70 research outputs found
Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.
No full textThe Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation
The multicopper ferroxidase hephaestin enhances intestinal iron absorption in mice
No full textHephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues
The D519G Polymorphism of Glyceronephosphate O-Acyltransferase Is a Risk Factor for Familial Porphyria Cutanea Tarda.
No full textBoth familial and sporadic porphyria cutanea tarda (PCT) are iron dependent diseases. Symptoms of PCT resolve when iron stores are depleted by phlebotomy, and a sequence variant of HFE (C282Y, c.843G>A, rs1800562) that enhances iron aborption by reducing hepcidin expression is a risk factor for PCT. Recently, a polymorphic variant (D519G, c.1556A>G, rs11558492) of glyceronephosphate O-acyltransferase (GNPAT) was shown to be enriched in male patients with type I hereditary hemochromatosis (HFE C282Y homozygotes) who presented with a high iron phenotype, suggesting that GNPAT D519G, like HFE C282Y, is a modifier of iron homeostasis that favors iron absorption. To challenge this hypothesis, we investigated the frequency of GNPAT D519G in patients with both familial and sporadic PCT. Patients were screened for GNPAT D519G and allelic variants of HFE (both C282Y and H63D). Nucleotide sequencing of uroporphyrinogen decarboxylase (URO-D) identified mutant alleles. Patients with low erythrocyte URO-D activity or a damaging URO-D variant were classified as familial PCT (fPCT) and those with wild-type URO-D were classified as sporadic PCT (sPCT). GNPAT D519G was significantly enriched in the fPCT patient population (p = 0.0014) but not in the sPCT population (p = 0.4477). Both HFE C282Y and H63D (c.187C>G, rs1799945) were enriched in both PCT patient populations (p<0.0001) but showed no greater association with fPCT than with sPCT.GNPAT D519G is a risk factor for fPCT, but not for sPCT
Double photoionization of alkaline earth atoms and photoelectron spectroscopy of reactive intermediates
No full textDouble photoionization (DPI) of the outermost s"2 electrons of Ca and Sr atoms in the vapour phase has been studied by measurements of the angular correlation between the directions of emission of the two electrons. This process has been studied using monochromatic radiation produced by the second generation Daresbury synchrotron radiation source (SRS). The extremely low cross sections of the DPI processes were enhanced by using incident radiation at wavelengths corresponding to autoionizing resonances of the neutral species. Measurements of DPI for Ca atoms have also been collected at a non resonant photon energy using the third generation Elettra SRS in Trieste. Discrepancies between the present experimental results and the limited existing theory are found for the non resonant process. Two-step double photoionization via intermediate excited ionic states has been studied in both Ca and Sr atoms. Measurements of angular distributions and angular correlations between the directions of emission of inner shell photoelectrons and the associated Auger electrons allowed detailed characterizations of the photoionization processes. Valence photoelectron spectroscopy of the short-lived reactive intermediates O_2(a"1#DELTA#_g), OH and OD has been performed using the Daresbury SRS. Autoionizing resonances have been assigned to members of series of Rydberg states converging towards higher ionic limits. Measurements of angular distributions of photoelectrons allowed further insight into the photoionization processes to be obtained. Suggestions are presented for further experimental investigations. (author)SIGLEAvailable from British Library Document Supply Centre-DSC:DXN040244 / BLDSC - British Library Document Supply CentreGBUnited Kingdo
Functional characterization of novel thioredoxin reductase and thioredoxin peroxidase in Drosophila
No full textMolecular oxygen is key to aerobic life, but is also converted into cytotoxic byproducts referred to as reactive oxygen species (ROS). In mammals, intracellular defense against ROS includes superoxide dismutase (Sod), catalase (Cat) and thiol-dependent redox systems, in which glutathione reductase (GR), thioredoxin reductase (TrxR) and the corresponding peroxidases are key enzymes. Mammalian TrxRs and glutathione peroxidases (GPxs) are selenium-containing enzymes. The fruit fly 'Drosophila' possesses most ROS-detoxifying enzymes reported for mammals with the possible exception of GR and GPx activities. This thesis presents an investigation of key 'Drosophila' antioxidant genes encoding TrxR-1 and thioredoxin peroxidase (TPx). I show that a single ' Drosophila' gene, termed 'Trxr-1', specifies cytoplasmic and mitochondrial non-selenocysteine-containing TrxRs that arise by alternative splicing. I generated transcript-specific mutants and used 'in vivo ' approaches to explore the biological activities of the two splicing variants by introducing the respective individual transgenes into ' Trxr-1' mutant flies. The results show that although the two respective TrxRs have similar biochemical properties, they cannot substitute for each other, 'in vivo. Trxr-1' null mutations result in larval death, whereas mutations causing reduced TrxR-1 activity reduce pupal eclosion and cause a severe shortening of the adult lifespan. I also provide genetic evidence for a functional interaction between TrxR-1, Sod1 and Cat, suggesting that the overall burden of ROS metabolism in 'Drosophila' is shared by the two defense systems. Finally, I report the 'in silico' identification of two non-selenium containing GPx-like genes in the ' Drosophila' genome and present an initial biochemical characterization of one of the two gene products. The results show that one of the GPx-like genes encodes a TPx rather than a GPx. Transgene-dependent overexpression of the TPx gene increases the resistance of individuals to experimentally-induced oxidative stress, but does not compensate for the loss of Cat, an enzyme which, like TPx, functions to eliminate intracellular hydrogen peroxide. Furthermore, transgene-derived overexpression of TPx in mutant flies lacking Sod1, an antioxidant enzyme which protects cells from superoxide radical toxicity, is detrimental. This contrasts to transgene-derived overexpression of Cat which can partially rescue the 'sod1' mutant. These observations indicate that TPx1 and Cat function in metabolically distinct pathways
Female and male gamete mitochondria are distinct and complementary in transcription, structure, and genome function
No full textRespiratory electron transport in mitochondria is coupled to ATP synthesis while generating mutagenic oxygen free radicals. Mitochondrial DNA mutation then accumulates with age, and may set a limit to the lifespan of individual, multicellular organisms. Why is this mutation not inherited? Here we demonstrate that female gametes—oocytes—have unusually small and simple mitochondria that are suppressed for DNA transcription, electron transport, and free radical production. By contrast, male gametes—sperm—and somatic cells of both sexes transcribe mitochondrial genes for respiratory electron carriers and produce oxygen free radicals. This germ-line division between mitochondria of sperm and egg is observed in both the vinegar fruitfly and the zebrafish—species spanning a major evolutionary divide within the animal kingdom. We interpret these findings as an evidence that oocyte mitochondria serve primarily as genetic templates, giving rise, irreversibly and in each new generation, to the familiar energy-transducing mitochondria of somatic cells and male gametes. Suppressed mitochondrial metabolism in the female germline may therefore constitute a mechanism for increasing the fidelity of mitochondrial DNA inheritance
We also CanFly! The 2nd MexFly drosophila research conference
No full textThe 2nd Mexican Drosophila Research Conference (MexFly) took place on June 30th and July 1st, 2016 in Mexico City, at the Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav). Principal investigators, postdocs, students, and technicians from Drosophila labs across Mexico attended. The guest speaker was Chris Rushlow from New York University, who presented work on Zelda, a key transcriptional activator of the early zygotic genome. Here we provide a brief report of the meeting, which sketches the present landscape of Drosophila research in Mexico. We also provide a brief historical note on one of the pioneers of the field in this country, Victor Salceda, personally trained by Theodosius Dobzhansky. Salceda presented at the meeting an update of his collaborative project with Dobzhansky on the distribution of Drosophila pseudoobscura chromosomal inversions, initiated over forty years ago
Biophysical and genetic analysis of iron partitioning and ferritin function in Drosophila melanogaster
No full textMetals have vital functions as prosthetic groups in enzymes, but in labile form they can propagate oxidative stress. The primary function of ferritin is to store bioavailable iron in the form of ferrihydrite. In animals, ferritin is also used to traffic and recycle iron, and to modulate intestinal iron absorption. However, the effect of ferritin accumulation on cellular iron bioavailability remains poorly understood. Moreover, putative in vivo interactions of ferritin with other metal ions have been proposed, but their physiological relevance remains unclear. Here, heterozygous mutant and overexpression ferritin strains of Drosophila melanogaster were subjected to dietary iron manipulations to study the dynamics of iron partition between ferritin and other proteins. Quantitative magnetic analysis of whole fly samples indicated that iron loading of the ferritin core varied in the different genotypes. Total paramagnetic iron content, a likely correlate of bioavailable iron, was reduced in flies overexpressing ferritin when compared with control white flies. Further, three-dimensional maps of the ferritin protein shell and iron core were obtained from single particle transmission electron microscopy imaging and confirmed the similarity between Drosophila and Trichoplusia ferritin structures. Purified Drosophila ferritin also contained small amounts of zinc and manganese. Flies that overexpressed ferritin accumulated in their bodies half the amount of manganese compared to their respective controls. Our results indicate that ferritin may be involved in the homeostasis of other divalent metals, besides iron, and that overexpression of ferritin, sometimes employed to rescue neurodegenerative models of disease, serves to limit divalent metal bio-availability in cells. © 2013 The Royal Society of Chemistry.This work was supported by the CONACYT project 179835 to F.M.Peer Reviewe
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