沙丁胺醇对黑腹果蝇基因组稳定性、细胞凋亡和蛋白表达的影响

【目的】已有研究表明,食用了饲喂以沙丁胺醇为主要成分的瘦肉精的动物肉类后,瘦肉精成分会在人体内富集,摄入过量沙丁胺醇会对生物体造成不良影响,但是,其具体毒性作用机理目前尚不明确。作为一种模式生物,黑腹果蝇Drosophila melanogaster与哺乳动物的基因具有较高的同源性,且具有繁殖周期短、方便进行遗传操作等优势。因此,我们通过研究过量沙丁胺醇对黑腹果蝇基因组稳定性、细胞凋亡和蛋白表达的影响,来探究它对生物体毒性作用的机理。【方法】将野生型黑腹果蝇3龄幼虫用含沙丁胺醇（120 μg/mL）的饲料饲喂2 h后,对幼虫翅成虫盘进行H2Av抗体免疫染色。选取rpr-lacZ转基因黑腹果蝇1龄幼虫用含沙丁胺醇（40 和120 μg/mL）的饲料饲喂,对幼虫翅成虫盘进行lacZ活性测定。提取沙丁胺醇处理后的野生型3龄幼虫总蛋白,采用SDS-PAGE比较对照组和实验组蛋白表达的差异,并通过质谱分析差异蛋白的氨基酸序列。【结果】沙丁胺醇处理后,经免疫荧光染色发现野生型黑腹果蝇幼虫翅成虫盘部分细胞中组蛋白H2Av的量有显著增加。随着沙丁胺醇浓度的增加,转rpr-lacZ报告基因黑腹果蝇成虫盘细胞lacZ活性增强。采用SDS-PAGE和质谱分析表明,沙丁胺醇处理后黑腹果蝇肌动蛋白（Actin-87E）和异柠檬酸脱氢酶表达量上升。【结论】沙丁胺醇处理会引起黑腹果蝇细胞核DNA损伤,对基因稳定性有显著影响,并且会促进细胞凋亡和蛋白表达的改变。沙丁胺醇可能通过促进肌肉收缩和加速生物体能量代谢这两方面来减少脂肪积蓄。     

Differential expression of lysosomal associated membrane protein (LAMP-1) during mammalian spermiogenesis

The mammalian acrosome is a secretory vesicle of mature sperms that plays an important role in fertilization. Recent evidence had pointed out that some components found at endosomes in somatic cells are associated with the developing acrosome during the early steps of spermiogenesis. Moreover, the mammalian acrosome contains many enzymes found within lysosomes in somatic cells. In this work, we studied the dynamics of some components of the endosome/lysosome system, as a way to understand the complex membrane trafficking circuit established during spermatogenesis. We show that the cation independent-mannose-6-phosphate receptor (CI-MPR) is transiently expressed in the cytoplasm of mid-stage spermatids (steps 5-11). On the other hand, gamma-adaptin, an adaptor molecule of a complex involved in trafficking from the Golgi to lysosomes, was expressed in cytoplasmic vesicles only in pachytene and Cap-phase spermatids (steps 1-5). Our major finding is that the lysosomal protein LAMP-1 is differentially expressed during spermiogenesis. LAMP-1 appears late in spermatogenesis (Acrosome-phase) contrasting with LAMP-2, which is present throughout the complete process. Both proteins appear to be associated with cytoplasmic vesicles and not with the developing acrosome. None of the studied proteins is present in epididymal spermatozoa. Our results suggest that the CI-MPR could be involved in membrane trafficking and/or acrosomal shaping during spermiogenesis.     

The novel estrogen-induced gene EIG121 regulates autophagy and promotes cell survival under stress

We previously identified a novel estrogen-induced gene, EIG121, as being differentially regulated in endometrioid and nonendometrioid endometrial carcinoma. The function of EIG121 was unknown. Using a tetracycline-inducible system, we found that overexpression of EIG121, but not of LacZ, caused a profound suppression of cell growth. Subcellular fractionation and immunofluroscent labeling indicated that EIG121 was a transmembrane protein localized in the plasma membrane-late endosome–lysosome compartments. Deletion of the putative transmembrane domain abolished the membrane association. In cells overexpressing EIG121, cytoplasmic vacuoles accumulated after EIG121 induction, and the autophagosome marker LC3 translocated into punctuate, dot-like structures. Electron microscopy revealed that in cells overexpressing EIG121, autophagosomes were markedly increased. Overexpression of EIG121 also increased the cells containing acidic vesicles and induced lysosomal degradation of long-lived proteins. In MCF-7 cells, both EIG121 and LC3 were rapidly degraded by a lysosomal mechanism after starvation. Knockdown of EIG121 blocked starvation-induced LC3 degradation. By itself, knockdown of EIG121 did not affect cell survival. When combined with starvation or cytotoxic agents, EIG121 knockdown greatly increased apoptosis. Our results suggest that EIG121 is associated with the endosome–lysosome compartments and may have an important role in autophagy. Under unfavorable conditions such as starvation and exposure to cytotoxic agents, EIG121 may protect cells from cell death by upregulating the autophagy pathway.     

Spatial and temporal relationships between cell death and tissue overgrowth in imaginal wing disks of the Drosophila mutant I(3)C43hs1

The temperature-sensitive mutant l(3)c43^hs1 is lethal at the restrictive temperature late in the last larval instar and has wing disks that show excessive growth when larvae are reared at 25°C. Such mutant disks give rise to defective wings showing duplications and deficiencies. Abnormal folding patterns are localized to the region between the wing pouch and the area where adepithelial cells are found; the disks retain an epithelial morphology. Apoptotic cell death is distributed throughout the wing disks without any obvious concentration of dead cells in a specific area. Cell death is seen as early as 12 hr after a shift to the restrictive temperature. Temperature shift experiments also show that cell death precedes the onset of overgrowth, but since the spatial distribution of death is not localized to the regions of abnormal folds, it is unlikely that cell death and overgrowth are causally related.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Drosophila syndecan regulates tracheal cell migration by stabilizing Robo levels

Here we identify a new role for Syndecan (Sdc), the only transmembrane heparan sulphate proteoglycan in Drosophila, in tracheal development. Sdc is required cell autonomously for efficient directed migration and fusion of dorsal branch cells, but not for dorsal branch formation per se. The cytoplasmic domain of Sdc is dispensable, indicating that Sdc does not transduce a signal by itself. Although the branch-specific phenotype of sdc mutants resembles those seen in the absence of Slit/Robo2 signalling, genetic interaction experiments indicate that Sdc also helps to suppress Slit/Robo2 signalling. We conclude that Sdc cell autonomously regulates Slit/Robo2 signalling in tracheal cells to guarantee ordered directional migration and branch fusion.     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.     

产气荚膜梭菌促进黑腹果蝇的生长和发育

【目的】黑腹果蝇Drosophila melanogaster肠道中栖生着众多微生物,通过分离和研究其内共生菌,以研究肠道菌群的多态性和作用。【方法】利用Hungate滚管技术从黑腹果蝇成虫肠道分离厌氧细菌;通过记录果蝇的发育历期和生长速率,检测该细菌对果蝇发育和生长的影响。【结果】首次从黑腹果蝇肠道内分离到一株产气荚膜梭菌Clostridium perfringens。该菌能够有效地定植到果蝇肠道内,是果蝇肠道共生菌。产气荚膜梭菌显著地缩短无菌果蝇的发育历期,将无菌果蝇成蛹天数由20 d缩短到8.1 d,羽化天数由30 d缩短到12.7 d。该菌还可以提高果蝇生长速率。【结论】本研究揭示了产气荚膜梭菌是果蝇的内共生菌,可以通过提高生长速率而有效地促进果蝇的生长和发育。     

The Drosophila Mst ortholog,hippo, restricts growth and cell proliferation and promotes apoptosis

Establishing and maintaining homeostasis is critical to the well-being of an organism and is determined by the balance of cell proliferation and death. Two genes that function together to regulate growth, proliferation, and apoptosis in Drosophila are warts (wts), encoding a serine/threonine kinase, and salvador (sav), encoding a WW domain containing Wts-interacting protein. However, the mechanisms by which sav and wts regulate growth and apoptosis are not well understood. Here, we describe mutations in hippo (hpo), which encodes a protein kinase most related to mammalian Mst1 and Mst2. Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator cyclin E and apoptosis inhibitor DIAP1. Hpo, Sav, and Wts interact physically and functionally, and regulate DIAP1 levels, likely by Hpo-mediated phosphorylation and subsequent degradation. Thus, Hpo links Sav and Wts to a key regulator of apoptosis.     

The acentriolar state of the Drosophila cell lines 1182

A Drosophila melanogaster cell line devoid of centrioles has been recently described. In order to achieve an easier characterization of these acentriolar cells, we used the monoclonal antibody Bx 63 of M. Frasch which recognizes the Drosophila centrosome. Although centrosomes are detected at every mitotic pole in Drosophila cells with centrioles, no such structure has been observed in 1182-4 acentriolar cells. The antigenic material is, however, present in these cells. Moreover, we noticed a certain proportion of acentriolar cells in 4 other 1182 lines. The lack of centrioles previously found only in the 1182-4 cells seems therefore not accidental and should be linked to their particular origin.     

Population Dynamics of Herpetomonas ampelophilae, with a Note on the Systematics of Herpetomonas from Drosophila spp.

SYNOPSIS Previous surveys of feral Drosophila melanogaster in Clarke County, Georgia, indicated 10 to 30% of the flies were parasitized with Herpetomonas ampelophilae. In the present study, herpetomoniasis in D. melanogaster reared in population cages rose from 0.01% to 90% in 14 days. The infection fluctuated between 5% and 100% during the 250 days in which the ageledeme was maintained.
Herpetomonas were found endotrophically, peritrophically, and in the Malpighian tubules. In population cages, although there were sporadic rises in percentages, peritrophic and Malpighian tube infections usually remained low. Endotrophic infections often reached 100%, with individual flies being heavily parasitized. Although Chatton felt that several species of Herpetomonas inhabit a single species of Drosophila , we suggest that all the Herpetomonas in D. melanogaster be referred to as H. ampelophilae.     

冻干胡蜂蜂蛹对果蝇寿命和繁殖性能的影响

本实验通过研究胡蜂蜂蛹冻干品对果蝇寿命和繁殖力的影响,证明胡蜂蜂蛹具有延缓衰老和增强繁殖性能的作用。采用乙醚麻醉法,收集8h（小时）内羽化的成虫,选择个体大小相近的雌雄果蝇,在不同浓度的培养基中饲养,每两天观察统计一次果蝇死亡数,直到全部死亡。结果表明,雌雄果蝇在5．0％实验组与对照组的平均寿命比较均具有极显著差异性（P〈0．01）,雌雄果蝇在0．2％和1．0％实验组与对照组比较平均寿命差异性显著（P〈0．05）,由此可见胡蜂蜂蛹可延长果蝇平均寿命;0．2％,1．0％组的果蝇子代数显著高于对照组果蝇子代数（P〈0．05）,5．0％组果蝇子代数极显著高于对照组果蝇子代数（P〈0．01）,由此可见冻干胡蜂蜂蛹可增强果蝇的繁殖能力。