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.

Human SNF2L Gene Is Regulated Constitutively and Inducibly in Neural Cells via a cAMP-Response Element

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.

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.

Probiotics may Reduce Inflammation by Enhancing Peroxisome Proliferator Activated Receptor gamma Activation in HT-29 Cells / 대한소화기학회지

BACKGROUND/AIMS: The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor highly expressed in the colon which plays an anti-inflammatory role through the inhibition of nuclear factor-kappaB (NF-kappaB) pathway. Probiotics have been shown to exert beneficial effects on inflammatory bowel diseases. However, the exact mechanism by which probiotics exert protection against intestinal inflammation is not well understood. The aims of this study were to evaluate the attenuation of inflammatory response by probiotics in intestinal epithelial cells and to study the association between probiotics and PPARgamma. METHODS: HT-29 human epithelial cells were stimulated with LPS (20microgram/mL) and probiotics, Lactobacillus casei (L. casei) (10(5)-10(7) cfu/mL), or with LPS (20microgram/mL) alone for 24 hours. Interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), toll-like receptor-4 (TLR-4) and PPARgamma mRNA expressions were assessed by RT-PCR. IL-8 protein secretion was measured by ELISA. HT-29 cells were transfected with tk promoter-luciferase plasmid containing a peroxisome proliferator response element (PPRE). After stimulation with L. casei or PPARgamma agonist (15d-PGJ2 or ciglitazone), luciferase activities were measured. RESULTS: LPS induced IL-8, COX-2, TLR-4 mRNA expression, and IL-8 protein secretion in HT-29 cells. Treatment with LPS and L. casei in comparison with LPS stimulation alone lowered IL-8, COX-2, TLR-4 mRNA expression, and IL-8 protein secretion. L. casei increased PPARgamma mRNA expression in dose-dependent manner. L. casei activated PPRE in HT-29 cells transfected with PPRE3-tk-luciferase construct. CONCLUSIONS: Probiotics, L. casei, suppresses the expression of inflammatory mediators in intestinal epithelial cells. The anti-inflammatory action of L. casei might be partially related to PPARgamma activation.

Weakening of the repressive YY-1 site on the thrombospondin-1 promoter via c-Jun/YY-1 interaction

Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767~+756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407~+756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.