STUDIES ON THE ANTITUMOR ACTIVITY AND ISOLATION OF THE GLYCOPROTEIN FROM THE GONAD OF CHLAMYS (AZUMAPECTEN) FARRERI

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IMPACT OF STARVATION ON SURVIVAL，MEAT CONDITION AND METABOLISM OF CHLAMYS FARRERI

The effects of 60-day starvation on survival rate. condition index (CI) , changes ofnutrient composition of different tissues, respiration and excretion of scallop ChlartnCs farreri were studiedin laboratory from Oct. 17 to Dec. 15 ,1997. Two groups (control and starvation with 200 individualseach) were cultured in two 2 m3 tanks, with 31 to 32 salinity water at 1 7 “C . Starvation effects were mea-sured alter 1 0, 20, 40 and 60 days. There was no mass mortality of scallops cf the two tanks and survivalrates of the control and starvation groups were 93 .5 % and 92.0 %, respectively. Starvation had strongeffect on the meat condition of the scallops, especially after 10 days; when relative lipid percentagedropped sharply while relative protein percentage increased. The impact of starvation on the oxygen consumption mtc (OCR) and the ammonia-N cxcretion mtc (AER) was obvious.     

Effects of different combinations of cryoprotectants on spermatozoon vitality of Chinese scallop,Chlamys farreri,during low temperature equilibrium

The effects of membrane penetrating anti—freezing agents (MPAAs), DMSO (dimethyl sulfoxide), glycerol, EG (ethylene glycol) and methanol in combination with different cryoprotective additives such as carbohydrates, macromoleules and inorganic compounds on the spermatozoon vitality of Chinese scallop,Chlamys farreri, during 1 h 0 °C equilibrium were investigated. When only MPAAs existed, the detrimental effects of different MPAAs ranked in the following order: DMSO<methanol<EG<glycerol. When carbohydrates were added into MPAAs solution, 5% glucose caused larger decrease of spermatozoon vitality than 2.4% lactose. 5% glucose or 2.4% lactose in 7.5% glycerol caused complete damage. 10% yolk was best in maintaining the spermatozoon vitality except when used in combination with 10% methanol. 10% milk significantly decreased spermatozoon vitality in EG and methanol and enhanced its vitality in glycerol, but did not significantly influence it in DMSO. Glycine MPAAs ranked in the following order. 10% yolk <10% yolk +2.4% lactose <2.4% lactose <2.0% citrate <10% milk <10% milk +2.0% citrate <5% glucose <80mmol/L glycine +55 mmol/L NaHCO₃. Contribution No. 2434 from the Institute of Oceanology, Chinese Academy of Sciences.     

Effects of benzo(a)pyrene exposure on the antioxidant enzyme activity of scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Scallop Chlamys farreri was exposed to different concentrations of benzo(a)pyrene (BaP) (0.5 μg/L, 1.0 μg/L, 10.0 μg/L and 50.0 μg/L) for 30 days in seawater. The 7-ethoxyresorufin O-deethylase (EROD) activity was significantly induced, and increased with the increasing BaP concentration. The glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx) activities increased in short time at low concentration of BaP, and was significantly depressed at high concentrations. Scallop gill was more sensitive to BaP than the digestive gland, and the digestive gland was the main tissue to deal with oxyradicals. The contents of malondialdehyde (MDA) increased with the exposure time and there was a positive correlation (concentration-effect) between the MDA content and the concentration of BaP. The biomarkers determined in this experiment had important roles in detoxification, and showed great potential as biomarkers for oxidative stress. Controlled laboratory experiments designed to simulate field exposure scenarios are particularly useful in ascertaining biomarkers suitable for use with complex contaminant mixtures in the marine environment. Supported by the Technology Development Program of Shandong (No. 2008GG1005010) and the Program of Introducing Talents of Discipline of Universities (111 Project, No. B08049)     

Effects of benzo（a）pyrene exposure on the antioxidant enzyme activity of scallop Chlamysfarreri

Scallop Chlamys farreri was exposed to different concentrations of benzo(a)pyrene (BaP) (0.5 μg/L, 1.0 μg/L, 10.0 μg/L and 50.0 μg/L) for 30 days in seawater. The 7-ethoxyresorufin O-deethylase (EROD) activity was significantly induced, and increased with the increasing BaP concentration. The glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx) activities increased in short time at low concentration of BaP, and was significantly depressed at high concentr...     

Characterization,expression and function analysis of DAX1 gene of scallop (Chlamys farreri jones and preston 1904) during its gametogenesis

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop (Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at mRNA and protein levels. The cDNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame (ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher (P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.     

Studies on extraction procedure and antioxidative activity of phlorotannins fromSargassum kjellmanianum

Known only in phaeophyceae, phlorotannins (brown algal polyphenols) are natural products with potential uses in pharmacology. This study yielded an extraction procedure to obtain high purity, high molecular weight phlorotannins fromSargassum kjellmanianum and revealed the characteristics of their infrared and flourescence spectra. The antioxidative activity of phlorotannins, which was about 2.6 times as strong as that of 0.02% BHT (tert-butyl-4-hydroxytoluene), showed potential for preventing oil rancidification. Contribution No. 2918 from the Institute of Oceanology, Chinese Academy of Sciences. The project was supported by The State Ninth-five Key Program of China. (No. 96-916-04-01).     

Effects of polypeptide from<Emphasis Type="Italic">Chlamys farreri</Emphasis> on amino acid content in guinea pig skin irradiated by chronic ultraviolet A and B

We examined the effects of polypeptide fromChlamys farreri (PCF) on the amount of hydroxyproline in guinea pig skin irradiated by chronic ultraviolet A (UVA) and ultraviolet B (UVB) radiation. PCF was applied locally before repeated exposure of guinea pig to UVA and UVB. The contents of hydroxyproline and other amino acids in guinea pig skin were determined by automatic amino acid analyzer. Our results showed that: (1) long-time UVA and UVB radiation can reduce dramatically the amounts of hydroxyproline, aspartic acid, threonine, glycine, phenylalanine and lysine in guinea pig skin in comparison with the control group (P<0.05); (2) Compared with model group, pre-treatment with 5% and 20% PCF prior to UVA and UVB radiation can inhibit the decline of amino acids content in guinea pig skin in a dose-dependent manner (P<0.05). As the decrease of hydroxyproline, glycine and lysine contents in the skin directly reflexes type I collagen degeneration, our results indicated that the chronic application of PCF can protect skin type I collagen against UV radiation, and thus protect skin from photoaging. Project No. 39970638 supported by the NSFC, and also supported by the Science and Technology Bureau of Qingdao (No: 2001-28-50).     

Polypeptide from Chlamys farreri inhibits UVB-induced apoptosis of HaCaT cells via iNOS/NO and HSP90

Polypeptide from Chlamys farreri(PCF) is a novel marine bioactive product that was isolated from the gonochoric Chinese scallop Chlamys farreri,and was found to be an effective antioxidant in our recent studies.In this study,we investigated the effect of PCF on ultraviolet B(UVB)-induced apoptosis of HaCaT cells and the intracellular signaling pathways involved.Pretreatment with the inducible nitric oxide synthase(iNOS) inhibitor S-methylisothiourea sulfate inhibited UVB-induced apoptosis,indicating that iN...     

Immunotoxicity effect of benzo[α]pyrene on scallop Chlamys farreri

The toxic effects of benzo[α]pyrene (B[α]P) at different concentrations (0.1, 0.5, 1, 2.5 and 7.5 μgL⁻¹) on scallop (Chlamy farreri) immune system were studied. The results showed that B[α]P had significant toxic effects on the haemocyte counts, neutral red uptake, phagocytosis, bacteriolytic and antibacterial activity (P<0.05), while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolytic activity in all B[α]P treatment groups as well as antibacterial activity in groups of 0.5, 1, 2.5 and 7.5 μgL⁻¹ B[α]P decreased significantly (P<0.05). Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentration-response to B[α]P. Bacteriolytic activity in 0.1μgL⁻¹ B[α]P treatment group and antibacterial activity in 0.1 μgL⁻¹ and 0.5 μgL- B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group (P<0.05), except for antibacterial activity in 0.1 μgL⁻¹ B[α]P treatment group (P>0.05). Thus, B[α] has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms.     

Isolation and pharmacological activities of bromophenols from Rhodomela confervoides

Four bromophenols were isolated from the extract of marine red alga,Rhodomela confervoides by column chromatography and HPLC methods. By means of spectroscopic methods inclding IR, MS, 1D, and 2D-NMR techniques, their structures were elucidated as (1) 3-bromo-4,5-dihydroxy benzoic acid methyl ester; (2) bis (2,3-dibromo-4,5-dihydroxybenzyl) ether; (3) 3,4-dibromo-5-(methoxymethyl)-1,2-benzenediol and (4) 3-bromo-4,5-dihydroxy-benzaldehyde. Compound 1 was first isolated from the algae in nature, and 1, 4 were found to have selective cytotoxic activities against KB, Bel 7402 and A549 cells, 2 showed powerful antibacterial activities againstStaphylococcus aurens andPseudomonas aeruginosa. Supported by National “863” Program (No. 2004AA625030, 2001AA620503), Quingdao marine sciences project (No. 04-2-NN-26) and Key Knowledge Innovative Project of the Chinese Academy of Sciences (KZCX3-SW-215).     

A study on pathogens of Chinese prawn (Penaeus Chinensis) virus diseases

This pathogenic study shows that the viral diseases of Chinese prawns (Penaeus chinensis, O'sbeck) is due to three kinds of viruses: epithelium envelope baculovirus ofPenaeus chinensis (EEBV-PC, detected by the authors in 1993), infections hypodermal and hematopoietic necrosis virus, and hepatopancreatic parvo-like virus, and that the first two viruses seem to be the main pathogens of the epidemic in the northern regions in 1993.     

Studies on color type variants from mutagenized protoplasts ofPorphyra haitanensis Chang et Zheng &P. Yezoensis ueda (rhodophycease)

Isolated protoplasts from thalli ofPorphyra haitanensis andPorphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04–0.09% of red type variants inP. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 0.31–1.11% inP. yezoensis. After irradiation with UV: (1) 3.5–10.5% of red type variants inP. yezoensis were obtained: (2) 0.5–2.0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained inP. haitanensis. Colchicine and UV’s mutangenic effects onP. yezoensis protoplasts were stronger than those onP. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced by colchicine treatment grew faster than the wild type thalli. The clones of vegetative propagation from protoplasts of red type variants were still red type thalli. The red type variants will be good materials for genetic studies and improvement ofPorphyra strains.     

Preliminary identification of three new isolates in genus Alexandrium (Dinophyceae) from China sea area

The 5.8S ribosomal DNA sequences (5.8S rDNA) and their flanking regions, internal transcribed spacer 1 and spacer 2 (ITS1 and ITS2) of three new isolates in genus Alexandrium (Alexandrium sp. qd1, Alexandrium sp. qd2, Alexandrium sp. gz) from China were amplified, sequenced, and subjected to phylogenetic analysis. Alexandrium sp. gz and Alexandrium sp. qd1 were grouped with high bootstrap values with four strains/species, i.e., A. catenella South Korea strain, A. catenella Japan strain, and two from China, Alexandrium sp. AC03 and Alexandrium sp. AN01 being proposed to be A. catenalla in a previous study. Then Alexandrium sp. gz and Alexandrium sp. qd1 were identified as Alexandrium catenella. As A. catenella was isolated from Qingdao and Guangzhou sea areas, it supposedly distributed at least in these two areas and was genetically different. Alexandrium sp. qd2 differed greatly from species in Alexandrium. It clustered with Symbiodinium californium, Symbiodinium sp. G15 and Gymnodinium sp. Zhao 01 with 100% bootstrap value; so Alexandrium sp. qd2 affiliates to genus Symbiodinium, and is probably a free-living Symbiodinium species.     

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)     

Plateau typeE(%)- pH curves of interfacial exchange between liquid and solid in seawater

A study on the curves of the exchange ratioE(%)- pH on the liquid-solid interface of the systems Zn (II) with clay minerals, (Kaolinite, illite and montmorillonite), Zn (II) with hydrous ferric oxides (amorphous ferric oxide, geothite and hematite), Zn (II) with hydrous manganese oxide (γ-MnOOH, manganite and δ-MnO₂) etc. in seawater resulted in the discovery of new plateau type exchange ratioE(%)-pH curves not yet reported in literature. The two factors that decide the growth, decline and the change of the “plateau type” curve are: (1) the inherent characteristics of systems, which can be explained by the exchange constants (i=1, 2, 3, etc.) of interfacial stepwise ion/coordination particle exchange between liquid and solid; and (2) . The theory of interfacial stepwise ion/coordination particle exchange in seawater was applied to explain the experimental results of the above study and a general formula was derived for the plateau type exchange ratioE(%)-pH curves. The theoretically simulated and calculated curves coincided well with the experimental results. This paper provides new and powerful experimental basis to support the theory of interfacial stepwise ion/coordination particle exchange in seawater. The project was supported by the National Natural Science Foundation of China.     

Hematological changes in white spot syndrome virus-infected shrimp, Fenneropenaeus chinensis (Osbeck)

The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV (white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifestations of hemocytes were similar among moribund shrimps infected via injection, feeding and by nature. Firstly, the total hemocyte counts (THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly, necrotic, broken and disintegrated cells were often observed, and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly, necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared, whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV, and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.     

Effects of Na+/K+ ratio of groundwaters on the gill ion-transport enzyme activity, plasma osmolality and growth of Cynoglossus semilaevis juveniles

The effects of environmental Na⁺/K⁺ ratio on the gill ion-transport enzyme activity, plasma osmolality and growth of Cynoglossus semilaevis juveniles were investigated. The results showed that, plasma osmolality was similar among flounder adapted to different Na⁺/K⁺ ratios of saline groundwaters (P>0.05), while the growth, gill Na⁺, K⁺-ATPase and HCO₃ ⁻-ATPase activities were affected by Na⁺/K⁺ ratio significantly (P<0.05). The gill Na⁺, K⁺-ATPase activity reached its maximum on day 3, then decreased gradually from day 3 to day 9 and remained constant from day 9 to day 15. The peak values of gill Na⁺, K⁺-ATPase activity were detected on day 3 for all Na⁺/K⁺ ratios of saline groundwaters, then the enzyme activities descended, and on day 9 the enzyme activities achieved steady state, and the gill HCO₃ ⁻-ATPase activity increased rapidly and achieved steady state after one day. During steady state, the gill Na⁺, K⁺-ATPase and HCO₃ ⁻-ATPase activity of Na⁺/K⁺ ratios 20 and 40 treatments were significantly higher than those in the control group (Na⁺/K⁺ ratio 27.5), while there were no significant differences between the Na⁺/K⁺ ratio 30 treatment and the control group; the gill Na⁺, K⁺-ATPase activity of Na⁺/K⁺ ratio 20 and 40 treatments were significantly higher than that for ratio 30 treatment, but there were no significant differences of gill HCO₃ ⁻-ATPase activity among these treatments. At the end of the 15-day experiment, the weight gain (%) and specific growth rate (SGR) of flounders maintained in seawater were significantly higher than those in groundwaters; significant differences also occurred among the treatments; Na⁺/K⁺ ratio 30 treatment had the highest values (33.7% and 1.94 respectively), which were significantly higher than those under Na⁺/K⁺ ratios 20 and 40 treatments. Therefore, for the saline groundwater used in this experiment, it is suggested that the Na⁺/K⁺ ratio be adjusted to approximately 30, i.e., as close to that of natural seawater as possible in the culture of flounder.     

First record of <Emphasis Type="Italic">Paraonella platybranchia</Emphasis> (Hartman, 1961) (Polychaeta: Paraonidae) from the Yellow Sea

This paper reports for the first time a species of polychaetous family Paraonidae, Paraonella platybranchia (Hartman, 1961), a rare species found previously only in the coast near the boundary between the United States and Mexico, and it was recently discovered in the samples collected in 1982 from Chinese side of the Yellow Sea. This is the first record of the species in China as well as in the Northwest Pacific. Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. O72715)     

Two New Species of the Genus finocladia Kylin （Chordaria-les, Phaeophyta） from Yellow Sea Coasts of China

Two new species of the genus Tinocladia Kylin, T. gracilis sp. nov. and T. microsporangii sp. nov,, are described. T. gracilis is characterized by its slender frond, variable size, infrequently branched medullary filaments, and short assimilating filaments with 2 - 7 cells. T. microsporangii is characterized by its medullary cells which are slightly larger than sub-cortical ones; narrow sub-cortical layer, usually consisting of 4- 6 cells and very small unilocular sporangia. The holotypes are deposited at the Herbarium of the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.