Mitochondrial lipid alterations during Fas- and radiation-induced apoptosis

In the present study, we investigated the dynamic alterations in mitochondrial lipids occurring during Fas- and radiation-induced cell death. Cross-linking of CD-95 on Fas-sensitive Jurkat cells produced rapid increases in two species of mitochondrial phosphatidylglycerol. By 2.5 h, phosphatidylglycerol decreases below basal levels, concomitant with an increase in mitochondrial ceramide. In addition, between 1.5 and 3.0 h after anti-Fas crosslinking, there is a continued loss of mitochondrial cardiolipin. When gamma irradiation was used to induce apoptosis, similar lipid changes occurred, although with somewhat slower kinetics. Fas-resistant Jurkat cells exhibited phosphatidylglycerol as the dominant lipid species in their mitochondria. Following Fas ligation, there is a transient decrease in phosphatidylglycerol, but cardiolipin and ceramide remained unchanged. The high basal levels of PG in Fas-resistant cells and the increase in PG levels in Fas-sensitive cells undergoing apoptosis was determined to be due to increased PGP synthase activity. Thus, critical mitochondrial lipids could potentially serve as novel targets in regulating the apoptotic process.     

The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal γ-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.     

The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.     

Modulation of ionizing radiation-induced apoptosis by bantam microRNA in Drosophila

In Drosophila, heterozygosity in the pro-apoptotic gene hid significantly reduces apoptosis that is induced by ionizing radiation (IR). Therefore, mechanisms that regulate Hid levels can potentially contribute to life-or-death decision of an irradiated cell. 3′UTR of hid mRNA contains 5 potential binding sites for bantam microRNA. Ectopic expression of ban attenuated apoptosis that results from ectopic expression of hid but the significance of this regulation under physiological conditions remained to be investigated. We report here that ban is needed to limit IR-induced apoptosis in larval imaginal discs. Using tubulin–EGFP ban sensors with ban consensus sequences in the 3′UTR, we find that EGFP decreases following IR, indicating that IR activates ban. Likewise, a tubulin–EGFP reporter with hid-3′UTR is repressed in irradiated discs and this repression requires ban consensus sites in the hid 3′UTR. ban mutant larvae show increased sensitivity to killing by IR, which is suppressed by a mutation in hid. These results can fit into a model in which IR activates ban and ban represses hid to limit IR-induced apoptosis. miRNAs have been shown previously to be induced by radiation but this is the first report that a miRNA is functionally important for radiation responses.     

The role of feedback during singing and flight in Drosophila melanogaster

ABSTRACT. During Drosophila courtship 'pulse song', muscle potentials occur at two points during the cycle of neuromuscular events which result in a sound pulse being produced. The dorsal longitudinal, second and third dorsal ventral and axillary muscles show potentials 18 ms before each sound pulse while the first dorsal ventral, basalar and sternobasalar muscles fire 3 ms after the onset of each pulse. The timing of these events remains unaltered in animals with the antennae removed, indicating that acoustic feedback is not an important factor. Courting vestigial flies, in the absence of detectable wing base movements, produce indirect muscle potentials at the appropriate song inter-pulse intervals. Thus proprioceptive feedback is also unimportant in determining the intervals between pulses. During putative 'sine song', 'pulse song' and flight in vestigial flies, however, the timing of basalar muscle potentials is abnormal. Also, if the wing is driven externally at a frequency different from that of normal flight, basalar and, to a lesser extent, first dorsal ventral muscles, are phase locked to the driving frequency. These two results suggest that the timing of those muscles which fire at the beginning of the sound pulses is set by proprioceptive feedback. A model of song production is proposed which takes into account the data from this and from previously published papers.     

The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.     

The role of ARK in stress-induced apoptosis in Drosophila cells

The molecular mechanisms of apoptosis are highly conserved throughout evolution. The homologs of genes essential for apoptosis in Caenorhabditis elegans and Drosophila melanogaster have been shown to be important for apoptosis in mammalian systems. Although a homologue for CED-4/apoptotic protease-activating factor (Apaf)-1 has been described in Drosophila, its exact function and the role of the mitochondrial pathway in its activation remain unclear. Here, we used the technique of RNA interference to dissect apoptotic signaling pathways in Drosophila cells. Inhibition of the Drosophila CED-4/Apaf-1-related killer (ARK) homologue resulted in pronounced inhibition of stress-induced apoptosis, whereas loss of ARK did not protect the cells from Reaper- or Grim-induced cell death. Reduction of DIAP1 induced rapid apoptosis in these cells, whereas the inhibition of DIAP2 expression did not but resulted in increased sensitivity to stress-induced apoptosis; apoptosis in both cases was prevented by inhibition of ARK expression. Cells in which cytochrome c expression was decreased underwent apoptosis induced by stress stimuli, Reaper or Grim. These results demonstrate the central role of ARK in stress-induced apoptosis, which appears to act independently of cytochrome c. Apoptosis induced by Reaper or Grim can proceed via a distinct pathway, independent of ARK.     

The protective role of parsley extract against vincristine mutagenicity in Drosophila melanogaster

In this study, Drosophila melanogaster males were treated with parsley plant extract and the anticancer drug vincristine (VCR) singly and in combined treatments (pre, co and post-treatments) to detect the mutagenic effects by using sex-linked recessive lethal test (SLRL) and estimation of cholinesterase enzyme (ChE) activities in order to compare the sensitivity of the two test systems. A wild type strain Oregon-R (Or-R) male flies of D. melanogaster were reared on a medium containing one concentration of each of VCR and parsley (4 ml/100 ml medium) in each single and combined treatment. Also the activity of ChE was estimated in some insects of the two generations: F1 females, F2 bar eye females (heterozygous) and F2 wild types males. The results indicate that both of parsley and vincristine did not cause significant increases of SLRL test in either the single or combined treatments. In contrast, estimation of ChE activities showed significant increase in all the broods within single and combination treatments, except female of the second generation of spermatid brood which treated with parsley and VCR at the same time. It is concluded that enzyme estimation is more sensitive than SLRL test for detection the mutagenic effect for parsley’s extract and vincristine.     

Effects of a chromosome-3 mutator gene on radiation-induced mutability in Drosophila melanogaster females

A series of X-irradiation experiments was carried out using Drosophila melanogaster females homozygous for a third chromosome mutator gene and females which had a similar genetic background except that the mutator-bearing third chromosomes were substituted by normal wild-type chromosomes. The mutator females had been previously shown by Gold and Green to manifest a higher level of radiation-induced mutability (as measured by the X-ray-induction of sex-linked recessive lethals) in their pre-meiotic germ cells compared to normal females at an exposure of 100 R. In the presence work, the sensitivity of the pre-meiotic germ cells of mutator and normal females to the X-ray induction (2000 R) of sex-linked recessive lethals was studied. In addition, experiments were conducted to examine the sensitivity of the immature (stage 7; prophase I of meiosis) oocytes of both kinds of females to the induction of dominant lethals, X-linked recessive lethals and X-chromosome losses. The result show that in pre-meiotic germ cells, the frequencies of radiation-induced recessive lethals are similar in both kinds of females. However, the proportion of these mutations that occur in clusters of size 3 and higher, is higher in mutator than in normal females. In stage-7 oocytes, the frequencies of radiation-induced dominant lethals and sex-linked recessive lethals were similar in both kinds of females. The X-loss frequencies however, were consistently higher in mutator females although statistical significance was obtained only at higher exposures (3000 and 3750 R) and not at lower ones (750-2250 R). Possible reasons for the discrepancy between the present results and those of Gold and Green with respect to pre-meiotic germ cells are discussed.     

The role of the guanine insertion enzyme in O-biosynthesis in Drosophila melanogaster.

Drosophila tRNA can be guanylated by a crude enzyme from rabbit reticulocytes. Guanylating activity is also present in crude extracts of adult Drosophila. A major product of this reaction as well as several minor ones were resolved by RPC-5 chromatography. The main substrate of both the Drosophila and rabbit reticulocyte enzymes was the non-Q-containing aspartic acid tRNA, tRNA2gammaAsp. The QU-lacking (gamma) forms of asparagine, histidine and tyrosine tRNAs were also substrates and gave rise to the minor products of the reaction. In contrast, the Q- or Q*-containing (delta) forms of these tRNAs appear not to be substrates. The evidence strongly suggests that the guanyating enzyme is involved in Q biosynthesis and would be better termed a guanine replacement or pre-Q insertion enzyme.     

The Drosophila melanogaster DNA Ligase IV gene plays a crucial role in the repair of radiation-induced DNA double-strand breaks and acts synergistically with Rad54

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.     

The role of sperm numbers in sperm competition and female remating in Drosophila melanogaster

Single mating productivities (used as estimates of the relative number of sperm transferred) are highly correlated with several parameters used to quantify sperm competition in D. melanogster. Matings that result in the transferal of large numbers of sperm are associated with longer delay of female remating than are matings that transfer fewer sperm. Males that transfer larger numbers of sperm also suffer a smaller proportional reduction in reproductive success (smaller COST) than males transferring fewer sperm. The number of sperm transferred by a female's second mate is not related to the COST to the first male. However, there is a high positive correlation between the number of sperm transferred by the second male and P₂ (the proportion of second male progeny following female remating). Thus, large sperm numbers apparently increase the reproductive success of males whether they mate with virgin or non-virgin females. Because female receptivity mediates these events, there is no need to invoke sperm displacement to explain the reproductive outcome of female remating.The timing of female remating is evaluated in terms of a receptivity-threshold model. This model suggests that female receptivity returns when some small, relatively constant, number of sperm remain in storage.