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1.
J Asian Nat Prod Res ; : 1-15, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32024387

RESUMO

We described the design, synthesis and antimicrobial activities of novel pleuromutilin derivatives with substituted piperazine substrate. Minimum inhibitory concentration (MIC) was used to evaluate the activity of the derivatives against six bacteria in vitro, and compound 8 was potent against Staphylococcus aureus and Staphylococcus epidermidis with the MIC value of 0.0625 µg/ml. 10a and 10 b showed similar activity to positive control drugs (tiamulin, erythromycin) against S. aureus with the MIC value of 0.125 µg/ml. The binding mode of compound 8 and tiamulin to the ribosome pocket showed the correlation between binding parameters and the antibacterial activity, and more bonds and stronger combination could effectively enhance the activity of compounds.

2.
Parasit Vectors ; 12(1): 592, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852494

RESUMO

BACKGROUND: Eimeria tenella is a highly pathogenic coccidian that causes avian coccidiosis. Both nitromezuril (NZL) and ethanamizuril (EZL) are novel triazine compounds with high anticoccidial activity, but the mechanisms of their action are still unclear. This study explored the response of E. tenella to NZL and EZL by the study of changes in protein composition of the second-generation merozoites. METHODS: Label-free quantification (LFQ) proteomics of the second-generation merozoites of E. tenella following NZL and EZL treatment were studied by LC-MS/MS to explore the mechanisms of action. The identified proteins were annotated and analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction (PPI) networks analysis. RESULTS: A total of 1430 proteins were identified by LC-MS/MS, of which 375 were considered as differential proteins in response to drug treatment (DPs). There were 26 only found in the NZL treatment group (N-group), 63 exclusive to the EZL treatment group (E-group), and 80 proteins were present in both drug groups. In addition, among the DPs, the abundant proteins with significantly altered expression in response to drug treatment (SDPs) were found compared with the C-group, of which 49 were upregulated and 51 were downregulated in the N-group, and 66 upregulated and 79 downregulated in the E-group. Many upregulated proteins after drug treatment were involved in transcription and protein metabolism, and surface antigen proteins (SAGs) were among the largest proportion of the downregulated SDPs. Results showed the top two enriched GO terms and the top one enriched pathway treated with EZL and NZL were related, which indicated that these two compounds had similar modes of action. CONCLUSIONS: LFQ proteomic analysis is a feasible method for screening drug-related proteins. Drug treatment affected transcription and protein metabolism, and SAGs were also affected significantly. This study provided new insights into the effects of triazine anticoccidials against E. tenella.


Assuntos
Coccidiose/veterinária , Coccidiostáticos/administração & dosagem , Eimeria tenella/crescimento & desenvolvimento , Merozoítos/efeitos dos fármacos , Doenças das Aves Domésticas/tratamento farmacológico , Proteínas de Protozoários/química , Triazinas/administração & dosagem , Animais , Galinhas , Coccidiose/tratamento farmacológico , Coccidiose/parasitologia , Eimeria tenella/efeitos dos fármacos , Eimeria tenella/genética , Eimeria tenella/metabolismo , Merozoítos/genética , Merozoítos/crescimento & desenvolvimento , Merozoítos/metabolismo , Doenças das Aves Domésticas/parasitologia , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espectrometria de Massas em Tandem
3.
Vet Parasitol ; 215: 88-91, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26790742

RESUMO

As an obligate intracellular apicomplexan parasite, Eimeria tenella (E. tenella) can rapidly invade chicken cecum epithelial cells and cause avian coccidiosis. Enolase, an essential enzyme that catalyzes the reversible conversion of 2-phosphoglycerate into phosphoenolpyruvate, plays a very important role in glycolysis. In this study, each chicken was inoculated with 8×10(4) sporulated E. tenella oocysts suspended in 1ml of distilled water to determine the effects of acetamizuril, a new triazine anticoccidial drug, on enolase in the second-generation merozoites of E. tenella. The chickens were divided into two groups: the untreatment group (challenged with E. tenella oocysts and provided with normal feed) and the treatment group (challenged with E. tenella oocysts and provided with 5mg/kg of acetamizuril by oral gavage at 96h after inoculation). The second-generation merozoites of E. tenella (mz-En) were obtained at 120h after inoculation. Subsequently, quantitative real-time PCR and Western blotting were conducted to detect the enolase changes in mz-En at the transcriptional and translational levels. The results showed that enolase mRNA expression was downregulated, and the translational level was decreased in the treatment group. In addition, the subcellular localization of enolase demonstrated that enolase was distributed primarily at the top of the mz-En and that the fluorescence intensity was weak after treatment with acetamizuril. These findings indicated that enolase may be a promising target to prevent coccidiosis.


Assuntos
Coccidiostáticos/farmacologia , Eimeria tenella/efeitos dos fármacos , Eimeria tenella/enzimologia , Merozoítos/efeitos dos fármacos , Merozoítos/enzimologia , Fosfopiruvato Hidratase/metabolismo , Triazinas/farmacologia , Animais , Galinhas , Coccidiose/parasitologia , Coccidiose/veterinária , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Fosfopiruvato Hidratase/genética , Doenças das Aves Domésticas/parasitologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética
4.
Parasitol Res ; 113(3): 903-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24346578

RESUMO

Diclazuril has long been used as an effective benzeneacetonitrile anticoccidial for the control of Eimeria tenella that causes coccidiosis. However, the molecular mechanism underlying the anticoccidial effects of diclazuril remains elusive. In this study, a proteomic analysis of the effect of diclazuril on second-generation merozoites of E. tenella was performed. Using two-dimensional gel electrophoresis and real-time quantitative polymerase chain reaction (RT-PCR), 13 target proteins were found to be significantly affected by diclazuril treatment, with 11 of these proteins being identified as annotated proteins from E. tenella or other Apicomplexa parasites. These proteins contribute to various functions, including metabolism, protein synthesis, and host cell invasion. Using RT-PCR, we identified the potential pattern of transcriptional regulation induced by diclazuril, and we suggest some promising targets for the intervention of E. tenella infection.


Assuntos
Coccidiostáticos/farmacologia , Eimeria tenella/efeitos dos fármacos , Nitrilos/farmacologia , Proteoma/análise , Triazinas/farmacologia , Animais , Galinhas/parasitologia , Eimeria tenella/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Merozoítos/efeitos dos fármacos
5.
J Sep Sci ; 36(9-10): 1667-76, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23495243

RESUMO

Several Acalypha australis Linn. species are used in traditional medicine in Southeast Asia. In this work, the ultra-performance liquid chromatography/mass spectrometry fingerprints and the antibacterial activities of A. australis Linn. were investigated. An in-depth discussion on the reliability of identifying and obtaining potentially active compounds by spectrum-effect relationship and semi-preparative high performance liquid chromatography was conducted. The result shows that gallic acid and a compound with molecular weight of 634.1 in the fingerprints were the main antibacterial compounds. Compared to the crude extract of A. australis Linn., both compounds increase the antibacterial efficacy 10 to 20 times. Compounds with molecular weights of 154.0, 292.0, and 485.1 in the fingerprints were the auxiliary antibacterial compounds. Through the entire isolation procedure, we obtained these antibacterial compounds with purities of 92.53, 87.98, 90.73, 89.36, and 88.14%, respectively. This work provides a general model of the combination of ultra-performance liquid chromatography/mass spectrometry fingerprinting and antibacterial test to study the spectrum-effect relationships of A. australis Linn. This model can be used to discover further the active compounds of this herb.


Assuntos
Antibacterianos/química , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Euphorbiaceae/química , Espectrometria de Massas/métodos , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Ácido Gálico/química , Ácido Gálico/isolamento & purificação , Ácido Gálico/farmacologia , Peso Molecular
6.
Parasitol Res ; 112(4): 1771-80, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23417098

RESUMO

Screening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in Eimeria tenella (EtPP5) in the anticoccidial activity of diclazuril against chicken coccidiosis, EtPP5 was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Ultrastructural changes in second-generation merozoites and mRNA expression level of EtPP5 were monitored by transmission electron microscopy (TEM) and quantitative real-time PCR, respectively. The results showed that the full length of the cloned EtPP5 cDNA (2,495 bp) encompassed a 1,647-bp open reading frame encoding a polypeptide of 548 residues with an estimated molecular mass of 60.82 kDa and a theoretical isoelectric point of 5.89. Molecular analysis of EtPP5 reveals the presence of a C-terminal phosphatase domain and an extended N-terminal tetratricopeptide repeat motif, a typical feature of protein phosphatases. The cDNA sequence has been submitted to the GenBank database with accession number JX987508. EtPP5 shared 89% homology with the published sequence of a PP5 ortholog of Toxoplasma gondii at the amino acid level (GenBank XP_002364442.1). TEM observed that diclazuril induced ultrastructural changes in second-generation merozoites. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtPP5 mRNA expression was significantly downregulated by 51.4% by diclazuril treatment. The high similarity of EtPP5 to previously described PP5 of other organisms, as well as its downregulated expression and connection with apoptosis in the second-generation merozoites induced by diclazuril, suggests that it could act an important role in understanding the signaling mechanism underlining the diclazuril-induced merozoites apoptosis.


Assuntos
Apoptose , Coccidiose/tratamento farmacológico , Coccidiostáticos/farmacologia , Eimeria tenella/enzimologia , Nitrilos/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Triazinas/farmacologia , Motivos de Aminoácidos , Animais , Galinhas , Clonagem Molecular , Coccidiose/parasitologia , Coccidiostáticos/uso terapêutico , DNA de Protozoário/química , DNA de Protozoário/genética , Eimeria tenella/efeitos dos fármacos , Eimeria tenella/genética , Eimeria tenella/ultraestrutura , Perfilação da Expressão Gênica , Ponto Isoelétrico , Masculino , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Peso Molecular , Nitrilos/uso terapêutico , Proteínas Nucleares/química , Fases de Leitura Aberta , Fosfoproteínas Fosfatases/química , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Triazinas/uso terapêutico
7.
Parasitol Res ; 111(4): 1447-55, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22752697

RESUMO

The receptor for activated C kinase (RACK) cDNA of second-generation merozoites of Eimeria tenella was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends, compared with other species, and then successfully expressed using the pET-28a vector in Escherichia coli BL21 (DE3) (EtRACK). Nucleotide sequence analysis revealed that the full length of the cloned cDNA (1,264 bp) encompassed a 957-bp open reading frame encoding a polypeptide of 318 residues with an estimated molecular mass of 34.94 kDa and a theoretical isoelectric point of 5.97. Molecular analysis of EtRACK reveals the presence of seven WD40 repeat motifs. EtRACK localizes to the cytoplasm and nucleus in second-generation merozoites of E. tenella. The cDNA sequence has been submitted to the GenBank Database with accession number JQ292804. EtRACK shared 98% homology with the published sequence of a RACK protein from Toxoplasma gondii at the amino acid level (GenBank XP_002370996.1). Recombinant protein expression was induced using 1 mM of isopropyl ß-D-1-thiogalactopyranoside in vitro at 30 °C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the 39.79-kDa fusion protein existed in unsolvable form. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtRACK mRNA expression in the treatment group was downregulated by 81.3% by diclazuril treatment. The high similarity of EtRACK to previously described RACKs of other organisms, as well as its downregulated expression in second-generation merozoites induced by diclazuril, suggests that it could play a key role in the signaling event that precedes protein secretion and parasite invasion. Moreover, the downregulation of EtRACK mRNA expression also enriches studies on the mechanism of action of diclazuril on E. tenella.


Assuntos
Eimeria tenella/genética , Nitrilos/metabolismo , RNA Mensageiro/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Ativação Transcricional/efeitos dos fármacos , Triazinas/metabolismo , Animais , Clonagem Molecular , DNA de Protozoário/química , DNA de Protozoário/genética , Escherichia coli/genética , Expressão Gênica , Perfilação da Expressão Gênica , Ponto Isoelétrico , Merozoítos , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Receptores de Quinase C Ativada , Receptores de Superfície Celular/química , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Toxoplasma/genética
8.
Carbohydr Polym ; 89(2): 461-6, 2012 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24750744

RESUMO

To evaluate the immune activation and reactive oxygen species scavenging activity of Cordyceps militaris polysaccharides (CMP) in vivo, 90 male BALB/c mice were randomly divided into six groups. The mice in the three experimental groups were given cyclophosphamide at 80 mg/kg/d via intraperitoneal injection and 17.5, 35, or 70 mg/kg body weight CMP via gavage. The lymphocyte proliferation, phagocytic index, and biochemical parameters were measured. The results show that the administration of CMP was able to overcome the CY-induced immunosuppression, significantly increased the spleen and thymus indices, and enhanced the spleen lymphocyte activity and macrophage function. CMP can also improve the antioxidation activity in immunosuppressed mice, significantly increase the superoxidase dismutase, catalase, and glutathione peroxidase levels and the total antioxidant capacity, and decrease the malondialdehyde levels in vivo.


Assuntos
Antioxidantes/farmacologia , Cordyceps , Polissacarídeos Fúngicos/farmacologia , Fatores Imunológicos/farmacologia , Animais , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclofosfamida/farmacologia , Carpóforos , Glutationa Peroxidase/metabolismo , Imunossupressão , Imunossupressores/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Malondialdeído/metabolismo , Camundongos Endogâmicos BALB C , Miocárdio/metabolismo , Fagocitose/efeitos dos fármacos , Baço/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Timo/efeitos dos fármacos
9.
Exp Parasitol ; 125(3): 264-70, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20138868

RESUMO

The effects of diclazuril on mRNA expression levels of invasion-related microneme genes were examined in second-generation merozoites of Eimeria tenella (E. tenella) by quantitative real-time (QRT) PCR. Diclazruil treatment of infected chickens significantly decreased the number of second-generation merozoites by 65.13%, and resulted in downregulation of EtMIC genes: EtMIC1 by 65.63%, EtMIC2 by 64.12%, EtMIC3 by 56.82%, EtMIC4 by 73.48%, and EtMIC5 by 78.17%. SEM images of caecum tissue from uninfected chickens showed regular intestinal villus structure. In infected chickens, a distinct loss of the superficial epithelium, with a flattened mucosa and large-area necrosis and anabrosis, was evident. In diclazruil-treated chickens, a decrease in merozoite number and a visibly improved appearance of the caeca were noted. These improvements appeared to be mediated in part by downregulation of the expression of invasion-related EtMIC genes in response to diclazuril.


Assuntos
Galinhas/parasitologia , Coccidiose/veterinária , Coccidiostáticos/uso terapêutico , Eimeria tenella/efeitos dos fármacos , Nitrilos/uso terapêutico , Doenças das Aves Domésticas/tratamento farmacológico , Triazinas/uso terapêutico , Animais , Ceco/parasitologia , Ceco/patologia , Ceco/ultraestrutura , Coccidiose/tratamento farmacológico , Coccidiose/parasitologia , Coccidiose/patologia , Coccidiostáticos/farmacologia , DNA Complementar/metabolismo , Eimeria tenella/genética , Eimeria tenella/patogenicidade , Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Glicoproteínas/metabolismo , Masculino , Merozoítos/efeitos dos fármacos , Merozoítos/metabolismo , Microscopia Eletrônica de Varredura , Nitrilos/farmacologia , Reação em Cadeia da Polimerase , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/patologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Distribuição Aleatória , Triazinas/farmacologia
10.
Parasitol Res ; 106(3): 571-6, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20020308

RESUMO

Actin depolymerizing factor (ADF) is an essential actin-binding protein that plays a key role in the control of actin dynamics and actin-based motility processes in intracellular parasites. To determine the effects of diclazuril on ADF gene of second-generation merozoites (mz-ADF) mRNA expression in Eimeria tenella, mz-ADF gene was cloned by RT-PCR from extracted RNA in second-generation merozoite of E. tenella and successfully expressed by pET-28a vector in Escherichia coli BL21(DE3). Results showed that the full length of the cloned cDNA sequence of the mz-ADF gene is 476 bp including an ORF of 375 bp. The sequence has 100% homology with a published sequence of sporozoite stage E. tenella ADF mRNA (GenBank EF195234.1). The recombinant protein was induced to be expressed by 1 mM isopropyl beta-D: -1-thiogalactopyranoside in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that 16.99 kDa fusion protein existed in solvable form. Compared with the infected/control group, mz-ADF mRNA expression level was downregulated by 63.86% in the infected/treatment group with the treatment of diclazuril. In conclusion, the data presented here indicate that mz-ADF gene participates in an important role in the invasion host of E. tenella. Downregulation of mz-ADF mRNA expression enrich the mechanism study of diclazuril on E. tenella.


Assuntos
Antiprotozoários/farmacologia , Galinhas/parasitologia , Destrina/metabolismo , Eimeria tenella/enzimologia , Expressão Gênica , Merozoítos/enzimologia , Nitrilos/farmacologia , Triazinas/farmacologia , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Destrina/genética , Regulação para Baixo , Escherichia coli/genética , Masculino , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Análise de Sequência de DNA , Homologia de Sequência
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