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1.
PLoS One ; 15(1): e0228134, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990932

RESUMO

Chronic lameness affects bovine welfare and has a negative economic impact in dairy industry. Moreover, due to the translational gap between traditional pain models and new drugs development for treating chronic pain states, naturally occurring painful diseases could be a potential translational tool for chronic pain research. We therefore employed liquid chromatography tandem mass spectrometry (LC-MS/MS) to stablish the proteomic profile of the spinal cord samples from lumbar segments (L2-L4) of chronic lame dairy cows. Data were validated and quantified through software tool (Scaffold® v 4.0) using output data from two search engines (SEQUEST® and X-Tandem®). Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis was performed to detect proteins interactions. LC-MS/MS identified a total amount of 177 proteins; of which 129 proteins were able to be quantified. Lame cows showed a strong upregulation of interacting proteins with chaperone and stress functions such as Hsp70 (p < 0.006), Hsc70 (p < 0.0079), Hsp90 (p < 0.015), STIP (p > 0.0018) and Grp78 (p <0.0068), and interacting proteins associated to glycolytic pathway such as; γ-enolase (p < 0.0095), α-enolase (p < 0.013) and hexokinase-1 (p < 0.028). It was not possible to establish a clear network of interaction in several upregulated proteins in lame cows. Non-interacting proteins were mainly associated to redox process and cytoskeletal organization. The most relevant down regulated protein in lame cows was myelin basic protein (MBP) (p < 0.02). Chronic inflammatory lameness in cows is associated to increased expression of stress proteins with chaperone, metabolism, redox and structural functions. A state of endoplasmic reticulum stress and unfolded protein response (UPR) might explain the changes in protein expression in lame cows; however, further studies need to be performed in order to confirm these findings.


Assuntos
Doenças dos Bovinos/genética , Dor Crônica/veterinária , Regulação da Expressão Gênica , Coxeadura Animal/genética , Proteína Básica da Mielina/genética , Proteínas do Tecido Nervoso/genética , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/fisiopatologia , Dor Crônica/genética , Dor Crônica/metabolismo , Dor Crônica/fisiopatologia , Indústria de Laticínios , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lactação/fisiologia , Coxeadura Animal/metabolismo , Coxeadura Animal/fisiopatologia , Anotação de Sequência Molecular , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Proteômica/métodos , Corno Dorsal da Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/fisiopatologia
2.
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
3.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 373-378, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31631606

RESUMO

Objective: To establish a radiomic model for predicting lymph node (LN) metastasis in patients with non-small cell lung cancer (NSCLC). Methods: The prediction model was developed using a training cohort comprising 100 patients with clinicopathologically confirmed NSCLC. Data were gathered from January 2014 to December 2015. Radiomic features of NSCLC were obtained from non-contrast and enhancement computed tomography (CT). Lasso-logistic regression models were established for data dimension reduction, feature selection, and radiomics signature building. Consistency coefficient ( ICCs) was used to evaluate the consistency between observer interior and interobserver.The consistency index (C-index)is used to evalutate the prediction of lymph node metastasis by using the radiomics signature, shown with the area under the receiver operating characteristic curve ( AUC).Multivariate logistic regression analyses were performed to develop the prediction model, considering radiomics signature and clinicopathologic risk factors. The radiomics model was validated in a validation cohort comprising 100 consecutive NSCLC patients from January 2016 to December 2017 in terms of its calibration and discrimination. AUC was used to evaluate the predictive effectiveness of the model, and Delong test was used to compare models. Hosmer-Lemeshow good of fit test was used to evaluate the calibration of prediction models.The results were represented by correction curves to compare the consistency between the predicted results of the model and the actual probability of LN metastasis. Results: The consistency between observer interior and interobserver was good, with ICCs higher than 0.75.The radiomics signature, including 22 selected features, was associated with LN metastasis. AUC was 0.781 in training cohort and 0.776 in validation cohort. The individualized prediction model identified radiomics signature, neuron specific enolase (CEA), cytokeratin 19 fragment antigen 21-1 (CYFRA21-1), and carbohydrate antigen 125 (CA125) as independent predictors. The model showed good discrimination, with 0.836 AUC in the training cohort, and 0.821 AUC in the validation cohort. The model in both the training and validation cohorts had good calibration,which demonstrated high consistency with the actual LN metastasis. Conclusion: The radiomics model incorporating radiomics signature and clinical risk factors can be conveniently used to facilitate preoperative individualized prediction of LN metastasis in patients with NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Metástase Linfática/diagnóstico , Antígenos de Neoplasias/genética , Antígeno Ca-125/genética , Humanos , Queratina-19/genética , Modelos Logísticos , Linfonodos , Proteínas de Membrana/genética , Fosfopiruvato Hidratase/genética , Valor Preditivo dos Testes , Estudos Retrospectivos
4.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502568

RESUMO

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (Km = 0.23 +/- 0.013 x 10-3 M; Vmax = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na+ ions inhibit the enzyme while increasing concentrations of K+ ions were stimulatory. In case of divalent cations, it was found that Mg2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn2+, Mn2+, Fe2+, Cu2+, Ni2+ and Ca2+) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg2+ and Cr2+. The circular dichroism data indicate that other than Hg2+, Ni2+ and to a certain extent Cu2+, none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.


Assuntos
Estabilidade Enzimática/efeitos dos fármacos , Metais/farmacologia , Fosfopiruvato Hidratase/genética , Conformação Proteica/efeitos dos fármacos , Catálise/efeitos dos fármacos , Dicroísmo Circular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Íons/química , Íons/farmacologia , Metais/química , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/isolamento & purificação , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade
5.
Int J Mol Sci ; 20(16)2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31416219

RESUMO

Breast cancer is a complex and heterogeneous disease: Several molecular alterations cause cell proliferation and the acquisition of an invasive phenotype. Extracellular matrix (ECM) is considered essential for sustaining tumor growth and matrix metalloproteinases (MMPs) have been identified as drivers of many aspects of the tumor phenotype. Mounting evidence indicates that both α-enolase (ENO1) and Myc promoter-binding protein-1 (MBP-1) also played pivotal roles in tumorigenesis, although as antagonists. ENO1 is involved in cell growth, hypoxia tolerance and autoimmune activities besides its major role in the glycolysis pathway. On the contrary, MBP-1, an alternative product of ENO1, suppresses cell proliferation and the invasive ability of cancer cells. Since an important task in personalized medicine is to discriminate a different subtype of patients with different clinical outcomes including chances of recurrence and metastasis, we investigated the functional relationship between ENO1/MBP-1 expression and MMP-2 and MMP-9 activity levels in both tissues and sera of breast cancer patients. We focused on the clinical relevance of ENO1 and MMPs (MMP-2 and MMP-9) overexpression in breast cancer tissues: The association between the higher ENO1, MMP-2 and MMP-9 expression with a worse prognosis suggest that the elevated ENO1 and MMPs expression are promising biomarkers for breast cancer. A relationship seems to exist between MBP-1 expression and the decrease in the activity levels of MMP-9 in cancer tissues and MMP-2 in sera. Moreover, the sera of breast cancer patients grouped for MBP-1 expression differentially induced, in vitro, cell proliferation and migration. Our findings support the hypothesis of patient's stratification based on ENO1, MBP-1 and MMPs expression. Elucidating the molecular pathways through which MBP-1 influences MMPs expression and breast cancer regression can lead to the discovery of new management strategies.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Fosfopiruvato Hidratase/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo
6.
Microb Pathog ; 135: 103651, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31398532

RESUMO

Bartonella infection is distributed worldwide with animal and public health. Recent studies have shown that host cells infection by Bartonella has a series of different infection stages, beginning with encounter and adherence to the cells. In this study, we expressed and purified recombinant Bartonella henselae (B. henselae) α-enolase. And we found that B. henselae α-enolase is highly conserved in Bartonella species. The interacting protein partners of B. henselae α-enolase were showed by String-11. The interactions between B. henselae α-enolase and human plasminogen were subsequently confirmed by ELISA, pull down, T7 phage display and molecular docking assays. And the plasminogen-binding sites of B. henselae α-enolase are predicted at 247FYKNGSYFY255. These findings will help elucidate and improve the understanding of the molecular mechanisms of Bartonella infection.


Assuntos
Bartonella/enzimologia , Bartonella/genética , Proteínas de Transporte/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificação , Plasminogênio/metabolismo , Sequência de Aminoácidos , Bartonella henselae/enzimologia , Bartonella henselae/genética , Sítios de Ligação , Proteínas de Transporte/química , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/classificação , Filogenia , Plasminogênio/química , Proteínas Recombinantes
7.
Mol Med Rep ; 20(2): 1725-1735, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257504

RESUMO

MicroRNAs (miRNAs) have been found to play important regulatory roles in certain neurodegenerative diseases. The aim of the present study was to investigate the effect of miRNA­153 (miR­153) on the neural differentiation of HT­22 cells. Overexpression of miR­153 induced the differentiation of HT­22 cells, increasing the number of protrusions and branches, reducing the S phase distribution of the cell cycle, and attenuating the cell proliferation rate as determined using the Cell Counting Kit­8 assay. Furthermore, miR­153 increased the expression of neuron­specific γ­enolase (NSE), neuronal nuclei (NeuN), and N­ethylmaleimide­sensitive fusion attachment protein 23 (SNAP23) and SNAP25 at the transcriptional and protein level by PCR and western blot analysis. Moreover, miR­153 caused obvious upregulation of peroxiredoxin 5 (PRX5), which has been found to protect neural cells from death and apoptosis. miR­153 promoted neural differentiation and protected neural cells by upregulating the neuron markers γ­enolase, neuronal nuclei, and the functional proteins SNAP23, SNAP25 and PRX5. Therefore, miR­153 may be a potential target for the treatment of certain neurodegenerative diseases.


Assuntos
Hipocampo/citologia , MicroRNAs/genética , Neurogênese , Fosfopiruvato Hidratase/genética , Animais , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/metabolismo , Camundongos , Regulação para Cima
8.
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
9.
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
10.
EBioMedicine ; 45: 251-260, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31201144

RESUMO

BACKGROUND: Statin-associated muscle symptoms (SAMS) are the major adverse effects of the class of widely used lipid-lowering agents, and the underlying mechanism remains elusive. In this study, we investigated the potential contribution and molecular mechanism of increased lactate production to SAMS in mice. METHODS: C57BL/6 J mice were administrated with lovastatin and exercise capacity and blood and muscle lactate levels were measured. A variety of metabolic and molecular experiments were carried out on skeletal muscle cell lines A-204 and C2C12 to confirm the in vivo findings, and to delineate the molecular pathway regulating lactate production by statins. FINDINGS: Blood lactate levels of mice treated with lovastatin increased 23% compared to the control group, which was reproduced in type II predominant glycolytic muscles, accompanied with a 23.1% decrease of maximum swim duration time. The in vitro evidence revealed that statins increased the expression of muscle specific glycolytic enzyme ß-enolase through promoting the degradation of basal p53 proteins, resulting in increased of lactate production. Co-administered with dichloroacetate (DCA), a reagent effective in treating lactic acidosis, reverted the elevated lactate levels and the decreased exercise capacity. INTERPRETATION: Elevated lactate production by statins through the p53/ß-enolase axis contributes to SAMS. FUND: This work was supported by grants from the Science and Technology Development Fund (FDCT) of Macau (Project codes: 034/2015/A1 and 0013/2019/A1).


Assuntos
Ácido Láctico/sangue , Músculo Esquelético/efeitos dos fármacos , Fosfopiruvato Hidratase/genética , Proteína Supressora de Tumor p53/genética , Animais , Ácido Dicloroacético/farmacologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lovastatina/efeitos adversos , Lovastatina/farmacologia , Camundongos , Músculo Esquelético/metabolismo , Condicionamento Físico Animal
11.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 732-739, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31103831

RESUMO

Theileria annulata enolase (TaENO) could be assessed as a druggable target for tropical theileriosis treatment. The parasite enzyme plays an important role in many cellular functions and carries some structural differences like dipeptide (262EK263) and pentapeptide (103EWGYC107) insertions from the host enzyme, Bos taurus enolase. In this study, the functional effects of these insertions on TaENO activity were analyzed by in vitro site-directed mutagenesis and in silico molecular docking analyses for the first time in the literature. In vitro results showed that, although the deletion of the pentapeptide insertion (TaENOΔEWGYC) reduced the enzyme activity slightly, the removal of the dipeptide insertion (TaENOΔEK) halted it. Also, molecular docking results revealed that the deletion of these insertions affected the substrate binding affinity of the mutant enzymes. The active site of TaENOΔEK exhibited a small decrease of substrate binding affinity compared to the active site of TaENOΔEWGYC relative to the wild type TaENO. Although we conclude that both regions could be evaluated as possible drug-binding sites to inhibit TaENO in further studies, these results indicate that the dipeptide insertion could be a more promising drug binding site than the pentapeptide insertion.


Assuntos
Dipeptídeos/química , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fosfopiruvato Hidratase/química , Proteínas de Protozoários/química , Theileria annulata/enzimologia , Animais , Domínio Catalítico/genética , Bovinos , Dipeptídeos/genética , Fosfopiruvato Hidratase/genética , Proteínas de Protozoários/genética , Especificidade por Substrato/genética , Theileria annulata/genética
12.
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
13.
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
14.
Molecules ; 24(4)2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30781735

RESUMO

Progastrin-releasing peptide (ProGRP), which is known to be highly specific and sensitive to small cell lung cancer (SCLC), has been proven to be a valuable substitute for neuron-specific enolase in SCLC diagnostics and monitoring, especially in its early stages. The detection of ProGRP levels also facilitates a selection of therapeutic treatments. For the fabrication of our proposed biosensor, titanium (IV) oxide microparticles were first used, followed by dispersing gold nanoparticles into chitosan and immobilizing them onto a carbon paste electrode (CPE) surface. The developed immunosensor exhibits a much higher biosensing performance in comparison with current methods, when it comes to the detection of ProGRP. Therefore, the proposed CPE/TiO2/(CS+AuNPs)/anti-ProGRP/BSA/ProGRP is excellent for the development of a compact diagnostics apparatus.


Assuntos
Biomarcadores Tumorais/sangue , Técnicas Biossensoriais , Fragmentos de Peptídeos/sangue , Carcinoma de Pequenas Células do Pulmão/sangue , Ouro/química , Humanos , Nanocompostos/química , Fosfopiruvato Hidratase/genética , Proteínas Recombinantes/sangue , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Titânio/química
15.
Scand J Clin Lab Invest ; 79(1-2): 71-74, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30727773

RESUMO

Tumor markers are noninvasive diagnostic tools for cancer. Their abnormal expression often occurs earlier than clinical symptoms or other detection signals. Appropriate reference intervals (RIs) of tumor markers are important for health evaluation, cancer diagnosis, therapy monitoring and prognosis assessment. In this study, we aimed to establish the RIs of cancer antigen 125 (CA125), CA15-3, CA19-9, CA72-4, alpha fetoprotein (AFP), carcino-embryonic antigen (CEA), neuron-specific enolase (NSE) and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) in apparently healthy Henan population. A total of 1705 apparently healthy participants (21-89 years) were recruited from five representative geographical regions in Henan province. Nonparametric 95th percentile intervals were used to define the RIs of CA125, CA15-3, CA19-9, CA72-4, AFP, CEA, NSE and CYFRA21-1. The test results of CA125, CA15-3, CA19-9, CA72-4, AFP, CEA, NSE and CYFRA21-1 can traceable to reference measurement procedures. The age- and gender-specific RIs of the tumor markers were established. We established age- and gender-specific RIs for CA125, CA15-3, CA19-9, CA72-4, AFP, CEA, NSE and CYFRA21-1. The newly established RIs should be more suitable for Henan population. It will be valuable for clinicians to make a medical diagnosis, therapeutic management decision and other physiological assessment.


Assuntos
Antígenos de Neoplasias/genética , Antígenos Glicosídicos Associados a Tumores/genética , Biomarcadores Tumorais/genética , Antígeno Ca-125/genética , Antígeno CA-19-9/genética , Antígeno Carcinoembrionário/genética , Queratina-19/genética , Mucina-1/genética , Fosfopiruvato Hidratase/genética , alfa-Fetoproteínas/genética , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/sangue , Antígenos Glicosídicos Associados a Tumores/sangue , Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Antígeno CA-19-9/sangue , Antígeno Carcinoembrionário/sangue , China , Feminino , Voluntários Saudáveis , Humanos , Queratina-19/sangue , Masculino , Pessoa de Meia-Idade , Mucina-1/sangue , Fosfopiruvato Hidratase/sangue , Gravidez , Valores de Referência , Fatores Sexuais , alfa-Fetoproteínas/metabolismo
16.
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
17.
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
18.
Glia ; 67(2): 393-403, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30485540

RESUMO

Transgenic Tg2576 mice expressing human amyloid precursor protein (hAPP) with the Swedish mutation are among the most frequently used animal models to study the amyloid pathology related to Alzheimer's disease (AD). The transgene expression in this model is considered to be neuron-specific. Using a novel hAPP-specific antibody in combination with cell type-specific markers for double immunofluorescent labelings and laser scanning microscopy, we here report that-in addition to neurons throughout the brain-astrocytes in the corpus callosum and to a lesser extent in neocortex express hAPP. This astrocytic hAPP expression is already detectable in young Tg2576 mice before the onset of amyloid pathology and still present in aged Tg2576 mice with robust amyloid pathology in neocortex, hippocampus, and corpus callosum. Surprisingly, hAPP immunoreactivity in cortex is restricted to resting astrocytes distant from amyloid plaques but absent from reactive astrocytes in close proximity to amyloid plaques. In contrast, neither microglial cells nor oligodendrocytes of young or aged Tg2576 mice display hAPP labeling. The astrocytic expression of hAPP is substantiated by the analyses of hAPP mRNA and protein expression in primary cultures derived from Tg2576 offspring. We conclude that astrocytes, in particular in corpus callosum, may contribute to amyloid pathology in Tg2576 mice and thus mimic this aspect of AD pathology.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/metabolismo , Encéfalo/patologia , Fatores Etários , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares , Neurônios/metabolismo , Neurônios/patologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
19.
Glia ; 67(4): 729-740, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30485546

RESUMO

Microglia, which are pathological effectors and amplifiers in the central nervous system, undergo various forms of activation. A well-studied microglial-induced pathological paradigm, spinal microglial activation following peripheral nerve injury (PNI), is a key event for the development of neuropathic pain but the transcription factors contributing to microglial activation are less understood. Herein, we demonstrate that MafB, a dominant transcriptional regulator of mature microglia, is involved in the pathology of a mouse model of neuropathic pain. PNI caused a rapid and marked increase of MafB expression selectively in spinal microglia but not in neurons. We also found that the microRNA mir-152 in the spinal cord which targets MafB expression decreased after PNI, and intrathecal administration of mir-152 mimic suppressed the development of neuropathic pain. Reduced MafB expression using heterozygous Mafb deficient mice and by intrathecal administration of siRNA alleviated the development of PNI-induced mechanical hypersensitivity. Furthermore, we found that intrathecal transfer of Mafb deficient microglia did not induce mechanical hypersensitivity and that conditional Mafb knockout mice did not develop neuropathic pain after PNI. We propose that MafB is a key mediator of the PNI-induced phenotypic alteration of spinal microglia and neuropathic pain development.


Assuntos
Regulação da Expressão Gênica/genética , Fator de Transcrição MafB/metabolismo , Microglia/metabolismo , Neuralgia/patologia , Medula Espinal/patologia , Animais , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Fator de Transcrição MafB/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/transplante , Neuralgia/tratamento farmacológico , Limiar da Dor/fisiologia , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/uso terapêutico
20.
Ophthalmology ; 126(1): 38-48, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30352225

RESUMO

PURPOSE: To find genetic contributions to glaucoma in African Americans. DESIGN: Cross-sectional, case-control study. PARTICIPANTS: One thousand eight hundred seventy-five primary open-angle glaucoma (POAG) patients and 1709 controls, self-identified as being of African descent (AD), from the African Descent and Glaucoma Evaluation Study (ADAGES) III and Wake Forest School of Medicine. METHODS: MegaChip genotypes were imputed to Thousand Genomes data. Association of single nucleotide polymorphisms (SNPs) with POAG and advanced POAG was tested by linear mixed model correcting for relatedness and population stratification. Genetic risk scores were tested by receiver operator characteristic curves (ROC-AUCs). MAIN OUTCOME MEASURES: Primary open-angle glaucoma defined by visual field loss without other nonocular conditions (n = 1875). Advanced POAG was defined by age-based mean deviation of visual field (n = 946). RESULTS: Eighteen million two hundred eighty-one thousand nine hundred twenty SNPs met imputation quality of r2 > 0.7 and minor allele frequency > 0.005. Association of a novel locus, EN04, was observed for advanced POAG (rs185815146 ß, 0.36; standard error, 0.065; P < 3×10-8). For POAG, an AD signal was observed at the 9p21 European descent (ED) POAG signal (rs79721419; P < 6.5×10-5) independent of the previously observed 9p21 ED signal (rs2383204; P < 2.3×10-5) by conditional analyses. An association with POAG in FNDC3B (rs111698934; P < 3.9×10-5) was observed, not in linkage disequilibrium (LD) with the previously reported ED SNP. Additional previously identified loci associated with POAG in persons of AD were: 8q22, AFAP1, and TMC01. An AUC of 0.62 was observed with an unweighted genetic risk score comprising 11 SNPs in candidate genes. Two additional risk scores were studied by using a penalized matrix decomposition with cross-validation; risk scores of 50 and 400 SNPs were identified with ROC of AUC = 0.74 and AUC = 0.94, respectively. CONCLUSIONS: A novel association with advanced POAG in the EN04 locus was identified putatively in persons of AD. In addition to this finding, this genome-wide association study in POAG patients of AD contributes to POAG genetics by identification of novel signals in prior loci (9p21), as well as advancing the fine mapping of regions because of shorter average LD (FNDC3B). Although not useful without confirmation and clinical trials, the use of genetic risk scores demonstrated that considerable AD-specific genetic information remains in these data.


Assuntos
Afro-Americanos/genética , Glaucoma de Ângulo Aberto/genética , Fosfopiruvato Hidratase/genética , Polimorfismo de Nucleotídeo Único , Idoso , Estudos de Casos e Controles , Estudos Transversais , Feminino , Frequência do Gene , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Glaucoma de Ângulo Aberto/diagnóstico , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Curva ROC
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