Development of a standard protocol for quantitative polymerase chain reaction to detect adenovirus 36, which is associated with obesity.

It has been previously reported that adenovirus 36 (Ad36) infection is associated with obesity in humans and other animals. However, there is no clinically available standard protocol to detect Ad36 DNA. In this study, we developed a method for quantitative and rapid detection of Ad36 DNA. Using a TaqMan probe quantitative polymerase chain reaction (qPCR), we identified that the E3 and E4orf1 regions specifically detect Ad36 DNA, because these regions did not show cross reactivity with other types of adenoviruses. The limit of detection was 379 copy/ml and 384 copy/ml for E3 and E4orf1 regions of Ad36, respectively. The %CV (coefficient of variation) for reproducibility of the assay using adenovirus reference material ranged from 1.07-13.02. After we developed the standard protocol to detect Ad36 DNA, we used mouse as a surrogate model to confirm its clinical applicability. We administered Ad36 to mice via intranasal and oral routes, with intraperitoneal administration as the positive control, to analyze the effect of infection route. Ad36 DNA could be detected in lungs, liver, pancreas, and epididymal fat tissue after intraperitoneal injection, whereas it was found only in lungs after intranasal injection. No Ad36 DNA was detectable in any tested organ after oral injection. This indicates that the main route of infection with Ad36 is intranasal, suggesting that Ad36 is a respiratory virus. The standard protocol for qPCR developed in this study is useful for clinical detection of Ad36 DNA. Keywords: adenovirus 36; real-time PCR; obesity.

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation.

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.

Synergistic effects of genetically engineered stem cells expressing cytosine deaminase and interferon-ß via their tumor tropism to selectively target human hepatocarcinoma cells.

Stem cells have received a great deal of attention for their clinical and therapeutic potential for treating human diseases and disorders. Recent studies have shown that it is possible to genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non-toxic prodrugs to toxic metabolites, selectively migrate toward tumor sites and reduce tumor growth. In this study, we evaluated whether these GESTECs are capable of migrating to hepatocarcinoma cells and examined the potential therapeutic efficacy of gene-directed enzyme prodrug therapy against liver cancer cells in cellular and animal models. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to Hep3B hepatocarcinoma cells. GESTECs, that is, HB1.F3.CD or HB1.F3.CD.interferon-ß (IFN-ß) cells, engineered to express a suicide gene, cytosine deaminase (CD), selectively migrated toward liver cancer cells. Treatment of Hep3B, human liver cancer cells, with the prodrug 5-fluorocytosine (5-FC) in the presence of HB1.F3.CD or HB1.F3.CD.IFN-ß cells resulted in the inhibition of Hep3B cell growth. In a xenografted mouse model injected with hepatocarcinoma, we investigated the therapeutic effect of these stem cells. For 9 weeks, the xenografted mice were treated with HB1.F3.CD or HB1.F3.CD.IFN-ß in the presence of 5-FC. A growth of tumor mass was inhibited about 40-50% in the mice treated with GESTECs and a prodrug. In addition, we further confirmed the cytotoxic effect on tumor cells by histological analysis and migratory effect of therapeutic stem cells. Taken together, GESTECs expressing a fusion gene encoding CD and IFN-ß may exert a synergistic antitumor effect on this type of tumor.

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models.

As human amniotic fluid-derived stem cells (hAFSCs) are capable of multiple lineage differentiation, extensive self-renewal and tumor targeting, they may be valuable for clinical anticancer therapies. In this study, we used hAFSCs as vehicles for targeted delivery of therapeutic suicide genes to breast cancer cells. hAFSCs were engineered to produce AF2.CD-TK cells in order to express two suicide genes encoding bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) that convert non-toxic prodrugs, 5-fluorocytosine (5-FC) and mono-phosphorylate ganciclovir (GCV-MP), into cytotoxic metabolites, 5-fluorouracil (5-FU) and triphosphate ganciclovir (GCV-TP), respectively. In cell viability test in vitro, AF2.CD-TK cells inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the 5-FC or GCV prodrugs, or a combination of these two reagents. When the mixture of 5-FC and GCV was treated together, an additive cytotoxic effect was observed in the cell viability. In animal experiments using female BALB/c nude mouse xenografts, which developed by injecting MDA-MB-231 cells, treatment with AF2.CD-TK cells in the presence of 5-FC and GCV significantly reduced tumor volume and weight to the same extent seen in the mice treated with 5-FU. Histopathological and fluorescent staining assays further showed that AF2.CD-TK cells were located exactly at the site of tumor formation. Furthermore, breast tissues treated with AF2.CD-TK cells and two prodrugs maintained their normal structures (for example, the epidermis and reticular layers) while breast tissue structures in 5-FU-treated mice were almost destroyed by the potent cytotoxicity of the drug. Taken together, these results indicate that AF2.CD-TK cells can serve as excellent vehicles in a novel therapeutic cell-based gene-directed prodrug system to selectively target breast malignancies.

Real-time RT-PCR of Hantaan virus RNA used for the detection of virus response to antiviral drugs.

Hantaan virus (HTN) is an important cause of hemorrhagic fever with renal syndrome (HFRS) in Korea. HTN RNA can be detected with the RT-PCR and the quantity of HTN RNA in infected cells can be measured by competitive RT-PCR. The current study used the real-time RT-PCR for the detection of viral RNA S gene in a more detailed fashion than in the previous study (Nam et al., Virus Genes 26, 31-38, 2003). A standard curve was generated with serial 10-fold dilutions of the HTN RNA. The sensitivity of RNA detection was approximately 10 PFU of HTN. The cells infected with HTN were treated with the antiviral drugs ribavirin, zidovudine, and amantadine. 24 hrs after infection, real-time RT-PCR was used to detect the HTN RNA synthesized in the infected cells. No viral RNA was detected in the HTN-infected cells treated with antiviral drugs, but HTN RNA was detected in untreated HTN-infected cells. This finding suggested that real-time RTPCR should be used for the detection of antiviral activity against HTN.

Polymorphisms at codons 129 and 219 of the prion protein gene (PRNP) are not associated with sporadic Alzheimer's disease in the Korean population.

Polymorphisms of prion protein gene (PRNP) at codons 129 and 219 play an important role in the susceptibility to Creutzfeldt-Jakob disease (CJD). Alzheimer's disease (AD) and prion diseases, such as CJD, are both characterized by the accumulation of abnormally folded proteins in the brain. An association between sporadic AD and the PRNP polymorphism at codon 129 has been reported in several studies, but other studies have failed to confirm an association. To investigate whether PRNP polymorphisms are associated with an increased risk for developing sporadic AD in the Korean population, we compared the genotype, allele, and haplotype frequencies of PRNP polymorphisms in 271 sporadic AD patients with those in 236 healthy Koreans. Our study does not show a significant difference in PRNP genotype, allele, and haplotype frequency at codons 129 and 219 between sporadic AD and normal controls. Analyses stratifying by age at disease onset, and gender also failed to reveal any association between these polymorphisms and sporadic AD. These results indicate that these PRNP polymorphisms have no direct influence on the susceptibility to sporadic AD in the Korean population.

Application of cDNA microarray technique to detection of gene expression in host cells infected with viruses.

cDNA microarray technique was used to monitor changes in mRNA levels in cells after Hantaan virus (HTNV) infection. The values of the ratio of medians for HTNV and Japanese encephalitis virus (JEV) at the early stage of infection were compared and found similar, suggesting that the same or similar genes are associated with the early events of infection with either virus. The reproducibility of values of the "ratio of medians" for HTNV was examined. We found that applying cluster analysis to the gene expression data groups efficiently together genes with the same function. Therefore, in analyzing the effects of viral infection on host cells by the cDNA microarray technique, clustering data appear to be necessary for gaining biological meaning from a dump of gene expression profiles obtained from virus-infected cells.

Antiplatelet mechanism of 2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone (NQ304), an antithrombotic agent.

The effects of 2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone (NQ304), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein IIb/IIIa and intracellular signals were investigated using human platelets. NQ304 inhibited thrombin-, arachidonic acid- and thapsigargin-induced aggregation of washed human platelets with the IC50 values of 22.2+/-0.7, 6.5+/-0.2, and 7.6+/-0.1 microM, respectively. NQ304 significantly inhibited fluorescein isothiocyanate-conjugated fibrinogen binding to human platelet surface glycoprotein IIb/IIIa receptor by 75%, but failed to inhibit the fibrinogen binding to purified glycoprotein IIb/IIIa receptor. This result suggests that NQ304 inhibit platelet aggregation by suppression of an intracellular pathway that involves exposure of the glycoprotein IIb/IIIa receptor, rather than by direct inhibition of fibrinogen-glycoprotein IIb/IIIa binding. NQ304 significantly inhibited thrombin-induced increase in intracellular Ca2+ mobilization at the dose of 30 microM and ATP secretion in a dose-dependent manner. It also inhibited thrombin- and arachidonic acid-induced thromboxane A2 formation in human platelet dose-dependently. In conclusion, the antiplatelet mechanism of NQ304 may be due to the reduction of the thromboxane A2 formation, inhibition of adenosine triphosphate release and intracellular calcium mobilization.