Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Nucleic Acids Res ; 47(18): 9829-9841, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31396626

RESUMO

Elevated temperatures activate a heat shock response (HSR) to protect cells from the pathological effects of protein mis-folding, cellular mis-organization, organelle dysfunction and altered membrane fluidity. This response includes activation of the conserved transcription factor heat shock factor 1 (HSF-1), which binds heat shock elements (HSEs) in the promoters of genes induced by heat shock (HS). The upregulation of protein-coding genes (PCGs), such as heat shock proteins and cytoskeletal regulators, is critical for cellular survival during elevated temperatures. While the transcriptional response of PCGs to HS has been comprehensively analyzed in a variety of organisms, the effect of this stress on the expression of non-coding RNAs (ncRNAs) has not been systematically examined. Here we show that in Caenorhabditis elegans HS induces up- and downregulation of specific ncRNAs from multiple classes, including miRNA, piRNA, lincRNA, pseudogene and repeat elements. Moreover, some ncRNA genes appear to be direct targets of the HSR, as they contain HSF-1 bound HSEs in their promoters and their expression is regulated by this factor during HS. These results demonstrate that multiple ncRNA genes respond to HS, some as direct HSF-1 targets, providing new candidates that may contribute to organismal survival during this stress.


Assuntos
Caenorhabditis elegans/genética , Fatores de Transcrição de Choque Térmico/genética , RNA não Traduzido/genética , Transcriptoma/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Fatores de Transcrição de Choque Térmico/química , Resposta ao Choque Térmico/genética , Regiões Promotoras Genéticas , Ativação Transcricional/genética
2.
PLoS Genet ; 14(6): e1007379, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29927939

RESUMO

Argonaute (AGO) proteins partner with microRNAs (miRNAs) to target specific genes for post-transcriptional regulation. During larval development in Caenorhabditis elegans, Argonaute-Like Gene 1 (ALG-1) is the primary mediator of the miRNA pathway, while the related ALG-2 protein is largely dispensable. Here we show that in adult C. elegans these AGOs are differentially expressed and, surprisingly, work in opposition to each other; alg-1 promotes longevity, whereas alg-2 restricts lifespan. Transcriptional profiling of adult animals revealed that distinct miRNAs and largely non-overlapping sets of protein-coding genes are misregulated in alg-1 and alg-2 mutants. Interestingly, many of the differentially expressed genes are downstream targets of the Insulin/ IGF-1 Signaling (IIS) pathway, which controls lifespan by regulating the activity of the DAF-16/ FOXO transcription factor. Consistent with this observation, we show that daf-16 is required for the extended lifespan of alg-2 mutants. Furthermore, the long lifespan of daf-2 insulin receptor mutants, which depends on daf-16, is strongly reduced in animals lacking alg-1 activity. This work establishes an important role for AGO-mediated gene regulation in aging C. elegans and illustrates that the activity of homologous genes can switch from complementary to antagonistic, depending on the life stage.


Assuntos
Proteínas Argonautas/fisiologia , Caenorhabditis elegans/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Longevidade/genética , MicroRNAs/fisiologia , RNA de Helmintos/fisiologia , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Genes de Helmintos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Mutação , Proteínas de Ligação a RNA/fisiologia , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais/fisiologia
3.
Opt Lett ; 44(17): 4223-4226, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31465367

RESUMO

We developed a short-range light detection and ranging system using a supercontinuum (SC) source spectrally tailored to cover the ro-vibrational transition energies of desired components of a flue gas. The system enables remote measurements of the gas parameters, including temperature and concentration which play a key role in the performance of combustion power plants. The technique requires only one inspection window and, thus, can be used in combustion units with limited access. It exploits differential absorption between specific wavelength bands of the gas absorption spectrum. The transmittance of an individual wavelength band is derived from the detected backscattered temporal intensity of the SC pulses. We demonstrate water vapor temperature measurement in the range of 400°C-900°C in a laboratory furnace with the use of only two wavelength bands. Using more than two wavelength bands, the technique can be further extended to simultaneously measure temperature and concentration. By varying the direction of the incident beam in a non-parallel plane, a full 3D profile is also obtainable.

4.
Opt Lett ; 43(20): 5094-5097, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30320828

RESUMO

We demonstrate cantilever-enhanced photoacoustic spectroscopy in the mid-infrared using a supercontinuum source. The approach is broadband and allows for higher photoacoustic signal intensity and an enhanced signal-to-noise ratio as compared to systems employing conventional black body radiation sources. Using this technique, we perform spectroscopic measurements of the full ro-vibrational band structure of water vapor at 1900 nm and methane at 3300 nm with relative signal enhancement factors of 70 and 19, respectively, when compared to measurements that use the black body radiation source. Our results offer a novel perspective for photoacoustic detection opening the door to sensitive broadband analyzers in the mid-infrared spectral region.

5.
Opt Express ; 23(19): 25225-34, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26406720

RESUMO

We investigate incoherent broadband cavity enhanced absorption spectroscopy using a tailored supercontinuum source. By tailoring the supercontinuum spectrum to match the high reflectivity bandwidth of the mirrors, we achieve an unprecedented spectral brightness of more than 7 dBm/nm at wavelengths where the effective absorption path length in the cavity exceeds 40 km. We demonstrate the potential of the source in spectrally broadband measurement of weak overtone transitions of carbon dioxide and methane in the near-infrared 1590 nm - 1700 nm range and evaluate its performance against that of a typical superluminescent diode source. Minimum detectable absorption coefficients (3σ) of 2.2 × 10(-9) cm(-1) and 6.2 × 10(-9) cm(-1) are obtained with the supercontinuum and the superluminescent diode sources, respectively. We further develop a spectral fitting method based on differential optical absorption spectroscopy to fully and properly account for the combined effect of absorption line saturation and limited spectral resolution of the detection. The method allows to cope with high dynamic range of absorption features typical of real-world multi-component measurements.

6.
Nature ; 459(7244): 274-7, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19444217

RESUMO

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have important roles in gene regulation, germ cell maintenance and transposon silencing. The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa. This class of small RNAs, known as qiRNAs because of their interaction with QDE-2, are about 20-21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5' end, and originate mostly from the ribosomal DNA locus. The production of qiRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. qiRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE-1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for qiRNAs in the DNA-damage response by inhibiting protein translation.


Assuntos
Dano ao DNA/genética , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genética , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Ribossômico/genética , DNA de Cadeia Simples , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Neurospora crassa/enzimologia , Biossíntese de Proteínas , RNA Fúngico/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Moldes Genéticos
7.
Proc Natl Acad Sci U S A ; 109(18): 7079-84, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22509017

RESUMO

The multitude of archaea and bacteria inhabiting extreme environments has only become evident during the last decades. As viruses apply a significant evolutionary force to their hosts, there is an inherent value in learning about viruses infecting these extremophiles. In this study, we have focused on one such unique virus-host pair isolated from a hypersaline environment: an icosahedral, membrane-containing double-stranded DNA virus--Salisaeta icosahedral phage 1 (SSIP-1) and its halophilic host bacterium Salisaeta sp. SP9-1 closely related to Salisaeta longa. The architectural principles, virion composition, and the proposed functions associated with some of the ORFs of the virus are surprisingly similar to those found in viruses belonging to the PRD1-adenovirus lineage. The virion structure, determined by electron cryomicroscopy, reveals that the bulk of the outer protein capsid is composed of upright standing pseudohexameric capsomers organized on a T = 49 icosahedral lattice. Our results give a comprehensive description of a halophilic virus-host system and shed light on the relatedness of viruses based on their virion architecture.


Assuntos
Bacteriófagos/genética , Bacteroidetes/virologia , Evolução Molecular , Bacteriófagos/patogenicidade , Bacteriófagos/fisiologia , Bacteriófagos/ultraestrutura , Sequência de Bases , Capsídeo/ultraestrutura , Microscopia Crioeletrônica , DNA Viral/genética , Meio Ambiente , Genoma Viral , Interações Hospedeiro-Patógeno , Imageamento Tridimensional , Dados de Sequência Molecular , Fases de Leitura Aberta , Solução Salina Hipertônica , Integração Viral
8.
Heliyon ; 10(13): e33439, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39040318

RESUMO

Background: Knowledge of prevalence and epidemiology of cystic fibrosis (CF) and healthcare resource use among Finnish people with CF is incomplete. Methods: We conducted a population-based matched cohort study using retrospective real-world data from linked Finnish national registries. Electronic healthcare data and drug purchases of 102 people with CF were analyzed between January 2015 and December 2019 (follow-up). A 5-fold control population was matched by sex, age, and place of residence. Comorbidities and medication use that occurred at any time during follow-up were assessed; annual rates of hospital service use were adjusted for follow-up. Results: The prevalence of CF in Finland was 1.85 per 100,000. Median age at diagnosis was 1 year, with 60 % diagnosed at age <2 years and 80 % at age <10 years. Median age at death in people with CF was 31.4 years (n < 5); no controls died. The most common comorbidities included chronic sinusitis (39.2 %), pneumonia (38.2 %), diabetes (20.6 %), and nasal polyps (18.6 %). The most purchased medications were antibiotics (99.0 %) and pancreatic enzymes (84.3 %). The annualized rate of hospital visits was higher in people with CF vs controls (outpatient: mean [SD], 17.4 [14.5] vs 0.9 [3.3]; median, 13.6 vs 0.4, respectively; inpatient: mean [SD], 1.0 [1.66] vs 0.03 [0.14]; median, 0.34 vs 0, respectively). Conclusions: The prevalence of CF in Finland is remarkably low, likely reflecting unique population characteristics and, in part, delayed diagnosis. Antibiotic use is frequent among Finnish people with CF. Inpatient hospital visits are >30 times higher in people with CF than matched controls.

9.
PLoS Biol ; 8(10)2010 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-20957187

RESUMO

The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.


Assuntos
DNA Helicases/metabolismo , Proteínas Fúngicas/metabolismo , Interferência de RNA , RNA de Cadeia Dupla , RNA Polimerase Dependente de RNA/metabolismo , RNA , Proteína de Replicação A/metabolismo , Animais , DNA Helicases/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Neurospora crassa/genética , Neurospora crassa/metabolismo , RNA/genética , RNA/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Polimerase Dependente de RNA/genética , Proteína de Replicação A/genética , Ribonucleases/genética , Ribonucleases/metabolismo
10.
J Biol Chem ; 285(38): 29367-74, 2010 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-20647305

RESUMO

QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases.


Assuntos
Proteínas Fúngicas/metabolismo , Neurospora crassa/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Mutagênese Sítio-Dirigida , Neurospora crassa/genética , RNA Polimerase Dependente de RNA/genética , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Nucleic Acids Res ; 37(4): 1182-92, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19129226

RESUMO

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage phi 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.


Assuntos
Bacteriófago phi 6/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , RNA/biossíntese , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA/química , RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , Moldes Genéticos , Proteínas Virais/genética
12.
Opt Express ; 18(2): 1234-9, 2010 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-20173946

RESUMO

We study experimentally the statistical fluctuations observed in a supercontinuum generated in the normal dispersion regime through cascaded stimulated Raman scattering. Specifically, we show that the statistical distribution of shot-to-shot spectral variations evolves from a quasi-Gaussian in the saturated regime for Stokes orders near the pump to a long-tailed extreme-value distribution for Stokes orders at a large separation from the pump in the unsaturated regime.


Assuntos
Modelos Estatísticos , Análise Espectral Raman/métodos , Simulação por Computador , Interpretação Estatística de Dados , Luz , Espalhamento de Radiação
13.
Nucleic Acids Res ; 36(22): 7059-67, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18986997

RESUMO

RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regulatory mechanism for RNA polymerases that has also been implicated in RNA recombination, has not been considered. Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling. The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure. This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.


Assuntos
Bacteriófago phi 6/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , RNA/biossíntese , Proteínas Virais/metabolismo , Bacteriófago phi 6/fisiologia , Cinética , Nucleotídeos/metabolismo , RNA/química , Temperatura , Transcrição Gênica , Replicação Viral
14.
J Gen Virol ; 90(Pt 10): 2468-2473, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19553393

RESUMO

Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage phi6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837-7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the phi6 RdRP (phi6-siRNAs) was considerably more effective than single-site siRNAs. The phi6-siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.


Assuntos
Bacteriófago phi 6/enzimologia , Enterovirus Humano B/fisiologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Animais , Linhagem Celular , Camundongos , Replicação Viral/fisiologia
15.
Eur J Hum Genet ; 15(2): 185-93, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17003839

RESUMO

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion of a dodecamer repeat in the CSTB promoter accounts for the majority of EPM1 disease alleles worldwide. We here describe a novel PCR protocol for detection of the dodecamer repeat expansion. We describe two novel EPM1-associated mutations, c.149G > A leading to the p.G50E missense change and an intronic 18-bp deletion (c.168+1_18del), which affects splicing of CSTB. The p.G50E mutation that affects the conserved QVVAG amino acid sequence critical for cathepsin binding fails to associate with lysosomes. This further supports the previously implicated physiological importance of the CSTB-lysosome association. Expression of CSTB mRNA and protein was markedly reduced in lymphoblastoid cells of the patients irrespective of the mutation type. Patients homozygous for the dodecamer expansion mutation showed 5-10% expression compared to controls. By combining database searches with RT-PCR we identified several alternatively spliced CSTB isoforms. One of these, CSTB2, was also present in mouse and was analyzed in more detail. In real-time PCR quantification, CSTB2 expression was less than 5% of total CSTB expression in all human adult and fetal tissues analyzed. In patients homozygous for the minisatellite mutation, the level of CSTB2 was reduced similarly to that of CSTB implicating regulation from the same promoter. The physiological significance of CSTB2 remains to be determined.


Assuntos
Cistatinas/genética , Epilepsias Mioclônicas Progressivas/genética , Síndrome de Unverricht-Lundborg/genética , Processamento Alternativo/genética , Cistatina B , Cistatinas/análise , Cistatinas/metabolismo , Análise Mutacional de DNA , Feminino , Expressão Gênica , Homozigoto , Humanos , Masculino , Repetições de Microssatélites , Mutação , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas/genética , RNA Mensageiro/análise
16.
BMC Neurosci ; 6: 27, 2005 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-15826318

RESUMO

BACKGROUND: The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by accumulation of autofluorescent material in many tissues, especially in neurons. Mutations in the CLN8 gene, encoding an endoplasmic reticulum (ER) transmembrane protein of unknown function, underlie NCL phenotypes in humans and mice. The human phenotype is characterized by epilepsy, progressive psychomotor deterioration and visual loss, while motor neuron degeneration (mnd) mice with a Cln8 mutation show progressive motor neuron dysfunction and retinal degeneration. RESULTS: We investigated spatial and temporal expression of Cln8 messenger ribonucleic acid (mRNA) using in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and northern blotting. Cln8 is ubiquitously expressed at low levels in embryonic and adult tissues. In prenatal embryos Cln8 is most prominently expressed in the developing gastrointestinal tract, dorsal root ganglia (DRG) and brain. In postnatal brain the highest expression is in the cortex and hippocampus. Expression of Cln8 mRNA in the central nervous system (CNS) was also analyzed in the hippocampal electrical kindling model of epilepsy, in which Cln8 expression was rapidly up-regulated in hippocampal pyramidal and granular neurons. CONCLUSION: Expression of Cln8 in the developing and mature brain suggests roles for Cln8 in maturation, differentiation and supporting the survival of different neuronal populations. The relevance of Cln8 up-regulation in hippocampal neurons of kindled mice should be further explored.


Assuntos
Encéfalo/metabolismo , Epilepsia/metabolismo , Hipocampo/metabolismo , Excitação Neurológica/metabolismo , Proteínas de Membrana/biossíntese , Neurônios/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Epilepsia/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Excitação Neurológica/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Regulação para Cima/fisiologia
17.
WormBook ; : 1-18, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24967700

RESUMO

Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteoma , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Imunoprecipitação , Espectrometria de Massas , Complexos Multiproteicos , Processamento de Proteína Pós-Traducional , Proteômica/métodos
18.
Curr Opin Cell Biol ; 24(3): 333-40, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22464106

RESUMO

During the past decade, it has become evident that small non-coding RNAs (ncRNAs) participate in widespread and essential regulatory mechanisms in most eukaryotic cells. Novel classes of small RNAs, their biogenesis pathways and cellular effects are continuously being described, and new properties of already established ncRNAs are still being discovered. As the list of small RNA molecules and their roles becomes more and more extensive, one can get lost in the midst of new information. In this review, we attempt to bring order to the small ncRNA transcriptome by covering some of the major milestones of recent years. We go through many of the new properties that have been attributed to already familiar RNA molecules, and introduce some of the more recent novel classes of tiny ncRNAs.


Assuntos
Expressão Gênica , Pequeno RNA não Traduzido/fisiologia , Regulação da Expressão Gênica , Humanos , MicroRNAs , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Transcriptoma
19.
RNA ; 13(3): 422-9, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17237359

RESUMO

The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.


Assuntos
Bacteriófago phi 6/enzimologia , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA de Cadeia Dupla/biossíntese , RNA Interferente Pequeno/biossíntese , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Animais , Linhagem Celular , Humanos , Interferência de RNA , RNA de Cadeia Dupla/farmacologia , RNA Interferente Pequeno/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA