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1.
Comput Struct Biotechnol J ; 20: 1132-1141, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35317227

RESUMO

As the most abundant post-translation modifications (PTMs), the phosphorylation usually occurred on the intrinsically disordered regions (IDRs). The regulation on the structures and interactions of IDRs induced by phosphorylation is critical to the function performing. The eukaryotic transcription factor 1 (Ets-1) is a member of transcription factor family, which participates in many important biological processes. The DNA-binding ability of Ets-1 is auto-inhibited by a disordered serine-rich region (SRR) on the Ets-1. The inhibition ability of SRR is greatly enhanced by the phosphorylation of the serine on the SRR. Nevertheless, the molecular mechanisms of the phosphorylation regulation on the structure and activity of Ets-1 are still unclear and under debates. By using both of the molecular simulations and biochemical experiments, we studied the molecule mechanism of phosphorylation regulation on the auto-inhibition of the Ets-1. The reasons of stabilization of Ets-1 core by phosphorylation on SRR region were elucidated. More important, the free energy landscapes (FEL) show that both of the steric hindrance and allosteric regulation are responsible for the DNA-binding inhibitory induced by phosphorylation, but the steric effects contribute greater than the allosteric regulation. The phosphorylation not only enhances the electrostatic interactions to facilitate the steric impedance, but also promotes the formation of hydrophobic residue clusters, which provide major driven force for the allosteric regulation. The structural basis of auto-inhibition of Ets-1 induced by the phosphorylation revealed in this study would great help the developing of inhibitor for the cancer therapy.

2.
Molecules ; 26(23)2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34885715

RESUMO

Antibiotics played an important role in controlling the development of enteric infection. However, the emergence of antibiotic resistance and gut dysbiosis led to a growing interest in the use of natural antimicrobial agents as alternatives for therapy and disinfection. Chitosan is a nontoxic natural antimicrobial polymer and is approved by GRAS (Generally Recognized as Safe by the United States Food and Drug Administration). Chitosan and chitosan derivatives can kill microbes by neutralizing negative charges on the microbial surface. Besides, chemical modifications give chitosan derivatives better water solubility and antimicrobial property. This review gives an overview of the preparation of chitosan, its derivatives, and the conjugates with other polymers and nanoparticles with better antimicrobial properties, explains the direct and indirect mechanisms of action of chitosan, and summarizes current treatment for enteric infections as well as the role of chitosan and chitosan derivatives in the antimicrobial agents in enteric infections. Finally, we suggested future directions for further research to improve the treatment of enteric infections and to develop more useful chitosan derivatives and conjugates.


Assuntos
Anti-Infecciosos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Quitosana/uso terapêutico , Gastroenteropatias/tratamento farmacológico , Antibacterianos/uso terapêutico , Anti-Infecciosos/química , Infecções Bacterianas/microbiologia , Quitosana/análogos & derivados , Quitosana/química , Gastroenteropatias/microbiologia , Humanos , Nanopartículas/química
3.
Front Microbiol ; 12: 779541, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34912319

RESUMO

Escherichia coli has been developed as the most common host for recombinant protein expression. Unfortunately, there are still some proteins that are resistant to high levels of heterologous soluble expression in E. coli. Protein and peptide fusion tags are one of the most important methods for increasing target protein expression and seem to influence the expression efficiency and solubility as well. In this study, we identify a short 15-residue enhancing solubility peptide, the PCDS (protocatechuate 3,4-dioxygenase solubility) tag, which enhances heterologous protein expression in E. coli. This PCDS tag is a 45-bp long sequence encoding a peptide tag involved in the soluble expression of protocatechuate 3,4-dioxygenase, encoded by the pcaHG98 genes of Pseudomonas putida NCIMB 9866. The 45-bp sequence was also beneficial for pcaHG98 gene amplification. This tag was shown to be necessary for the heterologous soluble expression of PcaHG98 in E. coli. Purified His6-PcaHG98e04-PCDS exhibited an activity of 205.63±14.23U/mg against protocatechuate as a substrate, and this activity was not affected by a PCDS tag. This PCDS tag has been fused to the mammalian yellow fluorescent protein (YFP) to construct YFP-PCDS without its termination codons and YFPt-PCDS with. The total protein expressions of YFP-PCDS and YFPt-PCDS were significantly amplified up to 1.6-fold and 2-fold, respectively, compared to YFP alone. Accordingly, His6-YFP-PCDS and His6-YFPt-PCDS had 1.6-fold and 3-fold higher soluble protein yields, respectively, than His6-YFP expressed under the same conditions. His6-YFP, His6-YFP-PCDS, and His6-YFPt-PCDS also showed consistent fluorescence emission spectra, with a peak at 530nm over a scanning range from 400 to 700nm. These results indicated that the use of the PCDS tag is an effective way to improve heterologous protein expression in E. coli.

4.
J Food Biochem ; 45(12): e13988, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34730252

RESUMO

Xylaneses are very common xylanolytic enzymes, which are widely used in food, papermaking, and other industries. In this study, a xylanase-encoding gene xyn1923, which encodes a protein of 1352 amino acids, was identified through the whole genome analysis of Microbacterium imperiale YD-01. Bioinformatics analysis showed that Xyn1923 only had maximum similarity of 37% with the reported xylanase from Alkalihalobacillus halodurans C-125, indicating that Xyn1923 was a novel xylanase. The enzymatic properties of Xyn1923 were systematically analyzed after purification. The results showed that the specific activity of the enzyme was 10.582 ± 0.413 U/mg, while the optimum pH and temperature of the enzyme were 7.0 and 70°C, respectively. The enzyme is stable in the pH range of 6.0-9.0, and the enzyme activity could maintain more than 85% of the original activity after 16 hr incubation at pH 9.0. The enzyme activity is relatively stable in the range of 30-60°C, and its enzyme activity could maintain more than 89% of the original activity after treatment at 60°C for 30 min. Low concentrations (≤1 mM) of Co2+ , Ba2+ , Fe2+ , and Fe3+ metal ions exerted a stimulatory effect on the activity of Xyn1923. And in contrast, high concentrations (≥2 mM) of the above metal ions inhibit the activity of Xyn1923. Mg2+ , Ag+ , Cu2+ , Ca2+ , Mn2+ , and Pb2+ ions showed a negative effect on the activity of Xyn1923. Enzyme kinetic studies showed that Km and Vmax values for xylan were 7.842 ± 0.538 mg/ml and 15.208 ± 0.822 U/mg, respectively. Xyn1923 was found to be a weakly alkaline thermophilic xylanase through an enzymatic property analysis. PRACTICAL APPLICATIONS: Xylanases are widely used in food and feed, biofuels, papermaking, and other industries. However, their use is limited by poor performance under the conditions of pH and temperature. Therefore, the discovery of xylanases with the capability of working efficiently at alkaline pH and high temperature is the priority for its industrial applications. In this study, a novel xylanase-encoding gene xyn1923 from Microbacterium imperiale YD-01 was cloned and heterologously expressed in Escherichia coli. Enzymatic properties of this novel xylanase were investigated, indicating that the robust thermal stability and alkali resistance of Xyn1923 make it a potential candidate for the food and paper industries.


Assuntos
Endo-1,4-beta-Xilanases , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Cinética , Microbacterium
5.
Curr Microbiol ; 77(12): 3945-3952, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33011835

RESUMO

Many organisms secrete xylanase, an import group of proteins hydrolyzing xylan, and thus are able to use xylan as their carbon source. In this study, we sequenced the whole genome of a bacterial strain, YD01, which was isolated from the sludge near the sewage discharge outlet of a papermill and showed high alkalic xylanase activity. Its genome consists of a chromosome and two plasmids. Six rRNA genes, 46 tRNA genes, 3136 CDSs as well as 955 repetitive sequences were predicted. 3046 CDSs were functionally annotated. Phylogenetic analysis on 16S rRNA shows that YD01 is a new species in Microbacterium genus and is taxonomically close to M. jejuense THG-C31T and M. kyungheense THG-C26T. A comparative study on phylogenetic trees of 16S rRNA and xylanase genes suggests that xylanase genes in YD01 may originate from horizontal gene transfer instead of ancestral gene duplication.


Assuntos
Ácidos Graxos , Esgotos , DNA Bacteriano/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
6.
Curr Microbiol ; 77(5): 846-854, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31932996

RESUMO

In this work, the high-level expression of the human lysozyme (HLY) was achieved by both optimization of the gene copy number and co-expression of the transcription factor Hac1p for the unfolded protein response (UPR) in the host strain Pichia pastoris KM71H. A series of recombinant constructs with various numbers of HLY expression cassettes was generated for the production of recombinant strains integrated with different copies of the HLY gene. The copy number of the HLY gene was determined by real-time quantitative polymerase chain reaction, and the recombinant strains of P. pastoris carrying one, two, three, four, or six copies of the HLY gene were obtained. Maximum extracellular protein and lysozyme enzyme activity reached 436.99 ± 26.08 µg/mL and 61,900 ± 2036.47 U/mL, respectively, in the recombinant strain HLYH4-3 with the four copies of the HLY gene after shaking flask fermentation. Moreover, the co-expression of the transcription factor Hac1p in the recombinant strains further enhanced the HLY yields. Extracellular protein and lysozyme enzyme activity, respectively, reached 517.82 ± 4.19 µg/mL and 78,600 ± 1134.95 U/mL by using the Hac1p co-expression strain HLYH4-3/Hac1p. These values are the highest recorded level of human lysozyme expressed by P. pastoris in shaking flask fermentation so far.


Assuntos
Dosagem de Genes , Muramidase/biossíntese , Pichia/genética , Fatores de Transcrição/genética , Técnicas de Cultura Celular por Lotes , Fermentação , Proteínas Fúngicas/genética , Regulação da Expressão Gênica , Humanos , Muramidase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Resposta a Proteínas não Dobradas
7.
Appl Microbiol Biotechnol ; 104(4): 1609-1619, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900556

RESUMO

The emergence of antibiotic-resistant beta-hemolytic Streptococcus agalactiae strains poses increasing threat to human beings globally. As an attempt to create a novel lysin with improved activity against S. agalactiae, a chimeric lysin, ClyV, was constructed by fusing the enzymatically active domain (EAD) from PlyGBS lysin (GBS180) and the cell wall binding domain (CBD) from PlyV12 lysin (V12CBD). Plate lysis assay combined with lytic kinetic analysis demonstrated that ClyV has improved activity than its parental enzymatic domain GBS180 against multiple streptococci. Biochemical characterization showed that ClyV is active from pH 7 to 10, with the optimum pH of 9, and is stable under NaCl concentration of < 500 mM. In a S. agalactiae infection model, a single intraperitoneally administration of 0.1 mg/mouse of ClyV protected 100% mice, while it was observed that ~ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180. Moreover, a high dose of 0.8 mg/mouse ClyV did not show any adverse effects to the health or survival rate of the mice. Considering the robust bactericidal activity and good safety profile of ClyV, it represents a potential candidate for the treatment of S. agalactiae infections.


Assuntos
Antibacterianos/farmacologia , Enzimas/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus agalactiae/efeitos dos fármacos , Animais , Enzimas/biossíntese , Enzimas/genética , Feminino , Injeções Intraperitoneais , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/biossíntese , Infecções Estreptocócicas/microbiologia
8.
ACS Synth Biol ; 8(9): 1991-1997, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31487454

RESUMO

Direct cloning of natural product pathways for efficient refactoring and heterologous expression has become an important strategy for microbial natural product research and discovery, especially for those kept silent or poorly expressed in the original strains. Accordingly, the development of convenient and efficient cloning approaches is becoming increasingly necessary. Here we presented an in vitro packaging mediated cloning approach that combines CRISPR/Cas9 system with in vitro λ packaging system, for targeted cloning of natural product pathways. In such a scheme, pathways of Tü3010 (27.4 kb) and sisomicin (40.7 kb) were respectively cloned, and stuR was further depicted to positively regulate Tü3010 production. In vitro packaging mediated approach not only enables to activate cryptic pathways, but also facilitates refactoring or interrogating the pathways in conjunction with various gene editing systems. This approach features an expedited, convenient, and generic manner, and it is conceivable that it may be widely adopted for targeted cloning of the natural product pathways.


Assuntos
Produtos Biológicos/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Bactérias/genética , Produtos Biológicos/química , Clonagem Molecular , Edição de Genes , Família Multigênica , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Repressoras/genética , Sisomicina/química , Sisomicina/metabolismo , Streptomyces/genética , Transativadores/genética
9.
Curr Microbiol ; 76(11): 1235-1237, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31432211

RESUMO

Chlorobenzenes are ubiquitously distributed, highly persistent, and toxic environmental contaminants. Pandoraea pnomenusa MCB032 was isolated as a new dominant chlorobenzene-utilizing strain from a functionally stable bioreactor during the treatment of chlorobenzenes when strain Burkholderia sp. JS150 disappeared. In study, we report the complete genome sequence of strain MCB032 which consists of a circular chromosome and three plasmids, which are ~ 6 Mb in length with 5450 open reading frames-12 encoding rRNAs and 77 encoding tRNAs. We further identified 17 putative genes encoding the enzymes involved in the methyl-accepting chemotaxis proteins in sensing chemical gradients during chemotaxis. The annotated complete genome sequence of this strain will provide genetic insights into the degradation of chlorinated aromatic compounds. The information will empower the elucidation of chlorobenzene affinity hierarchy and species succession in the bioreactor.


Assuntos
Burkholderiaceae/genética , Burkholderiaceae/metabolismo , Clorobenzenos/metabolismo , Genoma Bacteriano , Biodegradação Ambiental , Burkholderiaceae/isolamento & purificação , Plasmídeos/genética , Plasmídeos/metabolismo , Sequenciamento Completo do Genoma
10.
Plant Sci ; 283: 1-10, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128679

RESUMO

Colletotrichum higginsianum causes anthracnose disease in a wide range of cruciferous crops and has been used as a model system to study plant-pathogen interactions and pathogenicity of hemibiotrophic plant pathogens. Conidiation, hyphae growth, appressorial development and appressorial penetration are significant steps during the infection process of C. higginsianum. However, the mechanisms of these important steps during infection remain incompletely understood. To further investigate the mechanisms of the plant-C. higginsianum interactions during infection progress, we characterized Cyclase-Associated Protein (ChCAP) gene. Deletion of the ChCAP gene resulted in reduction in conidiation and hyphal growth rate. The pathogenicity of ΔChCAP mutants was significantly reduced with much smaller lesion on the infected leaves compared to that of wild type strain with typically water-soaked and dark necrotic lesions on Arabidopsis leaves. Further study demonstrated that the appressorial formation rate, turgor pressure, penetration ability and switch from biotrophic to necrotrophic phases decreased obviously in ΔChCAP mutants, indicating that the attenuated pathogenicity of ΔChCAP mutants was due to these defective phenotypes. In addition, the ΔChCAP mutants sectored on PDA with abnormal, dark color, vesicle-like colony morphology and hyphae tip. Moreover, the ΔChCAP mutants had a reduced intracellular cAMP levels and exogenous cAMP can partially rescue the defects of ΔChCAP mutants in appressorial formation and penetration rate, but not in colony morphology, conidial shape and virulence, indicating that ChCAP is a key component in cAMP signaling pathway and likely play other roles in biology of C. higginsianum. In summary, our findings support the role of ChCAP in regulating conidiation, intracellular cAMP level, hyphal growth, appressorial formation, penetration ability and pathogenicity of this hemibiotrophic fungus.


Assuntos
Colletotrichum/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Proteínas Fúngicas/fisiologia , Hifas/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimento , Arabidopsis/microbiologia , Colletotrichum/metabolismo , Colletotrichum/patogenicidade , Colletotrichum/fisiologia , Proteínas Fúngicas/metabolismo , Hifas/fisiologia , Filogenia , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Esporos Fúngicos/fisiologia , Estresse Fisiológico
11.
Curr Microbiol ; 76(9): 1092, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29574478

RESUMO

The original version of this article unfortunately contained a mistake in the Fig. S1 of supplementary material. It is corrected with this erratum.

12.
Front Microbiol ; 9: 2848, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30524413

RESUMO

Cyclohexylamine (CHAM) is widely used in various industries, but it is harmful to human beings and the environment. Acinetobacter sp. YT-02 can degrade CHAM via cyclohexanone as an intermediate. In this study, the cyclohexylamine oxidase (CHAO) gene from Acinetobacter sp. YT-02 was cloned. Amino acid sequence alignment indicated that the cyclohexylamine oxidase (CHAOYT-02) was 48% identical to its homolog from Brevibacterium oxydans IH-35A (CHAOIH-35). The enzyme was expressed in Escherichia coli BL21 (DE3), and purified to apparent homogeneity by Ni-affinity chromatography. The purified enzyme was proposed to be a dimer of molecular mass of approximately 91 kDa. The enzyme exhibited its maximum activity at 50°C and at pH 7.0. The enzyme was thermolabile as demonstrated by loss of important percentage of its maximal activity after 30 min incubation at 50°C. Metal ions Mg2+, Co2+, and K+ had certain inhibitory effect on the enzyme activity. The kinetic parameters K m and V max were 0.25 ± 0.02 mM and 4.3 ± 0.083 µM min-1, respectively. The biochemical properties, substrate specificities, and three-dimensional structures of CHAOYT-02 and CHAOIH-35 were compared. Our results are helpful to elucidate the mechanism of microbial degradation of CHAM in the strain YT-02. In addition, CHAOYT-02, as a potential biocatalyst, is promising in controlling CHAM pollution and deracemization of chiral amines.

13.
Mol Med Rep ; 17(6): 7513-7520, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29620189

RESUMO

The current study aimed to identify the effect and primary mechanism of Curcumol on the migration of nasopharyngeal carcinoma (NPC) cells in vitro and in vivo. Curcumol was dissolved in absolute ethyl alcohol and the experiment was performed in NPC 5­8F cells in vitro and in vivo. The effect of different concentrations of Curcumol on cell migration was determined using wound healing and Transwell assays. A cell counting kit­8 (CCK­8) assay was also performed in order to determine cell viability. Flow cytometry was used to detect the effect of Curcumol on apoptosis. The expression of epithelial­mesenchymal transition (EMT)­associated proteins and genes was evaluated by western blotting, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and ELISA. In addition, the antitumor activity of Curcumol was investigated in female BALB/C nude mice with orthotopic tumor implants. The results indicated that cell apoptosis was increased and the viability of NPC 5­8F cells was decreased following treatment with Curcumol at doses of 0.1, 0.2 and 0.4 µM/ml. The results of in vivo experiments indicated that tumor growth and weight were decreased following Curcumol administration. Furthermore, the results of western blotting and RT­qPCR demonstrated that Curcumol altered the level of E­cadherin and N­cadherin in a dose­dependent manner in vivo. Curcumol also regulated the secretion of protein markers in the serum that were associated with EMT and TGF­ß1 in the 5­8F xenograft mouse model. Thus, the results indicated that Curcumol induced TGF­ß1­mediated EMT arrest by regulating E­cadherin and N­cadherin, which may prevent further development of NPC.


Assuntos
Carcinoma/metabolismo , Carcinoma/patologia , Medicamentos de Ervas Chinesas/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Sesquiterpenos/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Carcinoma/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/genética , Fator de Crescimento Transformador beta1/genética , Carga Tumoral
14.
Curr Microbiol ; 75(3): 284-287, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29063968

RESUMO

Acinetobacter sp. YT-02, a Gram-negative bacterium isolated from the activated sludge from a sodium N-cyclohexylsulfamate production plant, has the ability to degrade cyclohexylamine. It was classified as a member of Acinetobacter sp., a Gram-negative bacterium, sharing a 16S rRNA gene sequence identity of 99% with Acinetobacter guangdongensis strain 1NM-4. It could degrade 10 mmol/L cyclohexylamine within 22 h. Based on the identified metabolite, the metabolic pathway of cyclohexylamine could be postulated as it was degraded via cyclohexanone. Draft genome sequence of this strain (2,993, 647 bp of chromosome length) is presented here. We further identified the genes encoding the enzymes involved in cyclohexylamine oxidation to cyclohexanone and the subsequent downstream metabolic pathway of cyclohexanone oxidation. Strain YT-02 has the potentiality to be applied in the treatment of the pollutant cyclohexylamine, and it could also be treated as a research material to study the degradation mechanism of cyclohexylamine.


Assuntos
Acinetobacter/genética , Acinetobacter/isolamento & purificação , Cicloexilaminas/metabolismo , Genoma Bacteriano , Acinetobacter/classificação , Acinetobacter/metabolismo , Sequência de Bases , Biodegradação Ambiental , DNA Bacteriano/genética , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Esgotos/microbiologia
15.
Cell J ; 19(3): 512-519, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28836414

RESUMO

OBJECTIVES: Taraxerol acetate has potent anti-cancer effects via the induction of apoptosis, autophagy, cell cycle arrest, and inhibition of cell migration. However, whether taraxerol induced apoptosis and its underlying mechanisms of action is not clear. In the present study, we assess the effects of taraxerol on the mitochondrial apoptotic pathway and determine the release of cytochrome c to the cytosol and activation of caspases. MATERIALS AND METHODS: In this experimental study, we mainly investigated the effect of taraxerol on HeLa cells. We tested cell viability by the MTT assay and morphologic changes, analyzed apoptosis by DAPI staining and flow cytometry. We also determined reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) using a Microplate Reader. In addition, the apoptotic proteins were tested by Western blot. RESULTS: Taraxerol enhanced ROS levels and attenuated the MMP (Δψm) in HeLa cells. Taraxerol induced apoptosis mainly via the mitochondrial pathway including the release of cytochrome c to the cytosol and activation of caspases 9 and 3, and anti-poly (ADPribose) polymerase (PARP). Taraxerol could induce the down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax. It suppressed the PI3K/ Akt signaling pathway. CONCLUSIONS: These results demonstrated that taraxerol induced cell apoptosis through a mitochondria-mediated pathway in HeLa cells. Thus, taraxerol might be a potential anticervical cancer candidate.

16.
J Biotechnol ; 251: 166-173, 2017 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-28472672

RESUMO

The Gram-negative strain of Pseudomonas plecoglossicida NyZ12 isolated from soil has the ability to degrade cyclohexylamine (CHAM). The genes encoding CHAM degradation by gram-negative bacteria, however, have not been reported previously. In this study, ORFs predicted to encode CHAM degradation by NyZ12 were identified by bioinformatics analysis. Differential expression of the proposed ORFs was analyzed via RNA-seq and quantitative reverse transcription-PCR (qRT-PCR), using RNA extracted from NyZ12 cultured with or without CHAM addition. One CHAM-inducible ORF, RK21_02867 predicted to encode a cyclohexanone monooxygenase (ChnB) was disrupted, as were five ORFs, RK21_00425, RK21_02631, RK21_04207, RK21_04637 and RK21_05539, that had weak homology to the only known cyclohexylamine oxidase (CHAO encoded by chaA) found in Brevibacterium oxydans IH-35A. We also found that a tandem array of five ORFs (RK21_02866-02870) shared homology with those in an operon responsible for oxidation of cyclohexanone to adipic acid, although the ORFs in strain NyZ12 were arranged in a different order with previously found in cyclohexane, cyclohexanol or cyclohexanone degradation strains. The ORFs in this cluster were all up-regulated when CHAM was supplied as the sole carbon source. When one of these five genes, RK21_02867 encoding cyclohexanone (CHnone) monooxygenase, was knocked out, NyZ12 could not grow on CHAM, but it accumulated equimolar amounts of CHnone. Our results show that strain NyZ12 metabolized CHAM directly to CHnone which was then further metabolized to adipate. Despite clearly identifying genes encoding the steps for metabolism of CHAM metabolites, not every one of the putative chaAs was differentially expressed in the presence of CHAM and deletion of each one individually did not completely eliminate the capacity of NyZ12 to degrade CHAM, though it did reduce its growth in several instances. Our results suggest that there is genetic redundancy encoding the initial step in the oxidation of CHAM to CHnone in NyZ12 and that its CHAOs differ considerably from the ChaA, originally described in Brevibacterium oxydans IH-35A.


Assuntos
Genoma Bacteriano , Pseudomonas/genética , Biodegradação Ambiental , Cicloexilaminas/metabolismo , Genes Bacterianos , Oxirredutases/genética , Oxirredutases/metabolismo , Pseudomonas/metabolismo
17.
Food Funct ; 8(1): 132-141, 2017 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-27921103

RESUMO

The aim of the present study was to examine the anti-inflammatory effect of solanesol and to elucidate the underlying mechanisms. Heme oxygenase-1 (HO-1) plays an important role in cytoprotection against oxidative stress and inflammation. Solanesol induced HO-1 expression both at the level of mRNA and proteins, resulting in increased HO-1 activity. Solanesol treatment enhanced the level of the phosphorylated form, nuclear translocation, ARE-binding, and transcriptional activity of Nrf2. p38 and Akt contributed to ARE-driven HO-1 expression. Solanesol activated both p38 and Akt, and treatments with SB203580 (a p38 kinase inhibitor), LY294002 (an Akt inhibitor), specific p38 siRNA and Akt siRNA suppressed the solanesol-induced activation of Nrf2, resulting in a decrease in HO-1 expression. Solanesol also elevated the autophagic protein LC3B-II level. SnPP (a HO-1 inhibitor) and HO-1 siRNA markedly abolished the anti-inflammatory effect of solanesol against LPS-induced cell damage. Likewise, SB203580, LY294002, 3-MA and Baf-A1 inhibited the solanesol-induced anti-inflammatory effect. These studies demonstrate that solanesol attenuates inflammation by HO-1 induction via p38 and Akt signaling. Thus, it is quite plausible that HO-1 induction by solanesol could trigger anti-inflammatory pathways including limiting LPS-stimulated cytokine production through autophagic signaling via p38 and Akt.


Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/imunologia , Heme Oxigenase-1/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Terpenos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Citocinas/genética , Heme Oxigenase-1/imunologia , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Células RAW 264.7 , Proteínas Quinases p38 Ativadas por Mitógeno/genética
18.
Toxicol In Vitro ; 29(3): 600-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25645596

RESUMO

In present study, we showed that the mRNA and protein levels of HO-1 and Hsp70 in solanesol-treated L02 cells were significantly increased. The induction of the HO-1 by solanesol is majorly achieved via enhancing the nuclear translocation and transactivity of Nrf2 through enhancement of Hsp90-Keap1 interaction, while solanesol-elevated Hsp70 is related with promoting the nuclear translocation of HSF1 through the involvement of chaperones interaction. Furthermore, the induction of HO-1 and Hsp70 by solanesol could protect against ethanol-induced liver injury, including significantly suppressing the elevation of the activities of LDH and AST, attenuating ethanol-induced increase of the MDA, ROS level and decrease of the GSH level. Moreover, solanesol also suppressed ethanol-induced apoptosis of L02 cells by inhibition of nuclear morphological damage, procaspase 3 and cleavage of caspase 3 and PARP, suggesting solanesol may be beneficial against ALD. Solanesol also promoted tBHQ-mediated protective effects. However, treatment cells with SnPP or PES markedly abrogated the protective effects of solanesol on ethanol-induced cell injury. These results strongly suggested that solanesol could protect ethanol-induced L02 cell damage, which might be attributed to the activation of HO-1 and Hsp70.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Etanol/toxicidade , Proteínas de Choque Térmico HSP70/biossíntese , Heme Oxigenase-1/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Terpenos/farmacologia , Antioxidantes/metabolismo , Depressores do Sistema Nervoso Central/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Humanos , Fígado/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Regulação para Cima/efeitos dos fármacos
19.
J Biotechnol ; 199: 29-30, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25701176

RESUMO

Pseudomonas plecoglossicida NyZ12 (CCTCC AB 2015057), a Gram-negative bacterium isolated from soil, has the ability to degrade cyclohexylamine. The complete genome sequence of this strain (6,233,254bp of chromosome length) is presented, with information about the genes of characteristic enzymes responsible for cyclohexylamine oxidation to cyclohexanone and the integrated gene cluster for the metabolic pathway of cyclohexanone oxidation to adipate.


Assuntos
Cicloexilaminas/metabolismo , Genoma Bacteriano/genética , Pseudomonas/genética , Pseudomonas/metabolismo , Dados de Sequência Molecular , Família Multigênica/genética
20.
World J Microbiol Biotechnol ; 31(2): 371-7, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25532745

RESUMO

A consortium comprised of an engineered Escherichia coli DH5α and a natural pentachlorophenol (PCP) degrader, Sphingobium chlorophenolicum ATCC 39723, was assembled for degradation of hexachlorobenzene (HCB), a persistent organic pollutant. The engineered E. coli strain, harbouring a gene cassette (camA (+) camB (+) camC) that encodes the F87W/Y96F/L244A/V247L mutant of cytochrome P-450cam (CYP101), oxidised HCB to PCP. The resulting PCP was then further completely degraded by ATCC 39723. The results showed that almost 40 % of 4 µM HCB was degraded by the consortium at a rate of 0.033 nmol/mg (dry weight)/h over 24 h, accompanied by transient accumulation and immediate consumption of the intermediate PCP, detected by gas chromatography. In contrast, in the consortium comprised of Pseudomonas putida PaW340 harbouring camA (+) camB (+) camC and ATCC 39723, PCP accumulated in PaW340 cells but could not be further degraded, which may be due to a permeability barrier of Pseudomonas PaW340 for PCP transportation. The strategy of bacterial co-culture may provide an alternative approach for the bioremediation of HCB contamination.


Assuntos
Cânfora 5-Mono-Oxigenase/genética , Escherichia coli/enzimologia , Hexaclorobenzeno/metabolismo , Pentaclorofenol/metabolismo , Sphingomonadaceae/metabolismo , Biodegradação Ambiental , Cânfora 5-Mono-Oxigenase/metabolismo , Cromatografia Gasosa , Técnicas de Cocultura , Escherichia coli/genética , Engenharia Genética , Consórcios Microbianos , Mutação
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