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1.
Ann N Y Acad Sci ; 1119: 274-88, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18056975

RESUMO

Human progeroid Werner syndrome provides the current best model for analysis of human aging, recapitulating many aspects of normal aging as a result of mutation of the WRN gene. This gene encodes a RecQ-type helicase with additional exonuclease activity. While biochemical studies in vitro have proven invaluable in determining substrate specificities of the WRN exonuclease and helicase, it has been difficult to dissociate the two key enzyme activities in vivo. We are developing Drosophila as a model system for analysis of WRN function; the suitability of Drosophila for extensive and sophisticated genetic manipulation permits us to investigate regulatory pathways and the impact of WRN loss at organismal, cellular, and molecular levels. BLASTP screening of the Drosophila genome with human WRN sequence allowed us to identify three RecQ helicases with strong homology to human WRN, a presumed helicase component of the spliceosome, and two DEAH-box putative RNA helicases with weaker WRN homology. None of these helicases contain a WRN-like exonuclease domain, but two potential WRN-like exonucleases in flies encoded by the loci CG7670 and CG6744 were also identified in the BLAST search. CG6744 and CG7670 are more closely related to human WRN than to each other. We have obtained a fly strain with a piggyBac insertional mutation within the CG6744 locus, which decreases expression of the encoded mRNA. Such flies show elevated levels of somatic recombination. We suggest that WRN-like exonuclease activity is critical in maintaining genomic integrity in flies.


Assuntos
Modelos Animais de Doenças , Proteínas de Drosophila/genética , Instabilidade Genômica/genética , RecQ Helicases/genética , Síndrome de Werner/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Exodesoxirribonucleases , Deleção de Genes , Humanos , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner
2.
Biochem Soc Symp ; (71): 157-76, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15777020

RESUMO

Aerobic cells produce reactive oxygen species as a consequence of normal cellular metabolism, and an array of antioxidant systems are in place to maintain the redox balance. When the redox equilibrium of the cell is upset by pro-oxidant environmental stimuli, adaptive responses to the redox stress take place, which can result in up-regulation of antioxidant proteins and detoxification enzymes. Over the past few years, it has become apparent that members of the CNC (cap 'n' collar)-basic leucine zipper family of transcription factors are principal mediators of defensive responses to redox stress. In mammals, the CNC family members nuclear factor-erythroid 2 p45-related factors 1 and 2 (Nrf1 and Nrf2) have been shown to be involved in the transcriptional up-regulation of cytoprotective genes including those encoding glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase, glutathione S-transferases and aldo-keto reductases. An evolutionarily conserved system exists in Caenorhabditis elegans, and it is possible that Drosophila melanogaster may also utilize CNC transcription factors to induce antioxidant genes in response to pro-oxidant chemicals. The advent of microarray and proteomic technologies has advanced our understanding of the gene batteries regulated by oxidative insult, but has highlighted the complexity of gene regulation by environmental factors. This review focuses on the antioxidant response to environmental stress, and the impact that microarrays and proteomics have made in this field.


Assuntos
Antioxidantes/metabolismo , Citoproteção/fisiologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/farmacologia , Exposição Ambiental/efeitos adversos , Fator 1 Nuclear Respiratório , Fatores Nucleares Respiratórios , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Proteínas Repressoras/metabolismo , Proteínas Repressoras/farmacologia , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismo
3.
J Res Natl Inst Stand Technol ; 95(1): 49-67, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-28179757

RESUMO

A direct spectroradiometric determination of the temperature of freezing gold was performed by measuring the spectral radiances of a gold blackbody relative to those of a laser-irradiated integrating sphere which was calibrated with absolute silicon detectors and an electrically calibrated radiometer. The measurements were performed at three laser wavelengths near 600 nm, and the temperature of the blackbody was calculated by substituting the measured spectral radiances into Planck's radiation formula. The result obtained, TAu=(1337.33± 0.34) K, is 0.25 K below the gold-point assignment in the International Practical Temperature Scale of 1968 (IPTS-68) and has been adopted in September 1990 as the new gold-point value in the International Temperature Scale of 1990 (ITS-90). The effect of this change in the gold-point assignment on pyrometric, radiometric, and photometric measurement services provided by the National Institute of Standards and Technology is assessed.

4.
J Res Natl Inst Stand Technol ; 96(6): 647-668, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-28184140

RESUMO

An intercomparison of spectral irradiance measurements by 12 national laboratories has been carried out between 1987 and 1990. The intercomparison was conducted under the auspices of the Comité Consultatif de Photometrie et Radiometrie (CCPR) of the Comité International des Poids et Mesures, and the National Institute of Standards and Technology (NIST) served as the pilot laboratory. The spectral range of the intercomparison was 250 to 2400 nm and the transfer standards used were commercial tungsten-halogen lamps of two types. The world-wide consistency of the results (one standard deviation) was on the order of 1% in the visible spectral region and 2 to 4% in the ultraviolet and infrared portions of the spectrum. The intercomparison revealed no statistically significant differences between spectral-irradiance scales based on blackbody physics and absolute detector radiometry.

5.
J Res Natl Inst Stand Technol ; 95(6): 621-629, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-28179797

RESUMO

Following an absolute NIST measurement of the freezing temperature of gold and the adoption of the International Temperature Scale of 1990 (ITS-90), NIST has adopted new measurement scales for the calibration services based on thermal radiometry. In this paper, the new scales are defined and compared to the ITS-90, and the effects of the scale changes on NIST measurement services in optical pyrometry, radiometry, and photometry are assessed quantitatively. The changes in reported calibration values are within quoted uncertainties, and have resulted in small improvements in accuracy and better consistency with other radiometric scales.

6.
J Res Natl Inst Stand Technol ; 108(3): 199-228, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-27413606

RESUMO

As part of a continuing effort to validate the radiometric scales assigned to integrating sphere sources used in the calibration of Earth Observing System (EOS) instruments, a radiometric measurement comparison was held in May 1998 at Raytheon/Santa Barbara Remote Sensing (SBRS). This comparison was conducted in support of the calibration of the Moderate Resolution Imaging Spectroradiometer (MODIS) and the Landsat 7 Enhanced Thematic Mapper Plus (ETM+) instruments. The radiometric scale assigned to the Spherical Integrating Source (SIS100) by SBRS was validated through a comparison with radiometric measurements made by a number of stable, well-characterized transfer radiometers from the National Institute of Standards and Technology (NIST), the National Aeronautics and Space Administration's Goddard Space Flight Center (NASA's GSFC), and the University of Arizona Optical Sciences Center (UA). The measured radiances from the radiometers differed by ±3 % in the visible to near infrared when compared to the SBRS calibration of the sphere, and the overall agreement was within the combined uncertainties of the individual measurements. In general, the transfer radiometers gave higher values than the SBRS calibration in the near infrared and lower values in the blue. The measurements of the radiometers differed by ±4 % from 800 nm to 1800 nm compared to the SBRS calibration of the sphere, and the overall agreement was within the combined uncertainties of the individual measurements for wavelengths less than 2200 nm. The results of the radiometric measurement comparison presented here supplement the results of previous measurement comparisons on the integrating sphere sources used to calibrate the Multi-angle Imaging SpectroRadiometer (MISR) at NASA's Jet Propulsion Laboratory (JPL), Pasadena, CA and the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) at NEC Corporation, Yokohama, Japan.

8.
Biogerontology ; 10(3): 267-77, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18956248

RESUMO

The premature human ageing Werner's syndrome is caused by loss or mutation of the WRN helicase/exonuclease. We have recently identified the orthologue of the WRN exonuclease in flies, DmWRNexo, encoded by the CG7670 locus, and showed very high levels of mitotic recombination in a hypomorphic PiggyBac insertional mutant. Here, we report a novel allele of CG7670, with a point mutation resulting in the change of the conserved aspartate (229) to valine. Flies bearing this mutation show levels of mitotic recombination 20-fold higher than wild type. Molecular modelling suggests that D229 lies towards the outside of the molecule distant from the nuclease active site. We have produced recombinant protein of the D229V mutant, assayed its nuclease activity in vitro, and compared activity with that of wild type DmWRNexo and a D162A E164A double active site mutant we have created. We show for the first time that DmWRNexo has 3'-5' exonuclease activity and that mutation within the presumptive active site disrupts exonuclease activity. Furthermore, we show that the D229V mutant has very limited exonuclease activity in vitro. Using Drosophila, we can therefore analyse WRN exonuclease from enzyme activity in vitro through to fly phenotype, and show that loss of exonuclease activity contributes to genome instability.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Exodesoxirribonucleases/metabolismo , Exonucleases/metabolismo , Mutação Puntual , RecQ Helicases/metabolismo , Animais , Ácido Aspártico , Domínio Catalítico , Clonagem Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Exonucleases/química , Exonucleases/genética , Instabilidade Genômica , Genótipo , Humanos , Cinética , Mitose , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenótipo , Conformação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Temperatura , Valina , Helicase da Síndrome de Werner
9.
Aging Cell ; 7(3): 418-25, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18346216

RESUMO

The premature human aging Werner syndrome (WS) is caused by mutation of the RecQ-family WRN helicase, which is unique in possessing also 3'-5' exonuclease activity. WS patients show significant genomic instability with elevated cancer incidence. WRN is implicated in restraining illegitimate recombination, especially during DNA replication. Here we identify a Drosophila ortholog of the WRN exonuclease encoded by the CG7670 locus. The predicted DmWRNexo protein shows conservation of structural motifs and key catalytic residues with human WRN exonuclease, but entirely lacks a helicase domain. Insertion of a piggyBac element into the 5' UTR of CG7670 severely reduces gene expression. DmWRNexo mutant flies homozygous for this insertional allele of CG7670 are thus severely hypomorphic; although adults show no gross morphological abnormalities, females are sterile. Like human WS cells, we show that the DmWRNexo mutant flies are hypersensitive to the topoisomerase I inhibitor camptothecin. Furthermore, these mutant flies show highly elevated rates of mitotic DNA recombination resulting from excessive reciprocal exchange. This study identifies a novel WRN ortholog in flies and demonstrates an important role for WRN exonuclease in maintaining genome stability.


Assuntos
Drosophila melanogaster/genética , Instabilidade Genômica , Alelos , Sequência de Aminoácidos , Animais , Camptotecina/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Exodesoxirribonucleases/genética , Humanos , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , RecQ Helicases/genética , Recombinação Genética/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Síndrome de Werner/genética , Síndrome de Werner/patologia , Helicase da Síndrome de Werner
10.
Appl Opt ; 46(15): 2870-80, 2007 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-17514232

RESUMO

The development of a radiation thermometer calibrated for spectral radiance responsivity using cryogenic, electrical-substitution radiometry to determine the thermodynamic temperatures of the Ag- and Au-freezing temperatures is described. The absolute spectral radiance responsivity of the radiation thermometer is measured in the NIST Spectral Irradiance and Radiance Responsivity Calibrations using Uniform Sources (SIRCUS) facility with a total uncertainty of 0.15% (k=2) and is traceable to the electrical watt, and thus the thermodynamic temperature of any blackbody can be determined by using Planck radiation law and the measured optical power. The thermodynamic temperatures of the Ag- and Au-freezing temperatures are determined to be 1234.956 K (+/-0.110 K) (k=2) and 1337.344 K(+/-0.129 K) (k=2) differing from the International Temperature Scale of 1990 (ITS-90) assignments by 26 mK and 14 mK, respectively, within the stated uncertainties. The temperatures were systematically corrected for the size- of-source effect, the nonlinearity of the preamplifier and the emissivity of the blackbody. The ultimate goal of these thermodynamic temperature measurements is to disseminate temperature scales with lower uncertainties than those of the ITS-90. These results indicate that direct disseminations of thermodynamic temperature scales are possible.

11.
Appl Opt ; 46(1): 25-35, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17167550

RESUMO

A new facility for measuring irradiance in the UV was commissioned recently at the National Institute of Standards and Technology (NIST). The facility uses the calculable radiation from the Synchrotron Ultraviolet Radiation Facility as the primary standard. To measure the irradiance from a source under test, an integrating sphere spectrometer-detector system measures both the source under test and the synchrotron radiation sequentially, and the irradiance from the source under test can be determined. In particular, we discuss the calibration of deuterium lamps using this facility from 200 to 400 nm. This facility improves the current NIST UV irradiance scale to a relative measurement uncertainty of 1.2% (k=2).

12.
J Biol Chem ; 278(47): 46369-77, 2003 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-12954617

RESUMO

Glutamate-cysteine ligase (GCL) has a key influence on glutathione homeostasis. It has been proposed that mammalian GCL is regulated by the redox environment, and we show here that cysteine residues in the Drosophila melanogaster GCL modifier subunit (DmGCLM) can form covalent interactions with the catalytic subunit (DmGCLC) and modify its activity. Candidate components of intersubunit disulfides (Cys213, Cys214, and Cys267) were identified using matrix-assisted laser desorption ionization time-of-flight spectroscopy of iodoacetamide-modified DmGCLM as well as examination of the evolutionary conservation of cysteines. Mutation of the 3 cysteine residues allowed DmGCLM to associate with DmGCLC, but inhibited the formation of intersubunit disulfides. This caused a 2-fold reduction in the catalytic efficiency of Drosophila GCL, although activity remained significantly higher than the catalytic subunit alone. The cysteine mutant was also more sensitive to inhibition by glutathione than the unmodified holoenzyme. Notably, human GCLM could substitute for DmGCLM in modification of DmGCLC activity. The role of DmGCLM in vivo was examined by analysis of a Drosophila mutant (l(3)L0580) containing a P-element insertion in Gclm. We found that the P-element is not responsible for the lethal phenotype and separated the recessive lethal mutation from the P-element by recombination. This yielded two fully viable and fertile recombinants bearing the P-element insertion, which Western and Northern blotting indicated is a severely hypomorphic allele of Gclm. Glutathione levels were approximately 2-fold lower in the GclmL0580 mutants than in control strains, demonstrating the importance of DmGCLM in the regulation of glutathione homeostasis in vivo.


Assuntos
Proteínas de Drosophila/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Homeostase , Animais , Catálise , Sequência Conservada , Dissulfetos , Proteínas de Drosophila/genética , Evolução Molecular , Glutamato-Cisteína Ligase/genética , Mutação , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
J Biol Chem ; 277(2): 1158-65, 2002 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-11698394

RESUMO

Glutamate-cysteine ligase (GCL) plays an important role in regulating glutathione homeostasis. In mammals, it comprises a catalytic (GCLC) and modifier (GCLM) subunit. The existence of a modifier subunit in invertebrates has not been described to date. We now demonstrate that GCL from Drosophila melanogaster has a functional modifier subunit (DmGCLM). A putative DmGCLM was obtained as an expressed sequence tag with 27% identity to human GCLM at the amino acid level. D. melanogaster GCLC (DmGCLC) and the candidate DmGCLM were expressed separately in Escherichia coli, purified, mixed, and then subjected to gel filtration, where they eluted as an approximately 140-kDa complex. DmGCLC co-immunoprecipitated with DmGCLM from S2 cell extracts, suggesting that they also associate in vivo. Enzyme kinetic analyses showed that DmGCLC has a K(m) for glutamate of 2.88 mm, but when complexed with DmGCLM, the K(m) for glutamate is 0.45 mm. Inhibition of DmGCLC activity by glutathione was found to be competitive with respect to glutamate (K(i) = 0.03 mm), whereas inhibition of the GCL complex was mixed (K(i) = 0.67 mm), suggesting allosteric effects. In accordance with this, DmGCLC and DmGCLM have the ability to form reversible intermolecular disulfide bridges. A further mechanism for control of D. melanogaster GCL was found to be induction of DmGCLC by tert-butylhydroquinone in S2 cells. DmGCLM levels were, however, unaffected by tert-butylhydroquinone.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Glutamato-Cisteína Ligase/metabolismo , Sequência de Aminoácidos , Animais , Antioxidantes/farmacologia , Butionina Sulfoximina/farmacologia , Células Cultivadas , Cromatografia em Gel , Meios de Cultura Livres de Soro , Cistamina/farmacologia , Proteínas de Drosophila/química , Drosophila melanogaster/citologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/fisiologia , Inibidores Enzimáticos/farmacologia , Etiquetas de Sequências Expressas , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutamato-Cisteína Ligase/química , Glutationa/farmacologia , Humanos , Hidroquinonas/farmacologia , Dados de Sequência Molecular , Subunidades Proteicas , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
14.
Development ; 130(13): 2997-3005, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12756181

RESUMO

At the transition from meiosis to cleavage mitoses, Drosophila requires the cell cycle regulators encoded by the genes, giant nuclei (gnu), plutonium (plu) and pan gu (png). Embryos lacking Gnu protein undergo DNA replication and centrosome proliferation without chromosome condensation or mitotic segregation. We have identified the gnu gene encoding a novel phosphoprotein dephosphorylated by Protein phosphatase 1 at egg activation. Gnu is normally expressed in the nurse cells and oocyte of the ovary and is degraded during the embryonic cleavage mitoses. Ovarian death and sterility result from gnu gain of function. gnu function requires the activity of pan gu and plu.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Meiose/fisiologia , Mitose/fisiologia , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Epistasia Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Infertilidade , Masculino , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/fisiologia , Ovário/citologia , Ovário/fisiologia , Fenótipo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transgenes
15.
EMBO Rep ; 3(1): 34-8, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11751581

RESUMO

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.


Assuntos
Mapeamento Cromossômico , Drosophila melanogaster/genética , Cromossomo X , Animais , Cromossomos Artificiais Bacterianos , Mapeamento de Sequências Contíguas , Sondas de DNA , Elementos de DNA Transponíveis , Feminino , Genes Essenciais , Genes de Insetos , Masculino , Mutagênese
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