Microarray analysis of gene expression in olive flounder liver infected with viral haemorrhagic septicaemia virus (VHSV).

The most fatal viral pathogen in olive flounder Paralichthys olivaceus, is viral hemorrhagic septicemia virus, which afflicts over 48 species of freshwater and marine fish. Here, we performed gene expression profiling on transcripts isolated from VHSV-infected olive flounder livers using a 13 K cDNA microarray chip. A total of 1832 and 1647 genes were upregulated and down-regulated over two-fold, respectively, after infection. A variety of immune-related genes showing significant changes in gene expression were identified in upregulated genes through gene ontology annotation. These genes were grouped into categories such as antibacterial peptide, antigen-recognition and adhesion molecules, apoptosis, cytokine-related pathway, immune system, stress response, and transcription factor and regulatory factors. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to the microarray data. In conclusion, these results may be useful for the identification of specific genes or for the diagnosis of VHSV infection in flounder.

HBx induces the proliferation of hepatocellular carcinoma cells via AP1 over-expressed as a result of ER stress.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and chronic hepatitis B virus (HBV) infection is the most common risk factor for HCC. The HBV proteins can induce oncogenic or synergy effects with a hyperproliferative response on transformation into HCC. CREBH (cAMP-responsive, element-binding protein H), activated by stress in the endoplasmic reticulum (ER), is an ER-resident transmembrane bZIP (basic leucine zipper) transcription factor that is specifically expressed in the liver. In the present study, we address the role played by CREBH activated by ER stress in HBV-induced hepatic cell proliferation. We confirmed CREBH activation by ER stress and showed that it occurred as a result of/via hepatitis B virus X (HBx)-induced ER stress. CREBH activated by HBx increased the expression of AP-1 target genes through c-Jun induction. Under pathological conditions such as liver damage or liver regeneration, activated CREBH may have an important role to play in hepatic inflammation and cell proliferation, as an insulin receptor with dual functions under these conditions. We showed that CREBH activated by HBx interacted with HBx protein, leading to a synergistic effect on the expression of AP-1 target genes and the proliferation of HCC cells and mouse primary hepatocytes. In conclusion, in HBV-infected hepatic cells or patients with chronic HBV, CREBH may induce proliferation of hepatic cells in co-operation with HBx, resulting in HCC.

Characterization, expression profile, and promoter analysis of the Rhodeus uyekii vitellogenin Ao1 gene.

The fish Vitellogenin (Vg) gene has been applied as a biomarker for exposure to estrogenic compounds in the aquatic environment. In this study, we cloned and characterized Vg cDNA from the Korean rose bitterling Rhodeus uyekii (Ru-Vg). The Ru-Vg cDNA encodes a 1424-amino-acid polypeptide that belongs to the VgAo1 family and contains a putative signal peptide, lipovitellin I, phosvitin, and lipovitellin II, but does not contain the vWFD domain or the C-terminal peptide. The deduced Ru-Vg protein has high amino acid identity (73.97%-32.17%) with fish Vg proteins. Pairwise alignment and phylogenetic analysis revealed that Ru-Vg is most closely related to Acheilognathus yamatsutae Vg. Ru-Vg transcripts were detected using quantitative polymerase chain reaction in all tissues tested, with the highest level of expression observed in the ovary. Ru-Vg mRNA was upregulated in R. uyekii hepatopancreas cells in response to treatment with 17ß-estradiol (E2) or 17α-ethinylestradiol (EE2). Luciferase reporter expression, driven by the 5'-regulatory region of the Ru-Vg gene spanning from -1020 bp to the start codon was induced by the estrogen receptor and was synergistically activated by treatment with E2 or EE2. These results suggest that R. uyekii and the Ru-Vg gene may be useful as biomarkers for exposure to E2 or EE2.

Fatty acids increase hepatitis B virus X protein stabilization and HBx-induced inflammatory gene expression.

The protein level of human hepatitis B virus (HBV) in infection is variable, depending on patient context. We previously reported that HBV X protein (HBx) induces hepatic lipid accumulation and inflammation. Here, we show that abnormal levels of hepatic fatty acids increase HBx protein stability during HBV expression, resulting in the potentiation of HBx-induced inflammation. Reactive oxygen species, Ca(2+) signaling and expression levels of various lipid metabolic genes were investigated in HBx-expressing cells and in HBx transgenic mice. Fatty acids, including palmitate, stearate and oleate, increased HBx protein stability by preventing proteasome-dependent degradation. Hepatic inflammation induced by a high fat diet (HFD) and HBx was measured based on the expression of interleukin-6 and tumor necrosis factor α. In addition, the protein level of HBx increased in HFD-HBx transgenic mice. Reactive oxygen species production and intracellular Ca(2+) signal activation play critical roles in fatty-acid-induced HBx stabilization. Abnormal levels of hepatic fatty acids resulted in synergistic induction of HBx protein and liver inflammatory gene expression through HBx protein stabilization. These results indicate that different fatty acid levels in the liver might affect HBV-induced pathogenesis.

Molecular characterization and expression analysis of a peroxiredoxin 1 cDNA from Korean rose bitterling (Rhodeus uyekii).

Peroxiredoxins (Prxs), also known as natural killer cell enhancing factors in fish, role as antioxidant proteins and participate in a variety of biological processes, including H2O2-mediated cell signaling, molecular chaperoning, and mitochondrial function. In this study, we isolated and characterized a Prx 1 cDNA from the Korean rose bitterling Rhodeus uyekii, and designated it RuPrx 1. The RuPrx 1 cDNA encodes a 197-amino-acid polypeptide that belongs to the class of typical 2-Cys Prxs that contain peroxidatic and resolving cysteines. The deduced RuPrx 1 protein shows strong homology (77.38-92.89 %) with Prx 1 proteins from other species, including fish, amphibians, and mammals, and it is most closely related to rainbow smelt Prx 1. RuPrx 1 mRNA was ubiquitously detected in all tested tissues and its expression was comparatively high in the brain, intestine, kidney, liver, ovary, stomach, and testis. Expression of RuPrx 1 mRNA in liver peaked 3 h post-infection with Aeromonas hydrophila and decreased 24 h post-infection while the expression in intestine decreased 24 h post-infection. These results suggest that RuPrx 1 is conserved through evolution and may play roles similar to its mammalian counterparts.

Hepatitis B virus X increases immune cell recruitment by induction of chemokine SDF-1.

Hepatitis B virus X protein is a major factor in the HBV-induced disease developments. Stromal cell-derived factor-1 is a small cytokine that is strongly chemotactic for lymphocytes. We explored the role of HBx on recruitment of HBV-induced virus-nonspecific immune cells into liver. Immune cell recruitment and SDF-1 expression level significantly increased in livers of HBx-transgenic mice and X-box binding protein-1 significantly increased SDF-1 gene expression. Finally, we confirmed that immune cell recruitment into liver tissues of HBx-TG mice was diminished by a chemokine receptor antagonist. Therefore, HBx increases ER stress-dependent SDF-1 expression and induces HBV-induced immune cell recruitment into liver.

Molecular and functional analyses of the fast skeletal myosin light chain2 gene of the Korean oily bitterling, Acheilognathus koreensis.

We identified and characterized the primary structure of the Korean oily bitterling Acheilognathus koreensis fast skeletal myosin light chain 2 (Akmlc2f), gene. Encoded by seven exons spanning 3955 bp, the deduced 168-amino acid AkMLC2f polypeptide contained an EF-hand calcium-binding motif and showed strong homology (80%-98%) with the MLC2 proteins of Ictalurus punctatus and other species, including mammals. Akmlc2f mRNA was highly enriched in skeletal muscles, and was detectable in other tissues. The upstream regions of Akmlc2f included a TATA box, one copy of a putative MEF-2 binding site and several putative C/EBPß binding sites. The functional activity of the promoter region of Akmlc2f was examined using luciferase and red fluorescent protein reporters. The Akmlc2f promoter-driven reporter expressions were detected and increased by the C/EBPß transcription factor in HEK293T cells. The activity of the promoter of Akmlc2f was also confirmed in the developing zebrafish embryo. Although the detailed mechanism underlying the expression of Akmlc2f remains unknown, these results suggest the muscle-specific expression of Akmlc2f transcript and the functional activation of Akmlc2f promoter by C/EBPß.

Tumor suppressor protein VHL inhibits Hedgehog-Gli activation through suppression of Gli1 nuclear localization.

The transcription factor Gli1 acts in the last known step of the Hedgehog signaling, and deregulation of Gli1 is implicated in human cancers. VHL protein is widely expressed in both fetal and adult tissues and acts as a tumor suppressor. Here, we demonstrate the molecular mechanism through which VHL inhibits the Hedgehog-Gli pathway. VHL decreased Gli1-mediated promoter transactivation as well as the expression of Hedgehog/Gli pathway target genes. Nuclear translocation of cytosolic Gli1 protein was inhibited by VHL via protein-protein interaction. These results indicate that overexpression of VHL may antagonize Hedgehog-Gli activation at the post-translational level in Hedgehog pathway-induced cancers.

Hepatic STAMP2 decreases hepatitis B virus X protein-associated metabolic deregulation.

Six transmembrane protein of prostate 2 (STAMP2) plays a key role in linking inflammatory and diet-derived signals to systemic metabolism. STAMP2 is induced by nutrients/feeding as well as by cytokines such as TNFα, IL-1ß, and IL-6. Here, we demonstrated that STAMP2 protein physically interacts with and decreases the stability of hepatitis B virus X protein (HBx), thereby counteracting HBx-induced hepatic lipid accumulation and insulin resistance. STAMP2 suppressed the HBx-mediated transcription of lipogenic and adipogenic genes. Furthermore, STAMP2 prevented HBx-induced degradation of IRS1 protein, which mediates hepatic insulin signaling, as well as restored insulin-mediated inhibition of gluconeogenic enzyme expression, which are gluconeogenic genes. We also demonstrated reciprocal expression of HBx and STAMP2 in HBx transgenic mice. These results suggest that hepatic STAMP2 antagonizes HBx-mediated hepatocyte dysfunction, thereby protecting hepatocytes from HBV gene expression.

Molecular characterization of tripartite motif protein 25 (TRIM25) involved in ERα-mediated transcription in the Korean rose bitterling Rhodeus uyekii.

Tripartite motif-containing 25 (TRIM25), also known as estrogen-responsive finger protein (EFP), plays an essential role in cell proliferation and innate immunity. In the present study, we isolated and characterized the TRIM25 cDNA of the Korean rose bitterling Rhodeus uyekii, designated RuTRIM25. It encodes an open reading frame of 669 amino acids containing an N-terminal RBCC motif composed of a RING domain, two B boxes, and a coiled-coil domain and a C-terminal B30.2 (PRY/SPRY) domain. RuTRIM25 shows strong homology (79.7%) to zebrafish TRIM25 and shared 32.4-28.8% homology with TRIM25 from other species, including mammals. RuTRIM25 mRNA was expressed ubiquitously. It was highly expressed in the ovary, spleen, and liver and moderately in the stomach and intestine of normal Korean rose bitterling. The intracellular localization of RuTRIM25 in HEK293T cells was diffusely localized in the cytoplasm and its RING domain deletion mutant (RuTRIM25ΔR) was detected diffusely with some aggregates in the cytoplasm. RuTRIM25, but not RuTRIM25ΔR, is ubiquitinated in vivo. Ectopic expression of RuTRIM25 synergistically activated the estrogen receptor (ER)-mediated luciferase reporter activity in a dose-dependent manner in HEK293T cells. Together, these results suggest that the RuTRIM25 regulates the ER-mediated transcription in fish similarly to its mammalian counterpart.

Characterization of Paralichthys olivaceus peroxisome proliferator-activated receptor-α gene as a master regulator of flounder lipid metabolism.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that play key roles in lipid and energy homeostasis. Paralichthys olivaceus PPARα (PoPPARα) cDNA was isolated by initial reverse transcription-polymerase chain reaction (RT-PCR) using conserved region among fish species and rapid amplification of cDNA ends (RACE). The full-length of PoPPARα cDNA is 2040-bp long encoding a polypeptide with 505 amino acids and containing a DNA binding domain (C4-type zinc finger) and a ligand-binding domain. PoPPARα was detected from 1 day post-hatch and was highly expressed in the stomach, liver, and intestine of continuously fed flounder, approximately 16 cm in size. PoPPARα mRNA expression was down-regulated in the kidney, stomach, and liver of the 4.5-month-old flounder after a 30 day food-deprivation period. PoPPARα activates the PPAR response element (PPRE)-driven reporter, and treatment with Wy14643, a PPARα agonist, augmented PoPPARα-stimulated peroxisome proliferator response element activity in HINAE and HepG2 cells. PoPPARα activated the expression of fatty acid ß-oxidation related genes such as carnitine palmitoyltransferase 1A, medium chain acyl-CoA dehydrogenase, and acyl-CoA oxidase 1 and inhibited the expression of sterol regulatory element binding protein and fatty acid synthase by competitively inhibiting LXR/RXR heterodimer formation. These results suggest that PoPPARα plays an important role in lipid metabolism of olive flounder and that it is functionally and evolutionarily conserved in olive flounder and mammals.

Hepatitis B virus X protein stimulates the Hedgehog-Gli activation through protein stabilization and nuclear localization of Gli1 in liver cancer cells.

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver diseases, which frequently results in hepatits, cirrhosis, fibrosis, and ultimately hepatocellular carcinoma (HCC). Recent studies have shown the activation of Hedgehog signaling in HCC. Here, we provide evidences that HBV induces Gli-directed gene transactivation. HBx increases the protein stability of Gli proteins, which are key transcription factors of the Hedgehog signaling pathway, and nucleus translocation of Gli1 through direct protein interaction of HBx and Gli1. This functional synergism of Gli1 protein by HBx increases the Hedgehog activation-directed gene expression. Taken together, these results suggest that HBV infection might induce hepatocellular carcinoma by modulating post-translational activation of the hedgehog signaling components.

Oxygenated derivatives of cholesterol promote hepatitis B virus gene expression through nuclear receptor LXRα activation.

Hepatitis B virus (HBV) gene expression and replication are regulated by the activation of a number of liver-enriched transcription factors dependent on intracellular and extracellular stimuli. However, the association between the metabolic events and HBV gene expression remains unclear. In this study, we assessed the effects of cholesterol metabolism on HBV viral replication and gene expression. Exposure of oxygenated derivatives of cholesterol (oxysterols) increased HBV gene expression and viral promoter activity. This increase in HBV transcription and replication was directed by nuclear receptor LXRα induction in the presence of oxysterols. In addition, HBV viral expression by oxysterol was inhibited through small heterodimer partner and sterol regulatory element-binding protein 2, key regulators of cholesterol synthesis. When IFNα and oxysterols were co-incubated, oxysterols and LXRα significantly reduced the anti-HBV effects of IFNα. These results point to a novel mechanism of oxysterol-mediated gene regulation in HBV replication and a potent mechanism underlying the failure of IFNα-based treatment.

Endoplasmic reticulum stress induced by hepatitis B virus X protein enhances cyclo-oxygenase 2 expression via activating transcription factor 4.

Chronic hepatitis B is a disease of the liver that can progress to cirrhosis and liver cancer. The HBx (hepatitis B virus X) protein of hepatitis B virus is a multifunctional regulator that induces ER (endoplasmic reticulum) stress by previously unknown mechanisms. ER stress plays a critical role in inflammatory induction and COX2 (cyclo-oxygenase 2) is an important mediator of this inflammation. In the present study, we demonstrate the molecular mechanisms of HBx on induction of ER stress and COX2 expression. In addition, HBx reduced expression of enzymes which are involved in mitochondrial ß-oxidation of fatty acids and the mitochondrial inner membrane potential. The reduction in intracellular ATP levels by HBx induced the unfolded protein response and COX2 expression through the eIF2α (eukaryotic initiation factor 2α)/ATF4 (activating transcription factor 4) pathway. We confirmed that ATF4 binding to the COX2 promoter plays a critical role in HBx-mediated COX2 induction. The results of the present study suggest that HBV infection contributes to induction of hepatic inflammation through dysfunction of cellular organelles including the ER and mitochondria.

Forest cover classification by optimal segmentation of high resolution satellite imagery.

This study investigated whether high-resolution satellite imagery is suitable for preparing a detailed digital forest cover map that discriminates forest cover at the tree species level. First, we tried to find an optimal process for segmenting the high-resolution images using a region-growing method with the scale, color and shape factors in Definiens(®) Professional 5.0. The image was classified by a traditional, pixel-based, maximum likelihood classification approach using the spectral information of the pixels. The pixels in each segment were reclassified using a segment-based classification (SBC) with a majority rule. Segmentation with strongly weighted color was less sensitive to the scale parameter and led to optimal forest cover segmentation and classification. The pixel-based classification (PBC) suffered from the "salt-and-pepper effect" and performed poorly in the classification of forest cover types, whereas the SBC helped to attenuate the effect and notably improved the classification accuracy. As a whole, SBC proved to be more suitable for classifying and delineating forest cover using high-resolution satellite images.

Estimation of effective plant area index for South Korean forests using LiDAR system.

Light Detection and Ranging (LiDAR) systems can be used to estimate both vertical and horizontal forest structure. Woody components, the leaves of trees and the understory can be described with high precision, using geo-registered 3D-points. Based on this concept, the Effective Plant Area Indices (PAI(e)) for areas of Korean Pine (Pinus koraiensis), Japanese Larch (Larix leptolepis) and Oak (Quercus spp.) were estimated by calculating the ratio of intercepted and incident LIDAR laser rays for the canopies of the three forest types. Initially, the canopy gap fraction (G ( LiDAR )) was generated by extracting the LiDAR data reflected from the canopy surface, or inner canopy area, using k-means statistics. The LiDAR-derived PAI(e) was then estimated by using G ( LIDAR ) with the Beer-Lambert law. A comparison of the LiDAR-derived and field-derived PAI(e) revealed the coefficients of determination for Korean Pine, Japanese Larch and Oak to be 0.82, 0.64 and 0.59, respectively. These differences between field-based and LIDAR-based PAI(e) for the different forest types were attributed to the amount of leaves and branches in the forest stands. The absence of leaves, in the case of both Larch and Oak, meant that the LiDAR pulses were only reflected from branches. The probability that the LiDAR pulses are reflected from bare branches is low as compared to the reflection from branches with a high leaf density. This is because the size of the branch is smaller than the resolution across and along the 1 meter LIDAR laser track. Therefore, a better predictive accuracy would be expected for the model if the study would be repeated in late spring when the shoots and leaves of the deciduous trees begin to appear.

Bile acids increase hepatitis B virus gene expression and inhibit interferon-alpha activity.

Hepatitis B virus (HBV) is a 3.2 kb DNA virus that preferentially replicates in the liver. A number of transcription factors, including nuclear receptors, regulate the activities of HBV promoters and enhancers. However, the association between these metabolic events and HBV replication remains to be clearly elucidated. In the present study, we assessed the effects of bile acid metabolism on HBV gene expression. Conditions associated with elevated bile acid levels within the liver include choleostatic liver diseases and an increased dietary cholesterol uptake. The results obtained in the present study demonstrate that bile acids promote the transcription and expression of the gene for HBV in hepatic cell lines; in addition, farnesoid X receptor alpha and the c-Jun N-terminal kinase/c-Jun signal transduction pathway mediate the regulatory effect of bile acids. Furthermore, an orphan nuclear receptor, small heterodimer partner protein, is also involved in the bile acid-mediated regulation of HBV gene expression. The bile acid-mediated promotion of HBV gene expression counteracts the antiviral effect of interferon-alpha.

Estimating stem volume and biomass of Pinus koraiensis using LiDAR data.

The objective of this study was to estimate the stem volume and biomass of individual trees using the crown geometric volume (CGV), which was extracted from small-footprint light detection and ranging (LiDAR) data. Attempts were made to analyze the stem volume and biomass of Korean Pine stands (Pinus koraiensis Sieb. et Zucc.) for three classes of tree density: low (240 N/ha), medium (370 N/ha), and high (1,340 N/ha). To delineate individual trees, extended maxima transformation and watershed segmentation of image processing methods were applied, as in one of our previous studies. As the next step, the crown base height (CBH) of individual trees has to be determined; information for this was found in the LiDAR point cloud data using k-means clustering. The LiDAR-derived CGV and stem volume can be estimated on the basis of the proportional relationship between the CGV and stem volume. As a result, low tree-density plots had the best performance for LiDAR-derived CBH, CGV, and stem volume (R (2) = 0.67, 0.57, and 0.68, respectively) and accuracy was lowest for high tree-density plots (R (2) = 0.48, 0.36, and 0.44, respectively). In the case of medium tree-density plots accuracy was R (2) = 0.51, 0.52, and 0.62, respectively. The LiDAR-derived stem biomass can be predicted from the stem volume using the wood basic density of coniferous trees (0.48 g/cm(3)), and the LiDAR-derived above-ground biomass can then be estimated from the stem volume using the biomass conversion and expansion factors (BCEF, 1.29) proposed by the Korea Forest Research Institute (KFRI).

Characterization of the flounder IL-6 promoter and its regulation by the p65 NF-kappaB subunit.

Interleukin (IL)-6 plays important roles in the regulation of the immune response and inflammation in many cell types and its induction by bacterial endotoxins or cytokines is regulated at the transcriptional level. The present study demonstrates the isolation and characterization of the flounder IL-6 promoter sequence and its transcriptional regulation in olive flounder (hirame) natural embryo (HINAE) cells. The promoter region (-400 to +79 bp from the transcription initiation site) of the flounder IL-6 gene contains putative cis-regulatory elements for CCAAT-enhancer-binding proteins (C/EBP; -346 to -355 bp and -166 to -160 bp), cAMP response element binding (CREB; -81 to -85 bp), the activator protein 1 (AP-1; -56 to -62 bp), and NF-kappaB (-39 to -48 bp). Transfection of p65 stimulated the PoIL-6-luc-WT reporter, but not the PoIL-6-luc-kappaB mt reporter, and treatment with LPS augmented p65-stimulated reporter activity in HINAE cells. In contrast, transfection of C/EBPbeta or c-Jun failed to induce synergistic effects in the LPS-driven PoIL-6-luc-WT reporter activity. These results give us new insight into the regulation of flounder IL-6 transcription by the p65 NF-kappaB subunit.

SHP (small heterodimer partner) suppresses the transcriptional activity and nuclear localization of Hedgehog signalling protein Gli1.

Gli (glioma-associated oncogene homologue) proteins act as terminal effectors of the Hedgehog signalling pathway, which is implicated in the development of many human malignancies. Gli activation is important for cell proliferation and anti-apoptosis in various cancers. Several studies have suggested that nuclear receptors have anti-cancer effects by inhibiting the activation of various oncoproteins. However, the involvement of nuclear receptors on the Hedgehog/Gli signalling pathway is poorly defined. In the present study we identified SHP (small heterodimer partner) as a nuclear receptor that decreased the expression of Gli target genes by repressing the transcriptional activity of Gli1. The inhibitory effect of SHP was associated with the inhibition of Gli1 nuclear localization via protein-protein interaction. Finally, SHP overexpression decreased the expression of Gli target genes and SHP knockdown increased the expression of these genes. Taken together, these results suggest that SHP can play a negative role in Hedgehog/Gli1 signalling.