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1.
Plant Sci ; 289: 110243, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31623796

RESUMO

Maize (Zea mays) seeds are the major source of starch all over the world and the excellent model for researching starch synthesis. Seed starch content is a typical quantitative phenotype and many reports revealed that the glycolytic enzymes are involved in regulating starch synthesis, however the regulatory mechanism is still unclear. Here, we present a comparative phosphoproteomic study of three maize inbred lines with different seed starch content. It reveals that abundances of 62 proteins and 63 phosphoproteins were regulated during maize seed development. Dynamics of 17 enzymes related to glycolysis and starch synthesis were used to construct a phosphorylation regulatory network of starch synthesis. It shows that starch synthesis and glycolysis in maize seeds utilize the same hexose phosphates pool coming from sorbitol and sucrose as carbon source, and phosphorylation of ZmENO1 are suggested to contribute to increase starch content, because it is positively related to seed starch content in different developmental stages and different lines, and the phosphor-mimic mutant (ZmENO1S43D) damaged its enzyme activity which is vital in glycolysis. Our results provide a new sight into regulatory process of seed starch synthesis and can be used in maize breeding for high starch content.


Assuntos
Regulação da Expressão Gênica de Plantas , Fosfoproteínas/genética , Fosfopiruvato Hidratase/genética , Proteínas de Plantas/genética , Proteoma/genética , Amido/metabolismo , Zea mays/metabolismo , Fosfoproteínas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Zea mays/enzimologia , Zea mays/genética , Zea mays/crescimento & desenvolvimento
2.
J Biochem Mol Toxicol ; 33(10): e22382, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31468657

RESUMO

Vanillic acid (VA) is found in high concentrations in various plants and used as traditional medicine for various diseases. The aim of the existing study is to illustrate the protective effects of VA against benzo(a)pyrene (B(a)P)-induced lung cancer in Swiss albino mice. B(a)P (50 mg/kg b.wt.) was given orally to induce lung cancer in mice. The body weight, tumor incidence, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and enzymatic/nonenzymatic antioxidants (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and glutathione) were estimated. Further histochemical investigation through hematoxylin and eosin staining was also carried out. B(a)P administered groups showed increased levels of serum pathological markers CEA, NSE along with reduced final body weight as well as decreased tissue enzymatic and nonenzymatic antioxidants activities, whereas VA treatment (200mg/kg/b.wt) along with B(a)P showed significantly reverted the above changes, which proves as prominent anticancer effects in experimentally induced lung cancer. Overall, these results suggest that VA has an efficient preventive action against B(a)P-induced lung cancer, and this is attributed to its free-radical scavenging antioxidant activities.


Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/prevenção & controle , Ácido Vanílico/farmacologia , Animais , Antioxidantes/metabolismo , Antígeno Carcinoembrionário/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-31263685

RESUMO

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma, whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase (Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.


Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Mycoplasma hyopneumoniae/enzimologia , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/isolamento & purificação , Animais , Cristalografia por Raios X , Células Epiteliais/microbiologia , Modelos Moleculares , Mycoplasma hyopneumoniae/metabolismo , Mycoplasma hyopneumoniae/patogenicidade , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificação , Plasminogênio/metabolismo , Pneumonia Suína Micoplasmática/microbiologia , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Suínos , Fatores de Virulência
4.
J Physiol Pharmacol ; 70(2)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31356182

RESUMO

Acute cerebral ischemia triggers local and systemic immune response. The aims of this project was to assess the blood serum concentration of the markers of inflammation and markers of the blood brain barrier damage on the first day of ischemic stroke, and the mutual correlations between these marker levels. Patients with first-in-life stroke were analysed according to: plasma concentration of the following markers on the first day of stroke: interleukin 2 (IL-2) and interleuki 6 (IL-6), S100B, tumor necrosis factor-α (TNF-α), progranulin (GRN), neuron specific enolase (NSE), urokinase-type plasminogen activator (uPA), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), C-reactive protein (CRP), leucocyte and thrombocyte counts; their neurological status on the first day of stroke (NIHSS) and their functional status at 30 days following stroke (mRS). The study included 138 patients with mean age: 73.11 ± 11.48. Patients with a higher score on the NIHSS showed significantly higher concentrations of TNF-α, white blood cells (WBC), CRP, NSE, IL-6 and S100B. Patients with a higher score on the modified Rankin Score (mRS) showed significantly higher concentrations of WBC, CRP, GRN, IL-6, S100B. Factors with an independent influence on the neurological status on the first day of stroke were: sex, WBC, total blood platelet (PLT) count, CRP, S100B and IL-6 levels. Atrial fibrillation, leukocyte count, CRP, NSA, uPA, IL-6 and S100B showed an independent impact on the functional status on the 30th day of stroke. Patients with symptomatic atherosclerosis, as compared to others, were older (P = 0.003) and had higher levels of CRP, IL-6, and S100B. In each case, the differences were statistically significant. We conclude that the concentration of Il-6 and S100B on the first day of stroke are important for both the neurological status and the functional status in the acute period of the disease. Increased CRP and leukocyte count are associated with a worse prognosis regarding the course of acute stroke. The expression of pro-inflammatory agents and markers of blood-brain barrier damage in the acute phase of stroke seem to be more prominent in patients with symptomatic atherosclerosis than in patients with no clinical features of atherosclerosis. The expression of inflammatory parameters may indicate the importance of the inflammatory process starting during the early days of ischemic stroke, for the post-stroke neurological deficit.


Assuntos
Biomarcadores/sangue , Barreira Hematoencefálica/patologia , Isquemia Encefálica/patologia , Inflamação/patologia , Acidente Vascular Cerebral/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aterosclerose/sangue , Aterosclerose/metabolismo , Aterosclerose/patologia , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/sangue , Isquemia Encefálica/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína C-Reativa/metabolismo , Feminino , Humanos , Inflamação/sangue , Interleucina-6/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfopiruvato Hidratase/metabolismo , Prognóstico , Estudos Prospectivos , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Neuron ; 103(2): 266-276.e4, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31153647

RESUMO

Motor skills improve with practice, requiring outcomes to be evaluated against ever-changing performance benchmarks, yet it remains unclear how performance error signals are computed. Here, we show that the songbird ventral pallidum (VP) is required for song learning and sends diverse song timing and performance error signals to the ventral tegmental area (VTA). Viral tracing revealed inputs to VP from auditory and vocal motor thalamus, auditory and vocal motor cortex, and VTA. Our findings show that VP circuits, commonly associated with hedonic functions, signal performance error during motor sequence learning.


Assuntos
Prosencéfalo Basal/fisiologia , Dopamina/metabolismo , Vias Neurais/fisiologia , Neurônios/fisiologia , Área Tegmentar Ventral/fisiologia , Acelerometria , Potenciais de Ação/fisiologia , Animais , Biofísica , Toxina da Cólera/metabolismo , Estimulação Elétrica/efeitos adversos , Tentilhões , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Comportamento Imitativo/fisiologia , Masculino , Movimento/fisiologia , Fosfopiruvato Hidratase/metabolismo , Tempo de Reação/fisiologia , Fatores de Tempo , Transdução Genética , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismo , Vigília
6.
Biochim Biophys Acta Proteins Proteom ; 1867(9): 794-801, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31202897

RESUMO

SIRT1 (Silent mating type information regulation 2 homolog 1) play a neuroprotective effect through deacetylation target proteins in various neuronal diseases. However, the precise mechanisms remain elusive. In this study, we aim to identify those novel interacting partners of SIRT1 in rat brain tissue. By using a pre-clear GST-Pull down assay followed by the LC-MS/MS analysis, we've identified potential SIRT1's interacting partners, which function annotation by GO and KEGG analysis indicating some metabolic pathways are among the most enriched. Then we confirmed two candidates Enolase-1 (and NSE (Neuron-Specific Enolase) in brain) and PKM (Pyruvate Kinase Muscle) are associated with SIRT1 in brain tissue lysis by co-immunoprecipitation. Furthermore, increase or decrease the SIRT1 enzyme activity by its agonist SRT1720 or antagonist EX527 could significantly affect the acetylation level of endogenous NSE and PKM, SIRT1 overexpression or knock out expreiments also showed the same results as use SIRT1's agonist or antagonist. Moreover, the acetylation changes on NSE or PKM could finally lead to affection on their catalytic activity. Taken together, our findings suggest that the function of SIRT1 binding proteins is enriched in metabolic pathways. NSE and PKM are new SIRT1 binding molecules. SIRT1 may regulate acetylation level of NSE and PKM through deacetylation and further regulate their catalytic activity. Our study provides new evidence for the involvement of SIRT1 in the mechanisms of metabolic regulation in central nervous system.


Assuntos
Encéfalo/enzimologia , Fosfopiruvato Hidratase/metabolismo , Piruvato Quinase/metabolismo , Sirtuína 1/metabolismo , Acetilação/efeitos dos fármacos , Animais , Carbazóis/farmacologia , Catálise/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Fosfopiruvato Hidratase/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Piruvato Quinase/genética , Ratos , Ratos Sprague-Dawley , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética
7.
Anal Chim Acta ; 1068: 18-27, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31072474

RESUMO

In this work, water-soluble pillar[6]arene functionalized PdPt porous core-shell octahedral nanodendrites (WP6@PdPt PCONs) were simply synthesized and used for the fabrication of NSE immunosensor. The newest generation of macrocyclic host and biomimetic nano-enzymes have been effectively integrated to achieve the robust immobilization of signal molecules by host-guest molecular recognition and sensitively catalytic amplification of electrochemical signals. The addition of Pd and the formation of WP6@PdPt PCONs unique bimetallic nanostructure are beneficial to changing Pt-based catalyst electronic structure, accelerating the electron transport, promoting the generation of synergistic catalysis effect. Compared with enzyme-based methods, the fabricated sandwich-type immunosensor demonstrates more advantages in robustness owing to the introduction of host-guest chemistry and biomimetic nano-enzymes. In the wide range from 0.0003 to 100.00 ng mL-1, a good linear relationship (R2 = 0.998) and a low LOD (0.095 pg mL-1, S/N = 3) can be obtained. The proposed immunosensor shows remarkable analytical performances in the measurements of selectivity, stability, reproducibility, and real sample analysis, which provided a promising approach for clinical detection of NSE in human serum. Besides, the successful synthesis of WP6@PdPt PCONs provides a new idea for the preparation of biosensors based on bionic materials, nanotechnology and host-guest molecule recognition.


Assuntos
Imunoensaio , Nanocompostos/química , Fosfopiruvato Hidratase/sangue , Compostos de Amônio Quaternário/química , Biocatálise , Técnicas Biossensoriais , Técnicas Eletroquímicas , Humanos , Paládio/química , Tamanho da Partícula , Fosfopiruvato Hidratase/metabolismo , Platina/química , Porosidade , Solubilidade , Propriedades de Superfície , Água/química
8.
Food Chem ; 289: 240-249, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955608

RESUMO

This study was designed to investigate the differences in the proteomes of high- and low-quality goat and bovine muscles. In total, 364 and 111 differentially expressed proteins (DEPs) were identified in the high-quality/low-quality bovine and goat samples, respectively. The bioinformatic analysis demonstrated that DEPs are involved in glycolysis, the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation. An analysis of quality-related DEPs showed that nicotinamide adenine dinucleotide dehydrogenase (NADH) and succinate dehydrogenase (SDH) could be potential biomarkers for colour, while enolase (ENO) can be used as a marker protein for tenderness qualities in goat. Lactate dehydrogenase (LDH) and 14-3-3 protein can indicate goat and bovine tenderness, while heat-shock proteins (HSPs) can also be used as tenderness marker proteins for different species. Glycerin-3-phosphate dehydrogenase (GPDH) can be used as a protein to detect fat content, and guanine ribonucleotide-binding protein (G protein) may be a flavour marker protein in bovine meat.


Assuntos
Proteoma/análise , Proteômica , Animais , Biomarcadores/metabolismo , Bovinos , Ciclo do Ácido Cítrico , Biologia Computacional , Glicólise , Cabras , Proteínas de Choque Térmico/metabolismo , L-Lactato Desidrogenase/metabolismo , Músculo Esquelético/metabolismo , Fosfopiruvato Hidratase/metabolismo , Succinato Desidrogenase/metabolismo
9.
J Biol Chem ; 294(22): 8930-8941, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-30952697

RESUMO

Bacillus anthracis is the causative agent of anthrax in humans, bovine, and other animals. B. anthracis pathogenesis requires differentiation of dormant spores into vegetative cells. The spores inherit cellular components as phenotypic memory from the parent cell, and this memory plays a critical role in facilitating the spores' revival. Because metabolism initiates at the beginning of spore germination, here we metabolically reprogrammed B. anthracis cells to understand the role of glycolytic enzymes in this process. We show that increased expression of enolase (Eno) in the sporulating mother cell decreases germination efficiency. Eno is phosphorylated by the conserved Ser/Thr protein kinase PrkC which decreases the catalytic activity of Eno. We found that phosphorylation also regulates Eno expression and localization, thereby controlling the overall spore germination process. Using MS analysis, we identified the sites of phosphorylation in Eno, and substitution(s) of selected phosphorylation sites helped establish the functional correlation between phosphorylation and Eno activity. We propose that PrkC-mediated regulation of Eno may help sporulating B. anthracis cells in adapting to nutrient deprivation. In summary, to the best of our knowledge, our study provides the first evidence that in sporulating B. anthracis, PrkC imprints phenotypic memory that facilitates the germination process.


Assuntos
Bacillus anthracis/fisiologia , Proteínas de Bactérias/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Esporos Bacterianos/metabolismo , Bacillus anthracis/enzimologia , Proteínas de Bactérias/genética , Cinética , Magnésio/metabolismo , Mutagênese Sítio-Dirigida , Fosfopiruvato Hidratase/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
10.
Epilepsy Res ; 151: 78-84, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30844661

RESUMO

Temporal lobe epilepsy (TLE) is the most common form of intractable epilepsy. Currently, the molecular mechanisms underlying epileptogenesis in TLE remain elusive; however, synaptic transmission may play an important role in the pathogenesis of epilepsy. Synaptic transmission is regulated by diverse mechanisms, including presynaptic modulators of synaptic vesicle formation and release, modulators of neurotransmission and distinct Ca2+ sensors. DOC2A, a novel Ca2+ sensor, can regulate spontaneous synaptic transmission and has been implicated in Ca2+-dependent neurotransmitter release. In this study, we demonstrate for the first time that DOC2A expression is significantly increased in human TLE and in two different rat models of TLE (pilocarpine- and kindling-induced) compared to the control groups. Localization of DOC2A in the human TLE patients and pilocarpine post-SE rat model was observed in neurons but not in astrocytes; DOC2A was also concentrated at the presynaptic terminals and colocalized with VMAT2. Our results suggest that the abnormal protein expression of DOC2A in epileptic brain tissue may play an important role in epilepsy.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Epilepsia do Lobo Temporal/patologia , Proteínas do Tecido Nervoso/metabolismo , Lobo Temporal/metabolismo , Adolescente , Adulto , Animais , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/induzido quimicamente , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Excitação Neurológica/patologia , Excitação Neurológica/fisiologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Agonistas Muscarínicos/toxicidade , Fosfopiruvato Hidratase/metabolismo , Pilocarpina/toxicidade , Lobo Temporal/efeitos dos fármacos , Adulto Jovem
11.
Brain Behav ; 9(3): e01221, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30714330

RESUMO

BACKGROUND: Pettigrew syndrome (PGS) is a rare X-linked mental retardation that caused by AP1S2 mutation. The pathogenesis of AP1S2 deficiency has remained elusive. The purpose of this study is to give a comprehensive overview of the phenotypic and genetic spectrum of AP1S2 mutations. METHODS: This study systematically analyzed clinical features and genetic information of a Chinese family with AP1S2 variation, and reviewed previously reported literatures with the same gene variation. RESULTS: We identified a new c.1-1 G>C mutation in AP1S2 gene from a four generation family with seven affected individuals and found the elevated neuron-specific enolase (NSE) in a patient. We summarized the clinical manifestation of 59 patients with AP1S2 mutation. We found that pathogenic point mutations affecting AP1S2 are associated with dysmorphic features and neurodevelopmental problems, which included highly variable mental retardation (MR), delayed in walking, abnormal speech, hypotonia, abnormal brain, abnormal behavior including aggressive behavior, ASD, self-abusive, and abnormal gait. Patients with splice site mutation were more likely to lead to seizures. By contrast, patients with nonsense mutations are more susceptible to microcephaly. CONCLUSION: Our findings suggest AP1S2 mutations contribute to a broad spectrum of neurodevelopmental disorders and are important in the etiological spectrum of PGS.


Assuntos
Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Doenças dos Gânglios da Base , Transtornos Dismórficos Corporais , Síndrome de Dandy-Walker , Retardo Mental Ligado ao Cromossomo X , Transtornos do Neurodesenvolvimento , Convulsões , Adulto , Doenças dos Gânglios da Base/complicações , Doenças dos Gânglios da Base/genética , Doenças dos Gânglios da Base/psicologia , Transtornos Dismórficos Corporais/etiologia , Transtornos Dismórficos Corporais/genética , Criança , Pré-Escolar , Síndrome de Dandy-Walker/complicações , Síndrome de Dandy-Walker/genética , Síndrome de Dandy-Walker/psicologia , Feminino , Humanos , Masculino , Retardo Mental Ligado ao Cromossomo X/complicações , Retardo Mental Ligado ao Cromossomo X/genética , Retardo Mental Ligado ao Cromossomo X/psicologia , Mutação , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/genética , Linhagem , Fosfopiruvato Hidratase/metabolismo , Convulsões/complicações , Convulsões/genética , Convulsões/psicologia
12.
ScientificWorldJournal ; 2019: 6264072, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30728755

RESUMO

Pluripotent stem cells are used in regenerative medicine and exist in various internal organs. However, there are a small number of reports of neural cells or neural stem cells existing in the spleen. In this study, we sought to identify possible neural stem cells in the mouse spleen. The spleens of ICR mice were removed and small specimens were incubated in Dulbecco's modified Eagle's medium with Nutrient Mixture F-12 containing either 10% fetal bovine serum (FBS), 20% FBS, 10% neonate bovine serum, or 10% fetal calf serum. Neural cell medium was also used. The cultured cells were investigated for expression of the neural cell markers neuron-specific enolase (NSE) and neurofilament 150 kDa (NF-150) by immunocytochemistry. Mouse spleens were also examined by immunohistochemistry for NSE, NF-150, NF-200, peripherin, and glial fibrillary acidic protein. Cells morphologically resembling neural cells were obtained and were positive for neural cell markers. Some of the cells generated sphere-like formations, which may have been neurospheres. Cell proliferation was best in medium containing 10% FBS. Cells positive for neural markers were observed in the subcapsular and perivascular regions of the spleen. The cells were round and present in much lower numbers than in cell culture. These cells are suspected neural stem cells and would be expected to differentiate into neural cells in cell culture. This report suggests the existence of neural stem cells in the mouse spleen.


Assuntos
Proliferação de Células/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Baço/citologia , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Filamentos Intermediários/metabolismo , Camundongos Endogâmicos ICR , Células-Tronco Neurais/metabolismo , Periferinas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Células-Tronco Pluripotentes/metabolismo
13.
Mol Pain ; 15: 1744806918820452, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30799680

RESUMO

Migraine is the seventh most disabling disorder globally, with prevalence of 11.7% worldwide. One of the prevailing mechanisms is the activation of the trigeminovascular system, and calcitonin gene-related peptide (CGRP) is an important therapeutic target for migraine in this system. Recent studies suggested an emerging role of pituitary adenylate cyclase-activating peptide (PACAP) in migraine. However, the relation between CGRP and PACAP and the role of PACAP in migraine remain undefined. In this study, we established a novel repetitive (one, three, and seven days) electrical stimulation model by stimulating dura mater in conscious rats. Then, we determined expression patterns in the trigeminal ganglion and the trigeminal nucleus caudalis of the trigeminovascular system. Electrical stimulation decreased facial mechanical thresholds, and the order of sensitivity was as follows: vibrissal pad >inner canthus >outer canthus (P < 0.001). The electrical stimulation group exhibited head-turning and head-flicks (P < 0.05) nociceptive behaviors. Importantly, electrical stimulation increased the expressions of CGRP, PACAP, and the PACAP-preferring type 1 (PAC1) receptor in both trigeminal ganglion and trigeminal nucleus caudalis (P < 0.05). The expressions of two vasoactive intestinal peptide (VIP)-shared type 2 (VPAC1 and VPAC2) receptors were increased in the trigeminal ganglion, whereas in the trigeminal nucleus caudalis, their increases were peaked on Day 3 and then decreased by Day 7. PACAP was colocalized with NEUronal Nuclei (NeuN), PAC1, and CGRP in both trigeminal ganglion and the trigeminal nucleus caudalis. Our results demonstrate that the repetitive electrical stimulation model can simulate the allodynia during the migraine chronification, and PACAP plays a role in the pathogenesis of migraine potentially via PAC1 receptor.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Terapia por Estimulação Elétrica/métodos , Transtornos de Enxaqueca/terapia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Núcleo Inferior Caudal do Nervo Trigêmeo/fisiologia , Animais , Feminino , Masculino , Transtornos de Enxaqueca/fisiopatologia , Nociceptividade/efeitos dos fármacos , Dinâmica não Linear , Fosfopiruvato Hidratase/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Fatores de Tempo
14.
Mol Pain ; 15: 1744806918825044, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30799685

RESUMO

BACKGROUND: The glutamate type 1 transporter (GLT1) plays a major role in glutamate homeostasis in the brain. Although alterations of GLT1 activity have been linked to persistent pain, the significance of these changes is poorly understood. Focusing on the rostral ventromedial medulla, a key site in pain modulation, we examined the expression and function of GLT1 and related transcription factor kappa B-motif binding phosphoprotein (KBBP) in rats after adjuvant-induced hind paw inflammation. RESULTS: After inflammation, GLT1 and KBBP showed an early upregulation and gradual transition to downregulation that lasted throughout the eight-week observation period. Nitration of GLT1 was reduced at 30 min and increased at eight weeks after inflammation, suggesting an initial increase and later decrease in transporter activity. Mechanical hyperalgesia and paw edema exhibited an initial developing phase with peak hyperalgesia at 4 to 24 h, a subsequent attenuating phase, followed by a late persistent phase that lasted for months. The downregulation of GLT1 occurred at a time when hyperalgesia transitioned into the persistent phase. In the rostral ventromedial medulla, pharmacological block with dihydrokainic acid and RNAi of GLT1 and KBBP increased nociception and overexpression of GLT1 reversed persistent hyperalgesia. Further, the initial upregulation of GLT1 and KBBP was blocked by local anesthetic block, and pretreatment with dihydrokainic acid facilitated the development of hyperalgesia. CONCLUSIONS: These results suggest that the initial increased GLT1 activity depends on injury input and serves to dampen the development of hyperalgesia. However, later downregulation of GLT1 fosters the net descending facilitation as injury persists, leading to the emergence of persistent pain.


Assuntos
Vias Aferentes/metabolismo , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Dor Crônica/patologia , Neuroglia/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Aprendizagem da Esquiva , Tronco Encefálico/fisiologia , Dor Crônica/induzido quimicamente , Modelos Animais de Doenças , Transportador 1 de Aminoácido Excitatório/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Adjuvante de Freund/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hiperalgesia/fisiopatologia , Imunoprecipitação , Masculino , Medição da Dor , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução Genética
15.
Curr Microbiol ; 76(4): 495-502, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30798378

RESUMO

Bacterial strain 71-2 with phosphate-solubilizing activity was isolated from tobacco rhizosphere and classified as Burkholderia cenocepacia based on sequence analyses of 16S rRNA and recA genes. To learn phosphate-solubilizing mechanisms of 71-2, mutants showing reduced solubilizing phosphate activity were obtained using the EZ-Tn5 transposon. Mutant 71-2-MT51 was reduced in the solubilizing phosphate content to 34.36% as compared with the wild-type strain 71-2. The disrupted gene in 71-2-MT51 was cloned and sequenced, and the putative protein from the gene shared 65.26% identity to protein sequences of enolase from Escherichia coli, which suggests the gene encodes an enzyme of enolase. Complementation analyzing showed that Eno was responsible for phosphate solubilizing for B. cenocepacia strain 71-2. To our knowledge, this is the first report of Eno involved in phosphate solubilizing in B. cenocepacia as well as in other bacteria.


Assuntos
Proteínas de Bactérias/genética , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Fosfatos/metabolismo , Fosfopiruvato Hidratase/genética , Proteínas de Bactérias/metabolismo , Burkholderia cenocepacia/classificação , Burkholderia cenocepacia/crescimento & desenvolvimento , DNA Bacteriano/genética , Teste de Complementação Genética , Mutagênese , Mutação , Fosfopiruvato Hidratase/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Recombinases Rec A/genética , Rizosfera , Análise de Sequência de DNA , Microbiologia do Solo , Tabaco/microbiologia
16.
Biochemistry ; 58(9): 1188-1197, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30714720

RESUMO

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, ß-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and ß-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 µg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring ß-thujaplicin and the α- and ß-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 µM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Fosfopiruvato Hidratase/antagonistas & inibidores , Tropolona/farmacologia , Antibacterianos/química , Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/enzimologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Tropolona/química
17.
Mol Pain ; 15: 1744806919831909, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30700204

RESUMO

Descending nociceptive modulation from the supraspinal structures has an important role in cancer-induced bone pain (CIBP). Midbrain ventrolateral periaqueductal gray (vlPAG) is a critical component of descending nociceptive circuits; nevertheless, its precise cellular and molecular mechanisms involved in descending facilitation remain elusive. Our previous study has shown that the activation of p38 MAPK in vlPAG microglia is essential for the neuropathic pain sensitization. However, the existence of potential connection between astrocytes and c-Jun N-terminal kinase (JNK) pathway in CIBP has not yet been elucidated. The following study examines the involvement of astrocyte activation and upregulation of p-JNK in vlPAG, using a CIBP rat model. Briefly, CIBP was mimicked by an intramedullary injection of Walker 256 mammary gland carcinoma cells into the animal tibia. A significant increase in expression levels of astrocytes in the vlPAG of CIBP rats was observed. Furthermore, stereotaxic microinjection of the astrocytic cytotoxin L-α-aminoadipic acid decreased the mechanical allodynia as well as established and reversed the astrocyte activation in CIBP rats. A significant increase in expression levels of p-JNK in astrocytes in vlPAG of CIBP rats was also observed. Moreover, the intrathecal administration of JNK inhibitors SP600125 reduced the expression of glial fibrillary acidic protein, while microinjection of the SP600125 decreased the mechanical allodynia of CIBP rats. These results suggested that CIBP is associated with astrocyte activation in the vlPAG that probably participates in driving descending pain facilitation through the JNK MAPK signaling pathway. To sum up, these findings reveal a novel site of astrocytes modulation of CIBP.


Assuntos
Astrócitos/patologia , Dor do Câncer/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Substância Cinzenta Periaquedutal/patologia , Animais , Antracenos/farmacologia , Peso Corporal/efeitos dos fármacos , Neoplasias Ósseas/complicações , Neoplasias Ósseas/patologia , Antígeno CD11b/metabolismo , Dor do Câncer/etiologia , Carcinoma/complicações , Carcinoma/patologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Hiperalgesia/etiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Substância Cinzenta Periaquedutal/metabolismo , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley
18.
J Neuroinflammation ; 16(1): 35, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760285

RESUMO

BACKGROUND: Extracellular accumulation of amyloid ß-peptide (Aß) is one of pathological hallmarks of Alzheimer's disease (AD) and contributes to the neuronal loss. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-inducible neurotrophic factor. Many groups, including ours, have proved that MANF rescues neuronal loss in several neurological disorders, such as Parkinson's disease and cerebral ischemia. However, whether MANF exerts its protective effect against Aß neurotoxicity in AD remains unknown. METHODS: In the present study, the characteristic expressions of MANF in Aß1-42-treated neuronal cells as well as in the brains of APP/PS1 transgenic mice were analyzed by immunofluorescence staining, qPCR, and Western blot. The effects of MANF overexpression, MANF knockdown, or recombination human MANF protein (rhMANF) on neuron viability, apoptosis, and the expression of ER stress-related proteins following Aß1-42 exposure were also investigated. RESULTS: The results showed the increased expressions of MANF, as well as ER stress markers immunoglobulin-binding protein (BiP) and C/EBP homologous protein (CHOP), in the brains of the APP/PS1 transgenic mice and Aß1-42-treated neuronal cells. MANF overexpression or rhMANF treatment partially protected against Aß1-42-induced neuronal cell death, associated with marked decrease of cleaved caspase-3, whereas MANF knockdown with siRNA aggravated Aß1-42 cytotoxicity including caspase-3 activation. Further study demonstrated that the expressions of BiP, ATF6, phosphorylated-IRE1, XBP1s, phosphorylated-eIF2α, ATF4, and CHOP were significantly downregulated by MANF overexpression or rhMANF treatment in neuronal cells following Aß1-42 exposure, whereas knockdown of MANF has the opposite effect. CONCLUSIONS: These findings demonstrate that MANF may exert neuroprotective effects against Aß-induced neurotoxicity through attenuating ER stress, suggesting that an applicability of MANF as a therapeutic candidate for AD.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/uso terapêutico , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Regulação para Cima/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Crescimento Neural/genética , Neuroblastoma/patologia , Fosfopiruvato Hidratase/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico
19.
Mol Pain ; 15: 1744806919827469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30638145

RESUMO

Chronic pain is a pathological manifestation of neuronal plasticity supported by altered gene transcription in spinal cord neurons that results in long-lasting hypersensitivity. Recently, the concept that epigenetic regulators might be important in pathological pain has emerged, but a clear understanding of the molecular players involved in the process is still lacking. In this study, we linked Dnmt3a2, a synaptic activity-regulated de novo DNA methyltransferase, to chronic inflammatory pain. We observed that Dnmt3a2 levels are increased in the spinal cord of adult mice following plantar injection of Complete Freund's Adjuvant, an in vivo model of chronic inflammatory pain. In vivo knockdown of Dnmt3a2 expression in dorsal horn neurons blunted the induction of genes triggered by Complete Freund's Adjuvant injection. Among the genes whose transcription was found to be influenced by Dnmt3a2 expression in the spinal cord is Ptgs2, encoding for Cox-2, a prime mediator of pain processing. Lowering the levels of Dnmt3a2 prevented the establishment of long-lasting inflammatory hypersensitivity. These results identify Dnmt3a2 as an important epigenetic regulator needed for the establishment of central sensitization. Targeting expression or function of Dnmt3a2 may be suitable for the treatment of chronic pain.


Assuntos
Dor Crônica/complicações , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética , Hiperalgesia/metabolismo , Inflamação/complicações , Células do Corno Posterior/metabolismo , Regulação para Cima/fisiologia , Animais , Capsaicina/farmacologia , Células Cultivadas , Dor Crônica/induzido quimicamente , Dor Crônica/patologia , Ciclo-Oxigenase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Modelos Animais de Doenças , Proteínas de Escherichia coli/metabolismo , Adjuvante de Freund/toxicidade , Lateralidade Funcional , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Medição da Dor , Fosfopiruvato Hidratase/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Medula Espinal/patologia , Regulação para Cima/efeitos dos fármacos
20.
PLoS One ; 14(1): e0206338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30653526

RESUMO

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Streptococcus pyogenes/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Cães , Modelos Moleculares , Fosfopiruvato Hidratase/química , Ligação Proteica , Multimerização Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Streptococcus pyogenes/química
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