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1.
Artigo em Inglês | MEDLINE | ID: mdl-32131529

RESUMO

Due to the increasing pollution by petroleum hydrocarbons (PHs), it is an important task to develop eco-friendly and highly efficient methods for remediating petroleum-contaminated soils. In this study, bioremediation technology was applied to remediate PHs contaminated soils, and the bacterial community structure and physicochemical characteristics of the soil treated using different bioremediation regimens were analyzed. Compared with the control condition (S0), the PHs removal efficiency of biostimulation (S2) and bioaugmentation (S3) was increased significantly. Combined biostimulation with bioaugmentation (S4) had the highest PHs removal efficiency, up to 60.14 ± 4.12%. Among all the selected remediation strategies (S1-S4, S1: soil moisture content: 25-30%), the bacterial alpha-diversity was higher than in S0. The genera Acinetobacter, Escherichia-Shigella, Bacteroides, Microbacterium, and Parabacteroides were found to greatly contribute to PHs' degradation. In the group S4, the PH-degraders and soil enzyme activity were higher than in the other remediation regimens, and these indices gradually decreased in the mid-to-later periods of all remediation tests. Additionally, the abundance of alkB and nah genes was increased by improving the environmental condition of the microorganism communities. Redundancy analysis (RDA) revealed that the total nitrogen (TN) and total phosphorus (TP) had a positive correlation with total PHs degradation. This study offers insights into the microbial community response to environmental factors during bioremediation, which shows a promoting effect in enhancing the efficiency of PHs remediation.

2.
Front Microbiol ; 9: 2788, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30519220

RESUMO

The production of pneumocandin B0 is limited by feedback inhibition. Here, low-temperature adaptive laboratory evolution (ALE) was used to improve the production capacity of Glarea lozoyensis by enhancing its membrane permeability. After 50 cycles of ALE, the pneumocandin B0 production of the endpoint strain (ALE50) reached 2131 g/L, which was 32% higher than the starting strain (ALE0). ALE50 showed a changed fatty acid composition of the cell membrane, which-+h increased its permeability by 14%, which in turn increased the secretion ratio threefold. Furthermore, ALE50 showed increased intracellular proline and acetyl-CoA concentrations, superoxide dismutase (SOD), and catalase (CAT) activity, as well as total antioxidant capacity. The slight biomass decrease in ALE50 was accompanied by decreased isocitrate dehydrogenase (ICDH) and glucose-6-phosphate dehydrogenase (G6PDH) activity. Finally, a putative model of the accumulation and secretion of pneumocandin B0 in ALE50 was established. ALE is a promising method to release intracellular feedback inhibition.

3.
Front Microbiol ; 9: 2352, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30364147

RESUMO

Pneumocandin B0 is an important antifungal drug precursor produced by filamentous fungus Glarea lozoyensis. The high broth viscosity of cultures of this organism results in lower oxygen solubility and higher energy consumption for agitation and aeration, which mostly caused by the morphologies of filamentous fungi in submerged culture. In this study, the effects of different seed medium nitrogen sources on morphology and fermentation behavior of G. lozoyensis were investigated, and cotton seed powder resulted in small, compact pellets. Moreover, pneumocandin B0 yield in Erlenmeyer flasks were increased by 22.9%. Furthermore, pneumocandin B0 yield in a 50-L fermenter reached 2,100 mg/L and the dissolved oxygen maintained above 30%. Additionally, activities of phosphofructokinase (PFK), isocitrate dehydrogenase (ICDH), glucose 6-phosphate dehydrogenase (G6PDH), and malic enzyme (ME) were increased by 87.5, 50, 41.6, and 10.7%, respectively. This study demonstrates the feasibility and advantages of using cotton seed powder for controlling the fungal morphology and improving the product yield in pneumocandin fermentations.

4.
Mol Cell Biochem ; 385(1-2): 311-8, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24091918

RESUMO

The Bcr-Abl oncoprotein is the cause of chronic myelogenous leukemia (CML). Crystal structure analysis suggests that Bcr30-63 is the core of the Bcr-Abl oligomerization interface for aberrant kinase activity; however, the precise role of other residues of Bcr1-72 excluding Bcr30-63 have not been evaluated. In this study, Bcr30-63 was named OD2 and other residues of Bcr1-72 were named OD1. Cytoplasmic transduction peptide (CTP) was used to carry molecules into cytoplasm. CTP-OD1 and CTP-OD2 fusion peptides were expressed from a cold-inducible expression system. Our results demonstrated that both fusion peptides could localize into the cytoplasm, specifically interact with the Bcr-Abl protein and further inhibit growth, induce apoptosis, and decrease the phosphorylation of Bcr-Abl in K562 cell lines. However, the viability of THP-1, a Bcr-Abl negative cell line, was unaffected. These results suggested that CTP-OD1 and CTP-OD2 may be an attractive therapeutic option to inhibit the activation of Bcr-Abl kinase in CML.


Assuntos
Apoptose/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/isolamento & purificação , Peptídeos Penetradores de Células/metabolismo , Ensaios Enzimáticos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Células K562 , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
5.
Appl Biochem Biotechnol ; 171(7): 1715-25, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23996118

RESUMO

A highly efficient oil-degrading bacteria JZX-01 was isolated from the oil-contaminated soil of the seacoast near the Boxi Offshore Oil Field of China. Morphological, physiological, and 16S rDNA gene sequence analyses indicated that JZX-01 was assigned to the genus Rhodococcus sp. This strain decomposed 65.27 ± 5.63 % of the crude oil in 9 days. Gas chromatography-mass spectrometry analysis showed that even the long-chain hydrocarbons (C31-C38) and branched alkanes (pristine and phytane), which were regarded as the stubborn ones, could be degraded. Further study showed that the bacteria still has good oil degradation ability at low temperatures as well as under high salt conditions. Moreover, JZX-01 was found to have a biosurfactant-producing capacity, which significantly favors the surface tension reduction and crude oil degradation. The promising isolated strain Rhodococcus sp. JZX-01 could be further used for the bioremediation of oil-polluted soil or seawater in a wide range of temperatures and high salt conditions.


Assuntos
Petróleo/metabolismo , Rhodococcus/isolamento & purificação , Rhodococcus/metabolismo , Biodegradação Ambiental , Hidrocarbonetos/metabolismo , Tensoativos/metabolismo
6.
Huan Jing Ke Xue ; 32(10): 3053-8, 2011 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-22279923

RESUMO

The biodegradation of phenol, m-cresol and 4-chlorophenol by the pure and mixed culture of Pseudomonas putida and Candida tropicalis was investigated. The results showed that,with the initial concentrations of 100, 50 and 60 mg x L(-1) for phenol, m-cresol and 4-chlorophenol, respectively,and an increase in the absolute value of the kinetic constant k of 19.0%, 2.6% and 46.4% for the degradation of phenol, m-cresol and 4-chlorophenol, respectively, was reached by dual culture compared with pure culture. The activity assays of phenol hydroxylase and catechol 1, 2-dioxygenase demonstrated phenol was degraded via the ortho-cleavage pathway in both of the two species. Further experiments via high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry techniques (LC-MS) suggested the degradation of three phenolic compounds could be accelerated due to co-metabolism by Pseudomonas putida of cis, cis-muconic acid, and 4-carboxymethylenebut-2-en-4-olide accumulated in phenol and 4-chlorophenol degradation, respectively, 3, 4-dihydroxybenzoic acid, 2-maleylacetate, and 3-oxoadipate in the degradation of m-cresol by Candida tropicalis in dual culture.


Assuntos
Candida tropicalis/metabolismo , Clorofenóis/metabolismo , Cresóis/metabolismo , Fenol/metabolismo , Pseudomonas putida/metabolismo , Biodegradação Ambiental , Candida tropicalis/crescimento & desenvolvimento , Clorofenóis/isolamento & purificação , Cresóis/isolamento & purificação , Técnicas de Cultura/métodos , Fenol/isolamento & purificação , Pseudomonas putida/crescimento & desenvolvimento , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias/química
7.
Protein Expr Purif ; 68(2): 167-76, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19573604

RESUMO

Cytoplasmic transduction peptide (CTP) offers exciting therapeutic opportunities for the treatment of many diseases caused by cytoplasmic functional molecules. It can transduce large, biologically active proteins into the cytoplasmic compartment of several mammalian cells. However, other intriguing features of CTP, including its activity in vitro, and distribution and tissue infiltration abilities in vivo, remain to be explored. The present study was initiated to (1) further confirm the cytoplasmic localization preference and the enzymatic activity of the transduced CTP-beta-gal in vitro and (2) examine the kinetics and tissue distribution of the CTP-beta-gal fusion protein in mice. A CTP-beta-gal fusion protein was expressed in Escherichia coli and either transduced into BaF3-BCR/ABL cells or administered intravenously into female Balb/C mice at a dose of 100 microg per mouse. Its localization in BaF3-BCR/ABL cells was evaluated by immunocytochemistry and in situ X-gal staining, and its distribution in various tissues was analyzed both by in situ X-gal staining and quantitative enzymatic activity assay. beta-Galactosidase enzyme activity was observed in BaF3-BCR/ABL cells and in all tissues tested, with peak activity occurring at 15 min in most tissues and at 24h in brain. These data will not only allow rational selection of delivery schedules for therapeutic CTP, but will also aid the use of CTP fusion protein transduction in the development of protein therapeutics targeting the cytoplasmic compartment both in vitro and in vivo.


Assuntos
Proteínas de Bactérias/metabolismo , Citoplasma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/metabolismo , Animais , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular Transformada , Sistemas de Liberação de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Distribuição Tecidual , beta-Galactosidase/administração & dosagem , beta-Galactosidase/química , beta-Galactosidase/genética
8.
Protein Expr Purif ; 64(2): 167-78, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19041400

RESUMO

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr-Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr-Abl proteins, and an intact OD in Bcr-Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr-Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr-Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr-Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni+-NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr-Abl, and potently inhibited the phospho-tyrosine pathways of Bcr-Abl oncoprotein at a low concentration of 4 microM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.


Assuntos
Citoplasma/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/isolamento & purificação , Peptídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Células Cultivadas , Clonagem Molecular , Proteínas de Fusão bcr-abl/química , Expressão Gênica , Humanos , Células K562 , Microscopia Confocal , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química
9.
Ai Zheng ; 26(10): 1058-63, 2007 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-17927873

RESUMO

BACKGROUND & OBJECTIVE: The bcr-abl fusion gene induced by reciprocal translocation of t(9; 22)(q34; q11) plays an important role in pathogenesis of chronic myeloid leukemia (CML). Using the strategy of activating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) by the dsRNA formed between the CML-specific bcr/abl fusion gene mRNA and the exogenous recombinant antisense RNA, this study was to investigate the effect of the activated PKR on the proliferation of leukemia cell line K562, and explore its possible mechanisms. METHODS: dsRNA analogue polyriboinosinic polyribocytidylic acid (PolyIC), retroviral vector containing 40 bp of bcr/abl fusion gene sequence (RV-40AS), RV-40AS and 2-aminopurine (2-AP), and retroviral vector containing green fluorescent protein sequence (RV-GFP) were transfected or infected into K562 cells respectively; ECV304 cells were used as control. Cell proliferation was determined by cell counting, MTT assay, and semisolid clone formation experiment. Cell cycle was analyzed by flow cytometry (FCM). The expression of PKR, phosphated PKR (p-PKR), eukaryotic initiation factor-2alpha (eIF2alpha), and phosphated eIF2alpha (p-eIF2alpha) was detected by Western blot. Total protein synthesis was studied by 3H-leucine incorporation. RESULTS: polyIC inhibited the proliferation of K562 cells and ECV304 cells unspecifically, while RV-40AS only inhibited the proliferation of K562 cells specifically. 2-AP blocked the inhibitory effect of RV-40AS on the proliferation of K562 cells. The S phase proportion was significantly lower in polyIC-and RV-40AS-treated K562 cells than in untreated cells [(37.26+/-2.35)% and (31.48+/-3.65)% vs. (58.53+/-5.42)%, P<0.05], while the G0/G1 phase proportion was significantly higher in polyIC-and RV-40AS-treated cells than in untreated cells [(50.97+/-2.18)% and (57.47+/-3.61)% vs. (36.44+/-4.20)%, P<0.05]. The expression of p-PKR and p-eIF2alpha in polyIC-and RV-40AS-treated K562 cells and polyIC-treated ECV304 cells was obviously up-regulated. The total protein synthesis level was significantly lower in RV-40AS-treated K562 cells than in untreated K562 cells [(3.5+/-1.9) cpm/ng vs. (26.8+/-2.6) cpm/ng, P<0.05]. CONCLUSION: Targeted activation of PKR could inhibit the proliferation of K562 cells through inhibiting protein synthesis, and arresting progression of cell cycle.


Assuntos
Ciclo Celular , Proliferação de Células , Ativação Enzimática , Poli I-C/farmacologia , eIF-2 Quinase/metabolismo , 2-Aminopurina/farmacologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Vetores Genéticos , Humanos , Células K562 , RNA Antissenso/farmacologia , RNA de Cadeia Dupla/farmacologia , RNA Mensageiro/metabolismo , Retroviridae/genética , Transfecção
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