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1.
Adv Exp Med Biol ; 1131: 131-161, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646509

RESUMO

Calcium (Ca2+) is a fundamental regulator of cell fate and intracellular Ca2+ homeostasis is crucial for proper function of the nerve cells. Given the complexity of neurons, a constellation of mechanisms finely tunes the intracellular Ca2+ signaling. We are focusing on the sarco/endoplasmic reticulum (SR/ER) calcium (Ca2+)-ATPase (SERCA) pump, an integral ER protein. SERCA's well established role is to preserve low cytosolic Ca2+ levels ([Ca2+]cyt), by pumping free Ca2+ ions into the ER lumen, utilizing ATP hydrolysis. The SERCA pumps are encoded by three distinct genes, SERCA1-3, resulting in 12 known protein isoforms, with tissue-dependent expression patterns. Despite the well-established structure and function of the SERCA pumps, their role in the central nervous system is not clear yet. Interestingly, SERCA-mediated Ca2+ dyshomeostasis has been associated with neuropathological conditions, such as bipolar disorder, schizophrenia, Parkinson's disease and Alzheimer's disease. We summarize here current evidence suggesting a role for SERCA in the neurobiology of neuropsychiatric and neurodegenerative disorders, thus highlighting the importance of this pump in brain physiology and pathophysiology.


Assuntos
Encéfalo , Retículo Endoplasmático , Doenças do Sistema Nervoso , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Encéfalo/enzimologia , Encéfalo/patologia , Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/fisiopatologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
2.
Adv Exp Med Biol ; 1131: 649-679, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646529

RESUMO

Multifunctional calcium/calmodulin-stimulated protein kinases control a broad range of cellular functions in a multitude of cell types. This family of kinases contain several structural similarities and all are regulated by phosphorylation, which either activates, inhibits or modulates their kinase activity. As these protein kinases are widely or ubiquitously expressed, and yet regulate a broad range of different cellular functions, additional levels of regulation exist that control these cell-specific functions. Of particular importance for this specificity of function for multifunctional kinases is the expression of specific binding proteins that mediate molecular targeting. These molecular targeting mechanisms allow pools of kinase in different cells, or parts of a cell, to respond differently to activation and produce different functional outcomes.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Regulação Enzimológica da Expressão Gênica , Terapia de Alvo Molecular , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Fosforilação
3.
Gene ; 721: 144097, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31493507

RESUMO

BACKGROUND: Polo-like kinase 1 (PLK1) is a potential prognostic marker in colorectal cancer (CRC). Nevertheless, the clinicopathological and prognostic roles of PLK1 in CRC are still undefined. Therefore, we performed a meta-analysis to investigate the clinicopathological and prognostic relevance of PLK1 expression in CRC patients. METHODS: Studies published between 2003 and 2016 were selected for the meta-analysis based on an electronic literature search (PubMed, EMBASE and Chinese databases). Studies that investigated the clinicopathological and prognostic impacts of PLK1 expression in CRC patients were included for this analysis. RESULTS: Eleven studies that enrolled 1147 CRC patients were included in our meta-analysis. The effect of PLK1 level on overall survival (OS) was reported in five studies, which included 702 patients. Ten studies investigated the clinicopathological role of PLK1 expression in CRC patients. Consequently, PLK1 overexpression was associated with poorer OS in CRC patients. Furthermore, the results revealed that higher PLK1 levels were also observed in CRC tissues compared with that of normal colorectal tissues. In addition, this meta-analysis also revealed positive correlations between PLK1 upregulation and lymph node metastasis or invasion. PLK1 overexpression was significantly correlated with advanced TNM stages and higher Dukes stages. CONCLUSION: This meta-analysis strongly supports the hypothesis that PLK1 might serve as an important factor in evaluating the biological behavior and prognosis of CRC.


Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Ciclo Celular/biossíntese , Neoplasias Colorretais , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Humanos , Metástase Linfática , Estadiamento de Neoplasias , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Taxa de Sobrevida
4.
Microbiol Res ; 229: 126319, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31479952

RESUMO

Methionine is critical for variety of metabolic processes in biological organisms, acting as a precursor or intermediate for many final products. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by MetE. Here, we use Salmonella enterica serovar Typhimurium LT2 to study the regulation of the metE+ gene by an anaerobically induced small non-coding RNA-FnrS, the expression of which is strictly dependent on the anaerobic regulator-FNR. The MetE-HA protein was expressed at an increased level in the fnrS- and hfq- deficient strains under anaerobic conditions. The Hfq protein is predicted to stabilize the binding between small RNA(s) and their target mRNA(s). A transcriptional (op) and translational (pr) metE::lacZ fusion gene were separately constructed, with the metE+-promoter fused to a lacZ reporter gene. In an anaerobic environment, the metE::lacZ (pr) fusion gene and reverse transcription-PCR identified that FnrS and/or FNR negatively regulate metE+ mRNA levels in the rich media. Analysis of FnrS revealed a sequence complementary to the 5' mRNA translational initiation region (TIR) of the metE+ gene. Mutation(s) predicted to disrupt base pairing between FnrS and metE+ TIR were constructed in fnrS, and most of those resulted in the loss of repressive activity. When compensatory mutation(s) were made in metE+ 5' TIR to restore base pairing with FnrS, the repressive regulation was completely restored. Therefore, in this study, we identified that in anaerobic phase, there is a repression of metE+ gene expression by FnrS and that base-paring, between both expressive transcripts, plays an important role for this negative regulation.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Metiltransferases/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Salmonella typhimurium/enzimologia , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Metiltransferases/química , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
5.
Nat Biotechnol ; 37(9): 1041-1048, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477922

RESUMO

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1-4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Animais , Clonagem Molecular , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Células HEK293 , Humanos , Petromyzon , Conformação Proteica , RNA , RNA Guia/genética , Streptococcus pyogenes , Transcriptoma
6.
Toxicol Lett ; 315: 77-86, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31470059

RESUMO

T-2 toxin is a major pollutant in crops and feedstuffs. Due to its high toxicity in a variety of organisms, T-2 toxin is of great concern as a threat to humans and to animal breeding. Overexpression of CYP1A1 may contribute to carcinogenesis, and CYP1A1 may be a promising target for the prevention and treatment of human malignancies. Therefore, it is essential to understand the regulatory mechanism by which T-2 toxin induces CYP1A1 expression in human cells. In this study, we confirmed that T-2 toxin (100 ng/mL) induced the expression of CYP1A1 in HepG2 cells through NRF1 and Sp1 bound to the promoter instead of through the well-recognized Aromatic hydrocarbon receptors (AhR). In cells treated with T-2 toxin, Sp1, but not NRF1, was significantly upregulated. However, T-2 toxin apparently promoted the interaction between NRF1 and Sp1 proteins, as revealed by IP analysis. Furthermore, in T-2 toxin-treated HepG2 cells, nuclear translocation of NRF1 was enhanced, while knockdown of Sp1 ablated NRF1 nuclear enrichment. Our results revealed that the upregulation of CYP1A1 by T-2 toxin in HepG2 cells depended on enhanced interaction between Sp1 and NRF1. This finding suggests the tumorigenic features of T-2 toxin might be related to the CYP1A1, which provides new insights to understand the toxicological effect of T-2 toxin.


Assuntos
Citocromo P-450 CYP1A1/efeitos dos fármacos , Citocromo P-450 CYP1A1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 1 Relacionado a NF-E2/genética , Fator de Transcrição Sp1/genética , Toxina T-2/toxicidade , Regulação para Cima/efeitos dos fármacos , Carcinoma/fisiopatologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Pesquisas com Embriões , Regulação Enzimológica da Expressão Gênica , Humanos , Rim , Neoplasias Hepáticas/fisiopatologia , Fator 1 Relacionado a NF-E2/efeitos dos fármacos , Fator 1 Relacionado a NF-E2/metabolismo , Fator de Transcrição Sp1/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo
7.
Toxicol Lett ; 314: 181-186, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31404594

RESUMO

The cytochrome P450 enzyme (CYP) family includes key enzymes for the metabolism of drugs and xenobiotics. Several animal models have been used to determine the metabolite profile of specific drugs. Among these are porcine microsomes prepared from Göttingen minipigs. However, CYP expression profile in microsomes from this pig breed is unknown. In the present study, we determined the mRNA and protein profiles of a comprehensive selection of CYPs in microsomes prepared from male and female Göttingen minipigs. Using RT-PCR, western blotting and mass spectroscopy, we found that the expression levels of CYP1A, CYP2A and CYP2E1 were significantly higher in females than males. Moreover, some of the transcription factors controlling CYP transcription also showed a sex-dependent expression pattern. Conversely, expression of CYP2B, CYP2D and CYP3A was comparable between sexes. The overall CYP expression distribution showed high similarity with what previously been reported in humans. In conclusion, our results suggest that Göttingen minipigs are a reliable model for studying CYPs.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/enzimologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Isoenzimas , Masculino , Microssomos/enzimologia , Modelos Animais , Fatores Sexuais , Especificidade por Substrato , Suínos , Porco Miniatura
8.
Exp Parasitol ; 205: 107748, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31442453

RESUMO

Trypanosoma cruzi (the causative agent of Chagas disease) presents a complex life cycle that involves adaptations in vertebrate and invertebrate hosts. As a protozoan parasite of hematophagous insects and mammalian hosts, T. cruzi is exposed to reactive oxygen species (ROS). To investigate the functionality of T. cruzi tartrate-resistant acid phosphatase type 5 (TcACP5), we cloned, superexpressed and purified the enzyme. Purified TcACP5 exhibited a Vmax and apparent Km for pNPP hydrolysis of 7.7 ±â€¯0.2 nmol pNP × µg-1 × h-1 and 169.3 ±â€¯22.6 µM, respectively. The pH dependence was characterized by sharp maximal activity at pH 5.0, and inhibition assays demonstrated its sensitivity to acid phosphatase inhibitors. Similar activities were obtained with saturating concentrations of P-Ser and P-Thr as substrates. The enzyme metabolizes hydrogen peroxide (H2O2) in vitro, and parasites superexpressing this enzyme were more resistant to oxidative stress promoted by H2O2. Taken together, these results suggest that TcACP5 plays a central role in phosphoryl transfer and redox reactions.


Assuntos
Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/fisiologia , Fosfatase Ácida Resistente a Tartarato/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Microscopia Confocal , Oxirredução , Especificidade por Substrato , Fosfatase Ácida Resistente a Tartarato/antagonistas & inibidores , Fosfatase Ácida Resistente a Tartarato/química , Transfecção , Trypanosoma cruzi/efeitos dos fármacos
9.
Zhonghua Fu Chan Ke Za Zhi ; 54(7): 464-469, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31365959

RESUMO

Objective: To evaluate the effects of parthenolide on estradiol-synthesizing enzyme, steroidogenic acute regulatory protein (StAR), and ER isoforms,VEGF in human endometriotic stromal cells. Methods: Primary endometriotic stromal cells were treated with different concentrations (1, 5, 10 and 20 µmol/L) of parthenolide. The mRNA of StAR, ER isoforms (ERα and ERß), PR, vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), tumour necrosis factor-α (TNFα), tumour necrosis factor receptor (TNFR) 1, TNFR2 were measured by real-time PCR. The levels of estradiol and progesterone in the cell supernatant were measured by ELISA. Results: Different concentrations of parthenolide could up-regulate the mRNA of StAR in primary endometriotic stromal cells (F=5.722, P<0.05); the mRNA of StAR in the group of 20 µmol/L was significantly higher than that of the control group [2.6±0.3 versus 1.0, P<0.01]. Different concentrations of parthenolide could down-regulate the mRNA of ERα (F=6.921, P<0.01); the mRNA of ERα in the group of 20 µmol/L and 10 µmol/L were significantly lower than those of the control group [0.2±0.3 versus 0.3±0.3 versus 1.0, all P<0.05]. Different concentrations of parthenolide could down-regulate the ratios of ERα/ERß mRNA levels (F=4.209, P<0.05). Different concentrations of parthenolide could up-regulate the mRNA of VEGF and TNFR1 (F=10.964, P<0.01; F=7.286, P<0.01). There were no statiscal significances with different concentrations of parthenolide on the mRNA of ERß, PR, IL-6, TNFα and TNFR2, and the levels of estradiol and progesterone in the cell supernatant (all P>0.05). Conclusions: Parthenolide may regulate the expression of estradiol-synthesizing enzyme, ER isoforms and angiogenesis in endometriotic stromal cells. Parthenolide may promote the development of endometriosis.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Endometriose , Endométrio/efeitos dos fármacos , Sesquiterpenos/farmacologia , Células Estromais/metabolismo , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endometriose/induzido quimicamente , Endometriose/genética , Endométrio/metabolismo , Endométrio/patologia , Estradiol , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoformas de Proteínas , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Fator de Necrose Tumoral alfa/metabolismo
10.
J Agric Food Chem ; 67(34): 9569-9578, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31385495

RESUMO

Acetyl-CoA synthetase (ACS) plays a key role in microalgal lipid biosynthesis and acetyl-CoA industrial production. In the present study, two ACSs were cloned and characterized from the oleaginous microalga Chromochloris zofingiensis. In vitro kinetic analysis showed that the Km values of CzACS1 and CzACS2 for potassium acetate were 0.99 and 0.81 mM, respectively. Moreover, CzACS1 and CzACS2 had outstanding catalytic efficiencies (kcat/Km), which were 70.67 and 79.98 s-1 mM-1, respectively, and these values were higher than that of other reported ACSs. CzACS1 and CzACS2 exhibited differential expression patterns at the transcriptional level under various conditions. Screening a recombinant library of 52 transcription factors (TFs) constructed in the present study via yeast one-hybrid assay pointed to seven TFs with potential involvement in the regulation of the two ACS genes. Expression correlation analysis implied that GATA20 was likely an important regulator of CzACS2 and that ERF9 could regulate two CzACSs simultaneously.


Assuntos
Acetato-CoA Ligase/metabolismo , Clorófitas/enzimologia , Regulação Enzimológica da Expressão Gênica , Microalgas/enzimologia , Acetato-CoA Ligase/química , Acetato-CoA Ligase/genética , Biocatálise , Clorófitas/química , Clorófitas/genética , Cinética , Metabolismo dos Lipídeos , Microalgas/química , Microalgas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Cancer Invest ; 37(8): 327-338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31423851

RESUMO

Little is known about the endocannabinoid (eCB) system in squamous cell carcinoma of the oral tongue (SCCOT). Here we have investigated, at the mRNA level, expression of genes coding for the components of the eCB system in tumour and non-malignant samples from SCCOT patients. Expression of NAPEPLD and PLA2G4E, coding for eCB anabolic enzymes, was higher in the tumour tissue than in non-malignant tissue. Among genes coding for eCB catabolic enzymes, expression of MGLL was lower in tumour tissue while PTGS2 was increased. It is concluded that the eCB system may be dysfunctional in SCCOT.


Assuntos
Biomarcadores Tumorais/genética , Endocanabinoides/genética , RNA Mensageiro/genética , Transdução de Sinais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Neoplasias da Língua/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Ciclo-Oxigenase 2/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fosfolipases A2 do Grupo IV/genética , Humanos , Masculino , Pessoa de Meia-Idade , Monoacilglicerol Lipases/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipase D/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Neoplasias da Língua/patologia , Adulto Jovem
12.
Genome Biol ; 20(1): 156, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387610

RESUMO

BACKGROUND: Methylation of nucleotides, notably in the forms of 5-methylcytosine (5mC) in DNA and N6-methyladenosine (m6A) in mRNA, carries important information for gene regulation. 5mC has been elucidated to participate in the regulation of fruit ripening, whereas the function of m6A in this process and the interplay between 5mC and m6A remain uncharacterized. RESULTS: Here, we show that mRNA m6A methylation exhibits dynamic changes similar to DNA methylation during tomato fruit ripening. RNA methylome analysis reveals that m6A methylation is a prevalent modification in the mRNA of tomato fruit, and the m6A sites are enriched around the stop codons and within the 3' untranslated regions. In the fruit of the ripening-deficient epimutant Colorless non-ripening (Cnr) which harbors DNA hypermethylation, over 1100 transcripts display increased m6A levels, while only 134 transcripts show decreased m6A enrichment, suggesting a global increase in m6A. The m6A deposition is generally negatively correlated with transcript abundance. Further analysis demonstrates that the overall increase in m6A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene SlALKBH2, which is regulated by DNA methylation. Interestingly, SlALKBH2 has the ability to bind the transcript of SlDML2, a DNA demethylase gene required for tomato fruit ripening, and modulates its stability via m6A demethylation. Mutation of SlALKBH2 decreases the abundance of SlDML2 mRNA and delays fruit ripening. CONCLUSIONS: Our study identifies a novel layer of gene regulation for key ripening genes and establishes an essential molecular link between DNA methylation and mRNA m6A methylation during fruit ripening.


Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Metilação de DNA , Retículo Endoplasmático/enzimologia , Frutas/genética , Frutas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/metabolismo , Metilação , Mutação , Motivos de Nucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estabilidade de RNA , RNA Mensageiro/química
13.
Gene ; 712: 143945, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31279712

RESUMO

Reactive oxygen species, generated in all the aerobic organisms, can cause oxidative stress. Excessive ROS may become a source of carcinogen due to DNA damage, lipid peroxidation, cell injury, and cell death. In order to prevent these adverse effects of ROS, antioxidant enzymes have evolved in aerobic organisms. Catalase is a major antioxidant enzyme that breaks down excessive H2O2 and inhibits apoptotic cell death. Here we molecularly characterized catalase from red-lip mullet. The cDNA sequence of LhCAT consists of an ORF of 1545 bp, which encodes a 527 amino acid peptide (~60 kDa). Based on bioinformatics analysis, LhCAT possesses a domain architecture characteristic of catalases, including a catalase proximal active site signature and a catalase proximal heme-ligand signature. It also has heme and NADPH binding sites homologous to previously described catalases. Pairwise alignment with its homologs revealed that LhCAT shares 95.1% identity with Oplegnathus fasciatus catalase and 97.4% similarity with Sparus aurata catalase. An uprooted phylogenetic tree demonstrated that LhCAT resides in a clade with catalases from other teleosts and exhibits a close relationship with Oplegnathus fasciatus catalase. Among twelve tissue types, we observed the highest LhCAT mRNA expression in the liver, followed by blood. Immune challenge by Lactococcus garvieae, or Poly I:C in the blood or spleen resulted in up-regulation at 24 h post injection. We also tested the antioxidant activity of recombinant LhCAT against hydrogen peroxide and found its optimal concentration to be 12.5 µg/mL. Collectively, these data suggested that LhCAT play an important role in antioxidant defense and immune response of red-lip mullet.


Assuntos
Catalase/metabolismo , Proteínas de Peixes/metabolismo , Smegmamorpha , Adjuvantes Imunológicos , Animais , Antioxidantes/metabolismo , Catalase/genética , DNA Complementar/genética , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Heme/química , Peróxido de Hidrogênio/química , Sistema Imunitário , Ligantes , Fígado/enzimologia , Estresse Oxidativo , Filogenia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para Cima
14.
Aquat Toxicol ; 214: 105230, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31306923

RESUMO

Brachionus spp. (Rotifera: Monogononta) are globally distributed in aquatic environments and play important roles in the aquatic ecosystem. The marine monogonont rotifer Brachionus plicatilis is considered a suitable model organism for ecology, evolution, and ecotoxicology. In this study, we assembled and characterized the B. plicatilis genome. The total length of the assembled genome was 106.9 Mb and the number of final scaffolds was 716 with an N50 value of 1.15 Mb and a GC content of 26.75%. A total of 20,154 genes were annotated after manual curation. To demonstrate the use of whole genome data, we targeted one of the main detoxifying enzyme of phase I detoxification system and identified in a total of 28 cytochrome P450 s (CYPs). Based on the phylogenetic analysis using the maximum likelihood, 28 B. plicatilis-CYPs were apparently separated into five different clans, namely, 2, 3, 4, mitochondrial (MT), and 46 clans. To better understand the CYPs-mediated xenobiotic detoxification, we measured the mRNA expression levels of 28 B. plicatilis CYPs in response to chlorpyrifos and 2-ethyl-phenanthrene. Most B. plicatilis CYPs were significantly modulated (P < 0.05) in response to chlorpyrifos and 2-ethyl-phenanthrene. In addition, xenobiotic-sensing nuclear receptor (XNR) response element sequences were identified in the 5 kb upstream of promoter regions of 28 CYPs from the genome of B. plicatilis, indicating that these XNR can be associated with detoxification of xenobiotics. Overall, the assembled B. plicatilis genome presented here will be a useful resource for a better understanding the molecular ecotoxicology in the view of molecular mechanisms underlying toxicological responses, particularly on xenobiotic detoxification in this species.


Assuntos
Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Clorpirifos/toxicidade , Sistema Enzimático do Citocromo P-450/genética , Genoma Helmíntico , Fenantrenos/toxicidade , Rotíferos/enzimologia , Rotíferos/genética , Animais , Organismos Aquáticos/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Anotação de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/genética , Rotíferos/efeitos dos fármacos , Testes de Toxicidade Aguda , Poluentes Químicos da Água/toxicidade
15.
Zoonoses Public Health ; 66(6): 603-617, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31264805

RESUMO

OBJECTIVES: This study investigates the frequency and characteristics of carbapenemase-producing Escherichia coli/Klebsiella pneumoniae (CPE/K) and extended-spectrum cephalosporinase-producing E. coli/K. pneumoniae (ESCE/K) in healthy humans and livestock in rural Cambodia. Additionally, household practices as risk factors for faecal carriage of ESCE/K are identified. METHODS: Faecal samples were obtained from 307 humans and 285 livestock including large ruminants, pigs and poultry living in 100 households in rural Cambodia in 2011. Each household was interviewed, and multilevel logistic model determined associations between household practices/meat consumption and faecal carriage of ESCE/K. CPE and ESCE/K were detected and further screened for colistin resistance genes. RESULTS: CPE/K isolates harbouring blaOXA-48 were identified in two humans. The community carriage of ESCE/K was 20% in humans and 23% in livestock. The same ESBL genes: blaCTX-M-15 , blaCTX-M-14 , blaCTX-M-27 , blaCTX-M-55 , blaSHV-2 , blaSHV-12 , blaSHV-28 ; AmpC genes: blaCMY-2 , blaCMY-42, blaDHA-1 ; and colistin resistance genes: mcr-1-like and mcr-3-like were detected in humans and livestock. ESCE/K was frequently detected in women, young children, pigs and poultry, which are groups in close contact. The practice of burning or burying meat waste and not collecting animal manure indoors and outdoors daily were identified as risk factors for faecal carriage of ESCE/K. CONCLUSIONS: Faecal carriage of E. coli and K. pneumoniae harbouring extended-spectrum cephalosporinase genes are common in the Cambodian community, especially in women and young children. Exposure to animal manure and slaughter products are risk factors for intestinal colonization of ESCE/K in humans.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Cefalosporinase/metabolismo , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Adolescente , Adulto , Animais , Proteínas de Bactérias/genética , Camboja , Cefalosporinase/genética , Criança , Pré-Escolar , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Escherichia coli/enzimologia , Fezes/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Lactente , Klebsiella pneumoniae/enzimologia , Gado/microbiologia , Masculino , Fatores de Risco , População Rural , Zoonoses , beta-Lactamases/genética
16.
Nat Biotechnol ; 37(9): 1070-1079, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31332326

RESUMO

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.


Assuntos
Adenosina Desaminase/metabolismo , Evolução Molecular Direcionada , Edição de Genes , Adenosina Desaminase/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Humanos , Mutação INDEL , Camundongos
17.
Aquat Toxicol ; 214: 105254, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31357109

RESUMO

Intracellular copper/zinc superoxide dismutase (icCuZnSOD) is a member of superoxide dismutase family that is capable of catalyzing the superoxide radicals into either hydrogen peroxide (H2O2) or ordinary molecular oxygen (O2). Unlike mammals, the study of icCuZnSOD in aquatic animals is still in the infancy stage. Here, we identified the cDNA of na-iccuznsod from yellow drum (Nibea albiflora, Richardson 1846) and obtained its fusion protein for the first time. The mRNA expressions of na-iccuznsod were investigated in different tissues, and the dominant distribution was found in head-kidney, followed by brain, liver, heart, and gill. The effects of ammonia-N/nitrite-N on the mRNA expressions of na-iccuznsod were investigated. Na-iccuznsod transcription levels showed a general tendency of an initial up-regulation followed by a down-regulation in liver, gill, and head-kidney when yellow drum were exposed to ammonia-N/nitrite-N at the lethal concentration 50 at 96 h post-treatment, suggesting the important role of Na-icCuZnSOD in eliminating reactive oxygen species (ROS) induced by ammonia-N/nitrite-N. In addition, the characteristics of Na-icCuZnSOD protein and its comparative analysis with Na-ecCuZnSOD were investigated. Na-icCuZnSOD protein showed high enzyme stabilities over a wide range of temperature (10 to 60 °C) and pH (4.9 to 11.0), indicating its broad in vitro applications in many industries. Furthermore, the comparative analysis of Na-icCuZnSOD and Na-ecCuZnSOD gives a new perspective for the study of their structure-function relationship. Collectively, the present study will advance our understanding of the toxicity of ammonia-N/nitrite-N on yellow drum through testing the mRNA expression of iccuznsod gene, and broaden our knowledge of the protein characteristics of icCuZnSOD from fish.


Assuntos
Amônia/toxicidade , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica , Nitritos/toxicidade , Perciformes/genética , Estresse Fisiológico/genética , Superóxido Dismutase/genética , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Temperatura Ambiente , Fatores de Tempo , Poluentes Químicos da Água/toxicidade
18.
Zoolog Sci ; 36(3): 223-230, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251491

RESUMO

Serotonin (i.e., 5-hydroxytryptamine [5-HT]) plays a key role in stress responses in vertebrates. In mammals and teleosts, tryptophan hydroxylase (Tph), a rate-limiting enzyme in the biosynthesis of 5-HT, includes two paralogs: Tph 1 and Tph 2. The response of the Tphs to stress has been reported in mammals, but less is known about the responses of these enzymes to stress in fish. In the present study, we examined whether heat stress affects the mRNA expression of these Tphs in the brain of medaka fish (Oryzias latipes). We also determined the concentration of 5-HT in the brain, the mRNA expression of heat shock protein 90 alpha (Hsp90α) in the liver, plasma cortisol concentration, and blood glucose concentration in medaka. Whole-body exposure to repeated heat stress significantly decreased the mRNA expression of Tph1 and Tph2 in male and female medaka, whereas single heat stress did not affect the expression of either of the mRNAs. The 5-HT concentration also decreased significantly after repeated heat stress sessions in both sexes, but did not decrease after a single heat stress session. After single and repeated heat stress sessions, Hsp90α mRNA expression increased in both sexes; however, increments in the concentrations of plasma cortisol and blood glucose occurred in male, but not in female, medaka. These results suggest that both types of Tphs are involved in reducing 5-HT in the brain and are reliable indicators of chronic stress response in both sexes. However, stress responses in plasma cortisol and blood glucose concentrations differ between male and female medaka.


Assuntos
Encéfalo/enzimologia , Resposta ao Choque Térmico , Oryzias/metabolismo , Triptofano Hidroxilase/metabolismo , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Triptofano Hidroxilase/genética
19.
Nat Commun ; 10(1): 2850, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253801

RESUMO

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Actinas/fisiologia , Linhagem Celular Tumoral , Membrana Celular , Exocitose/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinases da Matriz/genética , Microtúbulos , Fosfatos de Fosfatidilinositol/fisiologia , Transporte Proteico , Proteína 3 Associada à Membrana da Vesícula/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
20.
Nat Commun ; 10(1): 2853, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253808

RESUMO

Plant innate immunity restricts growth of bacterial pathogens that threaten global food security. However, the mechanisms by which plant immunity suppresses bacterial growth remain enigmatic. Here we show that Arabidopsis thaliana secreted aspartic protease 1 and 2 (SAP1 and SAP2) cleave the evolutionarily conserved bacterial protein MucD to redundantly inhibit the growth of the bacterial pathogen Pseudomonas syringae. Antibacterial activity of SAP1 requires its protease activity in planta and in vitro. Plants overexpressing SAP1 exhibit enhanced MucD cleavage and resistance but incur no penalties in growth and reproduction, while sap1 sap2 double mutant plants exhibit compromised MucD cleavage and resistance against P. syringae. P. syringae lacking mucD shows compromised growth in planta and in vitro. Notably, growth of ΔmucD complemented with the non-cleavable MucDF106Y is not affected by SAP activity in planta and in vitro. Our findings identify the genetic factors and biochemical process underlying an antibacterial mechanism in plants.


Assuntos
Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Serina Endopeptidases/metabolismo , Arabidopsis/imunologia , Proteínas de Bactérias/genética , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidases/genética
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