In excitable and endocrine organs, calcium influxes through the L-type voltage-gated calcium channel (VGCC) which is composed of four (alpha 1, alpha 2, beta, and gamma) subunits. Temporal and spatial expression of calcium channel activity is regulated by the transcription of alpha 1 subunit. To elucidate the genomic organization of the VGCC alpha 1D subunit gene, a genomic clone was isolated from the human genomic library and its sequence was analyzed. A 12 kb genomic clone contained the 5'-flanking regulatory region and first two exons was selected and the initiation site for alpha 1D mRNA synthesis was examined by primer extension analysis. The major initiation site was found at the -523 NT position in the translation initiation site. The TATA box could not be found above the transcription initiation site. The CAT vector construct containing the 2.5 kb upstream region had high CAT activity on transfection to NG108-15 and PC12 cells, which confers the neuronal expression of the alpha 1D gene.
Amino Acid Sequence ; Base Sequence ; Calcium Channels/genetics* ; Chloramphenicol O-Acetyltransferase/genetics ; Cloning, Molecular ; Gene Library ; Genetic Vectors ; Human ; Molecular Sequence Data ; Regulatory Sequences, Nucleic Acid* ; Restriction Mapping

Since Steeg, et al.(1988) identified NM23/NDP kinase as non -metastasis gene, other multiple functions of have reported. One of them, Postel, et al.(1993) suggested that transcription factor PuF, being encoded by NM23 -H2/NDP kinase gene, interacts with nuclease hypersensitive element located upstream of the c -myc gene. C -myc amplification and activation can be present in squamous cell carcinoma of the head and neck as well as in an increased metastatic propensity for individual tumor. To clarify the role of NM23/NDP kinase on c -myc expression, comparison of these two gene expressions in cell lines was done. No direct correlation of expression kinetics was found. A plasmid containing human c -myc fragment was cloned upstream of chloramphenicol acetyltransferase (CAT) gene. When murine melanoma cell line was cotransfected with a murine NM23 -M2 including expression vector and c -myc CAT, CAT activity was elevated, while no change of CAT activity was found in the cotransfectant of human NM23 -H2 and c -myc CAT. Data suggest that murine NM23 -M2 gene transactivates c -myc gene indirectly with a cellular factor in murine cell line which dose not work with human NM23 -H2 gene. Additionally, we found same kinetics of NM23 -H2/NDP kinase and c -myc expression change correlated with proliferation of PLC/PRF/5 which was induced by HGF.
Animals ; Carcinoma, Squamous Cell ; Cats ; Cell Line* ; Chloramphenicol O-Acetyltransferase ; Clone Cells ; Gene Expression ; Head ; Humans ; Kinetics ; Melanoma ; Neck ; Phosphotransferases* ; Plasmids ; Transcription Factors ; Transcriptional Activation

OBJECTIVETo investigate the space environment on the role of licorice mutagenesis analysis of proteins.

METHODLicorice (Glycyrrhiza uralensis) seeds were carried by a recoverable satellite for 18 days (the average radiation dose in the flight recovery module was 0.102 m x d(-1), the orbit semidiameter 350 km, gravity 10(-6)). After return, The satellite-flown seeds and the unflown seeds (ground control) were planted in the fields of experimental farm. The leaves of each group were used for studying the effects of space flight on CAT, SOD activity, the protein content and electrophoresis.

RESULTAfter the space flight, CAT, SOD activity of licorice increased in varying degrees, the difference was significant (P<0.05), two types of enzyme activity of sample from Ordos were higher than that from Hangjinqi. The protein content of licorice increased in a certain extent, the difference was significant (P<0.05), while protein electrophoresis also showed differences, weak new bands appeared.

CONCLUSIONThese results indicated that spaceflight has effect on protein of licorice, these changes may be used as a tool for accelerating the progress in G. uralensis breeding.

Chloramphenicol O-Acetyltransferase ; analysis ; metabolism ; Electrophoresis ; Extraterrestrial Environment ; Glycyrrhiza uralensis ; chemistry ; enzymology ; Plant Proteins ; analysis ; metabolism ; Spacecraft ; Superoxide Dismutase ; analysis ; metabolism

BACKGROUND: Sun exposure and therapeutic irradiation have been shown to induce alterations in extracellular matrix (ECM) proteins, including elastin, glycosaminoglycan and collagens. The integrity of the connective tissue mainly depends on balanced rates of matrix synthesis and degradation of the extracellular matrix. Therefore, matrix metalloproteinases (MMPs) may be involved in ultraviolet irradiation (UVR)-induced alterations in ECM proteins. OBJECTIVE: To evaluate the effects of UVA as well as UVB irradiations on ST-1 gene expression in cultured human skin fibroblasts. METHODS: After exposure of different doses of UVA and UVB on cultured human skin fibroblasts, we examined the expression of ST-1 gene by Northern blot analysis, chloramphenicol acetyltransferase (CAT) assay with CAT construct containing AP-1 binding site. Additionally, we carried out the gel mobility shift assay to investigate the effects of UVR on the DNA-binding activity of AP-1. RESULTS: After UVR on fibroblasts, the steady-state levels of ST-1 mRNA were in-creased in response to UVA and UVB by 2.5-fold and 4.2-fold, respectively, as compared with controls. Similar results were obtained by CAT assay showing that CAT activity increased as the UVA and UVB doses increased. Furthermore, gel mobility shift assay demonstrated that both UVA and UVB increased AP-1 DNA binding complexes. CONCLUSION: UVB as well as UVA up-regulated ST-1 gene expression at transcriptional levels in vitro. We speculate that modulation of MMPs, including ST-1, gene expression by UVR may contribute to the connective tissue damage related to photoaging and other photocutaneous disorders.
Animals ; Binding Sites ; Blotting, Northern ; Cats ; Chloramphenicol O-Acetyltransferase ; Collagen ; Connective Tissue ; DNA ; Elastin ; Electrophoretic Mobility Shift Assay ; Extracellular Matrix ; Fibroblasts* ; Gene Expression ; Humans* ; In Vitro Techniques ; Matrix Metalloproteinases ; RNA, Messenger ; Skin ; Solar System ; Transcription Factor AP-1

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p< or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p< or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.
Animal ; Binding Sites ; Breast Neoplasms ; Cell Division/drug effects ; Chloramphenicol O-Acetyltransferase/genetics ; Cyclins/*genetics ; Female ; Genes, Reporter ; HT29 Cells ; Human ; Mice ; *Panax ; Plant Extracts/pharmacology ; Protein p53/metabolism ; *RNA, Messenger ; *Trans-Activation (Genetics) ; Tumor Cells, Cultured

BACKGROUNDIt is still unclear whether viral genetic variability influences response to interferon (IFN)-alpha treatment. Recent reports suggest that IFN-alpha effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-alpha treatment.

METHODSThe HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines.

RESULTSThe T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-alpha/gamma and tumor necrosis factor (TNF)-alpha except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-alpha on HPRE.

CONCLUSIONSThere are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-alpha. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-alpha other than that of IFN-gamma or TNF-alpha on HPRE.

Chloramphenicol O-Acetyltransferase ; metabolism ; Genes, Regulator ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; pharmacology ; Plasmids ; Point Mutation ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUND: It has become generally accepted that cigarette smoking contributes to accelerated coronary and peripheral vascular disease, pulmonary fibrosis and periodontal disease. Moreover, it has been postulated that cigarette smoking causes skin-aging. Many of cutaneous manifestations of nicotine which is a major component of the particulate phase of tobacco smoke are related to its vasoconstrictive and thrombotic effects on the peripheral vascular system. How-ever, direct effect of nicotine on extracellular matrix (ECM) proteins including collagens is not well established. OBJECTIVE: To evaluate the effect of nicotine on type I collagen gene expression in cultured human skin fibroblasts. METHODS: After exposure to different doses of nicotine on cultured human skin fibroblasts, we examined the expressions of α1(I) procollagen gene and fibronectin gene by Northern blot analysis and chloramphenicol acetyltransferase (CAT) assay with CAT construct containing the 3.5 kb COL1A2 promoter. RESULTS: In Northern blot hybridization, steady-state levels of α1(I) procollagen mRNA were decreased 0.8-fold at 1 µg/mL of nicotine, 0.5-fold at 10 µg/mL and 0.2-fold at 100 µg/mL, compared to untreated control. Those of fibronectin mRNA were decreased 0.9-fold, 0.7-fold, and 0.3-fold, respectively. In CAT assay, the relative COL1A2 CAT activity was 1.0 in the untreated control, 0.7 at a concentration of 1 µg/mL of nicotine, 0.5 at 10 µg/mL, and 0.3 at 100 µg/mL. CONCLUSION: These results indicate that nicotine is a down-regulator of collagen gene expression at transcriptional level in vitro. We speculate that nicotine may contribute to the skin-aging by modulation of extracellular matrix gene expression including collagen as well as by its vasoconstrictive and thrombotic effects.
Animals ; Blotting, Northern ; Cats ; Chloramphenicol O-Acetyltransferase ; Collagen ; Collagen Type I ; Extracellular Matrix ; Fibroblasts* ; Fibronectins ; Gene Expression ; Humans* ; Nicotine* ; Periodontal Diseases ; Peripheral Vascular Diseases ; Procollagen ; Pulmonary Fibrosis ; RNA, Messenger ; Skin* ; Smoke ; Smoking ; Tobacco

BACKGROUND: The elastic fibers are a major fibrillar component of the extracellular matrix of several organs, and their presence provides elastic properties to these tissues. A variety of cytokines, growth factors, and hormones have been shown to modulate elastin gene expression. So far most interest increased the effects of external environment on elastin metabolism in the skin. It has become generally accepted that cigarette smoking contributes to accelerated coronary and peripheral vascular disease, pulmonary fibrosis and periodontal disease. Nicotine is a major component of the particulate phase of tobacco smoke. OBJECTIVE: Only little is known about the molecular and cellular mechanism underlying the effect of nicotine on the skin fibroblasts. Our study was performed to determine the effects of nicotine on elastin gene expression. METHOD: In this study, the effects of nicotine were examined by Northern blot hybridization, chloramphenicol acetyltransferase (CAT) assay, and laser scanning microscopy in cultured human fibroblasts. RESULTS: In Northern blot hybridization, steady-state levels of elastin mRNA were decreased 0.9-fold at 1 microgram/mL of nicotine, 0.7-fold at 10 microgram/mL and 0.5-fold at 100 microgram/mL, compared to untreated control. Nicotine caused a marked alteration in the elastin mRNA expression in a dose-related fashion. In CAT assay, the relative elastin CAT activity was 1.0 in the untreated control, 0.9 at a concenturation of 1 microgram/mL, 0.3 at 10 microgram/mL, and 0.2 at 100 microgram/mL. Nicotine caused a marked decrease on elastin promoter activity. In laser scanning microscopy, the immunosignal for elastin in nicotine-treated fibroblasts shows in less intense than in untreated control. CONCLUSION: These results indicate that nicotine may be a powerful down-regulator of elastin production, suggesting transcriptional depression of gene expression in cultured skin fibroblasts.
Animals ; Blotting, Northern ; Cats ; Chloramphenicol O-Acetyltransferase ; Cytokines ; Depression ; Elastic Tissue ; Elastin* ; Extracellular Matrix ; Fibroblasts* ; Gene Expression* ; Humans ; Intercellular Signaling Peptides and Proteins ; Metabolism ; Microscopy, Confocal ; Nicotine* ; Periodontal Diseases ; Peripheral Vascular Diseases ; Pulmonary Fibrosis ; RNA, Messenger ; Skin* ; Smoke ; Smoking ; Tobacco

BACKGROUND: Dehydroepiandrosterone(DHEA) and its sulfate ester dehydro- epiandrosterone sulfate(DHEA-S) are the steroids secreted most abdundantly by the human adrenal gland, but the physiologic role remains uncertain. Elastin is one of the major extracellular matrix components of the dermis and plays an important role in providing elasticity and resilience of the skin. Expression of elastin genes at transcriptional level is regulated and modulated by cytokines, vitamin D3, insulin-like growth factor-1, and steroids. But only little is known about the molecular and cellular mechanism underlying the effect of DHEA on the expression of elastin in cultured skin fibroblasts. OBJECTIVE: The purpose of this study was to examine the effect of DHEA on elastin gene expression in cultured skin fibroblasts. METHODS: In this study, the effects of DHEA were examined by Northern blot hybridization, chloramphenicol acetyltransferase assay(CAT), and laser scanning microscopy in cultured human fibroblasts. RESULTS: In Northern blot hybridization, levels of elastin mRNA were increased 1.5-fold at 1 nmol of DHEA, 4.2-fold at 0.1 mol and 6.5-fold at 10 mol, compared to untreated control. DHEA caused a alteration in the elastin mRNA expression in a dose-related fashion. In CAT assay, the relative mRNA CAT activity was 0.9 at a concentration of 1 nmol, 2.4 at 0.1 mol, and 2.7 at a 10 mol. DHEA caused a marked increase on elastin promotor activity. In confocal laser scanning microscopy, the immunosignal for elastin in DHEA-treated fibroblasts is more intense compared to the control. CONCLUSION: These results indicate that DHEA may be a powerful up-regulator of elastin production, suggesting transcriptional activation of gene expression in cultured skin fibroblasts.
Adrenal Glands ; Androsterone ; Animals ; Blotting, Northern ; Cats ; Chloramphenicol O-Acetyltransferase ; Cholecalciferol ; Cytokines ; Dehydroepiandrosterone* ; Dermis ; Elasticity ; Elastin* ; Extracellular Matrix ; Fibroblasts* ; Gene Expression ; Humans ; Microscopy, Confocal ; RNA, Messenger ; Skin* ; Steroids ; Transcriptional Activation

BACKGROUNDTo establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.

METHODSThe full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T. The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits. The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established. Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro. The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay.

RESULTSSDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000. The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells. Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method.

CONCLUSIONSThe established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities. Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene.

Animals ; Antibodies ; analysis ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase ; analysis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Genes, Reporter ; Male ; Papillomaviridae ; genetics ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; analysis ; biosynthesis ; immunology