Certain withanolides from Iochroma gesnerioides antagonize ecdysteroid action in a Drosophila melanogaster cell line

Sixteen withanolides isolated from Iochroma gesnerioides (Kunth) Miers (Solanaceae) have been assessed for their activities as ecdysteroid agonists and antagonists. None of the compounds showed any agonistic activity, but several showed significant antagonistic activity. With a 20-hydroxyecdysone concentration of 5 × 10^–8 M, the ED₅₀ values for 2,3-dihydro-3-methoxywithaferin A, 2,3-dihydro-3-methoxywithacnistine, 2,3-dihydro-3-methoxyiochromolide and 2,3-dihydro-3-hydroxywithacnistine are 3.5 × 10^-5 M, 1 × 10^–5 M, 5 × 10^–6 M and 2.5 × 10^–6 M, respectively.     

Ecdysteroid binding activity in embryos of Drosophila melanogaster

Ecdysteroid binding proteins have been found in nuclei of Drosophila melanogaster embryos. Comparison of results derived from Scatchard analysis, analogue binding competition, and sucrose gradient centrifugation has revealed no significant differences between the properties of the putative embryonic receptor and those of the receptor found in imaginal disks or Kc cells.     

Calmodulin-dependent and -independent apoptosis in cells of a Drosophila neuronal cell line

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca²⁺ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca²⁺ ions, although the origin of Ca²⁺ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca²⁺ ions whereas those by the other three chemicals utilized intracellular Ca²⁺ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).     

Characterization of the acid phosphatase and arylsulphatase activities in a tumorous blood cell line of Drosophila melanogaster

The lysosomal acid phosphatase (AP) and arylsulphatase (AS) activities in a tumorous blood cell line of Drosophila melanogaster have been characterized. The AP activity measured with para-nitrophenylphosphate as substrate had a pH optimum of pH 4. The K_m for this substrate was 0.1 mM. The activity was inhibited by l-tartrate, fluoride ions, DFP and anions such as phosphate and molybdate, but not by sulphydryl reagents nor by alkaline phosphatase inhibitors. The activity was adsorbed onto DEAE-Sephadex at pH 8 and eluted as one peak, when a NaCl gradient was applied to the column. It was not possible to separate the activity into several components by disc-gel electrophoresis or isoelectric focusing (pI of AP: 6.2). The mobility during disc-gel electrophoresis was not altered by prior incubation with bacterial neuraminidase. The AS was shown to hydrolyse the artificial substrates para-nitrophenylsulphate, para-nitrocatecholsulphate and acetylphenyl sulphate with pH optima of 6.8–7.2. The activity was detrimentally affected by various inorganic anions and could be totally inhibited by low concentrations of AgNO₃ (10⁻⁴ M). The AS bound to the anion exchanger more strongly than the AP, but in common with the AP, only one peak of activity could be detected by this and other separation techniques. The pI of the AS was 4.6. The AP and AS have similar apparent molecular weights (60–65,000) and sedimentation coefficients (6.6 and 5.8S, respectively).     

Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture

Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabeling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here, we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of long-terminal repeat retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data (called ngs_te_mapper2), which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.     

The effects of nutrition and methoprene treatment on ovarian ecdysteroid synthesis in Drosophila melanogaster

Radioimmunoassay of in vitro culture medium from ovaries of Drosophila melanogaster indicates that detectable ovarian ecdysteroid synthesis begins between 6 and 12 h after eclosion and reaches a peak between 24 and 30 h, when animals are reared at 25°C, 12 h photophase. Analysis of 24 and 72 h medium by a combination of high-performance liquid chromatography and radioimmunoassay demonstrates three ecdysteroid regions, two comigrating with known standards of ecdysone and 20-hydroxyecdysone and a third highly polar region containing one or more unidentified radioimmunoassay-active ecdysteroids. In 72 h medium the polar region comprises the majority of radioimmunoassay-active material while in 24 h medium the majority is in the ecdysone region. Provision of a nutritionally deficient diet to females at adult eclosion prevents the normal increase in vitellogenic-stage follicles and ovarian ecdysteroid synthesis. Methoprene treatment of such females stimulates a transient burst of ovarian ecdysteroid synthesis and the production of near normal numbers of vitellogenic oöcytes by 24 h, although by 48 h the number of vitellogenic oöcytes is less than normal.     

Evolutionary change in parasitoid resistance under crowded conditions in Drosophila melanogaster

Patterns of investment of limiting resources in such processes as competing for food and defense against natural enemies are shaped by trade-offs and constraints. In Drosophila melanogaster artificial selection for increased resistance to parasitoids results in a correlated decrease in larval competitive ability. Here we ask whether selection for competitive ability leads to a correlated reduction in parasitoid resistance. Replicated lines of D. melanogaster were maintained under crowded or uncrowded conditions for eight generations. As expected, the crowded lines evolved higher competitive ability (when tested against a common strain of fly). But instead of parasitoid resistance decreasing, we found a significant increase, and that this was associated with elevated densities of haemocytes in second-instar larvae. To understand these results we measured a variety of life-history traits in the two sets of lines. We find evidence that directly and indirectly selected changes in competitive ability are due to different mechanisms. We also ask why crowded conditions should select for increased resistance to parasitism, and conclude that it is unlikely to be due to correlated selection for resistance to other natural enemies, but might be due to correlated selection for better wound responses.     

Correlation of phenotypes of the apterous mutation in Drosophila melanogaster

The apterous (ap) mutant in Drosophila melanogaster exhibits phenotypes of wing deficiency, precocious adult death, and nonvitellogenic oocyte development. The latter phenotype previously has been shown to result from juvenile hormone (JH) deficiency in the adult stage. To explore the relationship between the hormone deficiency and the other phenotypes, the expression of each phenotype was measured in five alleles of ap (including a new, chemically-induced allele, ap^77f) as wing length, survival five days after eclosion, and initiation and progress of vitellogenic oocyte development. No correlation could be found between severity of wing phenotype and that of precocious adult death or nonvitellogenesis. However, the latter phenotypes were correlated in both ap homozygotes and allelic heterozygotes, since adults that survive have wild-type vitellogenesis, and those fated for precocious death fail to develop vitellogenic oocytes. These results indicate that no relationship exists between wing and JH deficiencies, but that precocious adult death is related to hormone deficiency — probably through pleiotropy, rather than through causality.     

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.     

Ecdysteroid control of metamorphosis in the differentiating adult leg structures of Drosophila melanogaster

During insect metamorphosis, the steroid hormone 20-hydroxyecdysone (20E) is responsible for coordinating the differentiation of adult structures. Several structures of the Drosophila melanogaster adult leg, the six distalmost joints, the bristles, and the pretarsal claws, were examined to investigate how 20E controls their development in vitro. Joints, bristles, and claws were dependent on 20E for differentiation between 20-22 and 24-26 h after puparium formation (APF). After 26-28 h APF, differentiation became hormone independent. Tissue-specific markers in 20E-free cultures showed that the bristle and joint cells had not undergone any further morphogenetic progression. In contrast, the pretarsi underwent partial differentiation. The concentration of 20E required for differentiation was structure specific; tarsal joints required higher concentrations of 20E (greater than 400 ng 20 E/ml) than pretarsal claws, bristles, and other joints (greater than 40 ng 20E/ml). The 20E precursor ecdysone (E) was also able to induce differentiation at concentrations over 700 ng E/ml, but did not show any synergistic interactions with 20E. Lastly, leg structures had a finite ability to respond to 20E; tarsal joints lost competence to respond after 32-34 h APF, while the remaining structures became incompetent after 44-46 h APF.     

Dynamics of the male germline stem cell population during aging of Drosophila melanogaster

Drosophila melanogaster has emerged as an important model system for the study of both stem cell biology and aging. Much is known about how molecular signals from the somatic niche regulate adult stem cells in the germline, and a variety of environmental factors as well as single point mutations have been shown to affect lifespan. Relatively little is known, however, about how aging affects specific populations of cells, particularly adult stem cells that may be susceptible to aging-related damage. Here we show that male germline stem cells (GSCs) are lost from the stem cell niche during aging, but are efficiently replaced to maintain overall stem cell number. We also find that the division rate of GSCs slows significantly during aging, and that this slowing correlates with a reduction in the number of somatic hub cells that contribute to the stem cell niche. Interestingly, slowing of stem cell division rate was not observed in long-lived methuselah mutant flies. We finally investigated whether two mechanisms that are thought to be used in other adult stem cell types to minimize the effects of aging were operative in this system. First, in many adult tissues stem cells exhibit markedly fewer cell cycles relative to transit-amplifying cells, presumably protecting the stem cell pool from replication-associated damage. Second, at any given time not all stem cells actively cycle, leading to 'clonal succession' from the reserve pool of initially quiescent stem cells. We find that neither of these mechanisms is used in Drosophila male GSCs.     

黑腹果蝇细胞谱系分析方法进展

对动物体内单个细胞的谱系进行分析有助于追踪其在发育过程中的作用,但是体内各种组织都是由很多形态、结构、功能各不相同的细胞构成的复杂系统,这种复杂性严重阻碍了对单个细胞的研究。嵌合克隆技术(Mosaic technique)和标记技术(Labeling technique)的出现为这一研究提供了强有力的手段。文章介绍了近几年来黑腹果蝇(Drosophila melanogaster)研究中常用的7种嵌合克隆标记方法,包括FRT介导的有丝分裂重组(FRT-mediated mitotic recombination)、MARCM(Mosaic analysis with a repressible cell marker)、TSG(Twin spotgenerator)、Twin-spot MARCM、Q-MARCM(Q system-based MARCM)、Coupled MARCM和G-TRACE(Gal4technique for real-time and clonal expression)技术,详述了这些技术的原理及应用,并对不同技术进行了对比。运用这些技术研究者可以从单细胞水平进行遗传学标记和操作,特别是在神经系统等复杂系统中追踪单个细胞的发育过程。果蝇中的这些技术也将为其他模式生物追踪细胞谱系提供参考。     

Nucleotide and copy-number polymorphism at the odorant receptor genes Or22a and Or22b in Drosophila melanogaster

In Drosophila, odorant receptors are encoded by an old and moderatelysized multigene family. Or22a and Or22b are two tandemly arrangedgenes of this family that have proved to be the result of arather young duplication. Nucleotide variation in the regionspanning both duplicates was surveyed in four natural populations(two African and two non-African) of Drosophila melanogasterand also analyzed in species of the melanogaster subgroup. Theintraspecific survey revealed a particular copy-number polymorphismin some of the studied populations, with the two genes (Or22aand Or22b) present in the long variant and a single chimericgene (Or22ab) present in the short variant. Estimated nucleotidediversity was higher in the short than in the long variant,despite the ancestral character of the latter variant in D.melanogaster. The general skew toward low-frequency variantsdetected in the non-African long variant and its reduced levelof silent polymorphism relative to divergence is consistentwith the recent fixation of an advantageous mutation at, ornearby, the Or22 long variant region. The nonnegligible frequencyof the short variant and the presence of a highly divergenthaplotype in the East African sample would point to direct orindirect selection for its maintenance in the species. Therewas evidence for a generally more rapid evolution of the Or22bcopy at both synonymous and nonsynonymous sites. However, anexcess of nonsynonymous substitutions was only detected in theearly history of this copy.     

Ultrastructure of the preparative phase of cell death in the larval fat body of Drosophila melanogaster

Progressive changes in the ultrastructure of the larval fat body of Drosophila melanogaster were studied during the third instar. In addition to electron microscopy, light microscopy and morphometric stereology were employed to evaluate the tissue at five 12-hr intervals: 48, 60, 72, 84, and 96 hr after hatching from the egg. Lipid and glycogen were found stored throughout the instar, whereas protein is stored in the form of cytoplasmic granules mainly during the final 24 hr. The cells increased in cross-sectional area, and there was a concomitant increase in the relative amounts of these substances. Based on morphological characteristics there were three types of protein granules which we called dense granules (D), heterogeneous granules (H), and autophagic vacuoles. The morphology, size range, time of appearance, and changes in frequency of these granules suggested that the H type arose from D granules, and that the autophagic vacuoles were derived from D and H types. Morphological evidence indicated D granules have the unusual characteristic of forming in the intercellular space before entering the cytoplasm.     

A transitional cell carcinoma cell line

Summary A transitional cell carcinoma cell line, COLO 232, was derived from a primary urinary bladder tumor in a Caucasian male. In culture, COLO 232 retained distinct uroepithelial phenotypic traits and produced both carcinoembryonic antigen and adrenocorticotropic hormone. COLO 232 had a chromosome mode of 58 and retained the X and Y chromosomes. Ten marker chromosomes were identified. COLO 232 will be of value for biochemical and immunological studies. Presented in part at the 28th Annual Meeting of the Tissue Culture Association, June 7, 1977. This work was supported by Grant No. CA 15018 awarded by the National Cancer Institute, DHEW, and the Mary B. and L. H. Marshall Fund.     

Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins

The objective of this investigation was to determine the effect of steroid hormones on the synthesis of progesterone in a stable porcine granulosa cell line, JC-410. We also examined the effect of steroid hormones on expression of the genes encoding the steroidogenic enzymes, cytochrome P450-cholesterol side chain cleavage (P450scc) and 3β-hydroxy-5-ene steroid dehydrogenase (3β-HSD). We observed that 48 h exposure of the JC-410 cells to estradiol-17β (estradiol), androstenedione, 5-dihydrotestosterone, levonorgestrel, and 5-cholesten-3β, 25-diol (25-hydroxycholesterol) resulted in stimulation of progesterone synthesis. 25-Hydroxycholesterol augmented progesterone synthesis stimulated by estradiol, 5-dihydrotestosterone, levonorgestrel and 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP). This increase in progesterone synthesis was additive with estradiol, 5-dihydrotestosterone and levonorgestrel, and synergistic with 8-Br-cAMP. Cholera toxin, progesterone, levonorgestrel and androstenedione increased P450scc mRNA levels, whereas estradiol had no effect. Cholera toxin, progesterone and levonorgestrel increased 3β-HSD mRNA levels, but estradiol and androstenedione had no effect. The results were interpreted to mean that estrogens, androgens and progestins regulate progesterone synthesis in the JC-410 cells. The effect of androgens appears to be mediated by stimulation of P450scc gene expression while progestins stimulate both P450scc and 3β-HSD gene expression. Our results support the concept that progesterone is an autocrine regulator of its own synthesis in granulosa cells.