RESUMEN
Backgroundand Objectives: Delay of reperfusion therapy is related to high mortality in cases of ST-segment elevation myocardial infarction (STEMI). Guidelines emphasize that the first-medical-contact-to-balloon (FMCTB) time should be within 90 min. A mobile cloud-based 12-lead electrocardiogram (MC-ECG) transmission system might be useful in such cases, especially in rural areas. Materials and Methods: From April 2019 to June 2021, both an MC-ECG transmission system and the conventional method in which a physician checks the ECG in a hospital (Conventional) were used for transport by emergency medical services in Shin-Yukuhashi Hospital, Fukuoka, Japan. During this period, 8684 consecutive patients were transported to this hospital. Among them, we investigated 48 STEMI patients. The MC-ECG group (n = 23) and the Conventional group (n = 25) were enrolled. Results: There was no significant difference in FMCTB time between the MC-ECG and Conventional groups (MC-ECG: 72.0 (60.5-107) min vs. Conventional: 80.0 (63.0-92.0) min, p = 0.77). The length of hospital stay in the MC-ECG group was significantly shorter than that in the Conventional group (12.0 (10.0-15.0) days vs. 16.0 (12.0-19.0) days, p = 0.039). The logistic regression model showed that patients' non-use of MC-ECG was associated with a risk of more than 15-day length of hospital stay with an adjusted odd ratio of 0.08 (95% CI: 0.013-0.55, p = 0.0098). Conclusions: Using the MC-ECG, the length of hospital stay in patients with STEMI was significantly reduced.
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Servicios Médicos de Urgencia , Infarto del Miocardio con Elevación del ST , Electrocardiografía , Hospitales , Humanos , Factores de TiempoRESUMEN
Patterning of vertebrate melanophores is essential for mate selection and protection from UV-induced damage. Patterning can be influenced by circulating long-range factors, such as hormones, but it is unclear how their activity is controlled in recipient cells to prevent excesses in cell number and migration. The zebrafish wanderlust mutant harbors a mutation in the sheddase bace2 and exhibits hyperdendritic and hyperproliferative melanophores that localize to aberrant sites. We performed a chemical screen to identify suppressors of the wanderlust phenotype and found that inhibition of insulin/PI3Kγ/mTOR signaling rescues the defect. In normal physiology, Bace2 cleaves the insulin receptor, whereas its loss results in hyperactive insulin/PI3K/mTOR signaling. Insulin B, an isoform enriched in the head, drives the melanophore defect. These results suggest that insulin signaling is negatively regulated by melanophore-specific expression of a sheddase, highlighting how long-distance factors can be regulated in a cell-type-specific manner.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Tipificación del Cuerpo , Insulina/metabolismo , Melanóforos/fisiología , Pigmentación , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Movimiento Celular/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica , Insulina/genética , Melanóforos/citología , Mutación , Fenotipo , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
A 78-year-old man with unstable angina showed 90% stenosis in the proximal left anterior descending artery. Pre-procedural intravascular ultrasound revealed ruptured plaque and attenuated plaque in the lesion. Under these conditions, two overlapping sirolimus-eluting stent (SES) implantation in this lesion resulted in slow flow which was recovered by intracoronary nitrates, nicorandil, and nitroprusside without further complications. When the patient showed up again 5 years later with recurrence of angina pectoris, angiography revealed a hazy ulcerated in-stent restenosis (ISR) at the site of the SES. Pre-procedural optical coherence tomography (OCT) imaging revealed multiple intimal ruptures, cavity formation behind the stent struts, a thin-cap fibroatheroma containing neointima surrounded by signal-poor, lipid-rich area in the proximal SES, suggesting the progression of neoatherosclerosis within SES. Importantly, there occurred slow flow again after balloon angioplasty for this lesion. We would suggest careful OCT examination is warranted to confirm development of neoatherosclerosis within the stent, and distal protection device should be considered to prevent slow flow phenomenon even in a patient with very late ISR.
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Angioplastia Coronaria con Balón/instrumentación , Fármacos Cardiovasculares/administración & dosificación , Reestenosis Coronaria/terapia , Estenosis Coronaria/terapia , Stents Liberadores de Fármacos , Fenómeno de no Reflujo/etiología , Sirolimus/administración & dosificación , Anciano , Angioplastia Coronaria con Balón/efectos adversos , Angiografía Coronaria , Circulación Coronaria , Reestenosis Coronaria/diagnóstico , Reestenosis Coronaria/etiología , Reestenosis Coronaria/fisiopatología , Estenosis Coronaria/diagnóstico , Estenosis Coronaria/fisiopatología , Dentadura Parcial Provisoria , Humanos , Masculino , Fenómeno de no Reflujo/diagnóstico , Fenómeno de no Reflujo/fisiopatología , Recurrencia , Retratamiento , Factores de Tiempo , Tomografía de Coherencia Óptica , Resultado del Tratamiento , Ultrasonografía IntervencionalRESUMEN
Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.
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Apoptosis/genética , Asparagina/metabolismo , Aspartatoamoníaco Ligasa/antagonistas & inhibidores , Citrato (si)-Sintasa/genética , Glutamina/deficiencia , Factor de Transcripción Activador 4/metabolismo , Asparagina/biosíntesis , Asparagina/química , Aspartatoamoníaco Ligasa/biosíntesis , Ácido Aspártico/biosíntesis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Ciclo del Ácido Cítrico , Humanos , Ácido Oxaloacético/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
BACKGROUND: (18)F-fluoro-2-deoxyglucose (FDG) positron emission tomography (PET) is assumed to be the most useful method for evaluating the viability of the myocardium. However, there are few reports regarding serial changes in (18)F-FDG-PET images of acute myocardial infarction (AMI). We evaluated serial changes in glucose-loaded (18)F-FDG-PET, (123)I-ß-methyl-p-iodophenyl-penta-decanoic acid (BMIPP) single-photon emission computed tomography (SPECT) and (99m)Tc-Tetrofosmin (TF) gated SPECT images in patients with AMI. METHODS AND RESULTS: We enrolled 7 consecutive patients with first anterior AMI who successfully underwent percutaneous coronary intervention (PCI). (18)F-FDG-PET images were obtained in the acute, subacute, chronic, mid-term and long-term phases. (123)I-BMIPP and (99m)Tc-TF SPECT images were obtained in the subacute, chronic, mid-term and long-term phases. We determined the total defect score (TDS) for each image. The TDS of the glucose-loaded (18)F-FDG-PET, (123)I-BMIPP and( 99m)Tc-TF SPECT images indicated significant serial decrease (P<0.001). Comparing these images, the TDS of the glucose-loaded (18)F-FDG-PET images was larger than that of the (123)I-BMIPP and (99m)Tc-TF SPECT images, and the TDS indicated (18)F-FDG-PET>(123)I-BMIPP>(99m)Tc-TF in all phases. CONCLUSIONS: The defect areas of glucose-loaded (18)F-FDG-PET images were significantly larger than those of (123)I-BMIPP and( 99m)Tc-TF SPECT images during 9 months follow-up of patients with successful PCI for anterior AMI. Additionally, the impairment of glucose metabolism was prolonged.
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Ácidos Grasos/administración & dosificación , Fluorodesoxiglucosa F18/administración & dosificación , Yodobencenos/administración & dosificación , Infarto del Miocardio/diagnóstico por imagen , Intervención Coronaria Percutánea , Tomografía de Emisión de Positrones , Radiofármacos/administración & dosificación , Tomografía Computarizada de Emisión de Fotón Único , Anciano , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/terapia , Compuestos Organofosforados , Compuestos de Organotecnecio , RadiografíaRESUMEN
Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.
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Adenosina Difosfato/biosíntesis , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Luminiscencia , Conteo por CintilaciónRESUMEN
BACKGROUND: Upper arm swelling and venous hypertension at arteriovenous fistula sites, and insufficiency of hemodialysis are induced by central venous lesions in chronic hemodialysis patients. Percutaneous transluminal angioplasty (PTA) for central venous lesions is first-choice treatment. Cardiac function can be evaluated by measuring the acute increase in venous return volume after PTA. METHODS: We studied 6 cases of successful PTA for central venous stenotic or occluded lesions, and evaluated cardiac function by Swan-Ganz (SG) catheter and ultrasound echocardiography (UCG) at pre-, post-PTA, and on the following day. RESULTS: Ejection fraction (EF) in 6 cases was 71.0 ± 5.5% on UCG. Two cases of subclavian venous stenosis, one case of subclavian venous occlusion, and three cases of brachiocephalic venous occlusion were enrolled. The reference diameter (RD) was 10.2 ± 4.9 mm, % diameter-stenosis (%DS) was 92.2 ± 12.2% at pre-PTA, and %DS at post-PTA was 21.7 ± 20.7%. There were no significant differences in pulmonary capillary-wedge, pulmonary artery, and right ventricular end-diastolic pressure in SG at pre- and post-PTA. The pressure of right atrium (RA) and cardiac output (CO) were significantly increased by PTA (RA pressure at pre-/post-PTA, 9.7 ± 2.9/11.7 ± 3.6 mmHg, p<0.05, CO at pre-/post-PTA 5.09 ± 2.07/5.45 ± 2.25 l/min, p<0.05). There were no significant differences in serial EF, left atrial and left ventricular diameters on UCG. However, the short-diameter of right ventricle (RV) and RA were significantly increased at post-PTA and recovered on the following day (RV short-diameter at pre-/post-/following-day PTA, 26.7 ± 3.5/32.5 ± 1.9/29.1 ± 1.7 mm, p<0.05; RA short-diameter at pre-/post-/following-day PTA, 30.2 ± 4.2/36.3 ± 2.4/32.1 ± 3.6mm, p<0.05). CONCLUSIONS: Volume overload after PTA for central venous stenotic or occluded lesions in chronic hemodialysis patients resulted in increased RA and RV diameters. These changes were transient and completely recovered by the following day. PTA for central venous lesions in patients with normal EF can be performed without clinical cardiac problems.
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Angioplastia , Corazón/fisiología , Hiperemia/terapia , Diálisis Renal , Anciano , Anciano de 80 o más Años , Venas Braquiocefálicas , Cateterismo de Swan-Ganz , Ecocardiografía , Femenino , Atrios Cardíacos/diagnóstico por imagen , Insuficiencia Cardíaca/etiología , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Volumen Sistólico , Vena SubclaviaRESUMEN
The restenosis rate of coronary stent has significantly decreased by implantation of the drug-eluting stent (DES). We often experienced the DES implantation for very small target vessels. The minimum size of DES in Japan and USA is 2.5 mm-diameter, but there were no reports of the expandability of DESs for the very small target vessels with reference diameter <2.2 mm. We clarify the expandable performance of 2.5 mm-DESs for very small target vessels with reference diameter <2.2 mm in vitro and vivo study. We studied 3 pieces in each kind of DES (Sirolimus-eluting stent; SES, Paclitaxel-eluting stent; PES, Zotarolimus-eluting stent; ZES and Everolimus-eluting stent; EES) in vitro and vivo study of the porcine coronary artery with reference diameter <2.2 mm. By using the delivery balloon, each stent was initially dilated with 3.5 atm. And the pressure of 0.5 atm. was applied until it reached the maximum pressure of 12 atm. The minimum pressure of the full expanded stent balloon was estimated as the minimum expandable pressure. The stent-inner diameter and area on each pressure were measured by IVUS. The average minimum expandable pressure (atm.) in vitro/vivo was 4.7/4.5 in SES, 7.2/6.8 in PES, 4.3/4.5 in ZES and 3.8/3.8 in EES. The inner diameter (mm) in vitro/vivo at minimum expandable pressure was 1.81 ± 0.07/1.84 ± 0.05 in SES, 2.31 ± 0.10/2.13 ± 0.13 in PES, 2.41 ± 0.13/1.98 ± 0.31 in ZES and 2.13 ± 0.11/1.88 ± 0.22 in EES. The stent inner-diameter (mm) of DESs at 8 atm. in vivo was 2.16/2.21/2.45/2.25 in SES/PES/ZES/EES. All kinds of DES could be delivered to very small target vessels with reference diameter <2.2 mm at the minimum expandable pressure in vivo study, but the stent which presented adequate stent inner-diameter at 8 atm. was only SES. We have to implant the 2.5 mm-DESs for very small target vessels according to the data based on this expandability of DESs to bail out threatening occlusion due to coronary dissection or elastic recoil.
RESUMEN
Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFRα) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z' value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFRα inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRα inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries.
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Bioensayo/métodos , Transformación Celular Neoplásica/efectos de los fármacos , Diseño de Fármacos , Microscopía/métodos , Neoplasias Experimentales/patología , Tecnología Farmacéutica/métodos , Células 3T3 , Animales , Línea Celular Tumoral , RatonesRESUMEN
One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Biblioteca de Péptidos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Quinasa Tipo Polo 1RESUMEN
Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488 fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells.
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Antineoplásicos/aislamiento & purificación , Caspasas/metabolismo , Diagnóstico por Imagen/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Clorometilcetonas de Aminoácidos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Inhibidores de Caspasas , Línea Celular Tumoral , Supervivencia Celular , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , Modelos Biológicos , Transfección , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.
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Acetil-CoA Carboxilasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Compuestos de Sulfhidrilo/análisis , Acetil-CoA Carboxilasa/biosíntesis , Animales , Acido Graso Sintasa Tipo I/metabolismo , Fluorescencia , Humanos , RatasRESUMEN
Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.
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Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Bases de Datos como Asunto , Relación Dosis-Respuesta a Droga , Etiquetas de Secuencia Expresada , Prueba de Complementación Genética , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nucleotidiltransferasas/química , Sistemas de Lectura Abierta , Fenotipo , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoRESUMEN
The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5'-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans. Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene (pct1+) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.