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1.
Two new cell lines have been established from the muscle and swim bladder tissues of barramundi, Lates calcarifer, and designated as BM (barramundi muscle) and BSB (barramundi swimbladder), respectively. The cells multiplied well at 28 °C in Leibovitz’s L‐15 medium supplemented with 10% foetal bovine serum, and have been continuously subcultured more than 100 times to date. Morphologically, BM cells were mostly fibroblastic, whereas BSB were mostly epithelial. Both cell lines were susceptible to grouper iridovirus (GIV) and displayed characteristics of apoptosis after viral infection. The induction of apoptosis was further assayed in GIV‐infected BM and BSB cells by various methods. The inhibition of cell growth by GIV was demonstrated by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. Morphological observations revealed typical apoptotic features in the infected cells, including cell shrinkage and rounding, chromosome condensation and formation of apoptotic body‐like vesicles. Chromosome fragmentation was detected by DNA laddering and TUNEL assays. Finally, the appearance of phosphotidylserine on the outer leaflet of apoptotic cell membranes was confirmed by annexin V staining. This is the first report of apoptosis induced by GIV in fish cells.  相似文献   

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4.
Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV‐2L gene, which encodes a protein of unknown function. GIV‐2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV‐2L protein by immunizing mice with GIV‐2L‐His‐tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV‐infected cells, we showed that GIV‐2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV‐2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.  相似文献   

5.
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.  相似文献   

6.
In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.  相似文献   

7.
Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria‐submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti‐(active caspase‐3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi‐infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi‐infected turbot compared with non‐infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase‐3‐positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.  相似文献   

8.
The caspase-3 appears to be a key protease in the apoptotic pathway. We identified caspase-3 complementary DNAs from the ovaries of the protandrous cinnamon clownfish (Amphiprion melanopus), and investigated its mRNA and proteins, and activity levels during the sex change (I, mature male; II, male at 90 days after removing of the female; and III, mature female). The nucleotide sequence of the caspase-3 cDNA was 969 base pairs in length with open reading frames encoding peptides of 282 amino acids. The caspase-3 mRNA and protein, and activity levels in stages of the mature gonad are higher than those of the development gonad stage. To understand the effect of gonadotropin-releasing hormone (GnRH) on gonad apoptosis, we examined expression of genes caspase-3 mRNA and activity level in immature cinnamon clownfish gonads after GnRH analogue (GnRHa). The findings support the hypothesis that caspase-3 expression is associated with both testicular and ovarian development, and suggests that it may play a role in the control of ovarian development in cinnamon clownfish. Also, we demonstrate that GnRH agonists stimulate caspase-3 production which can in turn stimulate apoptosis. The present study provides a framework for better understanding of the role of caspase-3 during sex change processes in fish.  相似文献   

9.
This study investigated the effect of continuous temperature decrease on hemocyte apoptosis of the white shrimp Litopenaeus vannamei. In the stress group, water temperature decreased from 26 to 17 °C at a rate of 1 °C/h. Shrimp kept at 26 ± 0.5 °C were used as control group. Total hemocyte count (THC), reactive oxygen species (ROS) production, cytoplasmic free-Ca2+ (CF-Ca2+) concentration, mitochondrial membrane potential (MMP), apoptotic cell ratio, and caspase-3 activity of L. vannamei hemocytes were determined when water temperature decreased to 23, 20, and 17 °C, respectively. Increased ROS production in hemocytes was observed when water temperature decreased to 20 and 17 °C. Decreased THC and cellular MMP, increased CF-Ca2+ concentration, apoptotic cell ratio, and caspase-3 activity were shown when water temperature decreased to 17 °C. These results indicate that water temperature decrease can induce oxidative stress on shrimp hemocytes and then cause mitochondria and caspase-3 mediated hemocyte apoptosis and THC reduction, when water temperature decreased to an unconformable level.  相似文献   

10.
Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.  相似文献   

11.
Lymphocystis disease virus (LCDV), a large icosahedral DNA virus classified to the iridovirus family, is the causative agent of lymphocystis, a disease which occurs in marine and freshwater fish species and is characterized by formation of papilloma-like lesions on the surface of the skin. In vitro, LCDV infection causes flounder gill cells, an adherent cell line, to exhibit an obvious cytopathic effect (CPE). In order to test whether apoptosis is responsible for the observed CPE, cells infected with LCDV at a multiplicity of infection (m.o.i.) of 5 PFU per cell were examined at various time intervals for the appearance of apoptotic signs. Nuclear fragmentation, DNA laddering and caspase activation were observed in the infected cells at the time (i.e. 10 days post-infection) when an intensive CPE was observed. These findings demonstrate that LCDV is capable of inducing apoptosis in vitro, which is different from the result of LCDV infection in vivo, and consequently suggest an intricate LCDV-host interaction.  相似文献   

12.
为探究黄鳝( Monopterus albus )Caspase-3在性腺性逆转发育过程中的功能和作用,实验扩增黄鳝 caspase-3 的部分序列,检测了其在不同发育时期性腺中mRNA表达水平、蛋白相对含量以及表达位置。通过PCR扩增获得黄鳝 caspase-3 基因cDNA序列,推导的氨基酸序列对比发现,黄鳝 caspase-3 与大黄鱼( Larimichthys crocea )和鳜( Siniperca chuatsi )亲缘关系最近。实时定量PCR ( qRT-PCR)结果显示, caspase-3 mRNA在黄鳝各发育时期性腺中均有表达,在卵黄生成期性腺中表达量最高,并伴随性腺向雄性发育表达量呈下降的趋势;而其蛋白相对含量在性腺性逆转过程中呈现逐渐上升的趋势,间性晚期最高。免疫组织化学染色结果显示,Caspase-3蛋白阳性信号定位于卵黄生成期卵母细胞细胞质、皮质小泡期卵母细胞的细胞核和颗粒细胞,以及各个发育时期性腺中初级生长期卵母细胞的细胞质和细胞核。综上,Caspase-3与黄鳝性逆转过程关系密切,推测其可能参与了性逆转过程中卵母细胞的凋亡过程。  相似文献   

13.
Grouper iridovirus (GIV) is one of the most serious pathogens in mariculture and causes high mortality rates in cultured groupers; then, effective medicines for controlling GIV infections are urgently needed. Viola philippica is a well‐known medicinal plant, and the application of V. philippica aqueous extracts against GIV infection was assessed by different methods in this study. The results showed that the working concentration of V. philippica aqueous extracts was 10 mg/ml. V. philippica aqueous extracts below 10 mg/ml have no significant cytotoxic effects on cell viability, while extracts over 15 mg/ml decreased cell viability and showed cytotoxic activity. V. philippica aqueous extracts had excellent inhibitory effects against GIV infection in vitro and in vivo. The possible antiviral mechanism of V. philippica was further analysed, which indicated that V. philippica did no damages to GIV particles, but it could disturb GIV binding, entry and replication in host cells. V. philippica had the best inhibitory effects against GIV during viral infection stage of binding and replication in host cells. Overall, the results suggest that appropriate concentration of V. philippica aqueous extracts has great antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling GIV infection in farmed groupers.  相似文献   

14.
任胜杰  吴青  张佐  袁文清  陈兰  郑曙明 《水产学报》2017,41(10):1609-1622
为研究黄芪多糖(APS)和当归多糖(ASP)对维氏气单胞菌诱导鲫细胞凋亡的影响,实验设阴性对照组、阳性对照组,黄芪多糖组和当归多糖组,通过对鲫用维氏气单胞菌攻毒处理,用流式细胞仪测定血细胞的凋亡比例和细胞周期变化,并用荧光显微镜和透射电镜观察凋亡细胞的形态。结果显示,维氏气单胞菌攻毒后阳性对照组鲫血细胞的细胞凋亡率均极显著高于多糖组和阴性对照组;多糖组能显著降低鲫的细胞凋亡率且黄芪多糖组效果更显著;攻毒后鲫肝细胞和肾脏淋巴细胞出现染色质凝集、细胞核固缩边集和细胞空泡化及凋亡小体;同阴性对照组相比;维氏气单胞菌攻毒可以引起鲫血细胞周期中S/G2+M期细胞比例极显著下降,sub-G1极显著升高,抑制细胞分裂诱发凋亡;多糖组则G0/G1期细胞极显著降低,S/G2+M期细胞极显著升高,sub-G1极显著降低,促进细胞分裂抑制凋亡。黄芪多糖和当归多糖添加量在1%时能抑制维氏气单胞菌攻毒引起的细胞凋亡。  相似文献   

15.
Rainbow trout gastroenteritis (RTGE) is an emerging disease that has acquired new relevance in European rainbow trout, Oncorhynchus mykiss (Walbaum), culture, because of the economic losses it causes. Disease aetiology and pathogenesis remain unclear. The lesions appear restricted to the gastrointestinal tract where extensive mucosal detachment associated with high numbers of segmented filamentous bacteria (SFB) can be detected. In this study, an RTGE outbreak in north-western Spain was investigated, and findings observed in diseased trout were compared with control fish. PAS stain and immunohistochemical assays with anti-CD3ε and anti-active caspase-3 antibodies were performed. The results showed that CD3ε+ inflammatory infiltrates were present in the intestine of diseased trout both in the lamina propria-submucosa and within the epithelium. Moreover, an increased number of caspase-3+ cells in the intestinal mucosa and also strong anti-caspase-3 immunoreactivity in desquamated cells in the gut lumen were observed. Changes in the number of goblet cells were also found, resulting in an increase or depletion of mucous cells depending on the severity of the intestinal lesions. These findings suggest that T cells and apoptosis play an important role in the development and pathogenesis of RTGE.  相似文献   

16.
To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post‐exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post‐clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC‐1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.  相似文献   

17.
喹乙醇诱导鲤肝细胞凋亡的研究   总被引：6，自引：0，他引：6  
汪开毓 《水产学报》2004,28(6):733-737
细胞凋亡(apoptosis)是受基因控制的细胞自主的有序死亡。凋亡细胞的形态特征主要表现为细胞核的染色质浓缩,边移,聚集在核膜下,进而核发生裂解,核碎片与细胞碎片有单层膜包裹形成凋亡小体(apoptosisbody)[1]。目前已发现某些物理因素(辐射、高温等)、细胞因子、病毒感染和  相似文献   

18.
Molecular characterization was carried out on an iridovirus isolated from yellow grouper, Epinephelus awoara . The major capsid protein (MCP) gene was located, sequenced and compared with homologous genes from other iridoviruses. The nucleotide sequence is 1392 bases long and contains a single open reading frame beginning at an ATG codon from the 5' end and terminating at a TAA codon at the 3' end. The open reading frame encodes a protein of 463 amino acids with a predicted molecular weight of 50 272 Da. Pairwise amino acid alignments detected a high degree of sequence identity between grouper iridovirus (GIV) MCP and the homologous genes of other iridoviruses. The MCP gene of GIV was most similar to the MCP gene from frog virus 3 (FV3) with 70% nucleotide and 73% amino acid sequence identity. The predicted molecular weight of the protein of this gene is comparable with the apparent weight obtained by SDS–PAGE. Pathogenicity of the GIV was investigated in yellow grouper by intraperitoneal injection of 107 and 104 TCID50 virus. Cumulative mortalities reached 100% within 11 and 25 days post-infection, respectively, while no grouper died in the control group. The molecular studies demonstrated that GIV is a member of the genus Ranavirus .  相似文献   

19.
Nervous necrosis virus (NNV) infection induces host cell apoptosis by an ill-understood process. We utilized a fusion between enhanced green fluorescent protein (EGFP) and the zfBcl-x(L) gene in GL-av cells to select for zfBcl-x(L) stable cell lines and to assess the effectiveness of the anti-apoptotic protein Bcl-x(L) in circumventing NNV-induced cell death. Stable EGFP and EGFP-Bcl-x(L)-expressing clones were obtained at high purity within 2.5-3 months. In the latter, the EGFP-Bcl-x(L) fusion protein (approximately 58.2 kDa, as ascertained by Western blot) was predominantly targeted to mitochondria. We assayed for apoptosis in red-spotted grouper NNV Tainan no. 1 (RGNNV TN1)-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different virus doses. NNV infection of NNV Bcl-x(L) GL-av cell line revealed a protective effect, with a decrease in TUNEL-positive cells of 7%, 8% and 31.8% at 24, 48 and 72 h, respectively. In addition, RGNNV infection of the Bcl-x(L) GL-av cell line revealed a protective effect, with an enhanced viability of 3%, 40% and 73% at 24, 48, and 72 h, respectively. We conclude that NNV-induced apoptotic cell death can be lessened in transgenic grouper fish cells.  相似文献   

20.
In this study, we exposed black sea bream, Mylio macrocephalus (Basilewsky), fibroblast (BSF) and silver sea bream, Sparus sarba Forsskål, fibroblast (SSF) cell lines to a recombinant Vibrio harveyi haemolysin (VHH) and investigated mechanisms involved in apoptosis. A decrease in mitochondrial membrane potential, followed by an increase in caspase 3 activity, occurred within 2–8 h of VHH exposure, in both cell lines; however, VHH did not alter cellular levels of reactive oxygen species. As heat shock protein 70 (HSP70) is known to prevent the onset of apoptosis in certain mammalian cells, we aimed to test whether such a protective effect is operative in VHH‐exposed fibroblasts. The amounts of HSP70 were elevated in SSF and BSF via an acute heat shock or an acute heat shock followed by a 6 h recovery. It was found that the VHH‐mediated reduction in mitochondrial membrane potential was suppressed in cells that had a 6 h post‐heat shock recovery, and the protective effect of heat shock‐induced HSP70 was attenuated following treatment of cells with the HSP70 inhibitor, quercetin. This study demonstrates how haemolysin causes cell death via induction of apoptosis and provides evidence as to the role of HSP70 as an anti‐apoptotic factor.  相似文献   

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