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1.
PLoS One ; 15(5): e0232006, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407331

RESUMO

BACKGROUND: The utility of ex vivo Magnetic resonance imaging proton density fat fraction (MRI-PDFF) in donor liver fat quantification is unknown. PURPOSE: To evaluate the diagnostic accuracy and utility in predicting early allograft dysfunction (EAD) of ex vivo MRI-PDFF measurement of fat in deceased donor livers using histology as the gold standard. METHODS: We performed Ex vivo, 1.5 Tesla MRI-PDFF on 33 human deceased donor livers before implantation, enroute to the operating room. After the exclusion of 4 images (technical errors), 29 MRI images were evaluable. Histology was evaluable in 27 of 29 patients. EAD was defined as a peak value of aminotransferase >2000 IU/mL during the first week or an INR of ≥1.6 or bilirubin ≥10 mg/dL at day 7. RESULTS: MRI-PDFF values showed a strong positive correlation (Pearson's correlation coefficient) when histology (macro-steatosis) was included (r = 0.78, 95% confidence interval 0.57-0.89, p<0.0001). The correlation appeared much stronger when macro plus micro-steatosis were included (r = 0.87, 95% confidence interval 0.72-0.94, p<0.0001). EAD was noted in 7(25%) subjects. AUC (Area Under the Curve) for macro steatosis (histology) predicted EAD in 73% (95% CI: 48-99), micro plus macro steatosis in 76% (95% CI: 49-100). AUC for PDFF values predicted EAD in 67(35-98). Comparison of the ROC curves in a multivariate model revealed, adding MRI PDFF values to macro steatosis increased the ability of the model in predicting EAD (AUC: 79%, 95% CI: 59-99), and addition of macro plus micro steatosis based on histology predicted EAD even better (AUC: 90%: 79-100, P = 0.054). CONCLUSION: In this pilot study, MRI-PDFF imaging showed potential utility in quantifying hepatic steatosis ex-vivo donor liver evaluation and the ability to predict EAD related to severe allograft steatosis in the recipient.


Assuntos
Fígado/diagnóstico por imagem , Imagem por Ressonância Magnética , Hepatopatia Gordurosa não Alcoólica/patologia , Idoso , Área Sob a Curva , Bilirrubina/análise , Biomarcadores/metabolismo , Feminino , Humanos , Coeficiente Internacional Normatizado , Fígado/patologia , Transplante de Fígado , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Projetos Piloto , Curva ROC , Transaminases/metabolismo , Transplante Homólogo
2.
Proc Natl Acad Sci U S A ; 117(23): 12784-12790, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461365

RESUMO

Fruit development normally occurs after pollination and fertilization; however, in parthenocarpic plants, the ovary grows into the fruit without pollination and/or fertilization. Parthenocarpy has been recognized as a highly attractive agronomic trait because it could stabilize fruit yield under unfavorable environmental conditions. Although natural parthenocarpic varieties are useful for breeding Solanaceae plants, their use has been limited, and little is known about their molecular and biochemical mechanisms. Here, we report a parthenocarpic eggplant mutant, pad-1, which accumulates high levels of auxin in the ovaries. Map-based cloning showed that the wild-type (WT) Pad-1 gene encoded an aminotransferase with similarity to Arabidopsis VAS1 gene, which is involved in auxin homeostasis. Recombinant Pad-1 protein catalyzed the conversion of indole-3-pyruvic acid (IPyA) to tryptophan (Trp), which is a reverse reaction of auxin biosynthetic enzymes, tryptophan aminotransferases (TAA1/TARs). The RNA level of Pad-1 gene increased during ovary development and reached its highest level at anthesis stage in WT. This suggests that the role of Pad-1 in WT unpollinated ovary is to prevent overaccumulation of IAA resulting in precocious fruit-set. Furthermore, suppression of the orthologous genes of Pad-1 induced parthenocarpic fruit development in tomato and pepper plants. Our results demonstrated that the use of pad-1 genes would be powerful tools to improve fruit production of Solanaceae plants.


Assuntos
Ácidos Indolacéticos/metabolismo , Mutação com Perda de Função , Partenogênese , Proteínas de Plantas/genética , Solanum melongena/genética , Transaminases/genética , Flores/genética , Flores/metabolismo , Flores/fisiologia , Homeostase , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solanum melongena/fisiologia , Transaminases/metabolismo
3.
Food Chem ; 321: 126691, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32251922

RESUMO

Low-alcohol Huangjiu (LAH), which contains reduced contents of ethanol and higher alcohols, is prepared by diluting original Huangjiu that has a high ethanol content, which leads to a weakened flavor (i.e., acidity). To increase acidity and reduce higher alcohols level in LAH, the gene ALD6 encoding aldehyde dehydrogenase was expressed in yeast HJ-1 under the control of the pPGK1 promoter and terminators with varying activities (tGIC1, tPGK1 and tCPS1) by scarless replacement at BAT2 locus, yielding the engineered strains HJΔB-AG, HJΔB-AP, and HJΔB-AC. The acetate concentration produced by HJΔB-AG, HJΔB-AP, and HJΔB-AC was 1.26-, 1.84-, and 2.51-fold of that of HJ-1, respectively. Furthermore, the concentration of higher alcohols produced by HJΔB-AG, HJΔB-AP, and HJΔB-AC decreased by 39.91%, 45.55%, and 52.80%, respectively. This study resulted in the creation of promising recombinant yeast strains and introduced a method that can be used for the high-quality production of LAH by acid-producing Saccharomyces cerevisiae.


Assuntos
Etanol/metabolismo , Microrganismos Geneticamente Modificados , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia , Acetatos/metabolismo , Álcoois , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Fermentação , Microbiologia de Alimentos , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões Terminadoras Genéticas , Transaminases/genética , Transaminases/metabolismo
4.
BMC Complement Med Ther ; 20(1): 32, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024513

RESUMO

BACKGROUND: Yigan mingmu oral liquid (YGMM) is a herbal medicine based on a famous Chinese herbal formula that has been used for sore eyes for more than 400 years. Eye health is closely associated with the liver based on TCM. This study aimed to investigate the hepatoprotective effect of YGMM against acute liver injury induced by alcohol in rats. METHODS: Experimental rats were administered with silymarin and YGMM through the gastric gavage during the entire experiment. Starting from the 11th day, the rats were administered orally with 14 ml/kg Red Star Erguotou Liquor, a popular brand, at 4 h after the dose of silymarin (100 mg/kg) and YGMM (1, 2.5 and 5 ml/kg in low, middle and high dosage group, respectively) once a day for 4 weeks except for the rats in the normal group. Biochemical parameters, including ALT, AST, TB, TG, T-SOD, GSH, and MDA were detected to evaluate the protective effect of YGMM. Pathological changes were observed through histopathological examination. RESULTS: Treatment with YGMM exhibited a significant protective effect by reversing the biochemical parameters (ALT, AST, TB, TG, and GSH) and histopathological changes. Histopathological examination by Oil Red O Staining Solution showed that lipid droplets were significantly reduced in the silymarin and YGMM groups (p < 0.001) when compared to alcohol group. CONCLUSIONS: YGMM exhibits a significant hepatoprotective activity against acute liver injury induced by alcohol in rats.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Plantas Medicinais , Animais , Modelos Animais de Doenças , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Transaminases/metabolismo
5.
Oxid Med Cell Longev ; 2020: 6325378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32064027

RESUMO

In addition to the lung, the liver is considered another major target for paraquat (PQ) poisoning. Hydrogen sulfide (H2S) has been demonstrated to be effective in the inhibition of oxidative stress and inflammation. The aim of this study was to investigate the protective effect of exogenous H2S against PQ-induced acute liver injury. The acute liver injury model was established by a single intraperitoneal injection of PQ, evidenced by histological alteration and elevated serum aminotransferase levels. Different doses of NaHS were administered intraperitoneally one hour before exposure to PQ. Analysis of the data shows that exogenous H2S attenuated the PQ-induced liver injury and oxidative stress in a dose-dependent manner. H2S significantly suppressed reactive oxygen species (ROS) generation and the elevation of malondialdehyde content while it increased the ratio of GSH/GSSG and levels of antioxidant enzymes including SOD, GSH-Px, HO-1, and NQO-1. When hepatocytes were subjected to PQ-induced oxidative stress, H2S markedly enhanced nuclear translocation of Nrf2 via S-sulfhydration of Keap1 and resulted in the increase in IDH2 activity by regulating S-sulfhydration of SIRT3. In addition, H2S significantly suppressed NLRP3 inflammasome activation and subsequent IL-1ß excretion in PQ-induced acute liver injury. Moreover, H2S cannot reverse the decrease in SIRT3 and activation of the NLRP3 inflammasome caused by PQ in Nrf2-knockdown hepatocytes. In summary, H2S attenuated the PQ-induced acute liver injury by enhancing antioxidative capability, regulating mitochondrial function, and suppressing ROS-induced NLRP3 inflammasome activation. The antioxidative effect of H2S in PQ-induced liver injury can at least partly be attributed to the promotion of Nrf2-driven antioxidant enzymes via Keap1 S-sulfhydration and regulation of SIRT3/IDH2 signaling via Nrf2-dependent SIRT3 gene transcription as well as SIRT3 S-sulfhydration. Thus, H2S supplementation can form the basis for a promising novel therapeutic strategy for PQ-induced acute liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Inflamação/metabolismo , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Isocitrato Desidrogenase/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Mitocôndrias/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas/metabolismo , Sulfetos/administração & dosagem , Superóxido Dismutase-1/metabolismo , Transaminases/metabolismo
6.
Medicine (Baltimore) ; 99(3): e18775, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32011470

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) with abnormal transaminase were main targeted disorder in clinical intervention. Acupuncture embedding has been used as a modified acupuncture therapy in current management, while no comprehensive summarization has been established. Hence, we conducted a systematic review and meta-analysis to evaluate the effectiveness and safety of acupoint embedding alone or in combination for NAFLD with abnormal transaminase, and to provide potential regimen for further verification. METHODS: Seven English and Chinese databases were systematically researched from inception to February 28, 2019, including PubMed, Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Chinese biomedical literature database (SinoMed), Chinese National Knowledge Infrastructure (CNKI), Chinese VIP information (VIP), and WanFang database. Academic dissertations were also searched as supplement. The searching terms included "nonalcoholic fatty liver disease," "acupoint embedding," "clinical trial," with their corresponding synonyms. Randomized controlled trials (RCTs) and quasi-RCTs involving acupoint embedding alone or in combination for adult patients with NAFLD with abnormal transaminase were included. The diagnosis of NAFLD should be confirmed by radiologic evidence. Two researchers independently completed predefined data sets extraction and quality assessment. STATA 15.0 was applied to estimate the combined effect presented as odds ratio or mean difference (MD) with a 95% confidence interval (CI). The primary outcome was the change of serum alanine aminotransferase (ALT). RESULTS: A total of fifteen studies with 1349 patients were included. Meta-analysis reported that acupoint embedding alone or in combination was superior to conventional medications on ALT change (MD: 16.58, 95%CI: [10.42, 22.74], P < .001). The benefits were also demonstrated in other outcomes, including aspartate aminotransferase, triglyceride, and total cholesterol, total efficacy rate and radiological efficacy rate. The safety profile of acupoint embedding was satisfactory. BL18 (Ganshu) was the most frequently utilized acupoint. CONCLUSION: To some extent, the systematic review supported the application of acupoint embedding in management of NAFLD, while further high-quality studies should be designed to evaluate the practical effect of acupoint embedding.


Assuntos
Pontos de Acupuntura , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/terapia , Transaminases/metabolismo , Humanos
7.
Nat Chem Biol ; 16(4): 415-422, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042199

RESUMO

In biotin biosynthesis, the conversion of pimeloyl intermediates to biotin is catalyzed by a universal set of four enzymes: BioF, BioA, BioD and BioB. We found that the gene homologous to bioA, the product of which is involved in the conversion of 8-amino-7-oxononanoate (AON) to 7,8-diaminononanoate (DAN), is missing in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We provide structural and biochemical evidence showing that a novel dehydrogenase, BioU, is involved in biotin biosynthesis and functionally replaces BioA. This enzyme catalyzes three reactions: formation of covalent linkage with AON to yield a BioU-DAN conjugate at the ε-amino group of Lys124 of BioU using NAD(P)H, carboxylation of the conjugate to form BioU-DAN-carbamic acid, and release of DAN-carbamic acid using NAD(P)+. In this biosynthetic pathway, BioU is a suicide enzyme that loses the Lys124 amino group after a single round of reaction.


Assuntos
Biotina/biossíntese , Oxirredutases/ultraestrutura , Synechocystis/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Diamino Aminoácidos/química , Diamino Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Biotina/metabolismo , Catálise , Clonagem Molecular , Cianobactérias/genética , Cianobactérias/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos , Oxirredutases/metabolismo , Synechocystis/genética , Transaminases/metabolismo
8.
Nat Cell Biol ; 22(2): 167-174, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029896

RESUMO

Branched-chain amino acid (BCAA) metabolism is potentially linked with development of pancreatic ductal adenocarcinoma (PDAC)1-4. BCAA transaminase 2 (BCAT2) was essential for the collateral lethality conferred by deletion of malic enzymes in PDAC and the BCAA-BCAT metabolic pathway contributed to non-small-cell lung carcinomas (NSCLCs) other than PDAC3,4. However, the underlying mechanism remains undefined. Here we reveal that BCAT2 is elevated in mouse models and in human PDAC. Furthermore, pancreatic tissue-specific knockout of Bcat2 impedes progression of pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1-Cre (KC) mice. Functionally, BCAT2 enhances BCAA uptake to sustain BCAA catabolism and mitochondrial respiration. Notably, BCAA enhances growth of pancreatic ductal organoids from KC mice in a dose-dependent manner, whereas addition of branched-chain α-keto acid (BCKA) and nucleobases rescues growth of KC organoids that is suppressed by BCAT2 inhibitor. Moreover, KRAS stabilizes BCAT2, which is mediated by spleen tyrosine kinase (SYK) and E3 ligase tripartite-motif-containing protein 21 (TRIM21). In addition, BCAT2 inhibitor ameliorates PanIN formation in KC mice. Of note, a lower-BCAA diet also impedes PDAC development in mouse models of PDAC. Thus, BCAT2-mediated BCAA catabolism is critical for development of PDAC harbouring KRAS mutations. Targeting BCAT2 or lowering dietary BCAA may have translational significance.


Assuntos
Adenocarcinoma/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , Neoplasias Pancreáticas/genética , Proteínas da Gravidez/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transaminases/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Aminoácidos de Cadeia Ramificada/farmacologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cetoácidos/metabolismo , Cetoácidos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/metabolismo , Transaminases/metabolismo
10.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952110

RESUMO

Maresin-1 (MaR1) is a specialized pro-resolving mediator, derived from omega-3 fatty acids, whose functions are to decrease the pro-inflammatory and oxidative mediators, and also to stimulate cell division. We investigated the hepatoprotective actions of MaR1 in a rat model of liver ischemia-reperfusion (IR) injury. MaR1 (4 ng/gr body weight) was administered prior to ischemia (1 h) and reperfusion (3 h), and controls received isovolumetric vehicle solution. To analyze liver function, transaminases levels and tissue architecture were assayed, and serum cytokines TNF-α, IL-6, and IL-10, mitotic activity index, and differential levels of NF-κB and Nrf-2 transcription factors, were analyzed. Transaminase, TNF-α levels, and cytoarchitecture were normalized with the administration of MaR1 and associated with changes in NF-κB. IL-6, mitotic activity index, and nuclear translocation of Nrf-2 increased in the MaR1-IR group, which would be associated with hepatoprotection and cell proliferation. Taken together, these results suggest that MaR1 alleviated IR liver injury, facilitated by the activation of hepatocyte cell division, increased IL-6 cytokine levels, and the nuclear localization of Nrf-2, with a decrease of NF-κB activity. All of them were related to an improvement of liver injury parameters. These results open the possibility of MaR1 as a potential therapeutic tool in IR and other hepatic pathologies.


Assuntos
Proliferação de Células/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Ácidos Docosa-Hexaenoicos/química , Ácidos Graxos Ômega-3/química , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/irrigação sanguínea , Fígado/fisiopatologia , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/fisiopatologia , Transaminases/metabolismo
11.
BMC Plant Biol ; 20(1): 9, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906853

RESUMO

BACKGROUND: Zygophyllum is an important medicinal plant, with notable properties such as resistance to salt, alkali, and drought, as well as tolerance of poor soils and shifting sand. However, the response mechanism of Zygophyllum spp. to abiotic stess were rarely studied. RESULTS: Here, we aimed to explore the salt-tolerance genes of Zygophyllum plants by transcriptomic and metabolic approaches. We chose Z. brachypterum, Z. obliquum and Z. fabago to screen for salt tolerant and sensitive species. Cytological observation showed that both the stem and leaf of Z. brachypterum were significantly thicker than those of Z. fabago. Then, we treated these three species with different concentrations of NaCl, and found that Z. brachypterum exhibited the highest salt tolerance (ST), while Z. fabago was the most sensitive to salt (SS). With the increase of salt concentration, the CAT, SOD and POD activity, as well as proline and chlorophyll content in SS decreased significantly more than in ST. After salt treatment, the proportion of open stomata in ST decreased significantly more than in SS, although there was no significant difference in stomatal number between the two species. Transcriptomic analysis identified a total of 11 overlapping differentially expressed genes (DEGs) in the leaves and roots of the ST and SS species after salt stress. Two branched-chain-amino-acid aminotransferase (BCAT) genes among the 11 DEGs, which were significantly enriched in pantothenate and CoA biosynthesis, as well as the valine, leucine and isoleucine biosynthesis pathways, were confirmed to be significantly induced by salt stress through qRT-PCR. Furthermore, overlapping differentially abundant metabolites showed that the pantothenate and CoA biosynthesis pathways were significantly enriched after salt stress, which was consistent with the KEGG pathways enriched according to transcriptomics. CONCLUSIONS: In our study, transcriptomic and metabolomic analysis revealed that BCAT genes may affect the pantothenate and CoA biosynthesis pathway to regulate the salt tolerance of Zygophyllum species, which may constitute a newly identified signaling pathway through which plants respond to salt stress.


Assuntos
Coenzima A/metabolismo , Metaboloma/genética , Tolerância ao Sal/genética , Transcriptoma/genética , Zygophyllum , Coenzima A/genética , Perfilação da Expressão Gênica , Genes de Plantas , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Estômatos de Plantas/citologia , Estômatos de Plantas/ultraestrutura , Transdução de Sinais/genética , Transaminases/genética , Transaminases/metabolismo , Zygophyllum/anatomia & histologia , Zygophyllum/genética , Zygophyllum/metabolismo
12.
J Biosci Bioeng ; 129(1): 99-103, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31320263

RESUMO

Bioamination methods using microorganisms have attracted much attention because of the increasing demand for environmentally friendly bioprocesses. n-Butylamine production from glucose in Escherichia coli was demonstrated in this study, which has never been reported because of the absence of n-butylamine-producing pathway in nature. We focused on a transaminase-mediated cascade for bioamination from an alcohol or aldehyde. The cascade can convert an alcohol or an aldehyde to the corresponding amine with l-alanine as an amine donor. Here, n-butyraldehyde, which is a metabolic intermediate in the n-butanol producing pathway, is a potential intermediate for producing n-butylamine using this cascade. Hence, the n-butanol-producing pathway and the transaminase-mediated cascade were combined into a synthetic metabolic pathway for producing n-butylamine from glucose. Firstly, we demonstrated the conversion of n-butanol to n-butylamine using a three enzyme-mediated cascade. n-Butanol was successfully converted to n-butylamine in 92% yield in the presence of l-alanine and ammonium chloride. Then, the n-butanol-producing pathway and transaminase-mediated cascade were introduced into E. coli. Using this system, n-butylamine was successfully produced from glucose as a carbon source at a concentration of 53.2 mg L-1 after 96 h cultivation using a ppc (phosphoenolpyruvate carboxylase)-deficient strain. To the best of our knowledge, this is the first report of the direct production of n-butylamine from glucose, and may provide a starting point for the development of microbial methods to produce other bioamines.


Assuntos
Butilaminas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glucose/metabolismo , Transaminases/metabolismo , 1-Butanol/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Etanol/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas , Transaminases/genética
13.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140322, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31740415

RESUMO

Biocatalysis, the use of enzymes in chemical transformations, is an important green chemistry tool. Cascade reactions combine different enzyme activities in a sequential set of reactions. Cascades can occur within a living (usually bacterial) cell; in vitro in 'one pot' systems where the desired enzymes are mixed together to carry out the multi-enzyme reaction; or using microfluidic systems. Microfluidics offers particular advantages when the product of the reaction inhibits the enzyme(s). In vitro systems allow variation of different enzyme concentrations to optimise the metabolic 'flux', and the addition of enzyme cofactors as required. Cascades including cofactor recycling systems and modelling approaches are being developed to optimise cascades for wider industrial scale use. Two industrially important enzymes, transaminases and carboxylic acid reductases are used as examples regarding their applications in cascade reactions with other enzyme classes to obtain important synthons of pharmaceutical interest.


Assuntos
Oxirredutases/metabolismo , Transaminases/metabolismo , Biocatálise , Coenzimas/metabolismo , Química Verde , Cinética , Microfluídica/métodos , Oxirredutases/antagonistas & inibidores , Transaminases/antagonistas & inibidores
14.
Appl Biochem Biotechnol ; 190(3): 880-895, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31515673

RESUMO

Recombinant proteins were often expressed with His-tag to simplify the purification process. Among them, transaminase was mostly expressed with fusion tags and widely used in the production of numerous amino moieties. However, the existence of the His-tag has been reported to affect various properties of different recombinant enzymes, while the effect on transaminase was rarely studied. In this paper, we investigated the effect of His-tag on transaminase based on the various activities of 4-aminobutyrate-2-oxoglutarate transaminase (GabT) when it was expressed in vector pETDuet-1. We found that His-tag did not affect the enantioselectivity, but decreased the catalytic activity to different extents according to its existence and location. Native GabT maintained the highest catalytic activity; GabT with C-terminal His-tag showed slightly lower activity than native GabT but about 2.2-fold higher than GabT with N-terminal His-tag. Besides, other fusion tags like T7-tag and S-tag inserted between N-His-tag and GabT can relieve the decreasing effect of His-tag on GabT activity. Furthermore, whole cell catalytic activity of several transaminases was improved by deleting the N-terminal His-tag. This study provided a strategy for the efficient expression of recombinant transaminase with improved catalytic activity and might attract attention to the effect of His-tag on other enzymatic properties.


Assuntos
Histidina/química , Transaminases/metabolismo , Catálise , Enteropeptidase/metabolismo , Plasmídeos , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Transaminases/química
15.
Proc Natl Acad Sci U S A ; 117(2): 1174-1180, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882449

RESUMO

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.


Assuntos
Alcaloides/biossíntese , Ascomicetos/enzimologia , Ascomicetos/metabolismo , Indolizidinas/metabolismo , Policetídeo Sintases/metabolismo , Fosfato de Piridoxal/metabolismo , Alcaloides/genética , Alcaloides/isolamento & purificação , Antibacterianos/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Catálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Hidroxilação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Família Multigênica , Filogenia , Policetídeo Sintases/classificação , Policetídeo Sintases/genética , Policetídeos , Fosfato de Piridoxal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Nat Commun ; 10(1): 5634, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822677

RESUMO

The blood-feeding behavior of Anopheles females delivers essential nutrients for egg development and drives parasite transmission between humans. Plasmodium growth is adapted to the vector reproductive cycle, but how changes in the reproductive cycle impact parasite development remains unclear. Here, we show that the bloodmeal-induced miR-276-5p fine-tunes the expression of branched-chain amino acid transferase to terminate the reproductive cycle. Silencing of miR-276 prolongs high rates of amino acid (AA) catabolism and increases female fertility, suggesting that timely termination of AA catabolism restricts mosquito investment into reproduction. Prolongation of AA catabolism in P. falciparum-infected females also compromises the development of the transmissible sporozoite forms. Our results suggest that Plasmodium sporogony exploits the surplus mosquito resources available after reproductive investment and demonstrate the crucial role of the mosquito AA metabolism in within-vector parasite proliferation and malaria transmission.


Assuntos
Anopheles/fisiologia , MicroRNAs/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Aminoácidos/metabolismo , Animais , Anopheles/efeitos dos fármacos , Sequência de Bases , Ecdisona/farmacologia , Corpo Adiposo/metabolismo , Feminino , Inativação Gênica , MicroRNAs/genética , Modelos Biológicos , Reprodução/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esteroides/metabolismo , Transaminases/metabolismo
17.
Anticancer Res ; 39(12): 6723-6730, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810937

RESUMO

BACKGROUND/AIM: Phosphoserine aminotransferase 1 (PSAT1) is an enzyme implicated in serine biosynthesis, and its overexpression has been linked to cancer cell proliferation. Therefore, targeting PSAT1 is considered to be an anticancer strategy. MATERIALS AND METHODS: The viability of non-small cell lung cancer (NSCLC) cells was measured by MTT assay. Protein and mRNA expression were determined by western blot and reverse transcription polymerase chain reaction, respectively. RESULTS: Glutamine-limiting conditions were generated through glutamine deprivation or CB-839 treatment, which induced PSAT1 expression in NSCLC cells. PSAT1 expression induced by glutamine-limiting conditions was regulated by activating transcription factor 4. Knock-down of PSAT1 enhanced the sensitivity of NSCLC cells to glutamine-limiting conditions. Interestingly, ionizing radiation induced PSAT1 expression, and knocking down PSAT1 increased cell sensitivity to ionizing radiation. CONCLUSION: Inhibiting PSAT1 might aid in the treatment of lung cancer, and PSAT1 may be a therapeutic target for lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Glutamina/metabolismo , Neoplasias Pulmonares/metabolismo , Transaminases/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Benzenoacetamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Introdução de Genes , Glutaminase/antagonistas & inibidores , Glutamina/antagonistas & inibidores , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , RNA Mensageiro/metabolismo , Tolerância a Radiação , Tiadiazóis/farmacologia , Transaminases/genética
18.
Chem Commun (Camb) ; 55(100): 15133-15136, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31789331

RESUMO

Herein we report the development of an efficient cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins by the residue-specific approach. This system makes use of common phenol derivatives and the tyrosine phenol lyase machinery to create various tyrosine analogues that impart desired properties on the target proteins. Biosynthesized 2-fluorotyrosine was incorporated into three industrially important enzymes which resulted in enhanced thermostability.


Assuntos
Engenharia de Proteínas , Tirosina Fenol-Liase/metabolismo , Tirosina/biossíntese , Biocatálise , Fluorometria , Oxirredutases/genética , Oxirredutases/metabolismo , Transaminases/genética , Transaminases/metabolismo , Tirosina/análogos & derivados , Tirosina Fenol-Liase/genética
19.
Biomolecules ; 10(1)2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31861280

RESUMO

Abstract: Many tumors readily convert l-glutamine to α-ketoglutarate. This conversion is almost invariably described as involving deamidation of l-glutamine to l-glutamate followed by a transaminase (or dehydrogenase) reaction. However, mammalian tissues possess another pathway for conversion of l-glutamine to α-ketoglutarate, namely the glutaminase II pathway: l-Glutamine is transaminated to α-ketoglutaramate, which is then deamidated to α-ketoglutarate by ω-amidase. Here we show that glutamine transaminase and ω-amidase specific activities are high in normal rat prostate. Immunohistochemical analyses revealed that glutamine transaminase K (GTK) and ω-amidase are present in normal and cancerous human prostate and that expression of these enzymes increases in parallel with aggressiveness of the cancer cells. Our findings suggest that the glutaminase II pathway is important in providing anaplerotic carbon to the tricarboxylic acid (TCA) cycle, closing the methionine salvage pathway, and in the provision of citrate carbon in normal and cancerous prostate. Finally, our data also suggest that selective inhibitors of GTK and/or ω-amidase may be clinically important for treatment of prostate cancer. In conclusion, the demonstration of a prominent glutaminase II pathway in prostate cancer cells and increased expression of the pathway with increasing aggressiveness of tumor cells provides a new perspective on 'glutamine addiction' in cancers.


Assuntos
Amidoidrolases/metabolismo , Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Liases/metabolismo , Próstata/enzimologia , Neoplasias da Próstata/enzimologia , Transaminases/metabolismo , Animais , Glutamina/análise , Humanos , Masculino , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Ratos , Ratos Sprague-Dawley
20.
Int J Mol Sci ; 21(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861687

RESUMO

Iron (Fe) is an essential element required for plant growth and development. Under Fe-deficientconditions, plants have developed two distinct strategies (designated as strategy I and II) to acquire Fe from soil. As a graminaceous species, rice is not a typical strategy II plant, as it not only synthesizes DMA (2'-deoxymugineic acid) in roots to chelate Fe3+ but also acquires Fe2+ through transporters OsIRT1 and OsIRT2. During the synthesis of DMA in rice, there are three sequential enzymatic reactions catalyzed by enzymes NAS (nicotianamine synthase), NAAT (nicotianamine aminotransferase), and DMAS (deoxymugineic acid synthase). Many transporters required for Fe uptake from the rhizosphere and internal translocation have also been identified in rice. In addition, the signaling networks composed of various transcription factors (such as IDEF1, IDEF2, and members of the bHLH (basic helix-loop-helix) family), phytohormones, and signaling molecules are demonstrated to regulate Fe uptake and translocation. This knowledge greatly contributes to our understanding of the molecular mechanisms underlying iron deficiency responses in rice.


Assuntos
Ferro/metabolismo , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/metabolismo , Ácido Azetidinocarboxílico/análogos & derivados , Ácido Azetidinocarboxílico/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/metabolismo , Estresse Fisiológico , Transaminases/metabolismo
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