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Yi-Ling Lin

Publications and source records attributed to Yi-Ling Lin.

36 records · Page 2Linked to original sources
PubMed2004

Sub-fragments of the envelope gene are highly protective against the Japanese encephalitis virus lethal infection in DNA priming--protein boosting immunization strategies.

The envelope (E) gene of Japanese encephalitis virus (JEV) plays a major protective role against JEV infection. In order to locate the part of E gene that is responsible for this protection, an N-terminal fragment EA (nucleotide number 933-1877 bp of JEV genome) and a C-terminal fragment EB (nucleotide number 1851-2330 bp of JEV genome) from E gene were prepared. Both of these fragments were used in the form of recombinant proteins (rEA and rEB) and plasmid DNA (pEA, pM15EA and pEB) for immunizations. Recombinant EA protein (rEA) was previously found to be non-protective because it was expressed in an insoluble form. Plasmid EA (pEA) was also found to be non-protective unless it is preceded by a 15 mer signal peptide derived from the very C-terminal of the membrane gene (M) of JEV to form pM15EA plasmid indicating the importance of the signal peptide in the expression of EA immunogenicity. Although pM15EA and pEB are both immunogenic and protective against JEV lethal infection, the protection by both fragments however is not optimal. Even when pM15EA and pEB were used together for immunization, maximum protection as those induced by control vaccine was not achieved. However, if individual fragments (EA or EB) were used in a DNA priming-protein boosting or protein priming-DNA boosting strategy, high levels of protection were achieved by both fragments. This was especially true for EA fragment where the level of protection against JEV lethal infection was equal to that induced by commercially available vaccine alone. The protection correlated very well with the neutralizing antibody titers and the T helper cell involved in this process in mainly the Th1 type.

Animals↗
PubMed2004

Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures.

We investigated the possibility of producing the therapeutic recombinant cytokine, Interferon-gamma (IFN-gamma), in transgenic rice cell (Oryza sativa, cultivar TNG67) suspension cultures. We tested expression of two vector constructs, each harboring an alphaAmy3 leader peptide and a C-terminus His 6 tag fused to a human IFN-gamma cDNA, one driven by a sucrose-starvation inducible promoter (rice alphaAmy3 promoter) and the other by a constitutive maize ubiquitin promoter, in rice cell suspensions, introduced via Agrobacterium tumefaciens. There was a significant difference in the amounts of recombinant IFN-gamma protein produced by the Ups and Amy cell lines, as cytosolic and secretory proteins respectively. Immunological analysis of IFN-gamma recombinant protein conferred a dose-dependent anti-dengue virus activity in human A549 cells, similar to the commercial product. We discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant IFN-gamma.

Cell Culture Techniques↗
PubMed2004

Replication-incompetent virions of Japanese encephalitis virus trigger neuronal cell death by oxidative stress in a culture system.

It has been shown that replication of the Japanese encephalitis virus (JEV) can trigger infected cells to undergo apoptosis. In the present study, it is further demonstrated that replication-incompetent virions of JEV, obtained by short-wavelength ultraviolet (UV) irradiation, could also induce host-cell death. It was found that UV-inactivated JEV (UV-JEV) caused cell death in neuronal cells such as mouse neuroblastoma N18 and human neuronal NT-2 cells, but not in non-neuronal baby hamster kidney BHK-21 fibroblast or human cervical HeLa cells. Only actively growing, but not growth-arrested, cells were susceptible to the cytotoxic effects of UV-JEV. Killing of UV-JEV-infected N18 cells could be antagonized by co-infection with live, infectious JEV, suggesting that virions of UV-JEV might engage an as-yet-unidentified receptor-mediated death-signalling pathway. Characteristically, mitochondrial alterations were evident in UV-JEV-infected N18 cells, as revealed by electron microscopy and a loss of membrane potential. N18 cells infected by UV-JEV induced generation of reactive oxygen species (ROS) as well as the activation of nuclear factor kappa B (NF-kappaB), and the addition of anti-oxidants or specific NF-kappaB inhibitors to the media greatly reduced the cytotoxicity of UV-JEV. Together, the results presented here suggest that replication-incompetent UV-JEV damages actively growing neuronal cells through a ROS-mediated pathway.

Animals↗
PubMed2004

Identification of Tbr-1/CASK complex target genes in neurons.

Tbr-1, a neuron-specific T-box transcription factor, plays a critical role in brain development. Here, we performed a computational search using the non-palindromic T-box binding sequence, namely the non-palindromic T-element, to determine the putative downstream target genes of Tbr-1. More than 20 identified genes containing the non-palindromic T-element in the 5' regulatory region were found expressed in brain. Luciferase reporter assays using cultured hippocampal neurons showed that overexpression of Tbr-1 and CASK-enhanced promoter activities of some of these putative target genes, including NMDAR subunit 2b (NR2b), glycine transporter, interleukin 7 receptor (IL-7R) and OX-2. Among these genes, NR2b promoter responded strongest to overexpression of Tbr-1 and CASK. Deletion of the non-palindromic T-elements from NR2b promoter impaired the induction by Tbr-1 and CASK. We also examined expression of these target genes in Tbr-1 knockout mice, it was found that NR2b expression was consistently downregulated. Similarly, both RNA and protein expression levels of NMDAR subunit 1 (NR1), which also contains the non-palindromic T-elements in its 5' regulatory region, were reduced in Tbr-1 knockout mice. We suggest that Tbr-1/CASK protein complex regulates expression of these downstream target genes and thus modulates neuronal activity and function.

Amino Acid Transport Systems, Neutral↗
PubMed2004

Blocking of the alpha interferon-induced Jak-Stat signaling pathway by Japanese encephalitis virus infection.

The induction of alpha/beta interferon (IFN-alpha/beta) is a powerful host defense mechanism against viral infection, and many viruses have evolved strategies to overcome the antiviral effects of IFN. In this study, we found that IFN-alpha had only some degree of antiviral activity against Japanese encephalitis virus (JEV) infection, in contrast to another flavivirus, dengue virus serotype 2, which was highly sensitive to IFN-alpha in the cultured cell system. JEV infection appeared to render cells resistant to IFN-alpha since the IFN-alpha-induced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) was gradually reduced as the JEV infection progressed. Since the biological activities of IFNs are triggered by the Janus kinase (Jak) signal transducer and activation of transcription (Stat) signaling cascade, we then studied the activation of Jak-Stat pathway in the virus-infected cells. The IFN-alpha-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 was suppressed by JEV in a virus replication and de novo protein synthesis-dependent manner. Furthermore, JEV infection blocked the tyrosine phosphorylation of IFN receptor-associated Jak kinase, Tyk2, without affecting the expression of IFN-alpha/beta receptor on the cell surface. Consequently, expression of several IFN-stimulated genes in response to IFN-alpha stimulation was also reduced in the JEV-infected cells. Overall, our findings suggest that JEV counteracts the effect of IFN-alpha/beta by blocking Tyk2 activation, thereby resulting in inhibition of Jak-Stat signaling pathway.

Animals↗
PubMed2004

Confocal laser scanning microscopy: I. An overview of principle and practice in biomedical research.

Evolving from conventional microscopic technologies, confocal microscopy has proved itself to play an important role in the biomedical research during the past decade. Confocal microscope has many advantages over traditional microscope including the ability to look deeply into inside cells with less photodamage and photobleach, reconstruct three-dimensional images, and chart intracellular dynamic events in the living cells. With these remarkable properties and the availability of fluorescent dyes for living cells, the confocal microscopy has been widely used in solving many unknown questions in biological and pharmacological fields. In clinics, confocal microscope has also served as an important tool to observe the living cells in skins and eyes. For anesthesiologists, confocal microscope has made possible novel experimental approaches for the effects of multiple anesthetic agents on cells. Furthermore, the technology of fibreoptical confocal endomicroscopy is now on its way of maturation. It will soon be the era for confocal microscopy to explore the "cell behavior" inside of intact living tissues.

Anesthetics↗
PubMed2003

Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice.

To evaluate the potential of DNA vaccine against dengue (DEN) infection, we characterize the protective efficacy and immune responses of mice intramuscularly injected with plasmid encoding DEN-2 non-structural protein 1 (NS1). Intravenously challenged by lethal DEN-2, mice vaccinated with NS1-DNA exhibited a delay onset of paralysis, a marked decrease of morbidity, and a significant enhancement of survival. In addition to a moderate increase of NS1-specific antibody titer from immunized mice measured by ELISA, a strong priming effect on anti-NS1 response was also noticed in plasmid NS1-vaccinated mice by radioimmunoprecipitation (RIP) or immunoblot analysis. Interestingly, newborn mice from NS1-DNA-immunized dam showed stronger resistance to viral challenge, as compared to those from vector DNA or PBS-immunized dams, indicating the protective role of NS1-specific antibody. In contrast to humoral immune response, DNA immunization can elicit strong cellular immune responses, including NS1-specific T cell proliferation and cytolytic activity. The NS1-DNA-induced protection can be further augmented by co-injection of plasmid encoding interleukin 12 (IL-12), suggesting an effector role of Th1 immunity against DEN infection. In summary, our results suggest the potential of NS1-DNA vaccine against DEN infection, and indicate both NS1-specific humoral and cellular immune responses contribute to the protection.

Animals↗
PubMed2003

Carvedilol, a new antioxidative beta-blocker, blocks in vitro human peripheral blood T cell activation by downregulating NF-kappaB activity.

OBJECTIVE: The activation of T lymphocytes contributes to the inflammatory process of atherosclerosis. Here we examined the effects of carvedilol, a new beta-blocker containing an antioxidative property, on the activation of T cells. METHODS: Human peripheral blood T cells were negatively selected from whole blood. Cytokines were measured by ELISA. The NF-kappaB and related protein activity was determined by electrophoretic mobility shift assays, Western blotting, kinase assays and transfection assays. RESULTS: Carvedilol was nontoxic at concentrations </=10 microM, however, higher dosages (>/=20 microM) induced T cell apoptosis. We demonstrated that carvedilol inhibited cytokine production from various stimuli-activated T cells. Carvedilol also suppressed the expression of T cell activation markers, including CD25, CD69 and CD71. Molecular investigation indicated that carvedilol specifically downregulated NF-kappaB but not activator protein 1 DNA-binding activity in activated T cells. The inhibitory effect was likely due to its antioxidative property. Meanwhile, carvedilol prevented stimuli-induced IkappaBalpha degradation. Such an effect was mediated through the inhibition of IkappaBalpha kinase activity. The inhibitory specificity on NF-kappaB by carvedilol was also demonstrated in transfection assays. CONCLUSIONS: Our results demonstrated a novel therapeutic mechanism of carvedilol in atherosclerosis, namely the inhibition of T cell activation via downregulating NF-kappaB activity.

Adrenergic beta-Antagonists↗
PubMed2003

Propofol reduces nitric oxide biosynthesis in lipopolysaccharide-activated macrophages by downregulating the expression of inducible nitric oxide synthase.

Nitric oxide is an active oxidant that contributes to the physiology and pathophysiology of macrophages. Propofol has been widely used in intravenous anesthesia. It possess antioxidant and immunomodulating effects. This study aimed to evaluate the effects of propofol on nitric oxide production in lipopolysaccharide-activated macrophages. Exposure of macrophages to propofol (25, 50 and 75 micro M), to lipopolysaccharide (0.5, 1, 1.5 and 2 ng/ml) or to a combination of propofol and lipopolysaccharide did not affect cell viability. However, propofol at 100 micro M led to significant cell death ( P<0.05). The levels of nitrite, an oxidative product of nitric oxide, were increased in lipopolysaccharide-treated macrophages in a concentration-dependent manner ( P<0.01), while propofol could concentration-dependently decrease the lipopolysaccharide-enhanced nitrite levels ( P<0.01). Immunoblotting analysis revealed that lipopolysaccharide increased the protein level of inducible nitric oxide synthase (iNOS). The co-treatment of propofol and lipopolysaccharide significantly reduced this lipopolysaccharide-induced iNOS protein (357+/-49 x 10(3) versus 92+/-6 x 10(3) arbitrary units, P<0.01). Analysis by reverse transcriptase-polymerase chain reaction showed that lipopolysaccharide induced mRNA of iNOS, but that the inductive effect was inhibited by propofol (95+/-7 x 10(2) versus 30+/-4 x 10(2) arbitrary units, P<0.01). This study has demonstrated that propofol, at therapeutic concentrations, could suppress nitric oxide biosynthesis by inhibiting iNOS expression in lipopolysaccharide-activated macrophages. The mechanism of suppression was at a pretranslational level.

Animals↗
PubMed2003

Propofol suppresses macrophage functions and modulates mitochondrial membrane potential and cellular adenosine triphosphate synthesis.

BACKGROUND: Propofol is an intravenous anesthetic agent that may impair host defense system. The aim of this study was to evaluate the effects of propofol on macrophage functions and its possible mechanism. METHODS: Mouse macrophage-like Raw 264.7 cells were exposed to propofol, at 3, 30 (a clinically relevant concentration), and 300 microm. Cell viability, lactate dehydrogenase, and cell cycle were analyzed to determine the cellular toxicity of propofol to macrophages. After administration of propofol, chemotactic, phagocytic, and oxidative ability and interferon-gamma mRNA production were carried out to validate the potential effects of propofol on macrophage functions. Mitochondrial membrane potential and cellular adenosine triphosphate levels were also analyzed to evaluate the role of mitochondria in propofol-induced macrophage dysfunction. RESULTS: Exposure of macrophages to 3 and 30 microm propofol did not affect cell viability. When the administered concentration reached 300 microm, propofol would increase lactate dehydrogenase release, cause arrest of cell cycle in G1/S phase, and lead to cell death. In the 1-h-treated macrophages, propofol significantly reduced macrophage functions of chemotactic and oxidative ability in a concentration-dependent manner. However, the suppressive effects were partially or completely reversed after 6 and 24 h. Propofol could reduce phagocytic activities of macrophages in concentration- and time-dependent manners. Exposure of macrophages to lipopolysaccharide induced the mRNA of interferon-gamma, but the induction was significantly blocked by propofol. Propofol concentration-dependently decreased the membrane potential of macrophage mitochondria, but the effects were descended with time. The levels of cellular adenosine triphosphate in macrophages were also reduced by propofol. CONCLUSIONS: A clinically relevant concentration of propofol can suppress macrophage functions, possibly through inhibiting their mitochondrial membrane potential and adenosine triphosphate synthesis instead of direct cellular toxicity.

Actins↗
PubMed2003

Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein.

Previously reported findings by our group showed that non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) was localized mainly in the JEV-induced convoluted membrane (CM), which has been proposed to originate from rough endoplasmic reticulum (rER), Golgi apparatus or the trans-Golgi network (TGN), and serves as a reservoir for viral proteins during virus assembly. Earlier findings indicated that NS3 of Kunjin virus interacts with microtubules. In addition, one of the Golgi-associated proteins, tumour susceptibility protein 101 (TSG101), associates with microtubules and is required for budding of retroviral particles. To clarify the association of NS3 with microtubules or with TSG101 during JEV assembly, we applied immunofluorescence, co-immunoprecipitation and immunoelectron microscopic methods. Virus infection, as well as transfection with an NS2B-NS3 expression plasmid, induced microtubule rearrangement. When cells were treated with colchicine, which interferes with microtubule polymerization, NS3 still associated with tubulin and TSG101. Furthermore, tubulin and TSG101 were co-localized with NS3 in the CM by immunogold labelling. Our observations indicate that microtubules and TSG101 associate with NS3, which is incorporated into the JEV-induced structure during JEV replication.

Animals↗
PubMed2002

Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability.

Kit/SCF signaling and Mitf-dependent transcription are both essential for melanocyte development and pigmentation. To identify Mitf-dependent Kit transcriptional targets in primary melanocytes, microarray studies were undertaken. Among identified targets was BCL2, whose germline deletion produces melanocyte loss and which exhibited phenotypic synergy with Mitf in mice. BCL2's regulation by Mitf was verified in melanocytes and melanoma cells and by chromatin immunoprecipitation of the BCL2 promoter. Mitf also regulates BCL2 in osteoclasts, and both Mitf(mi/mi) and Bcl2(-/-) mice exhibit severe osteopetrosis. Disruption of Mitf in melanocytes or melanoma triggered profound apoptosis susceptible to rescue by BCL2 overexpression. Clinically, primary human melanoma expression microarrays revealed tight nearest neighbor linkage for MITF and BCL2. This linkage helps explain the vital roles of both Mitf and Bcl2 in the melanocyte lineage and the well-known treatment resistance of melanoma.

Adenoviridae↗
PubMed2002

Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein.

Nitric oxide (NO) plays a crucial role in the physiological and pathophysiological regulations of osteoblast functions. This study is designed to evaluate the toxic effects of NO released by sodium nitroprusside (SNP), an NO donor, on neonatal Wistar rat calvarial osteoblasts from the analyses of cell viability, alkaline phosphatase (ALP) activity, cell morphology, apoptotic cells, terminal deoxynucleotidyl transferase-mediated dUTP nick end-label (TUNEL) assay, DNA ladder, and immunocytochemistry and Western blot for proapoptotic Bax protein. SNP increased the levels of nitrite, an oxidative product of NO, in the culture medium of osteoblasts in concentration- and time-dependent manners, and altered cell morphologies to round and shrinkage shapes. Administration of osteoblasts with SNP resulted in concentration- and time-dependent decreases of cell viability and ALP activity. Analysis of apoptotic cells revealed that SNP increased the percentages of osteoblasts processing apoptosis. Analyses of TUNEL and DNA ladder showed that SNP caused DNA fragmentation. Pretreatment with cycloheximide, an inhibitor of protein synthesis, partially blocked SNP-induced osteoblast apoptosis. Imunocytochemical and immunoblotting analyses revealed that SNP increased Bax protein in osteoblasts. This study suggests that SNP could increase the levels of NO in osteoblasts, and cause osteoblast apoptosis possibly through the de novo synthesis of proapoptotic Bax protein.

Alkaline Phosphatase↗
PubMed2002

Roles of the glucocorticoid receptor in resectable hepatocellular carcinoma.

BACKGROUND: The glucocorticoid receptor (GR) was discovered in the cytosol of hepatocellular carcinoma (HCC) cells more than 10 years ago. However, the influence of the GR on the prognosis of HCC after liver resection remains unclear. METHODS: Ninety-two consecutive patients with HCC who survived liver resection and who did not receive any preoperative neoadjuvant therapy were enrolled in this study. The GR level in cytosol of cancerous tissue was determined by the dextran-coated charcoal method. The clinicopathologic characteristics and long-term prognosis of patients with GR-positive tumors (GR-positive group) were compared with those of patients with GR-negative tumors (GR-negative group). RESULTS: GR was found in 63 patients (68.5%) with a mean +/- SEM concentration of 26.97 +/- 4.05 fmol/g protein. There were no significant differences in patient clinicopathologic characteristics between GR-positive and GR-negative groups. The 5-year disease-free and actuarial survival rates for GR-positive and GR-negative groups were 21.6% and 44.4% (P =.002) and 57.2% and 83.3% (P =.0003), respectively. After multivariate analysis was performed, GR positivity was found to be an independent prognostic factor of disease-free and actuarial survival after liver resection for HCC. CONCLUSIONS: The GR can be found in the cytosol of most HCCs and is an independent prognostic factor of HCC after liver resection. Patients with GR-positive HCC have lower survival rates than those with GR-negative HCC.

Carcinoma, Hepatocellular↗
PubMed2002

Antiviral effects of an iminosugar derivative on flavivirus infections.

Endoplasmic reticulum (ER) alpha-glucosidase inhibitors, which block the trimming step of N-linked glycosylation, have been shown to eliminate the production of several ER-budding viruses. Here we investigated the effects of one such inhibitor, N-nonyl-deoxynojirimycin (NN-DNJ), a 9-carbon alkyl iminosugar derivative, on infection by Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2). In the presence of NN-DNJ, JEV and DEN-2 infections were suppressed in a dose-dependent manner. This inhibitory effect appeared to influence DEN-2 infection more than JEV infection, since lower concentrations of NN-DNJ substantially blocked DEN-2 replication. Secretion of the flaviviral glycoproteins E and NS1 was greatly reduced, and levels of DEN-2 viral RNA replication measured by fluorogenic reverse transcription-PCR were also decreased, by NN-DNJ. Notably, the viral glycoproteins, prM, E, and NS1 were found to associate transiently with the ER chaperone calnexin, and this interaction was affected by NN-DNJ, suggesting a potential role of calnexin in the folding of flaviviral glycoproteins. Additionally, in a mouse model of lethal challenge by JEV infection, oral delivery of NN-DNJ reduced the mortality rate. These findings show that NN-DNJ has an antiviral effect on flavivirus infection, likely through interference with virus replication at the posttranslational modification level, occurring mainly in the ER.

1-Deoxynojirimycin↗
PubMed2002

Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response.

The malfunctioning of the endoplasmic reticulum (ER) of cells in hosts ranging from yeast to mammals can trigger an unfolded protein response (UPR). Such malfunctioning can result from a variety of ER stresses, including the inhibition of protein glycosylation and calcium imbalance. To cope with ER stresses, cells may rely on the UPR to send a signal(s) from the ER to the nucleus to stimulate appropriate cellular responses, including induction of chaperone expression. During Japanese encephalitis virus (JEV) infection, the lumen of the ER rapidly accumulates substantial amounts of viral proteins for virus progeny production. In the present study, we demonstrate that as evidenced by certain chaperone inductions, JEV infection triggers the UPR in fibroblast BHK-21 cells and in neuronal N18 and NT-2 cells, in which JEV results in apoptotic cell death. By contrast, no UPR was observed in apoptosis-resistant K562 cells infected by JEV. JEV infection also activates expression of CHOP/GADD153, a distinctive transcription factor often induced by the UPR, and appears to trigger activation of p38 mitogen-activated protein kinase, a posttranslational activator of CHOP. Ectopic enforcement of CHOP expression enhanced JEV-induced apoptosis, whereas treatment with a p38-specific inhibitor, SB203580, partially blocked JEV-induced apoptosis. Interestingly, bcl-2 overexpression and treatment with a pancaspase inhibitor, z-VAD-fmk, inhibited CHOP induction and diminished JEV-induced apoptosis, suggesting that Bcl-2 and caspases could be the upstream regulators of CHOP. Our results thus suggest that virus-induced ER stress may participate, via p38-dependent and CHOP-mediated pathways, in the apoptotic process triggered by JEV infection.

Animals↗
PubMedDate not supplied

Selection of immunodominant fragments from envelope gene for vaccine against Japanese encephalitis virus in DNA priming-protein boosting protocols.

Fragmentation of E gene of JEV into smaller fragments, none of the fragments either in plasmids form or in recombinant protein form can induce optimal protection against the virus infection. It is only when DNA priming-protein boosting strategies are used then the N-terminal E(A) and the C-terminal E(B) showed full protection against JEV as those induced by commercial vaccine, provided both fragments are preceded in the N-terminal by a signal peptide M(15) derived from C-terminal of prM gene in JEV genome. When the subfragments of E(A): E(A1) and E(A2) and E(B): E(B1) and E(B2) are tested, only E(A1) subfragment can replace E(A) in protein boosting to induce optimal protection against JEV, E(A2), E(B1), E(B2) in plasmid or protein forms are not. Therefore, along the E gene (978-2330 bp) N-terminal, E(A1) (978-1580 bp) and C-terminal E(B) (1851-2330 bp) are the most effective in inducing immunity against JEV but not the middle fragment E(A2) (1518-1877 bp) (see for orientation of E(A1), E(A2) and E(B) in E gene). Under the notion that molecular complexity determines the outcome of immune response of the host, E(B) being shorter, simpler in molecular structure and can be easily expressed in soluble form in E. coli (as opposed to insoluble E(A1)), E(B) probably will be the choice as a candidate vaccine to protect the host against JEV infection.

Animals↗