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1.
Curr Microbiol ; 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31982968

RESUMO

The soil organic carbon is associated with the plant quality and the microbial community structure. In the present study, carbon fertilizers were applied to paddy soil to elucidate the relationship between soil carbon and neutral aroma substances in both tobacco and soil microbiome by transcriptome sequencing and 16S rDNA-based analysis, respectively. Our results showed that (1) the increase in soil carbon content was closely correlated with the abundance of microorganisms belonging to two classes (which could potentially affect tobacco plants), namely Gammaproteobacteria and Chloroflexia, (2) soil carbon apparently affected tobacco neutral aroma substances, and (3) soil carbon improved neutral aroma substances by affecting the transcriptional processes of sesquiterpenoid and chlorophyll biosyntheses. These results suggest that increased soil carbon-especially active organic carbon-resulted in desirable improvements in aroma substances in tobacco leaves.

2.
Materials (Basel) ; 12(24)2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817600

RESUMO

High-temperature plastic flow of heat-resistant 2.25Cr-1Mo-0.25V steel was investigated by hot tension (at 500-650 °C) on a Gleeble 3800 machine. The strain rate of hot tension was set as 0.001-1 s-1. The constitutive relation of the steel was modeled by the introduction of the parameters termed "true activation energy" and "threshold stress". Then, the kinetics of high-temperature plastic flow was analyzed based on an Arrhenius equation modified by a "threshold stress". The stress exponent of the modified equation was equal to 5. True activation energy was estimated to be 132 kJ·mol-1. According to the slip band model, the basic mechanism behind the hot deformation of the steel was considered to be dislocation climbing, which was governed by grain boundary diffusion. This model proved to be successful in its analysis of the experimental results of hot tension tests.

3.
PLoS One ; 14(10): e0224556, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31671156

RESUMO

The application of biochar is one of the most useful methods for improving soil quality, which is of the utmost significance for the continuous production of crops. As there are no conclusive studies on the specific effects of biochar application on tobacco quality, this study aimed to improve the yield and quality of tobacco as a model crop for economic and genetic research in southern China, by such application. We used transcriptome sequencing to reveal the effects of applied biochar on tobacco development before and after topping. Our results showed that topping affected carbon and nitrogen metabolism, photosynthesis and secondary metabolism in the tobacco plants, while straw biochar-application to the soil resulted in amino acid and lipid synthesis; additionally, it affected secondary metabolism of the tobacco plants through carbon restoration and hormonal action, before and after topping. In addition to the new insights into the impact of biochar on crops, our findings provide a basis for biochar application measures in tobacco and other crops.

4.
PeerJ ; 7: e7576, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31565561

RESUMO

Background: The increasing demand for food production has resulted in the use of large quantities of chemical fertilizers. This has created major environmental problems, such as increased ammonia volatilization, N2O emission, and nitrogen (N) leaching from agricultural soil. In particular, the utilization rate of N fertilizer is low in subtropical southern parts of China due to high rainfall. This causes not only large financial losses in agriculture, but also serious environmental pollution. Methods: In this study, 16S rDNA-based analysis and static-chamber gas chromatography were used to elucidate the effects of continuous straw biochar application on the N pool and bacteria environment in two typical soil types, purple and paddy soils, in southern China. Results: Straw biochar application (1) improved the soil N pool in both rhizosphere and non-rhizosphere soils; (2) significantly reduced the emission of N2O, with no difference in emission between 1 and 2 years of application; (3) increased the abundance of N-processing bacteria in the soil and altered the bacterial community structure; and (4) improved the tobacco yield and N use efficiency in paddy soil. These findings suggest that, in southern China, the application of straw biochar can promote N transformation in purple and paddy soils and reduce the emission of the greenhouse gas N2O.

5.
Life Sci ; 234: 116742, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31401315

RESUMO

AIMS: The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor that is expressed in cases of non-small cell lung cancer (NSCLC). Previous studies demonstrated that M3R antagonists reduce the proliferation of NSCLC. However, how antagonists inhibit the NSCLC proliferation and migration is still little known. This study aims to investigate the mechanism of M3R involved in the growth of NSCLC. MAIN METHODS: The CRISPR/Cas9 was used to knock out (KO) the M3R gene. A real-time cell analyzer (RTCA) was used to record the proliferation of NSCLC cells. The migration and cell cycle of NSCLC cells were evaluated with scratch test and flow cytometry (FCM), respectively. Antibody microarray analysis was performed to detect the expression of proteins after antagonizing M3R and knocking out of M3R, subsequently some of these important proteins were verified by western blot. KEY FINDINGS: The proliferation and migration of NSCLC cells were inhibited by M3R antagonist R2-8018 and knocking out of M3R. Antagonism or knocking out of M3R reduced the phosphorylation of EGFR. Moreover, c-Src and ß-arrestin-1 are involved in the mechanism of how the inhibition of M3R affects EGFR in NSCLC. Further study demonstrated that PI3K/AKT and MEK/ERK signal pathways are involved in M3R-induced EGFR transactivation in NSCLC, and the molecules involved in the cell cycle progression and migration of NSCLC cells were identified. SIGNIFICANCE: This further understanding of the relationship between M3R and NSCLC facilitates the design of therapeutic strategy with M3R antagonist as an adjuvant drug for NSCLC treatment.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Receptor Muscarínico M3/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptor Muscarínico M3/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
R Soc Open Sci ; 6(7): 181499, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31417689

RESUMO

Soil carbon reserves are the largest terrestrial carbon pools. Common agricultural practices, such as high fertilization rates and intensive crop rotation, have led to global-scale environmental changes, including decreased soil organic matter, lower carbon/nitrogen ratios and disruption of soil carbon pools. These changes have resulted in a decrease in soil microbial activity, severe reduction in soil fertility and transformation of soil nutrients, thereby causing soil nutrient imbalance, which seriously affects crop production. In this study, 16S rDNA-based analysis and static chamber-gas chromatography were used to elucidate the effects of continuous application of straw biochar on soil carbon pools and the soil microbial environments of two typical soil types (purple and paddy soils) in southern China. Application of biochar (1) improved the soil carbon pool and its activity, (2) significantly promoted the release of soil CO2 and (3) improved the soil carbon environment. Soil carbon content was closely correlated with the abundance of organisms belonging to two orders, Lactobacillales and Bacteroidales, and, more specifically, to the genus Lactococcus. These results suggest that biochar affects the soil carbon environment and soil microorganism abundance, which in turn may improve the soil carbon pool.

7.
Molecules ; 24(15)2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31357453

RESUMO

Polymyxins are considered to be the last-line antibiotics that are used to treat infections caused by multidrug-resistant (MDR) gram-negative bacteria; however, the plasmid-mediated transferable colistin resistance gene (mcr-1) has rendered polymyxins ineffective. Therefore, the protein encoded by mcr-1, MCR-1, could be a target for structure-based design of inhibitors to tackle polymyxins resistance. Here, we identified racemic compound 3 as a potential MCR-1 inhibitor by virtual screening, and 26 compound 3 derivatives were synthesized and evaluated in vitro. In the cell-based assay, compound 6g, 6h, 6i, 6n, 6p, 6q, and 6r displayed more potent activity than compound 3. Notably, 25 µΜ of compound 6p or 6q combined with 2 µg·mL-1 colistin could completely inhibit the growth of BL21(DE3) expressing mcr-1, which exhibited the most potent activity. In the enzymatic assay, we elucidate that 6p and 6q could target the MCR-1 to inhibit the activity of the protein. Additionally, a molecular docking study showed that 6p and 6q could interact with Glu246 and Thr285 via hydrogen bonds and occupy well the cavity of the MCR-1 protein. These results may provide a potential avenue to overcome colistin resistance, and provide some valuable information for further investigation on MCR-1 inhibitors.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Desenho de Drogas , Fosfotransferases/química , Fosfotransferases/farmacologia , Proteínas de Bactérias/síntese química , Técnicas de Química Sintética , Simulação por Computador , Modelos Moleculares , Fosfotransferases/síntese química , Relação Estrutura-Atividade
8.
Brain Res ; 1717: 204-213, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30940440

RESUMO

Chronic neuropathic pain has demonstrated that coexisting psychiatric disorders are associated with disability and poorer treatment outcomes. Hyperpolarization-activated cyclic nucleotide-gated (HCN, Ih) channels play a major role in pain via hyperexcitability and facilitation of ectopic firing in neurons. Neuronal hyperexcitability contributes to pain maintenance and anxiety/depression. GABA-mediated inhibitory postsynaptic neurotransmission in the brain is impaired in the pathophysiology of chronic neuropathic pain with comorbidity mood disorders. Currently, interaction of HCN channels and GABAergic synaptic transmission inhibition in neuropathic pain and the associated comorbidity anxiety/depression mechanism remains relatively unknown. To address this, the HCN channel inhibitor, ZD7288, was administrated to Wistar Kyoto (WKY) rats after spared nerve injury (SNI). Our findings show that intracerebroventricular injection of ZD7288 concurrently attenuates co-existing nociceptive and depression-like behaviors, and increases glutamicacid decarboxylase (GAD67/65) expression and GABA levels in the hippocampus and thalamus with High-performance liquid chromatography technique. It suggests that inhibition of HCN channels is likely to decrease the hyperexcitability of neurons in rat SNI and improve the level of GABA. Further, HCN channel may offer a new strategy to alleviate both neuropathic pain and comorbidity for depression.

9.
Huan Jing Ke Xue ; 39(5): 2412-2419, 2018 May 08.
Artigo em Chinês | MEDLINE | ID: mdl-29965542

RESUMO

To identify the effect of biochar addition on soil abiotic and biotic properties and provide evidence for the soil improvement with biochar input, the soil physiochemical properties and fungal community were investigated in a cinnamon soil after 3-year biochar additions of 10, 20, and 40 t ·hm-2. The relationship between the fungal community and edaphic physicochemical characteristics was also analyzed. The results showed that soil pH, moisture, total nitrogen (TN), and total organic carbon (TOC) significantly increased but dissolved organic carbon (DOC) content and soil bulk density decreased with biochar addition. High-throughput sequencing results indicated that biochar amendment had little influence on fungal α diversity but significantly changed the fungal community structure. The taxonomic classification showed that the dominant fungal phyla were Ascomycota, Zygomycota, and Basidiomycota, and these phyla accounted for more than 90% of the total sequences. The relative abundance of Ascomycota and Basidiomycota increased, while the abundance of Zygomycota decreased with biochar addition. At the genus level, biochar addition increased the relative abundances of Alternaria, Conocybe, and Aspergillus but decreased the relative abundances of Actinomucor and Gibberella. Redundancy analysis (RDA) showed that soil DOC, pH, and moisture were key environmental factors leading to the shift in the soil fungal community composition. In summary, the application of biochar changed the soil physicochemical properties, which drove the ecological succession of soil fungal communities.


Assuntos
Carvão Vegetal/química , Fungos/classificação , Microbiologia do Solo , Solo/química
10.
CNS Neurosci Ther ; 23(12): 930-939, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28990373

RESUMO

INTRODUCTION: Sigma-1 receptors (Sig-1Rs) are unique endoplasmic reticulum proteins that have been implicated in both neurodegenerative and ischemic diseases, such as Alzheimer's disease and stroke. Accumulating evidence has suggested that Sig-1R plays a role in neuroprotection and axon outgrowth. The underlying mechanisms of Sig-1R-mediated neuroprotection have been well elucidated. However, the mechanisms underlying the effects of Sig-1R on axon outgrowth are not fully understood. METHODS: To clarify this issue, we utilized immunofluorescence to compare the axon lengths of cultured naïve hippocampal neurons before and after the application of the Sig-1R agonist, SA4503. Then, electrophysiology and immunofluorescence were used to examine voltage-gated calcium ion channel (VGCCs) currents in the cell membranes and growth cones. RESULTS: We found that Sig-1R activation dramatically enhanced the axonal length of the naïve hippocampal neurons. Application of the Sig-1R antagonist NE100 and gene knockdown techniques both demonstrated the effects of Sig-1R. The growth-promoting effect of SA4503 was accompanied by the inhibition of voltage-gated Ca2+ influx and was recapitulated by incubating the neurons with the L-type, N-type, and P/Q-type VGCC blockers, nimodipine, MVIIA and ω-agatoxin IVA, respectively. This effect was unrelated to glial cells. The application of SA4503 transformed the growth cone morphologies from complicated to simple, which favored axon outgrowth. CONCLUSION: Sig-1R activation can enhance axon outgrowth and may have a substantial influence on neurogenesis and neurodegenerative diseases.


Assuntos
Anisóis/farmacologia , Axônios/efeitos dos fármacos , Canais de Cálcio/metabolismo , Hipocampo/citologia , Neurônios , Propilaminas/farmacologia , Receptores sigma/agonistas , Análise de Variância , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Masculino , Potenciais da Membrana/efeitos dos fármacos , Morfolinas/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nootrópicos/farmacologia , Técnicas de Patch-Clamp , Piperazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Tubulina (Proteína)/metabolismo
11.
Front Pharmacol ; 8: 302, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28603497

RESUMO

The sigma-1 receptor is a 223 amino acids molecular chaperone with a single transmembrane domain. It is resident to eukaryotic mitochondrial-associated endoplasmic reticulum and plasma membranes. By chaperone-mediated interactions with ion channels, G-protein coupled receptors and cell-signaling molecules, the sigma-1 receptor performs broad physiological and pharmacological functions. Despite sigma-1 receptors have been confirmed to regulate various types of ion channels, the relationship between the sigma-1 receptor and N-type Ca2+ channel is still unclear. Considering both sigma-1 receptors and N-type Ca2+ channels are involved in intracellular calcium homeostasis and neurotransmission, we undertake studies to explore the possible interaction between these two proteins. In the experiment, we confirmed the expression of the sigma-1 receptors and the N-type calcium channels in the cholinergic interneurons (ChIs) in rat striatum by using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) and immunofluorescence staining. N-type Ca2+ currents recorded from ChIs in the brain slice of rat striatum was depressed when sigma-1 receptor agonists (SKF-10047 and Pre-084) were administrated. The inhibition was completely abolished by sigma-1 receptor antagonist (BD-1063). Co-expression of the sigma-1 receptors and the N-type calcium channels in Xenopus oocytes presented a decrease of N-type Ca2+ current amplitude with an increase of sigma-1 receptor expression. SKF-10047 could further depress N-type Ca2+ currents recorded from oocytes. The fluorescence resonance energy transfer (FRET) assays and co-immunoprecipitation (Co-IP) demonstrated that sigma-1 receptors and N-type Ca2+ channels formed a protein complex when they were co-expressed in HEK-293T (Human Embryonic Kidney -293T) cells. Our results revealed that the sigma-1 receptors played a negative modulation on N-type Ca2+ channels. The mechanism for the inhibition of sigma-1 receptors on N-type Ca2+ channels probably involved a chaperone-mediated direct interaction and agonist-induced conformational changes in the receptor-channel complexes on the cell surface.

12.
J Mol Neurosci ; 62(1): 17-27, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28341892

RESUMO

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Evidence suggests that AQP4 is associated with glutamate transporter-1 (GLT-1) for glutamate clearance and contributes to morphine dependence. Previous studies show that AQP4 deficiency changed the mu opioid receptor expression and opioid receptors' characteristics as well. In this study, we focused on whether AQP4 could form macromolecular complex with GLT-1 and mu opioid receptor (MOR) and participates in morphine dependence. By using immunofluorescence staining, fluorescence resonance energy transfer, and co-immunoprecipitation, we demonstrated that AQP4 forms protein complexes with GLT-1 and MOR in both brain tissue and primary cultured astrocytes. We then showed that the C-terminus of AQP4 containing the amino acid residues 252 to 323 is the site of interaction with GLT-1. Protein kinase C, activated by morphine, played an important role in regulating the expression of these proteins. These findings may help to reveal the mechanism that AQP4, GLT-1, and MOR form protein complex and participate in morphine dependence, and deeply understand the reason that AQP4 deficiency maintains extracellular glutamate homeostasis and attenuates morphine dependence, moreover emphasizes the function of astrocyte in morphine dependence.


Assuntos
Aquaporina 4/metabolismo , Astrócitos/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Dependência de Morfina/metabolismo , Receptores Opioides mu/metabolismo , Animais , Aquaporina 4/química , Sítios de Ligação , Encéfalo/metabolismo , Células Cultivadas , Masculino , Ligação Proteica , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley
13.
Front Cell Neurosci ; 10: 127, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27242438

RESUMO

In response to diverse stimuli, two-pore-domain potassium channel TREK-2 regulates cellular excitability, and hence plays a key role in mediating neuropathic pain, mood disorders and ischemia through. Although more and more input modalities are found to achieve their modulations via acting on the channel, the potential role of subunit interaction in these modulations remains to be explored. In the current study, the deletion (lack of proximal C-terminus, ΔpCt) or point mutation (G312A) was introduced into TREK-2 subunits to limit K(+) conductance and used to report subunit stoichiometry. The constructs were then combined with wild type (WT) subunit to produce concatenated dimers with defined composition, and the gating kinetics of these channels to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH (pHo) were characterized. Our results show that combination of WT and ΔpCt/G312A subunits reserves similar gating properties to that of WT dimmers, suggesting that the WT subunit exerts dominant and positive effects on the mutated one, and thus the two subunits controls channel gating via a concerted cooperative manner. Further introduction of ΔpCt into the latter subunit of heterodimeric channel G312A-WT or G312A-G312A attenuated their sensitivity to 2-APB and pHo alkalization, implicating that these signals were transduced by a cis-type mechanism. Together, our findings elucidate the mechanisms for how the two subunits control the pore gating of TREK-2, in which both intersubunit concerted cooperative and cis-type manners modulate the allosteric regulations induced by 2-APB and pHo alkalization.

14.
Front Cell Neurosci ; 10: 71, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27047336

RESUMO

The striatum is the most prominent nucleus in the basal ganglia and plays an important role in motor movement regulation. The cholinergic interneurons (ChIs) in striatum are involved in the motion regulation by releasing acetylcholine (ACh) and modulating the output of striatal projection neurons. Here, we report that muscarinic ACh receptor (M receptor) agonists, ACh and Oxotremorine (OXO-M), decreased the firing frequency of ChIs by blocking the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Scopolamine (SCO), a nonselective antagonist of M receptors, abolished the inhibition. OXO-M exerted its function by activating the Gi/o cAMP signaling cascade. The single-cell reverse transcription polymerase chain reaction (scRT-PCR) revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs. Among them, M2 receptors and HCN2 channels were the most dominant ones and expressed in every single studied cholinergic interneuron (ChI).Our results suggest that ACh regulates not only the output of striatal projection neurons, but also the firing activity of ChIs themselves by activating presynaptic M receptors in the dorsal striatum. The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibition on the excitability of ChIs. This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of cholinergic activity and ACh release in the dorsal striatum.

15.
Sci Rep ; 6: 21248, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26879043

RESUMO

TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically.


Assuntos
Ativação do Canal Iônico , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Domínios e Motivos de Interação entre Proteínas , Regulação Alostérica , Animais , Glicina/química , Humanos , Canais de Potássio de Domínios Poros em Tandem/química , Conformação Proteica , Isoformas de Proteínas
16.
Sci Rep ; 6: 18768, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26729652

RESUMO

Exploitation of surface plasmonic devices (SPDs) in the terahertz (THz) band is always beneficial for broadening the application potential of THz technologies. To clarify features of SPDs, a practical characterization means is essential for accurately observing the complex field distribution of a THz surface wave (TSW). Here, a THz digital holographic imaging system is employed to coherently exhibit temporal variations and spectral properties of TSWs activated by a rectangular or semicircular slit structure on metal foils. Advantages of the imaging system are comprehensively elucidated, including the exclusive measurement of TSWs and fall-off of the time consumption. Numerical simulations of experimental procedures further verify the imaging measurement accuracy. It can be anticipated that this imaging system will provide a versatile tool for analyzing the performance and principle of SPDs.

17.
Biochem Biophys Res Commun ; 463(3): 200-4, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-25998380

RESUMO

The corticotropin releasing factor (CRF) plays a central role in regulating the activities of hypothalamic-pituitary-adrenal (HPA) axis in the presence of a variety of stressful stimuli via binding to its type 1 receptors (CRFR1). Despite that many peptidic or non-peptidic antagonists of CRFR1 have been developed to serve as therapeutic tools to CRF-related pathologies, none of them have been utilized clinically. Targeting the extracellular domain 1 (EC1) of CRFR1, the CRF-binding site, represents a new strategy to inhibit the function of the receptor. However, no such agents have been identified up to now. Herein, by using an 87-amino acid fragment corresponding to the EC1 region as the bait, we screened the binding polypeptides from a phage display (Ph.D.-12) peptide library. After 3-round biopanning, positive clones were selected and the polypeptides carried by them were identified. 5 polypeptides were found to bind with the target specifically. Among them, the P7 exhibited the highest affinity. By evaluating the cAMP accumulation in the CRFR1 or CRFR2-expressing HEK293 cells, we demonstrated that P7 blocking the function of CRFR1, but not CRFR2. In addition, we also found that P7 and CRF act on CRFR1 competitively. Taken together, we reveal that P7, a novel polypeptide identified from phage display library, inhibits the function of CRFR1 effectively and specifically by binding at its EC1 domain. The new polypeptide might provide a promising agent for diagnostic or therapeutic utilities in CRF-related disorders.


Assuntos
Peptídeos/química , Peptídeos/farmacologia , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Sequência de Aminoácidos , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Receptores de Hormônio Liberador da Corticotropina/química
18.
PLoS One ; 9(4): e95503, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24743689

RESUMO

BACKGROUND: Abnormal release of neurotransmitters after microwave exposure can cause learning and memory deficits. This study investigated the mechanism of this effect by exploring the potential role of phosphorylated synapsin I (p-Syn I). METHODS: Wistar rats, rat hippocampal synaptosomes, and differentiated (neuronal) PC12 cells were exposed to microwave radiation for 5 min at a mean power density of 30 mW/cm2. Sham group rats, synaptosomes, and cells were otherwise identically treated and acted as controls for all of the following post-exposure analyses. Spatial learning and memory in rats was assessed using the Morris Water Maze (MWM) navigation task. The protein expression and presynaptic distribution of p-Syn I and neurotransmitter transporters were examined via western blotting and immunoelectron microscopy, respectively. Levels amino acid neurotransmitter release from rat hippocampal synaptosomes and PC12 cells were measured using high performance liquid chromatograph (HPLC) at 6 hours after exposure, with or without synapsin I silencing via shRNA transfection. RESULTS: In the rat experiments, there was a decrease in spatial memory performance after microwave exposure. The expression of p-Syn I (ser-553) was decreased at 3 days post-exposure and elevated at later time points. Vesicular GABA transporter (VGAT) was significantly elevated after exposure. The GABA release from synaptosomes was attenuated and p-Syn I (ser-553) and VGAT were both enriched in small clear synaptic vesicles, which abnormally assembled in the presynaptic terminal after exposure. In the PC12 cell experiments, the expression of p-Syn I (ser-553) and GABA release were both attenuated at 6 hours after exposure. Both microwave exposure and p-Syn I silencing reduced GABA release and maximal reduction was found for the combination of the two, indicating a synergetic effect. CONCLUSION: p-Syn I (ser-553) was found to play a key role in the impaired GABA release and cognitive dysfunction that was induced by microwave exposure.


Assuntos
Transtornos da Memória/metabolismo , Micro-Ondas/efeitos adversos , Memória Espacial/fisiologia , Sinapsinas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Masculino , Transtornos da Memória/genética , Células PC12 , Fosforilação , Ratos , Ratos Wistar
19.
J. physiol. biochem ; 70(1): 185-191, mar. 2014.
Artigo em Inglês | IBECS | ID: ibc-121617

RESUMO

Mas-related G protein-coupled receptor D (MrgD) is expressed almost exclusively in nociceptive primary sensory neurons and the neurons located in stratum granulosum of skin. More and more evidence suggest that MrgD plays an important role in pain sensation and/or transduction. Recent studies have demonstrated that the receptor is also involved in itch sensation in both mouse and human. In the present study, we identified a robust inward current in MrgD-expressing Xenopusoocytes by using â-alanine, a putative ligand of MrgD. The currents were sensitive to inhibitor of Ca2+-activated chloride channels (CaCCs) and intracellular Ca2+ chelator, suggesting they were produced by endogenous CaCCs. Furthermore, it was demonstrated that upon the application of phospholipase C (PLC) inhibitor, or antisense oligonucleotides of inositol trisphosphate receptor (IP3R), the â-alanine-induced currents were dramatically depressed. However, protein kinase C inhibitor did not display any visible effect on CaCC currents. In summary, our data suggest that the activation of MrgD promotes the open of endogenous CaCCs via Gq-PLC-IP3-Ca2+ pathway. The current findings reveal the functional coupling between MrgD and CaCCs in Xenopus oocytes and also provide a facile model to assay the activity of MrgD (AU)


Assuntos
Animais , Xenopus , Dor/fisiopatologia , Bloqueadores dos Canais de Cálcio/farmacocinética , Substâncias Protetoras/farmacocinética , Modelos Animais de Doenças/métodos , Canais de Cloreto/antagonistas & inibidores , Receptores de Superfície Celular/biossíntese
20.
J Physiol Biochem ; 70(1): 185-91, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24078284

RESUMO

Mas-related G protein-coupled receptor D (MrgD) is expressed almost exclusively in nociceptive primary sensory neurons and the neurons located in stratum granulosum of skin. More and more evidence suggest that MrgD plays an important role in pain sensation and/or transduction. Recent studies have demonstrated that the receptor is also involved in itch sensation in both mouse and human. In the present study, we identified a robust inward current in MrgD-expressing Xenopus oocytes by using ß-alanine, a putative ligand of MrgD. The currents were sensitive to inhibitor of Ca(2+)-activated chloride channels (CaCCs) and intracellular Ca(2+) chelator, suggesting they were produced by endogenous CaCCs. Furthermore, it was demonstrated that upon the application of phospholipase C (PLC) inhibitor, or antisense oligonucleotides of inositol trisphosphate receptor (IP3R), the ß-alanine-induced currents were dramatically depressed. However, protein kinase C inhibitor did not display any visible effect on CaCC currents. In summary, our data suggest that the activation of MrgD promotes the open of endogenous CaCCs via G(q)-PLC-IP3-Ca(2+) pathway. The current findings reveal the functional coupling between MrgD and CaCCs in Xenopus oocytes and also provide a facile model to assay the activity of MrgD.


Assuntos
Canais de Cloreto/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Oócitos/metabolismo , Receptores Acoplados a Proteínas-G/fisiologia , Animais , Sinalização do Cálcio , Células Cultivadas , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Potenciais da Membrana , Ratos , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta-Alanina/farmacologia , beta-Alanina/fisiologia
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