Ya-Hui Tsai

Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

The NS5A protein of the hepatitis C virus (HCV) is an integral component of the viral replicase. It also modulates cellular signaling and perturbs host interferon responses. The multifunctional characteristics of NS5A are mostly attributed to its ability to interact with various cellular proteins. This study aimed to identify the novel cellular factors that interact with NS5A and decipher the significance of this interaction in viral replication. The NS5A-interacting proteins were purified by the tandem affinity purification (TAP) procedure from cells expressing NS5A and identified by mass spectrometry. The chaperone protein Hsp72 was identified herein. In vivo protein-protein interaction was verified by co-immunoprecipitation and an in situ proximity ligation assay. In addition to NS5A, Hsp72 was also associated with other members of the replicase complex, NS3 and NS5B, suggesting that it might be directly involved in the HCV replication complex. Hsp72 plays a positive regulatory role in HCV RNA replication by increasing levels of the replicase complex, which was attributed either to the increased stability of the viral proteins in the replicase complex or to the enhanced translational activity of the internal ribosome entry site of HCV. The fact that the host chaperone protein Hsp72 is involved in HCV RNA replication may represent a therapeutic target for controlling virus production.

Micro-RNA-21 regulates the sensitivity to cisplatin in human neuroblastoma cells

BACKGROUND/PURPOSE Drug resistance often causes treatment failure in neuroblastomas. Increasing evidence has implicated that the micro-RNAs (miRNAs) are involved in the development of drug resistance. In this report, we aimed to investigate the role of miRNA in cisplatin resistance of neuroblastoma cells. MATERIALS AND METHODS The cell viability of the neuroblastoma cells after cisplatin treatment was analyzed. The expression of the miRNAs and phosphatase and tensin homolog (PTEN) messenger RNA in the neuroblastoma cells was studied by real-time polymerase chain reaction. Overexpression of miRNA or suppression of miRNA expression by antagomir was used to investigate the effects of miRNA on the cisplatin-induced cell death or proliferation. RESULTS The expression of miR-21 was increased in the cisplatin-resistant (CisR) neuroblastoma cells as compared with the parental cells, and the antagomir against miR-21 converted the resistant cells into sensitive ones. Ectopic expression of pre-miR-21 in parental cells resulted in decreased sensitivity to cisplatin treatment. In addition, overexpression of pre-miR-21 markedly increa sed the proliferation rate of neuroblastoma cells. The level of PTEN messenger RNA and protein in the CisR cells was lower than that in the parental cells. Transfection of pre-miR-21 into the parental cells reduced the PTEN expression, and transfection of anti-miR-21 into the CisR cells increased the PTEN expression. CONCLUSION Micro-RNA-21 regulated the drug resistance and proliferation in neuroblastoma cells.

Valproic acid affected the survival and invasiveness of human glioma cells through diverse mechanisms.

The effects of valproic acid (VPA) on the viability, apoptosis, and invasiveness of two glioma cells (A172 and T98G) and the underlying mechanisms were studied. VPA induced cytotoxicity and apoptosis, and suppressed the invasiveness of both cells. VPA increased the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 in A172 cells, but decreased it in T98G cells. siRNA blockade of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression partially reversed VPA-mediated effects in T98G cells, but had no effect on A172 cells. VPA increased the expression of phospho-JNK1 and phospho-ERK1/2 in A172 cells, but decreased it in T98G cells. Inhibition of JNK1 and/or ERK1/2 partially reversed the VPA effects in A172 cells. In conclusion, the effects of VPA (loss of viability, increased apoptosis, and decreased invasiveness) are, at least partly, mediated through the RECK-MMPs pathway in T98G cells and the mitogen-activated protein kinase pathways in A172 cells. The action of VPA seems to be cell type-specific in glioma cells.

The non-structural 5A protein of hepatitis C virus exhibits genotypic differences in interferon antagonism.

BACKGROUND/AIMS Patients infected with hepatitis C virus (HCV) genotype 2 or 3 usually respond better to interferon (IFN) treatment than those infected with genotype 1. In this study, we investigated whether the non-structural 5A protein (NS5A) of HCV genotypes 1 and 2 (1b-NS5A and 2a-NS5A, respectively) exerted differential counteractivities against IFN treatment. METHODS We compared the inhibitory effects of 1b-NS5As and 2a-NS5As on IFN activity. We also investigated the replication inhibition of HCV subgenomic replicons containing 1b-NS5A or 2a-NS5A in response to IFN treatment. RESULTS 1b-NS5As exerted more profound inhibitory effects on IFN activity than 2a-NS5As. The replication of the 2a-NS5A-containing replicons was more sensitive to IFN treatment than that of the 1b-NS5A-containing replicons. Deletion of the interferon sensitivity-determining region/protein kinase R-binding domain (PKR-BD), the V3 domain, or the C-terminus region of NS5A significantly abrogated its anti-IFN activity. Domain swapping between 1b-NS5A and 2a-NS5A in the V3 domain and/or the C-terminus region resulted in a transfer of their anti-IFN activity. CONCLUSIONS 1b-NS5As exert higher magnitudes of IFN antagonism than do 2a-NS5As. The V3 and the C-terminus regions are responsible for the differential anti-IFN effects. This phenomenon may partly explain the genotype-linked differences in the response of HCV to IFN treatment.

The Potential of Tetrandrine as a Protective Agent for Ischemic Stroke

Stroke is one of the leading causes of mortality, with a high incidence of severe morbidity in survivors. The treatment to minimize tissue injury after stroke is still unsatisfactory and it is mandatory to develop effective treatment strategies for stroke. The pathophysiology of ischemic stroke is complex and involves many processes including energy failure, loss of ion homeostasis, increased intracellular calcium level, platelet aggregation, production of reactive oxygen species, disruption of blood brain barrier, and inflammation and leukocyte infiltration, etc. Tetrandrine, a bisbenzylisoquinoline alkaloid, has many pharmacologic effects including anti-inflammatory and cytoprotective effects. In addition, tetrandrine has been found to protect the liver, heart, small bowel and brain from ischemia/reperfusion injury. It is a calcium channel blocker, and can inhibit lipid peroxidation, reduce generation of reactive oxygen species, suppress the production of cytokines and inflammatory mediators, inhibit neutrophil recruitment and platelet aggregation, which are all devastating factors during ischemia/reperfusion injury of the brain. Because tetrandrine can counteract these important pathophysiological processes of ischemic stroke, it has the potential to be a protective agent for ischemic stroke.

Bioactive saccharide-conjugated polypeptide micelles for acid-triggered doxorubicin delivery

The synthesis and self-assembly of lactobionolactone-conjugated poly(l-glutamic acid)-b-poly(l-phenylalanine) amphiphilic block copolypeptides (Lac-PGA-b-PPhe) and their evaluation for anticancer drug doxorubicin (DOX) delivery have been investigated. Lactobionolactone was functionalized with the azide group and successfully conjugated with the terminal alkyne groups on the polypeptides through click reaction and these amphiphilic glycopolypeptides self-assembled to form micelles with bioactive galactose units on the particle surface as confirmed by selective lectin binding experiments. Drug release experiments showed that DOX released faster from saccharide-conjugated micelles under acidic conditions than under neutral conditions. The DOX-loaded, saccharide-conjugated micelles exhibited higher cytotoxicity toward HepG2 tumor cells than free DOX and saccharide-free micelles loaded with DOX at low concentrations, suggesting that the saccharide-conjugated micelles can effectively bind to the cells through specific recognition and subsequently the higher uptake of saccharide-conjugated micelles led to higher drug release and cytotoxicity under pH-sensitive conditions.

HDAC Inhibitors and RECK Modulate Endoplasmic Reticulum Stress in Tumor Cells

In the tumor microenvironment hypoxia and nutrient deprived states can induce endoplasmic reticulum (ER) stress. If ER stress is not relieved, the tumor cells may become apoptotic. Therefore, targeting ER homeostasis is a potential strategy for cancer treatment. Various chemotherapeutic agents including histone deacetylase (HDAC) inhibitors can induce ER stress to cause cell death in cancers. Some HDAC inhibitors can prevent HDAC from binding to the specificity protein 1-binding site of the promoter of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and up-regulate RECK expression. Up-regulation of RECK expression by HDAC inhibitors has been observed in various cancer types. RECK is a tumor and metastasis suppressor gene and is critical for regulating tumor cell invasiveness and metastasis. RECK also modulates ER stress via binding to and sequestering glucose-regulated protein 78 protein, so that the transmembrane sensors, such as protein kinase RNA-like ER kinase are released to activate eukaryotic translational initiation factor 2α phosphorylation and enhance ER stress. Therefore, HDAC inhibitors may directly induce ER stress or indirectly induce this stress by up-regulating RECK in cancer cells.

Application of Biodegradable Polyhydroxyalkanoates as Surgical Films for Ventral Hernia Repair in Mice

The cytotoxicity and biosafety of poly-(3-hydroxybutyrate) (P3HB) and poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HBV) films were investigated in vitro using 3T3 fibroblast cells and in vivo through subcutaneous implantation of the film in mice. The in vitro test revealed that endotoxin-free P3HB and P3HBV films allowed cell attachment and growth. Film-soaked conditional media showed no significant inhibitory or cytotoxic effects on cell proliferation. The in vivo absorption test showed that both the P3HB and P3HBV films slowly degraded and that P3HB had a slower degradation rate than that of P3HBV. Applying a P3HB film in hernia repair demonstrated a favorable outcome: the film was able to correct the abdominal ventral hernia by inducing connective tissue and fat ingrowth and exhibited an extremely slow rate of degradation. Furthermore, the P3HB film demonstrated the advantage of lower intestinal adhesion to the ventral hernia site compared with the P3HBV and PP commercial films.

Application of three-dimensional imaging to the intestinal crypt organoids and biopsied intestinal tissues.

Two-dimensional (2D) histopathology is the standard analytical method for intestinal biopsied tissues; however, the role of 3-dimensional (3D) imaging system in the analysis of the intestinal tissues is unclear. The 3D structure of the crypt organoids from the intestinal stem cell culture and intestinal tissues from the donors and recipients after intestinal transplantation was observed using a 3D imaging system and compared with 2D histopathology and immunohistochemistry. The crypt organoids and intestinal tissues showed well-defined 3D structures. The 3D images of the intestinal tissues with acute rejection revealed absence of villi and few crypts, which were consistent with the histopathological features. In the intestinal transplant for megacystis microcolon intestinal hypoperistalsis syndrome, the donors intestinal tissues had well-developed nerve networks and interstitial cells of Cajal (ICCs) in the muscle layer, while the recipients intestinal tissues had distorted nerve network and the ICCs were few and sparsely distributed, relative to those of the donor. The 3D images showed a clear spatial relationship between the microstructures of the small bowel and the features of graft rejection. In conclusion, integration of the 3D imaging and 2D histopathology provided a global view of the intestinal tissues from the transplant patients.

The Zinc Deficiency and Long-Term Outcome in the Cases after Isolated Intestinal Transplantation in Taiwan

OBJECTIVES The small intestine is the primary site for absorption of dietary zinc. Intestinal transplant recipients are at high risk for zinc deficiency because of the long process of posttransplant adaptation. We initiated an intestinal transplant program in Taiwan in 2007. In this study, we aimed to retrospectively investigate the incidence of zinc deficiency in recipients after intestinal transplantation. METHODS Twenty-one isolated intestinal transplants were performed in 20 patients with 1 retransplantation. The level of serum zinc was monitored periodically, and zinc supplements were administered when zinc level was below 700 ng/mL. Twelve patients with graft above 1-year survival and with available related data were enrolled for the analysis of zinc deficiency. The levels of serum zinc were tracked, and the protocol of zinc supplementation is discussed herein. RESULTS The survival rates of 20 transplant recipients for 1 year, 3 years, and 5 years were 85%, 75%, and 65%, respectively. In the 12 grafts that survived longer than 1 year, we found that zinc deficiency was highest during the third (41.7%) to sixth (50%) month after transplantation. Sustained supplementation of zinc was required for over 70% of patients throughout the 3-year period to maintain their zinc level around the lower normal limit. CONCLUSION The outcome of isolated small bowel transplantation is promising. Periodical monitoring and sufficient dosing of zinc supplements should be considered into the posttransplant protocol to prevent zinc deficiency after intestinal transplantation.