CRM1-spike-mediated nuclear export of hepatitis B virus encapsidated viral RNA.

Hepatitis B virus (HBV) is a global pathogen. We report here that the cellular CRM1 machinery can mediate nuclear export of entire HBV core (HBc) particles containing encapsidated viral RNAs. Two CRM1-mediated nuclear export signals (NESCRM1) cluster at the conformationally flexible spike tips of HBc particles. Mutant NESCRM1 capsids exhibit strongly reduced associations with CRM1 and nucleoporin358 in vivo. CRM1 and NXF1 machineries mediate nuclear export of HBc particles independently. Inhibition of nuclear export has pleiotropic consequences, including nuclear accumulation of HBc particles, a significant reduction of encapsidated viral RNAs in the cytoplasm but not in the nucleus, and barely detectable viral DNA. We hypothesize an HBV life cycle where encapsidation of the RNA pregenome can initiate early in the nucleus, whereas DNA genome maturation occurs mainly in the cytoplasm. We identified a druggable target for HBV by blocking its intracellular trafficking.

HBV maintains electrostatic homeostasis by modulating negative charges from phosphoserine and encapsidated nucleic acids.

Capsid assembly and stability of hepatitis B virus (HBV) core protein (HBc) particles depend on balanced electrostatic interactions between encapsidated nucleic acids and an arginine-rich domain (ARD) of HBc in the capsid interior. Arginine-deficient ARD mutants preferentially encapsidated spliced viral RNA and shorter DNA, which can be fully or partially rescued by reducing the negative charges from acidic residues or serine phosphorylation of HBc, dose-dependently. Similarly, empty capsids without RNA encapsidation can be generated by ARD hyper-phosphorylation in insect, bacteria, and human hepatocytes. De-phosphorylation of empty capsids by phosphatase induced capsid disassembly. Empty capsids can convert into RNA-containing capsids by increasing HBc serine de-phosphorylation. In an HBV replicon system, we observed a reciprocal relationship between viral and non-viral RNA encapsidation, suggesting both non-viral RNA and serine-phosphorylation could serve as a charge balance buffer in maintaining electrostatic homeostasis. In addition, by comparing the biochemistry assay results between a replicon and a non-replicon system, we observed a correlation between HBc de-phosphorylation and viral replication. Balanced electrostatic interactions may be important to other icosahedral particles in nature.

The Correlation between Chitin and Acidic Mammalian Chitinase in Animal Models of Allergic Asthma.

Asthma is the result of chronic inflammation of the airways which subsequently results in airway hyper-responsiveness and airflow obstruction. It has been shown that an elicited expression of acidic mammalian chitinase (AMCase) may be involved in the pathogenesis of asthma. Our recent study has demonstrated that the specific suppression of elevated AMCase leads to reduced eosinophilia and Th2-mediated immune responses in an ovalbumin (OVA)-sensitized mouse model of allergic asthma. In the current study, we show that the elicited expression of AMCase in the lung tissues of both ovalbumin- and Der P2-induced allergic asthma mouse models. The effects of allergic mediated molecules on AMCase expression were evaluated by utilizing promoter assay in the lung cells. In fact, the exposure of chitin, a polymerized sugar and the fundamental component of the major allergen mite and several of the inflammatory mediators, showed significant enhancement on AMCase expression. Such obtained results contribute to the basis of developing a promising therapeutic strategy for asthma by silencing AMCase expression.

Resveratrol and its metabolites modulate cytokine-mediated induction of eotaxin-1 in human pulmonary artery endothelial cells.

Coronary heart disease (CHD) is a leading cause of death in many developed countries. Evidence has long implicated endothelial injury and inflammation as apical events in the pathogenesis of atherosclerosis, the primary cause of CHD. Numerous risk factors contribute to a damaged, inflamed endothelium. Conversely, cardioprotective agents targeting the dysfunctional endothelium have also been identified, notably from dietary sources. We have used cultured human pulmonary artery endothelial cells (HPAECs) to test the diet-mediated cardioprotective hypothesis. In this review, we summarize our recent findings on control of transcription and expression of inflammation biomarker eotaxin-1 in HPAECs exposed to single or combined proinflammatory cytokines interleukin-13 (IL-13) and tumor necrosis factor-α (TNF-α), and attenuation of the observed eotaxin-1 responses by prior or simultaneous treatment with resveratrol and its metabolites. Control of eotaxin-1 gene regulation may be considered an in vitro model to evaluate agents linking cardioprotection with endothelial cell damage and inflammation.

Control of stability of cyclin D1 by quinone reductase 2 in CWR22Rv1 prostate cancer cells.

Aberrant expression of cyclin D1, frequently observed in human malignant disorders, has been linked to the control of G(1)→S cell cycle phase transition and development and progression in carcinogenesis. Cyclin D1 level changes are partially controlled by GSK-3ß-dependent phosphorylation at threonine-286 (Thr286), which targets cyclin D1 for ubiquitination and proteolytic degradation. In our continuing studies on the mechanism of prostate cancer prevention by resveratrol, focusing on the role of its recently discovered target protein, quinone reductase 2 (NQO2), we generated NQO2 knockdown CWR22Rv1 using short hairpin RNA (shRNA)-mediated gene silencing approach. We found that, compared with cells expressing NQO2 (shRNA08), NQO2 knockdown cells (shRNA25) displayed slower proliferation and G(1) phase cell accumulation. Immunoblot analyses revealed a significant decrease in phosphorylation of retinoblastoma Rb and cyclin D1 in shRNA25 compared with shRNA08. Moreover, shRNA25 cells showed a 37% decrease in chymotrypsin-like proteasome activity. An increase in AKT activity was also observed in shRNA25, supported by a ∼1.5-fold elevation in phosphorylation and ∼50% reduction/deactivation of GSK-3α/ß at Ser21/9, which were accompanied by a decrease in phosphorylation of cyclin D1 at T286. NQO2 knockdown cells also showed attenuation of resveratrol-induced downregulation of cyclin D1. Our results indicate a hitherto unreported role of NQO2 in the control of AKT/GSK-3ß/cyclin D1 and highlight the involvement of NQO2 in degradation of cyclin D1, as part of mechanism of chemoprevention by resveratrol.

Control of eotaxin-1 expression and release by resveratrol and its metabolites in culture human pulmonary artery endothelial cells.

Population studies suggest that moderate red wine intake correlates with reduced risk of cardiovascular disease (CVD); cardioprotection may attribute to consumption of red wine polyphenol resveratrol. Since inflammation plays a key role in CVD, we investigated modulation of inflammation by resveratrol and its metabolites by determining the expression and release of chemokine, eotaxin-1, in cultured human pulmonary artery endothelial cells (HPAEC) treated with proinflammatory cytokines IL-13 and TNF-α. Up-regulation of eotaxin-1 gene expression by IL-13 or TNF-α was confirmed by RT-PCR, by reporter assays using eotaxin-1 gene promoter constructs, and by the changes in transcriptional factors STAT6 and NF-κB. Exposure to resveratrol suppressed IL-13 and TNF-α induced eotaxin-1 gene expression as well as attenuated the eotaxin-1 promoter activity, in coordination with inhibition of expression of JAK-1, reduction in phosphorylated-STAT6 and decreased p65 subunit of NF-κB. In addition, quantitative determination of eotaxin-1 release using enzyme-linked immunosorbent assay (ELISA) showed increased eotaxin-1 release in response to treatment by IL-13 and TNF-α, which was effectively inhibited by resveratrol. Whether resveratrol metabolites affected eotaxin-1 was also tested; piceatannol showed potency similar to resveratrol. We propose that control of eotaxin-1 expression and release by proinflammatory cytokines in HPAEC may be considered as an in vitro model for screening and discovering polyphenols with anti-inflammatory activities and cardioprotective potentials.

Using a fed-batch culture strategy to enhance rAAV production in the baculovirus/insect cell system.

Recombinant adeno-associated virus (rAAV) is one of the most promising vectors for human gene therapy. However, the production systems that are currently available have a limited capacity and cannot provide sufficient quantities of rAAV for preclinical or clinical trials. Many novel methods for improving rAAV production have been developed, but few researchers have focused on the culture process. In this study, we use a fed-batch culture system to enhance rAAV yield in the baculovirus/insect cell system. When the insect cells were co-infected with MOI=5 of Bac-GFP at a ratio of 1:9:9 (Bac-GFP: Bac-Rep: Bac-VP), the fed-batch culture achieved optimal rAAV yields. In batch culture, the optimal cell density for producing rAAV was found to be 1x10(6) cells/ml, and the highest rAAV yield (1.22x10(8) IVP/ml, 122 IVP/cell) occurred at day 5 post-infection. In the fed-batch culture, rAAV yield reached 2.13x10(8) IVP/ml at day 4 post-infection, and the highest rAAV yield was 2.40x10(8) IVP/ml (240 IVP/cell) at day 5 post-infection. The cost of the batch and fed-batch cultures is similar; however, the rAAV yield was 2.6-fold higher in the fed-batch culture system compared with that in the batch culture system. Therefore, here we demonstrated an economical and efficient strategy for rAAV production.

Colloid chitin azure is a dispersible, low-cost substrate for chitinase measurements in a sensitive, fast, reproducible assay.

Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of chitinase activity.

IL-6-transfected tumor cells modulate the status of CD8(+) and CD4(+) T cells to control tumor growth.

IL-6 is a proinflammatory cytokine secreted by tumor cells and immune cells to affect the development of cancer. This study demonstrates the effects of tumor-derived IL-6 on the malignancy of tumor cells and tumor immunity. The tumor cell line, EG7, was transfected with a mammalian expression vector encoding the full length of murine IL-6 to mimic IL-6-secreting tumor cells. Two IL-6 transfectants with low and high IL-6 production were compared in vitro and in vivo. While the in vitro proliferation rates of both transfectants and the parental line were similar, high expression of IL-6 induced a significant reduction in tumor growth in vivo. Concomitantly, there was an increase in IFN-gamma positive tumor-infiltrating lymphocytes and a decrease in the suppressive CD4(+)CD25(+)FoxP3(+) population. These results demonstrate the direct effects of tumor-derived IL-6 on cancer development and the induction of tumor immunity.

Inhibition of acidic mammalian chitinase by RNA interference suppresses ovalbumin-sensitized allergic asthma.

Asthma, a chronic helper T cell type 2-mediated inflammatory disease, is characterized by airway hyperresponsiveness and inflammation. Growing evidence suggests that increased expression of acidic mammalian chitinase (AMCase) may play a role in the pathogenesis of asthma. In the present study, we sought to develop an RNA interference approach to suppress allergic asthma in mice through silencing of AMCase expression. Mice sensitized with ovalbumin (OVA) were intratracheally administered a recombinant adeno-associated virus expressing short hairpin RNA (rAAV-shRNA) against AMCase. In OVA-sensitized mice, the development of allergic symptoms was significantly associated with elevated AMCase expression. After administration of rAAV-shRNA, there was a significant reduction of AMCase expression in the lung and in bronchoalveolar lavage fluid (BALF) cells of sensitized mice. Sensitized mice receiving rAAV-shRNA showed a significant improvement in allergic symptoms, including airway hyperresponsiveness (AHR), eosinophil infiltration, eotaxin, interleukin-13 secretion in BALF, and serum OVA-specific IgE level. Our data suggest the hyperexpression of AMCase in asthma can be suppressed by rAAV-mediated shRNA. Silencing AMCase expression by shRNA may be a promising therapeutic strategy in asthma.