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1.
Plant Cell Environ ; 39(1): 12-25, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25158995

RESUMO

Although mitochondrial alternative oxidase (AOX) has been proposed to play essential roles in high light stress tolerance, the effects of AOX on chlorophyll synthesis are unclear. Previous studies indicated that during greening, chlorophyll accumulation was largely delayed in plants whose mitochondrial cyanide-resistant respiration was inhibited by knocking out nuclear encoded AOX gene. Here, we showed that this delay of chlorophyll accumulation was more significant under high light condition. Inhibition of cyanide-resistant respiration was also accompanied by the increase of plastid NADPH/NADP(+) ratio, especially under high light treatment which subsequently blocked the import of multiple plastidial proteins, such as some components of the photosynthetic electron transport chain, the Calvin-Benson cycle enzymes and malate/oxaloacetate shuttle components. Overexpression of AOX1a rescued the aox1a mutant phenotype, including the chlorophyll accumulation during greening and plastidial protein import. It thus suggests that light intensity affects chlorophyll synthesis during greening process by a metabolic signal, the AOX-derived plastidial NADPH/NADP(+) ratio change. Further, our results thus revealed a molecular mechanism of chloroplast-mitochondria interactions.


Assuntos
Arabidopsis/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Respiração Celular , Clorofila/metabolismo , Cloroplastos/metabolismo , Genes Reporter , Luz , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , NADP/metabolismo , Oxirredutases/metabolismo , Fotossíntese , Fitol/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/metabolismo , Tetrapirróis/metabolismo
2.
J Neurosci ; 23(4): 1169-78, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12598605

RESUMO

Neuropathic pain is a common and often incapacitating clinical problem for which little useful therapy is presently available. Painful peripheral neuropathies can have many etiologies, among which are trauma, viral infections, exposure to radiation or chemotherapy, and metabolic or autoimmune diseases. Sufferers generally experience both pain at rest and exaggerated, painful sensitivity to light touch. Spontaneous firing of injured nerves is believed to play a critical role in the induction and maintenance of neuropathic pain syndromes. Using a well characterized nerve ligation model in the rat, we demonstrate that hyperpolarization-activated, cyclic nucleotide-modulated (HCN) "pacemaker" channels play a previously unrecognized role in both touch-related pain and spontaneous neuronal discharge originating in the damaged dorsal root ganglion. HCN channels, particularly HCN1, are abundantly expressed in rat primary afferent somata. Nerve injury markedly increases pacemaker currents in large-diameter dorsal root ganglion neurons and results in pacemaker-driven spontaneous action potentials in the ligated nerve. Pharmacological blockade of HCN activity using the specific inhibitor ZD7288 reverses abnormal hypersensitivity to light touch and decreases the firing frequency of ectopic discharges originating in Abeta and Adelta fibers by 90 and 40%, respectively, without conduction blockade. These findings suggest novel insights into the molecular basis of pain and the possibility of new, specific, effective pharmacological therapies.


Assuntos
Canais Iônicos/fisiologia , Proteínas do Tecido Nervoso , Neuralgia/etiologia , Neurônios/fisiologia , Potenciais de Ação , Animais , Linhagem Celular , Membrana Celular/química , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Condutividade Elétrica , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiopatologia , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Cinética , Masculino , Neuralgia/genética , Neuralgia/fisiopatologia , Neurônios/ultraestrutura , Canais de Potássio/fisiologia , Pirimidinas/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley
3.
J Neurosci ; 23(9): 3607-15, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12736331

RESUMO

Laser capture microdissection in combination with microarrays allows for the expression analysis of thousands of genes in selected cells. Here we describe single-cell gene expression profiling of CA1 neurons in the rat hippocampus using a combination of laser capture, T7 RNA amplification, and cDNA microarray analysis. Subsequent cluster analysis of the microarray data identified two different cell types: pyramidal neurons and an interneuron. Cluster analysis also revealed differences among the pyramidal neurons, indicating that even a single cell type in vivo is not a homogeneous population of cells at the gene expression level. Microarray data were confirmed by quantitative RT-PCR and in situ hybridization. We also report on the reproducibility and sensitivity of this combination of methods. Single-cell gene expression profiling offers a powerful tool to tackle the complexity of the mammalian brain.


Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Bacteriófago T7/genética , Contagem de Células , Análise por Conglomerados , Feminino , Hipocampo/citologia , Hibridização In Situ , Interneurônios/química , Interneurônios/metabolismo , Lasers , Neurônios/química , Células Piramidais/química , Células Piramidais/metabolismo , RNA Antissenso/análise , RNA Antissenso/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
4.
Methods Mol Med ; 99: 225-38, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15131341

RESUMO

In this chapter we cover what we have found to be a "best practice" for real-time polymerase chain reaction (PCR) for relative mRNA quantification. We describe our techniques for tissue-sample collection and freezing, sample handling for quick and reproducible extraction of total RNA, first strand cDNA synthesis, real-time PCR amplification, and template dilution and storage for PCR. We offer our insights on intron-spanning primer design for genes (when applicable), effective primer selection vs reaction optimization, and relative quantification and sample normalization using housekeeping genes. Comments are also provided on the choice of PCR reagents including fluorescent probes, prevention of PCR "carry over," and on the practical aspects of real-time PCR theory and interpretation.


Assuntos
Dor/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , DNA Complementar/genética , RNA/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação
5.
Comb Chem High Throughput Screen ; 12(1): 64-72, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19149492

RESUMO

Hyperpolarization- and Cyclic Nucleotide-gated (HCN) channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. The HCN family consists of four members (HCN1-4). HCN channels represent the molecular correlates of I(h) (also known as 'funny' I(f) and 'queer' I(q)), a hyperpolarization-activated current best known for its role in controlling heart rate and in the regulation of neuronal resting membrane potential and excitability. A significant body of molecular and pharmacological evidence is now emerging to support a role for these channels in the function of sensory neurons and pain sensation, particularly pain associated with nerve or tissue injury. As such, HCN channels may represent valid targets for novel analgesic agents. This evidence will be reviewed in this article. We will then summarize our efforts to develop and validate methods for screening for novel HCN channel blockers.


Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Descoberta de Drogas/métodos , Canais de Potássio/efeitos dos fármacos , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização
6.
Proc Natl Acad Sci U S A ; 99(13): 8573-8, 2002 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-12084918

RESUMO

The cloning of novel G protein-coupled receptors and the search for their natural ligands, a process called reverse pharmacology, is an excellent opportunity to discover novel hormones and neurotransmitters. Based on a degenerate primer approach we have cloned a G protein-coupled receptor whose mRNA expression profile indicates highest expression in the dorsal root ganglia, specifically in the subset of small neurons, suggesting a role in nociception. In addition, moderate expression was found in lung, hypothalamus, peripheral blood leukocytes, and ovaries. Guided by a receptor-activation bioassay, we identified adenine as the endogenous ligand, which activated the receptor potently and with high structural stringency. Therefore, we propose to name this receptor as the adenine receptor. Hormonal functions have already been demonstrated for adenine derivatives like 6-benzylaminopurine in plants and 1-methyladenine in lower animals. Here, we demonstrate that adenine functions as a signaling molecule in mammals. This finding adds a third family besides P1 and P2 receptors to the class of purinergic receptors.


Assuntos
Adenina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Gânglios Espinais/metabolismo , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , Primers do DNA , DNA Complementar , Dados de Sequência Molecular , Ratos , Receptores Purinérgicos/química , Receptores Purinérgicos/genética , Homologia de Sequência de Aminoácidos , Suínos
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