Cell death localization in situ in laboratory reared honey bee (Apis mellifera L.) larvae treated with pesticides

In this study, cell death detected by DNA fragmentation labeling and phosphatidylserine (PS) localization was investigated in the honey bee (Apis mellifera L.) midgut, salivary glands and ovaries after treating larvae with different pesticides offered via an artificial diet. To do this, honey bee larvae reared in an incubator were exposed to one of nine pesticides: chlorpyrifos, imidacloprid, amitraz, fluvalinate, coumaphos, myclobutanil, chlorothalonil, glyphosate and simazine. Following this, larvae were fixed and prepared for immunohistologically detected cellular death using two TUNEL techniques for DNA fragmentation labeling and Annexin V to detect the localization of exposed PS specific in situ binding to apoptotic cells. Untreated larvae experienced ∼10% midgut apoptotic cell death under controlled conditions. All applied pesticides triggered an increase in apoptosis in treated compared to untreated larvae. The level of cell death in the midgut of simazine-treated larvae was highest at 77% mortality and statistically similar to the level of cell death for chlorpyrifos (65%), imidacloprid (61%), myclobutanil (69%), and glyphosate (69%) treated larvae. Larvae exposed to fluvalinate had the lowest midgut columnar apoptotic cell death (30%) of any pesticide-treated larvae. Indications of elevated apoptotic cell death in salivary glands and ovaries after pesticide application were detected. Annexin V localization, indicative of apoptotic cell deletion, had an extensive distribution in the midgut, salivary glands and ovaries of pesticide-treated larvae. The data suggest that the tested pesticides induced apoptosis in tissues of honey bee larvae at the tested concentrations. Cell death localization as a tool for a monitoring the subclinical and sub-lethal effects of external influences on honey bee larval tissues is discussed.     

Stallion Semen Incubated with Hydrogen Peroxide Decreased DNA Fragmentation as Measured by the TUNEL Assay

Spermatozoa are highly specialized cells that are sensitive to environmental factors such as temperature and redox state. Hydrogen peroxide (H₂O₂) is a reactive oxygen species, and its presence is often associated with a decline in sperm function. The aim of this study was to evaluate the effect of H₂O₂ and heat stress on DNA damage, membrane integrity, and motility of stallion spermatozoa. Spermatozoa from three light-horse stallions were subjected to thermal stress, treatment with H₂O₂, or a combination of both. Treatments were organized using a 2 × 2 factorial design consisting of heat stress (control, 41°C) and potential oxidative stress (control, 50 μM H₂O₂) for 1 hour. The experiments were repeated independently on all stallions. Primary motility parameters were measured using a computer-assisted sperm analysis, whereas DNA damage was assessed using the TUNEL assay. To evaluate membrane integrity, an amine reactive dye was utilized. DNA and membrane integrity were simultaneously assessed in the same flow cytometry assay. Sperm incubated at 41°C were observed to have decreased motility (76.2% vs. 66.6%; P < .05) but not progressive motility (39.2% vs. 25.6%), membrane damaged (30.8% vs. 37.4%), or DNA damage (19.7% vs. 17.3%). Interestingly, when compared to control, treatments with H₂O₂ had decreased DNA damage (19.7% vs. 7.1%; P < .05), but did not affect any other parameter. Although our experiment demonstrated a favorable effect of 50 μM H₂O₂ on DNA damage, further studies need to be conducted to confirm these findings and to clarify any possible signaling mechanisms.     

Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line

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.     

尼罗罗非鱼Smoothened的鉴定、表达模式及在精巢中的作用

为探究Smoothened (Smo)信号在精巢不同细胞增殖与存活中的作用,实验分离鉴定了尼罗罗非鱼smo (命名为Onsmo),检测了其在不同组织中的表达分布及在精巢中的细胞表达模式,在尼罗罗非鱼精巢组织的体外培养体系中,用Smo特异性激动剂SAG或抑制剂环巴胺分别进行处理,EdU掺入法及TUNEL法检测了处理后生殖细胞(Vasa⁺)、Sertoli细胞(Amh⁺)与Leydig细胞(Cyp17a1⁺)增殖或凋亡情况。结果显示,Onsmo开放阅读框全长2 478 bp,编码825个氨基酸,含有7次跨膜结构域,与人SMO氨基酸一致性达77%;Onsmo表达于包括精巢在内的多个组织;在精巢中,Onsmo在多种不同类型细胞表达,包括精原细胞、精母细胞、Sertoli细胞以及Leydig细胞;在精巢组织的体外培养体系中,SAG处理对精原细胞增殖具有显著促进作用,而环巴胺处理对Sertoli细胞、Leydig细胞凋亡具有显著促进作用。研究表明,On Smo信号在尼罗罗非鱼精巢精原细胞增殖与体细胞存活中具有重要作用。该研究首次证实...     

TUNEL法和SCGE法评价奶牛性控冻精DNA损伤

末端转移酶介导的dUTP末端标记法(TUNEL)和单细胞凝胶电泳法(SCGE)常被用于检验细胞DNA的完整性。本研究采用这两种方法分别对性控、常规冻精进行检测,比较不同方法对精子DNA损伤的检测结果。结果表明:TUNEL法检测性控冻精DNA的损伤率为63.32±0.56%,显著高于(P0.01)常规冻精的31.67±1.03%;SCGE法检测性控冻精和常规冻精的DNA损伤率分别为64.00±0.68%和32.23±0.72%,差异极显著(P0.01)。而两种方法对性控冻精和常规冻精DNA损伤的检测结果间没有显著差异(P0.05),说明该两种方法均可用于冻精DNA损伤检测。     

Enhanced viability of a nervous necrosis virus-infected stable cell line over-expressing a fusion product of the zfBcl-xL and green fluorescent protein genes

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.     

家榆种子人工老化过程中的细胞形态学特征

以家榆种子为材料,在37℃、100%相对湿度下进行人工老化。应用电子显微镜、光学显微镜观察、细胞原位末端脱氧核苷酸转移酶标记(TUNEL)法检测家榆种子在人工老化条件下细胞发生凋亡的形态学特征。结果显示:老化第1天根冠少数几个细胞开始出现细胞凋亡;随着老化深入,生长点、子叶等多个部位开始有凋亡细胞出现;老化至第6天,部分凋亡细胞崩解,细胞质高度浓缩,剩余组织细胞膜的保护能力下降,出现真菌感染;同时,用外源Ca2+作为诱导剂对家榆种子进行处理,TUNEL法检测结果显示并未出现细胞凋亡。     

Comparative histomorphological and ultrastructural study of the luminal epithelium of the isthmus in laying and moulting domestic fowls (Gallus domesticus)

This study describes ciliated, nonciliated and mitochondrial luminal epithelial cells of the isthmus in laying and moulting domestic fowls using histological and ultrastructural techniques. The ciliated cells were nonsecretory, while numerous electron‐dense secretory granules were present in the nonciliated cells of laying birds. Mitochondrial cells, occurring in two morphologically distinct forms, constituted the third type of epithelial cell present in the isthmus. The SEM study showed that the luminal epithelium was dominated by ciliated cells, the cilia of which partially obscured adjacent nonciliated cells. The involution of the luminal epithelium in moulting birds occurred via autophagy, apoptosis and necrosis. Autophagic inclusions, which included autophagosomes and autolysosomes, were present in the early degenerative phases of ciliated, nonciliated and mitochondrial cells. Nonciliated cells underwent degeneration via apoptosis, which was characterized by nuclear and cytoplasmic condensation. Apoptotic and necrotic ciliated cells were evident during the intermediate and advanced stages of regression. The presence of apoptotic cell death was confirmed using the TUNEL assay. Loss of cilia via the formation of cilia packets was observed using TEM and SEM. Necrotic cell death occurred in mitochondrial cells during the intermediate and late stages of degeneration. In conclusion, the findings of the study on isthmus involution in moulting birds suggest that autophagy is a process confined to the early stages of degeneration, while apoptosis and/or necrosis occur in the terminal stages of regression.     

蚕的细胞凋亡相关基因及其检测技术

家蚕细胞凋亡是家蚕细胞的主动死亡过程,在家蚕细胞的发育和疾病的发生中起重要的作用,同时也是一种抵御病毒感染的天然性保护机制。而病毒也在长期进化过程中获得了适应性调节细胞凋亡的能力,或通过在病毒复制早期表达凋亡抑制产物,以阻止细胞凋亡达到繁殖子代病毒的目的,或通过晚期表达凋亡诱导物诱导细胞凋亡,以快速有效地释放子代病毒。根据细胞凋亡的典型特征,已经发展出ELISA法,流式细胞仪法等分子生物学检测方法,为更深入地了解细胞凋亡的机制打下了坚实的基础。     

口蹄疫病毒诱导BHK-21细胞产生凋亡的研究

试验利用口蹄疫病毒感染BHK-21细胞,通过MTT法、Hoechst 33258染色、原位末端标记技术(TUNEL)、流式细胞术和链特异性荧光定量RT-PCR,分别就口蹄疫病毒对BHK-21细胞生长的抑制作用、凋亡细胞的形态学和分子生物学特征、凋亡峰的出现和细胞周期的变化以及口蹄疫病毒基因组在BHK-21细胞内的复制情况进行了检测。结果表明:口蹄疫病毒可抑制BHK-21细胞的生长并诱导其产生凋亡,呈现典型的凋亡细胞特征,出现细胞凋亡峰并且细胞周期明显被阻滞在G1/G0期,同时对凋亡率和口蹄疫病毒基因组复制的关系做了初步研究。