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1.
Science ; 367(6482): 1105-1112, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139538

RESUMO

The mechanism by which psychological stress elicits various physiological responses is unknown. We discovered a central master neural pathway in rats that drives autonomic and behavioral stress responses by connecting the corticolimbic stress circuits to the hypothalamus. Psychosocial stress signals from emotion-related forebrain regions activated a VGLUT1-positive glutamatergic pathway from the dorsal peduncular cortex and dorsal tenia tecta (DP/DTT), an unexplored prefrontal cortical area, to the dorsomedial hypothalamus (DMH), a hypothalamic autonomic center. Genetic ablation and optogenetics revealed that the DP/DTT→DMH pathway drives thermogenic, hyperthermic, and cardiovascular sympathetic responses to psychosocial stress without contributing to basal homeostasis. This pathway also mediates avoidance behavior from psychosocial stressors. Given the variety of stress responses driven by the DP/DTT→DMH pathway, the DP/DTT can be a potential target for treating psychosomatic disorders.


Assuntos
Núcleo Hipotalâmico Dorsomedial/metabolismo , Comportamento Social , Estresse Psicológico/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Animais , Emoções/fisiologia , Feminino , Ácido Glutâmico/metabolismo , Homeostase , Masculino , Neurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Prosencéfalo/metabolismo , Transtornos Psicofisiológicos/terapia , Ratos , Ratos Endogâmicos LEC , Ratos Wistar , Transdução de Sinais
2.
Invest Ophthalmol Vis Sci ; 61(1): 1, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31995153

RESUMO

Purpose: Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. Methods: The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. Results: The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. Conclusions: These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Fosfotransferases/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas tau/metabolismo , Animais , Western Blotting , Células Cultivadas , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Regulação para Baixo , Técnica Indireta de Fluorescência para Anticorpo , Ácido Glutâmico/toxicidade , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Masculino , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Roscovitina/farmacologia , Transfecção , Enzimas Ativadoras de Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
3.
Phys Chem Chem Phys ; 22(5): 3008-3016, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957772

RESUMO

Infrared (IR) spectroscopy is commonly utilized for the investigation of protein structures and protein-mediated processes. While the amide I band provides information on protein secondary structures, amino acid side chains are used as IR probes for the investigation of protein reactions, such as proton pumping in rhodopsins. In this work, we calculate the IR spectra of the solvated aspartic acid, with both zwitterionic and protonated backbones, and of a capped form, i.e. mimicking the aspartic acid residue in proteins, by means of molecular dynamics (MD) simulations and the perturbed matrix method (PMM). This methodology has already proved its good modeling capabilities for the amide I mode and is here extended to the treatment of protein side chains. The computed side chain vibrational signal is in very good agreement with the experimental one, well reproducing both the peak frequency position and the bandwidth. In addition, the MD-PMM approach proposed here is able to reproduce the small frequency shift (5-10 cm-1) experimentally observed between the protonated and zwitterionic forms, showing that such a shift depends on the excitonic coupling between the modes localized on the side chain and on the backbone in the protonated form. The spectrum of the capped form, in which the amide I band is also calculated, agrees well with the corresponding experimental spectrum. The reliable calculation of the vibrational bands of carboxyl-containing side chains provides a useful tool for the interpretation of experimental spectra.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Proteínas/química , Espectrofotometria Infravermelho , Ácido Aspártico/química , Ácido Glutâmico/química , Teoria Quântica
4.
Aquat Toxicol ; 218: 105352, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31790938

RESUMO

Cadmium (Cd) affects plants and animal health seriously. Ca2+ signals in plant cells are important for adaptive responses to environmental stresses. Here we showed that 50 µM Cd shock stimulated the Ca2+ signal via modifying the instantaneous Ca2+ flux from influx of 17 pmol·cm-2·s-1 to the efflux of 240 pmol·cm-2·s-1 at 100 µm from rhizoid tip. And the Ca2+ signal transferred to the vein and mesophyll cell. The Ca addition decreased the accumulation of Cd. The gene expression of glutamate receptor-like (GLR) proteins, which is activated by Glu and triggers Ca2+ flux, was increased significantly by 24 h Cd stress. Glu content was increased under Cd stress and exogenous Glu triggered the Ca2+ signal in duckweed, while Ca2+ addition caused no influence to Glu content. GABA, which is synthesized from Glu and acts as an inhibitory neurotransmitter, has been decreased with 24 h Cd treatment. GABA addition increased the abscission rate and Glu addition decreased the abscission rate during Cd stress, suggesting that the Glu/GABA ratio is important for responding to Cd. This research shows the sight of the Glu, Ca2+, GABA signaling networks during Cd stress.


Assuntos
Araceae/efeitos dos fármacos , Cádmio/toxicidade , Sinalização do Cálcio/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Ácido gama-Aminobutírico/metabolismo , Animais , Araceae/metabolismo , Cádmio/metabolismo , Poluentes Químicos da Água/metabolismo
5.
Talanta ; 206: 120237, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514830

RESUMO

A new calibration approach based on the adaptation of Integrated Calibration Method (ICM) to consecutive two components analysis in separation science is presented. Consecutive ICM method (C-ICM) was conceptually developed and applied to determination of two excitatory amino acids - glutamate and aspartate in cerebrospinal fluids collected by the use of brain microdialysis from freely-moving animals. Both analytes as a neurotransmitters play an important role in formation of the memory trace, and thus the processes of learning and memory. Due to their low concentration and presence of interferences, considered analytical system - animal brain - was a big challenge. High-performance liquid chromatography (HPLC) with electrochemical detection (ECD) was used in all experimental work. The most important feature of proposed method is integration of interpolative and extrapolative ways to calculate analyte concentration in single calibration procedure, which consequently leads to obtain series of six estimations of analytical result. Comparison of individual estimations with each other allows for a more in-depth analysis of systematic errors. It was proved that C-ICM approach enables diagnosis and compensation of systematic errors induced by occurrence of interference effects and improvement of accuracy of analytical results. Most of all, it was demonstrated that application of this method is efficient and useful analytical tool in analysis of complicated biological samples in pharmacology and neuroscience.


Assuntos
Ácido Aspártico/líquido cefalorraquidiano , Ácido Glutâmico/líquido cefalorraquidiano , Animais , Calibragem , Cromatografia Líquida de Alta Pressão/métodos , Técnicas Eletroquímicas/métodos , Masculino , Camundongos Endogâmicos C57BL , Microdiálise , Neurotransmissores/líquido cefalorraquidiano , Ratos Wistar
6.
Food Microbiol ; 86: 103343, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31703887

RESUMO

The bacterial conversion of glutamine to glutamate is catalyzed by glutamine-amidotransferases or glutaminases. Glutamine deamination contributes to the formation of the bioactive metabolites glutamate, γ-aminobutyrate (GABA) and γ-glutamyl peptides, and to acid resistance. This study aimed to investigate the distribution of glutaminase(s) in lactobacilli, and to evaluate their contribution in L. reuteri to amino acid metabolism and acid resistance. Phylogenetic analysis of the glutaminases gls1, gls2 and gls3 in the genus Lactobacillus demonstrated that glutaminase is exclusively present in host-adapted species of lactobacilli. The disruption gls1, gls2 and gls3 in L. reuteri 100-23 had only a limited effect on the conversion of glutamine to glutamate, GABA, or γ-glutamyl peptides in sourdough. The disruption of all glutaminases in L. reuteri 100-23Δgls1Δgls2Δgls3 but not disruption of gls2 and gls3 eliminated the protective effect of glutamine on the survival of the strain at pH 2.5. Glutamine also enhanced acid resistance of L. reuteri 100-23ΔgadB and L. taiwanensis 107q, strains without glutamate decarboxylase activity. Taken together, the study demonstrates that glutaminases of lactobacilli do not contribute substantially to glutamine metabolism but enhance acid resistance. Their exclusive presence in host-adapted lactobacilli provides an additional link between the adaptation of lactobacilli to specific habitats and their functionality when used as probiotics and starter cultures.


Assuntos
Ácidos/metabolismo , Glutaminase/metabolismo , Lactobacillus reuteri/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Ácido Glutâmico/metabolismo , Glutaminase/genética , Glutamina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lactobacillus/enzimologia , Lactobacillus/genética , Lactobacillus/metabolismo , Lactobacillus reuteri/enzimologia , Lactobacillus reuteri/genética , Filogenia , Roedores/microbiologia
7.
Food Chem ; 305: 125440, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494496

RESUMO

Tea (Camellia sinensis) contains two active glutamate decarboxylases (CsGADs), whose unclear properties were examined here. CsGAD1 was 4-fold higher than CsGAD2 in activity. Their Km values for L-glutamate were around 5 mM. CsGAD1 and CsGAD2 performed best at 55 and 40 °C, respectively, and were both stimulated by calcium/calmodulin (Ca2+/CaM). Over 40 °C, their calmodulin-binding domains degraded. CsGADs were most active at pH 5.6, and were stimulated by Ca2+/CaM at pH 5.6-6.6, but inactivated at pH 3.6. Ca2+/CaM restored the CsGAD1 activity suppressed by inhibitors. CsGADs and CsCaM were localized to the cytosol. CsGAD1 was more highly expressed in most tissues, while CsGAD2 expression was more induced under stresses. The characteristics we first elucidated here revealed that CsGAD1 is the predominant isoform in tea plant, with CsGAD2 exhibiting a supplementary role under certain conditions. The information will contribute to regulation of GABA tea quality.


Assuntos
Camellia sinensis/enzimologia , Glutamato Descarboxilase/metabolismo , Proteínas de Plantas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Secas , Estabilidade Enzimática , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Cinética , Temperatura Ambiente
8.
Biosci Biotechnol Biochem ; 84(1): 118-125, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31516066

RESUMO

We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 - 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.


Assuntos
4-Aminobutirato Transaminase/química , Aminoácido Oxirredutases/química , Colorimetria/métodos , Ensaios Enzimáticos/métodos , Peroxidase/química , Ácido gama-Aminobutírico/análise , Ampirona/química , Ascorbato Oxidase/química , Catalase/química , Cromatografia Líquida , Ensaios Enzimáticos/economia , Compostos Férricos/química , Ácido Glutâmico/química , Peróxido de Hidrogênio/química , Lycopersicon esculentum/química , Acoplamento Oxidativo , Proteínas Recombinantes , Streptomyces/enzimologia , Espectrometria de Massas em Tandem
9.
Rev. bras. cancerol ; 66(1)20200129.
Artigo em Inglês | LILACS | ID: biblio-1049323

RESUMO

Introduction: Three vanadium complexes with orotic and glutamic acids, in their anion forms, were prepared and their in vitro cytotoxicity toward human lung fibroblasts (MRC-5), human hepatocellular carcinoma (HepG2) and human colorectal adenocarcinoma (Caco-2) are reported. Objective: Describe the synthesis and characterization of new vanadium complexes with orotic and glutamic acids, and test its antitumor activity against HepG2 and Caco-2. Method: The complexes were formulated as VO (oro), VO (α-glu) and VO (γ-glu) based on chemical, thermogravimetric analyses and infrared spectra. Results: Resazurin assay demonstrates its cytotoxicity against the HepG2 and Caco-2 cell lines with the IC50 ranging from 7.90 to 44.56 µmol.L-1. The cytotoxicity profiles indicate that the tumoral lines show more activity than the cells MRC-5, with selectivity indexes ranging from 1.58 to 8.96. Conclusion: The three complexes had better in vitro activity than cisplatin for both normal and cancer cell lines. The IC50 values are two to six times better for the cancer cell ines and five to seven times better for the normal cell lines. This study indicates that the complexes obtained are promising candidates for antitumor drugs.


Introdução: Foram preparados três complexos de vanádio com ácidos orótico e glutâmico, em suas formas aniônicas, e foi testada sua citotoxicidade in vitro para fibroblastos pulmonares humanos (MRC-5), carcinoma hepatocelular humano (HepG2) e adenocarcinoma colorretal humano (Caco-2). Objetivo: Descrever a síntese e caracterização de novos complexos de vanádio com ácidos orótico e glutâmico e testar sua atividade antitumoral contra HepG2 e Caco-2. Método: Os complexos foram formulados como VO (oro), VO (α-glu) e VO (γ-glu) com base em análises químicas, termogravimétricas e espectros no infravermelho. Resultados: O ensaio de resazurina demonstrou sua citotoxicidade contra as linhagens celulares HepG2 e Caco-2 com o IC50 variando de 7,90 a 44,56 µmol.L-1. Os perfis de citotoxicidade indicam que as linhas tumorais apresentam maior atividade que as células MRC-5, com índices de seletividade variando de 1,58 a 8,96. Conclusão: Os três complexos tiveram melhor atividade in vitro do que a cisplatina, tanto para linhagens celulares normais como cancerosas. Os valores de IC50 são de duas a seis vezes melhores para as linhagens celulares cancerosas e de cinco a sete vezes melhores para as linhagens celulares normais. Este estudo indica que os complexos obtidos são promissores candidatos a fármacos antitumorais.


Introducción: Tres complejos de vanadio con ácidos orótico y glutámico, en sus formas aniónicas, fueram preparados. Su citotoxicidad in vitro hacia los fibroblastos pulmonares humanos (MRC-5), el carcinoma hepatocelular humano (HepG2) y el adenocarcinoma colorrectal humano (Caco-2) son reportados. Objetivo: Los principales objetivos de este trabajo son describir la síntesis y caracterización de nuevos complejos de vanadio con ácidos orótico y glutámico y probar su actividad antitumoral contra el HepG2 y el Caco-2. Método: Los complejos fueron formulados como VO (oro), VO (α-glu) y VO (γ-glu) basados en análisis químicos, termogravimétricos y espectros infrarrojos. El ensayo de resazurina demuestra su citotoxicidad contra las líneas celulares HepG2 y Caco-2 con el IC50 que van de 7,90 a 44,56 µmol.L-1. Los perfiles de citotoxicidad indican que las líneas tumorales presentan mayor actividad que los MRC-5, con índices de selectividad que van de 1,58 a 8,96. Conclusión: Los tres complejos tuvieron mejor actividad in vitro que el cisplatino, tanto para líneas celulares normales como para líneas celulares cancerosas. Los valores del IC50 son de dos a seis veces mejores para las líneas celulares de cáncer y de cinco a siete veces mejores para las líneas celulares normales. Este estudio indica que los complejos obtenidos son candidatos prometedores para fármacos antitumorales.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Carcinoma Hepatocelular , Ácido Orótico , Técnicas In Vitro , Compostos de Vanádio , Ácido Glutâmico
10.
J Dairy Sci ; 103(1): 433-438, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733874

RESUMO

The objective of this study was to evaluate the potential benefits of supplementing glutamic acid in milk replacers (MR) with respect to calf performance, intestinal permeability, and metabolism. Sixty Holstein male calves (3 ± 1.3 d old and 45 ± 5.9 kg body weight) were individually housed and fed a control MR without AA supplementation (24.8% crude protein and 19.1% fat, dry matter basis), or MR supplemented with 0.3% glutamic acid (25.1% crude protein and 20.3% fat, dry matter basis). Animals followed the same MR feeding program and were weaned at 56 d of the study. The amount of starter concentrate offered was restricted to limit the effect of concentrate intake on calf metabolism. Individual daily consumption and weekly body weight were measured, and 4 h after the morning feeding, blood samples were obtained at 14 and 35 d to determine general biochemical parameters and plasma AA concentrations. On d 10 of the study, we conducted an intestinal permeability test by including 21 g of lactulose and 4.2 g of d-mannitol as markers in the MR. We found no differences in calf performance or in intestinal permeability (measured as lactulose:mannitol ratio). Serum glucose concentration was greater in unsupplemented calves than in Glu-supplemented calves. At 14 d, the proportion of plasma Leu was greater in Glu-supplemented calves; the proportion of Ile tended to be greater in Glu-supplemented calves; and the proportion of Met tended to be greater in unsupplemented calves. We observed no other differences. Small changes occurred in AA metabolism when supplementing calf MR with 0.3% glutamic acid, without leading to improvements in calf performance or changes in intestinal permeability.


Assuntos
Ração Animal/análise , Bovinos/crescimento & desenvolvimento , Dieta/veterinária , Ácido Glutâmico/administração & dosagem , Intestinos/efeitos dos fármacos , Substitutos do Leite/administração & dosagem , Aminoácidos/sangue , Fenômenos Fisiológicos da Nutrição Animal , Animais , Peso Corporal , Bovinos/metabolismo , Suplementos Nutricionais , Intestinos/fisiologia , Masculino , Metaboloma , Leite , Permeabilidade , Desmame
11.
Biomed Khim ; 65(5): 361-365, 2019 Aug.
Artigo em Russo | MEDLINE | ID: mdl-31666406

RESUMO

In the model of induced neuronal resistance to the toxic effect of glutamate (deprivation of trophic factors), exosome secretion is demonstrated. Exosomes are secreted at the development of resistance during deprivation and at the first 24 h after preconditioning, as was shown by dot blot of extracellular fluid using anti-CD63 antibody. The autophagy inhibitor bafilomycin (0.01 µM) significantly reduces the quantity of the secreted exosomes at the stage of autophagy induction and at 24 h after induction. At the same time, inhibition of autophagy during the deprivation of trophic factors prevents the development of resistance, but inhibition of autophagy during the first 24 h after deprivation does not affect the development of resistance. We suggest that the long-term effects of preconditioning may be mediated by exosome secretion.


Assuntos
Autofagia , Exossomos/metabolismo , Neurônios/citologia , Células Cultivadas , Ácido Glutâmico , Humanos
12.
Nutr Metab Cardiovasc Dis ; 29(12): 1353-1360, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31668457

RESUMO

BACKGROUND AND AIM: Circulating level of glutamate, a by-product of the catabolism of branched-chain amino acids, has been positively correlated with visceral adipose tissue accumulation and waist circumference (WC). The aim of the present study was to assess the potential of using glutamate level to identify individuals with abdominal obesity and a high cardiometabolic risk. METHODS AND RESULTS: The study sample included 99 men and 99 women. Fasting serum glutamate was measured using the Biocrates p180 kit. Anthropometric and metabolic variables were used to identify individuals with abdominal obesity (WC ≥ 95 cm in both sexes), the hypertriglyceridemic waist (HTW) phenotype and the metabolic syndrome (MetS). Mean (±SD) age was 34.1 ± 10.1 years, mean BMI was 29.0 ± 6.2 kg/m2 and mean WC was 92.7 ± 16.5 cm. Glutamate was strongly correlated with WC (r = 0.66 for men; r = 0.76 for women, both p < 0.0001) and multiple markers of metabolic dysfunction, particularly fasting triglyceride level (r = 0.59 for men; r = 0.57 for women, both p < 0.0001), HDL-cholesterol level (r = -0.45, p < 0.0001 in both sexes) and the HOMA-IR index (r = 0.65 for men; r = 0.60 for women, both p < 0.0001). Logistic regressions showed that glutamate had an excellent accuracy to identify individuals with abdominal obesity (ROC_AUC: 0.90 for both sexes), a good accuracy to identify those with the HTW phenotype (ROC_AUC: 0.82 for men; 0.85 for women) and fair-to-good accuracy for the MetS (ROC_AUC: 0.78 for men; 0.89 for women). CONCLUSION: Glutamate level may represent an interesting potential biomarker of abdominal obesity and metabolic risk.


Assuntos
Ácido Glutâmico/sangue , Síndrome Metabólica/sangue , Obesidade Abdominal/sangue , Adiposidade , Adolescente , Adulto , Biomarcadores/sangue , HDL-Colesterol/sangue , Estudos Transversais , Jejum/sangue , Feminino , Humanos , Masculino , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/etiologia , Pessoa de Meia-Idade , Obesidade Abdominal/complicações , Obesidade Abdominal/diagnóstico , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Fatores Sexuais , Triglicerídeos/sangue , Regulação para Cima , Circunferência da Cintura , Adulto Jovem
13.
World J Microbiol Biotechnol ; 35(11): 175, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673852

RESUMO

The important metabolic intermediate 5-aminolevulinic acid (ALA) is useful for cancer treatment or plant growth regulation and has consequently received much attention. In this study, we introduced the HemA1 and pgr7 genes from the higher plant Arabidopsis thaliana into recombinant Escherichia coli to overproduce extracellular 5-aminolevulinic acid via the C5 pathway. In the E. coli BL21 (DE3) strain background, the ALA concentration of the strain expressing both HemA1 and pgr7 was the highest and reached 3080.62 mg/L. Among the 7 tested hosts, ALA production was the highest in E. coli Transetta (DE3). In E. coli Transetta GTR/GBP, the expression levels of zwf, gnd, pgl and RhtA were upregulated. Glutamate induced the expression of the GltJ, GltK, GltL and GltS genes that are in involved in glutamate uptake. The recombinant E. coli Transetta GTR/GBP was able to produce 7642 mg/L ALA in modified minimal medium supplemented with 10 g/L glutamate and 15 g/L glucose after 48 h of fermentation at 22 °C. The results provide persuading evidence for the efficient production of ALA from glucose and glutamate in E. coli expressing A. thaliana HemA1 and pgr7. Further optimization of the fermentation process should be done to improve the ALA production to an industrially relevant level.


Assuntos
Aldeído Oxirredutases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Escherichia coli/genética , Ácido Glutâmico/metabolismo , Ácidos Levulínicos/metabolismo , Proteínas de Membrana/genética , Aldeído Oxirredutases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Fermentação , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Glucose/metabolismo , Proteínas de Membrana/metabolismo , Via de Pentose Fosfato , Proteínas Recombinantes
14.
J Food Sci ; 84(12): 3584-3593, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31721210

RESUMO

Maillard reaction intermediate (MGX) generated from glutathione and xylose in aqueous medium was prepared via the Maillard reaction performed under a two-stage temperature increase process. The purified MGX was identified by Fourier-transform infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance as N-(1-deoxy-d-xylulos-1-yl)-glutathione (Amadori compound, C15 H25 O10 N3 S) with five main isomers. The method of Maillard reaction performed under a two-stage temperature increase process was further verified by high-performance liquid chromatography. The optimal reaction time and temperature for the preparation of MGX was determined as 60 min at 90 °C. The yield of MGX was increased from 8.60% to 55.52% through thermal reaction coupled with vacuum dehydration. In addition, rapid and more Maillard-type volatile compounds were formed in MGX during thermal treatment than that in Maillard reaction products or glutathione-xylose mixture. Beside, MGX possessed more pleasing meat-like volatile profile compared with the Amadori compound of glutamic acid-xylose (AAX), cysteine-xylose (ACX), and glycine-xylose (AGX). Therefore, it suggested that the MGX had the potential to achieve a better flavor formation during thermal treatment. PRACTICAL APPLICATION: Maillard reaction intermediates, such as Amadori or Heyns rearrangement products (ARP or HRP), are important flavor precursors, which possess stable physicochemical properties, but tend to degrade into flavor compounds at high temperatures. Maillard reaction intermediate from glutathione and xylose acts as primary flavor enhancers to complete Maillard reaction to produce flavors in the subsequent thermal processing, which can significantly improve and stabilize the flavor quality of the meaty food, and deserves a very broad application prospects. The new developed method will be a significant theoretical basis on research preparation and properties of Maillard reaction intermediates in complex food systems.


Assuntos
Glutationa/química , Xilose/química , Cisteína/química , Aromatizantes/química , Ácido Glutâmico/química , Produtos Finais de Glicação Avançada/química , Glicina/química , Temperatura Alta , Reação de Maillard , Vácuo
15.
Invest Ophthalmol Vis Sci ; 60(14): 4759-4773, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738824

RESUMO

Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.


Assuntos
Diferenciação Celular/fisiologia , Colinérgicos/farmacologia , Células Ependimogliais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/embriologia , Transdução de Sinais/fisiologia , Esferoides Celulares/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Embrião de Galinha , Colina O-Acetiltransferase/metabolismo , Crioultramicrotomia , Ácido Glutâmico/farmacologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neurônios Retinianos/citologia , Esferoides Celulares/metabolismo , Fixação de Tecidos , Vimentina/metabolismo
16.
Adv Exp Med Biol ; 1190: 23-31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31760635

RESUMO

Regulation of differentiation and proliferation of Schwann cells is an essential part of the regulation of peripheral nerve development, degeneration, and regeneration. ZNRF1, a ubiquitin ligase, is expressed in undifferentiated/repair Schwann cells, directs glutamine synthetase to proteasomal degradation, and thereby increase glutamate levels in Schwann cell environment. Glutamate elicits subcellular signaling in Schwann cells via mGluR2 to modulate Neuregulin-1/ErbB2/3 signaling and thereby promote undifferentiated phenotype of Schwann cell.


Assuntos
Ácido Glutâmico/fisiologia , Bainha de Mielina/fisiologia , Nervos Periféricos/fisiologia , Transdução de Sinais , Proteínas de Transporte/fisiologia , Diferenciação Celular , Humanos , Receptores de AMPA/fisiologia , Células de Schwann
17.
World J Microbiol Biotechnol ; 35(12): 192, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31773365

RESUMO

Corynebacterium glutamicum is generally regarded as a safe microorganism, and widely used in the large-scale production of various amino acids and organic acids, such as L-glutamate, L-lysine and succinic acid. During the process of industrial fermentation, C. glutamicum is usually exposed to varying environmental stresses, such as variations in pH, salinity, temperature, and osmolality. Among them, pH fluctuations are regarded as one of the most frequent environmental stresses in microbial fermentation. In this review, we summarize the current knowledge of pH homeostasis mechanisms adopted by C. glutamicum for coping with low acidic pH and high alkaline pH stresses. Facing with low pH environments, C. glutamicum develops a variety of strategies to maintain intracellular pH homeostasis, such as lowering intracellular reactive oxygen species, the improvement of potassium transport, the regulation of mycothiol-related pathways, as well as the repression of sulfur assimilation. While during alkaline pH stresses, the Mrp-type Na+/H+ antiporters are shown to play a dominant role in conferring C. glutamicum cells resistance to alkaline pH. Furthermore, we also discuss the general strategies and prospects on metabolic engineering of C. glutamicum to improve alkaline or acid resistance.


Assuntos
Corynebacterium glutamicum/metabolismo , Homeostase , Concentração de Íons de Hidrogênio , Ácidos/metabolismo , Adaptação Biológica , Álcalis/metabolismo , Carboxiliases/metabolismo , Membrana Celular/metabolismo , Resistência a Medicamentos , Ácido Glutâmico/metabolismo , Lisina/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas , Prótons , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Ácido Succínico/metabolismo , Enxofre/metabolismo
18.
J Biomed Nanotechnol ; 15(12): 2321-2331, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31748014

RESUMO

Herein, we report the functional decoration of single-walled carbon nanotubes (swCNTs) with Pt dendrimer-encapsulated nanoparticles (Pt DENs) (dia. (1.78 ± 0.18) nm) for the amperometric sensing of glutamate. The functional decoration of swCNTs was carried out via electrochemical grafting of Pt DENs onto swCNTs, and subsequent cross-linking of glutamate oxidase (GluOx) enzymes to the grafted Pt DENs on swCNT surfaces. The critical role of Pt DENs as catalytic immobilization matrix allowed both the immobilization of GluOx enzymes while maintaining the enzymatic activity of GluOx, and the electrocatalytic oxidation of H2O2 generated enzymatically in the presence of glutamate. Taking advantage of Pt DENs as catalytic immobilization matrix, the resulting swCNTs films, denoted as GluOx/Pt DEN/swCNTs, were applied as amperometric sensing platforms that display superior analytical characteristics, including sensitivity, selectivity, stability, and reproducibility, to the non-catalytic counterpart (i.e., GluOx/swCNTs), which led to the promising application of GluOx/Pt DEN/swCNTs to the practical analysis of glutamate in real samples.


Assuntos
Nanotubos de Carbono , Dendrímeros , Ácido Glutâmico , Peróxido de Hidrogênio , Nanopartículas Metálicas , Platina , Reprodutibilidade dos Testes
19.
Nat Neurosci ; 22(12): 2000-2012, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31712775

RESUMO

Acquisition and extinction of learned fear responses utilize conserved but flexible neural circuits. Here we show that acquisition of conditioned freezing behavior is associated with dynamic remodeling of relative excitatory drive from the basolateral amygdala (BLA) away from corticotropin releasing factor-expressing (CRF+) centrolateral amygdala neurons, and toward non-CRF+ (CRF-) and somatostatin-expressing (SOM+) neurons, while fear extinction training remodels this circuit back toward favoring CRF+ neurons. Importantly, BLA activity is required for this experience-dependent remodeling, while directed inhibition of the BLA-centrolateral amygdala circuit impairs both fear memory acquisition and extinction memory retrieval. Additionally, ectopic excitation of CRF+ neurons impairs fear memory acquisition and facilities extinction, whereas CRF+ neuron inhibition impairs extinction memory retrieval, supporting the notion that CRF+ neurons serve to inhibit learned freezing behavior. These data suggest that afferent-specific dynamic remodeling of relative excitatory drive to functionally distinct subcortical neuronal output populations represents an important mechanism underlying experience-dependent modification of behavioral selection.


Assuntos
Complexo Nuclear Basolateral da Amígdala/fisiologia , Núcleo Central da Amígdala/fisiologia , Medo/fisiologia , Ácido Glutâmico/fisiologia , Animais , Condicionamento Clássico/fisiologia , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Extinção Psicológica/fisiologia , Reação de Congelamento Cataléptica/fisiologia , Camundongos Transgênicos , Vias Neurais/fisiologia , Somatostatina/genética , Somatostatina/metabolismo
20.
BMC Med Genet ; 20(1): 162, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31638929

RESUMO

BACKGROUND: Several studies have focused on the relationship between MMP-8 variants and cancer risk, but they have been unsuccessful in drawing reliable conclusions. METHODS: We employed odds ratio (OR) together with 95% confidence interval (CI) to assess the correlation between MMP-8 C-799 T, Lys460Thr, and Lys87Glu polymorphisms and cancer risk. We further employed in silico tools to evaluate the effect of MMP-8 expression on cancer susceptibility and overall survival time. RESULTS: A total of 8140 patients with malignant carcinoma and 10,529 healthy individuals (control) were enrolled. Overall, the analysis showed that the relationship between three MMP-8 variants and cancer susceptibility was not significant (allelic contrast, C-799 T: OR = 0.98, 95% CI = 0.92-1.04, Pheterogeneity = 0.068; Lys460Thr: OR = 0.94, 95% CI = 0.67-1.32, Pheterogeneity = 0.905; Lys87Glu: OR = 1.05, 95% CI = 0.93-1.18, Pheterogeneity = 0.968). Similar results were observed in subgroup analysis by ethnicity, cancer type, and source of control. In silico analysis indicated that MMP-8 expression was elevated in bladder cancer tissue compared to that in the control. However, both the higher and lower MMP-8 expression groups did not show an impact on the overall survival time of the patients. CONCLUSIONS: MMP-8 C-799 T, Lys460Thr, and Lys87Glu variants are not participant with the susceptibility of cancer.


Assuntos
Predisposição Genética para Doença , Ácido Glutâmico/genética , Lisina/genética , Metaloproteinase 8 da Matriz/genética , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Treonina/genética , Humanos , Neoplasias/enzimologia
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