Animals ; Antigens, Surface/genetics* ; Cell Communication/genetics* ; Copulation/physiology* ; Fallopian Tubes/metabolism* ; Female ; Fertilization/genetics* ; GPI-Linked Proteins/genetics* ; Gene Expression Regulation ; Genes, Reporter ; Green Fluorescent Proteins/metabolism* ; Litter Size ; Luminescent Proteins/metabolism* ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism* ; Reproduction/genetics* ; Signal Transduction ; Sperm Count ; Sperm Motility/genetics* ; Spermatozoa/metabolism* ; Uterus/metabolism*

Ebola virus, the cause of severe and fatal hemorrahagic fever in humans, belongs to filovirus family. This study was designed to establish a cell-based screening and evaluation system in the pharmacological study of antivirus compounds. Three reporter systems were established with recombinant pseudoviral luciferase of HIV core (pNL4-3.Luc.R(-)E(-)) packed with filovirus glycoprotein (EBOV-Zaire GP/HIV-luc, EBOV-Sudan GP/HIV-luc and Marburg GP/HIV-luc), which are required for virus entry of cells. The level of filovirus entry was determined by the expression of luciferase reporter gene in the infected cells. For screening of filovirus entry inhibitors, the vesicular stomatitis G packed pseudovirions (VSVG/HIV-luc) was used to determine the compound specificity. The results of known filovirus entry inhibitors demonstrated successful establishment of the new model systems, which would be useful in high throughput screening of anti-filovirus drugs in the future.
Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Ebolavirus ; drug effects ; physiology ; Genes, Reporter ; Glycoproteins ; genetics ; Hemorrhagic Fever, Ebola ; Humans ; Luciferases ; Viral Proteins ; genetics ; Virus Internalization ; drug effects

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.
Amphotericin B/*pharmacology ; Animals ; Antiprotozoal Agents/*pharmacology ; Drug Evaluation, Preclinical/instrumentation/*methods ; Female ; Gene Expression ; Genes, Reporter ; Green Fluorescent Proteins/genetics/*metabolism ; Humans ; Leishmania major/*drug effects/genetics/growth & development/physiology ; Leishmaniasis, Cutaneous/*parasitology ; Luciferases/genetics/*metabolism ; Mice

PURPOSE: Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. MATERIALS AND METHODS: Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 microg/40 microL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. RESULTS: Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. CONCLUSIONS: We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.
Animals ; Diabetes Mellitus, Experimental/complications ; Electroporation/*methods ; Erectile Dysfunction/*therapy ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy/*methods ; Luciferases/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Penile Erection/physiology ; Penis/*physiopathology ; Transfection

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.
Acanthamoeba castellanii/cytology/*genetics/physiology ; Amebiasis/*parasitology ; Amino Acid Sequence ; Autophagy ; Cell Membrane/metabolism ; DNA, Protozoan/chemistry/genetics ; Gene Dosage ; Gene Silencing ; Genes, Reporter ; Humans ; Molecular Sequence Data ; Phagosomes/metabolism ; Protein Isoforms ; Protozoan Proteins/*genetics/metabolism ; RNA, Messenger/genetics ; RNA, Protozoan/genetics ; RNA, Small Interfering/chemical synthesis/genetics ; Recombinant Fusion Proteins ; Sequence Alignment

Animals ; Electrochemical Techniques ; methods ; Gene Silencing ; Genes, Reporter ; Humans ; MicroRNAs ; blood ; genetics ; metabolism ; physiology ; Microarray Analysis ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

The present study was aimed at investigating the effect of activin on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the underlying molecular mechanism. The method of luciferase reporter gene was used. We firstly established a stable GH3 cell line which contains hGH gene promoter (-484 to 30 bp) and luciferase reporter gene by transfecting pGL3-484-Luc2 luciferase expression plasmid into GH3 cells using Lipofectamine transfection reagent. After treating these cells with activin or activin plus various signaling transduction activators, the concentration of GH in the medium and lysate of GH3 cells and luciferase activities in GH3 cells were measured. The results showed that activin (5 nmol/L, 50 nmol/L) decreased the secretion and synthesis of GH. The amounts of GH content in GH3 lysate and medium treated with 50 nmol/L activin were 82% and 59% of the control, respectively. Furthermore, activin (5, 50 nmol/L) reduced the luciferase expression in stable GH3 cells, with the expression being 77% and 69% of the control (P<0.001). Among the activators of intracellular signaling transduction pathways, mitogen-activated protein kinases kinase (MAPKK/MEK) activators C(6) ceramide (1 micromol/L) abolished completely the inhibitory effect of activin. Western blot analysis further confirmed the inhibition of phosphorylated MEK in GH3 cells. The inhibitory effect of activin was abrogated following the deletion of the fragment from -132 to -66 bp within the hGH gene promoter. These results indicate that activin decreases the activity of hGH gene promoter in rat pituitary GH3 cells. The intracellular MEK dependent signaling pathway and the promoter sequence that spans the -132 to -66 bp fragment of hGH gene are involved in the inhibitory effect of activin.
Activins ; physiology ; Animals ; Cell Line ; Cells, Cultured ; Genes, Regulator ; Genes, Reporter ; genetics ; Human Growth Hormone ; genetics ; Humans ; Luciferases ; genetics ; Promoter Regions, Genetic ; genetics ; Rats ; Somatotrophs ; cytology ; metabolism ; Transfection

OBJECTIVETo explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry.

METHODSA reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfectants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software.

RESULTSDose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX2,3 from 0 to 16 ng/mL.

CONCLUSIONpEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.

Animals ; Biological Assay ; Cell Line, Tumor ; Genes, Reporter ; physiology ; Green Fluorescent Proteins ; Harmful Algal Bloom ; physiology ; Humans ; Plasmids ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Shellfish ; analysis ; Sodium Channels ; Toxins, Biological ; chemistry ; toxicity

This study is to establish a cell-based pharmacological model targeting HIV-1 replication for compounds screening and to screen compounds randomly selected from compounds library by using this pseudotyped viral system. The cell-based HIV-1 replication pharmacological model was set up by HIV-1 core packed with vesicular stomatitis virus glycoprotein. The level of HIV-1 replication was presented by reporter genes expression (luciferase activity or percentage of GFP positive cells). When a compound has inhibitory effect on VSVG/HIV model, VSVG/MLV model would be used to test for specificity. Vesicular stomatitis virus glycoprotein can efficiently mediate HIV core into a wide range of host cells. Expression level of reporter genes showed dose-dependent manner with virion dilution. Among 500 compounds, three compounds dose-dependently inhibit HIV-1 replication, but not MLV replication. VSVG/HIV pseudotyped viral system can be used as a pharmacological model for HIV-1 replication inhibitor screening. Compounds 2-methylthio-5-(4-methylbenzo)amido-l,3,4-thiadiazole, N-(3-hydroxyphenyl)-2-(4-isobutylphenyl) propionamide, and N-(4-picolyl)-4-methylbenzenesulfonamide can specifically inhibit HIV-1 replication with IC50 of 1.92, 5.38, and 3.39 micromol L(-1) respectively.
Anti-HIV Agents ; pharmacology ; DNA Replication ; drug effects ; Didanosine ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; drug effects ; genetics ; HIV-1 ; drug effects ; physiology ; Humans ; Lamivudine ; pharmacology ; Pseudocowpox Virus ; Tumor Cells, Cultured ; Virus Replication ; drug effects ; Zidovudine ; pharmacology

The convergence of molecular and genetic disciplines with non-invasive imaging technologies has provided an opportunity for earlier detection of disease processes which begin with molecular and cellular abnormalities. This emerging field, known as molecular imaging, is a relatively new discipline that has been rapidly developed over the past decade. It endeavors to construct a visual representation, characterization, and quantification of biological processes at the molecular and cellular level within living organisms. One of the goals of molecular imaging is to translate our expanding knowledge of molecular biology and genomic sciences into good patient care. The practice of molecular imaging is still largely experimental, and only limited clinical success has been achieved. However, it is anticipated that molecular imaging will move increasingly out of the research laboratory and into the clinic over the next decade. Non-invasive in vivo molecular imaging makes use of nuclear, magnetic resonance, and in vivo optical imaging systems. Recently, an interest in Positron Emission Tomography (PET) has been revived, and along with optical imaging systems PET is assuming new, important roles in molecular genetic imaging studies. Current PET molecular imaging strategies mostly rely on the detection of probe accumulation directly related to the physiology or the level of reporter gene expression. PET imaging of both endogenous and exogenous gene expression can be achieved in animals using reporter constructs and radiolabeled probes. As increasing numbers of genetic markers become available for imaging targets, it is anticipated that a better understanding of genomics will contribute to the advancement of the molecular genetic imaging field. In this report, the principles of non-invasive molecular genetic imaging, its applications and future directions are discussed.
Animals ; Biological Processes ; Gene Expression ; Genes, Reporter ; Genetic Markers ; Genomics ; Molecular Biology ; Molecular Imaging* ; Optical Imaging ; Patient Care ; Physiology ; Positron-Emission Tomography