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1.
Microbiol Res ; 223-225: 137-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178047

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen with high clinical relevance for hospital infections of patients. Accumulating DNA sequencing results of clinical P. aeruginosa isolates have revealed frequent mutations in lasR gene, which encodes the highest arches component of quorum-sensing system (QS). We analyzed the sequencing data of lasR gene from a large collection of cystic fibrosis (CF) P. aeruginosa isolates. Our systematical analyses revealed that single nucleotide polymorphisms (SNPs) selection in lasR gene were largely constrained by codon-usage frequency. As a whole, SNP-substituted codons encoding unconserved amino acid resulted in unfavored codons with relatively low codon-usage frequency, while those associating with conserved amino acid were not strictly regulated in such way. These SNPs substitutions gives rise to diverse functional LasR isoforms and contributes to the relative growth fitness of recombinant lasR variant strains. Our survey reveals a novel pattern of SNPs selections in lasR gene of CF isolates. Our findings could be served as a powerful resource for understanding adaptive mechanism of clinical isolates under environmental constrains and developing anti-bacteria drugs for CF patients.


Assuntos
Proteínas de Bactérias/genética , Códon/genética , Fibrose Cística/microbiologia , Polimorfismo de Nucleotídeo Único/genética , Pseudomonas aeruginosa/genética , Transativadores/genética , Sequência de Aminoácidos , Regulação Bacteriana da Expressão Gênica , Isoformas de Proteínas , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Alinhamento de Sequência , Análise de Sequência de DNA
2.
mSystems ; 3(6)2018.
Artigo em Inglês | MEDLINE | ID: mdl-30505942

RESUMO

The rice blast fungus Magnaporthe oryzae poses a great threat to global food security. During its conidiation (asexual spore formation) and appressorium (infecting structure) formation, autophagy is induced, serving glycogen breakdown or programmed cell death function, both essential for M. oryzae pathogenicity. Recently, we identified an M. oryzae histone acetyltransferase (HAT) Gcn5 as a key regulator in phototropic induction of autophagy and asexual spore formation while serving a cellular function other than autophagy induction during M. oryzae infection. To further understand the regulatory mechanism of Gcn5 on M. oryzae pathogenicity, we set out to identify more Gcn5 substrates by comparative acetylome between the wild-type (WT) and GCN5 overexpression (OX) mutant and between OX mutant and GCN5 deletion (knockout [KO]) mutant. Our results showed that Gcn5 regulates autophagy induction and other important aspects of fungal pathogenicity, including energy metabolism, stress response, cell toxicity and death, likely via both epigenetic regulation (histone acetylation) and posttranslational modification (nonhistone protein acetylation). IMPORTANCE Gcn5 is a histone acetyltransferase that was previously shown to regulate phototropic and starvation-induced autophagy in the rice blast fungus Magnaporthe oryzae, likely via modification on autophagy protein Atg7. In this study, we identified more potential substrates of Gcn5-mediated acetylation by quantitative and comparative acetylome analyses. By epifluorescence microscopy and biochemistry experiments, we verified that Gcn5 may regulate autophagy induction at both the epigenetic and posttranslational levels and regulate autophagic degradation of a critical metabolic enzyme pyruvate kinase (Pk) likely via acetylation. Overall, our findings reveal comprehensive posttranslational modification executed by Gcn5, in response to various external stimuli, to synergistically promote cellular differentiation in a fungal pathogen.

3.
Am J Transl Res ; 10(10): 3099-3110, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30416653

RESUMO

Background: Postoperative pain has well defined and is perceived by patients as one of the most obnoxious aspects of surgical pain. The aim of this study was to determine whether the combination of Therapeutic ultrasound (TUS) and Curcumin (CUR) resulted in an enhancement of their pain relieving activities in a rat model of postoperative pain. Methods: We explored the effect of these treatment and their interaction with signal transduction pathways involved in inflammatory. In this study, TUS and CUR alone or in combination were administered prior to or simultaneously with or after the incisional surgery. Results: At the start time of administration, we observed that the TUS plus CUR treatment reduced the mean paw withdrawal threshold more efficiently than CUR alone. Then we demonstrated that TUS potentiates the antinociceptive effect of CUR in a rat model of chronic postoperative pain and that the combination could facilitate the recovery of surgical pain. However, preventive value was not statistically significant when the treatments were given prior to the incisional surgery. We provide evidence that TUS plus CUR administrations were safe and significantly reduced the ED50 compared to treatment with the single CUR treatment in rats. TUS plus CUR administrations decreases incisional surgery induced activation of inflammatory cells and down-regulation of chemokines and proinflammatory cytokines, MCP-1, MIP-1α, IL-1ß, and TNF-α through regulating Sirt1/NF-κB signaling pathway. Conclusions: Taken together, our results indicate that the combinations of TUS and CUR can be more effective in the anti-nociceptive effects than the treatment with CUR alone.

4.
Biochem Biophys Res Commun ; 505(4): 1090-1096, 2018 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-30314699

RESUMO

BACKGROUND: Nasopharyngeal carcinoma (NPC) is the most common type of head and neck cancers which is notable for its distinctive pattern of geographical distribution. HOTAIR has been reported to regulate nasopharyngeal carcinoma tumorigenesis and progression. However, the detailed mechanism underlying HOTAIR-promoted nasopharyngeal carcinoma remains not fully understood. METHODS: We used RT-qPCR approach to examine genes expression and mRNA level. MTT assay and soft agar assay were used to detect cell growth rate in culture and under suspended condition, respectively. Besides, we employed wound healing assay and transwell invasion assay to determine migration and invasion ability of nasopharyngeal carcinoma cells. We predicted direct downstream targets of miR-101 by bioinformatic analysis, which was confirmed by dual luciferase reporter assay. RESULTS: HOTAIR was upregulated in NPC tissues and cells. miR-101 inhibitor greatly enhanced HOTAIR knockdown-regulated cell proliferation, migration and invasion of CNE1 and CNE2 cells. miR-101 was shown to directly bind 3'-UTR of COX-2 and downregulate COX-2 expression. Finally, COX-2 overexpression was demonstrated to rescue the tumor phenotypes of nasopharyngeal carcinoma cells attenuated by HOTAIR knockdown or miR-101 mimic. CONCLUSIONS: Here, we highlight the importance of HOTAIR/miR-101/COX-2 axis in progression of nasopharyngeal carcinoma cells. Our findings provide a novel mechanism for explaining HOTAIR-induced nasopharyngeal carcinoma and help developing the therapeutical strategies by targeting HOTAIR.


Assuntos
Ciclo-Oxigenase 2/genética , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , RNA Longo não Codificante/metabolismo , Regulação para Cima , Movimento Celular , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Células HEK293 , Humanos , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia
5.
Oncotarget ; 8(31): 50747-50760, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28881600

RESUMO

Studies have demonstrated that curcumin (CUR) exerts its tumor suppressor function in a variety of human cancers including head and neck squamous cell carcinoma (HNSCC). However, the exact underlying molecular mechanisms remain obscure. Here, we aim to test whether CUR affects ATM/Chk2/p53 signaling pathway, leading to the induction of cell cycle arrest, inhibition of angiogenesis of HNSCC in vitro and in vivo. To this end, we conducted multiple methods such as MTT assay, Invasion assay, Flow cytometry, Western blotting, RT-PCR, and transfection to explore the functions and molecular insights of CUR in HNSCC. We observed that CUR significantly induced apoptosis and cell cycle arrest, inhibited angiogenesis in HNSCC. Mechanistically, we demonstrated that CUR markedly up-regulated ATM expression and subsequently down-regulated HIF-1α expression. Blockage of ATM production totally reversed CUR induced cell cycle arrest as well as anti-angiogenesis in HNSCC. Moreover, our results demonstrated that CUR exerts its antitumor activity through targeting ATM/Chk2/p53 signal pathway. In addition, the results of xenograft experiments in mice were highly consistent with in vitro studies. Collectively, our findings suggest that targeting ATM/Chk2/p53 signal pathway by CUR could be a promising therapeutic approach for HNSCC prevention and therapy.

6.
BMC Genomics ; 17: 354, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27185248

RESUMO

BACKGROUND: Sporisorium scitamineum causes the sugarcane smut disease, one of the most serious constraints to global sugarcane production. S. scitamineum possesses a sexual mating system composed of two mating-type loci, a and b locus. We previously identified and deleted the b locus in S. scitamineum, and found that the resultant SsΔMAT-1b mutant was defective in mating and pathogenicity. RESULTS: To further understand the function of b-mating locus, we carried out transcriptome analysis by comparing the transcripts of the mutant strain SsΔMAT-1b, from which the SsbE1 and SsbW1 homeodomain transcription factors have previously been deleted, with those from the wild-type MAT-1 strain. Also the transcripts from SsΔMAT-1b X MAT-2 were compared with those from wild-type MAT-1 X MAT-2 mating. A total of 209 genes were up-regulated (p < 0.05) in the SsΔMAT-1b mutant, compared to the wild-type MAT-1 strain, while 148 genes down-regulated (p < 0.05). In the mixture, 120 genes were up-regulated (p < 0.05) in SsΔMAT-1b X MAT-2, which failed to mate, compared to the wild-type MAT-1 X MAT-2 mating, and 271 genes down-regulated (p < 0.05). By comparing the up- and down-regulated genes in these two sets, it was found that 15 up-regulated and 37 down-regulated genes were common in non-mating haploid and mating mixture, which indeed could be genes regulated by b-locus. Furthermore, GO and KEGG enrichment analysis suggested that carbon metabolism pathway and stress response mediated by Hog1 MAPK signaling pathway were altered in the non-mating sets. CONCLUSIONS: Experimental validation results indicate that the bE/bW heterodimeric transcriptional factor, encoded by the b-locus, could regulate S. scitamineum sexual mating and/or filamentous growth via modulating glucose metabolism and Hog1-mediating oxidative response.


Assuntos
Basidiomycota/fisiologia , Meio Ambiente , Perfilação da Expressão Gênica , Reprodução Assexuada/genética , Transcriptoma , Metabolismo dos Carboidratos/genética , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Anotação de Sequência Molecular
7.
Nat Commun ; 6: 7576, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-26144867

RESUMO

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons.


Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Células-Tronco/fisiologia , Animais , Linhagem Celular , Humanos , MicroRNAs , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Tristetraprolina/genética , Tristetraprolina/metabolismo , Regulação para Cima
8.
Nucleic Acids Res ; 40(2): 787-800, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21948791

RESUMO

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.


Assuntos
Proteínas ELAV/genética , Poliadenilação , Regiões 3' não Traduzidas , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Proteínas ELAV/metabolismo , Retroalimentação Fisiológica , Humanos , Camundongos , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
9.
J Bacteriol ; 191(3): 735-46, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19060155

RESUMO

Type 3 (T3) effector proteins, secreted by nitrogen-fixing rhizobia with a bacterial T3 secretion system, affect the nodulation of certain host legumes. The open reading frame y4lO of Rhizobium sp. strain NGR234 encodes a protein with sequence similarities to T3 effectors from pathogenic bacteria (the YopJ effector family). Transcription studies showed that the promoter activity of y4lO depended on the transcriptional activator TtsI. Recombinant Y4lO protein expressed in Escherichia coli did not acetylate two representative mitogen-activated protein kinase kinases (human MKK6 and MKK1 from Medicago truncatula), indicating that YopJ-like proteins differ with respect to their substrate specificities. The y4lO gene was mutated in NGR234 (strain NGROmegay4lO) and in NGR Omega nopL, a mutant that does not produce the T3 effector NopL (strain NGR Omega nopLOmegay4lO). When used as inoculants, the symbiotic properties of the mutants differed. Tephrosia vogelii, Phaseolus vulgaris cv. Yudou No. 1, and Vigna unguiculata cv. Sui Qing Dou Jiao formed pink effective nodules with NGR234 and NGR Omega nopL Omega y4lO. Nodules induced by NGR Omega y4lO were first pink but rapidly turned greenish (ineffective nodules), indicating premature senescence. An ultrastructural analysis of the nodules induced by NGR Omega y4lO revealed abnormal formation of enlarged infection droplets in ineffective nodules, whereas symbiosomes harboring a single bacteroid were frequently observed in effective nodules induced by NGR234 or NGR Omega nopL Omega y4lO. It is concluded that Y4lO is a symbiotic determinant involved in the differentiation of symbiosomes. Y4lO mitigated senescence-inducing effects caused by the T3 effector NopL, suggesting synergistic effects for Y4lO and NopL in nitrogen-fixing nodules.


Assuntos
Rhizobium/crescimento & desenvolvimento , Simbiose/fisiologia , Western Blotting , Crotalaria/microbiologia , Crotalaria/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Genéticos , Pachyrhizus/microbiologia , Phaseolus/microbiologia , Phaseolus/ultraestrutura , Rhizobium/genética , Rhizobium/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/ultraestrutura , Simbiose/genética , Tephrosia/microbiologia , Tephrosia/ultraestrutura
10.
J Bacteriol ; 190(14): 5101-10, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18487326

RESUMO

Establishment of symbiosis between certain host plants and nitrogen-fixing bacteria ("rhizobia") depends on type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS). Here, we report that the open reading frame y4zC of strain NGR234 encodes a novel rhizobial type 3 effector, termed NopT (for nodulation outer protein T). Analysis of secreted proteins from NGR234 and T3SS mutants revealed that NopT is secreted via the T3SS. NopT possessed autoproteolytic activity when expressed in Escherichia coli or human HEK 293T cells. The processed NopT exposed a glycine (G50) to the N terminus, which is predicted to be myristoylated in eukaryotic cells. NopT with a point mutation at position C93, H205, or D220 (catalytic triad) showed strongly reduced autoproteolytic activity, indicating that NopT is a functional protease of the YopT-AvrPphB effector family. When transiently expressed in tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. Arabidopsis plants transformed with nopT showed chlorotic and necrotic symptoms, indicating a cytotoxic effect. Inoculation experiments with mutant derivatives of NGR234 indicated that NopT affected nodulation either positively (Phaseolus vulgaris cv. Yudou No. 1; Tephrosia vogelii) or negatively (Crotalaria juncea). We suggest that NopT-related polymorphism may be involved in evolutionary adaptation of NGR234 to particular host legumes.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/toxicidade , Rhizobium/fisiologia , Simbiose , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Arabidopsis/microbiologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Linhagem Celular , Crotalaria/microbiologia , Escherichia coli/genética , Deleção de Genes , Ordem dos Genes , Humanos , Peptídeo Hidrolases/genética , Phaseolus/microbiologia , Filogenia , Folhas de Planta/microbiologia , Transporte Proteico , Rhizobium/genética , Homologia de Sequência de Aminoácidos , Tephrosia/microbiologia , Tabaco/microbiologia
11.
Sheng Wu Gong Cheng Xue Bao ; 18(3): 300-3, 2002 May.
Artigo em Chinês | MEDLINE | ID: mdl-12192861

RESUMO

Primers were designed based on ompTS gene reported recently. With the specific primers, one target fragment about 1024 bp lacking the signal sequence of ompTS gene was amplified from A. hydrophila genomic DNA via PCR. The ompTS gene was hyperexpressed using gene fusion expression vector pRSET system, and the recombinant OMP exhibited a size of 39.9 kD with SDS-PAGE and Western blot analysis, which showed about 51% of total lysate proteins. Antibody to the purified recombinant OMP reacted not only to the recombinant OMP but also to the purified OMPs from A. hydrophila in ELISA and the 36.9 kD OMP in Western blot. The result indicates that the recombinant OMP has the same epitope with the nature one.


Assuntos
Aeromonas hydrophila/genética , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Sequência de Bases , Expressão Gênica , Masculino , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Coelhos , Proteínas Recombinantes/imunologia
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