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Chen P.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Chen P.,Chinese Academy of Fishery Sciences | Li J.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Li J.,Chinese Academy of Fishery Sciences | And 6 more authors.
Molecular Biology Reports | Year: 2012

Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. In the present study, a full-length cDNA sequence of catalase was cloned from the haemocytes of swimming crab Portunus trituberculatus by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end method. The catalase cDNA sequence contained 1,851 bp with an open reading frame of 1,551 bp encoding 516 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of P. trituberculatus catalase. The deduced catalase protein had a calculated molecular mass of 58.5 kDa with an estimated isoelectric point of 6.90. Multiple alignment analysis revealed that the deduced amino acid sequence of catalase shared high identity of 68-95 % with those of other species. Quantitative real-time RT-PCR analysis revealed that P. trituberculatus catalase transcript was strongly detected in haemocytes, hepatopancreas, heart, stomach, intestine, gill, ovary and muscle. The expression level of catalase transcripts both in haemocytes and hepatopancreas changed rapidly and dynamically after Vibrio alginolyticus challenging. These facts indicate that catalase was perhaps involved in the acute response against invading bacteria and was an inducible protein involved in the host innate immune response through elimination of H 2O 2 in crab. © 2012 Springer Science+Business Media Dordrecht.


Ma A.,Chinese Academy of Fishery Sciences | Ma A.,Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology | Ma A.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Wang X.,Chinese Academy of Fishery Sciences | And 2 more authors.
Chinese Journal of Oceanology and Limnology | Year: 2010

The morphology and structure of the olfactory organ of Cynoglossus semilaevis Günther are described. The oval olfactory sacs on both sides differ in size and in the number of lamellae, with those on the abocular side having smaller sacs and fewer lamellae than those on the ocular side. On the ocular side, the average ratio of sac length to eye diameter is 2.1 (i.e.>1) with an average of 91 lamellae, while on the abocular side, the values were 1.7 (i.e.>1) and 69, respectively. In addition, the surface morphology varies in different parts of the lamella. The frontal part, near the anterior nostril, is a non-sensory margin with cilia-free epidermal cells. Within this is an internal ciliated sensory area, which is intercalated with ciliated receptor cells and a few ciliated non-sensory cells. Additionally, some dense ciliated non-sensory cells make up a non-sensory area, which also contains cilia-free epidermal cells distributed in patches. In the rear of the olfactory sac near the posterior nostril, the lamellae differ in morphology from those of the frontal olfactory sac but are similar in having few ciliated receptor cells. In other words, the surface of the lamellae in the rear part of the olfactory sac is mainly non-sensory. At present, four types of lamellae (I, II, III and IV) have been recognized in relation to the pattern of the sensory epithelium. In this study, the frontal and rear lamellae resembled types I and IV, respectively, but are referred to as types I′ and IV′ because they are slightly less developed. Data on the ratio of length of lamellae to eye diameter, number of lamellae and the type of surface pattern of the lamellae show that the development of the olfactory system of C. semilaevis facilitates prey capture. © 2010 Chinese Society for Oceanology and Limnology, Science Press and Springer Berlin Heidelberg.


Tan Z.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Tan Z.,Chinese Academy of Fishery Sciences | Xing L.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Xing L.,Chinese Academy of Fishery Sciences | And 11 more authors.
Chinese Journal of Oceanology and Limnology | Year: 2011

The persistence of malachite green (MG), and its metabolite leucomalachite green (LMG), in fish tissues is still unclear, leading to many trade disputes. In this research, we established and evaluated an HPLC method that could detect MG and LMG simultaneously, and then investigated the persistence of these two toxins in the tissues of juvenile perch (Lateolabrax japonicus) post sub-chronic MG exposure at 1.0 mg/L. Exposure lasted for 2 h everyday and was repeated six times. The perch were then placed in MG-free seawater for 100 d to eliminate the toxins. Results show that MG accumulated in the tissues, including the gills, liver, muscle, blood and viscera, and then was metabolized rapidly to LMG. The concentrations of these two toxins increased significantly with the accumulation process. In general, the highest concentrations of MG and LMG in all tissue exceeded 1 000 μg/kg, except for MG in the muscle. The order of accumulation levels (highest to lowest) of MG was gill>blood>liver>viscera>muscle, while that of LMG was liver>blood>gill>viscera>muscle. High levels of MG or LMG could persist for several hours but decreased rapidly during the elimination process. The concentration of LMG was much higher than that of MG during the experiment, especially in the gill, liver and blood. Therefore, the three tissues play important roles in toxin accumulation, biotransformation, and elimination. Although the MG and LMG concentrations in muscle were much lower than in other tissues, the content still exceeded the European minimum required performance limit (MRPL), even after 2 400 h (100 d) of elimination. This demonstrates that it is extremely difficult to eliminate MG and LMG from tissues of perch, and therefore use of these toxins is of concern to public health. © 2011 Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg.


Li S.,Chinese Academy of Fishery Sciences | Li S.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Li S.,CAS Qingdao Institute of Oceanology | Wang L.,Chinese Academy of Fishery Sciences | And 18 more authors.
Journal of Separation Science | Year: 2016

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


Li J.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Chen P.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Liu P.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Gao B.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources | Wang Q.,Key Laboratory for Sustainable Utilization of Marine Fisheries Resources
Aquaculture | Year: 2010

A cytosolic manganese superoxide dismutase (cMnSOD) cDNA was cloned from the haemocytes of swimming crab Portunus trituberculatus using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the cMnSOD full-length cDNA consisted of 1106bp with an open reading frame of 861bp encoding a protein of 286 amino acids. Four conserved amino acids responsible for binding manganese (H110, H158, D243 and H247) and MnSOD signatures from 243 to 250 (DVWHHAYY) were observed. Sequence comparison showed that the cMnSOD deduced amino acid sequence of P. trituberculatus had similarity of 92%, 78%, 77% and 75% to that of blue crab Callinectes sapidus, white shrimp Litopenaeus vannamei, giant tiger prawn Penaeus monodon and freshwater prawn Macrobrachium rosenbergii, respectively. Quantitative real-time RT-PCR analysis revealed that P. trituberculatus cMnSOD transcript was strongly detected in haemocytes, hepatopancreas, heart, stomach, intestine, gill, ovary and muscle. RT-PCR analysis indicated that cMnSOD transcripts both in haemocytes and hepatopancreas increased at 3h and 6h after infection. These facts indicate that cMnSOD is potentially involved in the acute response against invading bacteria in P. trituberculatus. © 2010 Elsevier B.V.

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