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1.
J Adv Pract Oncol ; 12(7): 705-714, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34671500

RESUMO

PURPOSE: The objective of this study is to describe characteristics and short- and long-term outcomes of patients with hematologic malignancies who received cardiopulmonary resuscitation (CPR). METHODS: A retrospective review was conducted of all Code Blues at a large comprehensive cancer center. Demographic, clinical, and outcome variables were analyzed for patients with a hematologic malignancy who underwent CPR. RESULTS: Of 258 patients, 60.1% had leukemia. Outcomes included return of spontaneous circulation (70.2%), hospital survival (12%), and 90-day, 6-month, and 1-year survival rates of 9.8%, 8.2%, and 5.9%, respectively. Factors associated with hospital mortality included establishing a do not resuscitate order after CPR (p < .0001), location of CPR (p = .0004), cause of arrest (p = .0019), requiring vasopressors (p = .0130), mechanical ventilation (p = .0423), and acute renal failure post CPR (p = .0006). Although no difference in hospital survival between leukemia and non-leukemia patients was found, more non-leukemia patients were alive at 90 days (p = .0099), 6 months (p = .0023), and 1 year (p = .0119). CONCLUSIONS: Factors including organ dysfunction, location of CPR, and cause of arrest are associated with hospital mortality post CPR. However, immediate survival post CPR does not seem to be affected by a diagnosis of leukemia. These data should assist health care providers with discussions regarding advance care planning and goals of care after cardiac arrest.

2.
Astrobiology ; 13(6): 560-9, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23734755

RESUMO

Life beyond Earth may be based on RNA or DNA if such life is related to life on Earth through shared ancestry due to meteoritic exchange, such as may be the case for Mars, or if delivery of similar building blocks to habitable environments has biased the evolution of life toward utilizing nucleic acids. In this case, in situ sequencing is a powerful approach to identify and characterize such life without the limitations or expense of returning samples to Earth, and can monitor forward contamination. A new semiconductor sequencing technology based on sensing hydrogen ions released during nucleotide incorporation can enable massively parallel sequencing in a small, robust, optics-free CMOS chip format. We demonstrate that these sequencing chips survive several analogues of space radiation at doses consistent with a 2-year Mars mission, including protons with solar particle event-distributed energy levels and 1 GeV oxygen and iron ions. We find no measurable impact of irradiation at 1 and 5 Gy doses on sequencing quality nor on low-level hardware characteristics. Further testing is required to study the impacts of soft errors as well as to characterize performance under neutron and gamma irradiation and at higher doses, which would be expected during operation in environments with significant trapped energetic particles such as during a mission to Europa. Our results support future efforts to use in situ sequencing to test theories of panspermia and/or whether life has a common chemical basis.


Assuntos
Vida , Tolerância a Radiação , Marte
3.
Nature ; 475(7356): 348-52, 2011 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-21776081

RESUMO

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.


Assuntos
Genoma Bacteriano/genética , Genoma Humano/genética , Genômica/instrumentação , Genômica/métodos , Semicondutores , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/métodos , Escherichia coli/genética , Humanos , Luz , Masculino , Rodopseudomonas/genética , Vibrio/genética
4.
PLoS Biol ; 7(11): e1000238, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19901979

RESUMO

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Linhagem Celular , Fatores de Iniciação em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Genes , Genoma Humano , Humanos , Iniciação Traducional da Cadeia Peptídica , Estabilidade de RNA , Ribossomos/metabolismo
5.
Diabetes ; 57(7): 1896-904, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18420486

RESUMO

OBJECTIVE: The transcription factor nuclear factor-kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK) 1/2 are known to play decisive roles in cytokine-induced damage of rodent beta-cells. The upstream events by which these factors are activated in response to cytokines are, however, uncharacterized. The aim of the present investigation was to elucidate a putative role of the MAPK kinase kinase-1 (MEKK-1) in cytokine-induced signaling. RESEARCH DESIGN AND METHODS: To establish a functional role of MEKK-1, the effects of transient MEKK-1 overexpression in betaTC-6 cells, achieved by lipofection and cell sorting, and MEKK-1 downregulation in betaTC-6 cells and human islet cells, achieved by diced-small interfering RNA treatment, were studied. RESULTS: We observed that overexpression of wild-type MEKK-1, but not of a kinase dead MEKK-1 mutant, resulted in potentiation of cytokine-induced JNK activation, inhibitor of kappaB (IkappaB) degradation, and cell death. Downregulation of MEKK-1 in human islet cells provoked opposite effects, i.e., attenuation of cytokine-induced JNK and MKK4 activation, IkappaB stability, and a less pronounced NF-kappaB translocation. betaTC-6 cells with a downregulated MEKK-1 expression displayed also a weaker cytokine-induced iNOS expression and lower cell death rates. Also primary mouse islet cells with downregulated MEKK-1 expression were protected against cytokine-induced cell death. CONCLUSIONS: MEKK-1 mediates cytokine-induced JNK- and NF-kappaB activation, and this event is necessary for iNOS expression and cell death.


Assuntos
Citocinas/farmacologia , Ilhotas Pancreáticas/enzimologia , MAP Quinase Quinase Quinase 1/metabolismo , NF-kappa B/fisiologia , Animais , Morte Celular , DNA Complementar/genética , Indução Enzimática , Genes Reporter , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Fosforilação , RNA Interferente Pequeno/genética
6.
Endocrinology ; 149(6): 3046-53, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18308848

RESUMO

The aim of the present investigation was to characterize the role of the MAPK kinase kinase-1 (MEKK-1) in stress-induced cell death of insulin producing cells. We observed that transient overexpression of the wild type MEKK-1 protein in the insulin-producing cell lines RIN-5AH and betaTC-6 increased c-Jun N-terminal kinase (JNK) phosphorylation and augmented cell death induced by diethylenetriamine/nitroso-1-propylhydrazino)-1-propanamine (DETA/NO), streptozotocin (STZ), and hydrogen peroxide (H2O2). Furthermore, DETA/NO or STZ induced a rapid threonine phosphorylation of MEKK-1. Silencing of MEKK-1 gene expression in betaTC-6 and human dispersed islet cells, using in vitro-generated diced small interfering RNA, resulted in protection from DETA/NO, STZ, H2O2, and tunicamycin induced cell death. Moreover, in DETA/NO-treated cells diced small interfering RNA-mediated down-regulation of MEKK-1 resulted in decreased activation of JNK but not p38 and ERK. Inhibition of JNK by treatment with SP600125 partially protected against DETA/NO- or STZ-induced cell death. In summary, our results support an essential role for MEKK-1 in JNK activation and stress-induced beta-cell death. Increased understanding of the signaling pathways that augment or diminish beta-cell MEKK-1 activity may aid in the generation of novel therapeutic strategies in the treatment of type 1 diabetes.


Assuntos
Ilhotas Pancreáticas/enzimologia , MAP Quinase Quinase Quinase 1/metabolismo , Animais , Morte Celular , Sobrevivência Celular , Diabetes Mellitus Tipo 1/terapia , Citometria de Fluxo , Genes Reporter , Humanos , Insulinoma/enzimologia , Insulinoma/patologia , Ilhotas Pancreáticas/citologia , MAP Quinase Quinase Quinase 1/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas
7.
Endocrinology ; 149(1): 302-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17932218

RESUMO

Previous studies have indicated that the p38 MAPK participates in signaling events that lead to the death of the insulin-producing beta-cell. The aim of the present study was to elucidate the role of the TGF-beta-activated protein kinase 1-binding protein 1 (TAB1) in the cytokine-induced activation of p38. Levels of TAB1 mRNA and protein were analyzed by real-time PCR and immunoblotting, and TAB1 expression in mouse and human islet cells was down-regulated using lipofection of diced-small interfering RNA. TAB1 overexpression in beta-TC6 cells was achieved by transient transfections followed by fluorescence activated cell sorting. Phosphorylation of p38, c-Jun N-terminal kinase, and ERK was assessed by immunoblotting, and viability was determined using vital staining with bisbenzimide and propidium iodide. We observed that TAB1 is expressed in insulin-producing cells. Cytokine (IL-1beta + interferon-gamma)-stimulated p38 phosphorylation was significantly increased by TAB1alpha overexpression, but not TAB1beta overexpression, in beta-TC6 cells. The TAB1alpha-augmented p38 phosphorylation was paralleled by an increased cell death rate. Treatment of islet cells with diced-small interfering RNA specific for TAB1, but not for TGF-beta-activated kinase 1, resulted in lowered cytokine-induced p38 phosphorylation and protection against cell death. The cytokine-induced phosphorylation of c-Jun N-terminal kinase and ERK was not affected by changes in TAB1 levels. Finally, TAB1 phosphorylation was decreased by the p38 inhibitor SB203580. We conclude that TAB1alpha, but not TAB1beta, plays an important role in the activation of p38 in insulin-producing cells and therefore also in cytokine-induced beta-cell death.


Assuntos
Citocinas/farmacologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/fisiologia , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Transfecção
8.
Genome Biol ; 8(7): R142, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17640392

RESUMO

BACKGROUND: Iron uptake via endocytosis of iron-transferrin-transferrin receptor complexes is a rate-limiting step for cell growth, viability and proliferation in tumor cells as well as non-transformed cells such as activated lymphocytes. Signaling pathways that regulate transferrin uptake have not yet been identified. RESULTS: We surveyed the human signaling proteome for regulators that increase or decrease transferrin uptake by screening 1,804 dicer-generated signaling small interfering RNAs using automated quantitative imaging. In addition to known transport proteins, we identified 11 signaling proteins that included a striking signature set for the phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)-target of rapamycin (mTOR) signaling pathway. We show that the PI3K-mTOR signaling pathway is a positive regulator of transferrin uptake that increases the number of transferrin receptors per endocytic vesicle without affecting endocytosis or recycling rates. CONCLUSION: Our study identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a new regulator of iron-transferrin uptake and serves as a proof-of-concept that targeted RNA interference screens of the signaling proteome provide a powerful and unbiased approach to discover or rank signaling pathways that regulate a particular cell function.


Assuntos
Fosfatos de Fosfatidilinositol/fisiologia , Proteínas Quinases/fisiologia , Proteoma/fisiologia , Proteômica/métodos , Transferrina/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Proteínas Quinases/genética , Transporte Proteico/efeitos dos fármacos , Proteoma/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Serina-Treonina Quinases TOR , Transfecção
9.
Curr Biol ; 17(1): 85-91, 2007 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-17208191

RESUMO

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.


Assuntos
Centrossomo/metabolismo , Ciclina A/fisiologia , Ciclina B/fisiologia , Mitose/fisiologia , Membrana Nuclear/metabolismo , Núcleo Celular/metabolismo , Ciclina A2 , Ciclina B/metabolismo , Ciclina B1 , Ciclina B2 , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos
10.
Biochem J ; 393(Pt 1): 129-39, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16097952

RESUMO

The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced beta-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2'-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38alpha-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCdelta (protein kinase Cdelta), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1beta and interferon-gamma, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.


Assuntos
Citocinas/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , MAP Quinase Quinase 3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , MAP Quinase Quinase 3/genética , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/metabolismo , Camundongos , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno/genética
11.
J RNAi Gene Silencing ; 2(2): 181-94, 2006 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-19771225

RESUMO

Microarray studies have shown that individual synthetic small interfering RNAs (siRNAs) can have substantial off-target effects. Pools of siRNAs, produced by incubation of dsRNAs with recombinant Dicer or RNase III, can also be used to silence genes. Here we show that diced siRNA pools are highly complex, containing hundreds of different individual siRNAs. This high complexity could either compound the problem of off-target effects, since the number of potentially problematic siRNAs is high, or it could diminish the problem, since the concentration of any individual problematic siRNA is low. We therefore compared the off-target effects of diced siRNAs to chemically synthesized siRNAs. In agreement with previous reports, we found that two chemically synthesized siRNAs targeted against p38alpha MAPK (MAPK14) induced off-target changes in the abundance of hundreds of mRNAs. In contrast, three diced siRNA pools against p38alpha MAPK had almost no off-target effects. The off-target effects of a synthetic siRNA were reduced when the siRNA was diluted 3-fold in a diced pool and completely alleviated when it was diluted 30- or 300-fold, suggesting that when problematic siRNAs are present within a diced pool, their absolute concentration is too low to result in significant off-target effects. These data rationalize the observed high specificity of RNA interference in C. elegans and D. melanogaster, where gene suppression is mediated by endogenously-generated diced siRNA pools, and provide a strategy for improving the specificity of RNA interference experiments and screens in mammalian cells.

12.
Curr Biol ; 15(13): 1235-41, 2005 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-16005298

RESUMO

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/fisiologia , Moléculas de Adesão Celular , Retículo Endoplasmático/metabolismo , Fluorescência , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Jurkat , Proteínas de Membrana/genética , Mutação/genética , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/genética , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal
14.
Ann N Y Acad Sci ; 1040: 114-22, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15891014

RESUMO

RNA interference (RNAi) is emerging as a powerful and convenient tool for studying gene function and genetic variation. RNAi is mediated by 21- to 23-nucleotide-long, small interfering RNAs (siRNA) produced from larger double-stranded RNAs in vivo by the RNase III family enzyme Dicer. To overcome the problems associated with the use of predesigned synthetic siRNA molecules, a novel method utilizing the in vitro activity of recombinant Dicer has been developed recently. In nonislet cells, it has been demonstrated that a pool of siRNA, generated by Dicer from in vitro transcribed dsRNA (d-siRNA), mediates convenient, efficient, and reproducible gene silencing in various cell types. The aim of this study was to evaluate the ability of d-siRNA to silence endogenous gene expression in pancreatic islet cells. We observed that liposomal transfection mediates efficient transport of siRNA in up to 90% of dispersed islet cells and that d-siRNA mediates almost complete and nontoxic silencing of an endogenous mRNA, the messenger coding for the nonreceptor tyrosine kinase c-Abl. The approach described here using d-siRNA provides an important tool for elucidating gene function in further studies of pancreatic islets and diabetes pathophysiology.


Assuntos
Inativação Gênica/fisiologia , Ilhotas Pancreáticas/metabolismo , RNA Interferente Pequeno/biossíntese , Ribonuclease III/fisiologia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Ribonuclease III/biossíntese , Ribonuclease III/genética
15.
Nat Biotechnol ; 22(3): 306-12, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14990952

RESUMO

Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.


Assuntos
Técnicas Biossensoriais/métodos , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/métodos , Mitose/fisiologia , Animais , Técnicas Biossensoriais/instrumentação , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular , Desenho de Equipamento , Análise de Falha de Equipamento , Microscopia de Fluorescência/instrumentação , Microscopia de Vídeo/instrumentação , Ratos
16.
Nat Genet ; 36(2): 183-9, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14704668

RESUMO

Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme-generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 x 10(5) clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.


Assuntos
Enzimas de Restrição do DNA/metabolismo , Vetores Genéticos , Biblioteca Genômica , RNA Interferente Pequeno/metabolismo , Animais , Inativação Gênica/fisiologia , Camundongos , Mioblastos/metabolismo
17.
Neuron ; 39(2): 283-97, 2003 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-12873385

RESUMO

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.


Assuntos
Benzilaminas , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Neuritos/fisiologia , Isoformas de Proteínas/fisiologia , Sulfonamidas , Sinapses/fisiologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/classificação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Fracionamento Celular/métodos , Movimento Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Fluorescência Verde , Immunoblotting , Imuno-Histoquímica , Isoquinolinas/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Isoformas de Proteínas/genética , Interferência de RNA/fisiologia , Ratos , Sinapses/efeitos dos fármacos , Fatores de Tempo , Transfecção
18.
Nat Biotechnol ; 21(3): 324-8, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12592410

RESUMO

RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.


Assuntos
Interferência de RNA/fisiologia , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/química , Linhagem Celular , Endorribonucleases , Humanos , Rim/embriologia , Rim/metabolismo , Luciferases/biossíntese , Luciferases/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Ribonuclease III , Transfecção/métodos
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