Chemically-induced hepatocycte proliferation in the medaka (Oryzias latipes)

Several known mammalian carcinogens were examined for their ability to induce proliferation in hepatocytes of the medaka (Oryzias latipes). Compounds tested were N-nitrosodiethylamine (DEN), acrylonitrile (AN), 4-aminobiphenyl (4ABP) and methapyrilene (MP). Cell proliferation was assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry. Statistically significant increases in PCNA labeling indices (LIs) occurred following exposure to DEN, AN and 4ABP. Exposure to MP resulted in an apparent, but not statistically significant, LI increase. Proliferation, however, was not correlated with subsequent carcinogenesis except in the case of DEN. AN, 4ABP and MP were not carcinogenic to medaka in bioassays. Overall, cell proliferation appears to be a marker of chemically-induced cytotoxicity in medaka, but its relationship to carcinogenesis is presently not clear.     

Bromodeoxyuridine uptake in hydropic vacuolation and neoplasms in winter flounder liver

Winter flounder (Pleuronectes americanus) from Boston Harbor, Massachusett, undergo a progressive series of hepatic changes involving hydropic vacuolation of epithelial cells, biliary hyperplasia, and hepatocellular and cholangiocellular neoplasia. Severely affected fish often exhibit grossly visible lesions. To examine cell proliferation associated with these conditions, evidence for DNA synthesis was sought. Boston winter flounder livers were screened endoscopically to select fish with and without grossly visible hepatic lesions, and then injected intraperitoneally with bromodeoxyuridine (BrdU) 3 h before euthanasia. Incorporation of this nucleotide analog was visualized immunohistochemically in formalin-fixed, paraffin-embedded tissues. Basal epithelia in the gill and intestine stained strongly, as did renal hemopoietic epithelia. In contrast, renal tubular epithelia and cardiomyocytes did not stain. Normal appearing hepatocytes stained occasionally, whilst more frequent staining was observed in vacuolated and neoplastic hepatic epithelia. We conclude (1) that the BrdU assay as described here has potentially widespread application to the study of cell proliferation in fish tissues; (2) that vacuolated cells may actively proliferate in winter flounder liver from Boston Harbor; and (3) vacuolated cells may be involved in the neoplastic transformation of hepatic epithelia.     

Effects of several salt marsh plants on mouse spleen and thymus cell proliferation using mtt assay

In the present study, we have tested the effects of 21 salt marsh plants on cell proliferation of mouse immune cells (spleen and thymus) using MTT assay in culture. The methanolic extracts of six salt marsh plants (Rosa rugosa, Ixeris tamagawaensis, Artemisia capillaris, Tetragonia tetragonoides, Erigeron annus, and Glehnia littoralis) showed very powerful suppressive effects of mouse immune cell death and significant activities of cell proliferationin vitro. Especially, the methanolic extract ofRosa rugosa was found to have fifteen times compared to the control treatment, demonstrating that Rosa rugosa may have a potent stimulation effect on immune cell proliferation. These results suggest that several salt marsh plants includingRosa rugosa could be useful for further study as an immunomodulating agent.     

利用实时荧光定量PCR法测算红螯螯虾实体组织的细胞数量

螯虾是重要的水产养殖动物,也是研究甲壳动物疾病与免疫的一个良好模型.在研究不同组织的功能及受病原感染的影响时,需要了解组织内的细胞数量.但是,目前常用的细胞计数方法仅适用于单细胞悬液,无法用于实体组织.本研究以红螯螯虾( Cherax quadricarinatus)为对象,建立了基于基因拷贝数定量分析的实体组织细胞数...     

Requirement of endogenous iron for cytotoxicity caused by hydrogen peroxide in Euglena gracilis

It is widely thought that redox-active metals in cells such as iron or copper catalyze the reduction of hydrogen peroxide to toxic hydroxyl radicals or their equivalent. However, this has not been directly demonstrated in vivo. To probe this requirement, the freshwater microorganism Euglena gracilis was used. Its intracellular iron content can be modulated by the concentration of iron in the defined growth medium without effect on the proliferation rate of the cells. E. gracilis contains two large storage pools of cytosolic iron which can be monitored to assess cellular iron status. The toxicity of H₂O₂ in E. gracilis was inversely related to the amount of iron in the extracellular medium. At high levels of external iron, the metal carried out the Fenton reaction with H₂O₂ outside the cell, producing hydroxyl radicals as detected by electron-spin resonance spin-trapping experiments. This reaction reduced the amount of H₂O₂ that could diffuse into cells to cause toxicity. When cells with different intracellular iron content were placed in iron-deficient media, the the toxicity of H₂O₂, measured by inhibition of proliferation, was directly related to the concentration of internal iron. Using cells deficient in iron, this oxidant did not inhibit proliferation at low concentration but was somewhat effective at higher concentrations. Although the iron chelating agents, 1,10-phenanthroline and desferrioxamine, also depressed cytosolic iron, they were cytotoxic to E. gracilis and so could not be used unambiguously to examine the role of intracellular iron in the toxic effects of hydrogen peroxide.     

三疣梭子蟹(Portunus trituberculatus)乳化病的组织病理和超微病理研究

利用组织切片及超薄切片电镜技术对患“乳化病”三疣梭子蟹的鳃、肝胰腺、步足肌肉、心肌和性腺5种组织进行病理学分析。组织病理显示:乳化病的病变性质为变质性炎症,主要病变部位在肝胰腺,其病灶表现为水变性、脂肪变性和细胞坏死等症状,步足肌和心肌表现出肌纤维断裂、浑浊等症状。超微病理显示:在肝胰腺和步足肌组织中有大量细菌,肝胰腺细胞结构损伤主要表现为微绒毛损伤脱落、线粒体坏死、溶酶体增多及脂肪滴变性,肌纤维断裂、溶解,血隙中血淋巴细胞受严重侵染。     

半滑舌鳎(Cynoglossus semilaevis Günther)生长激素体外重组表达及活性分析

根据半滑舌鳎(Cynoglossus semilaevis Günther)生长激素(GH)基因的c DNA全长序列设计引物克隆得到其全长552个碱基成熟肽序列。利用RT-PCR方法将扩增片段克隆到原核表达载体p ET-28a上,实现了GH成熟肽在大肠杆菌BL21(DE3)中的融合表达。融合蛋白分子量为26 k Da,在IPTG诱导4h时目的蛋白表达量最高,占细菌总蛋白的41.5%,主要以包涵体形式存在。Western-blotting分析表明GH融合蛋白可特异性地被6×His抗体识别。诱导表达后的菌液沉淀经纯化和复性后,获得大小为26 k Da的纯化GH融合蛋白。以ELISA方法检测纯化后的GH融合蛋白显示其具有免疫学活性。本研究为认识半滑舌鳎生长轴的调控机制提供了基础资料。     

海萝抗氧化酶系与失水响应因子表达模式分析

潮间带红藻海萝(Gloiopeltis furcata)对失水胁迫具有很强的耐受能力。为探索海萝周期性失水过程中的响应机制,本研究在24 h内设计了两次连续的失水-复水循环处理,测定了海萝抗氧化酶活力的变化情况。同时,利用转录组测序技术,结合荧光定量PCR方法(qRT-RCR),对海萝失水响应基因的转录表达进行了验证。结果表明:海萝转录组共组装到32681条基因。与对照组相比,处理组共表达了7161条差异表达基因(differentially expressed genes, DEGs)。KEGG分析显示, DEGs分布在代谢、环境信息加工、有机体系统、遗传信息加工、细胞进程等方面。海萝抗氧化酶活力测定发现,抗氧化能力对海萝响应失水胁迫十分重要。过氧化氢酶(CAT)、硫氧还蛋白还原酶(TrxR)、超氧化物歧化酶(SOD)参与抗氧化过程,其中CAT酶活力对海萝抵抗失水胁迫尤为重要。此外,qRT-PCR结果显示,海萝中渗透调节物质红藻糖苷合成的相关基因(GfUGPase、GfGK、GfGPDH)只对初次失水处理有正响应。而热激蛋白70基因(GfHSP70)、碳酸酐酶基因(GfCA)、MYB蛋白编码基因(GfMYB)以及谷胱甘肽S转移酶基因(GfGST)在两次失水过程中其转录水平均有上调表达,它们也参与了海萝失水响应机制。     

凡纳滨对虾(Litopenaeus vannamei)Runt转录因子的基因克隆及其功能分析

Runt结构域转录因子家族(Runx)在哺乳动物体内存在三种基因型,分别起着造血,骨生成和控制上皮细胞增殖的作用。本文应用反转录和c DNA末端快速扩增(RACE)技术,首次在凡纳滨对虾(Litopenaeus vannamei)中克隆并测序了一种Runt(lvrunt)基因全长。lvrunt c DNA全长1754bp,含有1个663bp长的开放阅读框,编码221个氨基酸,理论分子量为23.6k Da,具有Runt-family结构域。对凡纳滨对虾鳃、肠、肝胰腺、类淋巴、心脏、肌肉组织和血淋巴lvrunt的转录特征进行分析,结果显示该基因几乎特异性地在血淋巴细胞中表达,而在其他组织中表达较低。肌肉注射重组造血激素蛋白(r AST)或白斑综合征病毒(WSSV)粗提液均可显著提高lvrunt在凡纳滨对虾体内的转录表达。上述结果提示,lvrunt主要在血细胞发挥作用,可能作为造血激素的下游基因起到调节血细胞增殖和分化的作用,并在受到病毒感染后,参与提升血细胞数量或者促进血细胞分化的过程。     

利用引物退火控制技术克隆日本囊对虾性腺差异表达基因

为了解日本囊对虾（Marsupenaeusjaponicus）性腺发育及分化的分子机制,本研究采用改良的引物退火控制技术克隆到了共8个精巢、卵巢的差异表达基因．其中4个为已知基因,分别为凝血酶敏感蛋白基因、皮质棒蛋白基因、增殖细胞核抗原基因和泛素缀合酶基因;4个为未知基因,分别命名为MjACP3-T、MjACP4一T、MjACPl5一T和MjACP4一O基因．采用实时定量PCR方法分析了4个未知基因在性腺发育过程的表达谱,结果表明：MjACP3一T、MjACP4．T和MjACPl5,T基因在日本囊对虾精巢和卵巢中的表达量有显著差异,且在各个时期精巢的表达量均高于卵巢,说明这3个基因可能在精巢的发育中起到某些重要作用．卵巢MjACP4一O基因的表达量变化较大,第5期卵巢的相对表达量达到最高（为1．38）,而其他各个阶段的变化较大,在第4期卵巢的相对表达量最低（为0．03）．在精巢中,MjACP4一O基因的表达量随着精巢逐渐发育成熟呈下降趋势．定量PCR结果与引物退火控制技术的结果有着很好的一致性．研究结果表明,引物退火控制技术是一种简单高效的克隆差异表达基因的技术．采用该技术所克隆到的8个精巢、卵巢的差异表达基因,为进一步了解日本囊对虾性腺发育分化的分子机制提供了基础材料．     

七鳃鳗鸟嘌呤转移因子Vav2分子克隆、表达及系统发育分析研究

鸟嘌呤转移因子Vav2为Vav家族成员,其在Ras-related GTP酶活性调节过程中发挥重要作用。本文首次从七鳃鳗体内克隆并表达出七鳃鳗Vav2分子（Lj-Vav2）。系统发育分析结果阐明,Lj-Vav2为脊椎动物Vav2基因家族的直系同源基因。进化动力学分析指明,CH结构域、DH结构域、PH结构域、C1结构域、SH3结构域、SH2结构域贯穿于脊椎动物Vav2家族的进化历程。实时定量PCR结果表明,Lj-Vav2分布于七鳃鳗心脏、肾脏、髓样组织、肝脏、鳃以及类淋巴细胞中,同时经过LPS刺激后淋巴细胞中Lj-Vav2的表达水平呈现明显上调趋势。综上,Lj-Vav2在七鳃鳗体内免疫防御过程中发挥重要作用。此外,我们的研究将为今后更好地阐明Lj-Vav2生物学功能奠定坚实的物质基础。     

原位杂交结合半薄切片对石鳖贝壳发育相关细胞的观察研究

多板纲软体动物(石鳖)生有8片重复排列的贝壳,与其他类群如腹足类、双壳类等有显著的区别,这些差异蕴含着重要的发育和演化意义。前期研究发现,红条毛肤石鳖(Acanthochitona rubrolineata)幼虫贝壳发育组织的细胞排列欠规则,在普通显微镜下不易识别单个细胞结构。本研究首先利用整装原位杂交手段鉴定出一些参与贝壳发育的细胞群体,发现表达engrailed的细胞分布于贝壳发育区中央区域的突起部位(脊)和边缘区域,其中脊区域的表达呈条纹状,边缘区域的表达则与贝壳发育区的外周吻合,但是对这些细胞的形态和排列模式等细节无法开展深入观察。接下来对石鳖幼虫进行了半薄切片,发现可以识别出壳板发育区细胞及脊细胞的大致轮廓和分布模式,但分辨率仍然受限。为了进一步提升分辨能力,结合了整装原位杂交实验及半薄切片技术,对表达engrailed基因的贝壳发育相关细胞开展了观察研究。结果显示,对整装原位杂交后的幼虫进行半薄切片,组织细胞结构保持完整,细胞轮廓清晰,能够清楚区分engrailed阳性细胞。观察表明,这些细胞具有较高的核质比,总体呈V形排列,且贝壳发育区中央部位与边缘部位的engraile...     

Quantification of cadmium-induced metallothionein in crustaceans by the silver-saturation method

Metallothionein (MT) has been proposed as a specific biochemical probe for metal exposure of aquatic organisms. It is recognized that its usefulness as a monitoring tool depends on the full understanding of its function and on the possibility of measuring its concentration in tissues. Therefore the study of MT in crustaceans is interesting from two different points of view: the need to understand mechanisms associated with the toxicology of metals, and the potential use of this protein for monitoring metal-contamined environments. Several methods have been developed for quantitative measurements of MT in biological samples. Immunochemical and electrochemical procedures are reliable techniques but they require sophisticated equipment and highly trained staff. In the present work, modifications to the silver-saturation method have been developed for the quantitative determination of MT in crustacean tissues, estimating silver concentrations by flame in an atomic absorption spectrophotometer. Cadmium and MT were measured both in Artemia and Procambarus clarkii after sublethal exposure to cadmium. An increase in MT content was recorded in crustacean tissues after 12 h of cadmium exposure. A high responsiveness was found in MT induction by cadmium in the two crustaceans and there was a clear relationship between cadmium concentration in water and MT levels in tissues. The silver-saturation assay may be a rapid assay technnique for biomonitoring purposes.     

Identification and classification of vertical chlorophyll patterns in the Benguela upwelling system and Angola-Benguela front using an artificial neural network

Information on the vertical chlorophyll structure in the ocean is important for estimating integrated chlorophyll a and primary production from satellite. For this study, vertical chlorophyll profiles from the Benguela upwelling system and the Angola-Benguela front were collected in winter to identify characteristic profiles. A shifted Gaussian model was fitted to each profile to estimate four parameters that defined the shape of the curve: the background chlorophyll concentration (B ₀), the height parameter of the peak (h), the width of the peak (σ) and the depth of the chlorophyll peak (z_m ). A type of artificial neural network called a self-organizing map (SOM) was then used on these four parameters to identify characteristic profiles. The analysis identified a continuum of chlorophyll patterns, from those with large surface peaks (>10 mg m^?3) to those with smaller near-surface peaks (<2 mg m^?3). The frequency of occurrence of each chlorophyll pattern identified by the SOM showed that the most frequent pattern (～12%) had a near-surface peak and the least frequent pattern (～2%) had a large surface peak. These characteristic profile shapes were then related to pertinent environmental variables such as sea surface temperature, surface chlorophyll, mixed layer depth and euphotic depth. Partitioning the SOM output map into environmental categories showed large peaks of surface chlorophyll dominating in water with cool temperature, high surface chlorophyll concentration and shallow mixed layer and euphotic depth. By contrast, smaller peaks of subsurface chlorophyll were in water with warmer temperature, lower surface chlorophyll concentration, intermediate mixed layer and deep euphotic depth. These relationships can be used semi-quantitatively to predict profile shape under different environmental conditions. The SOM analysis highlighted the large variability in shape of vertical chlorophyll profiles in the Benguela. This suggests that an ideal typical chlorophyll profile, as used in the framework of biogeochemical provinces, may not be applicable to this dynamic upwelling system.     

Standing stocks and production rates of phytoplankton and abundance of bacteria in the Seto Inland Sea, Japan

Standing stocks and production rates of phytoplankton and abundance of bacteria were investigated at 39 stations in the Seto Inland Sea, Japan during four crulses in October 1993, January, April and June 1994. Primary productivity was measured by the¹³C tracer method. Photosynthetic rate varied from 0.41 to 32.1 μgC/1/h with an average value of 4.67 μgC/l/h. Annual primary production was estimated to be 218 gC/m²/year. Annual primary production in this study was 1.8 times as high as the values which were previously reported at same area. The reason for the disagreement between our primary production value and previous values is not thought to be due to the difference of methods used for measuring primary production or the different Chl.a concentrations but to the method of estimating the primary production in the euphotic zone from thein vitro measurements. The distribution of bacterial cells in surface seawater was examined during the same cruises. Bacterial cell density ranged from 0.32 to 3.4×10⁶ cells/ml. The density was relatively high in the eutrophic regions of Hiroshima Bay and Osaka Bay In addition, a high density of bacteria was also observed in an area within Suo Nada where Chl.a was relatively low. The disparity between Chla and bacterial density in Suo Nada suggests that bacterial abundance can be controlled by the availability of substrates other than phytoplankton exudate.     

Spatial variability of the species composition,abundance, and productivity of the phytoplankton in the white sea in the late summer period

The species composition, cell concentration (N), and biomass (B) of the phytoplankton, as well as the chlorophyll a (Chl a) concentration, primary production (PP), and the concentrations of the dissolved inorganic micronutrients (phosphorus, silica, nitrogen as nitrite), were estimated for Kandalaksha Bay (KB), Dvina Bay (DB), and the basin (Bas) of the White Sea in August of 2004. The micronutrient concentrations were lower compared to the average long-term values for the summer period. The Chl a concentration varies from 0.9 to 2.0 mg/m³ for most of the studied areas, reaching up to 7.5 mg/m³ in the Northern Dvina River estuary. The surface water layer of the DB was the most productive area, where the PP reached up to 270–375 mg C/(m³ day). The phytoplankton biomass varied from 11 to 205 mg C/m³ with the highest values observed in the Bas and DB. Three groups of stations were defined during the analysis of the phytoplankton’s species composition similarity. The dinoflagellates Dinophysis norvegica and Ceratium fusus were particular to the phytoplankton assemblages in the KB; the diatom Ditylum brightwellii was particular to the upper and central parts of the DB. These three phytoplankton species were less abundant in the Bas.     

Climate change, reproductive performance and diet composition of marine birds in the southern California Current system, 1969–1997

We studied the effects of low-frequency climate change on the reproductive performance of 11 species of marine bird in the southern California Current system, 1969–1997. Reproductive performance of Brown Pelican (Pelecanus occidentalis) and Double-crested Cormorant (Phalacrocrax auritus) in southern California demonstrated an increase in the 1970s and early 1980s, attributable to recovery from organochlorine contamination (primarily DDE). Brandt's Cormorant (Phalacrocorax penicillatus) in central California was the only species to demonstrate a secular increase in performance through time, a pattern that remains unexplained. Ashy Storm-petrel (Oceanodroma homochroa) and Pelagic Cormorant (Phalacrocorax pelagicus) demonstrated curvilinear patterns of change, with decreasing reproductive performance in the past decade. All other species including Western Gull (Larus occidentalis), Pigeon Guillemot (Cepphus columba), Xantus's Murrelet (Synthiloboramphus hypoleucus), Common Murre (Uria aalge), Cassin's Auklet (Ptychoramphus aleuticus) and Rhinoceros Auklet (Cerorhinca monocerata) showed diminishing reproductive performance through time. Patterns of change for the murre and auklets were not significant, presumably because of a lack of reproductive variation for these species, which display a conservative breeding effort (i.e. single-egg clutches). Changes in the birds' abilities to provision young and maintain chick survival during May–July each year appeared most closely related to overall changes in reproductive performance. Dietary change indicated a decline in use of juvenile rockfish (Sebastes spp.) by marine birds in central California. There was also significant interannual variability in consumption of juvenile rockfish and the euphausiid Thysanoessa spinifera. Patterns of change in marine bird reproductive performance were generally concordant between southern and central California after considering the period of recovery for Brown Pelican and Double-crested Cormorant. The decline in reproductive performance and changes in diet composition do not appear directly related to the polarity reversal of the Pacific Decadal Oscillation in 1976/1977. Instead, reproductive performance and dietary characteristics indicate substantial change in the late 1980s, suggesting another regime-shift at that time.     

广泛分布于海洋生态系统中的壳状珊瑚藻——冈村石叶藻的形态解剖学和分子生物学描述

冈村石叶藻（Lithophyllum okamurae）是一种重要的壳状珊瑚藻,在海洋生态系统中具有提供初级生产力、产生碳酸盐沉积物以及为海洋生物提供栖息环境等功能。本文于三亚的热带海岸采集L. okamurae,首次基于形态解剖学特征和分子生物学方法对其进行了详细的描述。研究表明,L. okamurae的藻体为二组织性的拟薄壁组织结构,被分成三部分,包括：由1-3层呈扁平状正方形或长方形表皮细胞组成的表层,由一层正方形或长方形细胞组成的基层,以及由多层正方形或狭长形细胞组成的围层。在围层中可观察到栅状细胞,相邻藻丝细胞间通过次生纹孔相连,未见细胞融合。本文观察到L. okamurae的四种单孔生殖窠,即果孢子囊生殖窠、精子囊生殖窠、二分孢子囊生殖窠以及四分孢子囊生殖窠。其中,精子囊生殖窠通常浅埋于藻体中并微凸于藻体表面,果孢子囊生殖窠和无性生殖窠呈突出的圆锥形。基于18S rDNA、COI、rbcL和psbA基因序列的系统发育研究表明,L. okamurae和L. atlanticum在同一个进化枝上,亲缘关系最近。本文基于形态解剖学特征和分子生物学数据详细地对L. okamurae进行了描述,为海洋生态系统中藻类资源的利用和保护工作提供理论基础。