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1.
Nucleic Acids Res ; 47(8): 3970-3985, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30843055

RESUMO

RNA polymerase (RNAP), the transcription machinery, shows dynamic binding across the genomic DNA under different growth conditions. The genomic features that selectively redistribute the limited RNAP molecules to dictate genome-wide transcription in response to environmental cues remain largely unknown. We chose the bacterial osmotic stress response model to determine genomic features that direct genome-wide redistribution of RNAP during the stress. Genomic mapping of RNAP and transcriptome profiles corresponding to the different temporal states after salt shock were determined. We found rapid redistribution of RNAP across the genome, primarily at σ70 promoters. Three subsets of genes exhibiting differential salt sensitivities were identified. Sequence analysis using an information-theory based σ70 model indicates that the intergenic regions of salt-responsive genes are enriched with a higher density of σ70 promoter-like sites than those of salt-sensitive genes. In addition, the density of promoter-like sites has a positive linear correlation with RNAP binding at different salt concentrations. The RNAP binding contributed by the non-initiating promoter-like sites is important for gene transcription at high salt concentration. Our study demonstrates that hyperdensity of σ70 promoter-like sites in the intergenic regions of salt-responsive genes drives the RNAP redistribution for reprograming the transcriptome to counter osmotic stress.


Assuntos
DNA Bacteriano/genética , DNA Intergênico/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Cloreto de Potássio/farmacologia , Fator sigma/genética , Meios de Cultura/química , Meios de Cultura/farmacologia , DNA Bacteriano/metabolismo , DNA Intergênico/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Teoria da Informação , Modelos Genéticos , Pressão Osmótica , Regiões Promotoras Genéticas , Salinidade , Fator sigma/metabolismo , Transcrição Genética
2.
Exp Biol Med (Maywood) ; : 1535370218810892, 2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30400752

RESUMO

IMPACT STATEMENT: Liver fibrosis is a common wound-healing response to all kinds of liver injuries. Hepatic stellate cells (HSCs) activation is the key event during liver fibrogenesis. Thus, the elucidation of mechanisms for regulating HSCs activation is helpful for identifying novel anti-fibrotic targets and strategies. MAML1, an important component of Notch signal, functions in critical transcriptional coactivation in the Notch and Wnt/ß-catenin signal pathways. In the present study, we investigated the potential function of MAML1 during hepatic fibrogenesis in rats. Our results demonstrated that MAML1 participates in liver fibrosis through modulating HSCs activation via interrupting both the Notch and Wnt/ß-catenin signal transductions. Additionally, the inhibition of MAML1 markedly attenuated CCl4-induced hepatic fibrogenesis in rats. Our results shed a light for the exploitation of a new therapeutic strategy for hepatic fibrosis via targeting MAML1.

3.
Fungal Biol ; 122(6): 410-419, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29801784

RESUMO

Nitrogen starvation can induce cellular triacylglycerol (TAG) accumulation in different organisms with an unclear mechanism. In this study, we performed nutrient starvation and lipid droplet (LD) proteomics analyses of the filamentous fungus Metarhizium robertsii. Our results indicated that nitrogen starvation activated cell autophagic activity but inhibited the internalization of LDs into vacuoles for degradation. LD proteomic analyses identified an array of differentially accumulated proteins including autophagy-related (ATG) proteins, heat shock proteins, TAG metabolic and phospholipid biosynthetic enzymes when the fungus was grown in different nutrient conditions. In contrast to the highly activated MrATG8, the ATG proteins involved in vacuolar LD internalization were down-regulated after nitrogen starvation. Cellular TAG contents were increased in different ATG-gene null mutants of M. robertsii. In addition, TAG increase could be due to the up-regulation of TAG biogenesis along with the down-regulation of TAG catabolic enzymes in fungal cells after nitrogen deprivation. The data of this study benefit our understanding of the mechanism of nitrogen starvation induced TAG increase in different cells.


Assuntos
Autofagia , Gotículas Lipídicas/metabolismo , Metarhizium/metabolismo , Nitrogênio/deficiência , Estresse Fisiológico , Triglicerídeos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Vias Biossintéticas , Regulação para Baixo , Proteínas de Choque Térmico/metabolismo , Metarhizium/crescimento & desenvolvimento , Proteômica/métodos , Triglicerídeos/biossíntese , Triglicerídeos/genética , Regulação para Cima
4.
Environ Microbiol ; 20(1): 293-304, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29159973

RESUMO

Phosphatidylcholine (PC) plays an important role in maintaining membrane integrity and functionality. In this study, two key genes (Mrpct and Mrpem) putatively involved in the cytidine diphosphate (CDP)-choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways for PC biosynthesis were characterized in the insect pathogenic fungus Metarhizium robertsii. The results indicated that disruption of Mrpct did not lead to any reduction of total PC content but impaired fungal virulence and increased cellular accumulation of triacylglycerol. Deletion of Mrpem reduced PC content and impaired fungal conidiation and infection structure differentiation but did not result in virulence defects. Lipidomic analysis revealed that deletion of Mrpct and Mrpem resulted in dissimilar effects on increase and decrease of PC moieties and other phospholipid species accumulations. Interestingly, we found that these two genes played opposite roles in activation of cell autophagy when the fungi were grown in a nutrient-rich medium. The connection between PC metabolism and autophagy was confirmed because PC content was drastically reduced in Mratg8Δ and that the addition of PC could rescue null mutant sporulation defect. The results of this study facilitate the understanding of PC metabolism on fungal physiology.


Assuntos
Autofagia/genética , Citidina Difosfato Colina/genética , Metarhizium/genética , Metarhizium/metabolismo , Fosfatidilcolinas/biossíntese , Fosfatidiletanolamina N-Metiltransferase/genética , Animais , Citidina Difosfato Colina/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Genes Fúngicos/genética , Homeostase , Insetos/microbiologia , Metabolismo dos Lipídeos/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Fosfolipídeos/metabolismo , Virulência/genética
5.
Oncotarget ; 8(37): 60778-60788, 2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-28977825

RESUMO

The role of the Notch ligand Jagged1 in hepatic fibrosis remains to be elucidated. In the current study, we investigated the role of Jagged1 in the activation of hepatic stellate cells (HSCs) and development of hepatic fibrosis in rats. In vitro, Jagged1 in HSCs was downregulated and upregulated by Jagged1 siRNA and pcDNA3.1 Jagged1, respectively. The levels of epithelial-mesenchymal transition (EMT) markers and HSC activation markers were assessed using western blot analysis. The proliferation and migration capacity of HSCs were assessed using 5-ethynyl-2'-deoxyuridine (EdU) incorporation and Transwell migration assays. In vivo, a recombinant adeno-associated virus type 1 (rAAV1) vector carrying Jagged1 shRNA (rAAV1-Jagged1-shRNA) was constructed and transferred to rat livers via the tail vein. Reversion of liver fibrosis and the effect of Jagged1 signaling on EMT were studied using pathological, immunohistochemical and immunofluorescence methods. Our findings revealed that downregulation and upregulation of Jagged1 inhibited and promoted, respectively, HSC activation. The migratory capacity of HSCs was markedly restrained by Jagged1 siRNA. Furthermore, downregulation of Jagged1 suppressed EMT in HSCs. rAAV1-Jagged1-shRNA was generated to treat CCl4-induced hepatic fibrosis in rats. Treatment with rAAV1-Jagged1-shRNA reversed hepatic fibrosis by decreasing EMT. The results of the present study suggest that inhibition of Jagged1 is a potential treatment to ameliorate liver fibrosis.

7.
PLoS One ; 11(11): e0166808, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27875565

RESUMO

Macrophages play a key role in the pathogenesis of liver granuloma and fibrosis in schistosomiasis. However, the underlying mechanisms have not been fully characterized. This study revealed that the macrophages infiltrating the liver tissues in a murine model of Schistosoma japonica infection exhibited M2 functional polarization, and Notch1/Jagged1 signaling was significantly upregulated in the M2 polarized macrophages in vivo and in vitro. Furthermore, the blockade of Notch signaling pathway by a γ-secretase inhibitor could reverse macrophage M2 polarization in vitro and alleviate liver granuloma and fibrosis in the murine model of schistosomiasis. These results implied that the Notch1/Jagged1 signaling-dependent M2 polarization of macrophages might play an important role in liver granuloma and fibrosis in schistosomiasis, and the inhibition of Notch1/Jagged1 signaling might provide a novel therapeutic approach to administrate patients with schistosomiasis.


Assuntos
Cirrose Hepática/imunologia , Receptor Notch1/imunologia , Schistosoma japonicum/imunologia , Esquistossomose Japônica/imunologia , Transdução de Sinais/imunologia , Secretases da Proteína Precursora do Amiloide/imunologia , Animais , Modelos Animais de Doenças , Feminino , Proteína Jagged-1/imunologia , Cirrose Hepática/parasitologia , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Esquistossomose Japônica/patologia
8.
Sci Rep ; 5: 10625, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-26023866

RESUMO

Bax inhibitor 1 (BI-1) is a highly conserved protein originally identified as a suppressor of the proapoptotic protein Bax to inhibit cell death in animals and plants. The orthologs of BI-1 are widely distributed in filamentous fungi but their functions remain largely unknown. Herein, we report the identification and characterizations of MrBI-1, an ortholog of BI-1, in the entomopathogenic fungus Metarhizium robertsii. First, we found that MrBI-1 could partially rescue mammalian Bax-induced cell death in yeast. Deletion of MrBI-1 impaired fungal development, virulence and heat tolerance in M. robertsii. We also demonstrated that inactivation of MrBI-1 reduced fungal resistance to farnesol but not to hydrogen peroxide, suggesting that MrBI-1 contributes to antiapoptotic-like cell death via the endoplasmic reticulum stress-signaling pathway rather than the classical mitochondrium-dependent pathway. In particular, we found that unlike the observations in yeasts and plants, expression of mammalian Bax did not lead to a lethal effect in M. robertsii; however, it did aggravate the fungal apoptotic effect of farnesol. The results of this study advance our understanding of BI-1-like protein functions in filamentous fungi.


Assuntos
Adaptação Biológica , Apoptose , Proteínas Fúngicas/metabolismo , Temperatura Alta , Metarhizium/fisiologia , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Deleção de Genes , Peróxido de Hidrogênio/farmacologia , Metarhizium/classificação , Metarhizium/efeitos dos fármacos , Metarhizium/patogenicidade , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Fenótipo , Filogenia , Domínios e Motivos de Interação entre Proteínas , Esporos Fúngicos , Virulência , Proteína X Associada a bcl-2/metabolismo
9.
J Mol Cell Cardiol ; 81: 150-61, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25731682

RESUMO

Moderate enhanced reactive oxygen species (ROS) during early reperfusion trigger the cardioprotection against ischemia/reperfusion (I/R) injury, while the mechanism is largely unknown. Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) contributes to the cardioprotection but whether it is activated by ROS and how it regulates Ca(2+) homeostasis remain unclear. Here we investigated whether the ROS generated during early reperfusion protect the heart/cardiomyocyte against I/R-induced Ca(2+) overload and contractile dysfunction via the activation of JAK2/STAT3 signaling pathway by using a cardioprotective model of intermittent hypobaric hypoxia (IHH) preconditioning. IHH improved the postischemic recovery of myocardial contractile performance in isolated rat I/R hearts as well as Ca(2+) homeostasis and cell contraction in simulated I/R cardiomyocytes. Meanwhile, IHH enhanced I/R-increased STAT3 phosphorylation at tyrosine 705 in the nucleus and reversed I/R-suppressed STAT3 phosphorylation at serine 727 in the nucleus and mitochondria during reperfusion. Moreover, IHH improved I/R-suppressed sarcoplasmic reticulum (SR) Ca(2+)-ATPase 2 (SERCA2) activity, enhanced I/R-increased Bcl-2 expression, and promoted the co-localization and interaction of Bcl-2 with SERCA2 during reperfusion. These effects were abolished by scavenging ROS with N-(2-mercaptopropionyl)-glycine (2-MPG) and/or by inhibiting JAK2 with AG490 during the early reperfusion. Furthermore, IHH-improved postischemic SERCA2 activity and Ca(2+) homeostasis as well as cell contraction were reversed after Bcl-2 knockdown by short hairpin RNA. In addition, the reversal of the I/R-suppressed mitochondrial membrane potential by IHH was abolished by 2-MPG and AG490. These results indicate that during early reperfusion the ROS/JAK2/STAT3 pathways play a crucial role in (i) the IHH-maintained intracellular Ca(2+) homeostasis via the improvement of postischemic SERCA2 activity through the increase of SR Bcl-2 and its interaction with SERCA2; and (ii) the IHH-improved mitochondrial function.


Assuntos
Cálcio/metabolismo , Hipóxia/genética , Janus Quinase 2/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Depuradores de Radicais Livres/farmacologia , Regulação da Expressão Gênica , Hipóxia/metabolismo , Precondicionamento Isquêmico Miocárdico/métodos , Janus Quinase 2/genética , Masculino , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/antagonistas & inibidores , Fator de Transcrição STAT3/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Transdução de Sinais , Tiopronina/farmacologia
10.
Eukaryot Cell ; 14(4): 396-405, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25710964

RESUMO

Two-component signaling pathways generally include sensor histidine kinases and response regulators. We identified an ortholog of the response regulator protein Skn7 in the insect-pathogenic fungus Metarhizium robertsii, which we named MrSkn7. Gene deletion assays and functional characterizations indicated that MrSkn7 functions as a transcription factor. The MrSkn7 null mutant of M. robertsii lost the ability to sporulate and had defects in cell wall biosynthesis but was not sensitive to oxidative and osmotic stresses compared to the wild type. However, the mutant was able to produce spores under salt stress. Insect bioassays using these spores showed that the virulence of the mutant was significantly impaired compared to that of the wild type due to the failures to form the infection structure appressorium and evade host immunity. In particular, deletion of MrSkn7 triggered cell autolysis with typical features such as cell vacuolization, downregulation of repressor genes, and upregulation of autolysis-related genes such as extracellular chitinases and proteases. Promoter binding assays confirmed that MrSkn7 could directly or indirectly control different putative target genes. Taken together, the results of this study help us understand the functional divergence of Skn7 orthologs as well as the mechanisms underlying the development and control of virulence in insect-pathogenic fungi.


Assuntos
Autólise , Parede Celular/ultraestrutura , Proteínas Fúngicas/fisiologia , Metarhizium/fisiologia , Fatores de Transcrição/fisiologia , Animais , Quitinases/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Gafanhotos , Metarhizium/genética , Metarhizium/patogenicidade , Mariposas/microbiologia , Esporos Fúngicos/fisiologia , Fatores de Transcrição/genética , Ativação Transcricional , Virulência
11.
Fungal Genet Biol ; 81: 142-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25445307

RESUMO

Fungal polyketide synthases (PKSs) and their related gene clusters are highly diversified at both inter- and intra-specific levels. The most well characterized PKS enzymes include those responsible for the biosynthesis of polyketide pigments such as melanins. The genome of the insect pathogenic fungus Metarhizium robertsii contains 20 type I PKSs but none has been functionally characterized. In this study, two PKS genes (designated as MrPks1 and MrPKs2) showing homologies to those counterparts for the biosynthesis of heptaketide pigments and dihydroxynaphthalene (DHN)-melanins, respectively, were deleted in two different strains of M. robertsii. The results indicated that disruption of MrPks1 but not MrPks2 impaired fungal culture pigmentation and cell wall structure. In addition to the negative effect of the DHN-melanin pathway inhibitor, it was postulated that DHN-melanin would not be produced by M. robertsii. Various assays revealed that the stress resistance abilities against ultraviolet radiation, heat shock and oxidants, as well as virulence against insects were not impaired in ΔMrPks1 and ΔMrPks2 isolates when compared with the wild-type strain. Thus, the non-melanin pigment(s) produced by the fungus do not contribute to cell damage protection and pathogenicity in M. robertsii. Physiological differences were evident in the two examined wild-type strains. The results from this study advance the understanding of functional divergence of fungal PKSs.


Assuntos
Metarhizium/enzimologia , Metarhizium/metabolismo , Pigmentos Biológicos/biossíntese , Policetídeo Sintases/metabolismo , Animais , Parede Celular/metabolismo , Deleção de Genes , Insetos , Metarhizium/genética , Metarhizium/crescimento & desenvolvimento , Policetídeo Sintases/genética , Virulência
12.
Cardiovasc Res ; 105(2): 192-202, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25514931

RESUMO

AIMS: Uncoupling protein 3 (UCP3), located in the mitochondrial inner membrane, is cardioprotective, but its mechanisms of preserving mitochondrial function during ischaemia/reperfusion (I/R) are not fully understood. This study investigated whether UCP3 mediates/mimics the cardioprotection of H2O2 preconditioning (H2O2PC) against I/R injury and the downstream pathway that mediates H2O2PC- and UCP3-afforded cardioprotection. METHODS AND RESULTS: H2O2PC at 20 µM for 5 min significantly improved post-ischaemic functional recovery and reduced lactate dehydrogenase (LDH) release and infarct size with concurrently up-regulated UCP3 expressions in perfused rat hearts subjected to global no-flow I/R. These protections were blocked by UCP3 knockdown with short hairpin RNA but mimicked by UCP3 overexpression. Consistently, H2O2PC-attenuated I/R-induced cytosolic and mitochondrial Ca(2+) overload, Ca(2+) transient suppression, mitochondrial reactive oxygen species burst, and loss of mitochondrial inner membrane potential were reversed by UCP3 knockdown but mimicked by UCP3 overexpression. Moreover, co-immunoprecipitation assay revealed an interaction of UCP3 with the mitochondrial permeability transition pore (mPTP) component, adenine nucleotide translocator (ANT), while the cardioprotection induced by H2O2PC- and UCP3 overexpression in mitochondria, cardiac function, and cell survival was abolished by atractyloside, a mPTP opener binding to ANT, and partially inhibited by a PI3K/Akt inhibitor wortmannin. Furthermore, H2O2PC up-regulated the phosphorylation of Akt, and glycogen synthase kinase 3ß was blocked by UCP3 knockdown but mimicked by UCP3 overexpression. CONCLUSION: UCP3 mediates the cardioprotection of H2O2PC against I/R injury by preserving the mitochondrial function through inhibiting mPTP opening via the interaction with ANT and the PI3K/Akt pathway. Our findings reveal novel mechanisms of UCP3 in the cardioprotection.


Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , Peróxido de Hidrogênio/farmacologia , Canais Iônicos/metabolismo , Precondicionamento Isquêmico , Mitocôndrias Cardíacas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Animais , Masculino , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos Sprague-Dawley , Proteína Desacopladora 3
13.
Mol Microbiol ; 95(4): 576-89, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25441682

RESUMO

D-Xylose is the most abundant fermentable pentose in nature and can serve as a carbon source for many bacterial species. Since D-xylose constitutes the major component of hemicellulose, its metabolism is important for lignocellulosic biomass utilization. Here, we report a six-protein module for D-xylose signaling, uptake and regulation in solvent-producing Clostridium beijerinckii. This module consists of a novel 'three-component system' (a putative periplasmic ABC transporter substrate-binding protein XylFII and a two-component system LytS/YesN) and an ABC-type D-xylose transporter XylFGH. Interestingly, we demonstrate that, although XylFII harbors a transmembrane domain, it is not involved in D-xylose transport. Instead, XylFII acts as a signal sensor to assist the response of LytS/YesN to extracellular D-xylose, thus enabling LytS/YesN to directly activate the transcription of the adjacent xylFGH genes and thereby promote the uptake of D-xylose. To our knowledge, XylFII is a novel single transmembrane sensor that assists two-component system to respond to extracellular sugar molecules. Also of significance, this 'three-component system' is widely distributed in Firmicutes, indicating that it may play a broad role in this bacterial phylum. The results reported here provide new insights into the regulatory mechanism of D-xylose sensing and transport in bacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/metabolismo , Clostridium beijerinckii/genética , Clostridium beijerinckii/metabolismo , Xilose/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Proteínas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Transdução de Sinais
14.
Am J Physiol Heart Circ Physiol ; 306(11): H1569-81, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24705558

RESUMO

Inhibition of matrix metalloproteinases-2 (MMP-2) activation renders cardioprotection from ischemia/reperfusion (I/R) injury; however, the signaling pathways involved have not been fully understood. Intermittent hypobaric hypoxia (IHH) has been shown to enhance myocardial tolerance to I/R injury via triggering intrinsic adaptive responses. Here we investigated whether IHH protects the heart against I/R injury via the regulation of MMP-2 and how the MMP-2 is regulated. IHH (Po2 = 84 mmHg, 4-h/day, 4 wk) improved postischemic myocardial contractile performance, lactate dehydrogenase (LDH) release, and infarct size in isolated perfused rat hearts. Moreover, IHH reversed I/R-induced MMP-2 activation and release, disorders in the levels of MMP-2 regulators, peroxynitrite (ONOO(-)) and tissue inhibitor of metalloproteinase-4 (TIMP-4), and loss of the MMP-2 targets α-actinin and troponin I. This protection was mimicked, but not augmented, by a MMP inhibitor doxycycline and lost by the α1-adrenoceptor (AR) antagonist prazosin. Furthermore, IHH increased myocardial α1A-AR and α1B-AR density but not α1D-AR after I/R. Concomitantly, IHH further enhanced the translocation of PKC epsilon (PKCε) and decreased the release of mitochondrial cytochrome c due to I/R via the activation of α1B-AR but not α1A-AR or α1D-AR. IHH-conferred cardioprotection in the postischemic contractile function, LDH release, MMP-2 activation, and nitrotyrosine as well as TIMP-4 contents were mimicked but not additive by α1-AR stimulation with phenylephrine and were abolished by an α1B-AR antagonist chloroethylclonidine and a PKCε inhibitor PKCε V1-2. These findings demonstrate that IHH exerts cardioprotection through attenuating excess ONOO(-) biosynthesis and TIMP-4 loss and sequential MMP-2 activation via the activation of α1B-AR/PKCε pathway.


Assuntos
Hipóxia/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Traumatismo por Reperfusão/metabolismo , Actinina/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Doxiciclina/farmacologia , Masculino , Prazosina/farmacologia , Proteína Quinase C-épsilon/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/metabolismo
15.
Conf Proc IEEE Eng Med Biol Soc ; 2013: 4227-30, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24110665

RESUMO

Hand motion classification using surface electromyography (sEMG) has been widely studied for its applications in upper-limb prosthesis and human-machine interface etc. Pattern-recognition based control methods have many advantages, and the reported classification accuracy can meet the requirements of practical applications. However, the pattern instability of sEMG in actual use limited their real implementations, and limb position variations may be one of the potential factors. In this paper, we give a pilot study of the reverse effect of forearm rotations on hand motion classification, and the results show that the forearm rotations can substantially degrade the classifier's performance: the average intra-position error is only 2.4%, but the average interposition classification error is as high as 44.0%. To solve this problem, we use an extra accelerometer to estimate the forearm rotation angles, and the best combination of sEMG data and accelerometer outputs can reduce the average classification error to 3.3%.


Assuntos
Acelerometria/métodos , Eletromiografia/métodos , Antebraço/fisiologia , Rotação , Processamento de Sinais Assistido por Computador , Adulto , Membros Artificiais , Feminino , Mãos , Humanos , Masculino , Sistemas Homem-Máquina , Movimento , Projetos Piloto , Desenho de Prótese , Reprodutibilidade dos Testes
16.
Exp Biol Med (Maywood) ; 238(6): 600-9, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23918872

RESUMO

Liver fibrosis, a wound healing process following all kinds of liver injuries, is characterized by excessive deposition of extracellular matrix (ECM). Our previous study revealed that Notch3 might participate in liver fibrogenesis by regulating the activation of hepatic stellate cells (HSCs). The aim of this study was to assess the effects of Notch3 shRNA on hepatic fibrosis in a rat model induced by carbon tetrachloride (CCl4) and to clarify the mechanisms underlying those effects. Recombinant adeno-associated virus type 1 (rAAV1) vector carrying Notch3 shRNA (rAAV1-Notch3-shRNA) was generated and transferred to rat livers via the tail vein. The expression of Notch3, Jagged1, Hes1 and α-SMA were detected by real-time RT-PCR and immunofluorescence. The effects of rAAV1-Notch3-shRNA on fibrosis was investigated by pathological and immunohistochemical examination. Our findings showed that Notch3, Jagged1, Hes1 and α-SMA were downregulated. This downregulation was accompanied by improved hepatic fibrosis after the inhibition of Notch3 in vivo. rAAV1-Notch3-shRNA treatment reversed the epithelial-mesenchymal transition (EMT) in fibrotic livers by decreasing the expression of transforming growth factor ß1 (TGF-ß1) and vimentin in a line with the increased expression of E-cadherin. The inhibition of Notch3 was not found to play a role in hepatocyte proliferation. Rather, it inhibited hepatocyte apoptosis in vivo to some extent. The results of the present study suggest that the inhibition of Notch3 can protect hepatocytes from undergoing apoptosis and attenuate liver fibrogenesis. This may be a viable therapeutic option for hepatic fibrosis.


Assuntos
Dependovirus/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática/metabolismo , RNA Interferente Pequeno/genética , Receptores Notch/metabolismo , Animais , Caderinas/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Hepatócitos/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Masculino , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Notch3 , Receptores Notch/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo
17.
Cell Res ; 23(9): 1119-32, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23896987

RESUMO

Cardiovascular progenitor cells (CVPCs) derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold great promise for the study of cardiovascular development and cell-based therapy of heart diseases, but their applications are challenged by the difficulties in their efficient generation and stable maintenance. This study aims to develop chemically defined systems for robust generation and stable propagation of hPSC-derived CVPCs by modulating the key early developmental pathways involved in human cardiovascular specification and CVPC self-renewal. Herein we report that a combination of bone morphogenetic protein 4 (BMP4), glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 and ascorbic acid is sufficient to rapidly convert monolayer-cultured hPSCs, including hESCs and hiPSCs, into homogeneous CVPCs in a chemically defined medium under feeder- and serum-free culture conditions. These CVPCs stably self-renewed under feeder- and serum-free conditions and expanded over 10(7)-fold when the differentiation-inducing signals from BMP, GSK3 and Activin/Nodal pathways were simultaneously eliminated. Furthermore, these CVPCs exhibited expected genome-wide molecular features of CVPCs, retained potentials to generate major cardiovascular lineages including cardiomyocytes, smooth muscle cells and endothelial cells in vitro, and were non-tumorigenic in vivo. Altogether, the established systems reported here permit efficient generation and stable maintenance of hPSC-derived CVPCs, which represent a powerful tool to study early embryonic cardiovascular development and provide a potentially safe source of cells for myocardial regenerative medicine.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Coração/embriologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Cardiopatias/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miocárdio/citologia , Piridinas/farmacologia , Pirimidinas/farmacologia
18.
Arch Virol ; 158(12): 2543-52, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23836395

RESUMO

We compared nucleotide and deduced amino acid sequences of eight Japanese encephalitis virus (JEV) isolates derived from bats in China. We also compared the bat JEV isolates with other JEV isolates available from GenBank to determine their genetic similarity. We found a high genetic homogeneity among the bat JEVs isolated in different geographical areas from various bat species at different time periods. All eight bat JEV isolates belonged to genotype III. The mean evolutionary rate of bat JEV isolates was lower than those of isolates of other origin, but this difference was not statistically significant. Based on these results, we presume that the bat JEV isolates might be evolutionarily conserved. The eight bat JEV isolates were phylogenetically similar to mosquito BN19 and human Liyujie isolates of JEV. These results indicate that bats might be involved in natural cycle of JEV.


Assuntos
Quirópteros/virologia , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Genoma Viral , Animais , China , Análise por Conglomerados , Vírus da Encefalite Japonesa (Espécie)/classificação , Genótipo , Humanos , Dados de Sequência Molecular , Filogeografia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Fatores de Tempo , Proteínas Virais/genética
19.
Autophagy ; 9(4): 538-49, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23380892

RESUMO

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. Here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. Compared with the wild-type and gene-rescued mutant, Mratg8Δ is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. In addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Δ and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. In contrast to Mratg8Δ, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.


Assuntos
Autofagia , Metabolismo dos Lipídeos , Metarhizium/crescimento & desenvolvimento , Metarhizium/patogenicidade , Sequência de Aminoácidos , Animais , Autofagia/genética , Bombyx/microbiologia , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Glicina/metabolismo , Metarhizium/citologia , Metarhizium/genética , Dados de Sequência Molecular , Mutagênese/genética , Fenótipo , Esporos Fúngicos/citologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/ultraestrutura , Transcrição Genética , Virulência
20.
Zhonghua Gan Zang Bing Za Zhi ; 20(9): 677-82, 2012 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-23207232

RESUMO

OBJECTIVE: To investigate whether Notch signaling is activated in hepatic stellate cells (HSCs), and to determine whether manipulation of the Notch signaling pathway can effect the activation of HSCs. METHODS: The expression of Notch signaling components in unactivated or TGF-b1-activated HSC-T6 cells was detected by Taqman Probe-based gene expression analysis. Differential expression of Notch3 and Jagged1 was detected by immunofluorescence analysis. Notch3-mediated expression of the myofibroblastic markers, a-SMA and collagen I, was detected in HSC-T6 cells transfected with pcDNA3.1-N3ICD or Notch3 siRNA by Western blotting. RESULTS: Notch signaling components were expressed in both unactivated and activated HSC-T6 cells, but the TGF-b1-treated cells showed significantly higher expression levels of Jagged1 (3.9-fold, F = 2543.482), Notch3 (4.2-fold, F = 287.982), and HES1 (3.2-fold, F = 1719.851). Transfection-mediated over-expression of Notch3 led to significantly increased expression of a-SMA (6.8-fold, t = 13.157) and collagen I (5.5-fold, t = 9.810) (both P less than 0.01). Transient knock-down of Notch3 expression by siRNA decreased expression of the myofibroblastic markers (a-SMA by approximately 90%, t = 19.863 and collagen I by 84%, t = 10.376; both, P less than 0.01). Moreover, knock-down of Notch3 antagonized the TGF-b1-induced expression of a-SMA and collagen I. CONCLUSION: Notch signaling may participate in liver fibrogenesis by regulating HSC activation. Selective interruption of Notch3 may represent a new anti-fibrotic strategy to treat liver fibrosis.


Assuntos
Células Estreladas do Fígado/metabolismo , RNA Interferente Pequeno , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , RNA Mensageiro/genética , Ratos , Receptor Notch3 , Receptores Notch/genética , Proteínas Serrate-Jagged
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