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1.
J Sci Food Agric ; 102(1): 85-94, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031874

RESUMO

BACKGROUND: Epilactose, a potential prebiotics, was derived from lactose through enzymatic catalysis. However, production and purification of epilactose are currently difficult due to powerless enzymes and inefficient downstream processing steps. RESULTS: The encoding gene of cellobiose 2-epimerase (CE) from Caldicellulosiruptor sp. Rt8.B8 was cloned and expressed in Escherichia coli BL21(DE3). The enzyme was purified and it was suitable for industrial production of epilactose from lactose without by-products, because of high kcat (197.6 s-1 ) and preferable thermostability. The Rt8-CE gene was further expressed in the Bacillus subtilis strain. We successfully produced epilactose from 700 g L-1 lactose in 30.4% yield by using the recombinant Bacillus subtilis whole cells. By screening of a ß-galactosidase from Bacillus stearothermophilus (BsGal), a process for separating epilactose and lactose was established, which showed a purity of over 95% in a total yield of 69.2%. In addition, a mixed rare sugar syrup composed of epilactose and d-tagatose was successfully produced from lactose through the co-expression of l-arabinose isomerase and ß-galactosidase. CONCLUSION: Our study shed light on the efficient production of epilactose using a food-grade host expressing a novel CE enzyme. Moreover, an efficient and low-cost process was attempted to obtain high purity epilactose. In order to improve the utilization of raw materials, the production process of mixed syrup containing epilactose and d-tagatose with prebiotic properties produced from lactose was also established for the first time. © 2021 Society of Chemical Industry.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Celobiose/metabolismo , Dissacarídeos/biossíntese , Racemases e Epimerases/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/genética , Estabilidade Enzimática , Expressão Gênica , Temperatura Alta , Lactose/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
J Agric Food Chem ; 69(45): 13578-13585, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34736320

RESUMO

d-Allulose is considered an ideal alternative to sucrose and has shown tremendous application potential in many fields. Recently, most efforts on production of d-allulose have focused on in vitro enzyme-catalyzed epimerization of cheap hexoses. Here, we proposed an approach to efficiently produce d-allulose through fermentation using metabolically engineered Escherichia coli JM109 (DE3), in which a SecY (ΔP) channel and a d-allulose 3-epimerase (DPEase) were co-expressed, ensuring that d-fructose could be transported in its nonphosphorylated form and then converted into d-allulose by cells. Further deletion of fruA, manXYZ, mak, galE, and fruK and the use of Ni2+ in a medium limited the carbon flux flowing into the byproduct-generating pathways and the Embden-Meyerhof-Parnas (EMP) pathway, achieving a ≈ 0.95 g/g yield of d-allulose on d-fructose using E. coli (DPEase, SecY [ΔP], ΔFruA, ΔManXYZ, ΔMak, ΔGalE, ΔFruK) and 8 µM Ni2+. In fed-batch fermentation, the titer of d-allulose reached ≈23.3 g/L.


Assuntos
Escherichia coli , Frutose , Escherichia coli/genética , Fermentação , Racemases e Epimerases
3.
Medicine (Baltimore) ; 100(42): e27548, 2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34678889

RESUMO

RATIONALE: Progressive encephalopathy with brain edema and/or leukoencephalopathy-1 is an infantile, lethal neurometabolic disorder caused by a NAD(P)HX epimerase (NAXE) gene mutation. It is characterized by a fluctuating disease course with repeated episodes of improvement and regression. In this report, we present a rare case of NAXE gene mutation-related encephalopathy with unexpected neurological recovery and long survival time. PATIENT CONCERNS: A 20-month-old girl presented with progressively unsteady gait and bilateral hand tremors after a trivial febrile illness. Her disease rapidly progressed to consciousness disturbance, 4-limb weakness (muscle power: 1/5 on the Medical Research Council scale), and respiratory failure. The patient gradually recovered 2 months later. However, another episode of severe fever-induced encephalopathy developed 2 years after the initial presentation. DIAGNOSES: Results of laboratory investigations, including complete blood count, blood chemistry, inflammatory markers, and cerebral spinal fluid analysis were unremarkable. Electroencephalography and nerve conduction velocity studies yielded normal results. Brain magnetic resonance imaging on diffusion-weighted imaging revealed abnormal sysmmetric hyperintensity in the bilateral middle cerebellar peduncles. A genetic study using whole exome sequencing confirmed the diagnosis of NAXE gene mutation-related encephalopathy. INTERVENTIONS: Pulse therapy with methylprednisolone, intravenous immunoglobulin, coenzyme Q10, and carnitine were initially introduced. After a NAXE gene defect was detected, the vitamin B complex and coenzyme Q10 were administered. A continuous rehabilitation program was also implemented. OUTCOMES: NAXE gene mutation-related encephalopathy is usually regarded as a lethal neurometabolic disorder. However, the outcome in this case is better than that in the previous cases. She showed progressive neurological recovery and a longer survival time. The muscle power of the 4 limbs recovered to grade 4. At present (age of 5.5 years old), she can walk with an unsteady gait and go to school. LESSONS: Although NAXE gene mutation-related encephalopathy is rare, it should be considered as a differential diagnosis of early onset progressive encephalopathy.


Assuntos
Encefalopatias/genética , Encefalopatias/fisiopatologia , Racemases e Epimerases/genética , Suplementos Nutricionais , Feminino , Humanos , Lactente , Ubiquinona/análogos & derivados , Ubiquinona/uso terapêutico , Complexo Vitamínico B/uso terapêutico , Sequenciamento Completo do Exoma
4.
Compr Rev Food Sci Food Saf ; 20(6): 6012-6026, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34668314

RESUMO

D-allulose is the C-3 epimer of D-fructose, which rarely exists in nature, and can be biosynthesized from D-fructose by the catalysis of D-psicose 3-epimerase. D-allulose is safe for human consumption and was recently approved by the United States Food and Drug Administration for food applications. It is not only able be used in food and dietary supplements as a low-calorie sweetener, but also modulates a variety of physiological functions. D-allulose has gained increasing attention owing to its excellent properties. This article presents a review of recent progress on the properties, applications, and bioproduction progress of D-allulose.


Assuntos
Frutose , Racemases e Epimerases , Catálise , Humanos , Edulcorantes , Estados Unidos
5.
J Agric Food Chem ; 69(39): 11637-11645, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34569239

RESUMO

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T5060 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.


Assuntos
Ensaios de Triagem em Larga Escala , Racemases e Epimerases , Carboidratos Epimerases/metabolismo , Frutose , Concentração de Íons de Hidrogênio , Cinética
6.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34556581

RESUMO

d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunoprecipitation with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.


Assuntos
Encéfalo/fisiologia , Células-Tronco Neurais/fisiologia , Racemases e Epimerases/fisiologia , Serina/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/química
7.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548397

RESUMO

Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD+, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD+, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD+ and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.


Assuntos
Cisteína/química , Hidrolases/metabolismo , Lactobacillus plantarum/enzimologia , Níquel/metabolismo , Nucleotídeos/biossíntese , Piridinas/química , Racemases e Epimerases/metabolismo , Carboxiliases , Catálise , Cristalografia por Raios X , Hidrolases/química , Hidrólise , Modelos Moleculares , Conformação Proteica , Racemases e Epimerases/química , Especificidade por Substrato
8.
Int J Mol Sci ; 22(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34576170

RESUMO

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4-4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen-bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.


Assuntos
Clostridiales/enzimologia , Racemases e Epimerases/metabolismo , Cisteína/metabolismo , Simulação de Dinâmica Molecular , Racemases e Epimerases/genética , Temperatura
9.
Exp Eye Res ; 211: 108732, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34419444

RESUMO

The role of the N-Methyl-D-Aspartate Receptor (NMDAR) in the outer retina is unclear despite expression of the NMDAR-complex and its subunits in the outer retina. The flash-electroretinogram (fERG) offers a non-invasive measurement of the retinal field potentials of the outer retina that can serve to clarify NMDAR contribution to early retinal processing. The role of the NMDAR in retinal function was assessed using a genetic mouse model for NMDAR hypofunction (SR-/-), where the absence of the enzyme serine racemase (SR) results in an 85% reduction of retinal D-serine. NMDAR hypo- and hyperfunction in the retina results in alterations in the components of the fERG. The fERG was examined after application of exogenous D-serine to the eye in order to determine whether pre- and post-topical delivery of D-serine would alter the fERG in SR-/- mice and their littermate WT controls. Amplitude and implicit time of the low-frequency components, the a- and b-wave, were conducted. Reduced NMDAR function resulted in a statistically significantly delayed a-wave and reduced b-wave in SR-/- animals. The effect of NMDAR deprivation was more prominent in male SR-/- mice. A hyperfunction of the NMDAR, through exogenous topical delivery of 5 mM D-serine, in WT mice caused a significantly delayed a-wave implicit time and reduced b-wave amplitude. These changes were not observed in female WT mice. There were temporal delays in the a-wave and amplitude and a decrease in the b-wave amplitude and implicit time in both hypo- and NMDAR hyperfunctional male mice. These results suggest that NMDAR and D-serine are involved in the retinal field potentials of the outer retina that interact based on the animal's sex. This implicates the involvement of gonadal hormones and D-serine in retinal functional integrity.


Assuntos
Eletrorretinografia/efeitos dos fármacos , Retina/fisiologia , Serina/farmacologia , Animais , Feminino , Masculino , Visão Mesópica/fisiologia , Camundongos , Camundongos Knockout , Estimulação Luminosa , Racemases e Epimerases , Receptores de N-Metil-D-Aspartato/metabolismo
10.
Enzyme Microb Technol ; 149: 109850, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34311887

RESUMO

The rare sugar d-allulose is an attractive sucrose substitute due to its sweetness and ultra-low caloric value. It can be produced from D-fructose using d-allulose 3-epimerase (DAE) as the biocatalyst. However, most of the reported DAEs show low catalytic efficiency and poor thermostability, which limited their further use in food industrial. Here, a putative d-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense (HcDAE) was characterized, showing optimal activity at pH 8.0 and 70 °C in the presence of Mg2+. Saturation mutagenesis of Y7, C66, and I108, the putative residues responsible for substrate recognition at the O-4, -5, and -6 atoms of D-fructose was performed, and it yielded the triple mutant Y7H/C66L/I108A with improved activity toward D-fructose (345 % of wild-type enzyme). The combined mutant Y7H/C66L/I108A/R156C/K260C exhibited a half-half (t1/2) of 5.2 h at 70 °C and an increase of the Tm value by 6.5 °C due to the introduction of disulfide bridges between intersubunit with increased interface interactions. The results indicate that mutants could be used as industrial biocatalysts for d-allulose production.


Assuntos
Frutose , Racemases e Epimerases , Firmicutes , Concentração de Íons de Hidrogênio
11.
J Agric Food Chem ; 69(29): 8268-8275, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34231359

RESUMO

Cellobiose 2-epimerase (CE) can catalyze bioconversion of lactose to its prebiotic derivative epilactose. The catalytic property of a novel CE from Treponema brennaborense (Trbr-CE) was investigated. Trbr-CE showed the highest catalytic efficiency of epimerization toward lactose among all of the previously reported CEs. This enzyme's specific activity could reach as high as 208.5 ± 5.3 U/mg at its optimum temperature, which is 45 °C. More importantly, this enzyme demonstrated a considerably high activity at low temperatures, suggesting Trbr-CE as a promising enzyme for industrial low-temperature production of epilactose. This structurally flexible enzyme exhibited a comparatively high binding affinity toward substrates, which was confirmed by both experimental verification and computational analysis. Molecular dynamics (MD) simulations and binding free energy calculations were applied to provide insights into molecular recognition upon temperature changes. Compared with thermophilic CEs, Trbr-CE presents a more negative enthalpy change and a higher entropy change when the temperature drops.


Assuntos
Celobiose , Racemases e Epimerases , Lactose , Racemases e Epimerases/genética , Temperatura , Treponema
13.
Radiat Res ; 196(2): 213-224, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34087943

RESUMO

Ketogenic diets (KD) are high in fat and low in carbohydrates, forcing cells to utilize mitochondrial fatty acid oxidation for energy production. Since cancer cells demonstrate increased mitochondrial oxidative stress relative to normal cells, we hypothesized that a KD may selectively enhance metabolic oxidative stress in head and neck cancer cells, sensitizing them to radiation and platinum-based chemotherapy without causing increased toxicity in surrounding normal tissues. This hypothesis was tested in preclinical murine xenografts and in a phase 1 clinical trial (NCT01975766). In this study, mice bearing human head and neck cancer xenografts (FaDu) were fed either standard mouse chow or KetoCal® KD (90% fat, 8% carbohydrate, 2% protein) and exposed to ionizing radiation. Tumors were harvested from mice to test for glutathione, a biomarker of oxidative stress. In parallel, patients with locally advanced head and neck cancer were enrolled in a phase 1 clinical trial where they consumed KD and received radiation with concurrent platinum-based chemotherapy. Subjects consumed KetoCal KD via percutaneous endoscopic gastrostomy (PEG) tube and were also allowed to orally consume water, sugar-free drinks, and foods approved by a dietitian. Oxidative stress markers including protein carbonyls and total glutathione were assessed in patient blood samples both pre-KD and while consuming the KD. Mice bearing FaDu xenografts that received radiation and KD demonstrated a slight improvement in tumor growth rate and survival compared to mice that received radiation alone; however a variation in responses was seen dependent on the fatty acid composition of the diet. In the phase 1 clinical trial, a total of twelve patients were enrolled in the study. Four patients completed five weeks of the KD as per protocol (with variance in compliance). Eight patients did not tolerate the diet with concurrent radiation and platinum-chemotherapy (5 were patient decision and 3 were removed from study due to toxicity). The median number of days consuming a KD in patients who did not complete the study was 5.5 (range: 2-8 days). Reasons for discontinuation included "stress of diet compliance" (1 patient), grade 2 nausea (3 patients), and grade 3 fatigue (1 patient). Three patients were removed from the trial due to dose-limiting toxicities including: grade 4 hyperuricemia (2 patients) and grade 3 acute pancreatitis (1 patient). Median weight loss was 2.95% for the KD-tolerant group and 7.92% for patients who did not tolerate the diet. In conclusion, the ketogenic diet shows promise as a treatment combined with radiation in preclinical mouse head and neck cancer xenografts. A phase 1 clinical trial evaluating the safety and tolerability of KD demonstrated difficulty with diet compliance when combined with standard-of-care radiation therapy and cisplatin chemotherapy.


Assuntos
Dieta Cetogênica/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/dietoterapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , 3-Hidroxiacil-CoA Desidrogenases/efeitos dos fármacos , 3-Hidroxiacil-CoA Desidrogenases/efeitos da radiação , Acetil-CoA C-Aciltransferase/efeitos dos fármacos , Acetil-CoA C-Aciltransferase/efeitos da radiação , Adulto , Idoso , Animais , Isomerases de Ligação Dupla Carbono-Carbono/efeitos dos fármacos , Isomerases de Ligação Dupla Carbono-Carbono/efeitos da radiação , Quimiorradioterapia/efeitos adversos , Dieta Cetogênica/efeitos adversos , Enoil-CoA Hidratase/efeitos dos fármacos , Enoil-CoA Hidratase/efeitos da radiação , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Racemases e Epimerases/efeitos dos fármacos , Racemases e Epimerases/efeitos da radiação , Radiação Ionizante , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/efeitos da radiação
15.
Chem Soc Rev ; 50(10): 5952-5984, 2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34027955

RESUMO

Racemases and epimerases catalyse changes in the stereochemical configurations of chiral centres and are of interest as model enzymes and as biotechnological tools. They also occupy pivotal positions within metabolic pathways and, hence, many of them are important drug targets. This review summarises the catalytic mechanisms of PLP-dependent, enolase family and cofactor-independent racemases and epimerases operating by a deprotonation/reprotonation (1,1-proton transfer) mechanism and methods for measuring their catalytic activity. Strategies for inhibiting these enzymes are reviewed, as are specific examples of inhibitors. Rational design of inhibitors based on substrates has been extensively explored but there is considerable scope for development of transition-state mimics and covalent inhibitors and for the identification of inhibitors by high-throughput, fragment and virtual screening approaches. The increasing availability of enzyme structures obtained using X-ray crystallography will facilitate development of inhibitors by rational design and fragment screening, whilst protein models will facilitate development of transition-state mimics.


Assuntos
Inibidores Enzimáticos/metabolismo , Racemases e Epimerases/metabolismo , Regulação Alostérica , Biocatálise , Domínio Catalítico , Coenzimas/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Simulação de Dinâmica Molecular , Prótons , Racemases e Epimerases/antagonistas & inibidores , Especificidade por Substrato
16.
Cancer Sci ; 112(7): 2753-2769, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33932069

RESUMO

Reactive oxygen species (ROS) derived from aberrant tumor metabolism could contribute to tumor invasion and metastasis. NAD(P)HX Epimerase (NAXE), an epimerase that allows the repair of damaged forms of antioxidant NADPH, is a potential cellular ROS scavenger and its role in tumor development is still elusive. Here, we found that NAXE is significantly downregulated in hepatocellular carcinoma (HCC) tissues and cell lines. NAXE downregulation is associated with poor clinicopathological characteristics and is an independent risk factor for overall and disease-free survival of HCC patients after liver resection. In addition, low NAXE expression could identify worse prognosis of HCC patients before vascular invasion or in early stages of disease. In particularly, low NAXE expression in HCC is markedly associated with microvascular invasion (MVI) and its combination with MVI predicts poorer prognosis of HCC patients after liver resection. Furthermore, in vitro and in vivo experiments both showed that knockdown of NAXE expression in HCC cells promoted migration, invasion, and metastasis by inducing epithelial-mesenchymal transition (EMT), whereas NAXE overexpression causes the opposite effects. Mechanistically, low NAXE expression reduced NADPH levels and further caused ROS level increase and hypoxia-inducible factor-1α (HIF-1α) activation, thereby promoting invasion and metastasis of HCC by facilitating EMT. What is more, the tumor-promoting effect of NAXE knockdown in HCC xenograft can be abolished by giving mice N-acetyl-l-cysteine (NAC) in drinking water. Taken together, our findings uncovered a tumor suppressor role for NAXE in HCC by scavenging excessive ROS and inhibiting tumor-promoting signaling pathways, suggesting a new strategy for HCC therapy by targeting redox signaling.


Assuntos
Carcinoma Hepatocelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , Racemases e Epimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Análise de Variância , Animais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/secundário , Movimento Celular , Progressão da Doença , Intervalo Livre de Doença , Regulação para Baixo , Transição Epitelial-Mesenquimal , Feminino , Sequestradores de Radicais Livres/farmacologia , Xenoenxertos , Humanos , Neoplasias Hepáticas/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , NADP/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Oxirredução , Racemases e Epimerases/genética , Fatores de Risco
17.
PLoS One ; 16(4): e0248964, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33793635

RESUMO

Emerging studies indicate that APOA-I binding protein (AIBP) is a secreted protein and functions extracellularly to promote cellular cholesterol efflux, thereby disrupting lipid rafts on the plasma membrane. AIBP is also present in the mitochondria and acts as an epimerase, facilitating the repair of dysfunctional hydrated NAD(P)H, known as NAD(P)H(X). Importantly, AIBP deficiency contributes to lethal neurometabolic disorder, reminiscent of the Leigh syndrome in humans. Whereas cyclic NADPHX production is proposed to be the underlying cause, we hypothesize that an unbiased metabolic profiling may: 1) reveal new clues for the lethality, e.g., changes of mitochondrial metabolites., and 2) identify metabolites associated with new AIBP functions. To this end, we performed unbiased and profound metabolic studies of plasma obtained from adult AIBP knockout mice and control littermates of both genders. Our systemic metabolite profiling, encompassing 9 super pathways, identified a total of 640 compounds. Our studies demonstrate a surprising sexual dimorphism of metabolites affected by AIBP deletion, with more statistically significant changes in the AIBP knockout female vs male when compared with the corresponding controls. AIBP knockout trends to reduce cholesterol but increase the bile acid precursor 7-HOCA in female but not male. Complex lipids, phospholipids, sphingomyelin and plasmalogens were reduced, while monoacylglycerol, fatty acids and the lipid soluble vitamins E and carotene diol were elevated in AIBP knockout female but not male. NAD metabolites were not significantly different in AIBP knockout vs control mice but differed for male vs female mice. Metabolites associated with glycolysis and the Krebs cycle were unchanged by AIBP knockout. Importantly, polyamine spermidine, critical for many cellular functions including cerebral cortex synapses, was reduced in male but not female AIBP knockout. This is the first report of a systemic metabolite profile of plasma samples from AIBP knockout mice, and provides a metabolic basis for future studies of AIBP regulation of cellular metabolism and the pathophysiological presentation of AIBP deficiency in patients.


Assuntos
Fosfoproteínas/metabolismo , Racemases e Epimerases/metabolismo , Fatores Sexuais , Animais , Colesterol/metabolismo , Feminino , Metabolismo dos Lipídeos , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo
18.
Virchows Arch ; 479(2): 337-343, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33811532

RESUMO

Alpha-methylacyl-coenzyme A-racemase (AMACR), also known as p504s, is overexpressed in prostatic adenocarcinoma and is frequently used in combination with basal cell markers to aid in diagnosing difficult prostate adenocarcinoma cases. In this retrospective method comparison study, we examined the sensitivity and specificity of the ready-to-use anti-p504s (SP116) Rabbit Monoclonal Primary Antibody compared to the monoclonal rabbit anti-human AMACR clone 13H4 in prostatic adenocarcinoma samples. De-identified prostatic adenocarcinoma tissue samples were stained with either the SP116 or 13H4 antibody clone in combination with the VENTANA Basal Cell Cocktail (34ßE12+p63) and scored as positive or negative for prostatic adenocarcinoma. The scoring pathologist was blinded to the known historical diagnosis of each sample. The scoring pathologist correctly diagnosed each sample regardless of which p504s clone was used. Both assays using either clone were 100% concordant in their sensitivity and specificity. This study demonstrates that the ready-to-use anti-p504s (SP116) Rabbit Monoclonal Primary Antibody is equivalent to clone 13H4 concentrate when used according to package insert instructions in combination with the VENTANA Basal Cell Cocktail (34ßE12+p63) to aid pathologists in the diagnosis of prostatic adenocarcinoma.


Assuntos
Adenocarcinoma/imunologia , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/análise , Imuno-Histoquímica , Queratinas/análise , Neoplasias da Próstata/imunologia , Racemases e Epimerases/análise , Adenocarcinoma/patologia , Animais , Especificidade de Anticorpos , Humanos , Masculino , Valor Preditivo dos Testes , Neoplasias da Próstata/patologia , Coelhos , Reprodutibilidade dos Testes , Estudos Retrospectivos
19.
Sci Rep ; 11(1): 9031, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907230

RESUMO

Abnormalities in electroencephalographic (EEG) biomarkers occur in patients with schizophrenia and those clinically at high risk for transition to psychosis and are associated with cognitive impairment. Converging evidence suggests N-methyl-D-aspartate receptor (NMDAR) hypofunction plays a central role in the pathophysiology of schizophrenia and likely contributes to biomarker impairments. Thus, characterizing these biomarkers is of significant interest for early diagnosis of schizophrenia and development of novel treatments. We utilized in vivo EEG recordings and behavioral analyses to perform a battery of electrophysiological biomarkers in an established model of chronic NMDAR hypofunction, serine racemase knockout (SRKO) mice, and their wild-type littermates. SRKO mice displayed impairments in investigation-elicited gamma power that corresponded with reduced short-term social recognition and enhanced background (pre-investigation) gamma activity. Additionally, SRKO mice exhibited sensory gating impairments in both evoked-gamma power and event-related potential amplitude. However, other biomarkers including the auditory steady-state response, sleep spindles, and state-specific power spectral density were generally neurotypical. In conclusion, SRKO mice demonstrate how chronic NMDAR hypofunction contributes to deficits in certain translationally-relevant EEG biomarkers altered in schizophrenia. Importantly, our gamma band findings suggest an aberrant signal-to-noise ratio impairing cognition that occurs with NMDAR hypofunction, potentially tied to impaired task-dependent alteration in functional connectivity.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Eletroencefalografia , Feminino , Ritmo Gama , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Esquizofrenia/diagnóstico , Esquizofrenia/fisiopatologia , Filtro Sensorial , Comportamento Social
20.
J Microbiol Biotechnol ; 31(5): 756-763, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-33820885

RESUMO

Agarose is a linear polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass. However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in Vibrio sp. EJY3, a gram-negative marine bacterial species, we investigated AHG metabolism in Streptomyces coelicolor A3(2), an agarolytic gram-positive soil bacterium. Based on genomic data, the SCO3486 protein (492 amino acids) and the SCO3480 protein (361 amino acids) of S. coelicolor A3(2) showed identity with H2IFE7.1 (40% identity) encoding AHG dehydrogenase and H2IFX0.1 (42% identity) encoding 3,6-anhydro-L-galactonate cycloisomerase, respectively, which are involved in the initial catabolism of AHG in Vibrio sp. EJY3. Thin layer chromatography and mass spectrometry of the bioconversion products catalyzed by recombinant SCO3486 and SCO3480 proteins, revealed that SCO3486 is an AHG dehydrogenase that oxidizes AHG to 3,6-anhydro-L-galactonate, and SCO3480 is a 3,6-anhydro-L-galactonate cycloisomerase that converts 3,6-anhydro-L-galactonate to 2-keto-3-deoxygalactonate. SCO3486 showed maximum activity at pH 6.0 at 50°C, increased activity in the presence of iron ions, and activity against various aldehyde substrates, which is quite distinct from AHG-specific H2IFE7.1 in Vibrio sp. EJY3. Therefore, the catabolic pathway of AHG seems to be similar in most agar-degrading microorganisms, but the enzymes involved appear to be very diverse.


Assuntos
Galactose/análogos & derivados , NADPH Desidrogenase/metabolismo , Racemases e Epimerases/metabolismo , Streptomyces coelicolor/enzimologia , Aldeídos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Galactose/metabolismo , Concentração de Íons de Hidrogênio , Ferro , Redes e Vias Metabólicas , NADPH Desidrogenase/genética , Racemases e Epimerases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodófitas/química , Sefarose/metabolismo , Streptomyces coelicolor/metabolismo , Especificidade por Substrato , Temperatura
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