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1.
Infect Immun ; : IAI0031521, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34543119

RESUMO

Mycobacterium tuberculosis is a chronic infectious disease pathogen. To date, tuberculosis is a major infectious disease that endangers human health. To better prevent and treat tuberculosis, it is important to study the pathogenesis of M. tb. Based on early-stage laboratory research results, in this study, we verified the upregulation of sod2 in Bacillus Calmette-Guérin (BCG) and H37Rv infection. By detecting BCG/H37Rv intracellular survival in sod2-silenced and sod2- overexpressing macrophages, sod2 was found to promote the intracellular survival of BCG/H37Rv. Then, miR-495 was determined to be downregulated by BCG/H37Rv. BCG/H37Rv can upregulate sod2 expression by miR-495 to promote the intracellular survival of BCG/H37Rv through a decline in ROS levels. This study provides a theoretical basis for developing new drug targets and treating tuberculosis.

2.
J Microbiol ; 59(9): 854-860, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34382147

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen that places severe burdens on public health and animal husbandry. There are many pathogenic factors in E. coli. The type VI secretion system (T6SS) is a nano-microbial weapon that can assemble quickly and inject toxic effectors into recipient cells when danger is encountered. T6SSs are encoded in the genomes of approximately 25% of sequenced Gram-negative bacteria. When these bacteria come into contact with eukaryotic cells or prokaryotic microbes, the T6SS assembles and secretes associated effectors. In the porcine ExPEC strain PCN033, we identified four classic rearrangement hotspot (Rhs) genes. We determined the functions of the four Rhs proteins through mutant construction and protein expression. Animal infection experiments showed that the Δrhs-1CT, Δrhs-2CT, Δrhs-3CT, and Δrhs-4CT caused a significant decrease in the multiplication ability of PCN033 in vivo. Cell infection experiments showed that the Rhs protein is involved in anti-phagocytosis activities and bacterial adhesion and invasion abilities. The results of this study demonstrated that rhs1, rhs3, and rh4 plays an important role in the interaction between PCN033 and host cell. Rhs2 has contribution to cell and mice infection. This study helps to elucidate the pathogenic mechanism governing PCN033 and may help to establish a foundation for further research seeking to identify potential T6SS effectors.


Assuntos
Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/metabolismo , Doenças dos Suínos/microbiologia , Animais , Aderência Bacteriana , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/metabolismo , Feminino , Intestinos/microbiologia , Camundongos , Família Multigênica , Suínos
3.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198513

RESUMO

BACKGROUND: Pulmonary disease caused by Mycobacterium abscessus (M. abscessus) spreads around the world, and this disease is extremely difficult to treat due to intrinsic and acquired resistance of the pathogen to many approved antibiotics. M. abscessus is regarded as one of the most drug-resistant mycobacteria, with very limited therapeutic options. METHODS: Whole-cell growth inhibition assays was performed to screen and identify novel inhibitors. The IC50 of the target compounds were tested against THP-1 cells was determined to calculate the selectivity index, and then time-kill kinetics assay was performed against M. abscessus. Subsequently, the synergy of oritavancin with other antibiotics was evaluated by using checkerboard method. Finally, in vivo efficacy was determined in an immunosuppressive murine model simulating M. abscessus infection. RESULTS: We have identified oritavancin as a potential agent against M. abscessus. Oritavancin exhibited time-concentration dependent bactericidal activity against M. abscessus and it also displayed synergy with clarithromycin, tigecycline, cefoxitin, moxifloxacin, and meropenem in vitro. Additionally, oritavancin had bactericidal effect on intracellular M. abscessus. Oritavancin significantly reduced bacterial load in lung when it was used alone or in combination with cefoxitin and meropenem. CONCLUSIONS: Our in vitro and in vivo assay results indicated that oritavancin may be a viable treatment option against M. abscessus infection.


Assuntos
Antibacterianos/uso terapêutico , Lipoglicopeptídeos/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/fisiologia , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Imunossupressão , Espaço Intracelular/microbiologia , Lipoglicopeptídeos/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Células THP-1
4.
Molecules ; 26(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33915741

RESUMO

As an important zoonotic pathogen, Streptococcus suis (S. suis) can cause a variety of diseases both in human and animals, especially Streptococcal toxic shock-like syndrome (STSLS), which commonly appears in severe S. suis infection. STSLS is often accompanied by excessive production of inflammatory cytokines, which is the main cause of host death. Therefore, it is urgent to find a new strategy to relieve the damage caused by STSLS. In this study, we found, for the first time, that apigenin, as a flavonoid compound, could combine with ampicillin to treat severe S. suis infection. Studies found that apigenin did not affect the growth of S. suis and the secretion of suilysin (SLY), but it could significantly inhibit the hemolytic activity of SLY by directly binding to SLY and destroying its secondary structure. In cell assays, apigenin was found to have no significant toxic effects on effective concentrations, and have a good protective effect on S. suis-infected cells. More importantly, compared with the survival rate of S. suis-infected mice treated with only ampicillin, the survival rate of apigenin combined with an ampicillin-treated group significantly increased to 80%. In conclusion, all results indicate that apigenin in combination with conventional antibiotics can be a potential strategy for treating severe S. suis infection.


Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Apigenina/farmacologia , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus suis/efeitos dos fármacos , Ampicilina/química , Ampicilina/uso terapêutico , Animais , Antibacterianos/química , Apigenina/química , Apigenina/uso terapêutico , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/antagonistas & inibidores , Proteínas Hemolisinas/química , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/metabolismo , Relação Estrutura-Atividade , Resultado do Tratamento
5.
Mol Med Rep ; 23(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649799

RESUMO

Cytoglobin (Cygb) is a globin molecule that is ubiquitously expressed in all tissues and has a protective role under oxidative stress. It has also been demonstrated to be effective in the treatment of alcoholic fatty liver disease (AFLD). In order to study the molecular mechanisms underlying its beneficial effects for the treatment of alcoholic liver, two­dimensional electrophoresis and mass spectrometric analysis were performed on serum and liver tissues from an in vivo rat model of AFLD. A total of 26 differentially expressed proteins were identified in the serum and 20 differentially expressed proteins were identified in liver specimens. Using online bioinformatics tools, it was indicated that these differentially expressed proteins were primarily associated with pathways including binding and uptake of ligands by scavenger receptors, response to corticosteroid, plasma lipoprotein remodeling, regulation of complement cascade, hydrogen peroxide catabolic process, as well as response to nutrient and monosaccharide. The present results suggested that recombinant human Cygb exerts its role in the treatment of AFLD primarily through affecting nutrient metabolism, monocarboxylic acid biosynthesis, regulation of glutathione expression, plasma lipoprotein remodeling and removal of metabolic waste from the blood.


Assuntos
Biologia Computacional/métodos , Citoglobina/farmacologia , Fígado Gorduroso Alcoólico/tratamento farmacológico , Fígado Gorduroso Alcoólico/metabolismo , Proteoma/efeitos dos fármacos , Proteômica/métodos , Proteínas Recombinantes/farmacologia , Animais , Citoglobina/genética , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Proteoma/metabolismo , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33674433

RESUMO

Streptococcal toxic shock-like syndrome (STSLS) caused by the epidemic strain of Streptococcus suis leads to severe inflammation and high mortality. The life and health of humans and animals are also threatened by the increasingly severe antimicrobial resistance in Streptococcus suis There is an urgent need to discover novel strategies for the treatment of S. suis infection. Suilysin (SLY) is considered to be an important virulence factor in the pathogenesis of S. suis In this study, ellipticine hydrochloride (EH) was reported as a compound that antagonizes the hemolytic activity of SLY. In vitro, EH was found to effectively inhibit SLY-mediated hemolytic activity. Furthermore, EH had a strong affinity for SLY, thereby directly binding to SLY to interfere with the hemolytic activity. Meanwhile, it was worth noting that EH was also found to have a significant antibacterial activity. In vivo, compared with traditional ampicillin, EH not only significantly improved the survival rate of mice infected with S. suis 2 strain Sc19 but also relieved lung pathological damage. Furthermore, EH effectively decreased the levels of inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α]) and blood biochemistry enzymes (alanine transaminase [ALT], aspartate transaminase [AST], creatine kinase [CK]) in Sc19-infected mice. Additionally, EH markedly reduced the bacterial load of tissues in Sc19-infected mice. In conclusion, our findings suggest that EH can be a potential compound for treating S. suis infection in view of its antibacterial and antihemolysin activity.IMPORTANCE In recent years, the inappropriate use of antibiotics has unnecessarily caused the continuous emergence of resistant bacteria. The antimicrobial resistance of Streptococcus suis has also become an increasingly serious problem. Targeting virulence can reduce the selective pressure of bacteria on antibiotics, thereby alleviating the development of bacterial resistance to a certain extent. Meanwhile, the excessive inflammatory response caused by S. suis infection is considered the primary cause of acute death. Here, we found that ellipticine hydrochloride (EH) exhibited effective antibacterial and antihemolysin activities against S. suis in vitro In vivo, compared with ampicillin, EH had a significant protective effect on S. suis serotype 2 strain Sc19-infected mice. Our results indicated that EH, with dual antibacterial and antivirulence effects, will contribute to treating S. suis infections and alleviating the antimicrobial resistance of S. suis to a certain extent. More importantly, EH may develop into a promising drug for the prevention of acute death caused by excessive inflammation.


Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias/metabolismo , Elipticinas/uso terapêutico , Proteínas Hemolisinas/metabolismo , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus suis , Fatores de Virulência/metabolismo , Animais , Antibacterianos/farmacologia , Citocinas/sangue , Modelos Animais de Doenças , Elipticinas/farmacologia , Feminino , Hemólise/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Infecções Estreptocócicas/sangue , Streptococcus suis/efeitos dos fármacos , Streptococcus suis/crescimento & desenvolvimento , Streptococcus suis/metabolismo
7.
Biomed Pharmacother ; 132: 110804, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33017767

RESUMO

Colorectal cancer (CRC), initiated and maintained by colorectal cancer stem cells (CCSCs), ranks the third most common cancers and has drawn wide attentions worldwide. Therefore, targeting clearance of CCSCs has become an important strategy of CRC immunotherapy. Mucin1 (MUC1) is a tumor-associated cell surface antigen of CRC, but its role in CCSC vaccine remains unclear. In the study, we demonstrated that MUC1 may be a dominant antigen to exert antitumor immunity in CCSC vaccine. First, CCSCs were enriched from CT26 cell line via a serum-free sphere formation approach, and were identified by detecting expression of CD133, ALDH, and ALCAM. Then, the isolated CCSCs were frozen for 30 min and thawed for 30 min to prepare the cell lysate. The specific anti-MUC1 antibody was added to the cell lysate to neutralize the dominant antigen MUC1. Finally, mice were subcutaneously immunized with the cell lysate, followed by a challenge with CT26 cells at one week after final vaccination. Attractively, CCSC vaccine significantly activated the NK cells, T cells, and B cells, resulting in inhibiting the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+cells in tumor compared to CCSC vaccine with specific anti-MUC1 antibody. In addition, CCSC vaccine reduced expression of inflammatory factors in vaccinated mice. As expected, neutralizing antibody against MUC1 significantly impaired the antitumor efficacy of CCSC vaccine. Overall, CCSC vaccine could serve as a potent vaccine for CRC immunotherapy. The surface dominant antigen MUC1 may play a key role in regulating immunogenicity of CCSCs.


Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias Colorretais/prevenção & controle , Mucina-1/imunologia , Células-Tronco Neoplásicas/imunologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Feminino , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Vacinação
8.
Arch Biochem Biophys ; 692: 108522, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32781051

RESUMO

About one quarter of people worldwide are infected with tuberculosis, and multi-drug resistant tuberculosis (MDR-TB) remains a health threat. It is known that two-Component Signal Transduction Systems (TCSs) of Mycobacterium tuberculosis are closely related to tuberculosis resistance, but the mechanism by which orphan response protein Rv3143 regulates strain sensitivity to drug is still unclear. This study found that Rv3143 overexpression resulted in approximately two-fold increase in Mycobacterium smegmatis antibiotic sensitivity. Transcriptome sequencing indicated that 198 potential genes were regulated by Rv3143, affecting the sensitivity of the strain to rifampicin (RIF). MSMEG_4740 promoter binding with Rv3143, was screened out by surface plasmon resonance (SPR). Rv1524, the homologous gene of MSMEG_4740, belonging to the glycosyltransferase (Gtf) family, was related to cell wall modification. By measuring ethidium bromide (EB) accumulation, we found when Rv3143 or MSMEG_4740, or Rv1524 was overexpressed, the cell wall permeability of Mycobacterium smegmatis was increased. In addition, a combination of Rv3143 and RIF was observed. Our findings provide a new strategy for treating drug-resistant tuberculosis by increasing the expression of Rv3143 to enhance the strain sensitivity to antibiotics.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Mycobacterium smegmatis/metabolismo , Proteínas de Bactérias/genética , Parede Celular/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mycobacterium smegmatis/genética , Permeabilidade/efeitos dos fármacos , Rifampina/farmacologia , Transcriptoma/efeitos dos fármacos
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(7): 640-644, 2020 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-32727650

RESUMO

Objective To prepare the high-affinity and high-specificity mouse anti-human IgE monoclonal antibody (mAb) as a candidate immunosorbent. Methods BALB/c mice were immunized using recombinant antigen IgECepsilon2-4. Coated ELISA plate with IgECepsilon2-4 was used to screen positive cell lines by indirect ELISA, then coated ELISA plate with natural IgE, IgG, IgM, IgA, IgD to detect the affinity and specificity of serum-free culture supernatant of positive hybridoma cells with natural IgE. The cell line stably secreting specific anti-IgE monoclonal antibodies was expanded and inoculated into the abdominal cavity of BALB/c mice to prepare mAbs. The secreted mAbs were identified by ELISA kit followed by the identification of mAb subtypes. Results The 29 positive hybridoma cells were obtained after five cell fusions, of which 11 strains had strong affinity with natural IgE and 2 strains did not cross-react with other immunoglobulins 3E9 and 7B4. Conclusion The study successfully prepared mAbs against human IgE with high titer, affinity and specificity.


Assuntos
Imunoglobulina E/imunologia , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C
10.
Front Microbiol ; 11: 806, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32528422

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are the cause of a majority of human extraintestinal infections globally, resulting in enormous direct economic and medical costs. The plasmid-mediated, colistin-resistant gene mcr-1 has broken through the ultimate defense line against MDR Gram-negative pathogens. There is an urgent need to discover the new compound intended for colistin-resistant E. coli. In this study, antibacterial targets of ellipticine hydrochloride (EH) were confirmed by localized surface plasmon resonance (LSPR) and decatenation assay. The LSPR analysis exhibited good binding between EH and E. coli topoisomerase IV. In this study, a synergistic effect is obvious in the combination of EH and colistin, to which eight of ten strains showed synergy, while two isolates (20%) showed no difference. The bacteria enumeration analysis of EH treatment group suggested that the decreased bacterial titer can be observed in various tissues of infected mice. EH treatment significantly decreased the levels of a variety of pro-inflammatory factors, such as TNF-α and IL-6. Moreover, other related lesions, such as inflammatory cell infiltration, alveolar interstitial congestion, and edema were observed to be relieved to different extents. This study reveals the anti-E. coli potential activities and molecular mechanism of EH and the therapeutical effectiveness of EH application to animals. It provides us with a new option for fighting against multidrug-resistant ExPEC infections in the future.

11.
Molecules ; 25(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861925

RESUMO

Background: Tuberculosis remains a global disease that poses a serious threat to human health, but there is lack of new and available anti-tuberculosis agents to prevent the emergence of drug-resistant strains. To address this problem natural products are still potential sources for the development of novel drugs. Methods: A whole-cell screening approach was utilized to obtain a natural compound enniatin A1 from a natural products library. The target compound's antibacterial activity against Mycobacterium tuberculosis (M. tuberculosis) was evaluated by using the resazurin reduction micro-plate assay (REMA) method. The cytotoxicity of the compound against Vero cells was measured to calculate the selectivity index. The intracellular inhibition activity of enniatin A1 was determined. We performed its time-kill kinetic assay against M. tuberculosis. We first tested its synergistic effect in combination with the first and second-line anti-tuberculosis drugs. Finally, we measured the membrane potential and intracellular ATP levels of M. tuberculosis after exposure to enniatin A1. Results: We identified enniatinA1 as a potential antibacterial agent against M. tuberculosis, against which it showed strong selectivity. Enniatin A1 exhibited a time-concentration-dependent bactericidal effect against M. tuberculosis, and it displayed synergy with rifamycin, amikacin, and ethambutol. After exposure to enniatinA1, the membrane potential and intracellular ATP levels of M. tuberculosis was significantly decreased. Conclusions: Enniatin A1 exhibits the positive potential anti-tuberculosis agent characteristics.


Assuntos
Trifosfato de Adenosina/metabolismo , Antituberculosos/farmacologia , Depsipeptídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/agonistas , Chlorocebus aethiops , Depsipeptídeos/agonistas , Avaliação de Medicamentos , Sinergismo Farmacológico , Humanos , Células THP-1 , Células Vero
12.
ACS Appl Mater Interfaces ; 11(21): 19267-19276, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31067021

RESUMO

Heterogeneous Fe3O4 and Fe composites are highly desirable for microwave absorption application because of their complementary electromagnetic (EM) properties. With three-dimensional (3D) Fe2O3 as a sacrificing template, we realize the construction of Fe3O4/Fe composites with tunable chemical composition, and more importantly, these composites inherit the unique 3D microstructure from their precursor. The change in chemical composition produces significant impacts on the EM functions of these composites. On the one hand, dielectric loss can be improved greatly through positive interfacial polarization and reach the peak when the mass contents of Fe3O4 and Fe are 72.1 and 27.9 wt %, respectively. On the other hand, high Fe content slightly pulls down magnetic loss in the low-frequency range but favors strong magnetic loss in the high-frequency range because of the breakthrough of Snoek's limitation. The attenuation constant reveals that dielectric loss dominates overall consumption of incident EM waves. As a result, the optimized composite, F-350 (the reduction of Fe2O3 is conducted at 350 °C), shows the best microwave absorption performance, whose strongest reflection loss is -56.0 dB at 17.5 GHz and the effective bandwidth can cover the frequency range of 12.0-15.5 GHz with the thickness of 1.5 mm. Furthermore, an ultrawide effective bandwidth of 15.3 GHz can be achieved with the integrated thickness of 1.0-5.0 mm. Such a performance is superior to those of many reported Fe3O4/Fe composites, and a comparative analysis manifests that good microwave absorption of F-350 is also benefited from its unique 3D architecture.

13.
Chemosphere ; 211: 648-652, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30098560

RESUMO

Low concentrations of arsenic (As) contamination in aquatic environment is a worldwide issue, which is of great concern. To evaluate the impact of low concentrations of As on zebrafish, we measured the growth, antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), oxidative damage (malondialdehyde, MDA) and apoptosis-related genes (nrf2, p53 and c-jun) of adult zebrafish after exposing to different AsIII concentrations (0, 10, 50, 100 or 150 µg L-1) for 28 d. Results indicated that exposure to low AsIII concentrations decreased the zebrafish weight by 14%, increased the activities of SOD and CAT by 23-41% and 31-59%, decreased the contents of MDA by 29-54%, and modulated transcription of apoptosis related genes. Our study showed that chronic exposure to AsIII concentrations <150 µg L-1 generated oxidative stress and damage on zebrafish, and altered apoptosis-related genes in zebrafish.


Assuntos
Apoptose/genética , Arsenitos/química , Peixe-Zebra/genética , Animais , Estresse Oxidativo
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 34(3): 205-210, 2018 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-29773100

RESUMO

Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount, and then the recombinant protein was purified by Ni-NTA affinity chromatography. The interaction between high-affinity IgE receptor (FcepsilonR I) of KU812 cell surface and IgECepsilon2-4 was identified by immunofluorescence cytochemistry. The unknown proteins that specifically interacted with IgECepsilon2-4 were captured from human serum by SPR technique. Results The recombinant plasmid containing the signal peptide III showed the highest expression (6.2 mg/L). Highly purified recombinant protein IgECepsilon2-4 was obtained by affinity purification. Immunofluorescence cytochemistry showed that the recombinant protein IgECepsilon2-4 could be combined with the surface receptor of KU812 cells. Thirty-nine kinds of proteins which were likely to interact with IgECepsilon2-4 were captured from human serum by SPR. Conclusion We obtained the purified recombinant protein IgECepsilon2-4 that could be combined with KU812 cell surface receptor. Target fishing experiment revealed that the recombinant protein IgECepsilon2-4 was likely to interact with 39 kinds of proteins in human serum.


Assuntos
Células Eucarióticas/metabolismo , Imunoglobulina E/genética , Cadeias épsilon de Imunoglobulina/genética , Cadeias épsilon de Imunoglobulina/isolamento & purificação , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Células HEK293 , Humanos , Imunoglobulina E/análise , Imunoglobulina E/isolamento & purificação , Imunoglobulina E/metabolismo , Cadeias épsilon de Imunoglobulina/análise , Cadeias épsilon de Imunoglobulina/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção
15.
Genes (Basel) ; 9(3)2018 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-29495525

RESUMO

Abelmoschus esculentus (okra or lady's fingers) is a vegetable with high nutritional value, as well as having certain medicinal effects. It is widely used as food, in the food industry, and in herbal medicinal products, but also as an ornamental, in animal feed, and in other commercial sectors. Okra is rich in bioactive compounds, such as flavonoids, polysaccharides, polyphenols, caffeine, and pectin. In the present study, the concentrations of total flavonoids and polysaccharides in five organs of okra were determined and compared. Transcriptome sequencing was used to explore the biosynthesis pathways associated with the active constituents in okra. Transcriptome sequencing of five organs (roots, stem, leaves, flowers, and fruits) of okra enabled us to obtain 293,971 unigenes, of which 232,490 were annotated. Unigenes related to the enzymes involved in the flavonoid biosynthetic pathway or in fructose and mannose metabolism were identified, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. All of the transcriptional datasets were uploaded to Sequence Read Archive (SRA). In summary, our comprehensive analysis provides important information at the molecular level about the flavonoid and polysaccharide biosynthesis pathways in okra.

16.
Genome ; 61(2): 121-130, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29304291

RESUMO

Auxin plays key roles in regulating plant growth and development as well as in response to environmental stresses. The intercellular transport of auxin is mediated by the following four gene families: ATP-binding cassette family B (ABCB), auxin resistant1/like aux1 (AUX/LAX), PIN-formed (PIN), and PIN-like (PILS). Here, the latest assembled pepper (Capsicum annuum L.) genome was used to characterise and analyse the CaLAX and CaPIN gene families. Genome-wide investigations into these families, including chromosomal distributions, phytogenic relationships, and intron/exon structures, were performed. In total, 4 CaLAX and 10 CaPIN genes were mapped to 10 chromosomes. Most of these genes exhibited varied tissue-specific expression patterns assessed by quantitative real-time PCR. The expression profiles of the CaLAX and CaPIN genes under various abiotic stresses (salt, drought, and cold), exogenous phytohormones (IAA, 6-BA, ABA, SA, and MeJA), and polar auxin transport inhibitor treatments were evaluated. Most CaLAX and CaPIN genes were altered by abiotic stress at the transcriptional level in both shoots and roots, and many CaLAX and CaPIN genes were regulated by exogenous phytohormones. Our study helps to identify candidate auxin transporter genes and to further analyse their biological functions in pepper development and in its adaptation to environmental stresses.


Assuntos
Capsicum/genética , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Proteínas de Plantas/genética , Capsicum/metabolismo , Mapeamento Cromossômico , Genoma de Planta , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/metabolismo , Filogenia , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Transcriptoma
17.
J Cardiovasc Pharmacol Ther ; 23(2): 162-173, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28954528

RESUMO

Atherosclerosis is a chronic inflammatory vascular disease characterized by lipid accumulation and endothelial dysfunction. Cytoglobin has been shown to exert protective effects under oxidative stress conditions. The aim of this study was to determine whether recombinant human cytoglobin (rhCYGB) has protective effects against atherosclerosis. We intraperitoneally injected rhCYGB (0, 4, or 7 mg/kg BW) into the atherosclerotic rats daily for 60 days. The rhCYGB injections reduced low-density lipoprotein cholesterol (LDL-C) levels and increased high-density lipoprotein cholesterol levels in a dose-dependent manner, rhCYGB (7 mg/kg) significantly attenuated atherosclerosis. Blood proteins were separated by 2-dimensional electrophoresis and analyzed by mass spectrometry, and the majority of the proteins in question were participated in oxidative stress pathways and cardiovascular diseases. Human hepatocellular liver carcinoma cell line (HepG2) cells were treated with oleic acid (0.3 mmol/L), and Human acute monocytic leukemia cell line (THP-1) cells were incubated with oxidized LDL (ox-LDL; 50 µg/mL) to induce foam cell (FC) formation in vitro. Treatment with different concentrations of rhCYGB (0, 5, 10, and 15 µg/mL) significantly decreased the lipid droplet levels in HepG2 cells and cholesterol ester levels in FCs. Moreover, rhCYGB significantly increased superoxide dismutase and glutathione peroxidase activity and decreased malondialdehyde and nicotinamide adenine dinucleotide phosphate oxidase activity in cells. In addition, rhCYGB decreased NO and Reactive oxygen species (ROS) levels in FCs by functioning as an NO dioxygenase enzyme and ROS scavenger. Taken together, our findings indicate that rhCYGB prevented atherosclerosis by regulating lipid metabolism and oxidative stress. Our study provides insights into the possible usefulness of rhCYGB as an antiatherosclerosis agent.


Assuntos
Antioxidantes/farmacologia , Aorta Abdominal/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Citoglobina/farmacologia , Hipolipemiantes/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Ésteres do Colesterol/metabolismo , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Modelos Animais de Doenças , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Células Espumosas/patologia , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Placa Aterosclerótica , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Células THP-1
18.
PLoS One ; 12(6): e0177968, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28662027

RESUMO

Liver fibrosis, a common pathological process of chronic liver diseases, is the final stage of liver dysfunction that has severely deleterious impact on human health. Cytoglobin was first discovered in 2001 by proteomic analysis in rat stellate cells and was reported to play an important role in controlling tissue fibrosis. However, the mechanism by which cytoglobin inhibits or reverses the progression of fibrosis remains unclear. The present study examines the effect of recombinant human cytoblobin (rhCygb) in a rat model of liver fibrosis. Proteomic approaches were employed to identify differentially expressed proteins in the fibrosis model. Optimized conditions for two-dimensional gel electrophoresis were developed to provide improved protein detection and separation. A total of 43 spots were obtained and, through the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, 30 differentially expressed proteins were identified. Gene ontology term annotation and KEGG pathway analysis allowed us to explore the function of the represented proteins. Based on these results, we provide a theory of the molecular mechanism related to rhCygb reversion of fibrosis and which will assist in the identification of biomarkers in patient serum to improve early diagnosis of liver fibrosis.


Assuntos
Proteínas Sanguíneas/metabolismo , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional/métodos , Globinas/administração & dosagem , Cirrose Hepática/metabolismo , Animais , Citoglobina , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
BMC Genet ; 18(1): 33, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28388893

RESUMO

BACKGROUND: Auxin plays an important role in regulating plant growth and development as well as in the response of plants to abiotic stresses. Auxin is transported by three kinds of major protein families, including the AUXIN RESISTANT 1/LIKE AUX1 (AUX/LAX) influx carriers, the PIN-FORMED (PIN) efflux carriers and the ATP binding cassette B/P-glycoprotein/Multidrug-resistance (ABCB/MDR/PGP) efflux/condition carriers. The biological function of several auxin transporter genes has been well characterized in Arabidopsis thaliana. However, their function in response to exogenous auxin and abiotic stresses in watermelon (Citrullus lanatus. L) remained unknown. RESULTS: Here, the latest updated watermelon genome was used to characterise the ClLAX, ClPIN and ClABCB family genes from watermelon. The genome-wide analysis of the ClLAX, ClPIN and ClABCB family genes, including chromosome localisation, gene structure, and phylogenic relationships, was carried out. Seven ClLAXs, 11 ClPINs and 15 ClABCBs were mapped on 10 watermelon chromosomes. The expression profiles of the ClLAX, ClPIN and ClABCB genes under exogenous indole-3-acetic acid and various abiotic stresses (salt, drought, and cold stresses) treatments were performed by quantitative real-time PCR (qRT-PCR). The transcriptional level of majority ClLAX, ClPIN and ClABCB genes were changed by abiotic stresses in both shoots and roots. We also analysed the expression levels of ClLAX, ClPIN and ClABCB genes in graft response. CONCLUSION: Analysis of the expression patterns of ClLAX, ClPIN and ClABCB genes under salt, drought, cold treatment and grafting response helps us to understand the possible roles of auxin transporter genes in watermelon adaptation to environmental stresses.


Assuntos
Citrullus/genética , Citrullus/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Genômica , Melhoramento Vegetal , Estresse Fisiológico/genética , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Citrullus/metabolismo , Ácidos Indolacéticos/metabolismo , Especificidade de Órgãos , Filogenia
20.
Artigo em Inglês | MEDLINE | ID: mdl-28337427

RESUMO

Mycobacterium bovis (M. bovis), the most common pathogens of tuberculosis (TB), is virulent to human and cattle, and transmission between cattle and humans warrants reconsideration concerning food safety and public health. Recently, efforts have begun to analyze cellular proteomic responses induced by Mycobacterium tuberculosis (M. tb). However, the underlying mechanisms by which virulent M. bovis affects human hosts are not fully understood. For the present study, we utilized a global and comparative labeling strategy of isobaric tag for relative and absolute quantitation (iTRAQ) to assess proteomic changes in the human monocyte cell line (THP-1) using a vaccine strain and two virulent strains H37Rv and M. bovis. We measured 2,032 proteins, of which 61 were significantly differentially regulated. Ingenuity Pathway Analysis was employed to investigate the canonical pathways and functional networks involved in the infection. Several pathways, most notably the phagosome maturation pathway and TNF signaling pathway, were differentially affected by virulent strain treatment, including the key proteins CCL20 and ICAM1. Our qRT-PCR results were in accordance with those obtained from iTRAQ. The key enzyme MTHFD2, which is mainly involved in metabolism pathways, as well as LAMTOR2 might be effective upon M. bovis infection. String analysis also suggested that the vacuolar protein VPS26A interacted with TBC1D9B uniquely induced by M. bovis. In this study, we have first demonstrated the application of iTRAQ to compare human protein alterations induced by virulent M. bovis infections, thus providing a conceptual understanding of mycobacteria pathogenesis within the host as well as insight into preventing and controlling TB in human and animal hosts' transmission.


Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/química , Macrófagos/microbiologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/imunologia , Proteoma/análise , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Proteômica , Reação em Cadeia da Polimerase em Tempo Real
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