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1.
Front Immunol ; 10: 1852, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31440244

RESUMO

Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and ß-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.

2.
Infect Immun ; 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451616

RESUMO

Spiroplasma eriocheiris causes great economic losses in the crustacean aquaculture industry. However, the mechanism of S. eriocheiris infecting host cells was poorly studied. We established a spiroplasma-infected Drosophila Schneider 2 cells model and investigated its pathogenic mechanism. Firstly, S. eriocheiris could induce S2-cell apoptosis and necrosis, seriously decreased cell viability and increased intracellular reactive oxygen species (ROS) production. Further research found that S. eriocheiris could invade S2 cells and copies of intracellular spiroplasma were sharply increased at 12 h infection. In addition, S. eriocheiris could cause S2 cells to form typical inclusion bodies, exhibited large vacuoles. Secondly, S. eriocheiris were internalized into S2 cells and strongly inhibited through blocking clathrin-mediated endocytosis using chlorpromazine and dynasore. Inhibitors of macropinocytosis, protein kinase C and myosin II resulted in a significant reduction of S. eriocheiris in S2 cells. In contrast, disruption of cellular cholesterol by methyl-[gerds]-cyclodextrin and nystatin had no effect on S. eriocheiris infection. These results suggested that the entry of S. eriocheiris into S2 cells relied on clathrin-dependent endocytosis and micropinocytosis, but not via the caveolae-mediated endocytic pathway. In addition, intracellular number of S. eriocheiris were dramatically reduced after S2 cells were treated with cytoskeleton-depolymerizing agents including nocodazole and cytochalasin B. Thus, cellular infection by S. eriocheiris was related to microtubules and actin filaments. This research had successfully shown for the first time that S. eriocheiris could invade Drosophila S2 cells and provided a process for S. eriocheiris infection.

3.
Phytomedicine ; 64: 152899, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-31454649

RESUMO

BACKGROUND: P2Y12 receptor (P2Y12R) is a newly discovered Gi-coupled ADP receptor that plays critical role in platelet function. Ginsenosides are the main constituents responsible for most of pharmacological actions of ginseng, especially cardio-cerebrovascular protective efficacy that is closely related to the influence on platelet function. HYPOTHESIS/PURPOSE: To explore stereoselective effect of naturally abundant ginsenoside isomers, including the C-20 epimers of protopanaxadiol (PPD), protopanaxatriol (PPT), and their glycosides Rg2, Rg3, Rh1, Rh2 on P2Y12R in platelets. STUDY DESIGN/METHODS: Both in vitro assay and in silico molecular docking study were performed to investigate the stereoselective effects. RESULTS: In vitro assay using washed rat platelets revealed differential effects of ginsenoside isomers on ADP-induced platelet aggregation with the direction and degree of action varying with chemical structures. More to the point, the ginsenoside 20S-Rh2 but not its 20R-epimer was found to be the only one that could significantly promote in vitro platelets aggregation induced by ADP. The correlation analysis demonstrated that ginsenosides may have impact on P2Y12R related platelet functions through a cAMP-dependent pathway. Molecular docking stimulation further indicated that ginsenoside isomers could be potent substrate of P2Y12R with differential protein-ligand interaction that would be responsible for the stereoselective efficacy of C-20 ginsenoside epimers. Hydrogen bonding with Asp266 via the C-20 hydroxyl may provide ginsenosides with promoting effect on ADP-induced platelets aggregation, whereas interactions with Tyr105 could contribute to the promotion of inhibitory efficacy. CONCLUSION: Ginsenosides are potent P2Y12R substrate with stereoselective effects on P2Y12R-related platelet function, which result from their chemical diversity and are closely related to the different interaction ways as P2Y12R ligand.

4.
J Anat ; 235(2): 246-255, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31318052

RESUMO

The vomerovaginal canal (VVC) and palatovaginal canal (PVC) are two canals that open forward to the posterior wall of the pterygopalatine fossa (PPF). Although the anatomy and computed tomography (CT) appearances of the PVC have been well studied, the VVC has been rarely reported, especially in endoscopic examinations. Some studies have even failed to distinguish the PVC from the VVC on CT images. The purpose of this study was to demonstrate the anatomy of the VVC on endoscopy and reveal its differences from the PVC, and to analyse the relative positions of the VVC, PVC, and pterygoid canal on CT images. Ten dry skull bases were studied to observe the structures involved in the formation of the VVC. Dissection of four cadaveric heads was performed to demonstrate the anatomy of the VVC on endoscopy. Coronal CT image analysis in 70 patients was conducted to evaluate the distances and relative positions between the VVC, PVC, and pterygoid canal. The PVC and VVC were also compared on axial CT images. The osteological study showed the top wall of the VVC was the antero-inferior wall of the sphenoid sinus. The VVC may be a helpful landmark in endoscopic endonasal transpterygoid approaches. Steps and discrimination in the dissections of the VVC and PVC were described. The interval between the PVC and VVC could be observed on both coronal and axial CT images. The coronal CT images of patients showed differences in the positions and distances among the three canals at both the anterior and posterior apertures of the PVC. The VVC can be easily mistaken for the PVC if its existence is not suspected. The anatomical morphologies and trajectories of the VVC and PVC differed on both nasal endoscopy and CT. The existence of the VVC should be considered during surgery and CT diagnosis within this area.

5.
Molecules ; 24(11)2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167363

RESUMO

To discover new antiproliferative agents with high efficacy and selectivity, a new series of 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea derivatives (7a-7t) were designed, synthesized and evaluated for their antiproliferative activity against A549, HCT-116 and PC-3 cancer cell lines in vitro. Most of the target compounds demonstrated significant antiproliferative effects on all the selective cancer cell lines. Among them, the target compound, 1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{4-{{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methyl}thio}phenyl}urea (7i) was identified to be the most active one against three cell lines, which was more potent than the positive control with an IC50 value of 1.53 ± 0.46, 1.11 ± 0.34 and 1.98 ± 1.27 µM, respectively. Further cellular mechanism studies confirmed that compound 7i could induce the apoptosis of A549 cells in a concentration-dependent manner and elucidated compound 7i arrests cell cycle at G1 phase by flow cytometry analysis. Herein, the studies suggested that the 1-aryl-3-{4-[(pyridin-2-ylmethyl)thio]phenyl}urea skeleton might be regarded as new chemotypes for designing effective antiproliferative agents.

6.
Molecules ; 24(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832266

RESUMO

Nineteen ursolic acid analogues were designed, synthesized, and evaluated for their antiproliferative activity against the Hela and MKN45 cell lines. Some compounds containing a piperazine moiety displayed moderate to high levels of antitumor activities against the tested cancer cell lines. The most potent compound shares the IC50 value of 2.1 µM and 2.6 µM for the Hela and MKN45 cell lines, respectively. Further mechanism studies and in vivo antitumor studies have shown that it decreased the apoptosis regulator (BCL2/BAX) ratio, disrupted mitochondrial potential and induced apoptosis, and suppressed the growth of Hela xenografts in nude mice.


Assuntos
Antineoplásicos/química , Compostos Heterocíclicos/química , Neoplasias/tratamento farmacológico , Triterpenos/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desenho de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/síntese química , Humanos , Camundongos , Estrutura Molecular , Neoplasias/patologia , Nitrogênio/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Molecules ; 24(5)2019 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-30832453

RESUMO

3,4-dihydropyrimidin-2(1H)-one compounds (DHPMs) possess extensive biological activities and are mainly prepared via Biginelli reaction and N-alkylation. In the present study, selective alkylation of N¹ was investigated by using tetrabutylammonium hydroxide. In vitro cytotoxicity study on all synthesized compounds demonstrated that introduction of the aryl chain in the R³ as well as the low electron-donating group in the R¹ of DHPMs contributed to the anti-proliferative potency. A larger value of the partition coefficient (Log P) and suitable polar surface area (PSA) values were both found to be important in order to maintain the antitumor activity. The results from in vivo study indicated the great potential of compound 3d to serve as a lead compound for novel anti-tumor drugs to treat glioma. Pharmacophore study regarding the structure-activity relations of DHPMs were also conducted. Our results here could provide a guide for the design of novel bioactive 3,4-dihydropyrimidin-2(1H)-one compounds.


Assuntos
Antineoplásicos/farmacologia , Glioma/tratamento farmacológico , Pirimidinonas/farmacologia , Alquilação , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Catálise , Linhagem Celular Tumoral , Glioma/patologia , Humanos , Camundongos , Pirimidinonas/síntese química , Pirimidinonas/química , Solventes/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Molecules ; 24(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769844

RESUMO

Based on the analysis of epidermal growth factor receptor (EGFR) complexes with gefitinib with molecular docking, the scaffold-hopping strategy, combination of the active substructures, and structural optimization of EGFR inhibitors, a novel series of benzo[4,5]imidazo[2,1-b]thiazole derivatives was designed, synthesized, and evaluated for antitumor activity in human cancer cell lines and cellular toxicity against human normal cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and EGFR inhibitory activities in vitro. Some target compounds such as 2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(2-hydroxyphenyl)acetamide (D04) and 2-(benzo[4,5]imidazo[2,1-b]thiazol-3-yl)-N-(naphthalen-1-yl)acetamide (D08) have shown significant antitumor activity against the EGFR high-expressed human cell line HeLa. All the target compounds showed hardly any antitumor activity against the EGFR low-expressed human cell line HepG2, and nearly no cellular toxicity against the human normal cell lines HL7702 and human umbilical vein endothelial cell lines (HUVEC). The inhibitory activities against EGFR kinase in vitro of the three target compounds were greatly consistent with the anti-proliferative activities. The preliminary structure⁻activity relationships of the target compounds were summarized. Conclusively, the novel benzo[4,5]imidazo[2,1-b]thiazole derivatives as novel potential EGFR inhibitors may be used as the potential lead compounds for the development of antitumor agents.


Assuntos
Antineoplásicos/química , Neoplasias/tratamento farmacológico , Tiazóis/química , Acetamidas/química , Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Gefitinibe/química , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Simulação de Acoplamento Molecular , Tiazóis/síntese química , Tiazóis/farmacologia
9.
Fish Shellfish Immunol ; 87: 534-545, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30721776

RESUMO

Enterocytozoon hepatopenaei (EHP) causes hepatopancreatic microsporidiosis (HPM) in shrimp. HPM is not normally associated with shrimp mortality, but is associated with significant growth retardation. In this study, the responses induced by EHP were investigated in hepatopancreas of shrimp Litopenaeus vannamei using proteomics and metabolomics. Among differential proteins identified, several (e.g., peritrophin-44-like protein, alpha2 macroglobulin isoform 2, prophenoloxidase-activating enzymes, ferritin, Rab11A and cathepsin C) were related to pathogen infection and host immunity. Other proteomic biomarkers (i.e., farnesoic acid o-methyltransferase, juvenile hormone esterase-like carboxylesterase 1 and ecdysteroid-regulated protein) resulted in a growth hormone disorder that prevented the shrimp from molting. Both proteomic KEGG pathway (e.g., "Glycolysis/gluconeogenesis" and "Glyoxylate and dicarboxylate metabolism") and metabolomic KEGG pathway (e.g., "Galactose metabolism" and "Biosynthesis of unsaturated fatty acids") data indicated that energy metabolism pathway was down-regulated in the hepatopancreas when infected by EHP. More importantly, the changes of hormone regulation and energy metabolism could provide much-needed insight into the underlying mechanisms of stunted growth in shrimp after EHP infection. Altogether, this study demonstrated that proteomics and metabolomics could provide an insightful view into the effects of microsporidial infection in the shrimp L. vannamei.


Assuntos
Enterocytozoon/fisiologia , Metaboloma/imunologia , Penaeidae/genética , Penaeidae/imunologia , Proteoma/imunologia , Animais , Hepatopâncreas/imunologia , Penaeidae/metabolismo
10.
Org Biomol Chem ; 17(8): 2087-2091, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30702121

RESUMO

We herein describe an oxidative [4 + 1] annulation used to prepare 1,2,4-triazolo[4,3-a]pyridines in the presence of I2-DMSO. This protocol enables synthesis of triazolo[4,3-a]pyridine-quinoline linked diheterocycles via a direct oxidative functionalization of sp3 C-H bonds of 2-methyl-azaheteroarenes. The reaction shows a wide substrate scope and good functional group tolerance.

12.
Molecules ; 23(12)2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30558186

RESUMO

Antibiotic resistance has become a serious global problem that threatens public health. In our previous work, we found that ocotillol-type triterpenoid saponin showed good antibacterial activity. Based on preliminary structure-activity relationship, novel serious C-3 substituted ocotillol-type derivatives 7⁻26 were designed and synthesized. The in vitro antibacterial activity was tested on five bacterial strains (B. subtilis 168, S. aureus RN4220, E. coli DH5α, A. baum ATCC19606 and MRSA USA300) and compared with the tests on contrast. Among these derivatives, C-3 position free hydroxyl substituted compounds 7⁻14, showed good antibacterial activity against Gram-positive bacteria. Furthermore, compound 22 exhibited excellent antibacterial activity with minimum inhibitory concentrations (MIC) values of 2 µg/mL against MRSA USA300 and 4 µg/mL against B. subtilis. The structure-activity relationships of all current ocotillol-type derivatives our team synthesised were summarized. In addition, the prediction of absorption, distribution, metabolism, and excretion (ADME) properties and the study of pharmacophores were also conducted. These results can provide a guide to further design and synthesis works.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Ginsenosídeos/química , Bacillus subtilis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
13.
Nanoscale Res Lett ; 13(1): 368, 2018 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-30460496

RESUMO

A series of novel visible light driven all-solid-state Z-scheme BiOBr0.3I0.7/Ag/AgI photocatalysts were synthesized by facile in situ precipitation and photo-reduction methods. Under visible light irradiation, the BiOBr0.3I0.7/Ag/AgI samples exhibited enhanced photocatalytic activity compared to BiOBr0.3I0.7 and AgI in the degradation of methyl orange (MO). The optimal ratio of added elemental Ag was 15%, which degraded 89% of MO within 20 min. The enhanced photocatalytic activity of BiOBr0.3I0.7/Ag/AgI can be ascribed to the efficient separation of photo-generated electron-hole pairs through a Z-scheme charge-carrier migration pathway, in which Ag nanoparticles act as electron mediators. The mechanism study indicated that ·O2- and h+ are active radicals for photocatalytic degradation and that a small amount of ·OH also participates in the photocatalytic degradation process.

14.
Planta Med ; 2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30380571

RESUMO

Ginseng is a perennial herb that contains various medicinal substances. The major active constituents of ginseng are ginsenosides, which have multifarious biological activities. Some pharmacological activities are closely dependent on the stereoisomers derived from the configuration at C20. In this study, the in vitro anti-inflammatory activity of C20 epimeric ocotillol-type triterpenes (2, 3, 9: , and 10: ) and protopanaxadiol [20(S/R)-protopanaxadiol] were investigated. Epimers 2: and 3: were prepared starting from 20(S)-protopanaxadiol. Epimers 9: and 10: were synthesized from 20(R)-3-acetylprotopanaxadiol (7: ). The anti-inflammatory activity of 2, 3, 9, 10: , 20(S)-protopanaxadiol, and 20(R)-protopanaxadiol was evaluated in cultured mouse macrophage RAW 264.7 cells. The MTT assay was used to measure the cytotoxicity. RAW 264.7 cells were stimulated by lipopolysaccharide to release the inflammatory mediators nitric oxide, prostaglandin E2, TNF-α, and interleukin-6 and anti-inflammatory mediator interleukin-10. The effect of the compounds on the overproduction of nitric oxide, prostaglandin E2, TNF-α, interleukin-6, and interleukin-10 was determined using Griess and ELISA methods. The results demonstrated that the in vitro anti-inflammatory activities of C20 epimeric ocotillol-type triterpenes and protopanaxadiol were different. Both the 20S-epimers (2: and 3: ) and 20R-epimers (9: and 10: ) inhibited the release of inflammatory mediator nitric oxide, while mainly the 20S-epimers inhibited the release of inflammatory mediator prostaglandin E2, and the 20R-epimers inhibited the release of inflammatory cytokine TNF-α. Both the 20S-epimers [2, 3: , and 20(S)-protopanaxadiol] and 20R-epimers [9, 10: , and 20(R)-protopanaxadiol] inhibited the release of inflammatory cytokine interleukin-6, but mainly the 20S-epimers [2, 3: , and 20(S)-protopanaxadiol] increased the release of anti-inflammatory mediator interleukin-10.

15.
Fish Shellfish Immunol ; 82: 386-399, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30071344

RESUMO

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen of herpesviral hematopoietic necrosis disease of crucian carp. Our study aimed to investigate the molecular mechanisms and immune response at the mRNA and protein levels in head kidney during CyHV-2 infection. Three days after infection with CyHV-2, 7085 differentially expressed genes were identified by transcriptome sequencing, of which 3090 were up-regulated and 3995 were down-regulated. And 338 differentially expressed proteins including 277 up-regulated and 61 down-regulated were identified using tandem mass tag labeling followed by liquid chromatography tandem mass spectrometry. Notably, 128 differentially co-expressed genes at mRNA and protein levels (cDEGs) were reliably quantified, including 86 co-up-regulated and 42 co-down-regulated. In addition, 10 cDGEs in the above pathways were selected for qRT-PCR to confirm the validity of the transcriptome and proteome changes by showing that RIG-I, MDA5, LGP2, FAS, PKR and PKZ up-regulated and Integrin α, Integrin ß2, NCF2 and NCF4 down-regulated. This indicated that after CyHV-2 infection, the herpes simplex infection pathway, RIG-I like receptor signaling pathway, necroptosis pathway and p53 signaling pathway were activated and the phagosome pathway was suppressed. Our findings reveal the pathogenesis and the host immune mechanism of CyHV-2 infection of crucian carp.

16.
Acta Crystallogr C Struct Chem ; 74(Pt 6): 659-665, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29870000

RESUMO

3,5-Bis(arylidene)piperidin-4-one derivatives (BAPs) display good antitumour activity because of their double α,ß-unsaturated ketone structural characteristics. Reported BAPs have generally been symmetric and asymmetric BAPs have been little documented. Three asymmetric BAPs, namely (5E)-3-(4-tert-butylbenzylidene)-5-(4-fluorobenzylidene)-1-methylpiperidin-4-one, C24H26FNO, (5), (5E)-3-(4-tert-butylbenzylidene)-5-(3,5-dimethoxybenzylidene)-1-methylpiperidin-4-one, C26H31NO3, (6), and (5E)-3-{3-[(E)-(2,3-dihydroxybenzylidene)amino]benzylidene}-5-(2-fluorobenzylidene)-1-methylpiperidin-4-one, C27H23FN2O3, (12), were generated by Claisen-Schmidt condensation. They are characterized by NMR and FT-IR spectroscopies, and elemental analysis. Single-crystal structure analysis reveals that the two arylidene rings on both sides of the BAP structures adopt an E stereochemistry of the olefinic double bonds and the compounds are E,E isomers. Molecules of (5) and (12) generate one-dimensional chains through intermolecular hydrogen bonds, while compound (6) generates a two-dimensional network through hydrogen bonds. Preliminary cytotoxicities toward human liver hepatocellular carcinoma cell line (HepG2), human acute mononuclear granulocyte leukaemia (THP-1) and human normal hepatical cell line (LO2) were evaluated.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Cristalografia por Raios X , Humanos , Ligações de Hidrogênio , Estrutura Molecular , Espectroscopia de Infravermelho com Transformada de Fourier
17.
Sci Rep ; 8(1): 8577, 2018 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-29872058

RESUMO

Spiroplasma eriocheiris, a pathogen that causes mass mortality of Chinese mitten crab Eriocheir sinensis, is a wall less bacteria and belongs to the Mollicutes. This study was designed to investigate the effects of colchicine on S. eriocheiris growth, cell morphology, and proteins expression. We found that in the presence of colchicine, the spiroplasma cells lost their helicity, and the length of the cells in the experimental group was longer than that of the control. With varying concentrations of the colchicine treatment, the total time to achieve a stationary phase of the spiroplasma was increased, and the cell population was decreased. The virulence ability of S. eriocheiris to E. sinensis was effectively reduced in the presence of colchicine. To expound the toxical mechanism of colchicine on S. eriocheiris, 208 differentially expressed proteins of S. eriocheiris were reliably quantified by iTRAQ analysis, including 77 up-regulated proteins and 131 down-regulated proteins. Especially, FtsY, putative Spiralin, and NADH oxidase were down-regulated. F0F1 ATP synthase subunit delta, ParB, DNABs, and NAD(FAD)-dependent dehydrogenase were up-regulated. A qRT-PCR was conducted to detect 7 expressed genes from the iTRAQ results during the incubation. The qRT-PCR results were consistent with the iTRAQ results. All of our results indicate that colchicine have a strong impact on the cell morphology and cellular metabolism of S. eriocheiris.

18.
Fish Shellfish Immunol ; 79: 79-85, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29753143

RESUMO

Transferrin, a member of the iron binding superfamily protein, plays an extremely important role in the transport of iron in the biological process of cells. The result of preliminary proteomic study on E. sinensis hemocytes infected Spiroplasma eriocheiris showed the expression of transferrin (EsTF) and ferrin (EsFe) significantly changed. In addition, other reports have confirmed that transferrin, ferritin and iron are involved in the immune response of hosts. In order to validate the immune function of EsTF, the whole length of EsTF was successfully amplified by the gene cloning and RACE technique. The results showed that the full-length cDNA of the EsTF gene was 2748 bp, including a 2193 bp open reading frame which encodes 730 amino acids. The result of bioinformatics analysis showed EsTF contains two highly conserved TR_FER domains. Evolutionary analysis showed that EsTF has a close genetic relationship with other TFs of invertebrates. In addition, EsTF mRNA was highly transcripted in nerve and intestine tissues, followed by hemocytes. The expression of EsTF, EsFe1 and EsFe2 increased after exogenous supplemental of iron under the concentration of 100 nmol/L in water. After exogenous supplement of iron and injection with S. eriocheiris, these three gene transcription of mRNA levels were higher than that of PBS group, while lower than the S. eriocheiris group and the iron group. Besides, the copy number of S. eriocheiris in the experimental group was significantly reduced, and the death rate decreased. As can be seen, iron made transferrin and ferritin return to normal levels during the infection of S. eriocheiris and help the host maintain normal immunity levels to resist S. eriocheiris. These results further demonstrated that EsTF, EsFe1, EsFe2 and iron play a role in the immune defense mechanism of the crabs to resist S. eriocheiris infection.


Assuntos
Braquiúros/genética , Braquiúros/imunologia , Imunidade Inata/genética , Ferro/metabolismo , Spiroplasma/fisiologia , Transferrina/genética , Transferrina/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Hemócitos/imunologia , Hemócitos/microbiologia , Filogenia , Proteômica , Distribuição Aleatória , Transferrina/química
19.
Dev Comp Immunol ; 86: 1-8, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29709775

RESUMO

Cathepsin D (catD) belongs to a lysosomal aspartic protease superfamily. The full-length catD cDNA from the Chinese mitten crab Eriocheir sinensis (EscatD) was 2748 bp and contained a 1158-bp ORF encoding a protein of 385 amino acids, including a signal peptide and two N-glycosylation sites. Phylogenetic analysis showed that EscatD was clustered into a single group, together with other catD for crustaceans. Quantitative real-time PCR revealed that EscatD was expressed mainly in the eyes, hemocytes, intestine and nerve and was expressed weakly in heart, muscle and gills. After challenge with Spiroplasma eriocheiris, the expression of EscatD was significantly up-regulated from 1 d to 9 d. The copy number of S. eriocheiris in a silencing EscatD group was significantly higher than those in the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in an EscatD-dsRNA group. We further found that knockdown of EscatD by RNA interference resulted in a downward trend of expression levels of JNK, ERK, relish and p38 during the early stage, as well as a reduction in the expression of five antimicrobial peptides genes, namely, crusrin1, crustin2, ALF1, ALF2 and ALF3. The subcellular localization experiment suggested that recombinant EscatD was mainly located in the cytoplasm. The over-expression in Drosophila S2 cells indicated that EscatD could decrease the copy number of S. eriocheiris and increase cell viability. The above results demonstrated that EscatD plays an important immune role in E. sinensis to S. eriocheiris challenge.


Assuntos
Braquiúros/imunologia , Braquiúros/microbiologia , Catepsina D/imunologia , Spiroplasma/imunologia , Sequência de Aminoácidos , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Sequência de Bases , Interações Hospedeiro-Patógeno/imunologia , Filogenia
20.
Fish Shellfish Immunol ; 77: 438-444, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29625245

RESUMO

As a new-found aquaculture pathogen, Spiroplasma eriocheiris, has resulted in inconceivable economic losses in aquaculture. In the infection of S. eriocheiris, the Procambarus clakii hemocytes have indicated to be major target cells. What was designed to examine in our study is the hemocytes' immune response at the protein levels. Before the pathogen was injected and after 192 h of post-injection, the differential proteomes of the crayfish hemocytes were analyzed immediately by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatogramphytandem mass spectrometry (LC-MS/MS). This research had identified a total of 285 differentially expressed proteins. Eighty-three and 202 proteins were up-regulated and down-regulated, respectively, caused by the S. eriocheiris infection. Up-regulated proteins included alpha-2-macroglobulin (α2M), vitellogenin, ferritin, etc. Down-regulated proteins, involved with serine protease, peroxiredoxin 6, 14-3-3-like protein, C-type lectin, cdc42 homolog precursor, etc. The prophenoloxidase-activating system, antimicrobial action involved in the immune responses of P. clarkii is considered to be damaged due to S. eriocheiris infection. The present work could lay the foundation for future research on the proteins related to the susceptibility/resistance of P. clarkii to S. eriocheiris. In addition, it is helpful for our understanding molecular mechanism of disease processes in crayfishes.


Assuntos
Astacoidea/genética , Hemócitos/imunologia , Imunidade Inata/genética , Proteoma/imunologia , Spiroplasma/fisiologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/imunologia , Astacoidea/microbiologia , Proteômica
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