三氯生对雌性斑马鱼肝胰脏凋亡相关基因表达的影响

【目的】研究三氯生(TCS)对雌性硬骨鱼类肝胰脏损伤的相关分子机制。【方法】采用半静态水体接触染毒法,将雌性斑马鱼(Danio rerio)暴露于0、0.017、0.034、0.068 mg/L的TCS溶液42 d,采用普通PCR和实时荧光定量PCR技术测定雌性斑马鱼肝胰脏凋亡相关基因(Bcl-2、p53、MDM2、Bax)的表达情况。【结果】0.017~0.068 mg/L组的雌性斑马鱼肝胰脏Bcl-2、MDM2和Bax基因的表达极显著下调(P<0.01),p53基因的表达极显著上调(P<0.01)。【结论】0.017~0.068 mg/L的TCS显著影响雌性斑马鱼肝胰脏凋亡相关基因的表达,促进雌性斑马鱼肝胰脏细胞凋亡的发生。     

Development of novel microsatellite markers for Holothurian scabra (Holothuriidae), Apostichopus japonicas (Stichopodidae) and cross-species testing in other sea cucumbers

Thirty-five new microsatellite loci from the sea cucumbers H olothurian scabra(Jaeger, 1833) and Apostichopus japonicas(Selenka, 1867) were screened and characterized using the method of magnetic bead enrichment. Of the twenty-four polymorphic loci tested, eighteen were consistent with Hardy-Weinberg equilibrium after a modified false discovery rate(B-Y FDR) correction, whereas six showed statistically significant deviations(CHS2 and CHS11: P 0.014 790; FCS1, FCS6, FCS8 and FCS14: P 0.015 377). Furthermore, four species of plesiomorphous and related sea cucumbers(Holothurian scabra, Holothuria leucospilota, Stichopus horrens and Apostichopus japonicas) were tested for mutual cross-amplification using a total of ninety microsatellite loci. Although transferability and universality of all loci were generally low, the results of the cross-species study showed that the markers can be applied to identify individuals to species according to the presence or absence of specific microsatellite alleles. The microsatellite markers reported here will contribute to the study of genetic diversity, assisted breeding, and population conservation in sea cucumbers, as well as allow for the identification of individuals to closely related species.     

Identification of fucans from four species of sea cucumber by high temperature 1H NMR

Acidic polysaccharide, which has various biological activities, is one of the most important components of sea cucumber. In the present study, crude polysaccharide was extracted from four species of sea cucumber from three different geographical zones, Pearsonothuria graeffei (Pg) from Indo-Pacific, Holothuria vagabunda (Hv) from Norwegian Coast, Stichopus tremulu (St) from Western Indian Ocean, and Isostichopus badionotu (Ib) from Western Atlantic. The polysaccharide extract was separated and purified with a cellulose DEAE anion-exchange column to obtain corresponding sea cucumber fucans (SC-Fucs). The chemical property of these SC-Fucs, including molecular weight, monosaccharide composition and sulfate content, was determined. Their structure was compared simply with fourier infrared spectrum analyzer and identified with high temperature ¹H nuclear magnetic resonance spectrum analyzer (NMR) and room temperature ¹³C NMR. The results indicated that Fuc-Pg obtained from the torrid zone mainly contained 2,4-O-disulfated and non-sulfated fucose residue, whereas Fuc-Ib from the temperate zone contained non-, 2-O- and 2,4-O-disulfated fucose residue; Fuc-St from the frigid zone and Fuc-Hv from the torrid zone contained mainly non-sulfated fucose residue. The proton of SC-Fucs was better resolved via high temperature ¹H NMR than via room temperature ¹H NMR. The fingerprint of sea cucumber in different sea regions was established based on the index of anomer hydrogen signal in SC-Fucs. Further work will help to understand whether there exists a close relationship between the geographical area of sea cucumber and the sulfation pattern of SC-Fucs.     

总状蕨藻多糖提取工艺的研究

采用酶法提取与热水抽提相结合的方法研究了总状蕨藻多糖的提取工艺,结果显示,采用固液质量比1∶10,蛋白酶用量136.5 U/g,于60℃下酶法提取2.5 h后,再将固液质量比调至1∶20,在温度95℃条件下继续进行热水提取2.5 h,多糖得率为6.7%,比单用热水提取法提高了131.0%。     

Antioxidation activities of low-molecular-weight gelatin hydrolysate isolated from the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

Gelatin extracted from the body wall of the sea cucumber (Stichopus japonicus) was hydrolyzed with flavourzyme. Low-molecular-weight gelatin hydrolysate (LMW-GH) of 700–1700 Da was produced using an ultrafiltration membrane bioreactor system. Chemiluminescence analysis revealed that LMW-GH scavenges high free radicals in a concentration-dependent manner; IC₅₀ value for superoxide and hydroxyl radicals was 442 and 285 μg mL⁻¹, respectively. LMW-GH exhibited excellent inhibitory characteristics against melanin synthesis and tyrosinase activity in B16 cells. Furthermore, LMW-GH notably increased intracellular glutathione (GSH), which in turn suppressed melanogenesis. LMW-GH performs antioxidation activity, holding the potential of being used as a valuable ingredient in function foods, cosmetics and pharmaceuticals or nutriceuticals.     

Screen and effect analysis of immunostimulants for sea cucumber,Apostichopus japonicus

Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopus japonicus, a sea cucumber, and the lysozyme and superoxide dismutase (SOD) activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal's coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides (P<0.05). The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysaccharide, and burdock oligosaccharide on day 1 and day 2 after the injection (P<0.05). The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection (P<0.05), while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus.     

Screen and effect analysis of immunostimulants for sea cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopus japonicus, a sea cucumber, and the lysozyme and superoxide dismutase (SOD) activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal’s coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides (P<0.05). The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysaccharide, and burdock oligosaccharide on day 1 and day 2 after the injection (P<0.05). The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection (P<0.05), while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus. Supported by the National Key Technology Research and Development Program (863 Program, No. 2006BAD09A06) and the Special Fund of Chinese Central Government for Basic Scientific Research Operations in Commonweal Research Institutes (No. 02-2007B03)     

Rheological and Structural Properties of Sea Cucumber Stichopus japonicus During Heat Treatment

Changes in tissue structure, rheological properties and water content of raw and heated sea cucumber meat were studied. Sea cucumber Stichopus japonicus was heated at 25℃, 70℃ and 100℃ water for 5 min. The structural changes were observed using a light microscope and the rheological parameters （rupture strength, adhesive strength and deformation） determined using a texture meter. Microscopic photograph revealed that the structural change of heated meat was greater than that of raw meat. The rupture strength, adhesive strength and deformation of raw meat were smaller than those of the heated meat. Meanwhile, rheological parameters showed positive correlation with heating temperature. These changes are mainly caused by thermal denaturation and gelatinization of collagen during heating. These changes were also evidenced in observations using a light microscope and differential scanning calorimetry.     

Rheological and structural properties of sea cucumber Stichopus japonicus during heat treatment

Changes in tissue structure, rheological properties and water content of raw and heated sea cucumber meat were studied. Sea cucumber Stichopus japonicus was heated at 25°C , 70°C and 100°C water for 5 min. The structural changes were observed using a light microscope and the rheological parameters (rupture strength, adhesive strength and deformation) determined using a texture meter. Microscopic photograph revealed that the structural change of heated meat was greater than that of raw meat. The rupture strength, adhesive strength and deformation of raw meat were smaller than those of the heated meat. Meanwhile, rheological parameters showed positive correlation with heating temperature. These changes are mainly caused by thermal denaturation and gelatinization of collagen during heating. These changes were also evidenced in observations using a light microscope and differential scanning calorimetry.     

晚泥盆世大陆风化作用增强及其海洋环境效应:来自华南碳酸盐岩锶同位素的制约

上泥盆统弗拉斯阶和法门阶界线附近发生了大规模的生物灭绝, 其起因被认为是植物登陆导致的陆地化学风化作用加强及其引起的海洋水体缺氧所致, 但其直接证据还比较缺乏。运用广西地区广泛发育的浅海碳酸盐岩中锶元素(Sr)和同位素(87Sr/86Sr)、铀钍比值(U/Th)记录, 探究了从中泥盆统到下石炭统的陆地化学风化速率及海水氧化还原条件的变化。研究结果表明, 在晚泥盆世(法门阶早期)陆地化学风化作用加强, 导致海水中Sr浓度升高, 及更富集重的Sr同位素组分; 低的U/Th比值也显示该时期水体缺氧严重, 海水中U元素被还原到沉积物中, 导致海水中的U浓度降低。陆地表面化学风化的加强对晚泥盆世海洋水体氧化还原条件的波动以及海洋生物的灭绝有重要影响。      

Adverse Effect of TiO_2 Nanoparticles on Antioxidant System and Antitumor Activities of Macroalgae Gracilaria lemaneiformis

Recently, the widespread use of TiO_2 nanoparticles(nano-TiO_2) has attracted the global concern regarding their adverse effects to aquatic organisms. Nanoparticles may get to the aquatic environments and harm the growth of algae. This paper presents the studies of nano-TiO_2 concentrations influence on the photosynthetic characteristics, antioxidant system and antitumor activity of marine macroalgae Gracilaria lemaneiformis after exposed to 0, 5, 10, 20 or 40 mg L-1 of nano-TiO_2. Results showed that the photosynthetic pigment content of G. lemaneiformis obviously decreased and the antioxidant defense system was significantly disturbed after exposure to nano-TiO_2. Compared with control, the maximum level of malondialdehyde showed 3.3-fold increase for 15 days exposure to nano-TiO_2. The activity of superoxide dismutase and catalase enhanced obviously by 1.10-2.24-fold. The aqueous extract of G. lemaneiformis(AEGL) possesses anticancer activity against HepG2 liver cancer cells proliferation and the 48 h-cell inhibition exceeds 35%. But the toxicity of nano-TiO_2 would inhibit the anticancer activity of AEGL. When G. lemaneiformis was exposed to nano-TiO_2, the effect of the aqueous extract of G. lemaneiformis on the cell inhibition, reactive oxygen species production, apoptosis, cycle arrest of HepG2 cells significantly lessened, and which were linearly associated with nano-TiO_2 exposure concentration. Those results further confirmed that nano-TiO_2 can inhibit the anticancer activity of G. lemaneiformis. The results in this work imply that macroalgae can be an effective biomarker of nano-TiO_2 contaminant, and can be useful indicator to evaluate ecological effects of increasing pollutants in marine ecosystem.     

Fascaplysin sensitizes cells to TRAIL-induced apoptosis through upregulating DR5 expression

This study investigated the molecular mechanism of anti-tumor effect of fascaplysin, a nitrogenous red pigment firstly isolated from a marine sponge. Microarray analysis show that the TNF and TNF receptor superfamily in human umbilical vein endothelial cells (HUVEC) and human hepatocarcinoma cells (BEL-7402) were significantly regulated by fascaplysin. Western Blot results reveal that fascaplysin increased the expression of cleaved caspase-9, active caspase-3, and decreased the level of procaspase-8 and Bid. Flow cytometry and cytotoxicity tests indicate that fascaplysin sensitized cells to tumor necrosis-related apoptosis-inducing ligand-(TRAIL) induced apoptosis, which was markedly blocked by TRAIL R2/Fc chimera, a dominant negative form of TRAIL receptor DR5. Therefore, our results demonstrate that fascaplysin promotes apoptosis through the activation of TRAIL signaling pathway by upregulating DR5 expression.     

Weighted Correlation Network Analysis(WGCNA) of Japanese Flounder(Paralichthys olivaceus) Embryo Transcriptome Provides Crucial Gene Sets for Understanding Haploid Syndrome and Rescue by Diploidization

Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis(WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.     

方格星虫多糖对小鼠免疫活性的影响

研究了方格星虫多糖对小鼠免疫功能的影响。结果表明:方格星虫粗多糖(CSG)能够对抗环磷酰胺引起的小鼠胸腺萎缩和脾萎缩,明显拮抗白细胞减少,对正常小鼠免疫器官没有明显的增重作用;对正常小鼠腹腔巨噬细胞吞噬中性红能力有显著的增强作用。方格星虫多糖粗品(CSG)和精制多糖(SG1和SG2)能够促进小鼠脾淋巴细胞增殖,且能与ConA协同作用。     

Antiangiogenic activity of low-temperature lysozyme from a marine bacterium <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

We extracted marine low-temperature lysozyme (MLTL), a novel lysozyme, from a marine microorganism through fermentation. Our previous study suggested that a low molecular weight (16 kDa) may exert anti-tumor activity through antiangiogenesis. In this study, we extracted a high weight (39 kDa) and investigated its antiangiogenic activity in vivo and in vitro. Using zebrafish embryos as an in vivo study model, we found that treatment with MLTL significantly inhibited the growth of subintestinal vessels (SIVs) in a dose-dependent manner and that 400 μg/ml MLTL was sufficient to block the growth of SIVs. An in vitro study conducted using human umbilical vein endothelial cells (HUVECs) revealed that MLTL suppressed the proliferation, migration and tube formation of HUVECs in a dose-dependent manner. Interestingly, assays by flow cytometry and DNA electrophoresis indicated that MLTL was able to induce apoptosis of HUVECs. Moreover, further study demonstrated that the disruption of intracellular Ca²⁺ homeostasis may play an important role in MLTL induced apoptosis of HUVECs. Taken together, the results of this study demonstrate for the first time that MLTL inhibits angiogenesis through its pleiotropic effects on vascular endothelial cells and induces apoptosis through regulation of cellular Ca²⁺ levels. The results of this study also revealed a possible mechanism underlying the antiangiogenic effect of MLTL and suggested that MLTL may be a promising new antiangiogenic agent for use in cancer therapy.     

Investigation of the decline of Ulva prolifera in the Subei Shoal and Qingdao based on physiological changes

It has been a scientific consensus that Ulva prolifera green tide that break out annually between May and August in the recent decade off eastern coast of Yellow Sea. To investigate the ecological mechanism of the decline of U. prolifera green tide, we compared the physiological characteristics of U. prolifera samples collected in three stages of the green tide in Subei Shoal north Jiangsu, the initiation point, on May 10(early, the initiation), July 10(middle, the outbreak), and in Taipingjiao, Qingdao on August 10(late, the decline). Compared to the early samples, the middle, and late samples showed a lower chlorophyll content and photosynthetic e fficiency at a higher lipid peroxidation level. The middle and later samples had fewer chloroplasts in the cells of U. prolifera than those in the early samples. In addition, the cells of U. prolifera sampled in the late stage showed ruptured and deformed cell walls. Differences in physiological function were reflected in the growth rate. Both the middle and late U. prolifera samples presented negative growth rates. Correlation analysis results show that the rising temperature was mainly responsible for the local decline of U. prolifera green tide. The high light-intensity that could induce the photoinhibition was also an important factor for the decline. Therefore, U. prolifera population that remained in the Subei Shoal and those that northward drifted to Qingdao experienced the same decline processes: rising temperature and continuous high light conditions in the late phase of green tide reduced the photosynthetic capacity and destroyed the antioxidant system of the thallus, which lead to decrease of biomass. The only difference is that the temperature in the Subei Shoal rose earlier than that in Qingdao; thus, U. prolifera remaining in the Subei Shoal went disappeared first.     

Identification of three common Loliginidae squid species in the South China Sea by analyzing hard tissues with geometric outline method

The hard tissues of squid can provide important information for species identification. In this study, we used statolith and beak to identify three squid species including Uroteuthis duvaucelii, Loliolus beka, and U. edulis in the South China Sea. Because of the highly overlapping habitat and similar body morphology of the three squid species, we explored four different ways to identify them, by using statolith, upper beak, lower beak and a combination of statolith and beak. An outline geometric morphometric method and stepwise discriminant analysis were used to evaluate the most suitable method for the identification. We found that the combination of statolith and beak had the highest cross validation rate that was 75.0%, 87.5% and 88.7% for U. duvaucelii, L. beka and U. edulis, respectively. Using two beaks had similar results and the lowest cross validation rate was 60.0%, 50.0%, and 73.7% for the upper beak, 46.9%, 58.5% and 75.3% for the lower beak of U. duvaucelii, L. beka and U. edulis, respectively. Analyzing with the statolith had moderate cross validation which was 72.2%, 80.0%, and 87.7% for U. duvaucelii, L. beka and U. edulis, respectively. From the results it is suggested when the entire body of a squid is available, a combination of statolith and beak should be used for the identification. When only one hard tissue is available, species identification can be subjected to large errors.     

Growth inhibition to three red tide microalgae by extracts of Ulva pertusa

1 INTRODUCTION In recent decades, red tides that induced by in- creasing eutrophication occur frequently in coastal areas worldwide. This has caused serious problems in marine environment, aquaculture industries and human health (Qi et al., 1993; Horner e…     

Effects on the STAT3-shRNA in Non-Small-Cell Lung Cancer Therapy: Design,Induction of Apoptosis,and Conjugation with Chitosan-Based Gene Vectors

STAT3 plays a particularly important role in several cancer-related signal transduction pathways. Silencing STAT3 via RNA interference or small molecule inhibitors induces the apoptosis of tumor cells, thereby inhibiting the growth of the tumors. In this study, short-hairpin RNA sequences targeting the STAT3 genes were designed, synthesized, and then connected to pGPU6/GFP/Neo plasmids as the shRNA-expression vectors. The expression of STAT3-shRNA was analyzed by real-time PCR, western blotting, and cell apoptosis assay to study the growth and apoptosis of the cells. Then, the effect of STAT3 knockdown on the NCI-H1650 cells was studied in a tumor mouse model. The results revealed that, after an in vitro transfection, the proliferation of NCI-H1650 cells was inhibited, and the cells were induced to apoptosis. The mRNA and protein expression levels of STAT3 were downregulated in the STAT3-sh RNA group. In vivo, the tumor mass and volume in the STAT3-sh RNA group were significantly lower than in the other two groups. Both the in vivo and in vitro results demonstrated a long-period inhibiting effect on NSCLC, especially in vivo,when the tumor inhibition rate could reach 50% in the STAT3-shRNA group, which is an exciting outcome. Moreover, the study of the conjugation of STAT3-shRNA and chitosan-based vectors revealed that they could be combined steadily with good cytocompatibility and transfection efficiency. These results together provide convincing evidence for the application of STAT3-shRNA used in the treatment of non-small lung cancer, which could be a promoting prospect for the development of gene therapy.