Development and optimization of a cell screening system for farnesoid X receptor agonist / 生物工程学报

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

VIPR1 promoter methylation promotes transcription factor AP-2α binding to inhibit VIPR1 expression and promote hepatocellular carcinoma cell growth in vitro / 南方医科大学学报

OBJECTIVE@#To explore the transcriptional regulation mechanism and biological function of low expression of vasoactive intestinal peptide receptor 1 (VIPR1) in hepatocellular carcinoma (HCC).@*METHODS@#We constructed plasmids carrying wild-type VIPR1 promoter or two mutant VIPR1 promoter sequences for transfection of the HCC cell lines Hep3B and Huh7, and examined the effect of AP-2α expression on VIPR1 promoter activity using dual-luciferase reporter assay. Pyrosequencing was performed to detect the changes in VIPR1 promoter methylation level in HCC cells treated with a DNA methyltransferase inhibitor (DAC). Chromatin immunoprecipitation was used to evaluate the binding ability of AP-2α to VIPR1 promoter. Western blotting was used to assess the effect of AP-2α knockdown on VIPR1 expression and examine the differential expression of VIPR1 in the two cell lines. The effects of VIPR1 overexpression and knockdown on the proliferation, cell cycle and apoptosis of HCC cells were analyzed using CCK8 assay and flow cytometry. We also observed the growth of HCC xenograft with lentivirus-mediated over-expression of VIPR1 in nude mice.@*RESULTS@#Compared with the wild-type VIPR1 promoter group, co-transfection with the vector carrying two promoter mutations and the AP-2α-over-expressing plasmid obviously restored the luciferase activity in HCC cells (P < 0.05). DAC treatment of the cells significantly decreased the methylation level of VIPR1 promoter and inhibited the binding of AP-2α to VIPR1 promoter (P < 0.01). The HCC cells with AP-2α knockdown showed increased VIPR1 expression, which was lower in Huh7 cells than in Hep3B cells. VIPR1 overexpression in HCC cells caused significant cell cycle arrest in G2/M phase (P < 0.01), promoted cell apoptosis (P < 0.001), and inhibited cell proliferation (P < 0.001), while VIPR1 knockdown produced the opposite effects. In the tumor-bearing nude mice, VIPR1 overexpression in the HCC cells significantly suppressed the increase of tumor volume (P < 0.001) and weight (P < 0.05).@*CONCLUSION@#VIPR1 promoter methylation in HCC promotes the binding of AP-2α and inhibits VIPR1 expression, while VIPR1 overexpression causes cell cycle arrest, promotes cell apoptosis, and inhibits cell proliferation and tumor growth.

Metformin and lipopolysaccharide regulate transcription of NFATc2 gene via the transcription factor RUNX2 / 南方医科大学学报

OBJECTIVE@#To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene.@*METHODS@#The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription.@*RESULTS@#We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively.@*CONCLUSION@#pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.

The Establishment and Identification of Acute Myeloid Leukemia NOD-SCID-IL2rg / 中国实验血液学杂志

OBJECTIVE@#To establish the in vivo traceable acute myeloid leukemia mice model with Luciferase-Expressing KG1a Cells.@*METHODS@#KG1a cells with stable luciferase gene expression (called as KG1a-Luc cells) were constructed by lentivirus transfection, then sifted out by puromycin. Eighteen male NOD-SCID-IL2rg@*RESULTS@#KG1a cells expressing luciferase stably were successfully obtained. The tumor luminescence wildly spread at day 17 captured by in vivo imaging. The KG1a-Luc tumor cells could be detected in the peripheral blood of the mice, with the average percentage of (16.27±6.66)%. The morphology and pathology result showed that KG1a-Luc cells infiltrate was detected in bone marrow, spleens and livers. The survival time of the KG1a-Luc mice was notably shorter as compared with those in the control group, the median survival time was 30.5 days (95%CI: 0.008-0.260).@*CONCLUSION@#The acute myeloid leukemia NOD-SCID-IL2rg

The luciferase reporter system of the MMP12 endogenous promoter for investigating transcriptional regulation of the human MMP12 gene

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

ABSTRACT Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

Prevalência de dor crônica e sua associação com a situação sociodemográfica e atividade física no lazer em idosos de Florianópolis, Santa Catarina: estudo de base populacional / Prevalence of chronic pain and its association with the sociodemographic situation and physical activity in leisure of elderly in Florianópolis, Santa Catarina: population-based study

OBJETIVO: Estimar a prevalência de dor crônica e sua associação com a situação socioeconômica, demográfica e atividade física no lazer em idosos. MÉTODOS: Este estudo é parte do inquérito epidemiológico e transversal de base populacional e domiciliar EpiFloripa Idoso 2009-2010 realizado com 1.705 idosos (≥ 60 anos), residentes em Florianópolis, Santa Catarina. A partir da resposta afirmativa de dor crônica, foram investigadas as associações com as variáveis obtidas por meio de entrevista estruturada. Realizou-se a estatística descritiva, incluindo cálculos de proporções e intervalos de confiança 95% (IC95%). Na análise bruta e ajustada, empregou-se regressão de Poisson, estimando-se as razões de prevalência, com intervalos de confiança de 95% e valores p ≤ 0,05. RESULTADOS: Dentre os idosos investigados, 29,3% (IC95% 26,5 - 32,2) relataram dor crônica. Na análise ajustada, observou-se que as variáveis sexo feminino, menor escolaridade e pior situação econômica ficaram associadas significativamente com maior prevalência de dor crônica; ser fisicamente ativo no lazer ficou associado significativamente com menor prevalência do desfecho. CONCLUSÕES: Percebe-se que a dor crônica é um agravo que acomete considerável parcela de idosos, havendo desigualdades sociais na sua frequência e sendo beneficamente afetada pela atividade física no lazer. É necessário que políticas públicas de saúde subsidiem programas multidisciplinares de controle da dor incluindo a prática regular de atividade física, voltada especificamente à promoção da saúde do idoso, evitando assim que a dor crônica comprometa a qualidade de vida desta população. .

OBJECTIVE: To estimate the prevalence of chronic pain and its association with socioeconomic and demographic status, and leisure physical activity in the elderly population. METHODS: This study is part of an epidemiological cross-sectional population-based household survey called EpiFloripa Elderly 2009-2010, which was conducted with 1,705 elderly individuals (≥ 60 years) residents of Florianópolis, Santa Catarina. From the positive response to chronic pain, the associations with the variables were investigated through a structured interview. Descriptive statistics were conducted, including ratio calculation and 95% confidence intervals. In crude and adjusted analysis, Poisson regression was utilized, estimating prevalence ratios, with 95% confidence intervals and ≤ 0.05 p-values. RESULTS: Among the subjects, 29.3% (IC95% 26.5 - 32.2) reported chronic pain. Adjusted analysis showed that being female, having less years of schooling, and being in worse economic situation were significantly associated with a higher prevalence of chronic pain. Being physically active during leisure time was significantly associated with lower prevalence of the outcome. CONCLUSIONS: Therefore, it is clear that chronic pain affects a considerable amount of elderly individuals. Social inequalities are a harmful influence in these individuals' quality of life, inasmuch as those inequalities increase the frequency with which chronic pain afflicts them. At the same time, physical activity during leisure time decreases chronic pain frequency. It is fundamental that public health policies subsidize multidisciplinary pain management programs, which should include health targeted physical activity for the elderly, thus preventing the decrease in quality of life that chronic pain brings to this population. .

A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression

PURPOSE: Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects. MATERIALS AND METHODS: In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo. RESULTS: NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons. CONCLUSION: We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-gamma/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/µl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/µl group, 5 in 38 alive pups in the 1 ng/µl group and 1 in 21 alive pups in the 4 ng/µl group ( p < 0.1). The overall transgenic efficiency was higher for the 1 ng/µl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.

Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation

OBJECTIVE: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF). METHODS: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart. CONCLUSION: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia. .

Novel GATA5 loss-of-function mutations underlie familial atrial fibrillation

OBJECTIVE: This study aimed to identify novel GATA5 mutations that underlie familial atrial fibrillation. METHODS: A total of 110 unrelated patients with familial atrial fibrillation and 200 unrelated, ethnically matched healthy controls were recruited. The entire coding region of the GATA5 gene was sequenced in 110 atrial fibrillation probands. The available relatives of the mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional effect of the mutated GATA5 was characterized using a luciferase reporter assay system. RESULTS: Two novel heterozygous GATA5 mutations (p.Y138F and p.C210G) were identified in two of the 110 unrelated atrial fibrillation families. These missense mutations cosegregated with AF in the families and were absent in the 400 control chromosomes. A cross-species alignment of GATA5 protein sequence showed that the altered amino acids were completely conserved evolutionarily. A functional analysis revealed that the mutant GATA5 proteins were associated with significantly decreased transcriptional activation when compared with their wild-type counterpart. CONCLUSION: The findings expand the spectrum of GATA5 mutations linked to AF and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation, suggesting potential implications for the early prophylaxis and personalized treatment of this common arrhythmia.

Diagnostic Value of a Chimeric TSH Receptor (Mc4)-Based Bioassay for Graves' Disease

BACKGROUND/AIMS: Graves' disease (GD) is caused by thyroid-stimulating hormone receptor (TSHR) and thyroid-stimulating immunoglobulin (TSI). We used a recently introduced, technically enhanced TSI bioassay to assess its diagnostic value and determine the cut-off in patients in high iodine intake area. METHODS: In a cross-sectional setting, we collected serum from 67 patients with untreated GD, 130 with GD under treatment, 22 with GD in remission, 42 with Hashimoto's thyroiditis, 12 with subacute thyroiditis, 20 with postpartum thyroiditis, and 93 euthyroid controls. TSI was measured using the Thyretaintrade mark bioassay, which is based on Chinese hamster ovary cells transfected with chimeric TSHR (Mc4). TSI levels are reported as a specimen-to-reference ratio percentage (SRR%). RESULTS: The TSI levels in patients with GD (either treated or not) were significantly higher than those of the remaining patients (p < 0.05). The new bioassay showed a sensitivity of 97.0% and a specificity of 95.9% with a cut-off value of 123.0 SRR% for GD. A weak correlation was found between TSI and thyrotropin-binding inhibiting immunoglobulin (TBII) (rs = 0.259, p = 0.03), but no correlation was found between TSI and tri-iodothyronine or free thyroxine. CONCLUSIONS: The Mc4-CHO bioassay showed comparable diagnostic value for GD with the conventional TBII assay. We propose a cut-off of 123.0 SRR% in areas where iodine intake is high.

Aerosol delivery of kinase-deficient Akt1 attenuates Clara cell injury induced by naphthalene in the lungs of dual luciferase mice

Conventional lung cancer therapies are associated with poor survival rates; therefore, new approaches such as gene therapy are required for treating cancer. Gene therapies for treating lung cancer patients can involve several approaches. Among these, aerosol gene delivery is a potentially more effective approach. In this study, Akt1 kinase-deficient (KD) and wild-type (WT) Akt1 were delivered to the lungs of CMV-LucR-cMyc-IRES-LucF dual reporter mice through a nose only inhalation system using glucosylated polyethylenimine and naphthalene was administrated to the mice via intraperitoneal injection. Aerosol delivery of Akt1 WT and naphthalene treatment increased protein levels of downstream substrates of Akt signaling pathway while aerosol delivery of Akt1 KD did not. Our results showed that naphthalene affected extracellular signal-regulated kinase (ERK) protein levels, ERK-related signaling, and induced Clara cell injury. However, Clara cell injury induced by naphthalene was considerably attenuated in mice exposed to Akt1 KD. Furthermore, a dual luciferase activity assay showed that aerosol delivery of Akt1 WT and naphthalene treatment enhanced cap-dependent protein translation, while reduced cap-dependent protein translation was observed after delivering Akt1 KD. These studies demonstrated that our aerosol delivery is compatible for in vivo gene delivery.

Transcriptional analysis of the human PAX9 promoter

OBJECTIVES: PAX9 belongs to the Pax family of transcriptional factor genes. This gene is expressed in embryonic tissues such as somites, pharyngeal pouch endoderm, distal limb buds and neural crest-derived mesenchyme. Polymorphisms in the upstream promoter region of the human PAX9 have been associated with human non-syndromic tooth agenesis. In the present study, we verified the in vitro mRNA expression of this gene and the luciferase activity of two constructs containing promoter sequences of the PAX9 gene. MATERIAL AND METHODS: Embryonic tissues were obtained from digits, face, and midbrain/hindbrain regions. Fragments containing PAX9 promoter sequences were cloned into reporter plasmids and were transfected into the different cell cultures. mRNA were extracted from primary cell cultures. RESULTS: The semi-quantitative RT-PCR results showed that in vitro E13.5 limb bud and CNS cells express PAX9, but cells derived from the facial region do not. Moreover, the luciferase assay showed that protein activity of the constructed vector was weaker than pgl3 -basic alone. CONCLUSIONS: The present results suggest that the promoter sequences analyzed are not sufficient to drive PAX9 gene transcription.

Transcriptional Activity of Plasmodium Subtilisin-like Protease 2 (Pf-Sub2) 5'Untranslated Regions and Its Interaction with Hepatocyte Growth Factor

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants. In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf). Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5'untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to +12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5'untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.

Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

In vivo determination of the gap2 gene promoter activity in Giardia lamblia

A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.

Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ia. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.

The Agamma-195 (C->G) mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression