RESUMO
The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nucléolo Celular/metabolismo , Genes de Plantas/fisiologia , Genes de RNAr/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , RNA de Plantas/metabolismo , RNA Ribossômico 18S/biossíntese , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Nucléolo Celular/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Epigênese Genética/fisiologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Regiões Promotoras Genéticas/fisiologia , Protoplastos/citologia , Protoplastos/metabolismo , RNA de Plantas/genética , RNA Ribossômico 18S/genética , Transcrição Gênica/fisiologiaRESUMO
Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative ß-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.
Assuntos
Glycine max/fisiologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/enzimologia , Arabidopsis/genética , Citosol/enzimologia , Dioxigenases/metabolismo , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Fixação de Nitrogênio/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastídeos/enzimologia , Protoplastos/metabolismo , Interferência de RNA , Nódulos Radiculares de Plantas/genética , Xantofilas/análise , ZeaxantinasRESUMO
Uncontrolled growth of insulating lithium sulfide leads to passivation of sulfur cathodes, which limits high sulfur utilization in lithium-sulfur batteries. Sulfur utilization can be augmented in electrolytes based on solvents with high Gutmann Donor Number; however, violent lithium metal corrosion is a drawback. Here we report that particulate lithium sulfide growth can be achieved using a salt anion with a high donor number, such as bromide or triflate. The use of bromide leads to ~95 % sulfur utilization by suppressing electrode passivation. More importantly, the electrolytes with high-donor-number salt anions are notably compatible with lithium metal electrodes. The approach enables a high sulfur-loaded cell with areal capacity higher than 4 mA h cm-2 and high sulfur utilization ( > 90 %). This work offers a simple but practical strategy to modulate lithium sulfide growth, while conserving stability for high-performance lithium-sulfur batteries.
RESUMO
Human nucleolar phosphoprotein p140 (hNopp140) is a nucleolar phosphoprotein that can bind to doxorubicin, an anti-cancer agent. We have examined the interaction between hNopp140 and doxorubicin as well as the folding property of hNopp140. Also, the effects of ATP and phosphorylation on the affinity of hNopp140 to doxorubicin are investigated by affinity dependent co-precipitation and surface plasmon resonance methods. Doxorubicin preferentially binds to un-phosphorylated form of hNopp140 with a KD value of 3.3 x 10(-7) M. Furthermore, doxorubicin reduces the protein kinase CK2-dependent phosphorylation of hNopp140, indicating that doxorubicin may perturb the cellular function of hNopp140 by reducing the protein kinase CK2-dependent phosphorylation of hNopp140. Low contents of the secondary structures of hNopp140 and the fast rate of proteolysis imply that hNopp140 has a high percentage of flexible regions or extended loop structures.
Assuntos
Caseína Quinase II/metabolismo , Doxorrubicina/farmacologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Desnaturação ProteicaRESUMO
Hollow spheres consisting of porous CNT/CuO nanocomposite networks were prepared by aerosol process and their enhanced anodic properties for lithium-ion battery were investigated. Hollow spheres of CNT/CuO nanocomposites showed a 3D network wherein the length of the electron path was quite short compared with the agglomerated CNT/CuO nanocomposites. From electrochemical measurements, CuO itself shows poor discharge capacity and cycling performance due to its low electronic conductivity. In the CNT/CuO nanocomposite, enhanced discharge capacity was observed and showed similar values regardless of the morphology. With the addition of CNTs to CuO, CNTs can form a network that acts as an electron path-way in the insulating CuO matrix, leading to increased electrical conductivity. The morphology of nanocomposite affected cycle stability. Hollow spheres of CNT/CuO nanocomposite showed better cycle stability than that of agglomerated CNT/CuO nanocomposite. The hollow sphere of a CNT/CuO nanocomposite comprising a 3D network of CNTs can be applied as a high capacity anode material in Li-ion batteries.
RESUMO
Although carbon nanotubes (CNTs) have extraordinary mechanical, thermal, and electrical properties, application of CNTs remains limited due to their unique nano-sized tubular forms. CNT electrodes have relatively high sheet resistance, which does not meet the industrial requirements of various electrode materials. Thus, there are still challenges for improving the performance of CNTs in real applications, particularly in terms of satisfying industrial requirements. In this study, to utilize CNTs in bulk scale electrode applications, we developed a dry spinning technique. The dry spinning technique is a solid state fiber spinning technique that provides an adjustable aligned structure. The dry spinning approach also offers a facile and inexpensive fabrication process, factors which are favorable for industrial scalability for fabricating electrodes. We demonstrate a multilayer stacking process for enhancing the performance for Li-ion batteries. Multi-layer CNT textiles have low sheet resistance and a 3D woven structure provides high surface area. The fabricated 3D woven structured electrode delivers a higher reversible capacity of more than 400 mA hr/g with high cycle stabilities.
RESUMO
Symbiotic nodule formation on legume roots is characterized with a series of developmental reprograming in root tissues, including extensive proliferation of cortical cells. We examined a possible involvement of the target of rapamycin (TOR) pathway, a central regulator of cell growth and proliferation in animals and yeasts, during soybean nodule development. Our results show that transcription of both GmTOR and its key downstream effector, GmS6K1, are activated during nodulation, which is paralleled with higher kinase activities of these gene products as well. RNAi-mediated knockdown of GmS6K1 impaired the nodule development with severely reduced nodule weight and numbers. In addition, expression of a few nodulins including leghemoglobin was also decreased, and consequently nitrogen fixation was found to be reduced by half. Proteomic analysis of the GmS6K1-RNAi nodules identified glutamine synthetase (GS), an essential enzyme for nitrogen assimilation in nodules, as one of the proteins that are significantly down regulated. These results appear to provide solid evidence for a functional link between GmS6K1 and nodule development.
Assuntos
Glycine max/enzimologia , Proteínas de Plantas/genética , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Nódulos Radiculares de Plantas/enzimologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Plantas/metabolismo , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Glycine max/genética , Glycine max/crescimento & desenvolvimento , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Homeobox genes are essential regulators of plant development. ATHB23, a class I homeodomain leucine zipper gene of Arabidopsis, was found to be induced by treatment with the phytohormone gibberellin (GA). In order to clarify its role in development, we performed a histochemical analysis of transgenic plants containing a construct with a GUS::GFP reporter under the control of the 1.5 kb upstream region of ATHB23. The construct was mainly expressed in young leaves and the styles of flowers but not in mature leaves. Microscopic examination of young leaves revealed that it was expressed in the adaxial domain of leaf primordia and the rib meristem. Expression of ATHB23, like that of GA5 encoding GA 20-oxidase, was reduced in mutants related to adaxial-abaxial leaf polarity (phb-1d, se-2, and kan1 kan2). Reduced expression of the GUS::GFP reporter gene was also observed in an se-2 background. These results indicate that ATHB23 is under the control of GA and other activators such as PHB, and is involved in establishing polarity during leaf development.
Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/genética , Proteínas de Homeodomínio/genética , Zíper de Leucina/genética , Folhas de Planta/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/farmacologia , Proteínas de Homeodomínio/biossíntese , Meristema/metabolismo , MutaçãoRESUMO
Protein kinase CK2 is a ubiquitous protein kinase that can phosphorylate various proteins involved in central cellular processes, such as signal transduction, cell division, and proliferation. We have shown that the human nucleolar phosphoprotein p140 (hNopp140) is able to regulate the catalytic activity of CK2. Unphosphorylated hNopp140 and phospho-hNopp140 bind to the regulatory and catalytic subunits of CK2, respectively, and the interaction between hNopp140 and CK2 was prevented by inositol hexakisphosphate (InsP(6)). Phosphorylation of alpha-casein, genimin, or human phosphatidylcholine transfer protein-like protein by CK2 was inhibited by hNopp140, and InsP(6) recovered the suppressed activity of CK2 by hNopp140. These observations indicated that hNopp140 serves as a negative regulator of CK2 and that InsP(6) stimulates the activity of CK2 by blocking the interaction between hNopp140 and CK2.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Ácido Fítico/química , Caseína Quinase II/química , Caseínas/química , Catálise , Domínio Catalítico , Divisão Celular , Proliferação de Células , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/química , Fosfoproteínas/química , Fosforilação , Plasmídeos/metabolismo , Transdução de SinaisRESUMO
Aggregation of the Alzheimer's peptide Abeta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of Abeta aggregation from large libraries of inactive candidates. The screen uses a fusion of Abeta42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of Abeta42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Abeta42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic Abeta42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of Abeta42 aggregation and/or amyloidogenesis.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Peptídeos beta-Amiloides/química , Biofísica/métodos , Química Farmacêutica/métodos , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Humanos , Modelos Químicos , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Espectrometria de Fluorescência/métodos , Triazinas/químicaRESUMO
Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and beta-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and beta-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.
Assuntos
Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , Proteínas de Plantas/biossíntese , Tiorredoxinas/biossíntese , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Genes de Plantas , Peróxido de Hidrogênio/farmacologia , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Regiões Promotoras Genéticas , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Glycine max/genética , Simbiose , Tiorredoxinas/genéticaRESUMO
Low temperature induces a number of genes that encode the proteins promoting tolerance to freezing, mediated by ABA-dependent and ABA-independent pathways in plants. The cis-acting element called C/DRE is known to respond to low temperature independently of ABA action. To investigate the signalling and network of ABA-independent pathways, the transgenic Arabidopsis plants were generated containing several copies of the C/DRE derived from cor15a gene with a minimal promoter fused to a GUS reporter gene. The transgenic plants containing four copies of the C/DRE (4C/DRE-GUS) showed responsiveness to cold and drought treatments and were used for characterization of cold signalling and cross-talk. Cold-induced GUS expression was inhibited by okadaic acid at 1 nM, indicating that protein phosphatase 2A might act as a positive regulator. Light was shown to activate cold- and drought-induced GUS expression. Photo-reversibility of the GUS mRNA by red and far-red light with concomitant cold treatment suggests a role of phytochrome as a photoreceptor in mediating light signalling to activate the cold-induced gene expression through the C/DRE. Furthermore, GUS expression analysis in phyA or phyB or phyAphyB mutant backgrounds showed that phytochrome B is a primary photoreceptor responsible for the activation of cold-stress signalling in response to light. Light enhanced the induction kinetics of CBF1, 2, and 3 encoding the cognate transcription factors, and cor15a, in a consecutive manner compared to the dark condition in the cold, suggesting that the connection point between cold and light signalling mediated by phytochrome is at a higher step than the expression of CBF genes.