RESUMEN

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Asunto(s)

Técnicas de Amplificación de Ácido Nucleico , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/genética , Endonucleasas/metabolismo , Endonucleasas/genética

RESUMEN

In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.

RESUMEN

Kleine-Levin syndrome (KLS) is a rare sleep disorder characterized by periodic hypersomnia and behavioral or cognitive disturbances. Although prolonged emergence from general anesthesia and postoperative hypersomnia may occur in a patient with KLS, there is little information about the safe anesthetic management of these patients. We describe the case of a 22-year-old female previously diagnosed with KLS who was scheduled to have her third molars extracted under general anesthesia. Because the patient had symptoms of periodic hypersomnia and hyperphagia, the surgery was scheduled during a KLS crisis interval. General anesthesia was induced with propofol, remifentanil, and rocuronium, and maintained with desflurane and remifentanil. To prevent overuse of anesthetic agents, an electroencephalogram (EEG)-based depth of anesthesia monitor (SedLine; Masimo Corporation) was used intraoperatively. A neuromuscular monitor was also used to carefully titrate use of a neuromuscular blocking agent. After surgery, sugammadex was administered, and the patient quickly emerged within 10 minutes, as also confirmed by the EEG monitor. She had no KLS recurrence postoperatively. When anesthetizing patients with KLS, an EEG-based depth of anesthesia monitor and neuromuscular monitor may be warranted to ensure complete emergence from general anesthesia. In addition, elective surgery should be planned during crises intervals.

Asunto(s)

Anestesia Dental , Anestésicos Generales , Síndrome de Kleine-Levin , Adulto , Anestesia General , Electroencefalografía , Femenino , Humanos , Síndrome de Kleine-Levin/diagnóstico , Síndrome de Kleine-Levin/tratamiento farmacológico , Síndrome de Kleine-Levin/psicología , Adulto Joven

RESUMEN

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2-ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

Asunto(s)

Replicación del ADN/genética , Mutación , Complejo de Reconocimiento del Origen/genética , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Línea Celular Tumoral , Daño del ADN , Regulación de la Expresión Génica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Microscopía Fluorescente , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo

RESUMEN

Organisms are composed of various cell types with specific states. To obtain a comprehensive understanding of the functions of organs and tissues, cell types have been classified and defined by identifying specific marker genes. Statistical tests are critical for identifying marker genes, which often involve evaluating differences in the mean expression levels of genes. Differentially expressed gene (DEG)-based analysis has been the most frequently used method of this kind. However, in association with increases in sample size such as in single-cell analysis, DEG-based analysis has faced difficulties associated with the inflation of P-values. Here, we propose the concept of discriminative feature of cells (DFC), an alternative to using DEG-based approaches. We implemented DFC using logistic regression with an adaptive LASSO penalty to perform binary classification for discriminating a population of interest and variable selection to obtain a small subset of defining genes. We demonstrated that DFC prioritized gene pairs with non-independent expression using artificial data and that DFC enabled characterization of the muscle satellite/progenitor cell population. The results revealed that DFC well captured cell-type-specific markers, specific gene expression patterns, and subcategories of this cell population. DFC may complement DEG-based methods for interpreting large data sets. DEG-based analysis uses lists of genes with differences in expression between groups, while DFC, which can be termed a discriminative approach, has potential applications in the task of cell characterization. Upon recent advances in the high-throughput analysis of single cells, methods of cell characterization such as scRNA-seq can be effectively subjected to the discriminative methods.

Asunto(s)

Expresión Génica , Algoritmos , Análisis por Conglomerados , Simulación por Computador , Marcadores Genéticos , Humanos , Modelos Logísticos

RESUMEN

During duplication of the genome, eukaryotic cells may experience various exogenous and endogenous replication stresses that impede progression of DNA replication along chromosomes. Chemical alterations in template DNA, imbalances of deoxynucleotide pools, repetitive sequences, tight DNA-protein complexes, and conflict with transcription can negatively affect the replication machineries. If not properly resolved, stalled replication forks can cause chromosome breaks leading to genomic instability and tumor development. Replication stress is enhanced in cancer cells due, for example, to the loss of DNA repair genes or replication-transcription conflict caused by activation of oncogenic pathways. To prevent these serious consequences, cells are equipped with diverse mechanisms that enhance the resilience of replication machineries to replication stresses. This review describes DNA damage responses activated at stressed replication forks and summarizes current knowledge on the pathways that promote faithful chromosome replication and protect chromosome integrity, including ATR-dependent replication checkpoint signaling, DNA cross-link repair, and SLX4-mediated responses to tight DNA-protein complexes that act as barriers. This review also focuses on the relevance of replication stress responses to selective cancer chemotherapies.

Asunto(s)

Daño del ADN/genética , Replicación del ADN/genética , ADN/genética , Animales , Cromosomas/genética , Reparación del ADN/genética , Humanos , Proteínas/genética

RESUMEN

MyoD, a myogenic differentiation protein, has been studied for its critical role in skeletal muscle differentiation. MyoD-expressing myoblasts have a potency to be differentiated with proliferation of ectopic cells. However, little is known about the effect on chromatin structure of MyoD binding in proliferative myoblasts. In this study, we evaluated the chromatin structure around MyoD-bound genome regions during the cell cycle by chromatin immunoprecipitation sequencing. Genome-wide analysis of histone modifications was performed in proliferative mouse C2C12 myoblasts during three phases (G1, S, G2/M) of the cell cycle. We found that MyoD-bound genome regions had elevated levels of active histone modifications, such as H3K4me1/2/3 and H3K27ac, compared with MyoD-unbound genome regions during the cell cycle. We also demonstrated that the elevated H3K4me2/3 modification level was maintained during the cell cycle, whereas the H3K27ac and H3K4me1 modification levels decreased to the same level as MyoD-unbound genome regions during the later phases. Immunoblot analysis revealed that MyoD abundance was high in the G1 phase then decreased in the S and G2/M phases. Our results suggest that MyoD binding formed selective epigenetic memories with H3K4me2/3 during the cell cycle in addition to myogenic gene induction via active chromatin formation coupled with transcription.

Asunto(s)

Ciclo Celular , Proliferación Celular , Cromatina/química , Genoma , Músculo Esquelético/fisiología , Proteína MioD/metabolismo , Mioblastos/fisiología , Animales , Diferenciación Celular , Cromatina/genética , Cromatina/metabolismo , Ratones , Desarrollo de Músculos , Músculo Esquelético/citología , Proteína MioD/genética , Mioblastos/citología , Unión Proteica

RESUMEN

OBJECTIVES: We investigated the effect of the maze procedure with intensive pulmonary vein isolation (PVI) guided by ganglionated plexus (GP) mapping (the Maze with GP ablation group) on a long-term postoperative maintenance of sinus rhythm in patients with permanent atrial fibrillation (AF) and compared with that in patients undergoing the maze procedure with the conventional PVI (the Maze group). METHODS AND RESULTS: We investigated 48 patients who underwent the maze procedure with GP ablation for persistent AF and 43 patients who underwent the maze procedure. The Maze procedure was conducted by the endocardial application of bipolar radiofrequency ablation and cryoablation. Conventional PVI was applied three times for the entrance of right and left PVs, respectively. Intensive PVI for GP ablation was repeated six-to-eight times for both sides of PVs to cover the bilateral GP regions identified by GP mapping. The duration of permanent AF, the prevalence of concomitant primary heart diseases, and the postoperative follow-up period were comparable between the two groups. At discharge, 1 year, 5 years after the surgery, sinus rhythm was maintained in 74.4%, 61%, and 40.5% of the Maze group. In contrast, it was maintained in 93.7%, 88.9%, and 75.7% of the Maze with GP ablation group. The cumulative freedom rate from AF at 10 years after surgery was significantly higher in the Maze with GP ablation group. CONCLUSIONS: More intense PV isolation including adjacent GP may improve long-term results of maze procedure in patients with permanent AF.

Asunto(s)

Fibrilación Atrial , Ablación por Catéter , Venas Pulmonares , Fibrilación Atrial/cirugía , Humanos , Procedimiento de Laberinto , Periodo Posoperatorio , Venas Pulmonares/cirugía , Resultado del Tratamiento

RESUMEN

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA-protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4-XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4-ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.

Asunto(s)

Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Recombinasas/metabolismo , Estrés Fisiológico , Anafase , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Segregación Cromosómica , Cromosomas Humanos/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , Fase S

RESUMEN

BACKGROUND: Metformin is the most widely used oral antihyperglycemic agent for patients with type 2 diabetes mellitus (T2DM). Despite the possible benefits of metformin on diabetes mellitus (DM) and heart failure (HF), acute or unstable HF remains a precaution for its use. OBJECTIVE: The aim of the present prospective randomized controlled trial was to assess whether metformin treatment has beneficial effects on patients with T2DM with hypertension without overt HF. METHODS: A total of 164 patients (92 males, 72 females; median age 66 years) were included in this study. Patients with T2DM with a history of hypertension were randomized 1:1 to treatment for 1 year with either metformin (metformin-treated group) or other hypoglycemic agents (control group). The primary endpoints were changes in brain natriuretic peptide (BNP) levels, left ventricular (LV) mass index, and indicators of LV diastolic function. We also evaluated changes in both clinical findings and blood laboratory examination data. RESULTS: We observed no significant changes between baseline and 1-year post-treatment in LV mass index, BNP levels, or E/e' (early diastolic transmitral flow velocity/early diastolic mitral annular velocity; an indicator of LV diastolic function) in either the metformin-treated (n = 83) or the control (n = 81) groups. The metformin-treated group had a significant reduction of body mass index (BMI) and low-density lipoprotein cholesterol (LDL-C), but the control group did not. We determined that renal function, including serum creatinine and estimated glomerular filtration rate, deteriorated significantly in the control group but not in the metformin-treated group. CONCLUSION: LV mass and diastolic function were not affected after 1 year of metformin treatment in patients with T2DM. However, we observed benefits in terms of reductions in both BMI and LDL-C levels and preservation of renal function. TRIAL REGISTRATION: UMIN000006504. Registered 7 October 2011.

Asunto(s)

Diabetes Mellitus Tipo 2 , Ventrículos Cardíacos , Hipertensión , Metformina , Función Ventricular Izquierda/efectos de los fármacos , Anciano , Índice de Masa Corporal , LDL-Colesterol/sangre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Hipertensión/complicaciones , Hipertensión/diagnóstico , Hipertensión/metabolismo , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Masculino , Metformina/administración & dosificación , Metformina/farmacocinética , Péptido Natriurético Encefálico/sangre , Tamaño de los Órganos/efectos de los fármacos , Resultado del Tratamiento

RESUMEN

Glutamate-rich WD40 repeat containing 1 (GRWD1) functions as a histone chaperone to promote loading of the MCM replication helicase at replication origins. GRWD1 is overexpressed in several cancer cell lines, and GRWD1 overexpression confers tumorigenic potential in human cells. However, less is known concerning its oncogenic activity. Our previous analysis showed that GRWD1 negatively regulates the tumour suppressor p53 via the RPL11-MDM2-p53 and RPL23-MDM2-p53 axes. Here, we demonstrate that GRWD1 directly interacts with p53 via the p53 DNA-binding domain. Upon DNA damage, GRWD1 downregulation resulted in increased p21 expression. Conversely, GRWD1 co-expression suppressed several p53-regulated promoters. GRWD1 interacted with the p21 and MDM2 promoters, and these interactions required p53. By using the Human Cancer Genome Atlas database, we found that GRWD1 expression levels are inversely correlated with the expression levels of some p53-target genes. Interestingly, high GRWD1 expression in combination with low expression levels of some p53-target genes was significantly correlated with poor prognosis in skin melanoma patients with wild-type p53. Taken together, our findings suggest a novel oncogenic function of GRWD1 as a transcriptional regulator of p53 and that GRWD1 might be an attractive therapeutic target and prognostic marker in cancer therapy.

Asunto(s)

Proteínas Portadoras/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Células HCT116 , Humanos

RESUMEN

We previously reported the identification of a novel antimitotic agent with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-iodobenzohydrazide (code number NP-10). However, the mechanism(s) underlying the cancer cell-selective inhibition of mitotic progression by NP-10 remains unclear. Here, we identified NP-10-interacting proteins by affinity purification from HeLa cell lysates using NP-10-immobilized beads followed by mass spectrometry. The results showed that several mitosis-associated factors specifically bind to active NP-10, but not to an inactive NP-10 derivative. Among them, NUP155 and importin ß may be involved in NP-10-mediated mitotic arrest. Because NP-10 did not show antitumor activity in vivo in a previous study, we synthesized 19 NP-10 derivatives to identify more effective NP-10-related compounds. HMI83-2, an NP-10-related compound with a Cl moiety, inhibited HCT116 cell tumor formation in nude mice without significant loss of body weight, suggesting that HMI83-2 is a promising lead compound for the development of novel antimitotic agents.

Asunto(s)

Antineoplásicos/administración & dosificación , Neoplasias del Colon/tratamiento farmacológico , Proteínas de Complejo Poro Nuclear/metabolismo , Polietilenglicoles/administración & dosificación , beta Carioferinas/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HeLa , Humanos , Ratones , Ratones Desnudos , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Polietilenglicoles/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

BACKGROUND: Guidelines for peripheral arterial disease (PAD) recommend long-term antiplatelet therapy in symptomatic patients to reduce cardiovascular morbidity and mortality risk. Although diabetes is a known risk factor for PAD, PAD has been undertreated in these patients. This study aimed to evaluate risk factors for major amputation in patients with diabetes undergoing antiplatelet therapy for PAD.Methods and Results:This retrospective analysis of a 2-year observational cohort study (1,745 clinics in Japan, September 2009-2013) evaluated predictors of amputation in patients with diabetes undergoing antiplatelet therapy for PAD. Among 4,016 eligible patients, 52 had an amputation during follow-up. Amputation risk (Cox regression analysis) was predicted at baseline by history of lower extremity revascularization/amputation (hazard ratio [HR]: 2.92; 95% confidence interval [CI]: 1.39, 6.14), chronic kidney disease (HR: 4.19; 95% CI: 1.95, 8.97), and comorbid cerebrovascular and heart disease (HR: 3.32; 95% CI: 1.19, 9.30), and was unaffected by choice of oral antiplatelet therapy. In patients with PAD and diabetes, amputation event rate was highest for those with ankle-brachial pressure index (ABI) <0.40 and progressively decreased at higher ABI cut-offs. CONCLUSIONS: These findings inform real-world understanding of PAD in diabetic patients receiving antiplatelet therapy in Japan, and showed that ABI <0.4 was the strongest risk factor for amputation.

Asunto(s)

Amputación Quirúrgica , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Enfermedad Arterial Periférica/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/administración & dosificación , Administración Oral , Anciano , Anciano de 80 o más Años , Índice Tobillo Braquial , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiología , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/epidemiología , Inhibidores de Agregación Plaquetaria/efectos adversos , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento

RESUMEN

MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies.

Asunto(s)

Cisplatino/farmacología , Proteínas de Mantenimiento de Minicromosoma/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Ftalazinas/farmacología , Piperazinas/farmacología , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Femenino , Células HCT116 , Recombinación Homóloga/efectos de los fármacos , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/metabolismo , Compuestos Organoplatinos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos

RESUMEN

Surveillance of cardiovascular Events in Antiplatelet-treated arterioSclerosis Obliterans patients in JapaN (SEASON) is a 2-year, prospective, real-world, registry study conducted in Japan from 2009 to 2013. This post hoc analysis evaluated risk factors for limb ischemia in patients with peripheral arterial disease (PAD) and ankle-brachial index (ABI) <0.90. Vascular events were adjudicated by an Efficacy Endpoint Review Committee. Cox regression identified predictors of limb-specific peripheral vascular events (amputation, development of critical limb ischemia, and acute limb ischemia). Patients (n = 6565) were stratified according to ABI: normal (≥1.0; n = 1300), borderline (0.90 ≤ ABI ≤ 1.0; n = 776), and abnormal (<0.90; n = 4489). Compared to normal ABI, patients with ABI <0.90 had a significantly higher risk of any vascular event, all-cause death, and any limb-specific peripheral vascular event. Risk factors for limb-specific vascular events included history of lower extremity revascularization/amputation (adjusted hazard ratio: 2.18; 95% confidence interval [CI]: 1.49-3.20), chronic kidney disease (2.00; 1.33-3.00), diabetes (1.71; 1.16-2.52), and ABI <0.4 (4.45; 2.62-7.55) or <0.7 (1.78; 1.15-2.76). These findings from a Japanese real-world population confirm the increased vascular risk of patients with PAD and ABI <0.90 and identified risk factors for limb-specific peripheral vascular events.

Asunto(s)

Isquemia/epidemiología , Extremidad Inferior/irrigación sanguínea , Enfermedad Arterial Periférica/epidemiología , Enfermedad Aguda , Anciano , Anciano de 80 o más Años , Amputación Quirúrgica , Índice Tobillo Braquial , Enfermedad Crítica , Progresión de la Enfermedad , Femenino , Humanos , Isquemia/diagnóstico , Isquemia/mortalidad , Isquemia/terapia , Japón/epidemiología , Masculino , Persona de Mediana Edad , Enfermedad Arterial Periférica/diagnóstico , Enfermedad Arterial Periférica/mortalidad , Enfermedad Arterial Periférica/terapia , Inhibidores de Agregación Plaquetaria/uso terapéutico , Pronóstico , Estudios Prospectivos , Sistema de Registros , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo

RESUMEN

We have recently revealed that the proprioceptive signal from jaw-closing muscle spindles (JCMSs) is conveyed to the dorsal part of granular insular cortex rostroventrally adjacent to the rostralmost part of secondary somatosensory cortex (dGIrvs2) via the caudo-ventromedial edge (VPMcvm) of ventral posteromedial thalamic nucleus (VPM) in rats. However, it remains unclear to which cortical or subcortical structures the JCMS proprioceptive information is subsequently conveyed from the dGIrvs2. To test this issue, we injected an anterograde tracer, biotinylated dextranamine, into the electophysiologically identified dGIrvs2, and analyzed the resultant distribution profiles of labeled axon terminals in rats. Labeled terminals were distributed with an ipsilateral predominance. In the cerebral cortex, they were seen in the primary and secondary somatosensory cortices, lateral and medial agranular cortices and dorsolateral orbital cortex. In the basal ganglia, they were found in the caudate putamen, core part of accumbens nucleus, lateral globus pallidus, subthalamic nucleus, and substantia nigra pars compacta and pars reticulata. They were also observed in the central amygdaloid nucleus and extended amygdala (the interstitial nucleus of posterior limb of anterior commissure and the juxtacapsular part of lateral division of bed nucleus of stria terminalis). In the thalamus, they were seen in the reticular nucleus, ventromedial nucleus, core VPM, parvicellular part of ventral posterior nucleus, oval paracentral nucleus, medial and triangular parts of posterior nucleus, and zona incerta as well as the VPMcvm. These data suggest that the JCMS proprioceptive information through the dGIrvs2 is transmitted to the emotional 'limbic' regions as well as sensorimotor regions.

Asunto(s)

Corteza Cerebral/anatomía & histología , Corteza Cerebral/fisiología , Propiocepción/fisiología , Amígdala del Cerebelo/anatomía & histología , Amígdala del Cerebelo/fisiología , Animales , Ganglios Basales/anatomía & histología , Ganglios Basales/fisiología , Biotina/análogos & derivados , Dextranos , Potenciales Evocados , Cara/inervación , Lateralidad Funcional , Masculino , Boca/inervación , Vías Nerviosas/anatomía & histología , Vías Nerviosas/fisiología , Técnicas de Trazados de Vías Neuroanatómicas , Trazadores del Tracto Neuronal , Neuronas/citología , Neuronas/fisiología , Ratas Wistar , Tálamo/anatomía & histología , Tálamo/fisiología

RESUMEN

Antibodies are essential for characterizing various analytes. "Molecular-breeding" approaches enable rapid generation of antibody mutants with desirable antigen-binding abilities. Typically, prototype antibodies are converted to single-chain Fv fragments (scFvs), and random mutations are genetically introduced to construct molecular libraries with a vast diversity. Improved species therein are then isolated via phage display genotype-phenotype-connecting systems to separate them from a large excess of nonspecific scFvs. During these experiments, counting of phage particles is routinely performed. However, current methods depend on the time-consuming overnight cultivation of phage-infected bacteria on agar plates to estimate phage numbers as plaque-forming units (pfu) or colony-forming units, the results of which fluctuate considerably. Immunochemical systems capturing phage particles should be a more convenient and robust alternative. We therefore generated monoclonal antibodies against M13 filamentous phage, which is commonly used for phage display, by employing hybridoma technology. Combinatorial use of two such antibodies (Ab-M13#53 and #71; both specific to the major coat protein pVIII) enabled development of a sandwich enzyme-linked immunosorbent assay (ELISA) that could measure ca. 107-1010 phage pfu/mL. To construct a more convenient system, Ab-M13#71 was converted to the scFv form and further fused with an alkaline phosphatase variant. Using this fusion protein, the sandwich ELISA enabled rapid (within 90 min) and reliable phage counting without reducing the sensitivity, and the results were reasonably consistent with those of infection-based methods. The present anti-phage antibodies and scFvs might also enable visualization of individual phage particles by combining them with sensitive fluorescent staining.

Asunto(s)

Anticuerpos Monoclonales/genética , Bacteriófago M13/inmunología , Barajamiento de ADN/métodos , Anticuerpos de Cadena Única/genética , Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Hibridomas , Reproducibilidad de los Resultados , Anticuerpos de Cadena Única/inmunología

RESUMEN

Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1-binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1-interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitin-proteasome system. GRWD1 expression reversed the RPL23-mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis.

Asunto(s)

Proteínas Portadoras/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ribosómicas/metabolismo , Células HEK293 , Humanos , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos

RESUMEN

In metazoan cells, only a limited number of mini chromosome maintenance (MCM) complexes are fired during S phase, while the majority remain dormant. Several methods have been used to map replication origins, but such methods cannot identify dormant origins. Herein, we determined MCM7-binding sites in human cells using ChIP-Seq, classified them into firing and dormant origins using origin data and analysed their association with various chromatin signatures. Firing origins, but not dormant origins, were well correlated with open chromatin regions and were enriched upstream of transcription start sites (TSSs) of transcribed genes. Aggregation plots of MCM7 signals revealed minimal difference in the efficacy of MCM loading between firing and dormant origins. We also analysed common fragile sites (CFSs) and found a low density of origins at these sites. Nevertheless, firing origins were enriched upstream of the TSSs. Based on the results, we propose a model in which excessive MCMs are actively loaded in a genome-wide manner, irrespective of chromatin status, but only a fraction are passively fired in chromatin areas with an accessible open structure, such as regions upstream of TSSs of transcribed genes. This plasticity in the specification of replication origins may minimize collisions between replication and transcription.

Asunto(s)

Origen de Réplica , Composición de Base , Sitios de Unión , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Sitios Frágiles del Cromosoma , ADN/química , Genoma Humano , Células HeLa , Humanos , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción

RESUMEN

The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.

Asunto(s)

Western Blotting/métodos , Microscopía Fluorescente/métodos , Animales , Western Blotting/estadística & datos numéricos , Línea Celular , Colorantes Fluorescentes , Glutatión Transferasa/metabolismo , Aumento de la Imagen/métodos , Mediciones Luminiscentes , Ratones , Microscopía Fluorescente/estadística & datos numéricos , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad