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1.
J Immunol Methods ; 480: 112766, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32135162

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) studies suggest that antibody-dependent cellular cytotoxicity (ADCC) influences both virus acquisition and subsequent disease outcome. Technical issues with currently available assays, however, have limited the ability to comprehensively assess the impact of ADCC on transmission and disease progression. Commonly used ADCC assays use a target cell line, CEM.NKr-CCR5-Luc, that often does not support replication of relevant HIV-1 variants. Thus, the extent of ADCC responses against a large panel of HIV-1 strains often cannot be assessed using the currently available methods. We developed two new reporter cell-lines (MT4-CCR5-Luc and PM1-CCR5-Luc) to overcome these issues. MT4-CCR5-Luc cells are resistant, whereas PM1-CCR5-Luc cells are susceptible, to killing by a natural killer cell line, CD16+KHYG-1, in the absence of antibody. Polyclonal HIVIG gave similar ADCC estimates against HIV-1 isolate, NL4-3, regardless of which of the three cell lines were used as the targets. In contrast to CEM.NKr-CCR5-Luc and PM1-CCR5-Luc, however, MT4-CCR5-Luc target cells produce significantly higher luciferase after exposure to various HIV-1 strains, including transmitted founder variants and viruses incorporating specific envelopes of interest. This higher luciferase expression does not yield spurious results because ADCC estimates are similar when killing is assessed by both reporter protein expression and flow cytometry. Furthermore, ADCC estimates derived from MT4-CCR5-Luc cells are not skewed by non-antibody contents present in human plasma. In aggregate, the MT4-CCR5-Luc cell line can be used to estimate monoclonal antibody or plasma-induced ADCC responses against a diverse range of HIV-1 envelopes relevant for transmission and disease progression studies.


Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Linfocitos/virología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Línea Celular , Técnicas de Cocultivo , Genes Reporteros , Infecciones por VIH/inmunología , Infecciones por VIH/patología , VIH-1/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Luciferasas/biosíntesis , Luciferasas/genética , Linfocitos/inmunología
2.
Cell Transplant ; 28(12): 1542-1551, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31684762

RESUMEN

This study investigated the safety of a novel cell-labeling technology with mKATE and Renilla reniformis luciferase (mKATE-renLUC) and assessed the efficacy on tracking implanted human placental stromal cells (PSC) in an erectile dysfunction (ED) animal model. Human PSC were labeled with mKATE-renLUC using a lentivirus. Cell viability, apoptosis, proliferation, migration, surface marker expression and differentiation potential of the labeled PSC were evaluated and compared with non-labeled PSC. The paracrine profile of labeled cells was examined using an angiogenesis protein array. The brightness and duration of labeled cells with different densities were evaluated. An ED rat model was established and labeled PSC were injected into cavernosal tissue of the penis. The migration and distribution of transplanted PSC were monitored using an IVIS imaging system in real time. Implanted PSC were identified in isolated tissues via detection of mKATE fluorescence. The cell viability, morphology, proliferation, migration, surface marker expression and differentiation potential of mKATE-renLUC-labeled PSC were similar to those of non-labeled cells in vitro (no statistical difference p>0.05). Similar expressions of trophic factors were found between labeled and non-labeled PSC. The migration and distribution of PSC expressing renLUC were tracked in vivo using IVIS imaging system. mKATE-positive PSC were detected in penile, kidney, prostate and hepatic tissues using histological methods. This labeling technology provides a safe and effective cell-tracking approach with a brighter fluorophore and codon-optimized luciferase.


Asunto(s)
Movimiento Celular , Proliferación Celular , Rastreo Celular , Luciferasas , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Placenta/metabolismo , Animales , Femenino , Xenoinjertos , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Células Madre Mesenquimatosas/citología , Placenta/citología , Embarazo , Ratas
3.
J Virol ; 93(14)2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31068423

RESUMEN

Wild-type mammalian reoviruses (MRVs) have been evaluated as oncolytic agents against various cancers; however, genetic modification methods for improving MRV agents have not been exploited fully. In the present study, using MRV strain T1L, we generated a reporter MRV that expresses a NanoLuc luciferase (NLuc) gene and used it for noninvasive imaging of MRV infection in tumor xenograft mice. NLuc and a P2A self-cleaving peptide gene cassette were placed upstream of the L1 gene open reading frame to enable bicistronic expression of NLuc and the L1 gene product. BALB/c nude mice intranasally infected with MRV expressing NLuc (rsT1L-NLuc) displayed bioluminescent signals in the chest area at 4 days postinfection (dpi), which is consistent with natural MRV infection in the lung. Furthermore, to monitor tumor-selective infection by MRV, nude mice bearing human cancer xenografts were infected intravenously with rsT1L-NLuc. Bioluminescent signals were detected in tumors as early as 3 dpi and persisted for 2 months. The results demonstrate the utility of an autonomous replicating reporter MRV for noninvasive live imaging of replicating oncolytic MRV agents.IMPORTANCE Engineering of recombinant MRV for improved oncolytic activity has not yet been achieved due to difficulty in generating autonomous replicating MRV harboring transgenes. Here, we constructed a reporter MRV that can be used to monitor cancer-selective infection by oncolytic MRV in a mouse model. Among the numerous oncolytic viruses, MRV has an advantage in that the wild-type virus shows marked oncolytic activity in patients without any notable adverse effects. The reporter MRV developed herein will open avenues to the development of recombinant MRV vectors armed with anticancer transgenes.


Asunto(s)
Regulación Viral de la Expresión Génica , Luciferasas/biosíntesis , Mediciones Luminiscentes , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos/metabolismo , Orthoreovirus de los Mamíferos/metabolismo , Animales , Línea Celular Tumoral , Humanos , Luciferasas/genética , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Neoplasias/virología , Virus Oncolíticos/genética , Orthoreovirus de los Mamíferos/genética , Ensayos Antitumor por Modelo de Xenoinjerto
4.
Catheter Cardiovasc Interv ; 94(5): 669-676, 2019 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-30866153

RESUMEN

OBJECTIVES: To demonstrate coronary sinus (CS) retrograde catheterization as a practicable technique for delivering biologics into the heart. BACKGROUND: There are many options to deliver biologics into the heart. However, there is no single optimal technique when considering safety, biologic retention, and reproducibility. Retrograde delivery has the potential to address many of these concerns. This study evaluated retrograde CS infusion of luciferase-expressing plasmid in a porcine model using the Advance® CS Coronary Sinus Infusion Catheter and bioluminescence imaging to track the expression of the infused biological markers. METHODS: Plasmid was delivered retrograde into the CS in one of three infusion volumes. Twenty-four hours post-infusion, hearts were excised and underwent bioluminescence imaging to characterize the expression of the infusates. Heart and lung biopsies were also assessed for luciferase expression using RT-qPCR. RESULTS: Retrograde infusion was safe and successful in all nine test subjects. Luciferase detection was inconsistent in the low volume group. Bioluminescence was confined predominantly along the posterolateral left ventricle for medium volume infusions and was more broadly dispersed along the anterior side of the heart for high volume infusions. Tissue mRNA analysis corroborated the bioluminescence results, with the highest concentration of luciferase expression localized in the left ventricle. CONCLUSIONS: Retrograde CS infusion is a promising technique for delivering biological molecules to the heart. Specifically, this study demonstrated that the low pressure coronary venous system accommodates a wide range of infusion volumes and that biological infusates can be maintained in situ following the resumption of coronary venous flow.


Asunto(s)
Cateterismo Cardíaco , Seno Coronario , Técnicas de Transferencia de Gen , Luciferasas/administración & dosificación , Plásmidos/administración & dosificación , Animales , Infusiones Intravenosas , Luciferasas/biosíntesis , Luciferasas/genética , Mediciones Luminiscentes , Modelos Animales , Miocardio/metabolismo , Plásmidos/biosíntesis , Plásmidos/genética , ARN Mensajero/biosíntesis , Sus scrofa , Factores de Tiempo
5.
Tissue Eng Part A ; 25(1-2): 69-79, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-29638193

RESUMEN

The 5'-untranslated region (5'-UTR) of mRNA contains structural elements, which are recognized by cell-specific RNA-binding proteins, thereby affecting the translation of the molecule. The activation of an innate immune response upon transfection of mRNA into cells is reduced when the mRNA comprises chemically modified nucleotides, putatively by altering the secondary structure of the molecule. Such alteration in the 5'-UTR in turn may affect the functionality of mRNA. In this study, we report on the impact of seven synthetic minimalistic 5'-UTR sequences on the translation of luciferase-encoding unmodified and different chemically modified mRNAs upon transfection in cell culture and in vivo. One minimalistic 5'-UTR, consisting of 14 nucleotides combining the T7 promoter with a Kozak consensus sequence, yielded similar or even higher expression than a 37 nucleotides human alpha-globin 5'-UTR containing mRNA in HepG2 and A549 cells. Furthermore, also the kind of modified nucleotides used in in vitro transcription, affected mRNA translation when using different translation regulators (Kozak vs. translation initiator of short UTRs). The in vitro data were confirmed by bioluminescence imaging of expression in mouse livers, 6 h postintravenous injection of a lipidoid nanoparticle-formulated RNA in female Balb/c mice. Luciferase measurements from liver and spleen showed that minimal 5'-UTRs (3 and 7) were either equally effective or better than human alpha-globin 5'-UTR. These findings were confirmed with a human erythropoietin (hEPO)-encoding mRNA. Significantly, higher levels of hEPO could be quantified in supernatants from A549 cells transfected with minimal 5'-UTR7 containing RNA when compared to commonly used benchmarks 5'-UTRs. Our results demonstrate the superior potential of synthetic minimalistic 5'-UTRs for use in transcript therapies.


Asunto(s)
Regiones no Traducidas 5' , Luciferasas , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Células A549 , Animales , Femenino , Células Hep G2 , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones Endogámicos BALB C
6.
Macromol Biosci ; 19(2): e1800242, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30444317

RESUMEN

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide-based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low-transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan-alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold-mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex-loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN-γ secretion are seen with in vivo scaffold-mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold-mediated mRNA lipoplex delivery, while protein-based immunization did not elicit IgG production until 2 weeks post-injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.


Asunto(s)
Portadores de Fármacos/uso terapéutico , ARN Mensajero/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/uso terapéutico , Alginatos/química , Alginatos/uso terapéutico , Animales , Línea Celular , Proliferación Celular , Quitosano/química , Quitosano/uso terapéutico , Portadores de Fármacos/química , Femenino , Geles/química , Geles/uso terapéutico , Inmunización , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Luciferasas/biosíntesis , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Linfocitos T/citología , Vacunas Sintéticas/química
7.
Tissue Eng Part A ; 25(1-2): 145-158, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30047313

RESUMEN

IMPACT STATEMENT: The M3RNA (microencapsulated modified messenger RNA) platform is an approach to deliver messenger RNA (mRNA) in vivo, achieving a nonintegrating and viral-free approach to gene therapy. This technology was, in this study, tested for its utility in the myocardium, providing a unique avenue for targeted gene delivery into the freshly infarcted myocardial tissue. This study provides the evidentiary basis for the use of M3RNA in the heart through depiction of its performance in cultured cells, healthy rodent myocardium, and acutely injured porcine hearts. By testing the technology in large animal models of infarction, compatibility of M3RNA with current coronary intervention procedures was verified.


Asunto(s)
Técnicas de Transferencia de Gen , Infarto del Miocardio , Miocitos Cardíacos/metabolismo , ARN Mensajero , Animales , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mensajero/farmacología , Porcinos
8.
Mol Biol (Mosk) ; 52(5): 826-835, 2018.
Artículo en Ruso | MEDLINE | ID: mdl-30363058

RESUMEN

Reporter proteins find increasing application in biomedical studies in vitro and in vivo. However, to correctly interpret the results based on their use, it is important to understand whether reporter protein production is modulated in model cells and in what conditions such modulation may occur. Reporter activity was studied in Mel IL melanoma cells transiently transfected with a pCpG vector-based plasmid construct expressing firefly luciferase. Luciferase expression quickly dropped during the first two culture passages, which were followed by a quasi-stable period, when luciferase expression relatively slightly decreased with time. Phases of maximal and minimal luciferase production, which corresponded to the exponential and stationary growth phases, respectively, were observed during batch culture. When the medium was changed, luciferase production was stimulated in the stationary, but not exponential, cell growth phase. Severe hypoxia (0.1% O2) decreased the luciferase amount, suggesting substantial modulation of cell metabolism in total and luciferase production in particular. The targeted drug vemurafenib suppressed the luciferase production in Mel IL cells, whereas DMSO, which is often used as a drug solvent in experiments with cells, stimulated the luciferase production. Based on the results, it was hypothesized that modulation of reporter protein production in mammalian cells reflects the adaptation of intracellular metabolism to external conditions and may be a source of incorrect interpretations of experiments using reporter proteins.


Asunto(s)
Luciferasas/biosíntesis , Melanoma/enzimología , Línea Celular Tumoral , Genes Reporteros , Humanos , Transfección
9.
Mol Biol (Mosk) ; 52(1): 19-23, 2018.
Artículo en Ruso | MEDLINE | ID: mdl-29512631

RESUMEN

The translation of uncapped mRNAs encoding luciferase and green fluorescent protein in a cell-free translation system based on wheat germ extract has been studied. It turned out that two simultaneously translated (in one tube) different templates in a certain range of concentrations not only do not compete, but mutually enhance each other's translation. It has been shown that the synthesis of luciferase in the presence of mRNA that encodes green fluorescent protein is much more effective than in the translation of only luciferase mRNA at the same concentration. Similarly, the efficiency of the synthesis of green fluorescent protein increases in the presence of the template that encodes luciferase. It follows that the total effect of the concurrent translation of two different mRNAs exceeds the sum of the effects of the translation of each mRNA separately.


Asunto(s)
Sistema Libre de Células , Biosíntesis de Proteínas , ARN Mensajero/genética , Triticum/genética , Proteínas Fluorescentes Verdes/biosíntesis , Luciferasas/biosíntesis
10.
Methods Mol Biol ; 1706: 303-319, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29423806

RESUMEN

The genomic era, highlighted by large scale, genome-wide association studies (GWAS) for both common and rare diseases, have identified hundreds of disease-associated variants. However, most of these variants are not disease causing, but instead only provide information about a potential proximal functional variant through linkage disequilibrium. It is critical that these functional variants be identified, so that their role in disease risk can be ascertained. Luciferase assays are an invaluable tool for identifying and characterizing functional variants, allowing investigations of gene expression, intracellular signaling, transcription factors, receptor activity, and protein folding. In this chapter, we provide an overview of the different ways that luciferase assays can be used to validate functionality of a variant.


Asunto(s)
Genes Reporteros , Sitios Genéticos , Estudio de Asociación del Genoma Completo/métodos , Desequilibrio de Ligamiento , Luciferasas , Animales , Humanos , Luciferasas/biosíntesis , Luciferasas/genética
11.
J Virol ; 91(20)2017 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-28768857

RESUMEN

While the RNA-dependent RNA polymerase L protein of rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, has potential to be a therapeutic target for rabies, the molecular functions of this protein have remained largely unknown. In this study, to obtain a novel experimental tool for molecular function analysis of the RABV L protein, we established by using a reverse genetics approach an L gene-deficient RABV (Nishi-ΔL/Nluc), which infects, propagates, and correspondingly produces NanoLuc luciferase in cultured neuroblastoma cells transfected to express the L protein. trans-Complementation with wild-type L protein, but not that with a functionally defective L protein mutant, efficiently supported luciferase production by Nishi-ΔL/Nluc, confirming its potential for function analysis of the L protein. Based on the findings obtained from comprehensive genetic analyses of L genes from various RABV and other lyssavirus species, we examined the functional importance of a highly conserved L protein region at positions 1914 to 1933 by a trans-complementation assay with Nishi-ΔL/Nluc and a series of L protein mutants. The results revealed that the amino acid sequence at positions 1929 to 1933 (NPYNE) is functionally important, and this was supported by other findings that this sequence is critical for binding of the L protein with its essential cofactor, P protein, and thus also for L protein's RNA polymerase activity. Our findings provide useful information for the development of an anti-RABV drug targeting the L-P protein interaction.IMPORTANCE To the best of our knowledge, this is the first report on the establishment of an L gene-deficient, reporter gene-expressing virus in all species of the order Mononegavirales, also highlighting its applicability to a trans-complementation assay, which is useful for molecular function analyses of their L proteins. Moreover, this study revealed for the first time that the NPYNE sequence at positions 1929 to 1933 in the RABV L protein is important for L protein's interaction with the P protein, consistent with and extending the results of a previous study showing that the P protein-binding domain in the L protein is located in its C-terminal region, at positions 1562 to 2127. This study indicates that the NPYNE sequence is a promising target for the development of an inhibitor of viral RNA synthesis, which has high potential as a therapeutic drug for rabies.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Virales , Virus de la Rabia/enzimología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Animales , Línea Celular , ARN Polimerasas Dirigidas por ADN/química , Prueba de Complementación Genética , Luciferasas/biosíntesis , Luciferasas/genética , Lyssavirus/genética , Mutación , Fosfoproteínas/metabolismo , ARN Viral/genética , Virus de la Rabia/genética , Genética Inversa , Rhabdoviridae/genética , Proteínas Virales/química , Replicación Viral
12.
Methods Mol Biol ; 1631: 109-119, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28735393

RESUMEN

In order to understand plant stress tolerance and its application, it is important to identify the signaling components involved in the stress-regulated gene expression. One initial step for this is generation of a stress-inducible luminescent Arabidopsis and its use in genetic mutant screening. Here, we describe how to generate a transgenic Arabidopsis line harboring a single copy of the STABILIZED1 (STA1) promoter-driven luciferase transgene (STA1p-LUC) as an example. STA1 is a pre-mRNA splicing factor Prp6p homolog and is induced by cold and heat stresses. In addition, generation of the STA1p-LUC mutant pool and a luminescence imaging-based screening for STA1p-LUC deregulated mutants are described.


Asunto(s)
Arabidopsis , Regulación de la Expresión Génica de las Plantas/genética , Luciferasas , Mutación , Plantas Modificadas Genéticamente , Estrés Fisiológico , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Luciferasas/biosíntesis , Luciferasas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas/genética
13.
J Virol Methods ; 247: 38-44, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28532601

RESUMEN

Bioluminescence is a powerful tool in the study of viral infection both in vivo and in vitro. Foot-and-mouth disease virus (FMDV) has a small RNA genome with a limited tolerance to foreign RNA entities. There has been no success in making a reporter FMDV expressing a luciferase in infected cell culture supernatants. We report here for the first time a replication-competent FMDV encoding Nanoluciferase, named as Nano-FMDV. Nano-FMDV is genetically stable during serial passages in cells and exhibits growth kinetics and plaque morphology similar to its parental virus. There are applications for the use of Nano-FMDV such as real-time monitoring of FMDV replication in vitro and in vivo.


Asunto(s)
Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/fisiología , Genes Reporteros , Luciferasas/biosíntesis , Coloración y Etiquetado/métodos , Replicación Viral , Animales , Línea Celular , Inestabilidad Genómica , Mediciones Luminiscentes , Ensayo de Placa Viral , Cultivo de Virus , Virosis
14.
Toxicol In Vitro ; 45(Pt 3): 359-365, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28377212

RESUMEN

In vitro effect-based reporter assays are applied as biodetection tools designed to address nuclear receptor mediated-modulation. While such assays detect receptor modulating potential, cell viability needs to be addressed, preferably in the same well. Some assays circumvent this by co-transfecting a second constitutively-expressed marker gene or by multiplexing a cytotoxicity assay. Some assays, such as the CALUX®, lack this feature. The cytotoxic effects of unknown substances can confound in vitro assays, making the interpretation of results difficult and uncertain, particularly when assessing antagonistic activity. It's necessary to determine whether the cause of the reporter signal decrease is an antagonistic effect or a non-specific cytotoxic effect. To remedy this, we assessed the suitability of multiplexing a cell viability assay within the CALUX® transcriptional activation test for anti-androgenicity. Tests of both well-characterized anti-androgens and cytotoxic compounds demonstrated the suitability of this approach for discerning between the molecular mechanisms of action without altering the nuclear receptor assay; though some compounds were both cytotoxic and anti-androgenic. The optimized multiplexed assay was then applied to an uncharacterized set of polycyclic aromatic compounds. These results better characterized the mode of action and the classification of effects. Overall, the multiplexed protocol added value to CALUX test performance.


Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Supervivencia Celular/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Receptores Androgénicos/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Antagonistas de Andrógenos/farmacología , Animales , Disruptores Endocrinos/farmacología , Marcadores Genéticos , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Hidrocarburos Policíclicos Aromáticos/toxicidad
15.
Protein Expr Purif ; 133: 102-109, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28288897

RESUMEN

Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20-30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 µM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods.


Asunto(s)
Arabidopsis/citología , Arabidopsis/metabolismo , Crustáceos/genética , Luciferasas , Células Vegetales/metabolismo , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/genética , Crustáceos/enzimología , Glicosilación , Luciferasas/biosíntesis , Luciferasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética
16.
Methods Mol Biol ; 1582: 33-46, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28357660

RESUMEN

The retrovirus Human T-lymphotropic virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission, while cell-free infection of T-cells is inefficient. Substantial insights into the different routes of transmission have largely been obtained by imaging techniques or by flow cytometry. Recently, strategies to quantify infection events with HTLV-1 improved. In this chapter, we present two different methods to quantitate virus transmission. Both methods are based on measuring gene activity of luciferase with a cost-saving in-house luciferase assay. First, we established a reporter Jurkat T-cell line carrying a luciferase gene under the control of the HTLV-1 core promoter U3R. Upon co-culture with chronically HTLV-1-infected T-cell lines, reporter cells are infected, and upon expression of the viral transactivator Tax, the viral promoter is activated resulting in enhanced luciferase activity. However, this assay as presented here does not exclude cell fusion as the mechanism allowing intracellular Tax-dependent activation of luciferase gene expression. Therefore, we describe a second method, the single-cycle replication-dependent reporter system developed by Mazurov et al. (PLoS Pathog 6:e1000788, 2010) that allows quantitation of HTLV-1 infection in co-cultured cells. Taken together, both methods facilitate quantitation of HTLV-1 transmission and will help to unravel pathways required for cell-to-cell transmission on a quantitative basis.


Asunto(s)
Citometría de Flujo/métodos , Genes Reporteros , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/transmisión , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Infecciones por HTLV-I/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Células Jurkat , Luciferasas/biosíntesis , Luciferasas/genética
17.
Methods Mol Biol ; 1582: 47-55, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28357661

RESUMEN

Unlike HIV-1, HTLV-1 viral transmission requires cell-to-cell contacts, while cell-free virions are poorly infectious and almost absent from body fluids. Though the virus uses three nonexclusive mechanisms to infect new target cells: (1) MTOC polarization followed by formation of a virological synapse and viral transfer into a synaptic cleft, (2) genesis of a viral biofilm and its transfer of embedded viruses, or (3) HTLV-1 transmission using conduits. The Tax transactivator and the p8 viral proteins are involved in virological synapse and nanotube formation respectively.HTLV-1 transcription from the viral promoter (i.e., LTR) requires the Tax protein that is absent from the viral particle and is expressed after productive infection. The present chapter focuses on a series of protocols used to quantify HTLV-1 de novo infection of target cells. These techniques do not discriminate between the different modes of transmission, but allow an accurate measure of productive infection. We used cell lines that are stably transfected with LTR-GFP or LTR-luciferase plasmids and quantified Green Fluorescent Protein expression or luciferase activity, since both of them reflect Tax expression.


Asunto(s)
Productos del Gen tax/metabolismo , Genes Reporteros , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/transmisión , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Luciferasas/biosíntesis , Técnicas de Cocultivo , Productos del Gen tax/genética , Infecciones por HTLV-I/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Células Jurkat , Luciferasas/genética
18.
Drug Deliv ; 24(1): 641-650, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28282993

RESUMEN

Visualization of a drug delivery system could reveal the pharmacokinetic properties, which is essential for the design of a novel drug delivery system. In vivo optical imaging offers an advanced tool to monitor the drug release process and the therapeutic effect by the combination of fluorescence imaging and bioluminescence imaging. Multispectral fluorescence imaging can separate the drug and the carrier without interference. Herein, a dual fluorescent anti-tumor drug delivery system was monitored with the doxorubicin-loaded hydrogel to further explore the application of the porphyrin-incorporated hydrogel with four-arm PEG-PCL copolymer as a drug carrier, based on the beneficial fluorescence and good biocompatibility of the porphyrin incorporated hydrogel. Using nude mice bearing luciferase expressed hepatic tumor as models, the whole process from the drug delivery to the tumor therapeutic effects were real time visualized simultaneously after administration at interval from 0 to 18 d. The imaging results suggest that the fluorescence signals of the drug and the carrier can be separated and unmixed from the drug-loaded hydrogel successfully, avoiding the interference of the fluorescence signals. The tumor growth or inhibition can be real time tracked and analyzed quantitatively by bioluminescence imaging. Noninvasive continuous tracking the in vivo drug delivery process simultaneously is a potential trend for the precise drug delivery and treatment.


Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Portadores de Fármacos , Colorantes Fluorescentes/química , Neoplasias Hepáticas/tratamiento farmacológico , Imagen Óptica/métodos , Poliésteres/química , Polietilenglicoles/química , Porfirinas/química , Animales , Antibióticos Antineoplásicos/química , Línea Celular Tumoral , Doxorrubicina/química , Composición de Medicamentos , Hidrogeles , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Luciferasas/biosíntesis , Luciferasas/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Tecnología Farmacéutica/métodos , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos
19.
Glycoconj J ; 34(2): 157-161, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28194603

RESUMEN

Advanced glycation end products (AGEs) are stable end products of the Maillard reaction and accumulate with progressing ageing and degenerative diseases. Significant amounts of AGE-modified peptides are also consumed with processed food. AGEs bind to specific receptors, especially the receptor of AGEs (RAGE). Activation of RAGE then evokes intracellular signalling, finally resulting in the activation of the NF-κB transcription factor and therefore a proinflammatory state. We here analysed, whether NF-κB is activated in short term upon feeding an AGE-modified protein in-vivo. Transgenic mice expressing firefly luciferase under the control of an NF-κB responsive promoter were intraperitoneally injected or fed with AGE-modified- or control albumin and luciferase expression was analysed by in-vivo imaging and by in-vitro by determination of luciferase enzyme activity in heart, lung, gut, spleen, liver and kidney. In all organs, an activation of the luciferase reporter gene was observed in response to AGE-BSA feeding, however with different intensity and timing. The gut exhibited highest luciferase activity and this activity peaked 6-8 h post AGE-feeding. In heart and kidney, luciferase activity increased for up to 12 h post feeding. All other organs tested, exhibited highest activity at 10 h after AGE-consumption. Altogether, these data demonstrate that feeding AGE-modified protein resulted in a transient and systemic activation of the NF-κB reporter.


Asunto(s)
Albúminas/farmacología , Productos Finales de Glicación Avanzada/farmacología , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional/efectos de los fármacos , Animales , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Transgénicos , FN-kappa B/genética , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transgenes
20.
Protein Expr Purif ; 132: 68-74, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28108349

RESUMEN

Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.


Asunto(s)
Proteínas de Artrópodos , Crustáceos/genética , Luciferasas , Animales , Proteínas de Artrópodos/biosíntesis , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/aislamiento & purificación , Crustáceos/enzimología , Luciferasas/biosíntesis , Luciferasas/química , Luciferasas/genética , Luciferasas/aislamiento & purificación , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Solubilidad
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