Search results for "Luciferase"
showing 10 items of 67 documents
Single cell detection of latent cytomegalovirus reactivation in host tissue
2011
The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualiz…
Functional TSH receptor antibodies in children with autoimmune thyroid diseases
2018
The diagnostic value of the level of TSH receptor antibodies (TSHR-Ab) in the population of children with autoimmune thyroid diseases (AITDs) is still unknown. The aim of this cross-sectional study was to investigate the prevalence of TSHR-Ab in a paediatric cohort with AITD and healthy controls.A total of 240 serum samples were obtained from 205 patients with AITD, type 1 diabetes (T1D), juvenile arthritis (JA), and healthy controls (C). TSHR stimulating (TSI) and -blocking (TBI) immunoglobulins were measured in cell-based bioassays using CHO cells expressing a chimeric TSHR and a c-AMP response-element-dependent luciferase. TSI was reported as percentage of specimen-to-reference ratio (cu…
Development of aDrosophila melanogasterspliceosensor system forin vivohigh-throughput screening in myotonic dystrophy type 1
2014
AbstractAlternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1-MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the…
Inulin Derivatives Obtained <i>Via</i> Enhanced Microwave Synthesis for Nucleic Acid Based Drug Delivery
2015
A new class of therapeutic agents with a high potential for the treatment of different socially relevant human diseases is represented by Nucleic Acid Based Drugs (NABD), including small interfering RNAs (siRNA), decoy oligodeoxynucleotides (decoy ODN) and antisense oligonucleotides (ASOs). Although NABD can be engineered to be specifically directed against virtually any target, their susceptibility to nuclease degradation and the difficulty of delivery into target tissues severely limit their use in clinical practice and require the development of an appropriate nanostructured delivery system. For delivery of NABD, Inulin (Inu), a natural, water soluble and biocompatible polysaccharide, wa…
Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune r…
2010
Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5' mRNA cap analogs on the kinetics of the encoded protein, we found that m(2)(7,2'-O)Gpp(S)pG (beta-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as beta-S-ARCA(D1)-c…
APOBEC4 Enhances the Replication of HIV-1
2016
APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral L…
Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage
2021
The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation …
The unique complexity of the CYP3A4 upstream region suggests a nongenetic explanation of its expression variability.
2010
The individually variable and unpredictable expression of CYP3A4 compromises therapies with 50% of contemporary drugs. Gene variants explain only a fraction of this variability.We investigated the evolution of CYP3A4 transcriptional regulation by nuclear receptors such as the xenobiotics sensors PXR and CAR.The combination of a proximal ER6 element with XREM and CLEM represents the original scheme of CYP3A regulation by nuclear receptors in placental mammals. Among human CYP3A genes, this scheme is retained only in CYP3A4, whereas non-CYP3A4 genes lost these elements to a variable extent during primate evolution. In parallel, the number of elements outside XREM and CLEM potentially responsi…
Complex Contribution of the 3′-Untranslated Region to the Expressional Regulation of the Human Inducible Nitric-oxide Synthase Gene
2000
Cytokine stimulation of human DLD-1 cells resulted in a marked expression of nitric-oxide synthase (NOS) II mRNA and protein accompanied by only a moderate increase in transcriptional activity. Also, there was a basal transcription of the NOS II gene, which did not result in measurable NOS II expression. The 3′-untranslated region (3′-UTR) of the NOS II mRNA contains four AUUUA motifs and one AUUUUA motif, known to destabilize the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Luciferase reporter gene constructs containing the NOS II 3′-UTR showed a significantly reduced luciferase activity. The embryonic lethal abnormal vision (ELAV)-like protein HuR was found to b…
Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene.
2003
Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity c…