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1.
BMC Genomics ; 21(1): 880, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33297944

RESUMO

BACKGROUND: Late blight disease (LBD) caused by the pathogen Phytophthora infestans (PI), is the most devastating disease limiting potato (Solanum tuberosum) production globally. Currently, this disease pathogen is re-emerging and appearing in new areas at a very high intensity. A better understanding of the natural defense mechanisms against PI in different potato cultivars especially at the protein level is still lacking. Therefore, to elucidate potato proteome response to PI, we investigated changes in the proteome and leaf morphology of three potato cultivars, namely; Favorita (FA), Mira (MA), and E-malingshu N0.14 (E14) infected with PI by using the iTRAQ-based quantitative proteomics analysis. RESULTS: A total of 3306 proteins were found in the three potato genotypes, and 2044 proteins were quantified. Cluster analysis revealed MA and E14 clustered together separately from FA. The protein profile and related functions revealed that the cultivars shared a typical hypersensitive response to PI, including induction of elicitors, oxidative burst, and suppression of photosynthesis in the potato leaves. Meanwhile, MA and E14 deployed additional specific response mechanism different from FA, involving high induction of protease inhibitors, serine/threonine kinases, terpenoid, hormone signaling, and transport, which contributed to MA tolerance of LBD. Furthermore, inductions of pathogenesis-related proteins, LRR receptor-like kinases, mitogen-activated protein kinase, WRKY transcription factors, jasmonic acid, and phenolic compounds mediate E14 resistance against LBD. These proteins were confirmed at the transcription level by a quantitative polymerase chain reaction and at the translation level by western-blot. CONCLUSIONS: We found several proteins that were differentially abundant among the cultivars, that includes common and cultivar specific proteins which highlighted similarities and significant differences between FA, MA, and E14 in terms of their defense response to PI. Here the specific accumulation of mitogen-activated protein kinase, Serine/threonine kinases, WRKY transcription played a positive role in E14 immunity against PI. The candidate proteins identified reported in this study will form the basis of future studies and may improve our understanding of the molecular mechanisms of late blight disease resistance in potato.

2.
World J Clin Cases ; 8(18): 4266-4271, 2020 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-33024788

RESUMO

BACKGROUND: Cardiac resynchronization therapy (CRT) is a well-established therapy for patients with cardiomyopathy. CASE SUMMARY: The patient underwent left bundle branch area and left ventricular (reaching the left ventricular lateral vein through the coronary sinus) pacing. The optimal CRT was performed under the right bundle branch of the patient by adjusting the optimal a-v and v-v interphases to achieve the maximal benefit of the treatment. CONCLUSION: The patient was diagnosed with left bundle branch block and heart failure. A left bundle branch area pacemaker assisted in correcting the complete left bundle branch block. However, the shorter QRS wave shape after pacemaker implantation through the left bundle branch area indicated a complete right bundle branch block pattern. Hence, the left bundle branch area pacemaker is not always considered as the optimal treatment. The left bundle branch pacing with the optimization of cardiac resynchronization treatment may serve as a new CRT strategy.

3.
Medicine (Baltimore) ; 99(33): e21097, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32871980

RESUMO

INTRODUCTION: Substantial advances in cardiac pacing technology have been developed in the past decades. However, efforts to improve pacing technology to achieve physiological electrical activity, such as with cardiac resynchronization therapy, are underway. Permanent His bundle pacing, which directly stimulates the His-Purkinje network and electrically activates both ventricles, simulates physiological electric activity in the heart, and has been considered an ideal pacing strategy to treat arrhythmias. For patients with atrial fibrillation complicated by third-degree atrioventricular block (AVB), permanent His bundle pacing is a better option than conventional right ventricular apical or septal pacing, the latter of which may be associated with risks, such as heart failure. However, His bundle pacing exhibits some shortcomings, including elevated pacing threshold, dislocation, and abnormal sensing. CASE PRESENTATION: A 69-year-old female patient who had atrial fibrillation (AF) complicated by third-degree AVB and who was treated with permanent His bundle pacing combined with left bundle branch pacing. DIAGNOSIS: AF complicated by third-degree AVB. INTERVENTIONS: We used the left bundle branch as a backup pacing site to overcome any shortcomings related to permanent His bundle pacing. OUTCOMES: The patient recovered well without any events. CONCLUSION: We selected His bundle pacing as the primary pacing, but also used left bundle branch pacing as a backup approach. If His bundle pacing results in an increased sensing threshold, pacing threshold changes, or dislocations, left bundle branch pacing can compensate for dysfunction of permanent deficiencies in His bundle pacing, preserving physiological pacing.


Assuntos
Fibrilação Atrial/complicações , Fibrilação Atrial/terapia , Bloqueio Atrioventricular/complicações , Bloqueio Atrioventricular/terapia , Terapia de Ressincronização Cardíaca/métodos , Idoso , Fibrilação Atrial/diagnóstico por imagem , Fibrilação Atrial/fisiopatologia , Bloqueio Atrioventricular/diagnóstico por imagem , Bloqueio Atrioventricular/fisiopatologia , Fascículo Atrioventricular/fisiopatologia , Feminino , Humanos
4.
Mol Neurodegener ; 15(1): 40, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677986

RESUMO

Alzheimer's disease (AD) is the most common neurodegenerative disorder seen in age-dependent dementia. There is currently no effective treatment for AD, which may be attributed in part to lack of a clear underlying mechanism. Studies within the last few decades provide growing evidence for a central role of amyloid ß (Aß) and tau, as well as glial contributions to various molecular and cellular pathways in AD pathogenesis. Herein, we review recent progress with respect to Aß- and tau-associated mechanisms, and discuss glial dysfunction in AD with emphasis on neuronal and glial receptors that mediate Aß-induced toxicity. We also discuss other critical factors that may affect AD pathogenesis, including genetics, aging, variables related to environment, lifestyle habits, and describe the potential role of apolipoprotein E (APOE), viral and bacterial infection, sleep, and microbiota. Although we have gained much towards understanding various aspects underlying this devastating neurodegenerative disorder, greater commitment towards research in molecular mechanism, diagnostics and treatment will be needed in future AD research.

5.
Int J Mol Sci ; 20(1)2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30609684

RESUMO

In order to get a better understanding of protein association during Solanum tuberosum (cv. Sarpo Mira)⁻Phytophthora infestans incompatible interaction, we investigated the proteome dynamics of cv. Sarpo Mira, after foliar application of zoospore suspension from P. infestans isolate, at three key time-points: zero hours post inoculation (hpi) (Control), 48 hpi (EI), and 120 hpi (LI); divided into early and late disease stages by the tandem mass tagging (TMT) method. A total of 1229 differentially-expressed proteins (DEPs) were identified in cv. Sarpo Mira in a pairwise comparison of the two disease stages, including commonly shared DEPs, specific DEPs in early and late disease stages, respectively. Over 80% of the changes in protein abundance were up-regulated in the early stages of infection, whereas more DEPs (61%) were down-regulated in the later disease stage. Expression patterns, functional category, and enrichment tests highlighted significant coordination and enrichment of cell wall-associated defense response proteins during the early stage of infection. The late stage was characterized by a cellular protein modification process, membrane protein complex formation, and cell death induction. These results, together with phenotypic observations, provide further insight into the molecular mechanism of P. infestans resistance in potatos.


Assuntos
Resistência à Doença , Phytophthora infestans/patogenicidade , Proteínas de Plantas/genética , Proteoma/genética , Solanum tuberosum/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Solanum tuberosum/microbiologia
6.
Toxicon ; 153: 32-38, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30170166

RESUMO

The gamma-type inhibitor of snake venom phospholipase A2 (PLIγ) is expressed extensively in livers of both venomous and non-venomous snakes. It is not clear why PLIγs from different snake species possess diverse activities. To obtain high activity PLIγs and interpret the sequence-function relationships, we used DNA shuffling to hybridize the PLIγs of Sinonatrix annularis (saPLIγ) and Elaphe carinata (ecPLIγ). Chimera PLIγs (cPLIγ) of ∼550 bp were obtained by a series of gene manipulations including DNase I digestion, primer-free PCR, and PCR amplification with PLIγs primer pair. After successful insertion of cPLIγs into pCANTAB5e phage vector, the transformed TG1 strain of Esherichia coli was achieved. The cPLIγ phage library was produced and panned in a five-pace snake venom-coated immune tube. Three high affinity cPLIγ isoforms survived two rounds of panning. Prokaryote expression by the pET28c vector was employed for production of the three cPLIγs and the two parental PLIγs. These all showed anti-hemorrhage activity with cPLIγ 2 demonstrating superior inhibition to the parent PLIγs. Sequence alignment showed that the three kinds of cPLIγ were produced by gene splicing of S. annularis and E. carinata at different sites. Primary sequence changes brought regional changes in secondary and tertiary structure, which may explain the differences in PLIγ activity.


Assuntos
Colubridae/genética , Embaralhamento de DNA , Inibidores de Fosfolipase A2/química , Venenos de Serpentes/toxicidade , Sequência de Aminoácidos , Animais , Bacteriófagos/genética , Colubridae/metabolismo , Escherichia coli , Hemorragia/tratamento farmacológico , Fígado/metabolismo , Camundongos , Inibidores de Fosfolipase A2/isolamento & purificação , Inibidores de Fosfolipase A2/farmacologia , Isoformas de Proteínas , Alinhamento de Sequência , Venenos de Serpentes/antagonistas & inibidores
7.
Molecules ; 23(8)2018 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065214

RESUMO

Snake venom is a complex cocktail of toxins which induces a series of clinical and pathophysiological manifestations in victims, including severe local tissue damage and systemic alterations. Deinagkistrodon acutus (D. acutus) ranks among the "big four" life-threatening venomous species in China, whose venom possesses strong myotoxicity and hematotoxicity that often lead to permanent disability or muscle atrophy. Varespladib, an inhibitor of mammalian phospholipase A2 (PLA2), has been recently reproposed as an effective antidote against snakebite envenomation. The present study aimed at evaluating the protective role of varespladib on muscle regeneration in envenomed mice. Mice were grouped and subjected to inoculation with D. acutus venom or a mixture of venom and varespladib or control vehicle in the gastrocnemius muscle. Local injuries including hemorrhage, myonecrosis, ulceration, and systemic damages including general dysfunction, visceral failure, and inflammatory responses were observed at 1, 3, 7, 14, and 21 days. The results indicated that most of the muscle myonecrosis and hemorrhage were alleviated by varespladib. Besides, the pretreated mice recovered rapidly with lesser atrophy and muscle fibrosis. In conclusion, the findings of the present study suggested that varespladib is an effective antidote that could neutralize D. acutus venom and allow for earlier and improved rehabilitation outcome.


Assuntos
Acetatos/farmacologia , Antídotos/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Indóis/farmacologia , Necrose/tratamento farmacológico , Mordeduras de Serpentes/tratamento farmacológico , Úlcera/tratamento farmacológico , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/toxicidade , Crotalinae/fisiologia , Regulação da Expressão Gênica , Hemorragia/fisiopatologia , Hemorragia/prevenção & controle , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/lesões , Músculo Esquelético/inervação , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Necrose/patologia , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Fosfolipases A2 Citosólicas/antagonistas & inibidores , Fosfolipases A2 Citosólicas/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Mordeduras de Serpentes/patologia , Úlcera/patologia
8.
Toxicon ; 151: 89-95, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30003915

RESUMO

SaPLIγ is a natural phospholipase A2 (PLA2) inhibitor, isolated from Sinonatrix annularis, that has been demonstrated to protect against envenomation by other venomous snakes. As snake venom PLA2s and mammalian secretory PLA2s are similar, saPLIγ is thought to have potential to alleviate inflammatory reactions in which PLA2s act as a key enzyme for arachidonic acid release. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of action of saPLIγ in an animal model of carrageenan-induced acute inflammation. The results indicated that saPLIγ inhibited PLA2 subtypes extensively, especially IIA-PLA2, in a dose-dependent manner. Paw swelling in mice was reduced markedly by intraperitoneal saPLIγ 2.5 mg/kg, and the effect was significantly better than observed with dexamethasone at the same dose. Lower neutrophil infiltration and tissue edema was observed in the paws of saPLIγ-treated mice. Additionally, carrageenan-induced cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines (TNFα and IL-1ß) were also significantly down-regulated by saPLIγ in a dose-dependent manner. These results suggested that saPLIγ had effective anti-inflammatory effects in vivo, and these were produced by blocking mammalian IB, IIA, V and X sPLA2 subtypes.


Assuntos
Carragenina/toxicidade , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inibidores de Fosfolipase A2/farmacologia , Venenos de Serpentes/química , Animais , Colubridae , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Inibidores de Fosfolipase A2/administração & dosagem , Fosfolipases A2/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
9.
Molecules ; 23(2)2018 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-29439513

RESUMO

Phospholipase A2s (PLA2) is a major component of snake venom with diverse pathologic toxicities and, therefore, a potential target for antivenom therapy. Varespladib was initially designed as an inhibitor of mammal PLA2s, and was recently repurposed to a broad-spectrum inhibitor of PLA2 in snake venom. To evaluate the protective abilities of varespladib to hemorrhage, myonecrosis, and systemic toxicities that are inflicted by different crude snake venoms, subcutaneous ecchymosis, muscle damage, and biochemical variation in serum enzymes derived from the envenomed mice were determined, respectively. Varespladib treatment showed a significant inhibitory effect to snake venom PLA2, which was estimated by IC50 in vitro and ED50 in vivo. In animal models, the severely hemorrhagic toxicity of D. acutus and A. halys venom was almost fully inhibited after administration of varespladib. Moreover, signs of edema in gastrocnemius muscle were remarkably attenuated by administration of varespladib, with a reduced loss of myonecrosis and desmin. Serum levels of creatine kinase, lactate dehydrogenase isoenzyme 1, aspartate transaminase, and alanine transaminase were down-regulated after treatment with varespladib, which indicated the protection to viscera injury. In conclusion, varespladib may be a potential first-line drug candidate in snakebite envenomation first aid or clinical therapy.


Assuntos
Acetatos/farmacologia , Antivenenos/farmacologia , Venenos de Crotalídeos/toxicidade , Indóis/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Mordeduras de Serpentes/tratamento farmacológico , Alanina Transaminase/antagonistas & inibidores , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/antagonistas & inibidores , Aspartato Aminotransferases/sangue , Creatina Quinase/sangue , Crotalinae/fisiologia , Equimose/prevenção & controle , Edema/prevenção & controle , Feminino , Isoenzimas/antagonistas & inibidores , Isoenzimas/sangue , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/sangue , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Mordeduras de Serpentes/metabolismo , Mordeduras de Serpentes/fisiopatologia
10.
Can J Microbiol ; 62(8): 643-56, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27348421

RESUMO

Stipa purpurea is among constructive endemic species in the alpine steppe on the Qinghai-Xizang Plateau. To reveal the fungal community structure and diversity in the rhizosphere and roots of this important grass and to analyze the potential influence of different habitats on the structure of fungal communities, we explored the root endophyte and the directly associated rhizosphere communities of S. purpurea by using internal transcribed spacer rRNA cloning and sequencing methods. We found that the roots of S. purpurea are associated with a diverse consortium of Basidiomycota (59.8%) and Ascomycota (38.5%). Most fungi obtained from rhizosphere soil in S. purpurea have been identified as Ascomycetes, while the high proportion detected in roots were basidiomycetous endophytes. The species richness, diversity, and evenness of fungal assemblages were higher in roots than in the rhizosphere soil. Fungi inhabiting the rhizosphere and roots of S. purpurea are significantly different, and the rhizosphere and endophyte communities are largely independent with little overlap in the dominant phyla or operational taxonomic units. Taken together, these results suggested that a wide variety of fungal communities are associated with the roots and rhizosphere soil of S. purpurea and that the fungal assemblages are strongly influenced by different habitats.


Assuntos
Ascomicetos/isolamento & purificação , Basidiomycota/isolamento & purificação , Endófitos/isolamento & purificação , Poaceae/microbiologia , Rizosfera , Ascomicetos/genética , Basidiomycota/genética , Endófitos/genética , Lagos , Raízes de Plantas/microbiologia , Solo , Microbiologia do Solo
11.
Int J Biol Macromol ; 82: 607-13, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26522247

RESUMO

A polysaccharide termed Se-GP11 was extracted and purified from Se-enriched Grifola frondosa in our previous study. This study investigated the characterization, anti-tumor and immunomodulatory activity of Se-GP11. The results showed that Se-GP11 was composed of mannose, glucose and galactose with a molar ratio of 1:4.91:2.41. The weight-average molecular weight (Mw) and weight-average mean square radius (Rw) of Se-GP11 in 0.1M sodium chloride solution were 3.3×10(4)Da and 32.8 nm. Se-GP11 existed as a globular conformation with random coil structure. Se-GP11 had no anti-tumor activity against HepG-2 cells in vitro, and it significantly inhibited the growth of Heps tumor in vivo. Se-GP11 increased the relatively thymus and spleen weights as well as serum necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) levels. In addition, Se-GP11 promoted the phagocytosis and NO production of RAW264.7 as compared with that of the normal control group. The results revealed that the Se-GP11 may exhibit the anti-tumor through improving immunologic function of the tumor bearing mice.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/farmacologia , Grifola/química , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Selênio/química , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Chem Asian J ; 10(4): 865-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25377776

RESUMO

An efficient synthetic route toward the highly congested [5-6-7] tricyclic core of calyciphylline A-type alkaloids has been developed. This approach features a highly efficient intramolecular Diels-Alder cycloaddition to establish the aza-five-membered C ring as well as the C1 all-carbon quaternary center, and a subsequent cyclopropanation together with a ring-expansion reaction of the resulted adduct to construct the seven-membered D ring.

13.
Syst Appl Microbiol ; 37(5): 376-85, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24958606

RESUMO

A diverse array of bacteria that inhabit the rhizosphere and different plant organs play a crucial role in plant health and growth. Therefore, a general understanding of these bacterial communities and their diversity is necessary. Using the 16S rRNA gene clone library technique, the bacterial community structure and diversity of the rhizosphere and endophytic bacteria in Stellera chamaejasme compartments were compared and clarified for the first time. Grouping of the sequences obtained showed that members of the Proteobacteria (43.2%), Firmicutes (36.5%) and Actinobacteria (14.1%) were dominant in both samples. Other groups that were consistently found, albeit at lower abundance, were Bacteroidetes (2.1%), Chloroflexi (1.9%), and Cyanobacteria (1.7%). The habitats (rhizosphere vs endophytes) and organs (leaf, stem and root) structured the community, since the Wilcoxon signed rank test indicated that more varied bacteria inhabited the rhizosphere compared to the organs of the plant. In addition, correspondence analysis also showed that differences were apparent in the bacterial communities associated with these distinct habitats. Moreover, principal component analysis revealed that the profiles obtained from the rhizosphere and roots were similar, whereas leaf and stem samples clustered together on the opposite side of the plot from the rhizosphere and roots. Taken together, these results suggested that, although the communities associated with the rhizosphere and organs shared some bacterial species, the associated communities differed in structure and diversity.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biota , Endófitos/classificação , Endófitos/isolamento & purificação , Microbiologia do Solo , Thymelaeaceae/microbiologia , Bactérias/genética , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Endófitos/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Caules de Planta/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
14.
J Agric Food Chem ; 61(26): 6321-7, 2013 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-23738849

RESUMO

Trifolium pratense, a widespread legume forage plant, is reported to exhibit phytotoxic activity on other plants, but the active metabolites have not been clarified so far. A bioassay-guided fractionation of the root extracts led to the isolation of five isoflavonoids, which were elucidated by spectroscopic analysis. All of the purified compounds observably showed phytotoxic activities against Arabidopsis thaliana . Moreover, the inhibitory effects were concentration-dependent. The furan ring linked at C-4 and C-2' positions by an oxygen atom and a 1,3-dioxolane at C-4' and C-5' positions are considered to be critical factors for the phytotoxic activity. The concentrations of (6aR,11aR)-maackiain and (6aR,11aR)-trifolirhizin, concluded to be allelochemicals from soil around plants of T. pratense, were determined by HPLC and LC-MS to be 4.12 and 2.37 µg/g, respectively. These allelochemicals, which showed remarkable activities against the weed Poa annua may play an important role in assisting the widespread occurrence of T. pratense in nature.


Assuntos
Flavonoides/isolamento & purificação , Herbicidas/isolamento & purificação , Feromônios/isolamento & purificação , Extratos Vegetais/química , Exsudatos de Plantas/química , Raízes de Plantas/química , Trifolium/química , China , Flavonoides/análise , Flavonoides/química , Glucosídeos/análise , Glucosídeos/química , Glucosídeos/isolamento & purificação , Herbicidas/análise , Herbicidas/química , Compostos Heterocíclicos de 4 ou mais Anéis/análise , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/isolamento & purificação , Feromônios/análise , Feromônios/química , Extratos Vegetais/análise , Extratos Vegetais/isolamento & purificação , Exsudatos de Plantas/análise , Exsudatos de Plantas/isolamento & purificação , Raízes de Plantas/crescimento & desenvolvimento , Pterocarpanos/análise , Pterocarpanos/química , Pterocarpanos/isolamento & purificação , Solo/química , Trifolium/crescimento & desenvolvimento
15.
Proc Natl Acad Sci U S A ; 108(16): 6474-9, 2011 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-21464307

RESUMO

AKT activation requires phosphorylation of the activation loop (T308) by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and the hydrophobic motif (S473) by the mammalian target of rapamycin complex 2 (mTORC2). We recently observed that phosphorylation of the AKT hydrophobic motif was dramatically elevated, rather than decreased, in mTOR knockout heart tissues, indicating the existence of other kinase(s) contributing to AKT phosphorylation. Here we show that the atypical IκB kinase ε and TANK-binding kinase 1 (IKKε/TBK1) phosphorylate AKT on both the hydrophobic motif and the activation loop in a manner dependent on PI3K signaling. This dual phosphorylation results in a robust AKT activation in vitro. Consistently, we found that growth factors can induce AKT (S473) phosphorylation in Rictor(-/-) cells, and this effect is insensitive to mTOR inhibitor Torin1. In IKKε/TBK1 double-knockout cells, AKT activation by growth factors is compromised. We also observed that TBK1 expression is elevated in the mTOR knockout heart tissues, and that TBK1 is required for Ras-induced mouse embryonic fibroblast transformation. Our observations suggest a physiological function of IKKε/TBK1 in AKT regulation and a possible mechanism of IKKε/TBK1 in oncogenesis by activating AKT.


Assuntos
Quinase I-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fibroblastos/enzimologia , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/genética , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Miocárdio/enzimologia , Naftiridinas/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Companheira de mTOR Insensível à Rapamicina , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
J Clin Invest ; 120(8): 2805-16, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20644257

RESUMO

Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Fosfoproteínas/antagonistas & inibidores , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Cardiomegalia/etiologia , Cardiomiopatia Dilatada/etiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular , Sobrevivência Celular , Fatores de Iniciação em Eucariotos , Feminino , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Miocárdio/patologia , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Fosforilação , Proteínas , Serina-Treonina Quinases TOR
17.
J Cell Biol ; 184(6): 923-33, 2009 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-19307602

RESUMO

The insulin IGF-1-PI3K-Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca(2+) handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca(2+) channel (LTCC) protein density. The pore-forming channel subunit Ca(v)alpha1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca(v)alpha1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca(v)beta2, the LTCC chaperone for Ca(v)alpha1, antagonizes Ca(v)alpha1 protein degradation by preventing Ca(v)alpha1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca(2+) channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca(2+) entry, Ca(2+) handling, and contractility.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Cardiomiopatia Dilatada/enzimologia , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Motivos de Aminoácidos , Animais , Canais de Cálcio Tipo L/genética , Cardiomiopatia Dilatada/etiologia , Membrana Celular/enzimologia , Células Cultivadas , Sequência Conservada , Modelos Animais de Doenças , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Contração Miocárdica , Fosforilação , Estabilidade Proteica , Subunidades Proteicas , Transporte Proteico , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno , Fatores de Tempo , Transfecção
18.
Mol Hum Reprod ; 12(7): 451-60, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16772430

RESUMO

Placental trophoblast cells are unique endocrine cells that play vital roles during the processes of embryonic implantation and placentation. However, research into the function of human trophoblast has been largely restrained mainly due to a lack of adequate cell models. A normal placenta-origin cytotrophoblast cell line (NPC) was previously established by our group, but these cells showed replicating senescence after 50 population doublings (PDs). In this study, the human telomerase catalytic subunit gene (htert) was transferred into B6 strain of NPC cells, and strains with reconstituted telomerase activity (B6Tert) were established. It was shown that B6Tert-1 cells produce various biomarkers of normal extravillous cytotrophoblasts during the early weeks of gestation. Meanwhile, the cell invasiveness was inhibited by transforming growth factor beta (TGFbeta). However, their ability to form syncytium was relatively low when stimulated with fetal calf serum (FCS). The cells maintained normal cell growth properties and failed to elicit tumours in nude mice. They proliferated continuously with no signs of senescence until the final count at 210 PDs. The growth rate of B6Tert-1 cells was increased when compared with the parental cells, which results, at least partly, from facilitating release of the G1/S checkpoint during the cell-cycle regulation. This is the first report of immortalizing human normal cytotrophoblast (CTB) cells by activation of telomerase activity. The cells will provide an ideal in vitro model for the study of human extravillous trophoblast (EVT) functions and consequently the mechanisms of embryonic implantation and placentation.


Assuntos
Proteínas de Ligação a DNA/genética , Telomerase/genética , Telômero/metabolismo , Trofoblastos/metabolismo , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Cariotipagem , Queratinas/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Contraste de Fase/métodos , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Telômero/genética , Transfecção , Trofoblastos/citologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Vimentina/análise
19.
Acta Biochim Biophys Sin (Shanghai) ; 38(5): 335-41, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16680374

RESUMO

Saccharomyces cerevisiae Pif1p helicase is the founding member of the Pif1 subfamily that is conserved from yeast to human. The potential human homolog of the yeast PIF1 gene has been cloned from the cDNA library of the Hek293 cell line. Here, we described a purification procedure of glutathione S-transferase (GST)-fused N terminal truncated human Pif1 protein (hPif1deltaN) from yeast and characterized the enzymatic kinetics of its ATP hydrolysis activity. The ATPase activity of human Pif1 is dependent on divalent cation, such as Mg2+, Ca2+ and single-stranded DNA. Km for ATP for the ATPase activity is approximately 200 microM. As the ATPase activity is essential for hPif1's helicase activity, these results will facilitate the further investigation on hPif1.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/análise , DNA Helicases/análise , DNA Helicases/genética , Ativação Enzimática , Estabilidade Enzimática , Humanos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética
20.
Nucleic Acids Res ; 34(5): 1393-404, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16522649

RESUMO

Telomeres, the protein-DNA complexes at the ends of eukaryotic chromosomes, are essential for chromosome stability, and their maintenance is achieved by the specialized reverse transcriptase activity of telomerase or the homologous recombination pathway in most eukaryotes. Here, we identified a human helicase, hPif1 that inhibits telomerase activity. The primary sequence and biochemical analysis suggest that hPif1 is a potential homologue of Escherichia coli RecD, an ATP-dependent 5' to 3' DNA helicase. Ectopic expression of wild-type, but not the ATPase/helicase-deficient hPif1, causes telomere shortening in HT1080 cells. hPif1 reduces telomerase processivity and unwinds DNA/RNA duplex in vitro. hPif1 preferentially binds telomeric DNA in vitro and in vivo. We propose that the mechanism of hPif1's inhibition on telomerase involves unwinding of the DNA/RNA duplex formed by telomerase RNA and telomeric DNA, and RecD homologues in eukaryotes may have evolved gaining additional functions.


Assuntos
DNA Helicases/metabolismo , Telomerase/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Escherichia coli/química , Exodesoxirribonuclease V/química , Humanos , RNA/metabolismo , Homologia de Sequência de Aminoácidos , Telomerase/metabolismo , Telômero/metabolismo
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