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1.
BMC Plant Biol ; 19(1): 371, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438856

RESUMO

BACKGROUND: Propamocarb (PM) is one of the main pesticides used for controlling cucumber downy mildew. However, due to its volatility and internal absorption, PM can easily form pesticide residues on cucumber fruits that seriously endanger human health and pollute the environment. The breeding of new cucumber varieties with a low abundance of PM residues via genetic methods constitutes an effective strategy for reducing pesticide residues and improving cucumber safety and quality. To help elucidate the molecular mechanism resulting in a low PM residue abundance in cucumber, we used the cucumber cultivar 'D0351' (which has the lowest PM residue content) as the test material and identified genes related to low PM residue abundance through high-throughput tag-sequencing (Tag-Seq). RESULTS: CsMAPEG was constitutively expressed and showed both varietal and organizational differences. This gene was strongly expressed in 'D0351'. The expression levels of CsMAPEG in different cucumber tissues under PM stress were as follows: fruit>leaf>stem>root. CsMAPEG can respond to salicylic acid (SA), gibberellin (GA) and Corynespora cassiicola Wei (Cor) stress and thus plays an important regulatory role in plant responses to abiotic and biological stresses. The PM residue abundance in the fruits of CsMAPEG-overexpressing plants was lower than those found in antisense CsMAPEG plants and wild-type plants at all tested time points. The results revealed that CsMAPEG played a positive role in reducing the PM residue abundance. A CsMAPEG sense construct increased the contents of SOD, POD and GST in cucumber fruits, enhanced the degradation and metabolism of PM in cucumber, and thus effectively reduced the pesticide residue abundance in cucumber fruits. CONCLUSIONS: The expression patterns of CsMAPEG in cucumber cultivars with high and low pesticide residue abundances and a transgenic verification analysis showed that CsMAPEG can actively respond to PM stress and effectively reduce the PM residue abundance in cucumber fruits. The results of this study will help researchers further elucidate the mechanism responsible for a low PM residue abundance in cucumber and lay a foundation for the breeding of new agricultural cucumber varieties with low pesticide residue abundances.


Assuntos
Carbamatos/farmacologia , Cucumis sativus/genética , Fungicidas Industriais/farmacologia , Genes de Plantas , Resíduos de Praguicidas , Clonagem Molecular , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Perfilação da Expressão Gênica , Vetores Genéticos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transformação Genética
2.
Dokl Biochem Biophys ; 486(1): 192-196, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367819

RESUMO

A novel CYP74 clan gene CYP443С1 of the starlet sea anemone (Nematostella vectensis, Cnidaria) has been cloned, and the properties of the corresponding recombinant protein have been studied. Depending on the substrate, CYP443С1 exhibited double function hydroperoxide lyase/epoxyalcohol synthase activity.


Assuntos
Aldeído Liases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Anêmonas-do-Mar/enzimologia , Aldeído Liases/química , Aldeído Liases/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Anêmonas-do-Mar/genética , Alinhamento de Sequência
3.
World J Microbiol Biotechnol ; 35(8): 127, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375931

RESUMO

Aeromonas hydrophila is a Gram-negative bacterium that causes serious infections in aquaculture and exhibits significant multidrug resistance. The LysR-type transcriptional regulator (LTTR) family proteins are a well-known group of transcriptional regulators involved in diverse physiological functions. However, the role of LTTRs in the regulation of bacterial resistance to antibiotics is still largely unknown. In this study, to further investigate the role of four putative LTTR family proteins (A0KIU1, A0KJ82, A0KPK0, and A0KQ63) in antibiotic resistance in A. hydrophila, their genes were cloned and overexpressed in engineered Escherichia coli. After the optimization of experimental conditions including incubation time, temperature, and IPTG concentration, these proteins were successfully purified, and their specific antibodies against mice were obtained. Using western blot analysis, we found that these LTTR family proteins were downregulated in A. hydrophila following antibiotic treatment, indicating that they may be involved in the regulation of antibiotic resistance. Additionally, minimum inhibitory concentration (MIC) assays of chloramphenicol (CM), chlortetracycline (CTC), ciprofloxacin (CF), furazolidone (FZ), and balofloxacin (BF) in E. coli showed that overexpression of these LTTRs led to increased sensitivity to several antibiotics. To further validate their functional role in antibiotic resistance, we demonstrated that bacteria with loss of A0KQ63 (ΔAHA_3980) exhibited multi-drug resistance properties. Our results indicate that these LTTR family proteins may play an important role in the antibiotic resistance of A. hydrophila, and the that underlying mechanisms controlling antibiotic resistance should be further investigated.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Fatores de Transcrição/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Genes Bacterianos , Camundongos , Testes de Sensibilidade Microbiana , Fatores de Transcrição/análise , Fatores de Transcrição/genética
4.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
5.
J Enzyme Inhib Med Chem ; 34(1): 1439-1450, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31409157

RESUMO

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).


Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania infantum/enzimologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Catálise , Cromatografia de Afinidade , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração Inibidora 50 , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/isolamento & purificação , Timidilato Sintase/metabolismo
6.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
7.
Parasite ; 26: 39, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31294687

RESUMO

Glycogen synthase kinase 3 (GSK-3), which belongs to the serine/threonine kinase family, regulates glycogen metabolism, Wnt signaling, hormonal regulation, and embryonic development in many eukaryotes. Here, we cloned a complete open reading frame (ORF) of glycogen synthase kinase 3ß (GSK-3ß) from Haemaphysalis longicornis and characterized its transcriptional and functional status. The ORF of GSK-3ß possesses 1242 nucleotides encoding a mature protein of 413 amino acid residues. GSK-3ß nucleotide and protein sequences are highly conserved among different vertebrate and invertebrate animals, with identity between 47.8-100% and 63.2-88.7%, respectively. Sequence comparison showed one signature domain between the residues of 51 and 335 amino acids, which was identified as a protein kinase (serine/threonine). RT-PCR showed GSK-3ß mRNA present in all developmental stages of H. longicornis. Interestingly, a higher transcript level was observed in nymph and 7-day-old eggs compared with others by real-time PCR, indicating a role of GSK-3ß in the early stages of life. The functional status of GSK-3ß was characterized by RNA interference (RNAi) and caused significant (p < 0.05) reduction in feeding and reproduction, as well as an abnormality in eggs and hatching. Taken together, our results suggest that GSK-3ß may be an important candidate for a multiple antigen vaccine for controlling the tick population.


Assuntos
Glicogênio Sintase Quinase 3 beta/genética , Ixodidae/enzimologia , Ixodidae/genética , Animais , Clonagem Molecular , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Estágios do Ciclo de Vida , Masculino , Ninfa/genética , Oócitos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Mensageiro , Transdução de Sinais
8.
DNA Cell Biol ; 38(8): 773-785, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339741

RESUMO

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 µg/mL) was more sensitive when compared with HepG2 (IC50 1.9 µg/mL) and AGS (IC50 3.7 µg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.


Assuntos
ADP Ribose Transferases/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Proteínas Recombinantes/farmacologia , ADP Ribose Transferases/genética , Animais , Apoptose/fisiologia , Baculoviridae/genética , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Proteínas de Insetos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/genética , Células Sf9 , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
9.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1348-1358, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328491

RESUMO

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.


Assuntos
Streptomyces coelicolor , Biocatálise , Clonagem Molecular , Escherichia coli , Glucosiltransferases , Trealose
10.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
11.
Arch Insect Biochem Physiol ; 101(4): e21587, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31271487

RESUMO

The codling moth, Cydia pomonella, is a worldwide pest of pome fruits. Neuropeptides regulate most physiological functions in insects and represent new targets for the development of control agents. The only neuropeptides reported from the codling moth to date are the allatostatin A family peptides. To identify other neuropeptides and peptide hormones from codling moth, we analyzed head transcriptomes, identified 50 transcripts, and predicted 120 prepropeptides for the codling moth neuropeptides and peptide hormones. All transcripts were amplified, and these sequences were verified. One of the notable findings in this study is that diapause hormones (DHs) reported from Tortricid moths, including the codling moth, do not have the WFGPRL sequence in C-terminal ends in the pban genes. The C-terminal motif is critical to characterize insect DH peptides, and always conserved in pban/dh genes in Lepidoptera and many insect orders. Interestingly, the WFGPRL sequence was produced only from the capa gene in the codling moth. The allatostatin A-family encoding transcript predicted nine peptides, seven of which, as expected, are identical to those previously isolated from the moth. We also identified new codling moth orthologs of insect neuropeptides including CCHamides, allatostatin CC, RYamides, and natalisins. The information provided in this study will benefit future codling moth investigations using peptidoproteomics to determine peptide presence and functions.


Assuntos
Mariposas/metabolismo , Neuropeptídeos/metabolismo , Hormônios Peptídicos/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Regulação da Expressão Gênica , Neuropeptídeos/química , Hormônios Peptídicos/química
12.
World J Microbiol Biotechnol ; 35(8): 122, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346836

RESUMO

To promote enzymatic unhairing for leather production, a new unhairing enzyme is developed. The Keratinase (kerT) gene, which is amplified from B. amyloliquefaciens TCCC11319 by PCR, is expressed in B. subtilis WB600. The recombinant KerT reduces the collagenolytic protease content as well as improving the keratinase content effectively. Therefore, the improved keratinase leads to the obviously unhairing effect, whereas the low collagenolytic protease ensures the integrity of collagen fibers in hide. It represents, the leather grain surface isn't destroyed thereby the value of finished leather can be maintained. In addition, by analyzing the properties of KerT, tits activity isn't inhibited with Na+, K+ and Ca2+ which are commonly used in leather production. The freeze-dried fermentation broth can be used directly as unhairing enzyme without addition of traditional sulfide chemicals. By evaluating the properties of unhaired hide, the results show that the collagen degradation ability of this new unhairing enzyme is slightly and it does not cause any adverse effects on the leather quality. Besides, this unhairing enzyme doesn't further degrade collagen in the time range of 8 h to 24 h, thus it is safely and easy-control in actual production. In conclusion, the enzymatic unhairing method with recombinant KerT has the potential to be more sustainable and efficient alternative than current sulphur-lime method, and it does not require the further purification thereby saving the cost.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Bacteriano/isolamento & purificação , Peptídeo Hidrolases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Fragmentação do DNA , DNA Bacteriano/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
BMC Plant Biol ; 19(1): 316, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307394

RESUMO

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cátions/metabolismo , Clonagem Molecular , Eletrofisiologia , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
14.
BMC Plant Biol ; 19(1): 231, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159735

RESUMO

BACKGROUND: Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway. RESULTS: In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes indicate that Fm4CL-like 1 gene may play a role in the lignin synthesis pathway. Our study indicate that overexpression of Fm4CL-like 1 increases the lignin content of transgenic tobacco by 39.5% compared to WT, and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT. The xylem cell layer of transgenic line is increased by 40% compared to WT, the xylem cell wall thickness increased by 21.6% compared to the WT. Under mannitol-simulated drought stress, the root length of transgenic tobacco is 64% longer than WT, and the seed germination rate of the transgenic lines is 47% higher than that of WT. In addition, the H2O2 content in the transgenic tobacco was 22% lower than that of WT, while the POD and SOD content was higher than WT by 30 and 24% respectively, which showed Fm4CL-like 1 affect the accumulation of reactive oxygen species (ROS). The MDA content and relative conductivity was 25 and 15% lower than WT, respectively. The water loss rate is 16.7% lower than that of WT. The relative expression levels of stress-related genes NtHAK, NtAPX, NtCAT, NtABF2, and NtZFP were higher than those of WT under stress treatment. The stomatal apertures of OE (Overexpression) were 30% smaller than those of WT, and the photosynthetic rate of OE was 48% higher than that of WT. These results showed that the overexpression line exhibited stronger adaptability to osmotic stress than WT. CONCLUSIONS: Our results indicate that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis. Fm4CL-like 1 play an important role in osmotic stress by affecting cell wall and stomatal development.


Assuntos
Secas , Proteínas de Plantas/genética , Tabaco/fisiologia , Clonagem Molecular , Fraxinus/genética , Fraxinus/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Estresse Fisiológico/genética , Tabaco/genética
15.
J Med Microbiol ; 68(7): 1021-1032, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31188094

RESUMO

INTRODUCTION: The spread of carbapenem-resistant Acinetobacter baumannii has led to a worldwide healthcare problem. Carbapenem resistance in A. baumannii is mainly mediated by the acquisition of the carbapenem-hydrolyzing oxacillinase OXA-23. The phenotypic detection of carbapenem-producing A. baumannii is challenging and time-consuming. Hence, there is an unmet medical need for reliable and rapid diagnostic tools to detect OXA-23-producing Acinetobacter isolates to enable successful patient management. AIM: Development of an immunochromatographic lateral flow test (ICT) for the rapid and reliable detection of OXA-23-producing carbapenem-resistant Acinetobacter isolates. METHODOLOGY: For the development of an antibody-based ICT, we generated anti-OXA-23 monoclonal antibodies (MoAbs) and screened them sequentially for their ability to bind native OXA-23. Selected OXA-23-specific MoAbs were tested in different combinations for their capacity to capture and detect OXA-23His6 by sandwich enzyme-linked immunosorbent assay (ELISA) and ICT. A well-characterized collection of carbapenem-resistant Acinetobacter isolates with defined carbapenem resistance mechanisms were used to evaluate the specificity of the final OXA-23 ICT prototype. RESULTS: The antibody pairs best suited for the sandwich ELISA format did not match the best pairs in the ICT format selected during the development process of the final prototype OXA-23 ICT. This prototype was able to differentiate between OXA-23 subfamily-mediated carbapenem resistance and carbapenem-resistant Acinetobacter isolates overexpressing other OXAs with 100  % specificity and a turnaround time of 20 min from culture plate to result. CONCLUSION: With this rapid detection assay one can save 12-48 h of diagnostic time, which could help avoid inappropriate use of carbapenems and enable earlier intervention to control the transmission of OXA-23-producing carbapenem-resistant Acinetobacter isolates to other patients and healthcare workers.


Assuntos
Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Imunoensaio/métodos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/imunologia , Animais , Antibacterianos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias , Carbapenêmicos/metabolismo , Clonagem Molecular , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , beta-Lactamases/imunologia , beta-Lactamases/metabolismo
16.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 641-645, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204911

RESUMO

OBJECTIVE: To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region. METHODS: The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods. RESULTS: The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P<0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3. CONCLUSION: The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.


Assuntos
Antígenos de Neoplasias/genética , Proteínas Reguladoras de Apoptose/genética , Leucemia Monocítica Aguda , Adulto , Clonagem Molecular , Genes Reporter , Células HEK293 , Humanos , Leucemia Monocítica Aguda/genética , Luciferases , Regiões Promotoras Genéticas
17.
Biochemistry (Mosc) ; 84(2): 171-180, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31216976

RESUMO

Cytochromes P450 of the CYP74 family play a key role in the lipoxygenase cascade generating oxylipins (products of polyunsaturated fatty acid oxidation). The CYP74 family includes allene oxide synthases, hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases. In this work, we cloned the CYP74A88 gene from the Japanese buttercup (Ranunculus japonicus) and studied the properties of the encoded recombinant protein. The CYP74A88 enzyme specifically converts linoleic acid 9- and 13-hydroperoxides to oxiranyl carbinols 9,10-epoxy-11-hydroxy-12-octadecenoic acid and 11-hydroxy-12,13-epoxy-9-octadecenoic acid, respectively, which was confirmed by GC-MS analysis and kinetic studies. Therefore, the CYP74A88 enzyme is a specific epoxyalcohol synthase.


Assuntos
Biocatálise , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Ranunculus/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Japão
18.
Nat Commun ; 10(1): 2873, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253765

RESUMO

Green synthesis of precise inorganic nanomaterials is a major challenge. Magnetotactic bacteria biomineralise magnetite nanoparticles (MNPs) within membrane vesicles (magnetosomes), which are embedded with dedicated proteins that control nanocrystal formation. Some such proteins are used in vitro to control MNP formation in green synthesis; however, these membrane proteins self-aggregate, making their production and use in vitro challenging and difficult to scale. Here, we provide an alternative solution by displaying active loops from biomineralisation proteins Mms13 and MmsF on stem-loop coiled-coil scaffold proteins (Mms13cc/MmsFcc). These artificial biomineralisation proteins form soluble, stable alpha-helical hairpin monomers, and MmsFcc successfully controls the formation of MNP when added to magnetite synthesis, regulating synthesis comparably to native MmsF. This study demonstrates how displaying active loops from membrane proteins on coiled-coil scaffolds removes membrane protein solubility issues, while retains activity, enabling a generic approach to readily-expressible, versatile, artificial membrane proteins for more accessible study and exploitation.


Assuntos
Biomineralização , Nanopartículas de Magnetita , Proteínas/síntese química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas/química , Proteínas/metabolismo
19.
Nat Commun ; 10(1): 2885, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253769

RESUMO

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.


Assuntos
Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Adenosina Trifosfatases , Animais , Linhagem Celular , Clonagem Molecular , Microscopia Crioeletrônica , DNA/química , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli , Regulação da Expressão Gênica , Humanos , Insetos , Modelos Químicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
20.
Zoolog Sci ; 36(3): 215-222, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251490

RESUMO

Anurans occupy a wide variety of habitats of diverse salinities, and their osmoregulatory ability is strongly regulated by hormones. In this study, we compared the adaptability and hormonal responses to osmotic stress between two kajika frogs, Buergeria japonica (B.j.) and B. buergeri, (B.b.), which inhabit coastal brackish waters (BW) in the Ryukyu Islands and freshwater (FW) in the Honshu, respectively. Both hematocrit and plasma Na+ concentration were significantly higher in B.j. than in B.b. when both were kept in FW. After transfer to one-third seawater (simulating the natural BW environment), which is slightly hypertonic to their body fluids, their body mass decreased and plasma Na concentration increased significantly in both species. After transfer, plasma Na+ concentration increased significantly in both species. We examined the gene expression of two major osmoregulatory hormones, arginine vasotocin (AVT) and atrial natriuretic peptide (ANP), after partial cloning of their cDNAs. ANP mRNA levels were more than 10-fold higher in B.j. than in B.b. in FW, but no significant difference was observed for AVT mRNA levels due to high variability, although the mean value of B.j. was twice that of B.b. Both AVT and ANP mRNA levels increased significantly after transfer to BW in B.b. but not in B.j., probably because of the high levels in FW. These results suggest that B.j. maintains high plasma Na+ concentration and anp gene expression to prepare for the future encounter of the high salinity. The unique preparatory mechanism may allow B.j. wide distribution in oceanic islands.


Assuntos
Anuros/fisiologia , Ecossistema , Águas Salinas/química , Tolerância ao Sal/fisiologia , Animais , Fator Natriurético Atrial/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica/efeitos dos fármacos , Japão , Masculino , Osmorregulação/fisiologia , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cloreto de Sódio/farmacologia , Vasotocina/metabolismo
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