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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.
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
3.
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
4.
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
5.
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
6.
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
7.
Life Sci ; 238: 116898, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610193

RESUMO

AIMS: Learning and memory impairment is a common symptom in the early stages of various types of dementia. It is likely to reduce the incidence of dementia with correct intervention. α-Asarone is the main bioactive substance isolated from Acorus tatarinowii Schott and has been proven to improve memory dysfunction; however, at present, the specific underlying mechanism is poorly understood. The aim of the present study was to investigate the effect of α-asarone on ethanol-impaired cognitive ability and explore the underlying mechanism in mice. MAIN METHODS: A mouse model of impaired learning and memory was created by ethanol (2.0 g/kg, i.g.). α-Asarone (7.5, 15 or 30 mg/kg, i.p.) was delivered 10 min prior to ethanol administration. The behavioral effect of α-asarone was evaluated using the novel object recognition test. Glutamate (Glu) and γ-aminobutyric acid (GABA) levels in the hippocampus were determined by ELISA, and the protein expression levels of hippocampal GluR2, NMDAR2B, SYNΙ, GLT-1 and CaMKⅡ were detected by western blotting. KEY FINDINGS: Pretreatment with α-asarone significantly improved the behavioral performance, regulated the imbalance of Glu and GABA in the hippocampus and the abnormal expression of related proteins. A possible underlying mechanism is regulation of the calcium signaling cascade to correct functioning of related proteins, and thus, maintain the level of Glu. SIGNIFICANCE: Our results show that the improvement in learning and memory elicited by α-asarone may providing a possible novel candidate for the prevention of learning and memory impairment in the early stages of dementia.


Assuntos
Anisóis/farmacologia , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Fibrinolíticos/farmacologia , Hipocampo/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Animais , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurotransmissores/metabolismo
8.
Neurochem Res ; 44(11): 2449-2459, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31571097

RESUMO

Manganese (Mn) overexposure is a public health concern due to its widespread industrial usage and the risk for environmental contamination. The clinical symptoms of Mn neurotoxicity, or manganism, share several pathological features of Parkinson's disease (PD). Biologically, Mn is an essential trace element, and Mn in the brain is preferentially localized in astrocytes. This review summarizes the role of astrocytes in Mn-induced neurotoxicity, specifically on the role of neurotransmitter recycling, neuroinflammation, and genetics. Mn overexposure can dysregulate astrocytic cycling of glutamine (Gln) and glutamate (Glu), which is the basis for Mn-induced excitotoxic neuronal injury. In addition, reactive astrocytes are important mediators of Mn-induced neuronal damage by potentiating neuroinflammation. Genetic studies, including those with Caenorhabditis elegans (C. elegans) have uncovered several genes associated with Mn neurotoxicity. Though we have yet to fully understand the role of astrocytes in the pathologic changes characteristic of manganism, significant strides have been made over the last two decades in deciphering the role of astrocytes in Mn-induced neurotoxicity and neurodegeneration.


Assuntos
Astrócitos/metabolismo , Intoxicação por Manganês/fisiopatologia , Manganês/toxicidade , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Neurônios/metabolismo
9.
Acta Biochim Biophys Sin (Shanghai) ; 51(11): 1134-1141, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31650158

RESUMO

The widely used inhalation anesthetic, isoflurane, potentially induces neuronal injury in clinical practice. Previous studies showed multiple forms of cell death that resulted from isoflurane-induced cytotoxicity, but the precise underlying mechanism remains poorly understood. Ferroptosis has recently been identified as a non-apoptotic form of regulated cell death. Here, we found that ferroptosis inhibitors, ferrostatin-1 and deferoxamine mesylate (DFOM), showed great efficiency in maintaining cell viability in SH-SY5Y neuroblastoma cells exposed to a high concentration of isoflurane for 24 h. We also observed that cellular chelatable iron and lipid peroxidation were increased in a concentration-dependent manner in response to isoflurane. In addition, isoflurane upregulated Beclin1 phosphorylation, followed by the formation of a Beclin1-solute carrier family 7 member 11 (SLC7A11) complex, which affected the activity of cystine/glutamate antipoter and further regulated ferroptotic cell death. Accordingly, Beclin1 overexpression aggravated isoflurane-induced cell damage by upregulating ferroptosis. This phenomenon was significantly attenuated by silencing of Beclin1 in SH-SY5Y cells. These findings indicate that Beclin1 may regulate ferroptosis in a manner involving inhibition of glutamate exchange activity of system xc(-), which is implicated in isoflurane-induced toxicity. In particular, when isoflurane is administrated at high concentrations and for an extended duration, ferroptosis is more likely to play a crucial role in isoflurane-induced toxicity.


Assuntos
Proteína Beclina-1/fisiologia , Ácido Glutâmico/metabolismo , Ferro/metabolismo , Isoflurano/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cicloexilaminas/farmacologia , Desferroxamina/farmacologia , Humanos , Fenilenodiaminas/farmacologia
10.
Nat Commun ; 10(1): 4814, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645553

RESUMO

Sensory hypersensitivity is a common and debilitating feature of neurodevelopmental disorders such as Fragile X Syndrome (FXS). How developmental changes in neuronal function culminate in network dysfunction that underlies sensory hypersensitivities is unknown. By systematically studying cellular and synaptic properties of layer 4 neurons combined with cellular and network simulations, we explored how the array of phenotypes in Fmr1-knockout (KO) mice produce circuit pathology during development. We show that many of the cellular and synaptic pathologies in Fmr1-KO mice are antagonistic, mitigating circuit dysfunction, and hence may be compensatory to the primary pathology. Overall, the layer 4 network in the Fmr1-KO exhibits significant alterations in spike output in response to thalamocortical input and distorted sensory encoding. This developmental loss of layer 4 sensory encoding precision would contribute to subsequent developmental alterations in layer 4-to-layer 2/3 connectivity and plasticity observed in Fmr1-KO mice, and circuit dysfunction underlying sensory hypersensitivity.


Assuntos
Síndrome do Cromossomo X Frágil/metabolismo , Neurônios/metabolismo , Córtex Somatossensorial/metabolismo , Sinapses/metabolismo , Potenciais de Ação , Animais , Simulação por Computador , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Fenótipo , Córtex Somatossensorial/citologia
11.
Nat Commun ; 10(1): 4813, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645626

RESUMO

Cellular and circuit hyperexcitability are core features of fragile X syndrome and related autism spectrum disorder models. However, the cellular and synaptic bases of this hyperexcitability have proved elusive. We report in a mouse model of fragile X syndrome, glutamate uncaging onto individual dendritic spines yields stronger single-spine excitation than wild-type, with more silent spines. Furthermore, fewer spines are required to trigger an action potential with near-simultaneous uncaging at multiple spines. This is, in part, from increased dendritic gain due to increased intrinsic excitability, resulting from reduced hyperpolarization-activated currents, and increased NMDA receptor signaling. Using super-resolution microscopy we detect no change in dendritic spine morphology, indicating no structure-function relationship at this age. However, ultrastructural analysis shows a 3-fold increase in multiply-innervated spines, accounting for the increased single-spine glutamate currents. Thus, loss of FMRP causes abnormal synaptogenesis, leading to large numbers of poly-synaptic spines despite normal spine morphology, thus explaining the synaptic perturbations underlying circuit hyperexcitability.


Assuntos
Potenciais de Ação/fisiologia , Espinhas Dendríticas/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Ácido Glutâmico/metabolismo , Sinapses/metabolismo , Animais , Espinhas Dendríticas/ultraestrutura , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/patologia , Masculino , Camundongos , Camundongos Knockout , Neurogênese , Neurônios/metabolismo , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Córtex Somatossensorial/citologia , Sinapses/ultraestrutura
12.
Physiol Int ; 106(3): 250-260, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31564120

RESUMO

PURPOSE: Glioblastoma cells create glutamate-rich tumor microenvironment, which initiates activation of ion channels and modulates downstream intracellular signaling. N-methyl-D-aspartate receptors (NMDARs; a type of glutamate receptors) have a high affinity for glutamate. The role of NMDAR activation on invasion of glioblastoma cells and the crosstalk with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is yet to be explored. MAIN METHODS: LN18, U251MG, and patient-derived glioblastoma cells were stimulated with NMDA to activate NMDAR glutamate receptors. The role of NMDAR activation on invasion and migration and its crosstalk with AMPAR were evaluated. Invasion and migration of glioblastoma cells were investigated by in vitro trans-well Matrigel invasion and trans-well migration assays, respectively. Expression of NMDARs and AMPARs at transcript level was evaluated by quantitative real-time polymerase chain reaction. RESULTS: We determined that NMDA stimulation leads to enhanced invasion in LN18, U251MG, and patient-derived glioblastoma cells, whereas inhibition of NMDAR using MK-801, a non-competitive antagonist of the NMDAR, significantly decreased the invasive capacity. Concordant with these findings, migration was significantly augmented by NMDAR in both cell lines. Furthermore, NMDA stimulation upregulated the expression of GluN2 and GluA1 subunits at the transcript level. CONCLUSIONS: This study demonstrated the previously unexplored role of NMDAR in invasion of glioblastoma cells. Furthermore, the expression of the GluN2 subunit of NMDAR and the differential overexpression of the GluA1 subunit of AMPAR in both cell lines provide a plausible rationale of crosstalk between these calcium-permeable subunits in the glutamate-rich microenvironment of glioblastoma.


Assuntos
Glioblastoma/metabolismo , Glioblastoma/patologia , Invasividade Neoplásica/patologia , Receptores de N-Metil-D-Aspartato/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Maleato de Dizocilpina/farmacologia , Glioblastoma/tratamento farmacológico , Ácido Glutâmico/metabolismo , Humanos , Receptores de AMPA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
13.
Nat Commun ; 10(1): 4093, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501438

RESUMO

ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in this simple circuit, we focus on investigating the physiological properties and behavioral relevance of larval visual interneurons. We find that the ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Gαo signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, our studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system.


Assuntos
Drosophila melanogaster/fisiologia , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Vias Visuais/metabolismo , Animais , Comportamento Animal/efeitos da radiação , Cálcio/metabolismo , Drosophila melanogaster/efeitos da radiação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Interneurônios/efeitos da radiação , Larva/efeitos da radiação , Luz , Neurópilo/metabolismo , Neurópilo/efeitos da radiação , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/efeitos da radiação
14.
Biochem Mol Biol Educ ; 47(6): 620-631, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31520514

RESUMO

Bioinformatics was recently introduced as a module for both undergraduate and postgraduate biological sciences students at our institution. Our experience shows that inquiry-based hands-on exercises provide the most efficient approach to bioinformatic straining. In this article, we report a structural bioinformatics project carried out by Master degree students to determine structure-function relationships of the uncharacterized prokaryotic 5-oxoprolinase subunit A (PxpA). PxpA associates with the PxpBC complex to form a functional 5-oxoprolinase enzyme for conversion of 5-oxoproline to L-glutamate. Although the exact role of PxpA is yet to be determined, it has been demonstrated that PxpBC catalyses the first step of the reaction, which is phosphorylation of 5-oxoproline. Here, we provide evidence that PxpA is involved in the last two steps of the reaction:decyclization of the labile phosphorylated 5-oxoproline to the equally labile γ-glutamylphosphate, and subsequent dephosphorylation to L-glutamate. Structural bioinformatics analysis of four putative PxpA structures revealed that PxpA adopts a non-canonical TIM barrel fold with well-characterized TIM barrel enzyme features. These include a C-terminal groove comprising potentially essential conserved amino acid residues organized into putative motifs. Phylogenetic analysis suggests a relationship between taxonomic grouping and PxpA oligomerization. PxpA forms a tunnel upon ligand binding, thus suggesting that the PxpABC complex employs the mechanism of substrate channeling to protect labile intermediates. Ultimately, students were able to form a testable hypothesis on the function of PxpA, an achievement we consider encouraging other students to emulate. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):620-631, 2019.


Assuntos
Disciplinas das Ciências Biológicas/educação , Biologia Computacional/educação , Piroglutamato Hidrolase/química , Piroglutamato Hidrolase/metabolismo , Currículo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Humanos , Modelos Moleculares , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/metabolismo , Relação Estrutura-Atividade , Estudantes
15.
Biochimie ; 167: 61-67, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31520657

RESUMO

Ribosomal protein S6 in Escherichia coli is modified by ATP-dependent glutamate ligase RimK. Up to four glutamate residues are added to the C-terminus of S6 protein. In this work we demonstrated that unlike the majority of ribosome modifications in E. coli, oligoglutamylation of S6 protein is regulated and happens only in the stationary phase of bacterial culture. Only S6 protein incorporated into assembled small ribosomal subunits, but not newly made free S6 protein is a substrate for RimK protein. Overexpression of the rimK gene leads to the modification of S6 protein even in the exponential phase of bacterial culture. Thus, it is unlikely that any stationary phase specific factor is needed for the modification. We propose a model that S6 modification is regulated solely via the rate of ribosome biosynthesis at limiting concentration of RimK enzyme.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Peptídeo Sintases/metabolismo , Proteína S6 Ribossômica/metabolismo , Proteínas Ribossômicas/metabolismo , Escherichia coli/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo
16.
Int J Mol Sci ; 20(18)2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31540330

RESUMO

Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1G93A mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.


Assuntos
Esclerose Amiotrófica Lateral/genética , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Superóxido Dismutase-1/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Ácido Glutâmico/metabolismo , Glicina/administração & dosagem , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Camundongos , Mutação , Receptor de Glutamato Metabotrópico 5/genética , Receptores de Glutamato Metabotrópico/genética , Resorcinóis/administração & dosagem , Resorcinóis/farmacologia , Medula Espinal/metabolismo , Regulação para Cima
17.
Nat Commun ; 10(1): 4356, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554789

RESUMO

Itch is a distinct aversive sensation that elicits a strong urge to scratch. Despite recent advances in our understanding of the peripheral basis of itch, we know very little regarding how central neural circuits modulate acute and chronic itch processing. Here we establish the causal contributions of defined periaqueductal gray (PAG) neuronal populations in itch modulation in mice. Chemogenetic manipulations demonstrate bidirectional modulation of scratching by neurons in the PAG. Fiber photometry studies show that activity of GABAergic and glutamatergic neurons in the PAG is modulated in an opposing manner during chloroquine-evoked scratching. Furthermore, activation of PAG GABAergic neurons or inhibition of glutamatergic neurons resulted in attenuation of scratching in both acute and chronic pruritis. Surprisingly, PAG GABAergic neurons, but not glutamatergic neurons, may encode the aversive component of itch. Thus, the PAG represents a neuromodulatory hub that regulates both the sensory and affective aspects of acute and chronic itch.


Assuntos
Vias Neurais/fisiologia , Substância Cinzenta Periaquedutal/fisiologia , Prurido , Animais , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/fisiologia , Ácido Glutâmico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/citologia , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Substância Cinzenta Periaquedutal/citologia
18.
Int J Mol Sci ; 20(18)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546771

RESUMO

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aß oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas PrPC/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doença de Alzheimer/patologia , Animais , Ácido Glutâmico/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Glutamato/metabolismo
19.
Int J Mol Sci ; 20(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500132

RESUMO

Parkinson's disease is a progressive neurodegenerative disorder resulting from the degeneration of pigmented dopaminergic neurons in the substantia nigra pars compacta. It induces a series of functional modifications in the circuitry of the basal ganglia nuclei and leads to severe motor disturbances. The amino acid glutamate, as an excitatory neurotransmitter, plays a key role in the disruption of normal basal ganglia function regulated through the interaction with its receptor proteins. It has been proven that glutamate receptors participate in the modulation of neuronal excitability, transmitter release, and long-term synaptic plasticity, in addition to being related to the altered neurotransmission in Parkinson's disease. Therefore, they are considered new targets for improving the therapeutic strategies used to treat Parkinson's disease. In this review, we discuss the biological characteristics of these receptors and demonstrate the receptor-mediated neuroprotection in Parkinson's disease. Pharmacological manipulation of these receptors during anti-Parkinsonian processes in both experimental studies and clinical trials are also summarized.


Assuntos
Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Receptores de Glutamato/metabolismo , Animais , Ensaios Clínicos como Assunto , Descoberta de Drogas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Humanos , Terapia de Alvo Molecular , Neurotransmissores/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Receptores de Glutamato/genética , Transdução de Sinais/efeitos dos fármacos , Substância Negra/metabolismo , Transmissão Sináptica , Resultado do Tratamento
20.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480244

RESUMO

Major depressive disorder is one of the most common neuropsychiatric disorders worldwide. The treatment of choice that shows good efficacy in mood stabilization is based on selective serotonin reuptake inhibitors (SSRIs). Their primary mechanism of action is considered to be the increased synaptic concentration of serotonin through blockade of the serotonin transporter (SERT). In this study, we described an alternative mode of action of fluoxetine (FLX), which is a representative member of the SSRI class of antidepressants. We observed that FLX robustly decreases both glutamatergic and gamma-Aminobutyric acid (GABA)-ergic synaptic release in a SERT-independent manner. Moreover, we showed that this effect may stem from the ability of FLX to change the levels of main components of the SNARE (solubile N-ethylmaleimide-sensitive factor attachment protein receptor) complex. Our data suggest that this downregulation of SNARE fusion machinery involves diminished activity of protein kinase C (PKC) due to FLX-induced blockade of P/Q type of voltage-gated calcium channels (VGCCs). Taken together, by virtue of its inhibition at SERT, fluoxetine increases extracellular serotonin levels; however, at the same time, by reducing SNARE complex function, this antidepressant reduces glutamate and GABA release.


Assuntos
Fluoxetina/farmacologia , Ácido Glutâmico/metabolismo , Proteínas SNARE/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Canais de Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Feminino , Humanos , Modelos Neurológicos , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Proteína Quinase C/metabolismo , Ratos Wistar
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