[Show abstract][Hide abstract] ABSTRACT: A novel antistasin-like cDNA homologue named as Ab-Antistasin was isolated from the disk abalone Haliotis discus discus normalized cDNA library. The Ab-Antistasin (1398-bp) consisted of an 1185-bp open reading frame encoding 395 amino acid (aa) residues. The predicted molecular mass and isoelectric point of Ab-Antistasin was 44 kDa and 8.5, respectively, and showed highest identity (23.1%) to Hydra magnipapillata antistasin. The most striking feature of Ab-Antistasin is the 12-fold internal repeats (IR) of an antistasin-like domain. Ten of the 12 IR domains (26-27 aa) are highly conserved, with 6 cysteines and 1 glycine. Ab-Antistasin was comprised of three Bowman-Birk serine protease inhibitor family motifs. The recombinant Ab-Antistasin (rAb-Antistasin) was over-expressed in Escherichia coli and purified using a pMAL system. rAb-Antistasin (10 microM) was able to inhibit trypsin activity by 66% in a dose-dependent manner. Moreover, it exhibited low prolongation activity for coagulation in an APTT assay (86.0 s compared to control 42.0 s) with human blood. Endogenous Ab-Antistasin mRNA was found to be expressed in digestive tract, hepatopancreas, hemocytes, abductor muscle and mantle, with highest expression levels in digestive tract followed by hepatopancreas and hemocytes. Quantitative real time PCR results revealed that Ab-Antistasin transcription was significantly induced at 3 h post-infection (p.i.) after challenged by a mixture of bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes) in the abalone digestive tract; in the hemocytes, induction occurred at 6 and 12 h. The results indicated that Ab-Antistasin could play an important role in the immune responses of mollusks.
[Show abstract][Hide abstract] ABSTRACT: Abstract CuZn–superoxide dismutase (CuZn–SOD) is a key antioxidant enzyme playing a first line protective role against reactive oxygen species (ROS) by converting superoxide ( ) into H2O2. The CuZn–SOD gene was isolated from a whole abalone cDNA library and denoted as aCuZn–SOD. The full-length cDNA of aCuZn–SOD was 1021 bp, which contained 465-bp open reading frame (ORF) coding 154 amino acids. It contained highly conserved CuZn–SOD signature motif 1 (45GFHVHQFGDNT55) and motif 2 (139GNAGGRQACGVI150). Also, amino acid residues identified as Cu (His47, His49, His64, and His121) and Zn (His64, His72, His81, and ASP84) metal-binding sites were completely conserved in the aCuZn–SOD. The reverse transcription polymerase chain reaction (RT-PCR) results showed that aCuZn–SOD mRNA was expressed constitutively in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes in a tissue-specific manner. The aCuZn–SOD mRNA was significantly up-regulated (P radical generated by xanthine oxidase assay, showing CuZn–SOD is a functionally active antioxidant enzyme in disk abalone.
No preview · Article · Oct 2009 · Journal of the World Aquaculture Society
[Show abstract][Hide abstract] ABSTRACT: Natural fermentation was tested as a method of releasing active compounds during screening for potential anticoagulant activity
in three types of algae (Pachymeniopsis elliptica, Sargassum horneri, and Ulva pertusa). Freeze dried algae samples (2.5g) were fermented by adding 75g of sugar and 500mL of water and thereafter kept at room
temperature (25°C) for 3months. Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT)
were measured every 2weeks for 3months to determine the optimum time for the highest activity. Fermented P. elliptica, (which had the highest activity) was subjected to anion exchange chromatography (DEAE-cellulose) and sepharose 4B gel permeation
chromatography. The purified sample was analyzed by agarose-gel electrophoresis and polyacrylamide gel electrophoresis (PAGE)
to confirm the purification and to determine the molecular mass, respectively. The 360μg/mL of purified compound (Mwt>500,000Da)
had both APTT and PT activities (>1,000s). However, at the concentrations of 180μg/mL, purified compound and heparin showed
540 and >1,000s APTT activity, respectively. Though, the purified compound of P. elliptica considered as a weaker anticoagulant than heparin, this purified anticoagulant polysaccharide could be considered as a good
alternative source as an anticoagulant. Moreover, the technique of fermentation is an inexpensive and feasible, this purified
anticoagulant polysaccharide compound could be used in pharmaceutical and biomedical industry. Further investigations need
to be performed to determine the mechanism of this novel anticoagulant compound.
No preview · Article · Jul 2008 · European Food Research and Technology
[Show abstract][Hide abstract] ABSTRACT: Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by detoxification of hydrogen peroxide (H2O2). A gene coding for a putative catalase was isolated from the disk abalone (Haliotis discus discus) cDNA library and denoted as Ab-catalase. The full-length (2864 bp) Ab-catalase cDNA contained 1,503 bp open reading frame (ORF), encoding 501 amino acid residues with 56 kDa predicted molecular weight. The deduced amino acid sequence of Ab-catalase has characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDT358), NADPH and heme binding residues. Phylogenetic and pairwise identity results indicated that Ab-catalase is more similar to scallop (Chlamys farreri) catalase with 80% amino acid identity except for other reported disk abalone catalase sequences. Constitutive Ab-catalase expression was detected in gill, mantle, gonad, hemocytes, abductor muscle and digestive tract in tissue specific manner. Ab-catalase mRNA was up-regulated in gill and digestive tract tissues for the first 3h post injection of H2O2, showing the inducible ability of abalone catalase against oxidative stress generated by H2O2. The purified recombinant catalase showed 30,000 U/mg enzymatic activity against H2O2 and biochemical properties of higher thermal stability and broad spectrum of pH. Our results suggest that abalone catalase may play an important role in regulating oxidative stress by scavenging H2O2.
No preview · Article · Apr 2008 · Fish & Shellfish Immunology
[Show abstract][Hide abstract] ABSTRACT: Natural fermentation is a method of extracting anticoagulant compounds from red algae Schizymenia dubyi. Preliminary screening was carried out on the basis of the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) assays. The optimal fermentation time period for the highest APTT, PT, and TT values was found to be 8 weeks. Purification of the fermented sample by DEAE-anion exchange chromatography followed by Sepharose-4B chromatography resulted in a single polysaccharide, which was reconfirmed by a single spot on agarose gel electrophoresis. The purified sample had >1000s APTT activity at 130 μg/mL. The molecular weight estimated by polyaccrylamide gel electrophoresis was >500,000 Da. This is the first report indicating the presence of the anticoagulant compound in S. dubyi.
No preview · Article · Oct 2007 · Food Science and Technology International
[Show abstract][Hide abstract] ABSTRACT: The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O(2)(-) into the stabler H(2)O(2). In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 degrees C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 degrees C and completely heat inactivated at 80 degrees C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.
No preview · Article · Nov 2006 · Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The flesh and skin of Anguilla japonica and Conger myriaster were investigated for their antioxidant activity. Ethyl acetate and diethyl ether extracts (4 mg/mL) after extracting with methanol were tested for 2,2-diphenyl-1-picrylhidrazyl (DPPH) free radical scavenging (FRS) activity for flesh and skin of each eel species. The values were compared with -tocopherol and butylated hydroxytoluene (BHT). Extracts showing positive results, when tested for DPPH FRS, were examined for dose effect, hydrogen peroxide (H2O2), hydroxyl radical and superoxide scavenging assays and heat stability. All extracts of A. japonica showed a dose-dependent DPPH FRS and significant hydroxyl radical scavenging activities (>65%). The diethyl ether extract of the flesh of A. japonica showed the highest superoxide scavenging activity. Diethyl ether extracts of A. japonica were heat stable, and ethyl acetate extracts were stable up to 75C. Thus, it can be concluded that A. japonica is rich with heat stable and nonpolar antioxidants.
No preview · Article · Apr 2005 · Journal of Food Lipids
[Show abstract][Hide abstract] ABSTRACT: Ethyl acetate and diethyl ether extracts, previously extracted with methanol, were obtained from flesh and skin of Eptatretus burgeri (hag fish) and Enedrias nebulosus (white spotted eel). Eight different extracts (4 mg/mL) were tested for DPPH free radical scavenging activity and the values were compared with commercial antioxidants (α-tocopherol and BHT). All extracts of E. burgeri exhibited significantly positive results (> 65%) in scavenging DPPH radicals compared to E. nebulosus. Thus, only E. burgeri extracts were tested for dose effect, hydrogen peroxide, hydroxyl radical, superoxide scavenging assays and heat stability at 25, 50, 75 and 100 °C for 30 min. All extracts of E. burgeri showed higher DPPH radical scavenging activities by increasing concentration. Significantly higher results were observed for hydroxyl radical scavenging activity when compared with commercial antioxidants. Relatively moderate activity and very low activity were exhibited for superoxide scavenging and hydrogen peroxide activities, respectively. Diethyl ether extracts of E. burgeri were stable with increased temperature, while ethyl acetate extracts were stable up to 75 °C. Thus, there is a high potential for E. burgeri being rich with heat-stable antioxidants that can scavenge hydroxyl radicals.
No preview · Article · Jun 2004 · Food Science and Technology International