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1.
Cell ; 163(1): 230-45, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26365490

RESUMO

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.


Assuntos
Células-Tronco Embrionárias/virologia , Retrovirus Endógenos/genética , Provírus/genética , Animais , Fator 1 de Modelagem da Cromatina/genética , Fator 1 de Modelagem da Cromatina/metabolismo , Células-Tronco de Carcinoma Embrionário/virologia , Epigênese Genética , Camundongos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
2.
Hum Mol Genet ; 24(22): 6314-30, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26310625

RESUMO

The mutations of F-box protein 7 (FBXO7) gene (T22M, R378G and R498X) are associated with a severe form of autosomal recessive juvenile-onset Parkinson's disease (PD) (PARK 15). Here we demonstrated that wild-type (WT) FBXO7 is a stress response protein and it can play both cytoprotective and neurotoxic roles. The WT FBXO7 protein is vital to cell mitophagy and can facilitate mitophagy to protect cells, whereas mutant FBXO7 inhibits mitophagy. Upon stress, the endogenous WT FBXO7 gets up-regulated, concentrates into mitochondria and forms FBXO7 aggregates in mitochondria. However, FBXO7 mutations aggravate deleterious FBXO7 aggregation in mitochondria. The FBXO7 aggregation and toxicity can be alleviated by Proline, glutathione (GSH) and coenzyme Q10, whereas deleterious FBXO7 aggregation in mitochondria can be aggravated by prohibitin 1 (PHB1), a mitochondrial protease inhibitor. The overexpression of WT FBXO7 could lead to FBXO7 protein aggregation and dopamine neuron degeneration in transgenic Drosophila heads. The elevated FBXO7 expression and aggregation were identified in human fibroblast cells from PD patients. FBXO7 can also form aggregates in brains of PD and Alzheimer's disease. Our study provides novel pathophysiologic insights and suggests that FBXO7 may be a potential therapeutic target in FBXO7-linked neuron degeneration in PD.


Assuntos
Proteínas F-Box/genética , Mutação , Transtornos Parkinsonianos/genética , Animais , Células Cultivadas , Drosophila , Proteínas F-Box/metabolismo , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitofagia/genética , Transtornos Parkinsonianos/metabolismo , Gravidez , Proibitinas , Agregados Proteicos/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
3.
Electrophoresis ; 36(19): 2473-81, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26105009

RESUMO

Preparation of proteins from salt-gland-rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high-quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland-rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland-rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol-based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D-LC-MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol-based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high-throughput proteomic analyses involving LC-MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.


Assuntos
Avicennia/química , Proteínas de Plantas/análise , Proteoma/análise , Tolerância ao Sal/fisiologia , Plantas Tolerantes a Sal/química , Extração Líquido-Líquido , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteoma/química , Proteoma/isolamento & purificação , Proteômica
4.
Proteomics ; 14(21-22): 2545-57, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25236605

RESUMO

In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC-MS-based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two-aqueous-phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H(+) -ATPases, ATP-binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 (http://proteomecentral.proteomexchange.org/dataset/PXD000837).


Assuntos
Avicennia/química , Membrana Celular/química , Folhas de Planta/química , Proteínas de Plantas/análise , Avicennia/citologia , Avicennia/metabolismo , Membrana Celular/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteômica , Sais/metabolismo , Espectrometria de Massas em Tandem
5.
BMC Plant Biol ; 14: 291, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25404140

RESUMO

BACKGROUND: Salt stress is a major challenge for growth and development of plants. The mangrove tree Avicennia officinalis has evolved salt tolerance mechanisms such as salt secretion through specialized glands on its leaves. Although a number of structural studies on salt glands have been done, the molecular mechanism of salt secretion is not clearly understood. Also, studies to identify salt gland-specific genes in mangroves have been scarce. RESULTS: By subtractive hybridization (SH) of cDNA from salt gland-rich cell layers (tester) with mesophyll tissues as the driver, several Expressed Sequence Tags (ESTs) were identified. The major classes of ESTs identified include those known to be involved in regulating metabolic processes (37%), stress response (17%), transcription (17%), signal transduction (17%) and transport functions (12%). A visual interactive map generated based on predicted functional gene interactions of the identified ESTs suggested altered activities of hydrolase, transmembrane transport and kinases. Quantitative Real-Time PCR (qRT-PCR) was carried out to validate the expression specificity of the ESTs identified by SH. A Dehydrin gene was chosen for further experimental analysis, because it is significantly highly expressed in salt gland cells, and dehydrins are known to be involved in stress remediation in other plants. Full-length Avicennia officinalis Dehydrin1 (AoDHN1) cDNA was obtained by Rapid Amplification of cDNA Ends. Phylogenetic analysis and further characterization of this gene suggested that AoDHN1 belongs to group II Late Embryogenesis Abundant proteins. qRT-PCR analysis of Avicennia showed up-regulation of AoDHN1 in response to salt and drought treatments. Furthermore, some functional insights were obtained by growing E. coli cells expressing AoDHN1. Growth of E. coli cells expressing AoDHN1 was significantly higher than that of the control cells without AoDHN1 under salinity and drought stresses, suggesting that the mangrove dehydrin protein helps to mitigate the abiotic stresses. CONCLUSIONS: Thirty-four ESTs were identified to be enriched in salt gland-rich tissues of A. officinalis leaves. qRT-PCR analysis showed that 10 of these were specifically enriched in the salt gland-rich tissues. Our data suggest that one of the selected genes, namely, AoDHN1 plays an important role to mitigate salt and drought stress responses.


Assuntos
Avicennia/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Cloreto de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Avicennia/efeitos dos fármacos , Avicennia/fisiologia , Sequência de Bases , DNA Complementar/química , DNA Complementar/genética , Secas , Escherichia coli/genética , Escherichia coli/metabolismo , Etiquetas de Sequências Expressas , Redes Reguladoras de Genes , Dados de Sequência Molecular , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Tolerância ao Sal , Análise de Sequência de DNA , Cloreto de Sódio/farmacologia , Regulação para Cima
6.
Analyst ; 139(17): 4223-30, 2014 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-24961450

RESUMO

We describe an ultrasensitive electrochemical nucleic acid assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL)-related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation and used to label and amplify the target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The signal-amplified assay achieved a detection limit of 83 fM (5 × 10(-18) mol in 60 µL) targets oligonucleotides and has a 4-order-wide dynamic range of target concentration. The resulting assay allowed robust discrimination between the perfect match and a three-base mismatch sequence. When exposed to the full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of 1 × 10(-16) mol in 60 µL, corresponding to approximately 33 pg of the target gene. The high sensitivity and specificity of the assay enabled a PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15 in comparison to those obtained from negative cell line HL-60. The approach enables a simple, low-cost and ultrasensitive electrochemical nucleic acid detection in portable devices, point-of-care and early disease diagnostic applications.


Assuntos
Técnicas Eletroquímicas , Proteínas de Fusão bcr-abl/genética , Nanotubos de Carbono/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Sequência de Bases , Técnicas Biossensoriais , Linhagem Celular Tumoral , Enzimas Imobilizadas/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , RNA Mensageiro/genética
7.
Plant Cell Environ ; 36(8): 1410-22, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23336288

RESUMO

The specialized salt glands on the epidermis of halophytic plants secrete excess salts from tissues by a mechanism that is poorly understood. We examined the salt glands as putative salt and water bi-regulatory units that can respond swiftly to altering environmental cues. The tropical mangrove tree species (Avicennia officinalis) is able to grow under fluctuating salinities (0.7-50.0 dS m(-1)) at intertidal zones, and its salt glands offer an excellent platform to investigate their dynamic responses under rapidly changing salinities. Utilizing a novel epidermal peel system, secretion profiles of hundreds of individual salt glands examined revealed that these glands could secrete when exposed to varying salinities. Notably, rhythmic fluctuations observed in secretion rates were reversibly inhibited by water channel (aquaporin) blocker, and two aquaporin genes (PIP and TIP) preferentially expressed in the salt gland cells were rapidly induced in response to increasing salt concentration. We propose that aquaporins are involved and contribute to the re-absorption of water during salt removal in Avicennia officinalis salt glands. This constitutes an adaptive feature that contributes to salt balance of trees growing in saline environments where freshwater availability is limited.


Assuntos
Aquaporinas/metabolismo , Avicennia/fisiologia , Cloreto de Sódio/farmacologia , Água/metabolismo , Aquaporinas/genética , Avicennia/citologia , Avicennia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Meio Ambiente , Cloreto de Mercúrio/farmacologia , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/fisiologia , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Salinidade , Plântula/citologia , Plântula/efeitos dos fármacos , Plântula/fisiologia , Análise de Sequência de DNA , Cloreto de Sódio/metabolismo , Fatores de Tempo
8.
Transl Neurodegener ; 12(1): 44, 2023 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-37718439

RESUMO

A pathological feature of Parkinson's disease (PD) is the progressive loss of dopaminergic neurons and decreased dopamine (DA) content in the substantia nigra pars compacta in PD brains. DA is the neurotransmitter of dopaminergic neurons. Accumulating evidence suggests that DA interacts with environmental and genetic factors to contribute to PD pathophysiology. Disturbances of DA synthesis, storage, transportation and metabolism have been shown to promote neurodegeneration of dopaminergic neurons in various PD models. DA is unstable and can undergo oxidation and metabolism to produce multiple reactive and toxic by-products, including reactive oxygen species, DA quinones, and 3,4-dihydroxyphenylacetaldehyde. Here we summarize and highlight recent discoveries on DA-linked pathophysiologic pathways, and discuss the potential protective and therapeutic strategies to mitigate the complications associated with DA.


Assuntos
Dopamina , Doença de Parkinson , Humanos , Encéfalo , Neurônios Dopaminérgicos
9.
Nat Genet ; 55(2): 178-186, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36658435

RESUMO

Precision medicine promises to transform healthcare for groups and individuals through early disease detection, refining diagnoses and tailoring treatments. Analysis of large-scale genomic-phenotypic databases is a critical enabler of precision medicine. Although Asia is home to 60% of the world's population, many Asian ancestries are under-represented in existing databases, leading to missed opportunities for new discoveries, particularly for diseases most relevant for these populations. The Singapore National Precision Medicine initiative is a whole-of-government 10-year initiative aiming to generate precision medicine data of up to one million individuals, integrating genomic, lifestyle, health, social and environmental data. Beyond technologies, routine adoption of precision medicine in clinical practice requires social, ethical, legal and regulatory barriers to be addressed. Identifying driver use cases in which precision medicine results in standardized changes to clinical workflows or improvements in population health, coupled with health economic analysis to demonstrate value-based healthcare, is a vital prerequisite for responsible health system adoption.


Assuntos
Atenção à Saúde , Medicina de Precisão , Humanos , Singapura , Medicina de Precisão/métodos , Ásia
10.
Blood ; 116(17): 3286-96, 2010 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-20606168

RESUMO

In up to one-third of patients with acute myeloid leukemia, a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM), and this is thought to function in cancer pathogenesis. Here, we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases, caspase-6 and -8, through direct interaction with their cleaved, active forms, but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia.


Assuntos
Inibidores de Caspase , Mutação , Células Mieloides/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Apoptose , Caspase 6/metabolismo , Caspase 8/metabolismo , Diferenciação Celular , Linhagem Celular , Citoplasma/metabolismo , Células HeLa , Humanos , Nucleofosmina , Regulação para Cima
11.
J Gene Med ; 13(1): 26-36, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21259406

RESUMO

BACKGROUND: Combination therapy is usually desirable for successful cancer treatment, especially in cancers that are resistant to single forms of therapy. METHODS: To achieve an optimal therapeutic effect against glioblastoma, we tested a strategy that combines baculovirus-mediated transfer of the p53 tumor suppressor gene with the use of sodium butyrate, a histone deacetylase inhibitor. This strategy was designed based on the findings that the transduction efficiency of baculovirus in mammalian cells can be markedly enhanced by the addition of histone deacetylase inhibitors and that these inhibitors are effective in inducing cell cycle arrest, differentiation, or apoptosis in tumor cells. RESULTS: We observed a synergistic effect of the combination of the two treatments in provoking apoptosis in glioblastoma cells with mutant p53. In a mouse glioma xenograft model, the tumor inhibitory effect of baculovirus-expressed p53 was significantly enhanced by co-administration of sodium butyrate. CONCLUSIONS: These findings suggest a new approach to treat glioblastoma using baculovirus-mediated gene transfer in combination with administration of histone deacetylase inhibitor.


Assuntos
Butiratos/farmacologia , Terapia Combinada/métodos , Genes p53 , Terapia Genética , Vetores Genéticos/genética , Animais , Antineoplásicos/farmacologia , Apoptose , Baculoviridae/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Feminino , Regulação Viral da Expressão Gênica , Glioma/terapia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Neurochem Res ; 35(11): 1805-18, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20721623

RESUMO

In this study we demonstrate for the first time that short-lived intermediate glutathione (GSH) conjugates (5-S-GSH-DA-o-quinone and 2-S-GSH-DA-o-quinone) must have first formed when GSH reacted with dopamine (DA)-derived DA-o-quinones without enzymatic catalysis in solutions. These intermediate GSH-conjugates are unstable and would finally transform into reactive or non-reactive GSH-conjugates dependent on ambient reductive forces. We demonstrated that, under sufficient reductive force, the intermediate GSH-conjugates could be reduced and transform into non-reactive 5-S-GSH-DA and 2-S-GSH-DA. However, under insufficient reductive forces, the intermediate GSH-conjugates could cyclize spontaneously to form reactive 7-S-GSH-aminochrome (7-S-GSH-AM). The 7-S-GSH-AM is so reactive and toxic that it could further conjugate with another GSH to form non-reactive 4,7-bi-GSH-5,6-dihydroindole in solutions. Furthermore 7-S-GSH-AM could abrogate tyrosinase activity rapidly and even inhibit proteasome activity in solutions. However, 7-S-GSH-AM could undergo automatically internal rearrangement and transform into non-reactive 7-S-GSH-5,6-dihydroindole if it had not conjugated with GSH. Therefore, insufficient ambient reductive force, such as decreased GSH concentration, could lead to decreased GSH detoxification efficiency for toxic DA quinones. Based on findings in this study, we propose two potential detrimental positive feedback loops involving accelerated DA oxidation, increased GSH consumption and impaired GSH detoxification efficiency, as the potential underlying chemical explanation for dopaminergic neuron degeneration in Parkinson's disease.


Assuntos
Dopamina/análogos & derivados , Glutationa/análogos & derivados , Glutationa/química , Indóis/síntese química , Dopamina/química , Retroalimentação , Glutationa/síntese química , Glutationa/metabolismo , Indóis/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/metabolismo , Oxirredução
13.
Cell Rep ; 33(4): 108309, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33113365

RESUMO

cis-regulatory elements (CREs) regulate the expression of genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of their target genes in the cellular native environment. In this study, we perform a features-oriented CRISPR-utilized systematic (FOCUS) screen of OCT4-bound CREs using CRISPR-Cas9 to identify functional enhancers important for pluripotency maintenance in mESCs. From the initial 235 candidates tested, 16 CREs are identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncover a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos Facilitadores Genéticos/genética , Células-Tronco Pluripotentes/metabolismo , Animais
14.
J Neurochem ; 108(3): 601-10, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19046408

RESUMO

In this study, we demonstrated that transient transfection and over-expression of human mutant A53T alpha-synuclein (alpha-syn) could induce expression level- and time-dependent, non-apoptotic cell death in PC12 cells, while wild-type and mutant A30P alpha-syn could not. The non-apoptotic cell death induced by over-expression of A53T alpha-syn in PC12 cells was found to be dopamine (DA) related. It could be alleviated by nerve growth factor but not by chemicals that abrogate endoplasmic reticulum stress. Furthermore, PC12 cell death could be alleviated by N-acetyl-cysteine (NAC) as well as by L-cysteine; but not by cell permeable tyrosinase inhibitors. NAC could prevent DA auto-oxidation and tyrosinase-catalyzed DA oxidation, whereas L-cysteine could potently abrogate DA auto-oxidation but could not prevent tyrosinase-catalyzed DA oxidation. Both NAC and L-cysteine could increase the reduced and total GSH levels, and concurrently decrease the oxidized GSH level in PC12 cells. On the other hand, over-expression of human mutant A53T alpha-syn could decrease the reduced and total GSH levels, and increase the oxidized GSH level in the cells. Taken together, we concluded that auto-oxidation of endogenous DA aggravates non-apoptotic cell death induced by over-expression of human mutant A53T alpha-syn in PC12 cells.


Assuntos
Morte Celular/fisiologia , Dopamina/metabolismo , alfa-Sinucleína/biossíntese , alfa-Sinucleína/genética , Acetilcisteína/farmacologia , Animais , Benzimidazóis , Cromatografia Líquida de Alta Pressão , Cisteína/farmacologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Corantes Fluorescentes , Sequestradores de Radicais Livres/farmacologia , Glutationa/metabolismo , Humanos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Mutação , Fator de Crescimento Neural/farmacologia , Oxirredução , Células PC12 , Ratos , Sais de Tetrazólio , Tiazóis , Transfecção
15.
Neurochem Res ; 34(2): 316-26, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18600447

RESUMO

In this study, new procedure with improved tandem HPLC plus ESI-MS was utilized to decipher the protective role of glutathione (GSH) against dopamine (DA) oxidation. We demonstrated that auto-oxidation of DA could produce aminochrome (AM, a cyclized DA quinone), which could be effectively abrogated by reductants, especially by GSH. Furthermore GSH was demonstrated to be able to conjugate with AM to form various conjugates via condensation reactions without enzymatic catalysis. The GSH-AM conjugates tend to aggregate, possibly mediated by conjugated AM structures, but could be inhibited by GSH. We hypothesized that proteins conjugated by AM might facilitate Lewy body formation of Parkinson's disease (PD) in dopaminergic neurons via similar polymerization. We proposed that GSH could protect dopaminergic neurons against DA-induced toxicity via various mechanisms. The imbalance between DA oxidation and GSH protective capacity could be a key factor contributing to PD. Strategies to use GSH analogues, GSH inducers or to control DA oxidation might work to control PD onset and development.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dopamina/metabolismo , Glutationa/fisiologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Endopeptidase K/metabolismo , Oxirredução , Ribonuclease Pancreático/metabolismo
16.
Anal Chem ; 80(24): 9402-10, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19007191

RESUMO

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.


Assuntos
Ensaio de Amplificação de Sinal de DNA Ramificado , Eletroquímica , Proteínas de Fusão bcr-abl/genética , Leucemia Mieloide de Fase Crônica/genética , RNA Mensageiro/genética , Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Células Tumorais Cultivadas
17.
J Periodontol ; 79(3): 525-34, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18315436

RESUMO

BACKGROUND: Human periodontium contains different cell types that have various potential roles in hard and soft tissue regeneration. However, there is limited knowledge about how these diverse cell populations contribute to the regenerative process. In this study, we investigated the surface marker difference between different periodontal cells (alveolar osteoblasts [AOs], periodontal ligament fibroblasts [PDLFs], and gingival fibroblasts [GFs]) and their differentiation potential toward osteogenic and adipogenic phenotypes. METHODS: Periodontal cells (AOs, PDLFs, and GFs) from 14 subjects were isolated. The surface antigen expression pattern of cells was analyzed by cell flow cytometry, and the molecular and histologic characterizations under osteogenic and adipogenic inductions were monitored by reverse transcription-polymerase chain reaction, Western blot, and immunocytohistology. RESULTS: The cell phenotypes of AOs were verified by the high expressions of CD29 and CD49a, whereas PDLFs showed distinctively low levels of CD63 and CD73. Under adipogenic induction, limited AOs formed cube-shaped adipose-like cells, whereas PDLFs formed spindle-shaped adipose-like cells. All three cell types expressed baseline osteo-related genes. AOs demonstrated the highest osteogenic ability followed by PDLFs and GFs. CONCLUSIONS: Cells in alveolar bone and periodontal ligament contain osteogenic and adipogenic progenitors. These observations indicate a possible application for periodontium cells in hard or soft tissue regeneration.


Assuntos
Adipogenia , Células-Tronco Adultas/fisiologia , Processo Alveolar/citologia , Osteogênese , Ligamento Periodontal/citologia , Adipócitos/citologia , Adipogenia/genética , Adulto , Processo Alveolar/fisiologia , Antígenos de Superfície/análise , Calcificação Fisiológica , Diferenciação Celular , Forma Celular , Células Cultivadas , Feminino , Fibroblastos/fisiologia , Expressão Gênica , Humanos , Masculino , Células-Tronco Multipotentes/fisiologia , Osteoblastos/fisiologia , Osteogênese/genética , Ligamento Periodontal/fisiologia
18.
J Biomed Mater Res B Appl Biomater ; 84(1): 205-17, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17497685

RESUMO

AlloDerm, a processed acellular human tissue matrix, is used in a number of surgical applications for tissue repair and regeneration. In the present work, AlloDerm serves as a model system for studying gamma radiation-induced changes in tissue structure and stability as well as the effect of such changes on the cell-matrix interactions, including cell repopulation and matrix remodeling. AlloDerm tissue matrix was treated with 2-30 kGy gamma irradiation at room temperature. Gamma irradiation reduced the swelling of tissue matrix upon rehydration and caused significant structural modifications, including collagen condensation and hole formation in collagen fibres. The tensile strength of AlloDerm increased at low gamma dose but decreased with increasing gamma dosage. The elasticity of irradiated AlloDerm was reduced significantly. Calorimetric study showed that gamma irradiation destabilized the tissue matrix, resulting in greater susceptibility to proteolytic enzyme degradation. Although gamma irradiation did not affect in vitro proliferation of fibroblast cells, it promoted tissue degradation upon cell repopulation and influenced synthesis and deposition of new collagen.


Assuntos
Proliferação de Células/efeitos da radiação , Matriz Extracelular/efeitos da radiação , Engenharia Tecidual/métodos , Alicerces Teciduais , Fenômenos Biomecânicos , Varredura Diferencial de Calorimetria , Adesão Celular/efeitos da radiação , Células Cultivadas , Colágeno/efeitos da radiação , Elasticidade , Fibroblastos/efeitos da radiação , Liofilização , Raios gama , Temperatura Alta , Humanos , Imuno-Histoquímica , Pulmão/citologia , Desnaturação Proteica/efeitos da radiação , Resistência à Tração/efeitos da radiação
19.
Biomaterials ; 28(5): 814-24, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17045643

RESUMO

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone-calcium phosphate (mPCL-CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5 x 5 x 4mm(3)) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10 x 10 x 4mm(3)) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.


Assuntos
Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Transplante Ósseo/instrumentação , Transplante Ósseo/métodos , Células Estromais/citologia , Engenharia Tecidual/métodos , Fosfatase Alcalina/metabolismo , Animais , Substitutos Ósseos/química , Osso e Ossos/metabolismo , Teste de Materiais , Osteocalcina/biossíntese , Osteopontina/biossíntese , Próteses e Implantes , Ratos , Suínos
20.
Sci Rep ; 7(1): 10031, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28855698

RESUMO

Salinity affects growth and development of plants, but mangroves exhibit exceptional salt tolerance. With direct exposure to salinity, mangrove roots possess specific adaptations to tolerate salt stress. Therefore, studying the early effects of salt on mangrove roots can help us better understand the tolerance mechanisms. Using two-month-old greenhouse-grown seedlings of the mangrove tree Avicennia officinalis subjected to NaCl treatment, we profiled gene expression changes in the roots by RNA-sequencing. Of the 6547 genes that were differentially regulated in response to salt treatment, 1404 and 5213 genes were significantly up- and down-regulated, respectively. By comparative genomics, 93 key salt tolerance-related genes were identified of which 47 were up-regulated. Upon placing all the differentially expressed genes (DEG) in known signaling pathways, it was evident that most of the DEGs involved in ethylene and auxin signaling were up-regulated while those involved in ABA signaling were down-regulated. These results imply that ABA-independent signaling pathways also play a major role in salt tolerance of A. officinalis. Further, ethylene response factors (ERFs) were abundantly expressed upon salt treatment and the Arabidopsis mutant aterf115, a homolog of AoERF114 is characterized. Overall, our results would help in understanding the possible molecular mechanism underlying salt tolerance in plants.


Assuntos
Avicennia/genética , Regulação da Expressão Gênica de Plantas , Estresse Salino , Transcriptoma , Avicennia/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Transdução de Sinais
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