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1.
Am J Clin Nutr ; 111(3): 613-621, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31858113

RESUMO

BACKGROUND: Preconception nutrition sets the stage for a healthy pregnancy. Maternal fatty acids (FAs) are related to beneficial neonatal outcomes with DNA methylation proposed as a mechanism; however, few studies have investigated this association and none with preconception FAs. OBJECTIVES: We examined the relations of maternal plasma FA concentrations at preconception (n = 346) and 8 weeks of gestation (n = 374) with newborn DNA methylation. METHODS: The Effects of Aspirin in Gestation and Reproduction Trial (2006-2012) randomly assigned women with previous pregnancy loss to low dose aspirin or placebo prior to conception. We measured maternal plasma phospholipid FA concentration at preconception (on average 4 mo before pregnancy) and 8 weeks of gestation. Cord blood DNA from singletons was measured using the MethylationEPIC BeadChip. We used robust linear regression to test the associations of FA concentration with methylation ß-values of each CpG site, adjusting for estimated cell count using a cord blood reference, sample plate, maternal sociodemographic characteristics, cholesterol, infant sex, and epigenetic-derived ancestry. False discovery rate correction was used for multiple testing. RESULTS: Mean ± SD concentrations of preconception marine (20:5n-3+22:6n-3+22:5n-3) and ω-6 PUFAs, SFAs, MUFAs, and trans FAs were 4.7 ± 1.2, 38.0 ± 2.0, 39.4 ± 1.8, 11.6 ± 1.1, and 1.0 ± 0.4 % of total FA, respectively; concentrations at 8 weeks of gestation were similar. Preconception marine PUFA concentration was associated with higher methylation at GRAMD2 (P = 1.1 × 10-8), LOXL1 (P = 5.5 × 10-8), SIK3 (P = 1.6 × 10-7), HTR1B (P = 1.9 × 10-7), and MCC (P = 2.1 × 10-7) genes. Preconception SFA concentration was associated with higher methylation at KIF25-AS1 and lower methylation at SLC39A14; other associations exhibited sensitivity to outliers. The trans FA concentration was related to lower methylation at 3 sites and higher methylation at 1 site. FAs at 8 weeks of gestation were largely unrelated to DNA methylation. CONCLUSIONS: Maternal preconception FAs are related to newborn DNA methylation of specific CpG sites, highlighting the importance of examining nutritional exposures preconceptionally. This trial was registered at clinicaltrials.gov as NCT00467363.


Assuntos
Metilação de DNA , Ácidos Graxos/sangue , Gravidez/genética , Adolescente , Adulto , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Gravidez/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Receptor 5-HT1B de Serotonina/genética , Receptor 5-HT1B de Serotonina/metabolismo , Adulto Jovem
2.
J Agric Food Chem ; 67(45): 12502-12510, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31623431

RESUMO

Succinic acid (SA) is applied in the food, chemical, and pharmaceutical industries. 5-Hydroxyleucine (5-HLeu) is a promising precursor for the biosynthesis of antituberculosis drugs. Here, we designed a promising synthetic route for the simultaneous production of SA and 5-HLeu by combining l-leucine dioxygenase (NpLDO), l-glutamate oxidase (LGOX), and catalase (CAT). Two bioconversion systems: "a multienzyme cascade catalysis in vitro" (MECCS) and "whole-cell catalysis system" (WCCS) were constructed. A high-activity NpLDO mutant was screened by error-prone polymerase chain reaction (PCR) and showed 6.1-fold improvement of catalytic activity. After optimization of reaction conditions, MECSS yielded 3.15 g/L SA and 3.92 g/L 5-HLeu, while the production of SA and 5-HLeu by the most effective WCSS reached 15.12 and 18.83 g/L, respectively. This is the first attempt to use ferrous iron/α-ketoglutarate-dependent dioxygenases for the simultaneous production of SA and hydroxy-amino-acid. This research provides a tool for industrial production of food of high-value products from low-cost raw materials.


Assuntos
Aminoácido Oxirredutases/química , Proteínas de Bactérias/química , Catalase/química , Dioxigenases/química , Leucina/química , Nostoc/metabolismo , Ácido Succínico/química , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Catalase/genética , Catalase/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Leucina/metabolismo , Nostoc/enzimologia , Nostoc/genética , Ácido Succínico/metabolismo
3.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569601

RESUMO

BACKGROUND: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. METHODS: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1ß in the cartilage cell line ATDC5. Finally; the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. RESULTS: The adenovirus treatment upregulated LOXL2; Sox9; Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1ß-induced phospho-NF-κB/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-κB independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes; including the time and distance traveled on the treadmill and allodynia. CONCLUSION: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.


Assuntos
Envelhecimento/genética , Aminoácido Oxirredutases/genética , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/patologia , Adenoviridae/genética , Aminoácido Oxirredutases/metabolismo , Animais , Artrite Experimental , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Osteoartrite do Joelho/metabolismo , Transdução Genética
4.
Chem Biodivers ; 16(12): e1900467, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31556199

RESUMO

Pulmonary fibrosis (PF) is a chronic obstructive pulmonary disease without effective clinical drug treatment. Qing-Xuan Granule (QX) as a traditional Chinese patent medicine is clinically used to cure children's cough. This study was designed to investigate the effects of QX and possible molecular mechanisms for bleomycin-induced PF. The work used Western blotting and Q-PCR to explore the vitro and vivo mechanisms of QX treatment, while using HPLC-TOF/MS to explore the composition of QX. QX was given daily orally for two weeks after bleomycin intratracheal instillation. The protective effects of QX on lung function, inflammation, growth factors, hydroxyproline content and deposition of extracellular matrix were investigated. QX decreased expression of Col I and α-SMA in lung tissues by down-regulating TGF-ß1-Smad2/3 signaling and suppressed epithelial-mesenchymal transition and effectively reversed abnormal mRNA levels of MMP-1and TIMP-1 as well as LOXL-2 in lung tissues. HPLC-TOF/MS indicate that six substances could be the main active components, which were reported to protect against experimental lung disease.


Assuntos
Substâncias Protetoras/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Bleomicina/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Medicina Tradicional Chinesa , Camundongos , Camundongos Endogâmicos C57BL , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Fibrose Pulmonar/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
5.
Appl Microbiol Biotechnol ; 103(21-22): 8799-8812, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31522285

RESUMO

Bacitracin is a kind of macrocyclic dodecapeptide that produced by Bacillus, precursor supply served as a critical role in bacitracin production, here, the aim of this study wants to improve bacitracin production by enhancing Lysine (Lys) supply via metabolic engineering of B. licheniformis DW2, an industrial strain for bacitracin production. Firstly, exogenous addition of Lys was proven to be favorable for bacitracin production, and the strain LYS2 was attained through strengthening Lys synthetic pathways via overexpressing diaminopimelate decarboxylase LysA from B. licheniformis and diaminopimelate dehydrogenase DdH from Corynebacterium glutamicum, and the bacitracin produced by LYS2 was increased to 838.53 U/mL by 10.85%, compared with that of DW2 (756.45 U/mL). Secondly, oxaloacetate, the precursor of Lys, was accumulated by overexpressing pyruvate carboxylase PycA in LYS2, and 17.06% increase of bacitracin yield was attained in LYS3 (885.53 U/mL), compared with DW2. Thirdly, lysine decarboxylase gene yaaO was deleted to weaken Lys degradation, and the attained strain LYS4 showed further increased bacitracin production from 885.53 to 923.43 U/mL. Lastly, the transporter LysE was confirmed to act as a Lys exporter; LysP and YvsH were identified as the Lys importers in B. licheniformis DW2, and bacitracin yield was increased to 975.43 U/mL by 28.95% in final strain LYS5 via engineering the Lys transporters. Taken together, this study implied that metabolic engineering of Lys supply modules is an efficient strategy for enhancement production of bacitracin, and provided a promising strain of B. licheniformis for industrial production of bacitracin.


Assuntos
Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Bacitracina/biossíntese , Lisina/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Bacillus licheniformis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Corynebacterium glutamicum/enzimologia , Engenharia Metabólica , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo
6.
Cell Prolif ; 52(6): e12687, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31468594

RESUMO

OBJECTIVES: As one of the most life-threatening malignancies, gastric cancer is the third contributor of cancer mortalities globally. Increasing studies have proven the regulatory roles of lncRNAs in the development of diverse malignant tumours. But little is known about its function and molecular mechanism in gastric carcinoma. MATERIALS AND METHODS: RT-qPCR was performed to measure the expression pattern of LOXL1-AS1 in gastric cancer. To ascertain its definite role, CCK-8, EdU, Western blot, transwell and sphere formation assays were adopted. RNA pull-down, RIP, ChIP and luciferase reporter assays were carried out to investigate the molecular mechanism of LOXL1-AS1 in gastric carcinoma. RESULTS: LOXL1-AS1 was highly expressed in tissues and cells of gastric cancer. The upregulation of LOXL1-AS1 predicted poor prognosis in gastric carcinoma. Our findings demonstrated that LOXL1-AS1 accelerated the deterioration of gastric cancer by inducing cell proliferation, migration, EMT and stemness. Moreover, the expression of USF1 in gastric cancer was higher than in normal control and LOXL1-AS1 negatively modulated USF1. Functionally, LOXL1-AS1 acted as a ceRNA to upregulate USF1 via sponging miR-708-5p. Besides, we confirmed USF1 promoted the transcription of stemness marker SOX2. Rescue experiments testified the stimulative role of LOXL1-AS1/miR-708-5p/USF1 pathway in gastric cancer progression. It was also validated that LOXL1-AS1 facilitated cell growth of gastric carcinoma in vivo. CONCLUSIONS: Our study unravelled that LOXL1-AS1/miR-708-5p/USF1 pathway contributed to the development of gastric cancer.


Assuntos
Aminoácido Oxirredutases/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Fatores Estimuladores Upstream/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos
7.
Int J Oncol ; 55(3): 657-670, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322171

RESUMO

Cholangiocarcinoma (CCA) is the second most common hepatobiliary cancer after hepatocellular carcinoma. Antiangiogenic therapy has been administered to patients with CCA, but the benefits of this therapy remain unsatisfactory. Improved understanding of the molecular mechanisms underlying angiogenesis in CCA is required. In the present study, the expression of GATA­binding protein 6 (GATA6), lysyl oxidase­like 2 (LOXL2) and vascular endothelial growth factor A (VEGFA), in addition to the microvessel density (MVD), were evaluated by performing immunohistochemical staining of human CCA microarrays. The expression of GATA6/LOXL2 was associated with poor overall survival (P=0.01) and disease­free survival (P=0.02), and was positively associated with VEGFA expression (P=0.02) and MVD (P=0.04). In vitro, western blotting, reverse transcription­quantitative PCR analysis and ELISAs revealed that altered GATA6 and LOXL2 expression regulated the expression levels of secreted VEGFA. Co­immunoprecipitation demonstrated a physical interaction between GATA6 and LOXL2 in CCA cell lines, and the scavenger receptor cysteine­rich domain of LOXL2 interacted with GATA6, which regulated VEGFA mRNA expression and protein secretion, and promoted tube formation. In vivo analyses further revealed that GATA6/LOXL2 promoted VEGFA expression, angiogenesis and tumor growth. The GATA6/LOXL2 complex represents a novel candidate prognostic marker for stratifying patients with CCA. Drugs targeting this complex may possess great therapeutic value in the treatment of CCA.


Assuntos
Aminoácido Oxirredutases/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Fator de Transcrição GATA6/metabolismo , Microvasos/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Aminoácido Oxirredutases/genética , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Feminino , Fator de Transcrição GATA6/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Microvasos/metabolismo , Pessoa de Meia-Idade , Neoplasias Experimentais , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340433

RESUMO

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.


Assuntos
Aminoácido Oxirredutases/genética , Artrite/genética , Fissura Palatina/genética , Doenças do Tecido Conjuntivo/genética , Matriz Extracelular/genética , Perda Auditiva Neurossensorial/genética , Miopia/genética , Neoplasias/genética , Descolamento Retiniano/genética , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Artrite/enzimologia , Artrite/patologia , Fissura Palatina/enzimologia , Fissura Palatina/patologia , Colágeno/química , Colágeno/genética , Colágeno/metabolismo , Doenças do Tecido Conjuntivo/enzimologia , Doenças do Tecido Conjuntivo/patologia , Elastina/química , Elastina/genética , Elastina/metabolismo , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/química , Matriz Extracelular/enzimologia , Regulação da Expressão Gênica , Perda Auditiva Neurossensorial/enzimologia , Perda Auditiva Neurossensorial/patologia , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Miopia/enzimologia , Miopia/patologia , Neoplasias/enzimologia , Neoplasias/patologia , Especificidade de Órgãos , Descolamento Retiniano/enzimologia , Descolamento Retiniano/patologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo
9.
Mol Biotechnol ; 61(9): 650-662, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31201604

RESUMO

1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.


Assuntos
Aminoácido Oxirredutases/química , Aminoácidos Cíclicos/química , Cisteína/química , Etilenos/química , Lycopersicon esculentum/enzimologia , Óxidos de Nitrogênio/química , Proteínas de Plantas/química , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Substituição de Aminoácidos , Aminoácidos Cíclicos/metabolismo , Sítios de Ligação , Clonagem Molecular , Cisteína/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Etilenos/biossíntese , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Lycopersicon esculentum/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Óxidos de Nitrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Marcadores de Spin , Especificidade por Substrato
10.
Biochemistry ; 58(17): 2243-2249, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30945853

RESUMO

GoxA is a cysteine tryptophylquinone (CTQ)-dependent glycine oxidase that is a member of a family of LodA-like proteins. The electrochemical midpoint potential ( Em) values for the quinone/semiquinone couple and the semiquinone/quinol couple were determined to be 111 and 21, respectively. The Em value for the overall two-electron quinone/quinol couple was similar to those of CTQ- and tryptophan tryptophylquinone (TTQ)-bearing dehydrogenases. However, for the well-studied TTQ-dependent methylamine dehydrogenase, the quinone/semiquinone couple is more negative than the semiquinone/quinol couple, the opposite of what was determined for GoxA. The change in Em value for the two-electron quinone/quinol couple of CTQ in GoxA with pH indicates that the overall two-electron transfer process is associated with the transfer of one proton. Thus, the quinol is anionic. The data reported herein further suggest that in GoxA the CTQ semiquinone is neutral, in contrast to the TTQ-dependent dehydrogenases, in which it is an anionic TTQ semiquinone. These results are discussed in the context of the structure and function of this glycine oxidase, compared to that of the tryptophylquinone-dependent dehydrogenases.


Assuntos
Aminoácido Oxirredutases/química , Proteínas de Bactérias/química , Dipeptídeos/química , Indolquinonas/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Triptofano/análogos & derivados , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzoquinonas/química , Benzoquinonas/metabolismo , Dipeptídeos/metabolismo , Concentração de Íons de Hidrogênio , Hidroquinonas/química , Hidroquinonas/metabolismo , Indolquinonas/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Domínios Proteicos , Pseudoalteromonas/enzimologia , Pseudoalteromonas/genética , Pseudoalteromonas/metabolismo , Triptofano/química , Triptofano/metabolismo
11.
Int J Biol Macromol ; 133: 980-986, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31029626

RESUMO

Hemoglobin A1c (HbA1c) is a hemoglobin molecule in which the N-terminal valine residue of the ß subunit has been grafted with the glucose in blood. Its detection has important implications for the diagnosis of diabetes. Enzymatic colorimetric method using fructosyl peptide oxidase (FPO) is simple and rapid for HbA1c detection. A FPO mutant with enhanced activity was constructed and produced by E. coli; however, most of expressed mutant FPO was insoluble. Significantly enhanced expression solubility was achieved when cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of FPO mutant. Via the high affinity interaction between CBD and cellulose, the CBD fusion also facilitated the simultaneous purification and immobilization of FPO directly from E. coli cells lysate using bacterial cellulose (BC) nanofibrils as a matrix of very high specific area. A never-dried and water durable nanocellulose film with FPO activity could be easily obtained by collecting the FPO immobilized BC nanofibrils suspension on the surface of a microfiltration membrane. The activity of the ready-use FPO nanocellulose film was stable at least 7 days at room temperature for the detection of HbA1c level of 5.3-11% in blood samples.


Assuntos
Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/isolamento & purificação , Clostridium thermocellum/genética , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Técnicas Biossensoriais , Celulose/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Expressão Gênica , Hemoglobina A Glicada/análise , Humanos , Mutação , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
12.
Planta ; 249(6): 1903-1919, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30877435

RESUMO

MAIN CONCLUSION: Inoculation of endophytic Methylobacterium oryzae CBMB20 in salt-stressed rice plants improves photosynthesis and reduces stress volatile emissions due to mellowing of ethylene-dependent responses and activating vacuolar H+-ATPase. The objective of this study was to analyze the impact of ACC (1-aminocyclopropane-1-carboxylate) deaminase-producing Methylobacterium oryzae CBMB20 in acclimation of plant to salt stress by controlling photosynthetic characteristics and volatile emission in salt-sensitive (IR29) and moderately salt-resistant (FL478) rice (Oryza sativa L.) cultivars. Saline levels of 50 mM and 100 mM NaCl with and without bacteria inoculation were applied, and the temporal changes in stress response and salinity resistance were assessed by monitoring photosynthetic characteristics, ACC accumulation, ACC oxidase activity (ACO), vacuolar H+ ATPase activity, and volatile organic compound (VOC) emissions. Salt stress considerably reduced photosynthetic rate, stomatal conductance, PSII efficiency and vacuolar H+ ATPase activity, but it increased ACC accumulation, ACO activity, green leaf volatiles, mono- and sesquiterpenes, and other stress volatiles. These responses were enhanced with increasing salt stress and time. However, rice cultivars treated with CBMB20 showed improved plant vacuolar H+ ATPase activity, photosynthetic characteristics and decreased ACC accumulation, ACO activity and VOC emission. The bacteria-dependent changes were greater in the IR29 cultivar. These results indicate that decreasing photosynthesis and vacuolar H+ ATPase activity rates and increasing VOC emission rates in response to high-salinity stress were effectively mitigated by M. oryzae CBMB20 inoculation.


Assuntos
Etilenos/metabolismo , Methylobacterium/fisiologia , Oryza/microbiologia , Fotossíntese , Reguladores de Crescimento de Planta/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Carbono-Carbono Liases/genética , Carbono-Carbono Liases/metabolismo , Endófitos , Genótipo , Oryza/enzimologia , Oryza/genética , Oryza/fisiologia , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Salinidade , Estresse Salino , Estresse Fisiológico , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
13.
Georgian Med News ; (286): 32-36, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30829585

RESUMO

The aim of this study was to identify susceptibility variants of LOXL1 gene for Exfoliation Syndrome and Exfoliation Glaucoma by a case-control association study approach in Georgian population. Self-reported Georgian subjects were recruited between 2015 and 2017 at a specialized ophthalmic center. Patients underwent detailed ophthalmic examination to diagnose or exclude Exfoliation Syndrome (XFS) and Exfoliation Glaucoma (XFG). Patients underwent peripheral blood sampling. Genome-Wide Association Study (GWAS) was performed using Illumina OmniExpress Microarray. One hundred and thirty-two XFS patients and 195 healthy subjects were included into the study. Four LOXL1 variants were identified: rs2165241, rs3825942 (G153A), rs4886776 (R141L) and rs8042039 (G153D). Allele A of rs2165241 and allele G of rs3825942 are likely the main risk factors of disease development in Georgians with p=0.0001; OR= 5.8; 95% CI: 1.9986-16.9372 and p=0.002; OR=4.6; 95% CI: 1.7531-12.3146, respectively, both present in 96% of affected patients. The above-mentioned alleles are also encountered in more than 80% of healthy individuals. Two other SNPs have been described for the first time in exfoliation patients, though they appear to have no effect on the disease development in Georgian population. Two high-risk alleles of LOXL1 gene have been identified in Georgian population. As these SNPs are also very prevalent in healthy subjects, further studies are needed to identify the genetic mechanisms of exfoliation syndrome and exfoliation glaucoma.


Assuntos
Aminoácido Oxirredutases , Síndrome de Exfoliação , Predisposição Genética para Doença , Glaucoma de Ângulo Aberto , Polimorfismo de Nucleotídeo Único , Alelos , Aminoácido Oxirredutases/genética , Estudos de Casos e Controles , Síndrome de Exfoliação/genética , Estudo de Associação Genômica Ampla , Glaucoma de Ângulo Aberto/genética , Humanos
14.
Amino Acids ; 51(5): 813-828, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30900087

RESUMO

Lysyl oxidase-like 4 (LOXL4), a member of the LOX family proteins, catalyzes oxidative deamination of lysine residues in collagen and elastin, which are responsible for maintaining extracellular matrix homeostasis. In this study, the mRNA expression of LOXL4 in seven esophageal squamous cell carcinoma (ESCC) cell lines and 15 ESCC pairs of clinical samples were examined. Furthermore, LOXL4 protein levels in the ESCC cell lines were determined using western blotting. With the use of immunofluorescence, LOXL4 was observed to be localized primarily in the cytoplasm, but was also present in the nucleus. In addition, the results indicated that the upregulated expression of LOXL4 was associated with poor survival in patients with ESCC even following curative resection (P = 0.010). Similar Kaplan-Meier estimator curves for proteins that interact with LOXL4, SUV39H1 (P = 0.014) and COL2A1 (P = 0.011), were plotted. The analyses based on the protein-protein interaction network depicted the expression of LOXL4 and its associated proteins as well as their functions, suggesting that LOXL4 and its associated proteins may serve a significant role in the development and progression of ESCC. In conclusion, the results of the present study suggest that LOXL4 is a potential biomarker for patients with ESCC, as well as SUV39H1 and COL2A1, and high expression levels of these genes are associated with poor prognosis in patients with ESCC.


Assuntos
Aminoácido Oxirredutases/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Aminoácido Oxirredutases/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Mapas de Interação de Proteínas , Taxa de Sobrevida , Células Tumorais Cultivadas
15.
Neoplasia ; 21(4): 413-427, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30925417

RESUMO

Tumor lymphangiogenesis has been previously documented to predict regional lymph node metastasis and promote the spread to distant organs. However, the underlying mechanism initiating tumor lymphangiogenesis remains unclear. Here we described a novel role of tumor cell-derived Lysyl Oxidase-like protein 2 (LOXL2) in promoting lymphangiogenesis and lymph node metastasis in breast cancer. Immunohistochemistry (IHC) analysis of samples from breast cancer patients showed that the expression of LOXL2 was positively correlated with lymphatic vessel density and breast cancer malignancy. In animal studies, LOXL2-overexpressing breast cancer cells significantly increased lymphangiogenesis and lymph node metastasis, whereas knockdown of LOXL2 suppressed both processes. In order to study the mechanisms of lymphangiogenesis progression, we performed further in vitro investigations and the data revealed that LOXL2 significantly enhanced lymphatic endothelial cells (LECs) invasion and tube formation through directly activation of the Akt-Snail and Erk pathways. Moreover, LOXL2 also stimulated fibroblasts to secrete high level of pro- lymphangiogenic factors VEGF-C and SDF-1α. Taken together, our study elucidates a novel function of tumor cell secreted LOXL2 in lymphangiogenesis and lymph node metastasis, demonstrating that LOXL2 serves as a promising target for anti-lymphangiogenesis and anti-metastasis therapies for breast cancer.


Assuntos
Aminoácido Oxirredutases/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linfangiogênese/genética , Neovascularização Patológica/genética , Aminoácido Oxirredutases/metabolismo , Animais , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Camundongos , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo
16.
Int J Oncol ; 54(5): 1676-1690, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30816490

RESUMO

The aim of the present study was to investigate the effects of lysyl oxidase­like 2 (LOXL2) on the invasion, migration and epithelial­to­mesenchymal transition (EMT) of renal cell carcinoma (RCC) cells through the steroid receptor coactivator (Src)/focal adhesion kinase (FAK) signaling pathway. RCC tissues and adjacent normal tissues were collected from 80 patients with RCC. Immunohistochemistry was used to determine the positive expression rate of the LOXL2 protein. The expression levels of LOXL2 in the HK­2, 786­O, ACHN, Caki1 and A498 cell lines were detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The high LOXL2­expressing 786­O cells were selected for gene silencing experiments, whereas Caki1 cells, which exhibited low LOXL2 expression, were used for overexpression experiments. RT­qPCR and western blot analysis were applied to determine the expression of LOXL2, FAK, Src, matrix metalloproteinase (MMP)­9, epithelial (E)­cadherin, neuronal (N)­cadherin and vimentin. A MTT assay, a Transwell assay, a wound healing assay and flow cytometry were performed to detect cell proliferation, invasion, migration, cell cycle distribution and apoptosis, respectively. The protein expression rate of LOXL2 in RCC tissues was higher compared with that in adjacent normal tissues. Compared with adjacent normal tissues, the mRNA and protein expression levels of LOXL2, FAK, Src, MMP­9, N­cadherin and vimentin and the levels of FAK and Src phosphorylation were increased, while the mRNA and protein expression levels of E­cadherin were decreased in RCC tissues. Following the transfection of 786­O cells with small interfering (si) RNA against LOXL2, the mRNA and protein expression levels of FAK, Src, MMP­9, N­cadherin and vimentin and the levels of phosphorylated FAK and Src were notably decreased in the si­LOXL2 and PP2 inhibitor treated groups, while that of E­cadherin was substantially increased. Additionally, cell proliferation, invasion, migration and the percentage of RCC cells in the G1 phase were reduced, and cell apoptosis was increased. Additionally, Caki1 cells transfected with LOXL2 exhibited an opposite trend. In summary, these results indicate that LOXL2 silencing inhibits the invasion, migration and EMT in RCC cells through inhibition of the Src/FAK signaling pathway.


Assuntos
Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Transdução de Sinais , Adolescente , Adulto , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Regulação para Cima , Adulto Jovem , Quinases da Família src/metabolismo
17.
Biomed Res Int ; 2019: 4012590, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809541

RESUMO

Introduction: Radiotherapy is the mainstay in the treatment of prostate cancer. However, significant radioresistance of castration-resistant prostate cancer (CRPC) cells constitutes a main obstacle in the treatment of this disease. By using bioinformatic data mining methods, LOXL2 was found to be upregulated in both androgen-independent prostate cancer cell lines and radioresistant tumor samples collected from patients with prostate cancer. We speculate that LOXL2 may play an important role in the radioresistance of CRPC cells. Methods: The effect of LOXL2 knockdown on the radiosensitivity of androgen-independent prostate cancer cells lines was measured by the clonogenic assay and xenograft tumor experiments under in vitro and in vivo conditions, respectively. In studies on the mechanism, we focused on the EMT phenotype changes and cell apoptosis changes induced by LOXL2 knockdown in DU145 cells. The protein levels of three EMT biomarkers, namely, E-cadherin, vimentin, and N-cadherin, were measured by western blotting and immunohistochemical staining. Cell apoptosis after irradiation was measured by flow cytometry and caspase-3 activity assay. Salvage experiment was also conducted to confirm the possible role of EMT in the radiosensitization effect of LOXL2 knockdown in CRPC cells. Results: LOXL2 knockdown in CRPC cells enhanced cellular radiosensitivity under both in vitro and in vivo conditions. A significant reversal of EMT was observed in LOXL2-silenced DU145 cells. Cell apoptosis after irradiation was significantly enhanced by LOXL2 knockdown in DU145 cells. Results from the salvage experiment confirmed the key role of EMT process reversal in the radiosensitization effect of LOXL2 knockdown in DU145 cells. Conclusions: LOXL2 plays an important role in the development of cellular radioresistance in CRPC cells. Targeting LOXL2 may be a rational avenue to overcome radioresistance in CRPC cells. A LOXL2-targeting strategy for CRPC treatment warrants detailed investigation in the future.


Assuntos
Aminoácido Oxirredutases/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias de Próstata Resistentes à Castração/radioterapia , Tolerância a Radiação/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Exp Mol Med ; 51(2): 20, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30804321

RESUMO

Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and fetal morbidity and mortality with a prevalence of 6-8% of pregnancies. Although impaired trophoblast invasion in early pregnancy is known to be closely associated with preeclampsia, the underlying mechanisms remain elusive. Here we revealed that lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2) play a critical role in preeclampsia. Our results demonstrated that LOX and LOXL2 expression decreased in preeclamptic placentas. Moreover, knockdown of LOX or LOXL2 suppressed trophoblast cell migration and invasion. Mechanistically, collagen production was induced in LOX- or LOXL2-downregulated trophoblast cells through activation of the TGF-ß1/Smad3 pathway. Notably, inhibition of the TGF-ß1/Smad3 pathway could rescue the defects caused by LOX or LOXL2 knockdown, thereby underlining the significance of the TGF-ß1/Smad3 pathway downstream of LOX and LOXL2 in trophoblast cells. Additionally, induced collagen production and activated TGF-ß1/Smad3 were observed in clinical samples from preeclamptic placentas. Collectively, our study suggests that the downregulation of LOX and LOXL2 leading to reduced trophoblast cell migration and invasion through activation of the TGF-ß1/Smad3/collagen pathway is relevant to preeclampsia. Thus, we proposed that LOX, LOXL2, and the TGF-ß1/Smad3/collagen pathway can serve as potential markers and targets for clinical diagnosis and therapy for preeclampsia.


Assuntos
Aminoácido Oxirredutases/genética , Colágeno/metabolismo , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/metabolismo , Proteína-Lisina 6-Oxidase/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Trofoblastos/metabolismo , Aminoácido Oxirredutases/metabolismo , Biomarcadores , Linhagem Celular , Movimento Celular/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Pré-Eclâmpsia/diagnóstico , Gravidez , Proteína-Lisina 6-Oxidase/metabolismo , RNA Mensageiro/genética , Proteína Smad3/metabolismo
19.
Mol Cancer ; 18(1): 18, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30704479

RESUMO

BACKGROUND: Lysyl oxidase-like 4 (LOXL4) has been found to be dysregulated in several human malignancies, including hepatocellular carcinoma (HCC). However, the role of LOXL4 in HCC progression remains largely unclear. In this study, we investigated the clinical significance and biological involvement of LOXL4 in the progression of HCC. METHODS: LOXL4 expression was measured in HCC tissues and cell lines. Overexpression, shRNA-mediated knockdown, recombinant human LOXL4 (rhLOXL4), and deletion mutants were applied to study the function of LOXL4 in HCC. Exosomes derived from HCC cell lines were assessed for the ability to promote cancer progression in standard assays. The effects of LOXL4 on the FAK/Src pathway were examined by western blotting. RESULTS: LOXL4 was commonly upregulated in HCC tissues and predicted a poor prognosis. Elevated LOXL4 was associated with tumor differentiation, vascular invasion, and tumor-node-metastasis (TNM) stage. Overexpression of LOXL4 promoted, whereas knockdown of LOXL4 inhibited cell migration and invasion of HCC in vitro, and overexpressed LOXL4 promoted intrahepatic and pulmonary metastases of HCC in vivo. Most interestingly, we found that HCC-derived exosomes transferred LOXL4 between HCC cells, and intracellular but not extracellular LOXL4 promoted cell migration by activating the FAK/Src pathway dependent on its amine oxidase activity through a hydrogen peroxide-mediated mechanism. In addition, HCC-derived exosomes transferred LOXL4 to human umbilical vein endothelial cells (HUVECs) though a paracrine mechanism to promote angiogenesis. CONCLUSIONS: Taken together, our data demonstrate a novel function of LOXL4 in tumor metastasis mediated by exosomes through regulation of the FAK/Src pathway and angiogenesis in HCC.


Assuntos
Aminoácido Oxirredutases/genética , Carcinoma Hepatocelular/genética , Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Hepatócitos/metabolismo , Neoplasias Hepáticas/genética , Neovascularização Patológica/genética , Adulto , Idoso , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Exossomos/patologia , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Hepatócitos/patologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neovascularização Patológica/metabolismo , Neovascularização Patológica/mortalidade , Neovascularização Patológica/patologia , Comunicação Parácrina , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Quinases da Família src/genética , Quinases da Família src/metabolismo
20.
Appl Microbiol Biotechnol ; 103(4): 1725-1735, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30607487

RESUMO

Fructosyl peptide oxidase (FPOX, EC 1.5.3) belongs to the family of oxidoreductases, which is used as a diagnostic enzyme for diabetes mellitus. FPOX has activities toward Fru-ValHis and Fru-Lys as model compounds for hemoglobin A1c (HbA1c) and glycated albumin, respectively. However, when the concentration of HbA1c is measured, the activity toward Fru-Lys will cause interference. In this study, we focused on the substrate specificity engineering of FPOX from Eupenicillium terrenum through computational and experimental methods with characteristics more suitable for HbA1c measurement in the blood. Based on structural knowledge of E. terrenum FPOX (PDB ID 4RSL) and molecular modeling results, residues His-377, Arg-62, Lys-380, and Tyr-261 were selected as mutagenesis sites. The best mutant with lower binding energy, stronger hydrophobic interactions, and more hydrogen bonds with Fru-ValHis and higher binding energy toward Fru-Lys was selected for experimental studies. To investigate the conformational changes in FPOX due to the mutation, molecular dynamics simulation was also performed. The genes encoding of native and engineered variants were cloned into pET-22b(+) and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant enzymes were purified and their kinetic properties were studied. Substitution of Tyr261 with Trp resulted in a mutant enzyme with improved specificity for Fru-ValHis, a model compound of HbA1c. The specific activity of mutant FPOX increased by 5.1-fold to 145.2 ± 3.2 U/mg for Fru-ValHis and decreased by 13.7-fold to 1.3 U/mg ± 0.9 for Fru-Lys compared to the native variant. Kinetics analysis indicated that Tyr261Trp FPOX mutant had 11.7-fold increase in Kcat/Km for Fru-ValHis compared to the wild-type enzyme, while the Kcat/Km for Fru-Lys diminished by 22.4-fold. In summary, our computational and experimental results suggested that the engineered FPOX is a good candidate to efficient determination of HbA1c.


Assuntos
Aminoácido Oxirredutases/metabolismo , Eupenicillium/enzimologia , Hemoglobina A Glicada/análise , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/metabolismo , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/genética , Engenharia Genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato
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