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1.
Yakugaku Zasshi ; 143(2): 159-170, 2023.
Artigo em Japonês | MEDLINE | ID: mdl-36724929

RESUMO

We investigated the cytotoxicity on a mast cell line (C57 cells) of water-soluble extracts of coarse (>3 µm, PM>3) and fine (0.05-3 µm, PM0.05-3) atmospheric particulates collected from April 2016 to March 2019 in Fukuoka, Japan. We examined the direct cytotoxicity with punched-out membrane filter fragments of PM>3 and PM0.05-3 collected from April 2019 to March 2021, without extraction of the components. Also, cell proliferation and degranulation assays were conducted under conditions which caused no cytotoxicity with water-soluble extracts of PM>3 from FY2016 and PM>3 direct samples from FY2019. The findings revealed the significant direct cytotoxicity of many PM>3 and all PM0.05-3 samples, with higher cytotoxicity for PM0.05-3 (FY2019-2020). These results were different from the cytotoxicity effects of water-soluble extracts of PM>3 and PM0.05-3 samples (FY2016) in previous studies. In addition, inhibition of cell proliferation and induction of degranulation were significantly induced in a few PM>3 samples, showing a correlation with the suspended particulate matter (SPM) concentrations. This method using punched-out membrane filters is convenient and useful for assessing the direct effects of atmospheric particles on a small scale.


Assuntos
Poluentes Atmosféricos , Material Particulado , Material Particulado/toxicidade , Material Particulado/análise , Poluentes Atmosféricos/toxicidade , Poluentes Atmosféricos/análise , Tamanho da Partícula , Linhagem Celular , Água
2.
Biol Pharm Bull ; 44(1): 57-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33390550

RESUMO

Fine particulate matter (PM2.5) pollution causes serious health disorders, because PM2.5 becomes deposited in the tracheobronchial and alveoli regions. In the extrathoracic region, there are more deposits of coarse particulate matter than fine particulates. As adverse health issues caused by coarse particulates have not been well investigated, this study examined the cytotoxicity of water-soluble extracts of both fine (0.05-3 µm, PM0.05-3) and coarse (> 3 µm, PM>3) particulates collected from April 2016 to March 2019 in Fukuoka, Japan. Also evaluated were concentrations of NH4+ and SO42-, multi-components of well-known secondary generation substances. The findings revealed that PM>3 showed stronger cytotoxic effects on mast cell lines than PM0.05-3. Cytotoxic effects were observed at concentrations of over 15 mM of (NH4)2SO4 and over 30 mM of NH4Cl. In contrast, Na2SO4 caused few cytotoxic effects up to a concentration of 50 mM. The causative substances for this cytotoxicity may not have been NH4+ and SO42- because their PM>3 concentrations indicating the largest cytotoxic effects were 1 and 0.4 mM, respectively. The cytotoxicities of PM>3 and PM0.05-3 were the highest in the first half of FY2016. These cytotoxicities seem to be due to cross-border pollution, although this pollution has been declining in recent years. An increasing trend of cytotoxicity was observed in the second half of FY2018. This study showed that cytotoxicity and particulate concentrations are not always correlated. Thus, we should focus not only on the quantity of atmospheric particulate matter, but also on its quality.


Assuntos
Poluentes Atmosféricos/toxicidade , Citotoxinas/toxicidade , Mastócitos/efeitos dos fármacos , Tamanho da Partícula , Material Particulado/toxicidade , Água , Poluentes Atmosféricos/isolamento & purificação , Poluentes Atmosféricos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Citotoxinas/isolamento & purificação , Citotoxinas/metabolismo , Relação Dose-Resposta a Droga , Mastócitos/metabolismo , Camundongos , Material Particulado/isolamento & purificação , Material Particulado/metabolismo , Solubilidade , Água/metabolismo
3.
J Hazard Mater ; 385: 121526, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31732351

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to simply as "dioxin", is a persistent environmental pollutant. Because of its high environmental persistence and biological accumulation, humans and animals are often exposed to TCDD. Therefore, the harmful effects on humans and animals is a major concern. Although studies have elucidated the adverse estrogenic and anti-estrogenic effects of TCDD, it is unclear in which tissues TCDD exerts these effects in vivo. To investigate the estrogen-related effects of TCDD in various tissues, we generated an improved estrogen-responsive reporter transgenic mouse in which the luciferase gene luc2 is expressed in response to estrogenic signals. Using these mice, we clarified that TCDD inhibits estrogenic signaling in liver and kidney but enhances estrogenic signaling in the pituitary gland in the same individual. Expression of aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator, and estrogen receptor alpha mRNA was detected in liver, kidney, and pituitary gland, suggesting that the effects of TCDD on estrogenic signaling in these organs is independent of the expression pattern of these receptors. Thus, our results indicate that TCDD exerts both estrogenic and anti-estrogenic tissue-specific effects within the same individual.


Assuntos
Poluentes Ambientais/toxicidade , Moduladores de Receptor Estrogênico/toxicidade , Estrogênios/toxicidade , Dibenzodioxinas Policloradas/toxicidade , Animais , Linhagem Celular Tumoral , Poluentes Ambientais/farmacocinética , Moduladores de Receptor Estrogênico/farmacocinética , Estrogênios/farmacocinética , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Luciferases/genética , Camundongos Transgênicos , Dibenzodioxinas Policloradas/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual
4.
Biol Pharm Bull ; 39(8): 1381-6, 2016 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-27237601

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mRNA post-transcriptional regulation. The deregulation of miRNAs affects the expression of drug-metabolizing enzymes, drug transporters, and nuclear receptors, all of which are important in regulating drug metabolism. miRNA expression can be altered by several endogenous or exogenous agents, such as steroid hormones, carcinogens, and therapeutic drugs. However, it is unclear whether hepatic miRNA expression is regulated by nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are indispensable for the expression of the CYPs. Here we investigated the effects of the mouse PXR and CAR ligands pregnenolone-16α-carbonitrile (PCN) and 1,4-bis[(3,5-dichloropyridin-2-yl)oxy]benzene (TCPOBOP) on hepatic miRNA expression in mice. We found that the expression of 9 miRNAs was increased (>2-fold) and of 4 miRNAs was decreased (>50%) in response to PCN, while TCPOBOP treatment led to the up-regulation of 8 miRNAs and down-regulation of 6 miRNAs. Using several miRNA target prediction algorithms, we found that the predicted target genes included several lesser known Cyp genes (Cyp1a1, Cyp1b1, Cyp2b10, Cyp2c38, Cyp2u1, Cyp4a12a/b, Cyp4v3, Cyp17a1, Cyp39a1, and Cyp51). We analyzed the expression of these genes in response to PCN and TCPOBOP and found changes in their mRNA levels, some of which were negatively correlated with the expression of their corresponding miRNAs, suggesting that miRNAs may play a role in regulating Cyp enzyme expression. Further studies will be required to fully elucidate the miRNA regulatory mechanisms that contribute to modulating CYP expression.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , MicroRNAs/metabolismo , Carbonitrila de Pregnenolona/farmacologia , Piridinas/farmacologia , Animais , Fígado/metabolismo , Masculino , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo
5.
J Steroid Biochem Mol Biol ; 155(Pt B): 190-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25465476

RESUMO

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), are typical environmental contaminants and suspected endocrine-disrupting chemicals because they cause masculinization in female mollusks. In addition, previous studies have suggested that the endocrine disruption by organotin compounds leads to activation of peroxisome proliferator-activated receptor (PPAR)γ and retinoid X receptor (RXR). However, whether organotin compounds cause crucial toxicities in human development and reproduction is unclear. We here investigated the structure-dependent effect of 12 tin compounds on mRNA transcription of 3ß-hydroxysteroid dehydrogenase type I (3ß-HSD I) and progesterone production in human choriocarcinoma Jar cells. TBT, TPT, dibutyltin, monophenyltin, tripropyltin, and tricyclohexyltin enhanced progesterone production in a dose-dependent fashion. Although tetraalkyltin compounds such as tetrabutyltin increased progesterone production, the concentrations necessary for activation were 30-100 times greater than those for trialkyltins. All tested active organotins increased 3ß-HSD I mRNA transcription. We further investigated the correlation between the agonistic activity of organotin compounds on PPARγ and their ability to promote progesterone production. Except for DBTCl2, the active organotins significantly induced the transactivation function of PPARγ. In addition, PPARγ knockdown significantly suppressed the induction of mRNA transcription of 3ß-HSD I by all active organotins except DBTCl2. These results suggest that some organotin compounds promote progesterone biosynthesis in vitro by inducing 3ß-HSD I mRNA transcription via the PPARγ signaling pathway. The placenta represents a potential target organ for these compounds, whose endocrine-disrupting effects might cause local changes in progesterone concentration in pregnant women.


Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Disruptores Endócrinos/farmacologia , Compostos Orgânicos de Estanho/farmacologia , PPAR gama/genética , Progesterona/agonistas , Trofoblastos/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Humanos , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Gravidez , Progesterona/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Relação Estrutura-Atividade , Transcrição Gênica , Trofoblastos/metabolismo , Trofoblastos/patologia
6.
Metallomics ; 7(7): 1180-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25985376

RESUMO

Organotins, such as tributyltin (TBT) and triphenyltin (TPT), may disrupt endocrine activity in mammals arising from their ability to act as ligands for the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor γ (PPARγ). The structure of TBT is completely different from that of 9-cis retinoic acid (9cRA), an endogenous RXR ligand; and X-ray crystallographic studies have revealed that TBT and 9cRA have distinct binding interactions with human RXRα. Therefore, organotins and rexinoids likely activate RXR by different mechanisms. Here, we used human RXRα mutants to investigate which amino acid residues of the receptor are critical for transactivation induced by rexinoids and organotins. We found that 9cRA and a synthetic RXR agonist (LG100268) failed to activate R316A and L326A RXRα mutants. In contrast, all the tested organotins activated the R316A mutant, the L326A mutant, or both but failed to activate a C432A mutant. These results suggest that the importance of L326, which is located in the ß-strand, for rexinoid-induced transactivation of RXRα is comparable to that of R316; in contrast, C432 is critical for organotin-induced transactivation, whereas R316 and L326 are not required. We used a PPARγ/RXRα C432A heterodimer to determine whether TBT and TPT could activate the heterodimer by binding to PPARγ. We found that TBT and TPT activated the PPARγ/RXRα C432A heterodimer, which suggests that both compounds can activate the heterodimer through PPARγ. These findings indicate that the amino acid residues that are critical for organotin-induced transactivation of RXRα are distinct from those required for rexinoid-induced transactivation.


Assuntos
Compostos Orgânicos de Estanho/farmacologia , Receptor X Retinoide alfa/genética , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Compostos de Trialquitina/farmacologia , Alitretinoína , Linhagem Celular Tumoral , Humanos , Mutação Puntual , Receptor X Retinoide alfa/química
7.
Sci Rep ; 5: 8520, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25687586

RESUMO

Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT.


Assuntos
PPAR gama/química , PPAR gama/metabolismo , Ativação Transcricional , Sítios de Ligação , Linhagem Celular Tumoral , Disruptores Endócrinos/farmacologia , Humanos , Ligantes , Espectrometria de Massas , Modelos Moleculares , Mutação , Compostos Orgânicos de Estanho/farmacologia , PPAR gama/agonistas , PPAR gama/genética , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Compostos de Trialquitina/farmacologia
8.
Endocrinology ; 155(11): 4275-86, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25116705

RESUMO

Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms.


Assuntos
Evolução Molecular , Moluscos/genética , Receptores do Ácido Retinoico/genética , Tretinoína/metabolismo , Animais , Clonagem Molecular , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Moluscos/metabolismo , Filogenia , Ligação Proteica/genética , Estrutura Terciária de Proteína , Receptores do Ácido Retinoico/química , Homologia de Sequência
9.
Planta ; 240(1): 147-59, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24771022

RESUMO

Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k cat/K m of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k cat/K m of the recombinant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant RNS1. The magnitudes of the k cat/K m values or k cat values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.


Assuntos
Magnoliopsida/enzimologia , Ribonucleases/genética , Sequência de Aminoácidos , Droseraceae/enzimologia , Droseraceae/genética , Ácido Edético , Concentração de Íons de Hidrogênio , Cinética , Magnoliopsida/genética , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Ribonucleases/química , Ribonucleases/metabolismo , Sarraceniaceae/enzimologia , Sarraceniaceae/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato , Temperatura
10.
Shokuhin Eiseigaku Zasshi ; 54(4): 326-30, 2013.
Artigo em Japonês | MEDLINE | ID: mdl-24025212

RESUMO

We examined changes in the quality of drinking water stockpiled under various conditions for emergency use. The results indicated that the change in the quality of the stocked water was influenced mainly by the preservation period and not by the amount of water in the bottle. To maintain water quality, the amount of residual chlorine is less important than using sufficiently sterilized water, bottles and caps in the bottling process. Washing the bottles with a small amount of boiling water was not sufficient to ensure complete inhibition of microbial growth.


Assuntos
Água Potável , Contaminação de Alimentos/prevenção & controle , Conservação de Alimentos/métodos , Microbiologia da Água , Qualidade da Água , Cloro , Planejamento em Desastres , Água Potável/microbiologia , Esterilização , Fatores de Tempo
11.
Planta ; 238(5): 955-67, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23959189

RESUMO

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.


Assuntos
Drosera/enzimologia , Drosera/genética , Regulação da Expressão Gênica de Plantas , Ribonucleases/genética , Sarraceniaceae/enzimologia , Sarraceniaceae/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Western Blotting , Metilação de DNA/genética , Regulação Enzimológica da Expressão Gênica , Genes de Plantas/genética , Modelos Genéticos , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Ribonucleases/química , Ribonucleases/metabolismo , Homologia de Sequência de Aminoácidos
12.
J Biochem ; 154(2): 137-47, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23620598

RESUMO

The structure of the nucleosome has been solved at atomic resolution, and the genome-wide nucleosome positions have been clarified for the budding yeast Saccharomyces cerevisiae. However, the genome-wide three-dimensional arrangement of nucleosomal arrays in the nucleus remains unclear. Several studies simulated overall interphase chromosome architectures by introducing the putative persistence length of the controversial 30-nm chromatin fibres into the modelling and using data-fitting approaches. However, the genome-folding mechanism still could not be linked with the chromosome shapes, to identify which structures or properties of chromatin fibres or DNA sequences determine the overall interphase chromosome architectures. Here we demonstrate that the paths of nucleosomal arrays and the chromatin architectures themselves are determined principally by the physical properties of genomic DNA and the nucleus size in yeast. We clarified the flexibilities and persistence lengths of all linker DNAs of the organism, deduced their spatial expanses and simulated the architectures of all 16 interphase chromosomes in the nucleus, at a resolution of beads-on-a-string chromatin fibre. For the average spatial distance between two given loci in a chromosome, the model predictions agreed well with all experimental data reported to date. These findings suggest a general mechanism underlying the folding of eukaryotic genomes into interphase chromosomes.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromossomos Fúngicos/metabolismo , DNA Fúngico/metabolismo , Genoma Fúngico/fisiologia , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , DNA Fúngico/genética , Nucleossomos/genética , Saccharomyces cerevisiae/genética
13.
Biochemistry ; 52(8): 1344-53, 2013 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-23356538

RESUMO

DNA methylation in eukaryotes occurs on the cytosine bases in CG, CHG, and CHH (where H indicates non-G nucleotides) contexts and provides an important epigenetic mark in various biological processes. However, the structural and physical properties of methylated DNA are poorly understood. Using nondenaturing polyacrylamide gel electrophoresis, we performed a systematic study of the influence of DNA methylation on the conformation and physical properties of DNA for all CG, CHG, and CHH contexts. In the CG context, methylated multimers of the CG/CG-containing unit fragment migrated in gels slightly faster than their unmethylated counterparts. In the CHG context, both homo- and hemimethylation caused retarded migration of multimers of the CAG/CTG-containing fragment. In the CHH context, methylation caused or enhanced retarded migration of the multimers of CAA/TTG-, CAT/ATG-, CAC/GTG-, CTA/TAG-, or CTT/AAG-containing fragments. These results suggest that methylation increases DNA rigidity in the CG context and introduces distortions into several CHG and CHH sequences. More interestingly, we found that nearly all of the methylation repertoires in the CHG context and 98% of those in the CHH context in human embryonic stem cells were species that undergo conformational changes upon methylation. Similarly, most of the methylation repertoires in the Arabidopsis CHG and CHH contexts were sequences with methylation-induced distortion. We hypothesize that the methylation-induced properties or conformational changes in DNA may facilitate nucleosome formation, which provides the essential mechanism for alterations of chromatin density.


Assuntos
Metilação de DNA , DNA/química , DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Sequência de Bases , Ilhas de CpG , Eletroforese em Gel de Poliacrilamida , Células-Tronco Embrionárias/química , Humanos , Conformação de Ácido Nucleico
14.
Nucleic Acids Res ; 41(3): 1544-54, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23254334

RESUMO

Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg(2+) ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa- or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl(2). AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.


Assuntos
Montagem e Desmontagem da Cromatina , DNA/química , Nucleossomos/química , Animais , Sequência de Bases , Microscopia de Força Atômica , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Homologia de Sequência do Ácido Nucleico , Xenopus
15.
Biol Pharm Bull ; 35(4): 473-80, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22466549

RESUMO

Infection-associated inflammation can alter the expression levels and functions of cytochrome P450s (CYPs). Cyp gene expression is regulated by the activation of several nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). These receptors can be activated by xenobiotics, including medicines. Here, to study the xenobiotic-induced fluctuations in CYP during inflammation, we examined the effect of lipopolysaccharide (LPS) treatment on the level of mRNAs encoding hepatic CYPs induced by xenobiotic-activated nuclear receptors, in mice. Both the mRNA induction of Cyp genes and the metabolic activities of CYP proteins were examined. LPS treatment caused a significant decrease in the induced expression of the mRNAs for Cyp3a11, 2c29, 2c55, and 1a2, but not for Cyp2b10. To assess the CYP enzymatic activities, CYP3A-mediated testosterone 6ß-hydroxylation and the intrinsic clearance (CL(int)) of nifedipine in liver microsomes were measured in mice treated with the xenobiotic pregnenolone-16alpha-carbonitrile (PCN) with or without LPS administration. Both assays revealed that the CYP3A activity, which was induced by PCN, declined significantly after LPS treatment, and this decline correlated with the Cyp3a11 mRNA level. In addition, we found that the mRNAs for interleukin (IL)-1ß and tumor necrosis factor (TNF) α were increased after treatment with LPS plus xenobiotics. Our findings demonstrated that LPS treatment reduces the PXR- and AhR-mediated, and possibly CAR-mediated Cyp gene expression and further suggest that these decreases are dependent on inflammatory cytokines in the liver.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Xenobióticos/farmacologia , Animais , Benzo(a)pireno/farmacologia , Receptor Constitutivo de Androstano , Inflamação/induzido quimicamente , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptor de Pregnano X , Carbonitrila de Pregnenolona/farmacologia , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Hidrocarboneto Arílico/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores de Esteroides/agonistas
16.
Mol Biol Rep ; 39(4): 4989-96, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22160571

RESUMO

Histone eviction and deposition are critical steps in many nuclear processes. The histone H3/H4 chaperone Asf1p is highly conserved and is involved in DNA replication, DNA repair, and transcription. To identify the factors concerned with anti-silencing function 1 (ASF1), we purified Asf1p-associated factors from the yeast Saccharomyces cerevisiae by a GST pull-down experiment, and mass spectrometry analysis was performed. Several factors are specifically associated with Asf1p, including Vip1p. VIP1 is conserved from yeast to humans and encodes inositol hexakisphoshate and inositol heptakisphosphate kinase. Vip1p interacted with Asf1p as a dimer or in a complex with another protein(s). Deletion of VIP1 did not affect the interaction between Asf1p and other Asf1p-associated factors. An in vitro GST pull-down assay indicated a direct interaction between Asf1p and Vip1p, and the interaction between the two factors in vivo was detected by an immunoprecipitation experiment. Furthermore, genetic experiments revealed that VIP1 disruption increased sensitivity to 6-azauracil (6-AU), but not to DNA-damaging reagents in wild-type and ASF1-deleted strains. It is thought that 6-AU decreases nucleotide levels and reduces transcription elongation. These observations suggest that the association of Asf1p and Vip1p may be implicated in transcription elongation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Dano ao DNA , Replicação do DNA , Deleção de Genes , Ligação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transcrição Gênica
17.
Biochem Pharmacol ; 81(7): 866-72, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21276770

RESUMO

We previously demonstrated that prostaglandin (PG) E2 stimulates adhesion of mastocytoma P-815 cells (P-815 cells) to the Arg-Gly-Asp (RGD)-enriched matrix via the PGE2 receptor subtype EP4 [Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes, EP3 and EP4. J Biol Chem 2003;278:17977-81]. Here we investigated the role of various integrin subtypes in the induction of adherent activity in PGE(2)-stimulated P-815 cells. FACS analysis showed that P-815 cells express high levels of integrin α4, α5, ß1 and ß2 subunits and moderate levels of integrin αIIb, αv, ß3 and ß7 subunits. When treated with PGE2, the EP4 agonist ONO-AE1-329 or the cell permeable cAMP analogue, 8-Br-cAMP, P-815 cells showed markedly increased cell surface expression of integrin αIIb, αv and ß3 subunits, and these expressions were significantly reduced by addition of the protein synthesis inhibitor cycloheximide. Along with increased cell surface expression, mRNA and protein levels of the integrin ß3 subunit, but not of integrin αIIb and αv subunits, were simultaneously elevated. On the other hand, adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP was abolished by antibodies specific for integrin αv and ß3 subunits, but not by antibodies for integrin α4, α5, ß1, ß2 and ß7 subunits. Moreover, treatment with tirofiban, an integrin αIIbß3 antagonist, or eptifibatide, an integrin αvß3/αIIbß3 antagonist resulted in a decrease in adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP. These results suggest that de novo synthesis of the integrin ß3 subunit plays a pivotal role in PGE2-induced adhesion of P-815 cells to the RGD-enriched matrix through EP4-mediated cAMP signaling.


Assuntos
Adesão Celular/efeitos dos fármacos , Dinoprostona/farmacologia , Fibronectinas/metabolismo , Integrina beta3/biossíntese , Mastocitoma/patologia , Oligopeptídeos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Separação Celular , Citometria de Fluxo , Humanos , Mastocitoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Mol Biol Rep ; 38(1): 37-47, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20306228

RESUMO

Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.


Assuntos
DNA/química , DNA/genética , Células-Tronco Embrionárias/metabolismo , Conformação de Ácido Nucleico , Transativadores/genética , Transformação Genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Cromatina/genética , Desoxirribonuclease I/metabolismo , Células-Tronco Embrionárias/citologia , Genes Reporter/genética , Loci Gênicos/genética , Genoma/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Histonas/metabolismo , Camundongos , Modelos Moleculares , Regiões Promotoras Genéticas/genética , Transcrição Gênica
19.
Water Res ; 44(8): 2409-18, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20117817

RESUMO

This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) alpha in the Lake Biwa-Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RARalpha agonist contamination in the river basins. RARalpha agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6 ng-atRA/L and 23.5 ng-atRA/L being observed in Lake Biwa-Yodo River and Ina River, respectively. The results indicated that RARalpha agonists are always present and widespread in the rivers. Comparative investigation of RARalpha and estrogen receptor alpha agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARalpha agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RARalpha agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RARalpha agonists in the rivers. Although a trial conducted to identify RARalpha agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography-mass spectrometry analysis revealed that the major causative contaminants responsible for the RARalpha agonistic activity were not RAs (natural RAR ligands) and 4-oxo-RAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RARalpha agonists with high activity in the rivers.


Assuntos
Monitoramento Ambiental , Receptores do Ácido Retinoico/agonistas , Poluentes Químicos da Água/análise , Japão , Rios , Técnicas do Sistema de Duplo-Híbrido , Leveduras
20.
J Biochem Mol Toxicol ; 24(1): 60-5, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20175144

RESUMO

A cDNA of rat liver thioltransferase was cloned and then expressed using pMAL-c expression vector in Escherichia coli. Recombinant rat liver thioltransferase was expressed as a fusion protein with maltose-binding protein and then purified by amylose resin column chromatography to be homogeneity on 12.5% SDS-polyacrylamide gel electrophoretic analysis. The expressed proteins were shown as two bands at around 53 and 41 kDa, suggesting that the high molecular one was a fusion protein of recombinant thioltransferase (11.7 plus 41 kDa) and the other (smaller one) was a maltose-binding protein (41 kDa). A recombinant thioltransferase catalyzed a thiol/disulfide exchange reaction in the same way as thioltransferases purified from various sources. Compared with wild type, the mutants C23A, C26A, C79A, and C83A showed 0%, 17%, 82%, and 86% in the enzymatic activity, respectively. In addition, wild-type-transfected bacteria expressed in bacterial cells showed a strong resistance to H(2)O(2) treatment as well as the case of active mutants (C79A and C83A), but inactive mutants (C23A and C26A) showed no resistance to H(2)O(2) treatment as same as mocktransfection. Thioltransferase can be important for survival of bacterial cells under oxidative stress.


Assuntos
Cisteína/genética , Glutarredoxinas/genética , Mutação , Estresse Oxidativo/genética , Sequência de Aminoácidos , Animais , Sobrevivência Celular/genética , Cisteína/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Biblioteca Gênica , Glutarredoxinas/química , Glutarredoxinas/isolamento & purificação , Glutarredoxinas/metabolismo , Peróxido de Hidrogênio/toxicidade , Fígado , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transformação Bacteriana
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