Indicators of oxidative stress in long-lived individuals belonging to the municipality of Santa Clara

Aim: To determine indicators of oxidative stress in long-lived individuals. Methods: 120 subjects were studied and two groups were formed: 50 individuals older than 85 years of nuclear families and 70 adults under 60 years old taken as a control group, all belonging to the municipality of Santa Clara. Indicators of antioxidant defense status included enzymatic activities superoxide dismutase (SOD) and catalase (CAT), as well as reduced glutathione (GSH) concentrations. The determinations were made with the use of spectrophotometric techniques, and the comparisons between the groups were made through the statistical program SPSS with a level of significance of 95 percent. Results: The activity of the antioxidant enzyme SOD and GSH levels showed significant differences when comparing both study groups. In the case of the SOD enzyme, the group of long-lived individuals showed a significant reduction in their activity compared to the controls, while GSH levels also decreased in this group. The CAT enzyme activity showed no significant differences between the two study groups. Conclusions: The decrease in enzymatic activity SOD accompanied by a decrease in GSH levels could be an indicator of a state of oxidative imbalance in individuals older than 85 years, which increases their susceptibility to the action of reactive oxygen species(AU)

Effects of space flight on protein content and electrophoresis in Glycyrrhiza uralensis / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the space environment on the role of licorice mutagenesis analysis of proteins.</p><p><b>METHOD</b>Licorice (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.</p><p><b>RESULT</b>After 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.</p><p><b>CONCLUSION</b>These 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.</p>

Transcriptional activation of insulin-like growth factor binding protein 6 by 17beta-estradiol in SaOS-2 cells

Osteoblasts can synthesize the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs), which may either enhance or attenuate IGF-stimulated bone cell proliferation. Since estrogen induced osteoblastic differentiation and proliferation through an estrogen-responsive gene in target cells, we investigated the effects of estrogen on IGFBP-6 expression in the human osteoblastic-like cell line SaOS-2. Expressions of IGFBP-6 protein and mRNA increased 2.8 and 2-fold, respectively, in the presence of 17-beta-estradiol (E2) (0.01 to 1 micrometer) and estrogen receptor (ER) in SaOS-2 cells. On the other hand, E2 induced a 2-fold increase in SaOS-2 cell proliferation. To identify genomic sequences associated with estrogen responsiveness, the 5'-promoter region (-44 to +118) of the IGFBP-6 gene was cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. E2 induced a 3-fold increase in CAT activity in SaOS-2 cells transiently transfected with this construct. Identification of the estrogen-responsive element (ERE) [5'-CCTTCA CCTG-3'] (-9 to +1) in this IGFBP-6 gene promoter region was confirmed using electromobility shift assays and deletion analysis. This functional ERE was important for E2-induced trans-activation of the IGFBP-6 gene. These results demonstrate that E2 exhibits a positive effect on IGFBP-6 gene transcription through estrogen-liganded ER binding to the functional ERE in the IGFBP-6 gene promoter in SaOS-2 cells.

Marcadores de estrés oxidativo en ratas senescentes / Oxidative stress markers in aged rats

Se realizó un estudio sobre los marcadores de daño oxidativo y de defensa antioxidante en órganos de ratas jóvenes, adultas y senescentes; mediante los cuales se evidenció un incremento con el tiempo de la peroxidación lipídica en hígado, corazón, riñón y páncreas; así como una disminución en la actividad de la enzima antioxidante catalasa en todos los órganos, excepto el páncreas. Los cambios que se producen en el envejecimiento se han asociado a un desequilibrio en los mecanismos oxidantes y antioxidantes en las células. Los resultados demostraron un estado de estrés oxidativo que pudiera favorecer la aparición de enfermedades asociadas al envejecimiento.

A study of oxidative damage and anti-oxidizing defense markers in organs from young, adult and aged rats was conducted. It was evident that lipid peroxidation in liver, heart, kidneys and pancreas increases with the time as well as anti-oxidizing enzyme catalase reduces its action in all the organs except for pancreas. Changes at the time of aging have been associated with an imbalance in oxidizing and anti-oxidizing mechanisms within the cells. The results showed a state of oxidative stress that favors the occurrence of aging-related diseases.

An analysis on transcriptional regulation activity of human XBP1 gene 5' upstream DNA sequences / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.</p><p><b>METHODS</b>Six kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.</p><p><b>RESULTS</b>The reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.</p><p><b>CONCLUSION</b>The XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.</p>

Immediate-early inducible function in upstream region of junB gene / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To analyze the upstream region of radiation-induced junB gene.</p><p><b>METHODS</b>Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/beta-actin measured by quantitative Northern blot hybridization.</p><p><b>RESULTS</b>CAT mRNA expression containing 900 bp and 1560 bp junB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation.</p><p><b>CONCLUSIONS</b>590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.</p>

Preliminary identification and analysis of point mutations correlated with response to interferon-alpha in hepatitis B virus post-transcriptional regulatory elements / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>There 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.</p>

Primary functional analysis of CK13 gene 5' flanking region / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of cytokeratin 13 (CK13) gene expression control and the effects of different motifs of CK13 gene 5' flanking region on its transcriptional activity.</p><p><b>METHODS</b>The molecular clone technique and reporter gene analysis were used to assay the effects of different motifs of 513 bp of CK13 gene 5' flanking region on its transcriptional activity. The pCAT enhancer vectors with different motifs of CK13 gene 5' flanking region were constructed and transferred to HeLa cells with the help of lipofectin. The instant CAT expression of different clones was detected and the effects of different motifs of the CK13 gene 5' flanking region on its transcriptional activity were evaluated.</p><p><b>RESULTS</b>119 bp from -nt.325 to -nt.207 upstream of the first ATG of CK13 gene 5' flanking region included a silent element. 113 bp region from -nt.206 to -nt.94 included an enhanced element.</p><p><b>CONCLUSION</b>513 bp of CK13 gene 5' flanking region includes a silent element and an enhanced element. Further locating these cis elements and detecting the related trans reaction factors may unveil some important clues to the details of the mechanisms for the CK13 gene expression and tissue-specific expression.</p>

Caracterización molecular de mecanismos de resistencia a cloranfenicol en cepas de shigella flexneri aisladas en niños chilenos con diarrea aguda / Molecular characterization of resistance mechanisms to chloramphenicol in shigella flexneri strains isolated from chilean children with acute diarrhea

Background: Chloramphenicol is one of the therapeutic options for shigellosis, but resistance to this antimicrobial is increasing. Aim: To characterize molecular mechanisms conferring resistance to chloramphenicol (CmR) in Shigella flexneri strains isolated from Chilean children with acute diarrhea. Material and methods: Thirty one Shigella filexneri strains, including 22 with the CmR phenotype were analyzed. Strains were tested for antimicrobial susceptibility by plate dilution and for the presence of an internal fragment of the cat gene encoding for chloramphenicol o-acetyl-transferase, by polymerase chain reaction and Southern blot analysis. Results: All CmR strains had a minimal inhibitory concentration over 64 µg/ml and amplified the internal fragment of the cat gene. Southern blot analyses indicated that this gene was located in the bacterial chromosome. Conclusions: Resistance to chloramphenicol in this group of Shigella flexneri strains was mediated by a chromosomally located cat gene

The Effect of Dehydroepiandrosterone on Expression of Elastin in Cultured Skin Fibroblasts / 대한피부과학회지

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.

Improved expression by cytomegalovirus promoter/enhancer and behavior of vascular endothelial growth factor gene after myocardial injection of naked DNA

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart

Improved expression by cytomegalovirus promoter/enhancer and behavior of vascular endothelial growth factor gene after myocardial injection of naked DNA

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart

Establishment of a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase gene / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>SDS-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.</p><p><b>CONCLUSIONS</b>The 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.</p>

Identification of AP1 cis-element and transcriptional effect on cytokeratin 13 gene expression / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the type of CTGAATCA from -nt.199 to -nt.192 of the cytokeratin 13(CK13) gene 5' flanking region and determine its transcriptional effect on CK13 gene expression.</p><p><b>METHODS</b>The CAT systems were used to assess the effects of different motifs of CK13 gene 5' flanking region on transcription. The clones of pCAT-enhancer with the total length, -nt.207 to +nt.63 and the same length of -nt.207 to +nt.63, but the T, G of -nt.198, -nt.197 being changed to A, T of the CK13 gene 5' flanking region, were constructed and transferred to HeLa cells with the help of lipofectin. Then work was done to detect the instant CAT expression of different clones and evaluate the effects of CTGAATCA of the 5' flanking region on CK13 gene expression. The type of the cis-element of CTGAATCA was identified with electrophoretic mobility shift assay (EMSA) and competition-EMSA.</p><p><b>RESULTS</b>CTGAATCA in the CK13 gene 5' flanking region is an AP1 cis-element by EMSA and competition-EMSA, it promotes CK13 gene expression.</p><p><b>CONCLUSION</b>CTGAATCA from -nt.199 to nt.192 of the CK13 gene 5' flanking region is an AP1 reaction element, not a cAMP reaction element. It promotes transcriptional activity of CK13 gene 5' flanking region.</p>

Polymorphisms and functions of the aldose reductase gene 5' regulatory region in Chinese patients with type 2 diabetes mellitus / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function.</p><p><b>METHODS</b>The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins.</p><p><b>RESULTS</b>Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P < 0.05) respectively, and the frequencies of WT/C (-12) G were 10.5% and 2.5% (P > 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P < 0.025). The relative transcription activities of the wild-type, the C (-12) G and the C (-106) T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors.</p><p><b>CONCLUSION</b>The polymorphisms C (-12) G and C (-106) T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.</p>

Rapid assay for detection of chloramphenicol acetyl transferase in Haemophilus influenzae.

BACKGROUND & OBJECTIVES: Haemophilus influenzae causes a variety of life threatening infections in humans. Early detection of antimicrobial resistance is of importance in the treatment and management of infection. The modified Slack's method, a simple assay, has been evaluated in this study for the early detection of chloramphenicol resistance. METHODS: Fifty isolates of H. influenzae from invasive and non-invasive sites were included. Antimicrobial susceptibility testing was done by disc diffusion method and minimum inhibitory concentration (MIC) determination was performed for chloramphenicol only. Modified Slack's method was used to test for the production of chloramphenicol acetyl transferase (CAT). RESULTS: Invasive isolates showed higher degree of resistance to chloramphenicol (72%) compared to non-invasive ones (28%). One hundred per cent association was found between results of disc diffusion, MIC and CAT production amongst strains resistant to chloramphenicol. INTERPRETATION & CONCLUSION: The findings suggested that chloramphenicol still remains the drug of choice for treatment for non-invasive infection caused by H. influenzae. Modified Slack's method is a simple, rapid, inexpensive and reliable method for the detection of chloramphenicol resistance amongst H. influenzae.

The Effect of Nicotine on Elastin Gene Expression in Cultured Skin Fibroblasts / 대한피부과학회지

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.

American Ginseng Transcriptionally Activates p21 mRNA in Breast Cancer Cell Lines

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.

Nicotine Down-regulates COL1A2 Promoter in Cultured Human Skin Fibroblasts

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.

Cloning and characterization of 5'-upstream region of human phospholipase C-beta2 gene

5'-upstream region of the phospholipase C-beta2 gene, 810 bp, was cloned and characterized. S1 nuclease mapping and primer extension analyses revealed that a single transcriptional start site locates at 284 nucleotides upstream from the beginning of translation. The 5-upstream region lacks both TATA motif and typical initiator sequence, but retains GC-rich segment. Two putative regulatory regions, a negative region (-636/-588) and a positive region (-98/ -13) were identified in the upstream region of PLC-beta2 gene. We suggest that the transcription of PLC-beta2 may be regulated by binding of regulatory proteins to the negative and/or positive regulatory regions located in the upstream of the gene.