DNA ; DNA Packaging

A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by coexpression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Suprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4- dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evlauate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.
Adsorption ; Animals ; Antibodies ; COS Cells ; DNA ; Helper Viruses ; HIV-1* ; Membranes ; Plasmids ; Product Packaging ; RNA ; Virion

Osteosarcoma is the most common primary bone tumor, generally affecting young people. While the etiology of osteosarcoma has been largely unknown, recent studies have suggested that cyclooxygenase-2 (COX-2) plays a critical role in the proliferation, migration, and invasion of osteosarcoma cells. To understand the mechanism of action of COX-2 in the pathogenesis of osteosarcoma, we compared gene expression patterns between three stable COX-2-overexpressing cell lines and three control cell lines derived from U2OS human osteosarcoma cells. The data showed that 56 genes were upregulated, whereas 20 genes were downregulated, in COX-2-overexpressed cell lines, with an average fold-change > 1.5. Among the upregulated genes, COL1A1, COL5A2, FBN1, HOXD10, RUNX2, and TRAPPC2are involved in bone and skeletal system development, while DDR2, RAC2, RUNX2, and TSPAN31are involved in the positive regulation of cell proliferation. Among the downregulated genes, HIST1H1D, HIST1H2AI, HIST1H3H, and HIST1H4C are involved in nucleosome assembly and DNA packaging. These results may provide useful information to elucidate the molecular mechanism of the COX-2-mediated malignant phenotype in osteosarcoma.
Cell Line* ; Cell Proliferation ; Cyclooxygenase 2 ; DNA Packaging ; Gene Expression* ; Humans ; Nucleosomes ; Osteosarcoma* ; Phenotype

OBJECTIVE: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. METHODS: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. RESULTS: The sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). CONCLUSION: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.
Apoptosis ; Centrifugation* ; Centrifugation, Density Gradient ; Chromatin* ; DNA Fragmentation ; DNA* ; Methods ; Product Packaging* ; Semen ; Semen Analysis ; Sperm Motility ; Spermatozoa*

OBJECTIVE: To eliminate the potential problem of adenovirus contamination during recombinant adeno-associated virus (rAAV) vector production, we investigated new rAAV production method by a triple transfection of vector plasmid, packaging plasmid, and adenovirus helper plasmid. METHODS: This study was carried by triple transfection for the production of recombinant adeno-associated virus vector. This new production system was conducted with a plasmid construct which contained a mini-adenovirus genome capable of propagation of rAAV in the presence of adeno-assoceated viral rep and cap genes. To examine the helper functions of adenoviral plasmid on the production of rAAV vector, we carried out cotransfection with three plasmids, AAV vector, packaging construct, and adenovirus helper plasmids. The optimized plasmid quantity of transfection by calcium phosphate precipitation method was 25 micro gram of total plasmid DNA per 10 cm diameter plate of 293 cell. RESULTS: We found that rAAV yields peaked at 48 hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/mL based on the quantification of viral DNA. CONCLUSION: We constructed recombinant AAVp53 without adenovirus contamination. We thought that we construct high titer rAAVp53 particle through HPLC column in the near future.
Adenoviridae ; Calcium ; Chromatography, High Pressure Liquid ; Dependovirus* ; DNA ; DNA, Viral ; Genetic Therapy* ; Genome ; Humans* ; Plasmids ; Product Packaging ; Transfection ; Uterine Cervical Neoplasms*

An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene. HSV-1 protease is essential for DNA packaging and virus maturation. To perform high throughput for potent inhibitors, the efficient production of larger amounts of highly purified enzyme and protease activity assay method must be established. In this report, expression in E. coli and purification of the protease gene of HSV-1 strain F was investigated. The protease gene was cloned pET28, and the nucleotide sequence of protease catalytic domain of HSV-1 compared strain F with other strains (KOS and CL101). In these results the F strain was different in base sequence. However, the amino acid sequence was identifical. The HSV-1 protease was purified with His-tagged affinity column. The analysis of HSV-1 protease activity Was performed by high performance liquid chromatography.
Amino Acid Sequence ; Base Sequence ; Catalytic Domain ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Clone Cells ; DNA Packaging ; Drug Therapy ; Herpes Simplex* ; Herpesvirus 1, Human* ; Simplexvirus*

A system for the expression of synthetic hantavirus-like luciferase RNA was developed using Hantaan (HTN) virus as a helper virus. The hantavirus-like luciferase RNA was constructed by the deletion of the coding region in HTN virus (76~118) S genome and by replacement of a luciferase gene. PCR was performed using primers designed to amplify the whole region of hantavirus-like foreign gene. The resulting PCR product was placed under the control of the T7 RNA polymerase promoter for in vitro transcription. The produced hantavirus-like luciferase RNA was transfected into Vero-E6 cell infected with HTN virus using lipofectin. The level of expression was measured using a luminometer. The hantavirus-like luciferase RNA was allowed to amplify and express. And also, the hantavirus-like luciferase RNA was packaged into HTN virions. The 5' terminal and 3' terminal conserved sequences of HTN virus genome were sufficient to provide the signals for RNA amplification and packaging. This suggests that the RNA promoter region for hantavirus RNA synthesis is located in 3' terminal region. The luciferase activity was analyzed from the progeny virus-infected cells in order to examine if the 5' and 3' terminal sequences play a role in regulating a packaging pathway while genome of HTN virus is packaged. The luciferase activity was detectable in every cell passage. However, the activity of luciferase was decreased gradually after each passage. The fact that the hantavirus-like luciferase RNA can be packaged into progeny virus suggests that the 5' and 3' terminal sequences of HTN virus genome play an important role in regulating a packaging pathway.
Clinical Coding ; Conserved Sequence ; DNA-Directed RNA Polymerases ; Genome ; Hantavirus* ; Helper Viruses* ; Luciferases ; Polymerase Chain Reaction ; Product Packaging ; Promoter Regions, Genetic ; RNA* ; Virion

Human contains large number of human endogenous retroviruses (HERVs) in its genome. One of the HERV families, HERV-K, entered human genome most recently and includes many members with full-length intact proviruses. Normally, these proviruses do not express but infrequently they seem to express in cancers or autoimmune disease patients. To investigate expression mechanisms of these endogenous retroviruses, a DNA copy of HERV-K was cloned and its expression was studied. The transfection of the full-length clone into human cell lines did not produce any detectable viral capsid protein, Gag, and the transcription from its own promoter in LTR was extremely poor. The transcription was less than 10 percent compare to the exogenous retrovirus. However, when the Gag coding region was cloned under CMV promoter, Gag could be expressed efficiently and secreted as particles, probably virus like particles. The efficient expression also required a nuclear export signal. The expressed Gag could also package its own genomic RNA. These results indicate that the LTR of HERV-K is normally not active but its genes have a potential to express and possibly produce infectious particles.
Autoimmune Diseases ; Capsid Proteins ; Cell Line ; Clinical Coding ; Clone Cells* ; DNA* ; Endogenous Retroviruses ; Genome ; Genome, Human ; Humans* ; Nuclear Export Signals ; Product Packaging* ; Proviruses ; Retroviridae ; RNA ; Transfection

BACKGROUND: Leptin gene is known to be related to obesity in human and animals and complete genetic defect of the gene in ob/ob mouse has been identified. Therefore, ob/ob mouse is widely used as an animal model for the study of etiology and therapy of obesity. The main biological function of leptin was thought to involve in the regulation of food intake and weight gain, however, the regulatory mechanisms by which leptin functions in the weight reduction and lowering the blood glucose level are uncertain. In the present study, retroviral-mediated leptin gene transduction into ob/ob mouse was attempted for the correction of biochemical parameters of obesity. METHODS: Leptin cDNA was inserted into pLXSN retroviral vector (pLXSN-lep) and recombinant leptin expressing retrovirus particles (3 X10 CFU/mL) were produced in psi2 ecotropic packaging cells and subsequent transfection into PA317 amphotropic packaging cells. The leptin expressing recombinant viruses (LER) were transduced into NIH3T3 mouse fibroblasts and insertion of leptin cDNA into chromosomal DNA of PA317 and MH3T3 mouse fibroblasts was confirmed by Southern blot hybridizations. Leptin mRNA and its protein expressed in the cells were identified by Northern blot hybridization and Western blot immunodetection method, respectively. LER were injected I. P. into ob/ob mice, and body weight, food intake, serum leptin level and blood glucose level were measured. RESULTS: Expression of leptin was identified in PA317 and NIH3T3 mouse fibroblasts transduced with LER. Leptin content in sera of mice transfused with LER was drastically increased after 1 week and decreased to the almost basal level at 3 weeks after the transfusion. The body weight as well as food intake of ob/ob mouse transduced by LER decreased for the first 3 weeks and slightly increased thereafter. The reduction of both body weight and food intake in ob/ob mice transduced with LER was observed with the concomitant increase of serum leptin level, indicating that retroviral-mediated transduction of leptin gene in ob/ob mouse in vivo produced a biologically active leptin protein and released it into blood circulation. CONCLUSION: A transient expression of leptin cDNA in ob/ob mice by a retroviral-mediated transduction was performed and further studies are required for long term expression of the gene in vivo.
Animals ; Blood Circulation ; Blood Glucose ; Blotting, Northern ; Blotting, Southern ; Blotting, Western ; Body Weight ; DNA ; DNA, Complementary ; Eating ; Fibroblasts ; Humans ; Leptin* ; Mice ; Mice, Obese* ; Models, Animal ; Obesity ; Product Packaging ; Retroviridae ; RNA, Messenger ; Transfection ; Weight Gain ; Weight Loss ; Zidovudine*

Prostate cancer is one of the most common diseases in men worldwide. Surgery, radiation therapy, and hormonal therapy are effective treatments for early-stage prostate cancer. However, the development of castration-resistant prostate cancer has increased the mortality rate of prostate cancer. To develop novel drugs for castration-resistant prostate cancer, the molecular mechanisms of prostate cancer progression must be elucidated. Among the signaling pathways regulating prostate cancer development, recent studies have revealed the importance of noncanonical wingless-type MMTV integration site family (WNT) signaling pathways, mainly that involving WNT5A, in prostate cancer progression and metastasis; however, its role remains controversial. Moreover, chromatin remodelers such as the switch/sucrose nonfermentable (SWI/SNF) complex and chromodomain helicase DNA-binding proteins 1 also play important roles in prostate cancer progression through genome-wide gene expression changes. Here, we review the roles of noncanonical WNT signaling pathways, chromatin remodelers, and epigenetic enzymes in the development and progression of prostate cancer.
Male ; Humans ; Wnt Signaling Pathway ; Chromatin ; Prostatic Neoplasms, Castration-Resistant ; Chromatin Assembly and Disassembly