RESUMO
Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.
Assuntos
Membrana Celular , Venenos de Cnidários , Citosol , Esfingomielinas , Esfingomielinas/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Animais , Venenos de Cnidários/metabolismo , Venenos de Cnidários/genética , Humanos , Anêmonas-do-Mar/metabolismo , Anêmonas-do-Mar/genética , Colesterol/metabolismoRESUMO
Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and the Golgi lipid order defined by cholesterol. Single-molecule imaging of TBK1 reveals that STING clustering enhances the association with TBK1. We thus provide quantitative proof-of-principle for the signaling STING scaffold, reveal the mechanistic role of STING palmitoylation in the STING activation, and resolve the long-standing question of the requirement of STING translocation for triggering the innate immune signaling.
Assuntos
Lipoilação , Rede trans-Golgi , Rede trans-Golgi/metabolismo , Microscopia , Imagem Individual de Molécula , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Colesterol , Análise por Conglomerados , Imunidade InataRESUMO
This study was undertaken to investigate the effects of oral L-arginine administration and exercising training on the NO concentration emanating from rat tail and NOx in plasma. Obese (fa/fa) Zucker rats (n = 22) were divided into four groups: (1) oral L-arginine administration (A) (n = 6), (2) exercise training (E), (3) exercise training + L-arginine administration (E + A) (n = 5), and (4) non-exercise training + non-L-arginine administration (N) (n = 6). The control (+/+) Zucker rats (n = 22) were also divided into the same four groups. The body weight of the E + A and the A groups was significantly lower than that of the N group. The NO concentration emitted from the tail was higher in the L-arginine (E + A and A) groups than in the non-L-arginine (E and N) groups in both obese and control rats. Exercise training did not affect the skin gas NO concentration in either obese or control rats. Plasma NOx concentrations in four obese rats were significantly higher than those observed in control rats. Exercise training did not influence the level of plasma NOx in obese or control rats. In conclusion, this study confirmed that L-arginine administration increases the skin gas NO concentration and obesity increases the plasma NOx level. The plasma NOx concentrations were not affected by L-arginine administration or exercise training in obese or control rats.
Assuntos
Arginina/metabolismo , Óxido Nítrico/sangue , Obesidade/fisiopatologia , Pele/metabolismo , Animais , Arginina/farmacologia , Masculino , Condicionamento Físico Animal , Ratos , Ratos Zucker , CaudaRESUMO
The cultivated strawberry (Fragaria × ananassa) is an octoploid (2n = 8x = 56) of the Rosaceae family whose genomic architecture is still controversial. Several recent studies support the AAA'A'BBB'B' model, but its complexity has hindered genetic and genomic analysis of this important crop. To overcome this difficulty and to assist genome-wide analysis of F. × ananassa, we constructed an integrated linkage map by organizing a total of 4474 of simple sequence repeat (SSR) markers collected from published Fragaria sequences, including 3746 SSR markers [Fragaria vesca expressed sequence tag (EST)-derived SSR markers] derived from F. vesca ESTs, 603 markers (F. × ananassa EST-derived SSR markers) from F. × ananassa ESTs, and 125 markers (F. × ananassa transcriptome-derived SSR markers) from F. × ananassa transcripts. Along with the previously published SSR markers, these markers were mapped onto five parent-specific linkage maps derived from three mapping populations, which were then assembled into an integrated linkage map. The constructed map consists of 1856 loci in 28 linkage groups (LGs) that total 2364.1 cM in length. Macrosynteny at the chromosome level was observed between the LGs of F. × ananassa and the genome of F. vesca. Variety distinction on 129 F. × ananassa lines was demonstrated using 45 selected SSR markers.
Assuntos
Mapeamento Cromossômico , Fragaria/genética , Ligação Genética , Genoma de Planta , Repetições de Microssatélites , Cromossomos de Plantas/genética , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Etiquetas de Sequências Expressas , Loci Gênicos , Marcadores Genéticos , Polimorfismo Genético , Análise de Sequência de DNA , TranscriptomaRESUMO
Herpes zoster (HZ)-induced abdominal wall pseudohernia has been frequently reported, but there has been no report describing HZ-induced trunk muscle paresis leading to functional problems. We describe a 73-year-old man with T12 and L1 segmental paresis caused by HZ presenting with abdominal wall pseudohernia, scoliosis, and standing and gait disturbance who responded well to a systematic rehabilitation approach. He first noticed a right abdominal bulge in the 6th postherpetic week, which was gradually accompanied by right convex thoracolumbar scoliosis, pain, and standing and gait disturbance in the 12th week. Needle electromyography revealed abnormal spontaneous activities at rest in the right T12 myotomal muscles, and motor unit recruitment was markedly decreased. We arranged an outpatient rehabilitation program consisting of using a soft thoracolumbosacral orthosis for pain relief and trunk stability, muscle reeducation of the paretic abdominal muscles, strengthening of the disused trunk and extremity muscles, and gait exercise. Based on electromyographic findings, we instructed him in an effective method of muscle reeducation. After 4 months of rehabilitation, he showed marked improvement and became an outdoor ambulator. We suggest that electromyography is a useful tool to evaluate clinical status and devise an effective rehabilitation program in patients with HZ trunk paresis.
Assuntos
Transtornos Neurológicos da Marcha/virologia , Hérnia Abdominal/virologia , Herpes Zoster/complicações , Paresia/reabilitação , Paresia/virologia , Escoliose/virologia , Idoso , Transtornos Neurológicos da Marcha/diagnóstico , Transtornos Neurológicos da Marcha/reabilitação , Hérnia Abdominal/diagnóstico , Hérnia Abdominal/terapia , Herpes Zoster/diagnóstico , Herpes Zoster/terapia , Humanos , Masculino , Paresia/diagnóstico , Escoliose/diagnóstico , Escoliose/terapiaRESUMO
ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of > or =29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase , Metiltransferases/química , Proto-Oncogenes , Fatores de Transcrição , Transcrição Gênica , Western Blotting , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Células HeLa , Histona Metiltransferases , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células K562 , Espectrometria de Massas , Metilação , Metiltransferases/metabolismo , Proteína de Leucina Linfoide-Mieloide , Testes de Precipitina , Ligação Proteica , Proteínas Metiltransferases , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Coloração pela PrataRESUMO
Cyclin K, a newly recognized member of the "transcription" cyclin family, may play a dual role by regulating CDK and transcription. Using cDNA microarray technology, we found that cyclin K mRNA was dramatically increased in U373MG, a glioblastoma cell line deficient in wild-type p53, in the presence of exogenous p53. An electrophoretic mobility-shift assay showed that a potential p53-binding site (p53BS) in intron 1 of the cyclin K gene could indeed bind to p53 protein. Moreover, a heterologous reporter assay revealed that the p53BS possessed p53-dependent transcriptional activity. Colony-formation assays indicated that overexpression of cyclin K suppressed growth of T98G, U373MG and SW480 cells. The results suggested that cyclin K may play a role in regulating the cell cycle or apoptosis after being targeted for transcription by p53.
Assuntos
Ciclinas/metabolismo , Genes p53/genética , Transcrição Gênica , Adenoviridae/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Northern Blotting , DNA Complementar/metabolismo , Doxorrubicina/farmacologia , Raios gama , Biblioteca Gênica , Vetores Genéticos , Humanos , Íntrons , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/farmacologia , Ligação Proteica , Fatores de Tempo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Raios UltravioletaRESUMO
Interferon regulatory factors (IRFs) regulate transcription of interferon genes through DNA sequence-specific binding to these targets. Using a differential display method for examining gene expression in p53-defective cells infected with adenovirus containing wild-type p53, we found that expression of interferon regulatory factor 5 (IRF-5) mRNA was increased in the presence of exogenous p53. An electrophoretic mobility-shift assay showed that a potential p53 binding site (p53BS) detected in exon 2 of the IRF-5 gene could in fact bind to p53 protein. Moreover, a heterologous reporter assay revealed that the p53BS possessed p53-dependent transcriptional activity. Expression of IRF-5 was induced in p53+/+ cells (MCF7 and NHDF), but not inp53-/- cells (H1299) when DNA was damaged by gamma-irradiation, UV-radiation, or adriamycin treatment in a wild-type p53-dependent manner. These results suggest that IRF-5 is a novel p53-target, and that it might mediate the p53-dependent immune response.
Assuntos
Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Divisão Celular , Humanos , Fatores Reguladores de Interferon , Dados de Sequência Molecular , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
A cDNA microarray analysis indicated that Semaphorin3B (Sema3B), a gene whose product is involved in axon guidance and axonal repulsion, is inducible by p53. Introduction of exogenous p53 into a glioblastoma cell line lacking wild-type p53 (U373MG) dramatically induced expression of Sema3B mRNA. An electrophoretic mobility shift assay and a reporter assay confirmed that a potential p53 binding site present in the promoter region had p53-dependent transcriptional activity. Expression of endogenous Sema3B was induced in response to genotoxic stresses caused by adriamycin treatment or UV irradiation in a p53-dependent manner. Ectopic expression of Sema3B in p53-defective cells reduced the number of colonies in colony formation assays. These results suggest that Sema3B might play some role in regulating cell growth as a mediator of p53 tumor-suppressor activity.
Assuntos
Neoplasias da Mama/genética , Glioblastoma/genética , Neoplasias Pulmonares/genética , Glicoproteínas de Membrana/genética , Proteína Supressora de Tumor p53/fisiologia , Sequência de Bases , Sítios de Ligação , Northern Blotting , Neoplasias da Mama/metabolismo , Ensaio de Unidades Formadoras de Colônias , Sequência Consenso , Dano ao DNA , Primers do DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Luciferases/metabolismo , Neoplasias Pulmonares/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Semaforinas , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/efeitos da radiação , Raios UltravioletaRESUMO
To search for p53 target genes throughout the human genome, we applied a cDNA microarray system using adenovirus-mediated transfer of p53 into p53-deficient U373MG (glioblastoma) cells. In this manner, we detected dozens of genes that appeared to be regulated by wild-type p53. We describe here characterization of one such gene, termed CABC1 [chaperone-activity of bc1 complex in Schizosaccharomyces pombe (ABC1)-like], which encodes a 647-amino acid peptide with significant sequence similarity to activity of bc1 complex (ABC1) in Arabidopsis thaliana and S. pombe. The CABC1 product was located in mitochondria, and colony-formation assays with cancer cell lines indicated its ability to suppress cell growth. Inhibition of CABC1 expression by transfection with antisense oligonucleotide significantly reduced the apoptotic response induced by wild-type p53. These results suggest that CABC1 may play an important role in mediating p53-inducible apoptosis through the mitochondrial pathway.