Affinity isolation and mass spectral analysis of 1,10-phenanthroline (OP)-stimulated UV-damaged-DNA binding proteins expressed in zebrafish (Danio rerio) embryos

Our earlier studies indicated the high expression of a UV-damaged-DNA binding activity in zebrafish (Danio rerio) embryos at 12?h postfertilization (hpf). Two 30- to 35-kDa polypeptides homologous to the N-terminal lipovitellin 1 (Lv1) domain of the 150-kDa zebrafish vitellogenin 1 (zfVg1) were identified as the damage recognition factors in zebrafish extracts, and the metal-chelating agent 1,10-phenanthroline (OP) was found to inhibit the embryonic UV-damaged-DNA binding activity. This study further explored the DNA damage-sensing components in 12 hpf zebrafish extracts. UV-damaged-DNA binding proteins were enriched from zebrafish extracts by isoelectrofocusing. Both OP-sensitive and OP-stimulated, UV-damaged-DNA binding activities were detected in fractionated zebrafish extracts. Two-dimensional gel electrophoresis of proteins captured by an immobilized oligonucleotide carrying a UV-induced (6-4)photoproduct (6-4PP) revealed a 25-kDa polypeptide as the major 6-4PP-binding factor in an OP-stimulated fraction. Three 25-kDa factors that bound weakly to 6-4PPs were also isolated. The four polypeptides having pIs between 7.0 and 7.3 were unreactive to an anti-zfVg1 antibody targeting the Lv1 domain. Mass spectral analysis showed the appearance of amino acid sequences LPIIVTTYAK and IPEITMSK in all 25-kDa polypeptides and sequences exactly matching those contained in the four factors exist only in the C-terminal Lv2 domain of zfVg1, reflecting the origination of these factors from enzymatic cleavage of the Lv2 domain at slightly different positions. The OP-stimulated fraction produced a much stronger UV-dependent DNA incision activity in the presence than in the absence of OP, suggesting the association of these factors with DNA damage repair under metal-deficient conditions.     

HSP90抑制剂根赤壳菌素对斑马鱼胚胎发育的影响

为研究热休克蛋白90(heat shock protein 90,HSP90)在斑马鱼(Danio rerio)胚胎发育中的作用,本实验采用2μmol/L、5μmol/L、10μmol/L的HSP90抑制剂根赤壳菌素(radicicol)对斑马鱼发育期胚胎进行处理,监测斑马鱼胚胎不同发育时期两个HSP90功能抑制标志基因BAG3(BCL2-associated athanogene3)和HSPB1(heat shock protein beta-1)的mRNA的表达水平,并观察不同发育时期的胚胎发育状况。结果如下:(1)实时荧光定量PCR结果显示,BAG3和HSPB1 mRNA水平在根赤壳菌素处理胚胎12 hpf(hours post-fertilization,hpf)或24 hpf后显著增高,Western blot检测到5μmol/L根赤壳菌素处理胚胎24 hpf后HSP70表达上调,证明该实验条件下HSP90功能受到抑制;(2)根赤壳菌素处理后斑马鱼胚胎发育变缓,胚胎成活率统计显示:2μmol/L、5μmol/L、10μmol/L根赤壳菌素处理24 hpf胚胎成活率分别是95%、77%、35%,成活率随根赤壳菌素浓度的增高而降低;(3)5μmol/L根赤壳菌素处理72 hpf可见部分个体色素沉积减少、心包膜增大、肌肉萎缩等发育畸形。研究结果证明HSP90在斑马鱼胚胎发育过程中发挥重要的作用,根赤壳菌素在5μmol/L时已达到较佳抑制效果且成活率较高,而且胚胎发育出现了各种形态学变化,为后期研究HSP90调节斑马鱼胚胎发育奠定了基础。     

UVB辐射导致斑马鱼早期胚胎发育异常和DNA损伤的初步研究

为研究UVB辐射对斑马鱼(Danio rerio)早期胚胎发育的影响,采用波长302nm,强度分别为120、240、310、420μW/cm2的UVB照射斑马鱼早期发育阶段的胚胎,照射时间分别为1、3、5、10min,用于研究UVB照射对形态学及DNA损伤的影响;将原肠期、体节期胚胎及孵化后2d仔鱼经UVB照射,设立未经UVB照射组为对照组,观察记录UVB照射后胚胎发育情况,统计死亡率、畸形率;应用单细胞凝胶电泳(SCGE)技术检测不同UVB照射条件对DNA损伤的影响。结果表明:(1)UVB照射对斑马鱼早期胚胎发育形态有明显的影响,能够造成尾部弯曲、围心腔扩大、脊柱扭曲等多种畸形甚至死亡;(2)在检测UVB照射与DNA损伤的关系时,发现UVB照射对胚胎细胞中的DNA能够产生比较明显的损伤,且DNA损伤随照射强度的增加和照射时间的延长而加剧。在所研究的3个发育阶段中,原肠胚受损最为严重,但未发现明显的剂量累积效应。结论认为,UVB照射对斑马鱼早期胚胎发育有明显影响,不同的照射强度和时间造成的影响差异显著;不同发育阶段的斑马鱼胚胎对UVB照射的耐受程度也不同。     

尼罗罗非鱼整胚原位杂交技术的建立和初步应用

为了研究尼罗罗非鱼胚胎发育和器官形成过程中基因功能和基因表达图式,本研究建立了尼罗罗非鱼的整胚原位杂交流程。尼罗罗非鱼的胚胎具有卵黄大、不透明、色素出现早等特点,因此现有鱼类整胚原位杂交方法不能完全适用于尼罗罗非鱼的胚胎,故本研究做了相应的调整和优化:通过提高H2O2的浓度和添加KOH,改良了尼罗罗非鱼胚胎的色素去除方法;使用冷丙酮代替蛋白酶K在提高胚胎通透性的同时保持胚胎完整;减少了探针回收和抗体回收后的洗涤次数,完善了结果的图像采集和胚胎保存方案。使用尼罗罗非鱼的重组激活基因Rag1作为探针基因,整胚原位杂交结果显示Rag1基因表达的位置与已报道的斑马鱼和日本青鳉的Rag1基因在胚胎中的表达位置高度保守,胚胎完整,基因表达位置清晰可见,表明此套尼罗罗非鱼的整胚原位杂交技术流程成功有效。     

斑马鱼早期胚胎发育囊胚sphere时期的蛋白组学研究

斑马鱼(Danio rerio)早期胚胎发育囊胚时期是胚胎发育过程的关键时期,利用i TRAQ蛋白质质谱分析技术,检测斑马鱼早期胚胎发育过程中囊胚sphere时期的蛋白质表达情况,并分析该时期表达的蛋白质的相应功能和参与调控的生物过程。以野生型斑马鱼发育至sphere时期即4 hpf的胚胎为样本,利用i TRAQ标记与LC-MS/MS串联质谱技术,结合数据库比对,对该时期表达的蛋白质进行定性和定量的鉴定分析。检测结果共鉴定到的总蛋白数为1 178个,利用生物信息学进行功能分析,发现这些蛋白广泛参与了细胞信号传递、细胞运动和细胞骨架构建、细胞增殖、细胞分化、物质合成与代谢等各项重要的生命活动过程。研究表明,利用i TRAQ标记的方法可以对4hpf时期的斑马鱼胚胎中的蛋白质进行有效的分离和鉴定,并初步建立了斑马鱼早期发育关键阶段sphere时期的蛋白质组表达图谱,以期为斑马鱼胚胎发育过程中蛋白质组学和调控机制的研究提供参考。     

Stress-induced apoptosis by heat shock, UV and γ-ray irradiation in zebrafish embryos detected by increased caspase activity and whole-mount TUNEL staining

SUMMARY: Environmental stress-induced apoptosis in zebrafish Danio rerio embryos was characterized by assaying caspase-3-like activity and whole-mount terminal deoxynucleotidyl nick-end labeling (TUNEL) staining. Severe stress conditions, such as heat shock at 39°C for 1 h, ultraviolet light at 10–100 mJ/cm² and γ-ray irradiation at 5–20 Gy induced extensive apoptosis in embryos. Apoptotic cells were observed after the bud and 1-somite stages in normal embryos by TUNEL staining, and after stress treatment many TUNEL-positive cells were found in the enveloping and deep cell layers and the larval fin. The caspase-3-like activity increased severalfold during stress-induced apoptosis in a dose-dependent manner. These findings indicate that apoptotic pathways, mediated by caspase-3-like activity, play a major role in zebrafish embryogenesis under stress conditions.     

Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryos

A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time‐consuming procedure. This report addresses this need, considering that the development of protocols for lipid‐based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid‐based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP‐N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection.     

Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses

Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.     

Effects of embryonic exposure to α‐lipoic acid or ascorbic acid on hatching rate and development of zebrafish (Danio rerio)

This study investigates the effects of embryonic exposure to two different antioxidants on growth and development in fish. Zebrafish (Danio rerio) embryos (100 per group) were exposed to lipoic acid (LA, 6–12 μM) or ascorbic acid (AA, 100–200 μM) and the hatching rate, standard lengths (SL) at hatching, development and growth post‐hatching monitored. The SLs at hatching were increased (P<0.05) in both antioxidant‐exposed groups relative to the controls, with no effect on yolk reserves. This enhanced development persisted up to 15 days post hatching. At hatching, cell proliferation rates (P<0.0005) and basic fibroblast growth factor (P<0.001), were greater in the antioxidant‐exposed fish than in the controls (0 μM antioxidant); no oxidative DNA damage was detected (P>0.05). Activity of the endogenous antioxidant enzyme superoxide dismutase was greater (P<0.001) in LA‐treated fish than in the controls. The results suggest that embryonic treatment of zebrafish with LA or AA during embryogenesis enhanced cell proliferation, leading to increased somatic growth in the larval stages, persisting into the juvenile stage. The findings support the treatment of embryonic fish with antioxidants for enhanced results in aquaculture.     

Organization and promoter analysis of a tiger shrimp Penaeus monodon single WAP domain-containing protein gene

The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb 3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone (GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish.     

Expression patterns of acidic and basic fibroblast growth factor in loach fish embryos

Heparin-binding fractions were taken from the heparin sepharose columns on which extracts of loach fish (Misgurnus anguillicaudatus) embryos from blastula, gastrula, 4–8 and 12–16 somites stages were applied. These heparin-binding fractions, except the fraction derived from 12–16 somite embryos, showed potent mitogenic activity on fibroblast-like cells derived from caudal fin blastema of goldfish. Western blot analysis of these heparin-binding fractions was carried out using monoclonal antibodies against human acidic and basic fibroblast growth factors (FGF-1 and-2). An immunoreactive FGF-1 band at 16 kD was detected in the heparin-binding fraction derived from embryos in each stage of blastula, gastrula and 4–8 somites. An immunoreactive FGF-2 band at 17 kDa was detected exclusively in the heparin-binding fraction derived from 4–8 somite embryos. In the heparin-binding fraction derived from 12–16 somite embryos neither immunoreactive FGF-1 nor-2 member band was detectable.     

环境污染物对斑马鱼早期胚胎转座子转录活性的影响

转座子在生物基因组中占据重要的组成部分,是基因组中可移动和扩展的重要元件,其转座活性受外界环境因子调控。为了探讨环境应激对基因组转座子表达活性的影响,用环境污染物二噁英(TCDD)和重金属Cu~(2+)或Cd~(2+)处理斑马鱼早期胚胎,通过荧光定量PCR(q RT-PCR)比较处理前后9种结构完整的转座子转录活性变化,这9种转座子分别是DNA转座子Tc1家族的Tc-a、Tc-b、Tc-c、Tc-d、Tc-e,反转录转座子病毒家族ZB-ERV-1、ZB-ERV-2、反转录转座子LINE家族L1-323和L1-21。结果发现,TCDD使8个转座子转录活性明显下调,1个转座子显著上调;Cu~(2+)导致7个转座子上调,2个显著下调;Cd~(2+)导致6个转座子上调,3个显著下调。研究表明,斑马鱼转座子转录活性受环境因素影响显著,环境胁迫可能是转座子活性变化的原因之一。本研究对了解和评估环境因子对鱼类转座子活性的影响作用具有重要的意义,为进一步研究生物基因组的进化机制提供参考。     

Establishing a zebrafish transgenic line expressing tilapia lysozyme with enhanced antibacterial activity

In recent years, infectious disease caused by Streptococcus agalactiae has increased in aquaculture, threatening the healthy development of tilapia and resulting in substantial economic losses. Cultivating disease‐resistant tilapia by transgenic technology is one strategy that has been developed to address this issue. Here, we used a transposon system to investigate gene expression and tissue lytic activity in transgenic zebrafish expressing the tilapia lysozyme. The transpose vector contained the Tgf2 transposon skeleton, tilapia heat shock protein 70 promoter (Hsp70) and two target genes (tilapia C‐type lysozyme 3 [lysozyme‐C3] and green fluorescent protein [GFP]). The transgenic zebrafish F₀ generation was obtained by microinjecting the donor vector and transposase mRNA into fertilized zebrafish eggs. There was 45% green fluorescence in the zebrafish F₀ generation, which was spread over the entire body based on microscope observation. The F₀ generation was reared to sexual maturity, at which point individuals were mated with wild‐type zebrafish to produce the F₁ generation. Tilapia lysozyme‐C3 expression was detected in the liver of F₁ generation by RT‐PCR and western blotting, but was not detected in the skin, intestine, or muscle. Significantly higher liver bacteriolysis activity was detected in transgenic zebrafish compared with wild‐type zebrafish. Therefore, transgenic zebrafish may have an increased potential for bacterial resistance.     

Biochemical Characterization and Comparison of Pancreatic Lipases from the Pacific Bluefin Tuna,Thunnus orientalis; Totoaba,Totoaba macdonaldi; and Striped Bass,Morone saxatilis

Lipolytic activity from pancreatic crude extracts from the Pacific bluefin tuna (PBT), Thunnus orientalis; totoaba, Totoaba macdonaldi; and striped bass, Morone saxatilis, were biochemically characterized to detect possible differences in the digestive capacity of each fish. The maximum activity was at temperatures ranging from 35 to 45 C and at pH 8.0; the activity decreased significantly at a pH lower than 7.0. Lipases from PBT and totoaba showed an increase in activity with the presence of sodium taurocholate (NaTC; 6 mM). However, no increase in activity could be observed when a mixture of bile salts or natural bile extracts was used in the reaction mixture, which was observed in the presence of NaTC. In contrast, the lipase activity from striped bass increased accordingly when NaCl was added at different levels (0–1.5 M). Calcium was not required in the reaction mixture for any of the fish species tested. We concluded that all three lipases have different physicochemical properties and ionic strength and emulsifier requirements to obtain their maximum activity. Striped bass was far too sensitive to NaCl, while totoaba and PBT were sensitive to NaTC. The differences in activity shown here suggest the possibility of studying the use of different additives on feed formulations to maximize lipase efficiency.     

Development of Cre–loxP technology in zebrafish to study the regulation of fish reproduction

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.     

Metallothionein induction in cultured fibroblasts and liver of a marine flatfish,the turbot,Scophthalmus maximus

Intraperitoneal injection of turbot with Cd induced the synthesis of a low molecular weight hepatic Cd-binding protein and a 500bp mRNA, which hybridised to a plaice metallothionein (MT) cRNA probe. The Cd-binding protein displayed cross-reactivity in a competitive ELISA with antiserum raised against rainbow trout MT and had the characteristic amino acid composition, metal stoichiometry and spectral characteristics of a Class I MT. Only one isoform was apparent on ion exchange chromatography. Southern blot analysis of DNA cleaved with four restriction enzymes suggested that only a single MT gene is present in turbot.In an established turbot fibroblast cell line, Cd induced MT mRNA and MT levels in a dose and time-dependent manner. MT was also induced by Cu, Hg and Zn but not Pb exposure. Physiological concentrations of glucocorticoids and sex hormones did not induce MT synthesis, although at high concentrations a positive response to corticosterone, dexamethasone, hydrocortisone or progesterone was observedin vitro indicating the possible presence of a functional steroid regulatory element in the fish MT gene.Abbreviations used AMP adenosine monophosphate - CHSE chinook salmon embryo - DMSO dimethyl sulphoxide - HPLC high performance liquid chromatography - KB kenacid blue - MT metallothionein - Mr molecular weight (kDaltons) - NR neutral red - PEG polyethylene glycol - RTH rainbow trout hepatoma - SDS sodium dodecyl sulphate - SSC 0.15M NaCl, 0.015M sodium citrate - TF turbot fibroblast