[Show abstract][Hide abstract] ABSTRACT: Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.
[Show abstract][Hide abstract] ABSTRACT: Misexpression Suppressor of Ras 4 (MESR4), a plant homeodomain (PHD) finger protein with nine zinc-finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. Here we show that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti-MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E-F region, which is known to contain the histone gene cluster. We identified P-element insertions in the MESR4 locus, which were homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene was greatly reduced, and the level of EGFR signal-dependent double phosphorylated ERK (dp-ERK) remained low. However, in the context of wing vein formation, genetic interaction experiments suggested that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggest that MESR4 is a novel chromatin-binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Drosophila provides a powerful genetic model to analyze lipid metabolism. Drosophila has an adipose-like organ called the fat body, which plays a crucial role in energy homeostasis. Here, we conducted a fat body-specific misexpression screen to identify genes involved in lipid metabolism. We found that over-expression of a nuclear protein with nine C2H2 type zinc-finger motifs and a PHD-finger, Misexpression suppressor of ras 4 (MESR4), reduces lipid accumulation in the fat body, whereas MESR4 knockdown increases it. We further show that MESR4 up-regulates the expression of major lipases, which may account for the reduction in lipid storage in the fat body and the release of free fatty acids (FFAs) in the body. These results suggest that MESR4 acts as an important upstream regulator of energy homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.
Full-text · Article · Jan 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: A peptide aptamer that changes fluorescence upon binding to verotoxin was selected in vitro using ribosome display with a tRNA carrying an environment-sensitive fluorescent probe. The aptamer specifically bound to verotoxin with a dissociation constant (K
d) of 3.94 ± 1.6 µM, and the fluorescence decreased by 78 % as the verotoxin concentration was increased. The selected peptide can be used for detection of verotoxin.
No preview · Article · Nov 2014 · Biotechnology Letters
[Show abstract][Hide abstract] ABSTRACT: We established a method for bioluminescence imaging (BLI) to track real-time gene expression in live Drosophila embryos. We constructed a transgenesis vector containing multiple cloning sites and enhanced green-emitting luciferase (ELuc; Emerald Luc), a brighter and pH-insensitive luciferase for promoter analysis. To evaluate the utility of BLI using an ELuc reporter together with an optimized microscope system, we visualized the expression pattern of armadillo (arm), a member of the Wnt pathway in Drosophila, throughout embryogenesis. We generated transgenic flies carrying the arm:: ELuc fusion gene, and successfully performed BLI continuously for 22 h in the same embryos. Our study showed, for the first time, that arm::Eluc expression was dramatically increased in the anterior midgut rudiment, myoblasts of the dorsal/lateral musculature, and the posterior spiracle after stage 13, and the cephalic region at stage 17. To further demonstrate the application of our BLI system, we revealed that arm transcriptional activity in embryos was modulated inversely by treatment with ionomycin or 6-bromoindirubin-3-oxime (BIO), an inhibitor and activator of Wnt/β-catenin signaling, respectively. Therefore, our microscopic BLI system is useful for monitoring gene expression in live Drosophila embryos, and for investigating regulatory mechanisms by using chemicals and mutations that might affect expression.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-014-8000-8) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Several lines of evidence associate misregulated genetic expression with risk factors for diabetes, Alzheimer's, and other diseases that sporadically develop in healthy adults with no background of hereditary disorders. Thus, we are interested in genes that may be expressed normally through parts of an individual's life, but can cause physiological defects and disease when misexpressed in adulthood.
We attempted to identify these genes in a model organism by arbitrarily misexpressing specific genes in adult Drosophila melanogaster, using 14,133 Gene Search lines. We identified 39 "reduced-lifespan genes" that, when misexpressed in adulthood, shortened the flies' lifespan to less than 30% of that of control flies. About half of these genes have human orthologs that are known to be involved in human diseases. For about one-fourth of the reduced-lifespan genes, suppressing apoptosis restored the lifespan shortened by their misexpression. We determined the organs responsible for reduced lifespan when these genes were misexpressed specifically in adulthood, and found that while some genes induced reduced lifespan only when misexpressed in specific adult organs, others could induce reduced lifespan when misexpressed in various organs. This finding suggests that tissue-specific dysfunction may be involved in reduced lifespan related to gene misexpression. Gene ontology analysis showed that reduced-lifespan genes are biased toward genes related to development.
We identified 39 genes that, when misexpressed in adulthood, shortened the lifespan of adult flies. Suppressing apoptosis rescued this shortened lifespan for only a subset of the reduced-lifespan genes. The adult tissues in which gene misexpression caused early death differed among the reduced-lifespan genes. These results suggest that the cause of reduced lifespan upon misexpression differed among the genes.
[Show abstract][Hide abstract] ABSTRACT: Insulin/insulin-like growth factor (IGF) plays an important role as a systemic regulator of metabolism in multicellular organisms. Hyperinsulinemia, a high level of blood insulin, is often associated with impaired physiological conditions such as hypoglycemia, insulin resistance, and diabetes. However, due to the complex pathophysiology of hyperinsulinemia, the causative role of excess insulin/IGF signaling has remained elusive. To investigate the biological effects of a high level of insulin in metabolic homeostasis and physiology, we generated flies overexpressing Drosophila insulin-like peptide 2 (Dilp2), which has the highest potential of promoting tissue growth among the Ilp genes in Drosophila. In this model, a UAS-Dilp2 transgene was overexpressed under control of sd-Gal4 that drives expression predominantly in developing imaginal wing discs. Overexpression of Dilp2 caused semi-lethality, which was partially suppressed by mutations in the insulin receptor (InR) or Akt1, suggesting that dilp2-induced semi-lethality is mediated by the PI3K/Akt1 signaling. We found that dilp2-overexpressing flies exhibited intensive autophagy in fat body cells. Interestingly, the dilp2-induced autophagy as well as the semi-lethality was partially rescued by increasing the protein content relative to glucose in the media. Our results suggest that excess insulin/IGF signaling impairs the physiology of animals, which can be ameliorated by controlling the nutritional balance between proteins and carbohydrates, at least in flies.
Full-text · Article · Apr 2014 · Frontiers in Physiology
[Show abstract][Hide abstract] ABSTRACT: Titanium was treated with 3,4-dihydroxy-L-phenylalanine (DOPA) or dopamine to immobilize bone morphogenetic protein-2 (BMP2), a biomolecule. DOPA and dopamine solutions turned into suspensions, and precipitates were produced at high pH. Both treatments produced a brown surface on titanium that was thicker at high pH than low pH. Dopamine produced a thicker layer than DOPA. The hydrophobicity of the surfaces increased after treatment with dopamine independent of pH. Furthermore, there were more amino groups in the layers formed at pH 8.5 than pH 4.5 in both treatments. Dopamine treatment produced more amino groups in the layer than DOPA. BMP2 was immobilized on the treated surfaces via a coupling reaction using carbodiimide. More BMP2 was immobilized on surfaces treated at pH 8.5 than pH 4.5 in both treatments. The immobilized BMP induced specific signal transduction and alkali phosphatase, a differentiation marker. Thus, the present study demonstrates that titanium treated with DOPA or dopamine can become bioactive via the surface immobilization of BMP2, which induces specific signal transduction.
[Show abstract][Hide abstract] ABSTRACT: A peptide that binds and emits fluorescence in response to conformational change in a target protein was developed by in vitro selection using tRNA carrying a fluorogenic amino acid. This technology could prove to be useful for the development of separation-free immunoassays and bio-imaging analyses.
Full-text · Article · Dec 2013 · Chemical Communications
[Show abstract][Hide abstract] ABSTRACT: Ascorbic acid (AA) functions as an electron donor and scavenges reactive oxygen species such as superoxide, singlet oxygen, and hydroxyl radicals in vitro. However, little is known about the effect of an AA deficiency on protein and lipid oxidation levels in the liver. Therefore, we measured the levels of protein carbonyl and thiobarbituric acid reactive substances (TBARS) in livers from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are deficient in AA, because they lack the SMP30/GNL gene, which is essential for the biosynthesis of AA in vivo. To track the effect of an AA deficiency, at 30 d of age, mice were divided into the following four groups: AA (-) SMP30/GNL KO, AA (+) SMP30/GNL KO, AA (-) wild type (WT), and AA (+) WT. The AA (+) groups were given water containing 1.5 g/L AA, whereas the AA (-) groups received water without AA for 57 d. All mice were fed an AA-free diet. Subsequently, protein carbonyl levels in livers from AA (-) SMP30/GNL KO mice were significantly higher than those from the other three groups; however, TBARS levels were not significantly different among the four groups. Therefore, AA must act as an anti-oxidant for proteins but might not directly protect lipid oxidation in the liver.
No preview · Article · Dec 2013 · Journal of Nutritional Science and Vitaminology
[Show abstract][Hide abstract] ABSTRACT: An epidermal growth factor (EGF) derivative with affinity for apatite and titanium surfaces was designed using a peptide moiety derived from salivary statherin, a protein that adheres to hydroxyapatite. Since the active sequence has two phosphoserine residues, the EGF derivative was prepared by organic synthesis, and a 54 residue peptide was successfully prepared using this method. Circular dichroism spectra indicated that the conformation of EGF was not significantly altered by the addition of the affinity peptide sequence and the mitogenic activity was only slightly reduced when compared with the wild-type protein. However, the binding affinity of the modified EGF to hydroxyapatite and titanium was significantly higher than the unmodified EGF. The phosphate groups in the affinity sequence contributed to the affinity of modified EGF to both apatite and titanium. The modified EGF significantly enhanced the growth of cells on hydroxyapatite and titanium. It was also demonstrated that the bound EGF enhanced the signal transduction for longer periods than unbound EGF. In conclusion, the modified EGF had significantly higher binding affinity for apatite and titanium than soluble EGF, and the bound EGF significantly enhanced cell growth by long-lasting activation of intracellular signal transduction.
[Show abstract][Hide abstract] ABSTRACT: The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The effect of an AA deficiency on catecholamine biosynthesis in adult mice in vivo is unknown. Therefore, we quantified catecholamine and the expression of catecholamine synthetic enzymes in the adrenal glands of senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice placed in an AA-deficient state.
At 30 days of age, mice were divided into the following 4 groups: AA (-) SMP30/GNL KO, AA (+) SMP30/GNL KO, AA (-) wild type (WT), and AA (+) WT. The AA (+) groups were given water containing 1.5 g/L AA, whereas the AA (-) groups received water without AA until the experiment ended. In addition, all mice were fed an AA-depleted diet. Catecholamine levels were measured by a liquid chromatographic method. Tyrosine hydroxylase, dopa decarboxylase, dopamine β-hydroxylase, and phenylethanolamine N-methyltransferase mRNA expression levels were measured with the quantitative real-time polymerase chain reaction (qPCR). Tyrosine hydroxylase and dopamine β-hydroxylase protein levels were quantified by Western blot analysis.
In the adrenals of AA (-) SMP30/GNL KO mice, noradrenaline and adrenaline levels decreased significantly compared to other three groups of mice, although there were no significant differences in dopamine β-hydroxylase or phenylethanolamine N-methyltransferase mRNA content. Moreover, there was no significant difference in their dopamine β-hydroxylase protein levels. On the other hand, AA depletion did not affect dopamine levels in adrenal glands of mice.
An AA deficiency decreases the noradrenaline and adrenaline levels in adrenal glands of mice in vivo.
No preview · Article · Mar 2013 · European Journal of Nutrition
[Show abstract][Hide abstract] ABSTRACT: Theories of lifespan evolution are a source of confusion amongst aging researchers. After a century of aging research the dispute over whether the aging process is active or passive persists and a comprehensive and universally accepted theoretical model remains elusive. Evolutionary aging theories primarily dispute whether the aging process is exclusively adapted to favor the kin or exclusively non-adapted to favor the individual. Interestingly, contradictory data and theories supporting both exclusively programmed and exclusively non-programmed theories continue to grow. However, this is a false dichotomy; natural selection favors traits resulting in efficient reproduction whether they benefit the individual or the kin. Thus, to understand the evolution of aging, first we must understand the environment-dependent balance between the advantages and disadvantages of extended lifespan in the process of spreading genes. As described by distinct theories, different niches and environmental conditions confer on extended lifespan a range of fitness values varying from highly beneficial to highly detrimental. Here, we considered the range of fitness values for extended lifespan and develop a fitness-based framework for categorizing existing theories. We show that all theories can be classified into four basic types: secondary (beneficial), maladaptive (neutral), assisted death (detrimental), and senemorphic aging (varying between beneficial to detrimental). We anticipate that this classification system will assist with understanding and interpreting aging/death by providing a way of considering theories as members of one of these classes rather than consideration of their individual details.
Full-text · Article · Mar 2013 · Frontiers in Genetics
[Show abstract][Hide abstract] ABSTRACT: Aconitase catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, and its deficiency in humans is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. Here we investigated the effect of gene knockout and knockdown of the mitochondrial aconitase Acon in Drosophila. Acon-knockout flies were homozygous lethal, indicating that Acon is essential for viability. RNA interference-generated Acon-knockdown flies exhibited a variety of phenotypes, such as reduced locomotor activity, a shortened lifespan, and increased cell death in the developing brain. Metabolomic analysis revealed that acetyl-CoA, citrate/isocitrate, and cis-aconitate were significantly increased, while most metabolites of glycolysis and the TCA cycle were reduced. Reduced ATP and increased triacylglyceride suggested that lipids were used as an energy source because of the impaired glycolysis and TCA cycle. The Acon-knockdown model should facilitate further understanding of the pathophysiology of m-aconitase deficiency in humans.
Full-text · Article · Feb 2013 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of l-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-d-glucitol and d-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of l-gulonate in an extended (or all-trans) conformation; l-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.