Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well. Supported by the Award Foundation of Scientific Research for Excellent Young and Middle-age Scientist of Shandong Province (No. 2006BS07003) and the Knowledge Innovation Project of Chinese Academy of Sciences (KZCX2-YW-R-104)     

Immune response of <Emphasis Type="Italic">Pseudosciaena crocea</Emphasis> to the injection of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of Vibrio alginolyticus in density of 2×10⁷ CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly increased the number of erythrocytes, leucocytes, lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of large yellow croaker, and significantly reduced the number of peripheral blood granulocytes as compared with those in the control group. No significant difference in acid phosphytase and superoxide dismutase activity of serum was detected between the two groups. It is suggested that non-specific immune factors including leucocytes and anti-bacteria substance in peripheral blood played important role at the initial stage of infection, and specific immune factors such as antibody then played important role in response to anti-infection at the latter stage. Supported by the National High Technology Research and Development Program of China (863 program, No. 2007AA09Z115) and Technology Program of Xiamen (No. 3502Z73019)     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and stone flounder <Emphasis Type="Italic">Kareius bicoloratus</Emphasis>

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems. Supported by National Natural Science Foundation of China (No. 30600455) and the National Basic Research Program of China (973 Program, No.2004CB117402)     

The antitumor effect of bromophenol derivatives <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">Leathesia nana</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis>

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3), 2,2′,3,3′-tetrabromo-4,4′,5,5′-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTT assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S₁₈₀-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC₅₀ below 10 μg/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%, respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2007AA09Z410) and Knowledge Innovation Program of Chinese Academy of Sciences (No. KZCX2-YW-209)     

Toxic effects of several phthalate esters on the embryos and larvae of abalone Haliotis diversicolor supertexta

As the most widely used plasticizers in the world, phthalate esters (PAEs) are potential endocrine disruption compounds (EDCs). In the present study, the toxicity of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), di (2-ethylhexyl) phthalate (DEHP) on embryogenesis and larvae development of the marine univalve Haliotis diversicolor supertexta was examined in laboratory. The results show that the malformation of embryos appeared during the experiment, such as embryos died or lysed, small transparent flocculent rings studded on the periphery of the embryo, and the larvae could failed to hatch. In embryo toxic test, embryos incubated at the highest concentration of DMP, DEP and DBP solutions showed significantly high abnormal rate compared with the control, while DEHP solutions displayed no significant difference. In larval toxic test, in all concentrations of DMP, DEP and DBP solutions, larval settlement rates were low significantly than that of the control. Similarly, DEHP solutions show nearly no effect on the larval settlement. The order of toxicity on embryos and larvae is DBP>DEP>DMP>DEHP. Being a simple and easy stimulation to indoor spawn, sensitive to environmental factors, and short culture time, the embryos of H. diversicolor supertexta can be used to indicate toxicity of the PAEs. Supported by National Natural Science Foundation of China (No. 40476057)     

<Emphasis Type="Italic">p</Emphasis>CO<Subscript>2</Subscript> and carbon fluxes across sea-air interface in the Changjiang Estuary and Hangzhou Bay

Partial pressure of CO2 （pCO2） was investigated in the Changjiang （Yangtze River） Estuary, Hangzhou Bay and their adjacent areas during a cruise in August 2004, China. The data show that pCO2 in surface waters of the studied area was higher than that in the atmosphere with only exception of a patch east of Zhoushan Archipelago. The pCO2 varied from 168 to 2 264 μatm, which fell in the low range compared with those of other estuaries in the world. The calculated sea-air CO2 fluxes decreased offshore and varied from -10.0 to 88.1 mmol m^-2 d^-1 in average of 24.4 ± 16.5 mmol m^-2 d^-1. Although the area studied was estimated only 2 × 10^4 km^2, it emitted （5.9 ± 4.0） × 10^3 tons of carbon to the atmosphere every day. The estuaries and their plumes must be further studied for better understanding the role of coastal seas playing in the global oceanic carbon cycle.     

Efficient expression of green fluorescent protein （GFP） mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.     

Inheritance mode of microsatellite loci and their use for kinship analysis in the Pacific oyster （Crassostrea gigas）

Five full-sib families of the Pacific oyster （Crassostrea gigas） larvae were used to study the mode of inheritance at eight microsatellite loci, and the feasibility of these markers for kinship estimate was also examined. All eight microsatellite loci were compatible with Mendelian inheritance. Neither evidence of sex-linked barriers to transmission nor evidence of major barriers to fertilization between gametes from the parents was shown. Three of the eight loci showed the presence of null alleles in four families, demonstrating the need to conduct comprehensive species-specific inheritance studies for microsatellite loci used in population genetic studies. Although the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance, offspring from five full-sib families were unambiguously discriminated in the neighbor-joining dendrogram. This result indicates that the microsatellite markers may be capable of discriminating between related and unrelated oyster larvae in the absence of pedigree information, and is applicable to the investigation of the effective number of parents contributing to the hatchery population of the Pacific oyster.     

Genetic improvement on Chinese shrimp （Fenneropenaeus chinensis）： growth and viability performance in F1 hybrids of different populations

Fenneropenaeus chinensis distributed in the Yellow Sea and Bohai Sea of China and the west coast of the Korean Peninsula. Different geographical populations represent potentially different genetic resources. To learn further the characteristics of different geographical population, crosses among two wild and three farmed populations were produced. The two wild populations were from the Yellow Sea and Bohai Sea （WYP）, and the west coast of the Korean Peninsula and coast （WKN）. The three farmed populations included the offspring of first generation of wild shrimp from coast in Korea （FKN）, the Huang Hai （the Yellow Sea in Chinese） No.1 （HH1）, and JK98. The phenotypes growth and survival rates of these populations were compared to confirm the feasibility for crossbreeding. The body length （BL）, carapace length （CL）. carapace width （CW）, height of the second and third abdominal segment （HST）, width of the second and third abdominal segment （WST）, length of the first abdominal segment （LF）, length of the last abdominal segment （LL）, live body weight （BW）, and survival rate were measured, Different combinations were statistically performed with ANOVA and Duncan＇s Multiple Range Test. The results show that the survival rate of JK98（♀）×WKN（♂） was the highest, followed by WYP（♀）×WKN（♂）, FKN（♀）×WYP（♂）, FKN（♀）×HHI（♂） and WYP（♀）×FKN（♂）; the body weight of FKN（♀）×HHI（♂） was the highest, followed by FKN（♀）×WYP（♂）, WYP（♀）×WKN（♂）, WYP（♀）×FKN（♂） and JK98（♀）×WKN（♂）; the total length had the same ranking as the body weight. All growth traits in hybrids JK98（♀）×WKN（♂） were the lowest among all combinations. F1 hybrids had significant difference （P〈0.05） in BL, CL, HST, LL, and BW; and insignificant difference （P〉0.05） in other growth traits and survival rate. The results of Duncan＇s Multiple Range Test are that BL and CL of J     

Characterization of defensin gene from abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals＇ acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides （including six Cys）, molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar （70% in common） to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin （hd-def）, a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.