and the concentration of nitric oxide to 213 +/- 30.2 micromol/L (P < 0.05, vs control group). The apoptotic cell rate increased significantly to 17.85 +/- 0.78%, while the necrotic cell rate decreased to 3 2.99 +/- 0.83% (P < 0.05,compared with the 5-FU group). N(omega)-nitro-L-Arginine methyl ester (L-NAME), the antagonist of L-Arg, can block the apoptotic effects of endogenous nitric oxide.
CONCLUSIONS5-FU had a synergistic effects with L-Arg by increasing the production of endogenous nitric oxide. Endogenous nitric oxide plays an important role in the process where 5-FU induces apoptosis in liver carcinoma cells. L-Arg may be a good adjuvant for chemotherapy with 5-FU.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Arginine ; pharmacology ; Fluorouracil ; pharmacology ; Humans ; Immunohistochemistry ; Liver Neoplasms ; pathology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; analysis ; Nitric Oxide Synthase Type II ; Tumor Cells, Cultured
9.The Role of Nitric Oxide in Ocular Surface Cells.
Jae Chan KIM ; Gun Sic PARK ; Jin Kook KIM ; Young Myeong KIM
Journal of Korean Medical Science 2002;17(3):389-394
The role of nitric oxide (NO) in the ocular surface remains unknown. We investigated the conditions leading to an increase of NO generation in tear and the main sources of NO in ocular surface tissue. We evaluated the dual action (cell survival or cell death) of NO depending on its amount. We measured the concentration of nitrite plus nitrate in the tears of ocular surface diseases and examined the main source of nitric oxide synthase (NOS). When cultured human corneal fibroblast were treated with NO producing donor with or without serum, the viabilities of cells was studied. We found that the main sources of NO in ocular surface tissue were corneal epithelium, fibroblast, endothelium, and inflammatory cells. Three forms of NOS (eNOS, bNOS, and iNOS) were expressed in experimentally induced inflammation. In the fibroblast culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblast cells caused by serum deprivation in a dose dependent manner up to 500 micrometer SNAP, but a higher dose decreased cell viability. This study suggested that NO might act as a doubleedged sword in ocular surface diseases depending on the degree of inflammation related with NO concentration.
Animals
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Apoptosis/drug effects/physiology
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Aqueous Humor/metabolism
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Blood Proteins/pharmacology
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Cell Survival/drug effects/physiology
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Cells, Cultured
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Epithelium, Corneal/*cytology/*enzymology
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Fibroblasts/cytology/enzymology
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Humans
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Nitric Oxide/biosynthesis/*physiology
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Nitric Oxide Donors/pharmacology
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Nitric Oxide Synthase/metabolism
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Nitric Oxide Synthase Type I
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Nitric Oxide Synthase Type II
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Nitric Oxide Synthase Type III
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Penicillamine/*analogs & derivatives/pharmacology
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Peroxynitrous Acid/biosynthesis
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Rabbits
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Tears/metabolism
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Uveitis/metabolism
10.A study of the down-regulation effect of hepatitis B virus pre-protein S2 on inducible nitric oxide synthase gene promoter.
Feng-jin GUO ; Jun CHENG ; Dong JI ; Yan LIU ; Li-ying ZHANG ; Fang-zhou SONG
Chinese Journal of Hepatology 2005;13(10):749-753
OBJECTIVESTo investigate the regulating effect of HBV pre-S2 protein on iNOS gene promoter and the molecular biological mechanisms of pre-S2 protein in HBV pathogenicity.
METHODSPolymerase chain reaction (PCR) technique was employed to amplify the sequence of iNOS promoter and 3 deletion mutants using HepG2 genomic DNA as the template, and the products were cloned into the pGEM-T vector. The iNOS gene and 3 deletion mutants were cut from T- iNOS by Kpn I and Xho I, and then cloned into pCAT3-Basic. The resulting vectors were named p1-iNOSp, p2-iNOSp, p3-iNOSp, and p4-iNOSp. Each of the reporter vectors was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-pre-S2 by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-Basic were used as a negative control. The activity of CAT in HepG2 cells transfected was detected by an ELISA kit 48 hours after the transfection, which reflected the regulating effect of HBV pre-S2 protein on iNOS gene promoter activity.
RESULTSThe expressive vector pcDNA3.1(-)-pre-S2 and report vector pCAT3-iNOSp were constructed and confirmed by restriction enzyme digestion and sequencing. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells could down-regulate the activity of p1-iNOSp, p3-iNOSp, and the inhibition rate was 54.7% and 79.5%, respectively. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells had no regulatory effects on p2-iNOSp and p4-iNOSp.
CONCLUSIONIt is suggested that HBV pre-S2 protein can down-regulate iNOS gene promoter.
Down-Regulation ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; Humans ; Nitric Oxide Synthase Type II ; genetics ; Promoter Regions, Genetic ; genetics ; Protein Precursors ; biosynthesis ; genetics ; Transcriptional Activation ; Transfection