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Sample records for laser-ad pikaajaline uuring

  1. Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. Is the tRNA(Leu(UUR)) gene an etiologic hot spot?

    PubMed Central

    Moraes, C T; Ciacci, F; Bonilla, E; Jansen, C; Hirano, M; Rao, N; Lovelace, R E; Rowland, L P; Schon, E A; DiMauro, S

    1993-01-01

    We identified two patients with pathogenic single nucleotide changes in two different mitochondrial tRNA genes: the first mutation in the tRNA(Asn) gene, and the ninth known mutation in the tRNA(Leu(UUR)) gene. The mutation in tRNA(Asn) was associated with isolated ophthalmoplegia, whereas the mutation in tRNA(Leu(UUR)) caused a neurological syndrome resembling MERRF (myoclonus epilepsy and ragged-red fibers) plus optic neuropathy, retinopathy, and diabetes. Both mutations were heteroplasmic, with higher percentages of mutant mtDNA in affected tissues, and undetectable levels in maternal relatives. Analysis of single muscle fibers indicated that morphological and biochemical alterations appeared only when the proportions of mutant mtDNA exceeded 90% of the total cellular mtDNA pool. The high incidence of mutations in the tRNA(Leu(UUR)) gene suggests that this region is an "etiologic hot spot" in mitochondrial disease. Images PMID:8254046

  2. The Mitochondrial tRNALeu(UUR) A3302G Mutation may be Associated With Insulin Resistance in Woman With Polycystic Ovary Syndrome.

    PubMed

    Ding, Yu; Zhuo, Guangchao; Zhang, Caijuan

    2016-02-01

    The aim of this study was to investigate the role of mitochondrial DNA (mtDNA) mutations in polycystic ovary syndrome (PCOS) with insulin resistance (IR), and to explore the possible maternally effects on PCOS. We performed clinical, genetic, and molecular characterization of a Han Chinese family with maternally inherited IR, and we further investigated the possible relationship between mitochondrial genetic background, copy number, and IR. Most strikingly, members from the first and second generation of this family exhibited the type 2 diabetes mellitus (T2DM) with IR, while the member in the third generation of this family manifested the PCOS. Sequence analysis of the complete mitochondrial genome showed the presence of a homoplasmic A3302G in the acceptor arm of transfer RNA(Leu(UUR)) (tRNA(Leu(UUR))) gene. This mutation disrupted the highly conserved base pairing (2T-71A) and resulted a failure in mt-tRNA metabolism. Analysis of the mitochondrial copy number showed that the patients with PCOS and IR had lower copy number than the health controls, suggesting that mitochondrial dysfunction may be involved in the pathogenesis of IR. Taken together, the A3302G mutation was a pathogenic mutation associated with IR in this Chinese family.

  3. Mouse somatic mutation orthologous to MELAS A3302G mutation in the mitochondrial tRNA(Leu(UUR)) gene confers respiration defects.

    PubMed

    Shimizu, Akinori; Enoki, Shunkei; Ishikawa, Kaori; Mito, Takayuki; Obata, Kanae; Nagashima, Ruriko; Yonekawa, Hiromichi; Nakada, Kazuto; Hayashi, Jun-Ichi

    2015-11-27

    We searched for mtDNA harboring somatic mutations in mouse B82 cells, and found an A2748G mutation orthologous to the A3302G mutation in tRNA(Leu(UUR)) gene reported in a patient with MELAS, the most prevalent mitochondrial disease. We isolated subclones of B82 cells until we obtained one subclone harboring >95% A2748G mtDNA. Cytoplasmic transfer of A2748G mtDNA resulted in cotransfer of A2748G mtDNA and respiration defects into mouse ES cells. Thus, A2748G mtDNA is responsible for respiration defects, and the ES cells harboring A2748G mtDNA may be useful for generation of transmitochondrial mice harboring A2748G mtDNA as potential disease models of MELAS.

  4. The expanding clinical phenotype of the tRNA{sup Leu(UUR)} A{r_arrow}G mutation at np 3243 of mitochondrial DNA: Diabetic embryopathy associated with mitochondrial cytopathy

    SciTech Connect

    Feigenbaum, A.; Chitayat, D.; Robinson, B.; MacGregor, D.; Myint, T.

    1996-04-24

    We describe a family which demonstrates and expands the extreme clinical variability now known to be associated with the A{r_arrow}G transition at nucleotide position 3243 of the mitochondrial DNA. The propositus presented at birth with clinical manifestations consistent with diabetic embryopathy including anal atresia, caudal dysgenesis, and multicystic dysplastic kidneys. His co-twin was normal at birth, but at 3 months of life, presented with intractable seizures later associated with developmental delay. The twins` mother developed diabetes mellitus type I at the age of 20 years and gastrointestinal problems at 22 years. Since age 19 years, the maternal aunt has had recurrent strokes, seizures, mental deterioration and deafness, later diagnosed as MELAS syndrome due to the tRNA{sup Leu(UUR)} A{r_arrow}G mutation. A maternal uncle had diabetes mellitus type I, deafness, and normal intellect, and died at 35 years after recurrent strokes. This pedigree expands the known clinical phenotype associated with tRNA{sup Leu(UUR)} A{r_arrow}G mutation and raises the possibility that, in some cases, diabetic embryopathy may be due to a mitochondrial cytopathy that affects both the mother`s pancreas (and results in diabetes mellitus and the metabolic dysfunction associated with it) and the embryonic/fetal and placental tissues which make the embryo more vulnerable to this insult. 33 refs., 1 tab.

  5. Prevalence and clinical characterization of Japanese diabetes mellitus with an A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA{sup Leu (UUR)} gene

    SciTech Connect

    Odawara, Masato; Sasaki, Kayoko; Yamashita, Kamejiro

    1995-04-01

    An A-to-G mutation at nucleotide position 3243 of the mitochondrial genome has been associated with insulin-dependent diabetes mellitus (IDDM) and with noninsulin-dependent diabetes mellitus (NIDDM) with deafness. We investigated the prevalence of this mutation in Japanese patients with IDDM, NIDDM, and impaired glucose tolerance (IGT) and in nondiabetic control individuals, and we identified it in 3 of 300 patients with NIDDM or IGT (1.0%). None of these individuals had significant sensorineural hearing loss. None of the 94 IDDM or the 115 nondiabetic control subjects was positive for this mutation. Oral glucose tolerance test revealed that a 57-yr-old male with this mutation was rather hyperinsulinemic in the fasting state. The insulin secretion in this patient decreased with age; he did not complain of any hearing disorder, although audiometry revealed a slight elevation of hearing threshold at high frequencies. In conclusion, we found that a mitochondrial gene mutation at nucleotide position 3243 was present in about 1% of NIDDM patients including those patients with IGT. The subtype of diabetes mellitus with this mutation may have a clinical profile similar to that found in patients with NIDDM commonly seen in outpatient clinics. 25 refs., 2 figs., 1 tab.

  6. Soviet Military Doctrine: An Overview,

    DTIC Science & Technology

    2014-09-26

    toay, an gai best be summ~~aie savctigteetbhmn fmltr foce vlh anfgh, uviean ’na uur vt Duigte eid1r417, h oiesrpd- ul tary 1htorists ever since the mid...from even a marginal military superiority over one’s opponent. In 1972, retired Soviet Army Colonel V. M. Kulish noted: • • . it. must bj borne in

  7. Investigations of Photochemistry Using Holography and Photoacoustic Spectroscopy.

    DTIC Science & Technology

    1984-09-28

    Donald M. Burland PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT. PROJECT. TASK CD International Business Machines, Inc. AE OKUI UUR San...Hans Coufal Institution: International Business Machines Corporation San Jose Research Laboratory 5600 Cottle Road, San Jose, CA 95193 " Funding

  8. Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains

    PubMed Central

    2012-01-01

    Background Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. Results We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75−0.78 Mbp genomes and UUR serovars were 0.84−0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. Conclusions Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native

  9. Inrichting Gemeenschappelijke Ops-Room Voor DTO, JCG en C2000 (A Common Operations Room for DTO, JCG and C2000)

    DTIC Science & Technology

    2007-02-01

    een 3D digitale eisen en wensen leiden tot nieuwe Beschrijving van de mockup en een 2D plattegrond van de uitgangspunten en tot herontwerp...initiele ontwerp is gemodelleerd in een 3D digitale mockup en 2D plattegrond van de ruimte. De unifornie werkplek, met gescheiden toegang tot secure en...geplaatst kunnen worden. Digitale 24-uurs klokken, minstens 5 of 6 tijdzones tegeijik. Er is 66n crisisruimate opgenomen in het ontwerp. Onduidelijk is

  10. Military Health Service System Ambulatory Work Unit (AWU).

    DTIC Science & Technology

    1988-04-01

    probability of a value outside 2.0 standard deviations ranges from .0456 for A-3 -Al" 122 NILITRY HEALTH SERVICE SYSTEM AMULRTORY MORKe UurNT CANdU )(U) ARMY...00 2 w w >4 &4~ ~z til 0n0 rz~ co00 P - xU) I : 0 ) ) %z E-4 z U) HHw H~~ ~ ~ 0 olf4 E1E- 004 H U) UDm 0 4 E- H wE- ~ H E-4 H 04 H Z U H Q z HO H % x z

  11. Display Techniques for Advanced Crew Stations (DTACS). Phase 1. Display Techniques Study.

    DTIC Science & Technology

    1984-03-01

    HYBRID LIQUID CRYSTAL MATRIX MODULE . 0 P R E V I O U S L I Q U I D C R Y S T A L M A T R I C E S 0 .’.z A POSSIBLE ;’UURE LI QUID CRYSTAL w J800 xL ...These transmissions can be sustained in a pipeline mode of operation. Analog Input/Output Analog Input The analog input unit must be capable of...with respect to the axis maintaining focus across the image. Up to 50% keystone correction will be required. The minimum 99 R display brightness

  12. Study of Improved Aluminum Materials for Vehicular Armor

    DTIC Science & Technology

    1977-04-07

    WT l.u" ~ 4; ~sf __________5W A"t 1550-SZOOF gu4PA*i.sI. AD oor. EMDApttat lesrAmEt> Tro SOOT UU~r 0 Mr’bkXI"~ USIUf. O.ZftbUCTIOU5 Fat PASS AM 5OOF...little effect on ductility and f-acture toughness of Ai-6%Zn-3%Mg alloy. However, H 128 S-&; j Ink +~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~F7 M"++++• ,+I

  13. MELAS Syndrome with Cardiac Involvement: A Multimodality Imaging Approach

    PubMed Central

    Massobrio, Laura; Rubegni, Anna; Nesti, Claudia; Castiglione Morelli, Margherita; Boccalini, Sara; Galletto Pregliasco, Athena; Budaj, Irilda; Deferrari, Luca; Rosa, Gian Marco; Valbusa, Alberto

    2016-01-01

    A 49-year-old man presented with chest pain, dyspnea, and lactic acidosis. Left ventricular hypertrophy and myocardial fibrosis were detected. The sequencing of mitochondrial genome (mtDNA) revealed the presence of A to G mtDNA point mutation at position 3243 (m.3243A>G) in tRNALeu(UUR) gene. Diagnosis of cardiac involvement in a patient with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes syndrome (MELAS) was made. Due to increased risk of sudden cardiac death, cardioverter defibrillator was implanted. PMID:27891257

  14. Experimental Studies of Graphite-Epoxy and Boron-Epoxy Angle Ply Laminates in Shear,

    DTIC Science & Technology

    1977-09-01

    SPECIMENS tr15 0 BIEP I.- TEST 63 RUN 6SHERjR STRtiN MNII*N") ~iTEST 535 RUN 7 STEST 536 RUIN a uu .OU3 .00’i � gull 0 .00 W- -,-mou .0 ! .02 01...0.9 SERSTRAIN (No) FIG. 16A SHEAR RESPONSE OF PLANAR SPECIMENS 1300 BIEP X03 I-z I7 .rr~~5Qr~csc o~i~70 SHE~f SIRAIN (N/tN TEST 18:SHR 53S5NS OUN 1UUR

  15. On the Computational Complexity of Branch and Bound Search Strategies.

    DTIC Science & Technology

    1979-11-01

    PROjEcT. TASK Naval Postgraduate School /AES UI UUR Monterey, CA 93940 I L. CONTROLLING OFFICE NAME AND ADDIRESSV National Science Foundation 3o 79...we are given a statement and take its length as the size of the instance. Here the feasible set is the set of all legal proof sequences in the theory ...nodes at depth d in the generated tree is the random variable of interest and it is in- terpreted as the size of the population at time d. The theory of

  16. The isolated carboxy-terminal domain of human mitochondrial leucyl-tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells

    PubMed Central

    Perli, Elena; Giordano, Carla; Pisano, Annalinda; Montanari, Arianna; Campese, Antonio F; Reyes, Aurelio; Ghezzi, Daniele; Nasca, Alessia; Tuppen, Helen A; Orlandi, Maurizia; Di Micco, Patrizio; Poser, Elena; Taylor, Robert W; Colotti, Gianni; Francisci, Silvia; Morea, Veronica; Frontali, Laura; Zeviani, Massimo; d'Amati, Giulia

    2014-01-01

    Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA genes. Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no effective treatment is available at present. We show that three human mt aminoacyl-tRNA syntethases, namely leucyl-, valyl-, and isoleucyl-tRNA synthetase are able to improve both viability and bioenergetic proficiency of human transmitochondrial cybrid cells carrying pathogenic mutations in the mt-tRNAIle gene. Importantly, we further demonstrate that the carboxy-terminal domain of human mt leucyl-tRNA synthetase is both necessary and sufficient to improve the pathologic phenotype associated either with these “mild” mutations or with the “severe” m.3243A>G mutation in the mt-tRNALeu(UUR) gene. Furthermore, we provide evidence that this small, non-catalytic domain is able to directly and specifically interact in vitro with human mt-tRNALeu(UUR) with high affinity and stability and, with lower affinity, with mt-tRNAIle. Taken together, our results sustain the hypothesis that the carboxy-terminal domain of human mt leucyl-tRNA synthetase can be used to correct mt dysfunctions caused by mt-tRNA mutations. PMID:24413190

  17. Age and growth comparisons of Hovsgol grayling (Thymallus nigrescens Dorogostaisky, 1923), Baikal grayling (T. baicalensis Dybowski, 1874), and lenok (Brachymystax lenok Pallas, 1773) in lentic and lotic habitats of Northern Mongolia

    USGS Publications Warehouse

    Tsogtsaikhan, Pureviin; Mendsaikhan, Budiin; Jargalmaa, Ganzorigiin; Ganzorig, Batsaikhanii; Weidel, Brian C.; Filosa, Christopher; Free, Christopher; Young, Talia; Jensen, Olaf P.

    2017-01-01

    Despite concern over the conservation status of many Mongolian salmonids and the importance of their ecological role in Mongolia's aquatic ecosystems, little is known about their basic biology. Hovsgol grayling (Thymallus nigrescens) is endemic to Lake Hovsgol, Mongolia and listed as endangered on the Mongolian Red List. Baikal grayling (T. baicalensis) and lenok (Brachymystax lenok) are found in lakes and rivers throughout the Selenge drainage. A detailed study of the age and growth of these three salmonids was conducted based on 1,682 samples collected from July 2006 to July 2013 in Lake Hovsgol, its outlet the Eg River, and one of the Eg's largest tributaries, the Uur River. Our results suggest that Hovsgol grayling in particular can reach a much older maximum age (17 years in our samples) than previously believed based on aging from scales. Female Hovsgol grayling were heavier at a given length than their male counterparts. Lenok had a greater average length-at-age in Lake Hovsgol compared to the rivers and greater weight-at-length in the warmer Uur River than in the Eg; female lenok from the rivers had a greater average length-at-age than their male counterparts. This study provides critical new information for the management and conservation of these threatened salmonid species in Mongolia.

  18. Mitochondrial gene arrangement of the horseshoe crab Limulus polyphemus L.: conservation of major features among arthropod classes

    NASA Technical Reports Server (NTRS)

    Staton, J. L.; Daehler, L. L.; Brown, W. M.; Jacobs, D. K. (Principal Investigator)

    1997-01-01

    Numerous complete mitochondrial DNA sequences have been determined for species within two arthropod groups, insects and crustaceans, but there are none for a third, the chelicerates. Most mitochondrial gene arrangements reported for crustaceans and insect species are identical or nearly identical to that of Drosophila yakuba. Sequences across 36 of the gene boundaries in the mitochondrial DNA (mtDNA) of a representative chelicerate. Limulus polyphemus L., also reveal an arrangement like that of Drosophila yakuba. Only the position of the tRNA(LEU)(UUR) gene differs; in Limulus it is between the genes for tRNA(LEU)(CUN) and ND1. This positioning is also found in onychophorans, mollusks, and annelids, but not in insects and crustaceans, and indicates that tRNA(LEU)(CUN)-tRNA(LEU)(UUR)-ND1 was the ancestral gene arrangement for these groups, as suggested earlier. There are no differences in the relative arrangements of protein-coding and ribosomal RNA genes between Limulus and Drosophila, and none have been observed within arthropods. The high degree of similarity of mitochondrial gene arrangements within arthropods is striking, since some taxa last shared a common ancestor before the Cambrian, and contrasts with the extensive mtDNA rearrangements occasionally observed within some other metazoan phyla (e.g., mollusks and nematodes).

  19. Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria.

    PubMed

    Karicheva, Olga Z; Kolesnikova, Olga A; Schirtz, Tom; Vysokikh, Mikhail Y; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A; Martin, Robert P; Entelis, Nina; Tarassov, Ivan

    2011-10-01

    Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.

  20. Ultrashort Pulse Inscription of Photonic Structures in ZnSe and GaAs for Mid Infrared Applications

    DTIC Science & Technology

    2013-04-10

    pumped at 1928 nm with a continuous wave thulium fibre laser in the configuration shown in Figure 12(a). Continuous wave lasing was observed and a...investigated. Using a co-propagating 2300 nm laser diode and 1928 nm thulium fibre pump laser the internal gain of the waveguides was measured. By...experimental setup. The waveguide was then pumped with the 1928 nm thulium fiber laser (AdValue Photonics) and the output observed using an infrared camera

  1. On the Validity of Beurling Theorems in Polydiscs.

    DTIC Science & Technology

    1986-09-01

    DOCUMENTATION PAGE ta. mPocaTsecLJnIt CLASSIFICATION 1b.~ RESTRICTIVE MAPIKINGS 1UNC LAS SIFI ED 20, S&CLNITY’ CLASSIFICATION AUJT1400ITY 3 .WTI...ONANIAIN9EOR UURS 5. MONITORING ORGANIZATION REPORT NuMEEII Technical Report No. 154 AFOM-R-h 8 7 - 0 0 73 ". NAME6 OFP 0EfPORMgNG ORGANIZATION p. PFIC ...OFFICE SYMNEOL ~ I~A (.r’OL.~i’ 1 3 ; 2 307AFOSR/NM 00 FORM 1473,83 APR EDITION OP0 1 JAN 7251 OSSOLE’C UINCLAS:IF:E’: %, A6dOSR-TR o 8 7 - 0073

  2. Complete mitochondrial genome of Cygnus olor (Aves, Anseriformes, Anatidae).

    PubMed

    Park, Chang Eon; Park, Gun-Seok; Kwak, Yunyoung; Hong, Sung-Jun; Khan, Abdur Rahim; Jung, Byung Kwon; Park, Yeong-Jun; Kim, Jong-Guk; Park, Hee Cheon; Shin, Jae-Ho

    2016-09-01

    The complete mitochondrial genome of Cygnus olor (Aves, Anseriformes, Anatidae) was revealed in this study. Total 16 739 base pairs (bp) of this mitogenome encoded genes for 13 protein coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and a D-loop (control region). The 12S rRNA and 16S rRNA genes are located between tRNA-Phe and tRNA-Leu (UUR) and segmentalized by the tRNA-Val. D-loop is located between tRNA-Glu and tRNA-Phe. The overall base composition of C. olor is G + C: 47.8%, A + T: 52.2%, apparently with a slight AT bias. Following the phylogenetic analysis, the C. olor was closed to Anser cygnoides.

  3. Blast Operational Overpressure Model (BOOM): An Airblast Prediction Method

    DTIC Science & Technology

    1987-04-01

    Air Force Weapons Laboratory, Kirtland Air Force Base, New Mexico, June 1985. 9. Walpole , R.E., and Myers, R.H., Probability and Statistics for...v5THEN KFT) "H(l) LET RR=C1 S39: INPUT "WNC DIR 320:IF Ir)24G0THEN ,;:NPTWD( SP LET RR=C3 38IPT"N P 380: NH =Pt4.RR*IS:XH (KT) "iWS(I) u( NH +PH)/2...460: PD=ATN (U/U)/R THEN GOTO 11 AD 62 A9 I D(TE 1140:FwH(J)Vl000 LET:I PC<POTHEN -RW(I))/HD:C 48:LETZ LE=1.. P =C+I:TT(C)=T NH : A A -, E F .P D*F4T

  4. Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNA(Lys) gene.

    PubMed

    Shimizu, Akinori; Mito, Takayuki; Hayashi, Chisato; Ogasawara, Emi; Koba, Ryusuke; Negishi, Issei; Takenaga, Keizo; Nakada, Kazuto; Hayashi, Jun-Ichi

    2014-02-25

    We generated transmitochondrial mice (mito-mice) that carry a mutation in the tRNA(Lys) gene encoded by mtDNA for use in studies of its pathogenesis and transmission profiles. Because patients with mitochondrial diseases frequently carry mutations in the mitochondrial tRNA(Lys) and tRNA(Leu(UUR)) genes, we focused our efforts on identifying somatic mutations of these genes in mouse lung carcinoma P29 cells. Of the 43 clones of PCR products including the tRNA(Lys) or tRNA(Leu(UUR)) genes in mtDNA of P29 cells, one had a potentially pathogenic mutation (G7731A) in the tRNA(Lys) gene. P29 subclones with predominant amounts of G7731A mtDNA expressed respiration defects, thus suggesting the pathogenicity of this mutation. We then transferred G7731A mtDNA into mouse ES cells and obtained F0 chimeric mice. Mating these F0 mice with C57BL/6J (B6) male mice resulted in the generation of F1 mice with G7731A mtDNA, named "mito-mice-tRNA(Lys7731)." Maternal inheritance and random segregation of G7731A mtDNA occurred in subsequent generations. Mito-mice-tRNA(Lys7731) with high proportions of G7731A mtDNA exclusively expressed respiration defects and disease-related phenotypes and therefore are potential models for mitochondrial diseases due to mutations in the mitochondrial tRNA(Lys) gene. Moreover, the proportion of mutated mtDNA varied markedly among the pups born to each dam, suggesting that selecting oocytes with high proportions of normal mtDNA from affected mothers with tRNA(Lys)-based mitochondrial diseases may be effective as a primary prevention for obtaining unaffected children.

  5. Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease

    PubMed Central

    Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Álvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A

    2012-01-01

    BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. PMID:22747838

  6. Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNALys gene

    PubMed Central

    Shimizu, Akinori; Mito, Takayuki; Hayashi, Chisato; Ogasawara, Emi; Koba, Ryusuke; Negishi, Issei; Takenaga, Keizo; Nakada, Kazuto; Hayashi, Jun-Ichi

    2014-01-01

    We generated transmitochondrial mice (mito-mice) that carry a mutation in the tRNALys gene encoded by mtDNA for use in studies of its pathogenesis and transmission profiles. Because patients with mitochondrial diseases frequently carry mutations in the mitochondrial tRNALys and tRNALeu(UUR) genes, we focused our efforts on identifying somatic mutations of these genes in mouse lung carcinoma P29 cells. Of the 43 clones of PCR products including the tRNALys or tRNALeu(UUR) genes in mtDNA of P29 cells, one had a potentially pathogenic mutation (G7731A) in the tRNALys gene. P29 subclones with predominant amounts of G7731A mtDNA expressed respiration defects, thus suggesting the pathogenicity of this mutation. We then transferred G7731A mtDNA into mouse ES cells and obtained F0 chimeric mice. Mating these F0 mice with C57BL/6J (B6) male mice resulted in the generation of F1 mice with G7731A mtDNA, named “mito-mice-tRNALys7731.” Maternal inheritance and random segregation of G7731A mtDNA occurred in subsequent generations. Mito-mice-tRNALys7731 with high proportions of G7731A mtDNA exclusively expressed respiration defects and disease-related phenotypes and therefore are potential models for mitochondrial diseases due to mutations in the mitochondrial tRNALys gene. Moreover, the proportion of mutated mtDNA varied markedly among the pups born to each dam, suggesting that selecting oocytes with high proportions of normal mtDNA from affected mothers with tRNALys-based mitochondrial diseases may be effective as a primary prevention for obtaining unaffected children. PMID:24510903

  7. Defects in RNA metabolism in mitochondrial disease.

    PubMed

    Siira, Stefan J; Shearwood, Anne-Marie J; Bracken, Cameron P; Rackham, Oliver; Filipovska, Aleksandra

    2017-04-01

    The expression of mitochondrially-encoded genes requires the efficient processing of long precursor RNAs at the 5' and 3' ends of tRNAs, a process which, when disrupted, results in disease. Two such mutations reside within mt-tRNA(Leu(UUR)); a m.3243A>G transition, which is the most common cause of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), and m.3302A>G which often causes mitochondrial myopathy (MM). We used parallel analysis of RNA ends (PARE) that captures the 5' terminal end of 5'-monophosphorylated mitochondrial RNAs to compare the effects of the m.3243A>G and m.3302A>G mutations on mitochondrial tRNA processing and downstream RNA metabolism. We confirmed previously identified RNA processing defects, identified common internal cleavage sites and new sites unique to the m.3243A>G mutants that do not correspond to transcript ends. These sites occur in regions of predicted RNA secondary structure, or are in close proximity to such regions, and may identify regions of importance to the processing of mtRNAs.

  8. Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation.

    PubMed

    Schild, Christof; Hahn, Dagmar; Schaller, André; Jackson, Christopher Benjamin; Rothen-Rutishauser, Barbara; Mirkovitch, Jelena; Nuoffer, Jean-Marc

    2014-07-01

    Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNA(Leu(CUN))) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNA(Leu(CUN)) and tRNA(Leu(UUR)) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNA(Leu(CUN)) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.

  9. Screening for mtDNA diabetes mutations in Pima Indians with NIDDM.

    PubMed

    Sepehrnia, B; Prezant, T R; Rotter, J I; Pettitt, D J; Knowler, W C; Fischel-Ghodsian, N

    1995-03-27

    More than half of the Pima Indians over age 35 years have non-insulin-dependent (type II) diabetes mellitus (NIDDM). Extensive data indicate the importance of maternal diabetes in determining their risk for diabetes. Generally, the risk of having NIDDM is higher in patients with affected mothers than affected fathers. This has been attributed to intrauterine factors, but recently mitochondrial inheritance has been raised as an alternative hypothesis. In other populations, several families and individuals with diabetes due to a mitochondrial DNA point mutation at nucleotide 3243 in the tRNA(leu(UUR)) gene have been described, as has one family with a 10.4 kb mitochondrial DNA duplication/deletion. We tested whether these specific mitochondrial gene mutations could explain a portion of the excess maternal transmission seen in the Pima Indians. Mitochondrial DNA obtained from blood lymphocytes of 148 Pima Indians with NIDDM was screened both for the point mutation at nt 3243, and the 10.4 kb duplication/deletion. Neither of these mutations was detected, and although a small proportion of the excess maternal transmission in Pima Indians could still be due to yet undescribed mitochondrial mutations or imprinted nuclear genes, our data support the role of the intrauterine environment in this population.

  10. Variational solution of the Schrödinger equation using plane waves in adaptive coordinates: The radial case.

    PubMed

    Pérez-Jordá, José M

    2010-01-14

    A new method for solving the Schrödinger equation is proposed, based on the following details. First, a map u=u(r) from Cartesian coordinates r to a new coordinate system u is chosen. Second, the solution (orbital) psi(r) is written in terms of a function U depending on u so that psi(r)=/J(u)/(-1/2)U(u), where /J(u)/ is the Jacobian determinant of the map. Third, U is expressed as a linear combination of plane waves in the u coordinate, U(u)= sum (k)c(k)e(ik x u). Finally, the coefficients c(k) are variationally optimized to obtain the best energy, using a generalization of an algorithm originally developed for the Coulomb potential [J. M. Perez-Jorda, Phys. Rev. B 58, 1230 (1998)]. The method is tested for the radial Schrödinger equation in the hydrogen atom, resulting in micro-Hartree accuracy or better for the energy of ns and np orbitals (with n up to 5) using expansions of moderate length.

  11. Round and pointed-head grenadier fishes (Actinopterygii: Gadiformes) represent a single sister group: evidence from the complete mitochondrial genome sequences.

    PubMed

    Satoh, Takashi P; Miya, Masaki; Endo, Hiromitsu; Nishida, Mutsumi

    2006-07-01

    The gene order of mitochondrial genomes (mitogenomes) has been employed as a useful phylogenetic marker in various metazoan animals, because it may represent uniquely derived characters shared by members of monophyletic groups. During the course of molecular phylogenetic studies of the order Gadiformes (cods and their relatives) based on whole mitogenome sequences, we found that two deep-sea grenadiers (Squalogadus modificatus and Trachyrincus murrayi: family Macrouridae) revealed a unusually identical gene order (translocation of the tRNA(Leu (UUR))). Both are members of the same family, although their external morphologies differed so greatly (e.g., round vs. pointed head) that they have been placed in different subfamilies Macrouroidinae and Trachyrincinae, respectively. Additionally, we determined the whole mitogenome sequences of two other species, Bathygadus antrodes and Ventrifossa garmani, representing a total of four subfamilies currently recognized within Macrouridae. The latter two species also exhibited gene rearrangements, resulting in a total of three different patterns of unique gene order being observed in the four subfamilies. Partitioned Bayesian analysis was conducted using available whole mitogenome sequences from five macrourids plus five outgroups. The resultant trees clearly indicated that S. modificatus and T. murrayi formed a monophyletic group, having a sister relationship to other macrourids. Thus, monophyly of the two species with disparate head morphologies was corroborated by two different lines of evidence (nucleotide sequences and gene order). The overall topology of the present tree differed from any of the previously proposed, morphology-based phylogenetic hypotheses.

  12. The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA

    PubMed Central

    Hyvärinen, Anne K.; Pohjoismäki, Jaakko L. O.; Reyes, Aurelio; Wanrooij, Sjoerd; Yasukawa, Takehiro; Karhunen, Pekka J.; Spelbrink, Johannes N.; Holt, Ian J.; Jacobs, Howard T.

    2007-01-01

    The mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNALeu(UUR) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA). Electrophoretic mobility shift assays (EMSA) and SELEX, using mitochondrial protein extracts from cells induced to overexpress mTERF, revealed novel, weaker mTERF-binding sites, clustered in several regions of mtDNA, notably in the major non-coding region (NCR). Such binding in vivo was supported by mtDNA immunoprecipitation. Two-dimensional neutral agarose gel electrophoresis (2DNAGE) and 5′ end mapping by ligation-mediated PCR (LM-PCR) identified the region of the canonical mTERF-binding site as a replication pause site. The strength of pausing was modulated by the expression level of mTERF. mTERF overexpression also affected replication pausing in other regions of the genome in which mTERF binding was found. These results indicate a role for TERF in mtDNA replication, in addition to its role in transcription. We suggest that mTERF could provide a system for coordinating the passage of replication and transcription complexes, analogous with replication pause-region binding proteins in other systems, whose main role is to safeguard the integrity of the genome whilst facilitating its efficient expression. PMID:17884915

  13. Phylogenetic relationships of Iberian Aphodiini (Coleoptera: Scarabaeidae) based on morphological and molecular data.

    PubMed

    Cabrero-Sañudo, Francisco-José; Zardoya, Rafael

    2004-06-01

    A phylogeny of Iberian Aphodiini dung beetles was reconstructed based on morphological and molecular data. The data set included a total of 84 variable characters from wing venation, mouthparts, genitalia, and external morphology, as well as mitochondrial partial cytochrome c oxidase I (COI), complete tRNA-Leu (UUR), and partial cytochrome c oxidase II (COII) gene nucleotide sequences (1210 positions). Phylogenetic trees based on molecular data were relatively more resolved than those based on morphological characters. The Bayesian analysis of combined molecular and morphological data provided resolution not achieved by each data set separately. Ammoecius and Aphodius are the first lineages that branch off from the tree, followed by Acrossus, Nimbus, and Heptaulacus. The remaining studied taxa are recovered in a more derived clade that lacks internal resolution. Reconstructed trees based on molecular data showed relatively short internal nodes that were weakly supported. Such pattern may reflect a rapid radiation at the origin of the tribe Aphodiini, but also saturation of mutational changes. Several tests were conducted to discern between both competing hypotheses, as well as to assess the effect of incomplete taxon sampling.

  14. Simultaneous screening of multiple mutations by invader assay improves molecular diagnosis of hereditary hearing loss: a multicenter study.

    PubMed

    Usami, Shin-ichi; Nishio, Shin-ya; Nagano, Makoto; Abe, Satoko; Yamaguchi, Toshikazu

    2012-01-01

    Although etiological studies have shown genetic disorders to be a common cause of congenital/early-onset sensorineural hearing loss, there have been no detailed multicenter studies based on genetic testing. In the present report, 264 Japanese patients with bilateral sensorineural hearing loss from 33 ENT departments nationwide participated. For these patients, we first applied the Invader assay for screening 47 known mutations of 13 known deafness genes, followed by direct sequencing as necessary. A total of 78 (29.5%) subjects had at least one deafness gene mutation. Mutations were more frequently found in the patients with congenital or early-onset hearing loss, i.e., in those with an awareness age of 0-6 years, mutations were significantly higher (41.8%) than in patients with an older age of awareness (16.0%). Among the 13 genes, mutations in GJB2 and SLC26A4 were mainly found in congenital or early-onset patients, in contrast with mitochondrial mutations (12S rRNA m.1555A>G, tRNA(Leu(UUR)) m.3243A>G), which were predominantly found in older-onset patients. The present method of simultaneous screening of multiple deafness mutations by Invader assay followed by direct sequencing will enable us to detect deafness mutations in an efficient and practical manner for clinical use.

  15. Thermoelectric Conversion of Waste Heat to Electricity in an IC Engine Powered Vehicle

    SciTech Connect

    2012-01-31

    The thermoelectric generator shorting system provides the capability to monitor and short-out individual thermoelectric couples in the event of failure. This makes the series configured thermoelectric generator robust to individual thermoelectric couple failure. Open circuit detection of the thermoelectric couples and the associated short control is a key technique to ensure normal functionality of the TE generator under failure of individual TE couples. This report describes a five-year effort whose goal was the understanding the issues related to the development of a thermoelectric energy recovery device for a Class-8 truck. Likely materials and important issues related to the utility of this generator were identified. Several prototype generators were constructed and demonstrated. The generators developed demonstrated several new concepts including advanced insulation, couple bypass technology and the first implementation of skutterudite thermoelectric material in a generator design. Additional work will be required to bring this system to fruition. However, such generators offer the possibility of converting energy that is otherwise wasted to useful electric power. Uur studies indicate that this can be accomplished in a cost-effective manner for this application.

  16. Impaired mitochondrial Ca{sup 2+} homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

    SciTech Connect

    Kleist-Retzow, Juergen-Christoph von ||. E-mail: juergen-christoph.vonkleist@uk-koeln.de; Hue-Tran Hornig-Do; Schauen, Matthias; Eckertz, Sabrina; Tuan Anh Duong Dinh; Stassen, Frank; Lottmann, Nadine; Bust, Maria; Galunska, Bistra; Wielckens, Klaus; Hein, Wolfgang; Beuth, Joseph; Braun, Jan-Matthias; Fischer, Juergen H.; Ganitkevich, Vladimir Y. |; Maniura-Weber, Katharina; Wiesner, Rudolf J. |

    2007-08-15

    Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca{sup 2+} homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A > G and 3302A > G in tRNA{sup Leu(UUR)}, as well as Rho{sup 0} cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP + ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.

  17. THE IRON PROJECT: High-Energy-Density (HED) Plasma Opacities and Diagnostics

    NASA Astrophysics Data System (ADS)

    Gokce, Yasin; Bostelmann, T.; Nahar, S.; Pradhan, A.; Bailey, J.

    2014-05-01

    The composition of the Sun, the benchmark for astronomical objects, has been a longstanding problem for the last few decades. The abundances of common elements in the Sun, such as, carbon, nitrogen, oxygen, supported by helioseismology are at discrepant by up to 50% higher from those derived from state-of-the-art spectroscopy and elaborate 3-D radiative transfer models. The uncertainty is compounded by recent experiments at the Sandia National Laboratory on the Z-pinch inertial confinement fusion device which is able to re-create the HED plasma conditions existing at the solar radiative-convection zone boundary. Measured monochromatic iron opacities disagree with all known theoretical opacities models. The abundance problem and potential solution are related to radiative opacities. Uur continued investigation of the problem will be presented. We will also present collision strengths of carbon-like silicon which shows new resonances in the low energy region introduced by relativistic effects in the Breit-Pauli R-matrix method. Line intensity ratios of this ion, obtained for optically allowed transitions as seen in astronomical spectra, are the diagnostics for the density and termperature of the plasmas will be reported. Partial support of NSF, DOE.

  18. Screening for mtDNA diabetes mutations in Pima Indians with NIDDM

    SciTech Connect

    Sepehrnia, B.; Prezant, T.R.; Rotter, J.I.

    1995-03-27

    More than half of the Pima Indians over age 35 years have non-insulin-dependent (type II) diabetes mellitus (NIDDM). Extensive data indicate the importance of maternal diabetes in determining their risk for diabetes. Generally, the risk of having NIDDM is higher in patients with affected mothers than affected fathers. This has been attributed to intrauterine factors, but recently mitochondrial inheritance has been raised as an alternative hypothesis. In other populations, several families and individuals with diabetes due to a mitochondrial DNA point mutation at nucleotide 3243 in the tRNA{sup leu(UUR)} gene have been described, as has one family with a 10.4 kb mitochondrial DNA duplication/deletion. We tested whether these specific mitochondrial gene mutations could explain a portion of the excess maternal transmission seen in the Pima Indians. Mitochondrial DNA obtained from blood lymphocytes of 148 Pima Indians with NIDDM was screened both for the point mutation at nt 3243, and the 10.4 kb duplication/deletion. Neither of these mutations was detected, and although a small proportion of the excess maternal transmission in Pima Indians could still be due to yet undescribed mitochondrial mutations or imprinted nuclear genes, our data support the role of the intrauterine environment in this population. 32 refs, 21 figs.

  19. Unusual occurrence of intestinal pseudo obstruction in a patient with maternally inherited diabetes and deafness (MIDD) and favorable outcome with coenzyme Q10.

    PubMed

    Bergamin, Carla S; Rolim, Luiz Clemente; Dib, Sergio A; Moisés, Regina S

    2008-11-01

    Maternally inherited diabetes and deafness (MIDD) has been related to an A to G transition in the mitochondrial tRNA Leu (UUR) gene at the base pair 3243. This subtype of diabetes is characterized by maternal transmission, young age at onset and bilateral hearing impairment. Besides diabetes and deafness, the main diagnostic features, a wide range of multisystemic symptoms may be associated with the A3243G mutation. Organs that are most metabolically active, such as muscles, myocardium, retina, cochlea, kidney and brain are frequently affected. Gastrointestinal tract symptoms are also common in patients with mitochondrial disease and constipation and diarrhea are the most frequent manifestations. However, there are few prior reports of intestinal pseudo obstruction in MIDD patients. Here we report the case of a patient with MIDD associated with the mtDNA A3243G mutation who developed chronic intestinal pseudo obstruction, and the introduction of Coenzyme Q10 as adjunctive therapy led to a solution of the pseudo obstruction.

  20. Advanced laser sensing receiver concepts based on FPA technology.

    SciTech Connect

    Jacobson, P. L.; Petrin, R. R.; Jolin, J. L.; Foy, B. R.; Lowrance, J. L.; Renda, G.

    2002-01-01

    The ultimate performance of any remote sensor is ideally governed by the hardware signal-to-noise capability and allowed signal-averaging time. In real-world scenarios, this may not be realizable and the limiting factors may suggest the need for more advanced capabilities. Moving from passive to active remote sensors offers the advantage of control over the illumination source, the laser. Added capabilities may include polarization discrimination, instantaneous imaging, range resolution, simultaneous multi-spectral measurement, or coherent detection. However, most advanced detection technology has been engineered heavily towards the straightforward passive sensor requirements, measuring an integrated photon flux. The need for focal plane array technology designed specifically for laser sensing has been recognized for some time, but advances have only recently made the engineering possible. This paper will present a few concepts for laser sensing receiver architectures, the driving specifications behind those concepts, and test/modeling results of such designs.

  1. Metabolically induced heteroplasmy shifting and L-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

    PubMed Central

    Desquiret-Dumas, Valerie; Gueguen, Naig; Barth, Magalie; Chevrollier, Arnaud; Hancock, Saege; Wallace, Douglas C; Amati-Bonneau, Patrizia; Henrion, Daniel; Bonneau, Dominique; Reynier, Pascal; Procaccio, Vincent

    2012-01-01

    The m.3243A>G variant in the mitochondrial tRNALeu (UUR) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of L-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of L-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and L-arginine therapy may constitute promising therapeutic strategies against MELAS. PMID:22306605

  2. Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

    PubMed

    Aboklaish, Ali F; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I; Spiller, O Brad

    2014-11-01

    While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models.

  3. Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.

    PubMed Central

    Enríquez, J A; Attardi, G

    1996-01-01

    Analysis by acid polyacrylamide/urea gel electrophoresis of 14 individual mitochondrial tRNAs (mt-tRNAs) from human cells has revealed a variable decrease in mobility of the aminoacylated relative to the nonacylated form, with the degree of separation of the two forms not being correlated with the mass, polar character, or charge of the amino acid. Separation of the charged and uncharged species has been found to be independent of tRNA denaturation, being observed also in the absence of urea. In another approach, electrophoresis through a perpendicular denaturing gradient gel of several individual mt-tRNAs has shown a progressive unfolding of the tRNA with increasing denaturant concentration, which is consistent with an initial disruption of tertiary interactions, followed by the sequential melting of the four stems of the cloverleaf structure. A detailed analysis of the unfolding process of charged and uncharged tRNALys and tRNALeu(UUR) has revealed that the separation of the two forms of these tRNAs persisted throughout the almost entire range of denaturant concentrations used and was lost upon denaturation of the last helical domain(s), which most likely included the amino acid acceptor stem. These observations strongly suggest that the electrophoretic retardation of the charged species reflects an aminoacylation-induced conformational change of the 3'-end of these mt-tRNAs, with possible significant implications in connection with the known role of the acceptor end in tRNA interactions with the ribosomal peptidyl transferase center and the elongation factor Tu. Images Fig. 1 Fig. 2 Fig. 3 PMID:8710865

  4. Whole mitochondrial genome screening in maternally inherited non-syndromic hearing impairment using a microarray resequencing mitochondrial DNA chip.

    PubMed

    Lévêque, Marianne; Marlin, Sandrine; Jonard, Laurence; Procaccio, Vincent; Reynier, Pascal; Amati-Bonneau, Patrizia; Baulande, Sylvain; Pierron, Denis; Lacombe, Didier; Duriez, Françoise; Francannet, Christine; Mom, Thierry; Journel, Hubert; Catros, Hélène; Drouin-Garraud, Valérie; Obstoy, Marie-Françoise; Dollfus, Hélène; Eliot, Marie-Madeleine; Faivre, Laurence; Duvillard, Christian; Couderc, Remy; Garabedian, Eréa-Noël; Petit, Christine; Feldmann, Delphine; Denoyelle, Françoise

    2007-11-01

    Mitochondrial DNA (mtDNA) mutations have been implicated in non-syndromic hearing loss either as primary or as predisposing factors. As only a part of the mitochondrial genome is usually explored in deafness, its prevalence is probably under-estimated. Among 1350 families with non-syndromic sensorineural hearing loss collected through a French collaborative network, we selected 29 large families with a clear maternal lineage and screened them for known mtDNA mutations in 12S rRNA, tRNASer(UCN) and tRNALeu(UUR) genes. When no mutation could be identified, a whole mitochondrial genome screening was performed, using a microarray resequencing chip: the MitoChip version 2.0 developed by Affymetrix Inc. Known mtDNA mutations was found in nine of the 29 families, which are described in the article: five with A1555G, two with the T7511C, one with 7472insC and one with A3243G mutation. In the remaining 20 families, the resequencing Mitochip detected 258 mitochondrial homoplasmic variants and 107 potentially heteroplasmic variants. Controls were made by direct sequencing on selected fragments and showed a high sensibility of the MitoChip but a low specificity, especially for heteroplasmic variations. An original analysis on the basis of species conservation, frequency and phylogenetic investigation was performed to select the more probably pathogenic variants. The entire genome analysis allowed us to identify five additional families with a putatively pathogenic mitochondrial variant: T669C, C1537T, G8078A, G12236A and G15077A. These results indicate that the new MitoChip platform is a rapid and valuable tool for identification of new mtDNA mutations in deafness.

  5. MELAS syndrome: Clinical manifestations, pathogenesis, and treatment options.

    PubMed

    El-Hattab, Ayman W; Adesina, Adekunle M; Jones, Jeremy; Scaglia, Fernando

    2015-01-01

    Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. MELAS syndrome is a multi-organ disease with broad manifestations including stroke-like episodes, dementia, epilepsy, lactic acidemia, myopathy, recurrent headaches, hearing impairment, diabetes, and short stature. The most common mutation associated with MELAS syndrome is the m.3243A>G mutation in the MT-TL1 gene encoding the mitochondrial tRNA(Leu(UUR)). The m.3243A>G mutation results in impaired mitochondrial translation and protein synthesis including the mitochondrial electron transport chain complex subunits leading to impaired mitochondrial energy production. The inability of dysfunctional mitochondria to generate sufficient energy to meet the needs of various organs results in the multi-organ dysfunction observed in MELAS syndrome. Energy deficiency can also stimulate mitochondrial proliferation in the smooth muscle and endothelial cells of small blood vessels leading to angiopathy and impaired blood perfusion in the microvasculature of several organs. These events will contribute to the complications observed in MELAS syndrome particularly the stroke-like episodes. In addition, nitric oxide deficiency occurs in MELAS syndrome and can contribute to its complications. There is no specific consensus approach for treating MELAS syndrome. Management is largely symptomatic and should involve a multidisciplinary team. Unblinded studies showed that l-arginine therapy improves stroke-like episode symptoms and decreases the frequency and severity of these episodes. Additionally, carnitine and coenzyme Q10 are commonly used in MELAS syndrome without proven efficacy.

  6. Analysis of the mitochondrial genome of cheetahs (Acinonyx jubatus) with neurodegenerative disease.

    PubMed

    Burger, Pamela A; Steinborn, Ralf; Walzer, Christian; Petit, Thierry; Mueller, Mathias; Schwarzenberger, Franz

    2004-08-18

    The complete mitochondrial genome of Acinonyx jubatus was sequenced and mitochondrial DNA (mtDNA) regions were screened for polymorphisms as candidates for the cause of a neurodegenerative demyelinating disease affecting captive cheetahs. The mtDNA reference sequences were established on the basis of the complete sequences of two diseased and two nondiseased animals as well as partial sequences of 26 further individuals. The A. jubatus mitochondrial genome is 17,047-bp long and shows a high sequence similarity (91%) to the domestic cat. Based on single nucleotide polymorphisms (SNPs) in the control region (CR) and pedigree information, the 18 myelopathic and 12 non-myelopathic cheetahs included in this study were classified into haplotypes I, II and III. In view of the phenotypic comparability of the neurodegenerative disease observed in cheetahs and human mtDNA-associated diseases, specific coding regions including the tRNAs leucine UUR, lysine, serine UCN, and partial complex I and V sequences were screened. We identified a heteroplasmic and a homoplasmic SNP at codon 507 in the subunit 5 (MTND5) of complex I. The heteroplasmic haplotype I-specific valine to methionine substitution represents a nonconservative amino acid change and was found in 11 myelopathic and eight non-myelopathic cheetahs with levels ranging from 29% to 79%. The homoplasmic conservative amino acid substitution valine to alanine was identified in two myelopathic animals of haplotype II. In addition, a synonymous SNP in the codon 76 of the MTND4L gene was found in the single haplotype III animal. The amino acid exchanges in the MTND5 gene were not associated with the occurrence of neurodegenerative disease in captive cheetahs.

  7. The Australian fresh water isopod (Phreatoicidea: Isopoda) allows insights into the early mitogenomic evolution of isopods.

    PubMed

    Kilpert, Fabian; Podsiadlowski, Lars

    2010-03-01

    The complete mitochondrial (mt) genome sequence of the Australian fresh water isopod Eophreatoicus sp.-14 has been determined. The new species is a member of the taxon Phreatoicidea, a clade of particular interest, as it is often regarded as the sister group to all other Isopoda. Although the overall genome organization of Eophreatoicus sp.-14 conforms to the typical state of Metazoa--it is a circular ring of DNA hosting the usual 37 genes and one major non-coding region--it bears a number of derived characters that fall within the scope of "genome morphology". Earlier studies have indicated that the isopod mitochondrial gene order is not as conserved as that of other crustaceans. Indeed, the mt genome of Eophreatoicus sp.-14 shows an inversion of seven genes (including cox1), which is as far as we know unique. Even more interesting is the derived arrangement of nad1, trnL(CUN), rrnS, control region, cob, trnT, nad5 and trnF that is shared by nearly all available isopod mt genomes. A striking feature is the close proximity of the rearranged genes to the mt control region. Inferable gene translocation events are, however, more suitable to trace the evolution of mt genomes. Genes like nad1/trnL(CUN) and nad5/trnF, which retained their adjacent position after being rearranged, were most likely translocated together. A very good example for the need to understand the mechanisms of translocations is the remolding of trnL(UUR) to trnL(CUN). Both tRNA genes are adjacent and have a high sequence similarity, probably the result of a gene duplication and subsequent anticodon mutation. Modified secondary structures were found in three tRNAs of Eophreatoicus sp.-14, which are all characterized by the loss of the DHU-arm. This is common to crustaceans for tRNA Serine(AGY), while the arm-loss in tRNA Cysteine within Malacostraca is only shared by other isopods. Modification of the third tRNA, Isoleucine, is not known from any other related species. Nucleotide frequencies of

  8. The complete mitochondrial genome of Flustra foliacea (Ectoprocta, Cheilostomata) - compositional bias affects phylogenetic analyses of lophotrochozoan relationships

    PubMed Central

    2011-01-01

    Background The phylogenetic relationships of the lophophorate lineages, ectoprocts, brachiopods and phoronids, within Lophotrochozoa are still controversial. We sequenced an additional mitochondrial genome of the most species-rich lophophorate lineage, the ectoprocts. Although it is known that there are large differences in the nucleotide composition of mitochondrial sequences of different lineages as well as in the amino acid composition of the encoded proteins, this bias is often not considered in phylogenetic analyses. We applied several approaches for reducing compositional bias and saturation in the phylogenetic analyses of the mitochondrial sequences. Results The complete mitochondrial genome (16,089 bp) of Flustra foliacea (Ectoprocta, Gymnolaemata, Cheilostomata) was sequenced. All protein-encoding, rRNA and tRNA genes are transcribed from the same strand. Flustra shares long intergenic sequences with the cheilostomate ectoproct Bugula, which might be a synapomorphy of these taxa. Further synapomorphies might be the loss of the DHU arm of the tRNA L(UUR), the loss of the DHU arm of the tRNA S(UCN) and the unique anticodon sequence GAG of the tRNA L(CUN). The gene order of the mitochondrial genome of Flustra differs strongly from that of the other known ectoprocts. Phylogenetic analyses of mitochondrial nucleotide and amino acid data sets show that the lophophorate lineages are more closely related to trochozoan phyla than to deuterostomes or ecdysozoans confirming the Lophotrochozoa hypothesis. Furthermore, they support the monophyly of Cheilostomata and Ectoprocta. However, the relationships of the lophophorate lineages within Lophotrochozoa differ strongly depending on the data set and the used method. Different approaches for reducing heterogeneity in nucleotide and amino acid data sets and saturation did not result in a more robust resolution of lophotrochozoan relationships. Conclusion The contradictory and usually weakly supported phylogenetic