Chulhong Oh

Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide-stimulated RAW 264.7 macrophages.

In this study, potential anti-inflammatory effect of fucoxanthin isolated from brown algae was assessed via inhibitory effect of nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW 264.7 macrophage cells. The Myagropsis myagroides was selected for further experiments due to its profound NO inhibitory effect, and was partitioned with different organic solvents. Highest NO inhibitory effect was detected in the chloroform fraction, and the active compound was identified as fucoxanthin, a kind of carotenoid available in brown algae evidenced high correlation with the inhibitory effect of NO production (r(2)=0.9511). Though, fucoxanthin significantly inhibited the NO production, it slightly reduced the prostaglandin E(2) (PGE(2)) production. The inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions were inhibited by fucoxanthin. Further, RT-PCR analysis indicated that the iNOS and COX-2 mRNA expressions were suppressed by fucoxanthin. Moreover, the release of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), and the mRNA expression levels of those cytokines were reduced by the addition of fucoxanthin in a dose-dependent manner. Hence, these results suggest that the use of fucoxanthin may be a useful therapeutic approach for the various inflammatory diseases.

Inhibitory effect of diphlorethohydroxycarmalol on melanogenesis and its protective effect against UV-B radiation-induced cell damage

In this study, potential inhibitory effect of 21 species of marine algae on melanogenesis was assessed via tyrosinase inhibitory effect. The Ishige okamurae extract tested herein evidenced profound tyrosinase inhibitory effect, compared to that exhibited by other marine algae extracts. Thus, I. okamurae was selected for use in further experiments, and was partitioned with different organic solvents. Profound tyrosinase inhibitory effect was detected in the ethyl acetate fraction, and the active compound was identified as the carmalol derivative, diphlorethohydroxycarmalol (DPHC), which evidenced higher levels of activity than that of commercial whitening agent. Intracellular reactive oxygen species (ROS) induced by ultraviolet (UV)-B radiation was reduced by the addition of DPHC and cell viability was dose-dependently increased. Moreover, DPHC demonstrated strong protective properties against UV-B radiation via damaged DNA tail length and morphological changes in fibroblast. Hence, these results indicate that DPHC isolated from I. okamurae has potential whitening effects and prominent protective effects on UV-B radiation-induced cell damages which might be used in pharmaceutical and cosmeceutical industries.

Molecular evidence for the existence of lipopolysaccharide-induced TNF-α factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus)

The lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel family nuclear factor kappaB (Rel/NF-kB) are two important transcription factors which play major roles in the regulating inflammatory cytokine, apoptosis and immune related genes. Here, we report the discovery of disk abalone LITAF (AbLITAF) and Rel/NF-kB (AbRel/NF-kB) homologues and their immune responses. Full-length cDNA of AbLITAF consists of 441 bp open reading frame (ORF) that translates into putative peptide of 147 aa. Analysis of AbLITAF sequence showed it has characteristic LITAF (Zn(+2)) binding domain with two CXXC motifs. Phylogenetic analysis results further revealed that AbLITAF is a member of LITAF family. AbRel/NF-kB is 584 aa protein that contains several characteristic motifs including Rel homology domain (RHD), Rel protein signature, DNA binding motif, nuclear localization signal (NLS) and transcription factor immunoglobulin - like fold (TIG) similar to their invertebrate and vertebrate counterparts. Tissue specific analysis results showed that both AbLITAF and AbRel/NF-kB mRNA was expressed ubiquitously in all selected tissues in constitutive manner. However, constitutive expression of AbLITAF was higher than AbRel/NF-kB in all tissues except mantle. Upon immune challenge by bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemoragic septicemia virus (VHSV), AbLITAF showed the significant up-regulation in gills while AbRel/NF-kB transcription was not change significantly. Based on transcriptional response against immune challenge, we could suggest that regulation of TNF-alpha expression may have occurred mainly by LITAF activation rather than NF-kB in disk abalone. The cumulative data from other molluscs and our data with reference to TNF-alpha, LITAF and Rel/NF-kB from disk abalone provide strong evidence that LITAF and NF-kB are independent pathways likely to occur throughout the Phylum mollusca.

Allograft inflammatory factor-1 in disk abalone (Haliotis discus discus): molecular cloning, transcriptional regulation against immune challenge and tissue injury.

Here, we report the identification and characterization of allograft inflammatory factor-1 (AIF-1) from disk abalone Haliotis discus discus that was denoted as AbAIF-1. The full-length cDNA of AbAIF-1 consists of a coding region (453 bp) for 151 amino acids with a 17 kDa molecular mass. Analysis of AbAIF-1 sequence showed that it shares characteristic two EF hand Ca(+2)-binding motifs. Results from phylogenetic analysis further confirm that AbAIF-1 is a member of the AIF-1 family similar to invertebrate and vertebrate counterparts suggesting it has high evolutional conservation. Tissue-specific expression and transcriptional regulation of AbAIF-1 were analyzed after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes), viral hemorrhagic septicemia virus (VHSV) immune challenge and during tissue injury by quantitative real-time PCR. It is shown that the expression of AbAIF-1 mRNA was expressed ubiquitously in all selected tissues in constitutive manner showing the highest level in hemocytes. Upon bacteria and VHSV challenge, AbAIF-1 showed the significant up-regulation in hemocytes than gills. After the tissue injury in shell and mantle, AbAIF-1 and antioxidant selenium-dependant glutathione peroxidase (SeGPx) transcripts were significantly upregulated in abalone hemocytes. Taken together, these findings suggest that AIF-1 could response against the pathogenic challenge or tissue injury in abalone like mollusks. Also, AbAIF-1 may involve in wound healing and shell repair after the tissue injury of abalone.

Chromene induces apoptosis via caspase-3 activation in human leukemia HL-60 cells

In this study, the potent anti-tumor effects of brown algae on human leukemia HL-60 cells were investigated. The Sargassum siliquastrum extract among the 14 species of brown algae exhibited profound growth inhibitory effect on HL-60 cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, therefore, S. siliquastrum was selected for use in further experiments. The highest inhibitory activity of S. siliquastrum on HL-60 cells was detected in the chloroform fraction, and the active compound was identified as a kind of chromene, sargachromanol E (SE). SE treatment showed significant growth inhibitory effects on HL-60 cells in a dose-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies, fragmented DNA ladder, and the accumulation of DNA in the sub-G(1) phase of cell cycle. SE induced apoptosis was accompanied by downregulation of Bcl-xL, upregulation of Bax, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, z-DEVD-fmk, a caspase-3 inhibitor, significantly inhibited cell cytotoxicity, apoptotic characteristics such as apoptotic bodies, sub-G(1) DNA content, and cleavage of PARP induced by SE. These results suggest that SE exerts its growth inhibitory effects on HL-60 cells through caspase-3-mediated induction of apoptosis. Therefore, SE offers promising chemotherapeuric potential to prevent cancers such as human leukemia.

Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream (Oplegnathus fasciatus).

Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca(2+) binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.

Characterization of a novel molluscan MyD88 family protein from manila clam, Ruditapes philippinarum.

Myeloid differentiation factor 88 (MyD88) is a universal adaptor protein which is required for signal transduction of TLR/IL-1R family. In this study, a novel molluscan MyD88 family member protein (named as RpMyD88) was identified from manila clam, Ruditapes philippinarum. It was identified using BLAST algorithm from GS-FLX™ sequencing data. The cDNA of RpMyD88 consists of 1416 bp open reading frame (ORF) encoding 471 amino acid residues. The RpMyD88 contains death domain and Toll/interleukin-1 receptor (TIR) domain which are typical features of MyD88 family proteins. The predicted amino acid sequence of RpMyD88 shares 27% identity with scallop MyD88. The expression level of RpMyD88 mRNA was investigated in healthy and challenged clams by quantitative real-time RT-PCR. The RpMyD88 gene expression is ubiquitous in all selected tissues. The RpMyD88 mRNA was strongly expressed in hemocyte, gill and mantle. In contrast, it was weakly expressed in siphon, foot and adductor muscle. RpMyD88 was up-regulated in gill and hemocyte after immune challenge with both Vibrio tapetis and LPS challenge. All results considered, sequence characterization, comparison and gene expression data suggesting that MyD88-dependent signaling pathway is presence in manila clam and RpMyD88 plays an important role in innate immune response against bacteria.

Protective effect of diphlorethohydroxycarmalol isolated from Ishige okamurae against high glucose-induced-oxidative stress in human umbilical vein endothelial cells.

In the present study, the protective effect of diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae, a brown algae, on high glucose-induced-oxidative stress was investigated using human umbilical vein endothelial cells (HUVECs). High concentration of glucose (30 mM) treatment induced cytotoxicity whereas DPHC prevented cells from high glucose-induced damage; restoring cell viability was significantly increased. In addition, the lipid peroxidation, intracellular reactive oxygen species (ROS), and nitric oxide (NO) levels induced by high glucose treatment were effectively inhibited by addition of DPHC in a dose-dependent manner. DPHC also suppressed the over-expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins as well as nuclear factor-kappa B (NF-kappaB) activation induced by high glucose in HUVECs. These finding indicate that DPHC might be used as potential pharmaceutical agent which will reduce the damage caused by high glucose-induced-oxidative stress associated with diabetes.

Molluscan death effector domain (DED)-containing caspase-8 gene from disk abalone (Haliotis discus discus): Molecular characterization and expression analysis

The caspase family represents aspartate-specific cysteine proteases that play key roles in apoptosis and immune signaling. In this study, we cloned the first death effector domain (DED)-containing molluscan caspase-8 gene from disk abalone (Haliotis discus discus), which is named as hdCaspase-8. The full-length hdCaspase was 2855 bp, with a 1908 bp open reading frame encoding 636 amino acids. The hdCaspase-8 had 72 kDa predicted molecular mass with an estimated isoelectric point (PI) of 6.0. The hdCaspase-8 amino acid sequence contained the characteristic feature of an N-terminal two DED, a C-terminal catalytic domain and the caspase family cysteine active site ⁵¹³KPKLFFLQACQG⁵²⁴. Phylogenetic analysis results showed that hdCaspase-8 is more similar to the invertebrate Tubifex tubifex (sludge worm) caspase-8. Real-time RT-PCR results showed that hdCaspase-8 constitutively and ubiquitously expressed in all tested tissue of unchallenged disk abalone. The basal expression level of hdCaspase-8 in gill tissue was higher than all other tested tissues. The hdCaspase-8 mRNA expression in gill and hemocytes was significantly up-regulated by exposure to bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Listeria monocytogenes) and VHSV (viral hemorrhagic septicemia virus), as compared to control animals. These results suggest that hdCaspase-8 may be involved in immune response reactions in disk abalone.

Molecular characterization, gene expression analysis and biochemical properties of α-amylase from the disk abalone, Haliotis discus discus.

The present study reports the molecular characterization, cloning, expression, and biochemical characterization of alpha-amylase identified from the disk abalone, Haliotis discus discus cDNA library. The full length of the alpha-amylase cDNA was 1650 bp, and it encoded a polypeptide of 511 amino acids. The predicted HdAmyI molecular mass of mature protein was 54 kDa and the estimated isoelectric point (pI) was 8.3. The alpha-amylase gene showed its characteristic motifs, catalytic sites, substrate binding sites and conserved regions with other known species of alpha-amylases. Purified recombinant HdAmyI exhibited a relatively low activity of 0.1 U/mg protein towards 1% starch. HdAmyI had an optimum temperature and pH of 50 degrees C and 6.5, respectively. It also demonstrated stability in a wide range of temperatures and pH. Tissue-specific mRNA expression results showed that HdAmyI is expressed only in the digestive tract and hepatopancreas, with the highest levels in the hepatopancreas. Over 8 weeks of starvation, alpha-amylase transcription was decreased significantly relative to basal levels. However, after starvation, mRNA transcription was increased and returned to normal level by the 2nd week of feeding, suggesting that the alpha-amylase mRNA expression changes according to variations in food availability at the transcriptional level in disk abalone.