RESUMO
OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of ß-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with ß-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3' untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes.
Assuntos
Adaptação Fisiológica/genética , Regulação da Expressão Gênica de Plantas , Oryza/crescimento & desenvolvimento , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Dedos de Zinco , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Metais/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Oryza/efeitos dos fármacos , Oryza/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Peptídeos/metabolismo , Fenótipo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , RNA de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Sódio/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Dedos de Zinco/genéticaRESUMO
Responses to NaCl stress were investigated in phototrophically grown Alphaproteobacterium Rhodobacter sphaeroides by transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems, proVWX and betS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute in R. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operon otsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors, crpO (RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. When crpO was provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network in R. sphaeroides.
Assuntos
Lipídeos de Membrana/metabolismo , Metaboloma , Pressão Osmótica , Rhodobacter sphaeroides/efeitos dos fármacos , Cloreto de Sódio/metabolismo , Estresse Fisiológico , Transcriptoma , Betaína/metabolismo , Análise Mutacional de DNA , Rhodobacter sphaeroides/fisiologia , Transdução de Sinais , Cloreto de Sódio/toxicidade , Fatores de Tempo , Trealose/metabolismoRESUMO
The formation of radical species was examined in roots of soybean seedlings exposed to aluminum (Al). Electron spin resonance (ESR) spectra of root homogenates with the spin-trapping reagent 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) indicated the presence of carbon-centered radicals in plants not exposed to Al. Plants exposed to 50 microM Al showed a similar spectrum, with increased signal intensity. These radicals were likely produced through a H-atom abstraction reaction by hydroxyl (*OH) radicals, the synthesis of which was initiated by the formation of superoxide (O2*-) anions. The increased production of the carbon-centered radicals may be responsible for the lipid peroxidation in Al-treated roots.
Assuntos
Alumínio/toxicidade , Carbono/metabolismo , Radicais Livres/metabolismo , Glycine max/metabolismo , Raízes de Plantas/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Radical Hidroxila/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Pirróis/metabolismo , Corantes de Rosanilina/metabolismo , Glycine max/efeitos dos fármacosRESUMO
We developed a large cell culture surface with a nanostripe structure by paving polydimethylsiloxane (PDMS) replicas of a glass mold. The stripe structure has a height of 180 nm and top width of 500 nm with 400-nm intervals between stripes. Human stomach cancer SH-10-TC cells cultured on the surface changed their morphology to elongated shapes parallel to the nanostripes. In addition, cell motility parallel to the stripes was greatly enhanced. These findings strongly suggest that the nanostripe structure affected the cell physiology.
Assuntos
Técnicas de Cultura de Células , Movimento Celular , Dimetilpolisiloxanos/química , Nanoestruturas , Adesão Celular , Dimetilpolisiloxanos/síntese química , Vidro , Humanos , Células Tumorais CultivadasRESUMO
Reoptimization of analytical conditions was performed for a high-performance liquid chromatographic (HPLC) detection system for Cu(I) chelators based on the dequenching of Cu(I)-bathocuproine disulfonate complexes that occurs in the presence of Cu(I) chelators. The revision corrects for emission and excitation wavelengths that were in fact second-order light of the actual optimal wavelengths and for the composition of the postcolumn solution. These revisions resulted in an order of magnitude decrease in detection limits of phytochelatins, a class of cysteine-rich, heavy metal-binding peptides. The revised technique is capable of phytochelatin quantitation at femtomole quantities.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cobre/química , Ligantes , Metais/química , Fenantrolinas/química , Fitoquelatinas/químicaRESUMO
Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (Oxy), ferredoxin (Fd), and ferredoxin reductase (Red) components and is a member of the Rieske nonheme iron oxygenases. Rieske nonheme iron oxygenases are divided into five subclasses (IA, IB, IIA, IIB, and III) based on the number of constituents and the nature of their redox centers. Each component of a class IIB CARDO from Nocardioides aromaticivorans IC177 was purified, and the interchangeability of the electron transfer reactions with each component from the class III CARDOs was investigated. Despite the fact that the Fds of both classes are Rieske-type, strict specificities between the Oxy and Fd components were observed. On the other hand, the Fd and Red components were interchangeable, even though the Red components differ in cofactor composition; the class IIB Red contains flavin-adenine-dinucleotide (FAD)- and NADH-binding domains, whereas the class III Red has a chloroplast-type [2Fe-2S] cluster in addition to the FAD- and NADH-binding domains. The crystal structures of the class IIB Oxy and Fd components were compared to the previously reported Fd:Oxy complex structure of class III CARDO. This comparison suggested residues in common between class IIB and class III CARDOs that are important for interactions between Fd and Oxy. In the class IIB CARDOs, these included His75 and Glu71 in Fd and Lys20 and Glu357 in Oxy for electrostatic interactions, and Phe74 and Pro90 in Fd and Trp21, Leu359, and Val367 in Oxy for hydrophobic interactions. The residues that formed the interacting surface but were not conserved between classes were thought to be necessary to form the appropriate geometry and to determine electron transfer specificity between Fd and Oxy.
Assuntos
Actinomycetales/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dioxigenases/química , Dioxigenases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Dioxigenases/isolamento & purificação , Ferredoxinas/química , Ferredoxinas/isolamento & purificação , Ferredoxinas/metabolismo , Modelos Biológicos , Modelos Moleculares , Oxigenases/química , Oxigenases/isolamento & purificação , Oxigenases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/isolamento & purificaçãoRESUMO
We developed a novel microbioassay system equipped with a gradient mixer of two solutions, and we applied the microfluidic system to an anti-cancer agent test using living animal cells on a microchip. A microchannel for the gradient mixing of two solutions and eight other microchannels for cell assay were fabricated on a poly(dimethylsiloxane) substrate using a soft-lithography method. The functions necessary for this bioassay, i.e., cell culturing, chemical stimulation, cell staining, and fluorescence determination, were integrated into the microfluidic chip. Eight gradient concentrations of the fluorescein solution, ranging from 1 to 98 microg/ml, were archived at 0.1 microl/min on a microchip. A stomach cancer cell line was cultured, and a cell viability assay was conducted using 5-Fluorouracil as an anti-cancer agent on the microchip. Cell viability changed according to the estimated concentration of the agent solution. With the microbioassay system, an anti-cancer agent test was conducted using living cells simultaneously in eight individual channels with the gradient concentration of the agent on a microchip.
Assuntos
Antineoplásicos/análise , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Animais , Antineoplásicos/farmacologia , Bioensaio/instrumentação , Bioensaio/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dimetilpolisiloxanos/química , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Microquímica/instrumentação , Microquímica/métodos , Sensibilidade e Especificidade , Silicones/químicaRESUMO
The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components.