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1.
Nucleic Acids Res ; 49(15): 8900-8922, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34370034

RESUMO

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/virologia , Proteínas de Arabidopsis/fisiologia , Proteínas do Capsídeo/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Núcleo Celular/metabolismo , Doenças das Plantas/virologia , RNA Viral/química , DNA Polimerase Dirigida por RNA/metabolismo
2.
Genome Biol ; 22(1): 82, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33706811

RESUMO

BACKGROUND: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. RESULTS: Here we combine iCLIP and 3'-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3'UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3'UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3'UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3'UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3'UTRs. CONCLUSIONS: We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.

3.
Virologie (Montrouge) ; 24(4): 246-273, 2020 Aug 01.
Artigo em Francês | MEDLINE | ID: mdl-32795981

RESUMO

The nuclear export of mRNAs is a complex process, involving the participaton of numerous proteins, the recruitement of which starts during the early steps of mRNAs biosynthesis and maturation. This strategy allows the cell to export only mature and non-defective transcripts to the cytoplasm where they are directed to the translational machinery. The vast majority of mRNAs is exported by the dimeric transport receptor TAP-p15, which is mainly recruited by the large multiprotein complex TREX-1. Other mRNAs that do not display all typical features of a mature transcript use variants of the TAP-p15 export pathway or recruit the alternative export receptor CRM1. Most DNA viruses, retroviruses, and influenza viruses, the mRNAs of which are synthesized in the nucleus, also use TAP-p15 and/or CRM1 to export their mRNAs. The highjacking of the cellular export machinery by viral mRNAs usually involves the presence of constitutive structural elements that directly load cellular export factors and/or viral adaptor proteins. Associated with the host export machinery, viral mRNAs escape host surveillance, are efficiently exported in the cytoplasm in order to be translated, and thus make possible the progress toward the later events of the virus life cycles.


Assuntos
Núcleo Celular , RNA Viral , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
4.
Sci Rep ; 10(1): 10694, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612181

RESUMO

Cellular transitions during development and stress response depend on coordinated transcriptomic and proteomic alterations. Pollen is particular because its development is a complex process that includes meiotic and mitotic divisions which causes a high heat sensitivity of these cells. Development and stress response are accompanied by a reprogramming of the transcriptome, e.g. by post-transcriptional regulation via miRNAs. We identified known and potentially novel miRNAs in the transcriptome of developing and heat-stressed pollen of Solanum lycopersicum (tomato). The prediction of target mRNAs yielded an equal number of predicted target-sites in CDS and 3'UTR regions of target mRNAs. The result enabled the postulation of a possible link between miRNAs and a fine-tuning of transcription factor abundance during pollen development. miRNAs seem to play a role in the pollen heat stress response as well. We identified several heat stress transcription factors and heat shock proteins as putative targets of miRNAs in response to heat stress, thereby placing these miRNAs as important elements of thermotolerance. Moreover, for members of the AP2, SBP and ARF family members we could predict a miRNA-mediated regulation during development via the miR172, mir156 and mir160-family strengthening the current concept of a cross-connection between development and stress response in plants.


Assuntos
Lycopersicon esculentum/genética , MicroRNAs/genética , Pólen/crescimento & desenvolvimento , Pólen/genética , Termotolerância/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Lycopersicon esculentum/fisiologia , Transcriptoma/genética
5.
BMC Genomics ; 19(1): 447, 2018 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884134

RESUMO

BACKGROUND: Pollen development is central for plant reproduction and is assisted by changes of the transcriptome and proteome. At the same time, pollen development and viability is largely sensitive to stress, particularly to elevated temperatures. The transcriptomic and proteomic changes during pollen development and of different stages in response to elevated temperature was targeted to define the underlying molecular principles. RESULTS: The analysis of the transcriptome and proteome of Solanum lycopersicum pollen at tetrad, post-meiotic and mature stage before and after heat stress yielded a decline of the transcriptome but an increase of the proteome size throughout pollen development. Comparison of the transcriptome and proteome led to the discovery of two modes defined as direct and delayed translation. Here, genes of distinct functional processes are under the control of direct and delayed translation. The response of pollen to elevated temperature occurs rather at proteome, but not as drastic at the transcriptome level. Heat shock proteins, proteasome subunits, ribosomal proteins and eukaryotic initiation factors are most affected. On the example of heat shock proteins we demonstrate a decoupling of transcript and protein levels as well as a distinct regulation between the developmental stages. CONCLUSIONS: The transcriptome and proteome of developing pollen undergo drastic changes in composition and quantity. Changes at the proteome level are a result of two modes assigned as direct and delayed translation. The response of pollen to elevated temperature is mainly regulated at the proteome level, whereby proteins related to synthesis and degradation of proteins are most responsive and might play a central role in the heat stress response of pollen.


Assuntos
Adaptação Fisiológica/genética , Perfilação da Expressão Gênica , Resposta ao Choque Térmico/genética , Lycopersicon esculentum/fisiologia , Pólen/fisiologia , Proteômica , Lycopersicon esculentum/genética , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo
7.
PLoS One ; 12(12): e0189062, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29253877

RESUMO

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.


Assuntos
Brassica napus/virologia , Caulimovirus/metabolismo , Caulimovirus/patogenicidade , Transativadores/química , Transativadores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Caulimovirus/ultraestrutura , Corpos de Inclusão Viral/metabolismo , Corpos de Inclusão Viral/ultraestrutura , Proteínas Mutantes/metabolismo , Fenótipo , Domínios Proteicos , Protoplastos/metabolismo , Transcrição Reversa/genética , Relação Estrutura-Atividade , Virulência , Replicação Viral
8.
DNA Res ; 24(2): 205-217, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28025318

RESUMO

Alternative splicing (AS) is a key control mechanism influencing signal response cascades in different developmental stages and under stress conditions. In this study, we examined heat stress (HS)-induced AS in the heat sensitive pollen tissue of two tomato cultivars. To obtain the entire spectrum of HS-related AS, samples taken directly after HS and after recovery were combined and analysed by RNA-seq. For nearly 9,200 genes per cultivar, we observed at least one AS event under HS. In comparison to control, for one cultivar we observed 76% more genes with intron retention (IR) or exon skipping (ES) under HS. Furthermore, 2,343 genes had at least one transcript with IR or ES accumulated under HS in both cultivars. These genes are involved in biological processes like protein folding, gene expression and heat response. Transcriptome assembly of these genes revealed that most of the alternative spliced transcripts possess truncated coding sequences resulting in partial or total loss of functional domains. Moreover, 141 HS specific and 22 HS repressed transcripts were identified. Further on, we propose AS as layer of stress response regulating constitutively expressed genes under HS by isoform abundance.


Assuntos
Processamento Alternativo , Temperatura Alta , Lycopersicon esculentum/genética , Pólen/genética , Estresse Fisiológico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/fisiologia , Proteínas de Plantas/genética , Pólen/metabolismo , Pólen/fisiologia , Transdução de Sinais
10.
PLoS One ; 10(7): e0132665, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26162084

RESUMO

The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splicing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5' region and suggested that the main role of CaMV splicing is to downregulate expression of open reading frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.


Assuntos
Processamento Alternativo/genética , Caulimovirus/genética , Transcriptoma/genética , Sequência de Aminoácidos , Sequência de Bases , Caulimovirus/isolamento & purificação , Caulimovirus/patogenicidade , Simulação por Computador , Sequência Conservada/genética , Dados de Sequência Molecular , Mutação/genética , Sítios de Splice de RNA/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
Front Microbiol ; 6: 219, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25852675

RESUMO

Cyanobacteria are photosynthetic prokaryotes important for many ecosystems with a high potential for biotechnological usage e.g., in the production of bioactive molecules. Either asks for a deep understanding of the functionality of cyanobacteria and their interaction with the environment. This in part can be inferred from the analysis of their genomes or proteomes. Today, many cyanobacterial genomes have been sequenced and annotated. This information can be used to identify biological pathways present in all cyanobacteria as proteins involved in such processes are encoded by a so called core-genome. However, beside identification of fundamental processes, genes specific for certain cyanobacterial features can be identified by a holistic genome analysis as well. We identified 559 genes that define the core-genome of 58 analyzed cyanobacteria, as well as three genes likely to be signature genes for thermophilic and 57 genes likely to be signature genes for heterocyst-forming cyanobacteria. To get insights into cyanobacterial systems for the interaction with the environment we also inspected the diversity of the outer membrane proteome with focus on ß-barrel proteins. We observed that most of the transporting outer membrane ß-barrel proteins are not globally conserved in the cyanobacterial phylum. In turn, the occurrence of ß-barrel proteins shows high strain specificity. The core set of outer membrane proteins globally conserved in cyanobacteria comprises three proteins only, namely the outer membrane ß-barrel assembly protein Omp85, the lipid A transfer protein LptD, and an OprB-type porin. Thus, we conclude that cyanobacteria have developed individual strategies for the interaction with the environment, while other intracellular processes like the regulation of the protein homeostasis are globally conserved.

12.
Bioinform Biol Insights ; 9: 1-17, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25698879

RESUMO

Ribosome biogenesis involves a large inventory of proteinaceous and RNA cofactors. More than 250 ribosome biogenesis factors (RBFs) have been described in yeast. These factors are involved in multiple aspects like rRNA processing, folding, and modification as well as in ribosomal protein (RP) assembly. Considering the importance of RBFs for particular developmental processes, we examined the complexity of RBF and RP (co-)orthologs by bioinformatic assignment in 14 different plant species and expression profiling in the model crop Solanum lycopersicum. Assigning (co-)orthologs to each RBF revealed that at least 25% of all predicted RBFs are encoded by more than one gene. At first we realized that the occurrence of multiple RBF co-orthologs is not globally correlated to the existence of multiple RP co-orthologs. The transcript abundance of genes coding for predicted RBFs and RPs in leaves and anthers of S. lycopersicum was determined by next generation sequencing (NGS). In combination with existing expression profiles, we can conclude that co-orthologs of RBFs by large account for a preferential function in different tissue or at distinct developmental stages. This notion is supported by the differential expression of selected RBFs during male gametophyte development. In addition, co-regulated clusters of RBF and RP coding genes have been observed. The relevance of these results is discussed.

13.
Virologie (Montrouge) ; 19(3): 119-139, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33065908

RESUMO

As a pararetrovirus and because of the non-canonical translation of its polycistronic pregenomic 35S RNA, Cauliflower mosaic virus (CaMV) is an original model system that has been extensively studied. Recent advances have improved our understanding of CaMV aphid transmission, cell-to-cell movement, protein expression and virus counter-defense strategy against host plant defense. Since P6/TAV is involved in many aspects of viral pathogenesis as well as in some replication steps, it is considered as the key player of CaMV infectious cycle. This paper reviews our current knowledge on CaMV multiplication and pathogenesis, with special emphasis on steps in which P6/TAV has a major role.

14.
EMBO J ; 32(8): 1087-102, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23524850

RESUMO

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Arabidopsis , Ácidos Indolacéticos/metabolismo , Fases de Leitura Aberta , Fosforilação , Polirribossomos/metabolismo , Processamento de Proteína Pós-Traducional
15.
J Cell Physiol ; 228(2): 330-40, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22718137

RESUMO

Natural glycosaminoglycans (GAGs) and chemically modified GAG derivatives are known to support osteogenic differentiation of mesenchymal stromal cells (MSC). This effect has mainly been described to be mediated by increasing the effectiveness of bone anabolic growth factors such as bone morphogenetic proteins (BMPs) due to the binding and presentation of the growth factor or by modulating its signal transduction pathway. In the present study, the influence of chondroitin sulfate (CS) and two chemically over-sulfated CS derivatives on osteogenic differentiation of human mesenchymal stromal cells (hMSC) and on BMP-2 and transforming growth factor ß1 (TGF-ß1) signalling was investigated. Over-sulfated CS derivatives induced an increase of tissue non-specific alkaline phosphatase (TNAP) activity and calcium deposition, whereas collagen synthesis was slightly decreased. The BMP-2-induced Smad1/5 activation was inhibited in the presence of over-sulfated CS derivatives leading to a loss of BMP-2-induced TNAP activity and calcium deposition. In contrast, the TGF-ß1-induced activation of Smad2/3 and collagen synthesis were not affected by the over-sulfated CS derivatives. BMP-2 and TGF-ß1 did not activate the extracellular signal-regulated kinase 1/2 or mitogen-activated protein kinase p38 in hMSC. These data suggest that over-sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP-2 and TGF-ß1 signalling, and offer therefore an interesting approach for the improvement of bone healing.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Sulfatos de Condroitina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Adulto , Fosfatase Alcalina/biossíntese , Cálcio/metabolismo , Sulfatos de Condroitina/metabolismo , Colágeno/biossíntese , Feminino , Humanos , Masculino , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/biossíntese
16.
J Immunol ; 188(11): 5283-92, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22544926

RESUMO

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.


Assuntos
Endotélio Vascular/imunologia , Antígeno HLA-A2/fisiologia , Linfócitos T Citotóxicos/imunologia , Ligação Competitiva/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Antígeno HLA-A2/biossíntese , Antígeno HLA-A2/metabolismo , Humanos , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/metabolismo , Epitopos Imunodominantes/fisiologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Ligação Proteica/imunologia , Espectrometria de Massas por Ionização por Electrospray , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia
17.
Brain Res ; 1452: 18-28, 2012 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-22444273

RESUMO

Extensive data reporting the neurogenerative, neuroprotective and neuroregenerative potential of erythropoietin (EPO), mainly on RNA level, can be found in the literature. However, there is still a poor knowledge on the response of neuronal progenitor cells (NPC) upon stimulation with EPO in terms of the protein species involved. Herein, the effect of EPO on the proliferation of human mesencephalic NPC (hmNPC) under normoxia is monitored using cellular assays and proteomic analysis (two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry). The administration of EPO increased the proliferation of hmNPC within 4 days after application. It positively influenced the cell-cycle progression by affecting the G2 phase of the cell cycle. A proteomic analysis of the protein expression in hmNPC cultures 4 days after EPO treatment identified 8 proteins differentially expressed in EPO-treated cultures. It is likely that one or more of the identified proteins are involved in cellular pathways that promote cell proliferation and differentiation of hmNPC under normoxia. Their further characterization could provide cellular targets for the development of new therapeutic agents to treat CNS injury. Moreover, as EPO signaling is hypoxia-inducible, our findings may also indicate the beneficial effect of EPO to mimic hypoxia, while bypassing its negative effects, to culture human fetal midbrain-derived progenitor cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Eritropoetina/farmacologia , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Fetais/citologia , Humanos , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Receptores da Eritropoetina/metabolismo
18.
EMBO J ; 30(7): 1343-56, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21343906

RESUMO

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator-viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV-TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP--a novel and specific target of TOR/S6K1--in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes.


Assuntos
Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Proteínas Virais/metabolismo , Arabidopsis , Proteínas de Arabidopsis , Fator de Iniciação 3 em Eucariotos/metabolismo , Imunoprecipitação , Fosfatidilinositol 3-Quinases , Ligação Proteica , Mapeamento de Interação de Proteínas , Ribossomos/metabolismo , Técnicas do Sistema de Duplo-Híbrido
19.
Pharmacol Rep ; 63(6): 1435-41, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22358091

RESUMO

Although paracetamol is known to have a damaging effect, this pharmaceutical is widely applied to pregnant and lactating women. Despite substantial progress in our understanding of its hepatotoxicity, some mechanisms, particularly of its embryonal and developmental toxicity, are still unknown. Thus, cell culture assays that investigate its toxicity are of particular interest. We assessed the effects of acute paracetamol treatment on cell viability (LDH assay, MTT assay), glutathione content (GSH assay), metabolic status (albumin and urea assays) and telomerase activity using rat embryonic liver cells (RLC-18 cells). Incubation with low (6 mmol/l) and high (15 mmol/l) concentrations of toxin for 24 h leads to 20% and 50% cytotoxicity, respectively. Paracetamol exerted its toxicity in a similar pathway (depletion of GSH stores) as in adult liver cells, producing damage at the cellular level. Interestingly, paracetamol treatment significantly enhanced telomerase activity. Mechanisms involved in paracetamol-induced inhibition of cell senescence should be further elucidated. Telomerase activity in RLC-18 cells offers unique opportunities for examining basic biologic mechanisms. Our findings should encourage further studies to investigate a link between telomerase activity and toxicity, implying a role of impaired telomerase activity in human pathology.


Assuntos
Acetaminofen/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Telomerase/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Fígado/embriologia , Ratos , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
20.
Environ Sci Technol ; 44(13): 5277-82, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20527915

RESUMO

Mobile air conditioning (MAC) systems are the second-largest energy consumers in cars after driving itself. While different measurement series are available to illustrate their behavior in hot ambient conditions, little data are available for lower temperatures. There are also no data available on diesel vehicles, despite these being quite common in Europe (up to 70% of the fleet in some countries). In the present study, six representative modern diesel passenger cars were tested. In combination with data from previous measurements on gasoline cars, a new model was developed - EEMAC = Empa Emission model for Mobile Air Conditioning systems - to predict emissions from air conditioning. The measurements obtained show that A/C activity still occurs at temperatures below the desired interior temperature. The EEMAC model was applied to the average meteorological year of a central European region and compared with the US EPA MOBILE6 model. As temperatures in central Europe are often below 20 degrees C (the point below which the two models differ), the overall results differ clearly. The estimated average annual CO(2) output according to EEMAC is six times higher than that of MOBILE6. EEMAC also indicates that around two-thirds of the fuel used for air conditioning could be saved by switching the MAC system off below 18 degrees C.


Assuntos
Poluentes Atmosféricos/análise , Dióxido de Carbono/química , Ar Condicionado , Poluição do Ar/prevenção & controle , Monóxido de Carbono/análise , Poluentes Ambientais/química , Europa (Continente) , Combustíveis Fósseis , Gasolina/economia , Veículos Automotores , Óxidos de Nitrogênio/análise , Temperatura , Emissões de Veículos
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