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1.
PLoS Genet ; 15(8): e1008136, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31381575

RESUMO

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis.

2.
Brain Res Bull ; 153: 15-23, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31400495

RESUMO

Recent animal studies on heterochronic parabiosis (a technique combining the blood circulation of two animals) have revealed that young blood has a powerful rejuvenating effect on brain aging. Circulating factors, especially growth differentiation factor 11 (GDF11) and C-C motif chemokine 11 (CCL11), may play a key role in this effect, which inspires hope for novel approaches to treating age-related cerebral diseases in humans, such as neurodegenerative and neurovascular diseases. Recently, attempts have begun to translate these astonishing and exciting findings from mice to humans and from bench to bedside. However, increasing reports have shown contradictory data, questioning the capacity of these circulating factors to reverse age-related brain dysfunction. In this review, we summarize the current research on the role of young blood, as well as the circulating factors GDF11 and CCL11, in the aging brain and age-related cerebral diseases. We highlight recent controversies, discuss related challenges and provide a future outlook.

3.
Biosci Biotechnol Biochem ; : 1-11, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31382820

RESUMO

Quinoa crude polysaccharides (QPS) were extracted from Chenopodium quinoa Willd. The soluble non-starch polysaccharide fraction (QPS1) was subsequently purified by DEAE-52 cellulose and Sephadex G-50 gel chromatography, using QPS as raw materials. Its chemical structure was identified using FT-IR, NMR, AFM, SEM and Congo red staining. High performance gel permeation chromatography (HPGPC) was used to determine molecular weight, and composition by HPLC. QPS1, with a molecular weight of 34.0 kDa, was mainly composed of mannose, rhamnose, galacturonic acid, glucose, galactose, xylose and arabinose at a molar ratio of 2.63:2.40:1.64:6.28:1.95:2.48:5.01. In addition, we evaluated the ameliorative effects of QPS1 on the improvement of anti-cyclophosphamide (CTX)-induced immunosuppression in ICR mice. The result exhibited significantly immune-enhancing activity: QPS1 successfully improved the content of IFN-γ, IL-6, IFN-ɑ, IgM and lysozyme (LYSO) in serum for three weeks, enhanced the phagocytic function of mononuclear macrophages and ameliorated delayed allergy in mice.

4.
Gut ; 68(10): 1846-1857, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31154396

RESUMO

OBJECTIVE: APOBEC3B (A3B), a cytidine deaminase acting as a contributor to the APOBEC mutation pattern in many kinds of tumours, is upregulated in patients with hepatocellular carcinoma (HCC). However, APOBEC mutation patterns are absent in HCC. The mechanism of how A3B affects HCC progression remains elusive. DESIGN: A3B -promoter luciferase reporter and other techniques were applied to elucidate mechanisms of A3B upregulation in HCC. A3B overexpression and knockdown cell models, immunocompetent and immune-deficient mouse HCC model were conducted to investigate the influence of A3B on HCC progression. RNA-seq, flow cytometry and other techniques were conducted to analyse how A3B modulated the cytokine to enhance the recruitment of myeloid--derived suppressor cells (MDSCs) and tumour--associated macrophages (TAMs). RESULTS: A3B upregulation through non-classical nuclear factor-κB (NF-κB)signalling promotes HCC growth in immunocompetent mice, associated with an increase of MDSCs, TAMs and programmed cell death1 (PD1) exprssed CD8+ T cells. A CCR2 antagonist suppressed TAMs and MDSCs infiltration and delayed tumour growth in A3B and A3BE68Q/E255Q- expressing mouse tumours. Mechanistically, A3B upregulation in HCC depresses global H3K27me3 abundance via interaction with polycomb repressor complex 2 (PRC2) and reduces an occupancy of H3K27me3 on promoters of the chemokine CCL2 to recruit massive TAMs and MDSCs. CONCLUSION: Our observations uncover a deaminase-independent role of the A3B in modulating the HCC microenvironment and demonstrate a proof for the concept of targeting A3B in HCC immunotherapy.

5.
Cell Death Dis ; 10(5): 360, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043581

RESUMO

Transforming growth factor ß (TGF-ß) serves critical functions in brain injury, especially in cerebral ischemia; however, apart from its neuroprotective effects, its role in regulating neurogenesis is unclear. TGF-ß acts in different ways; the most important, canonical TGF-ß activity involves TGF-ß receptor I (TßRI) or the activin receptor-like kinase 5 (ALK5) signaling pathway. ALK5 signaling is a major determinant of adult neurogenesis. In our previous studies, growth arrest and DNA damage protein 45b (Gadd45b) mediated axonal plasticity after stroke. Here, we hypothesized that ALK5 signaling regulates neural plasticity and neurological function recovery after cerebral ischemia/reperfusion (I/R) via Gadd45b. First, ALK5 expression was significantly increased in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Then, we knocked down or overexpressed ALK5 with lentivirus (LV) in vivo. ALK5 knockdown reduced axonal and dendritic plasticity, with a concomitant decrease in neurological function recovery. Conversely, ALK5 overexpression significantly increased neurogenesis as well as functional recovery. Furthermore, ALK5 mediated Gadd45b protein levels by regulating Smad2/3 phosphorylation. Finally, ALK5 coimmunoprecipitated with Gadd45b. Our results suggested that the ALK5 signaling pathway plays a critical role in mediating neural plasticity and neurological function recovery via Gadd45b after cerebral ischemia, representing a new potential target for cerebral I/R injury.

6.
Neuroscience ; 410: 293-304, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31075313

RESUMO

Medullary dorsal horn (MDH), the homolog of spinal dorsal horn, plays essential roles in processing of nociceptive signals from orofacial region toward higher centers, such as the ventral posteromedial thalamic nucleus (VPM) and parafascicular thalamic nucleus (Pf), which belong to the sensory-discriminative and affective aspects of pain transmission systems at the thalamic level, respectively. In the present study, in order to provide morphological evidence for whether neurons in the MDH send collateral projections to the VPM and Pf, a retrograde double tracing method combined with immunofluorescence staining for substance P (SP), SP receptor (SPR) and Fos protein was used. Fluoro-gold (FG) was injected into the VPM and the tetramethylrhodamine-dextran (TMR) was injected into the Pf. The result revealed that both FG- and TMR-labeled projection neurons were observed throughout the entire extent of the MDH, while the FG/TMR double-labeled neurons were mainly located in laminae I and III. It was also found that some of the FG/TMR double-labeled neurons within lamina I expressed SPR and were in close contact with SP-immunoreactive (SP-ir) terminals. After formalin injection into the orofacial region, 41.4% and 34.3% of the FG/TMR double-labeled neurons expressed Fos protein in laminae I and III, respectively. The present results provided morphological evidence for that some SPR-expressing neurons within the MDH send collateral projections to both VPM and Pf and might be involved in sensory-discriminative and affective aspects of acute orofacial nociceptive information transmission.

7.
Eur J Pharmacol ; 854: 9-21, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-30951716

RESUMO

Colitis-associated cancer (CAC) has a close relationship with ulcerative colitis (UC). Therapeutic effect of Schisandrin B (SchB) on UC and CAC remains largely unknown. We investigated the preventative effect of SchB on the dextran sulphate sodium (DSS) model of UC and azoxymethane (AOM)/DSS model of CAC. Furthermore, focal adhesion kinase (FAK) activation and influence on commensal microbiota are important for UC treatment. Impact on FAK activation by SchB in UC development was evaluated in vivo and vitro. We also conducted 16S rRNA sequencing to detect regulation of gut microbiota by SchB. Enhanced protection of intestinal epithelial barrier by SchB through activating FAK contributed to protective effect on colon for the fact that protection of SchB can be reversed by inhibition of FAK phosphorylation. Furthermore, influence on gut microbiota by SchB also played a significant role in UC prevention. Our results revealed that SchB was potent to prevent UC by enhancing protection of intestinal epithelial barrier and influence on gut microbiota, which led to inhibition of CAC. SchB was potential to become a new treatment for UC and prevention of CAC.

8.
Brain Res ; 1715: 224-234, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928506

RESUMO

Previous studies have demonstrated that both dorsal raphe nucleus (DR) and insular cortex (IC) are critical for somatic sensory information transmission and regulation, especially for pain, and neurons in the DR send projection fibers to the IC. However, whether these ascending connections are involved in the processing of itch sensation remains unknown. In order to provide evidence for that, fluoro-gold (FG) retrograde tracing combined with immunofluorescent histochemical staining was performed for revealing the chemical nature of the projection neurons and the FOS expression induced by acute itch stimulation via intradermal histamine or chloroquine injection in the mouse. Both FOS- and p-ERK-containing neurons were increased in the DR and IC in the acute itch mice compared to those in the sham group. After FG was injected into the IC, FG-labeled retrograde neuronal cell bodies were observed in the whole extent of the brainstem, especially in the DR. About 81% of the total number of FG-labeled neurons in the DR showed serotonin (5-HT)-immunopositive staining. About 32% FG-labeled 5-HT-ergic neurons within DR expressed FOS in chroloquine-induced acute itch, whereas only 6% FG-labeled 5-HT-ergic neurons within DR expressed FOS in histamine-induced acute itch. These results provide morphological evidence for that there are 5-HT-ergic projections from the DR to IC which might be involved in the sensory information processing of acute itch. These results are helpful for understanding functional roles of 5-HT-ergic ascending projection under the condition of acute itch.

9.
J Mech Behav Biomed Mater ; 94: 10-18, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30851656

RESUMO

Natural structural materials featuring fine hierarchical architectures often display remarkable mechanical properties. To inherit the microstructures of biological materials, nickel-plated luffa sponges were fabricated through electrochemical deposition using natural luffa sponges as templates. Four groups of samples were processed based on nickel electroless and electroplating, and then characterized by X-ray diffraction and optical/scanning electron microscopy. Axial compression tests were performed to characterize the mechanical properties of the nickel-plated samples to compare with those of the original natural sponges. Results showed that a uniform layer of nickel was formed on the luffa fibers by electroless plating; conversely, by electroplating the nickel only minimal deposits were found on the inner luffa wall due to the uneven current distribution over the surface of sponge. Accordingly, electroless plating was deemed to be far more effective for metal deposition of materials with complex structures, such as luffa sponge. Alkali treatments prior to plating were found to be critical for subsequent mechanical performance and energy absorption capacity. The mechanical properties of nickel-plated samples surpass those of original luffa sponges, with the enhancement efficiency, i.e., the ratio of specific stiffness and strength, being highest for electroless-plated samples with a prior alkali treatment. Specifically, their energy absorption capacity was far superior to that in other comparable materials. Using a power scaling law, an empirical relationship was derived which indicated that the bending-dominated behavior of the nickel-plated luffa sponges was similar to that of open-cell foams. We believe that other artificially "bio-inherited materials" could be successfully processed and developed in this manner. The superior properties of bio-inherited materials that we obtained in this work may provide inspiration for future research efforts on bio-inspired structural materials.

10.
Front Physiol ; 9: 1511, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30420813

RESUMO

Spinal cord stimulation (SCS) has been shown to improve the consciousness levels of patients with disorder of consciousness (DOC). However, the underlying mechanisms of SCS remain poorly understood. This study recorded resting-state electroencephalograms (EEG) from 16 patients with minimally conscious state (MCS), before and after SCS, and investigated the mechanisms of SCS on the neuronal dynamics in MCS patients. Detrended fluctuation analysis (DFA), combined with surrogate data method, was employed to measure the long-range temporal correlations (LRTCs) of the EEG signals. A surrogate data method was utilized to acquire the genuine DFA exponents (GDFAE) reflecting the genuine LRTCs of brain activity. We analyzed the GDFAE in four brain regions (frontal, central, posterior, and occipital) at five EEG frequency bands [delta (1-4 Hz), theta (4-8 Hz), alpha (8-13 Hz), beta (13-30 Hz), and gamma (30-45 Hz)]. The GDFAE values ranged from 0.5 to 1, and showed temporal and spatial variation between the pre-SCS and the post-SCS states. We found that the channels with GDFAE spread wider after SCS. This phenomenon may indicate that more cortical areas were engaged in the information integration after SCS. In addition, the GDFAE values increased significantly in the frontal area at delta, theta, and alpha bands after SCS. At the theta band, a significant increase in GDFAE was observed in the occipital area. No significant change was found at beta or gamma bands in any brain region. These findings show that the enhanced LRTCs after SCS occurred primarily at low-frequency bands in the frontal and occipital regions. As the LRTCs reflect the long-range temporal integration of EEG signals, our results indicate that information integration became more "complex" after SCS. We concluded that the brain activities at low-frequency oscillations, particularly in the frontal and occipital regions, were improved by SCS.

11.
Circ Genom Precis Med ; 11(7): e002099, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29997225

RESUMO

BACKGROUND: Intracranial aneurysm (IA) is usually a late-onset disease, affecting 1% to 3% of the general population and leading to life-threatening subarachnoid hemorrhage. Genetic susceptibility has been implicated in IAs, but the causative genes remain elusive. METHODS: We performed next-generation sequencing in a discovery cohort of 20 Chinese IA patients. Bioinformatics filters were exploited to search for candidate deleterious variants with rare and low allele frequency. We further examined the candidate variants in a multiethnic sample collection of 86 whole exome sequenced unsolved familial IA cases from 3 previously published studies. RESULTS: We identified that the low-frequency variant c.4394C>A_p.Ala1465Asp (rs2298808) of ARHGEF17 was significantly associated with IA in our Chinese discovery cohort (P=7.3×10-4; odds ratio=7.34). It was subsequently replicated in Japanese familial IA patients (P=0.039; odds ratio=4.00; 95% confidence interval=0.832-14.8) and was associated with IA in the large Chinese sample collection comprising 832 sporadic IA-affected and 599 control individuals (P=0.041; odds ratio=1.51; 95% confidence interval=1.02-Inf). When combining the sequencing data of all familial IA patients from 4 different ethnicities (ie, Chinese, Japanese, European American, and French-Canadian), we identified a significantly increased mutation burden for ARHGEF17 (21/106 versus 11/306; P=8.1×10-7; odds ratio=6.6; 95% confidence interval=2.9-15.8) in cases as compared with controls. In zebrafish, arhgef17 was highly expressed in the brain blood vessel. arhgef17 knockdown caused blood extravasation in the brain region. Endothelial lesions were identified exclusively on cerebral blood vessels in the arhgef17-deficient zebrafish. CONCLUSIONS: Our results provide compelling evidence that ARHGEF17 is a risk gene for IA.

12.
Accid Anal Prev ; 106: 285-296, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28654844

RESUMO

In pedestrian-vehicle accidents, pedestrians typically suffer from secondary impact with the ground after the primary contact with vehicles. However, information about the fundamental mechanism of pedestrian head injury from ground impact remains minimal, thereby hindering further improvement in pedestrian safety. This study addresses this issue by using multi-body modeling and computation to investigate the influence of vehicle front-end shape on pedestrian safety. Accordingly, a simulation matrix is constructed to vary bonnet leading-edge height, bonnet length, bonnet angle, and windshield angle. Subsequently, a set of 315 pedestrian-vehicle crash simulations are conducted using the multi-body simulation software MADYMO. Three vehicle velocities, i.e., 20, 30, and 40km/h, are set as the scenarios. Results show that the top governing factor is bonnet leading-edge height. The posture and head injury at the instant of head ground impact vary dramatically with increasing height because of the significant rise of the body bending point and the movement of the collision point. The bonnet angle is the second dominant factor that affects head-ground injury, followed by bonnet length and windshield angle. The results may elucidate one of the critical barriers to understanding head injury caused by ground impact and provide a solid theoretical guideline for considering pedestrian safety in vehicle design.


Assuntos
Acidentes de Trânsito/estatística & dados numéricos , Traumatismos Craniocerebrais/etiologia , Veículos Automotores , Pedestres , Caminhada/lesões , Análise de Variância , Fenômenos Biomecânicos , Traumatismos Craniocerebrais/prevenção & controle , Humanos
13.
Eur J Pharmacol ; 811: 117-124, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28595903

RESUMO

Although judicious use of tyrosine kinase inhibitors that target BCR-ABL constitutes an effective strategy for the control of chronic myeloid leukemia (CML), drug resistance is observed due to kinase domain mutations, among which a major one is BCR-ABLT315I. In this study, we identified SHC004-221A1 as a potent inhibitor of T315I and other BCR-ABL mutants. Biochemical assays demonstrated that SHC004-221A1 has an inhibitory effect on all selected BCR-ABL mutants. In vitro studies showed that SHC004-221A1 inhibited the proliferation of tumor cell lines carrying native and T315I mutant BCR-ABL. Signaling pathway analysis revealed that SHC004-221A1 inhibited the phosphorylation of STAT5 and CrkL, which contributed to the apoptosis of CML cells. In vivo studies indicated that SHC004-221A1 suppressed BCR-ALBT315I-driven tumor growth in mice. Taken together, the results of this study suggested that SHC004-221A1 may be a promising BCR-ABLT315I inhibitor for the treatment of CML.


Assuntos
Fluorbenzenos/farmacologia , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Purinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Fluorbenzenos/uso terapêutico , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Camundongos , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
J Pharm Biomed Anal ; 140: 81-90, 2017 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-28343077

RESUMO

Thirteen unknown impurities in flomoxef sodium were separated and characterized by liquid chromatography coupled with high resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF MS)with positive and negative modes of electrospray ionization method for further improvement of official monographs in pharmacopoeias. The fragmentation patterns of impurities in flomoxef in the negative ion mode were studied in detail, and new negative-ion fragmentation regularities were discovered. Chromatographic separation was performed on a Kromasil C18 column (250mm×4.6mm, 5µm). The mobile phase consisted of (A) ammonium formate aqueous solution (10mM)-methanol (84:16, v/v) and (B) ammonium formate aqueous solution (10mM)-methanol (47:53, v/v). In order to determine the m/z values of the molecular ions and formulas of all detected impurities, full scan LC-MS in both positive and negative ion modes was firstly executed to obtain the m/z value of the molecules. Then LC-MS2 and LC-MS3 were carried out on target compounds to obtain as much structural information as possible. Complete fragmentation patterns of impurities were studied and used to obtain information about the structures of these impurities. Structures of thirteen unknown degradation products in flomoxef sodium were deduced based on the high resolution MSn data with both positive and negative modes. The forming mechanisms of degradation products in flomoxef sodium were also studied.


Assuntos
Contaminação de Medicamentos , Cefalosporinas , Cromatografia Líquida , Isomerismo , Espectrometria de Massas , Sódio
15.
Int J Cardiol ; 214: 246-53, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27077543

RESUMO

BACKGROUND: CXXC-type zinc-finger protein CXXC5 has been reported to be associated with the development of cardiovascular disease. Recently, through signaling pathway screening we found that CXXC5 activated Tgfß and myocardial differentiation signaling pathways simultaneously. Although the physiological and pathological function of CXXC5 in many organs has been well elucidated, its function in heart remains unclear. METHODS AND RESULTS: Here, we found that zebrafish CXXC5 and SMAD were interacting through ZF-CXXC and MH1 domain. Over-expression of CXXC5 in cardiomyocyte increased the luciferase report activity of Tgfß signaling pathway. Spatiotemporal expression profile of cxxc5 showed that it consistently expressed during cardiogenesis. Knockdown of cxxc5 in zebrafish displayed looping defects, cardiac dysplasia, pericardial edema, and decreased contraction ability, accompanied with down-regulation of members referring to cardiac looping downstream genes of Tgfß signaling pathway, such as nkx2.5, hand2, and has2. Co-injection of hand2 mRNA with cxxc5 morpholino rescued the cardiac looping detects. CONCLUSION: Our study is the first to provide an in vivo evidence for cxxc5 regulating heart development and cardiac looping via Tgfß related signaling pathway. This finding suggested that CXXC5 may serve as a possible marker that has potential diagnostic and prognostic value in fetus with congenital heart disease.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Ligação Proteica , Ratos , Transdução de Sinais , Proteínas Smad/química , Proteínas de Peixe-Zebra/química
16.
Neurochem Res ; 41(6): 1263-73, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26738991

RESUMO

Purinergic P2X3 receptors (P2X3Rs) play extensive roles in nerve cells in the central nervous system, particularly in hyperexcitability and calcium (Ca(2+)) influx. However, the role of P2X3Rs in epilepsy has not been previously investigated. To determine the relationship between P2X3Rs and epilepsy, the expression and cellular location of P2X3Rs in patients with intractable temporal lobe epilepsy (TLE) and in a lithium chloride-pilocarpine-induced chronic rat model of epilepsy were assessed. Furthermore, the function of P2X3Rs was assessed in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to evaluate the expression levels of P2X3Rs in brain tissues from TLE patients and an epileptic rat model, whereas immunofluorescence labeling was applied to determine the distribution of target proteins. Whole-cell recording was subsequently performed to identify the influence of P2X3Rs on seizure-like discharges. P2X3Rs were located at the cell bodies and dendrites of neurons with significantly increased expression in the TLE patients and epileptic rat model. In vitro, P2X3R activation accelerated sustained repetitive firing, whereas P2X3R inhibition led to relatively low-frequency discharges. To the best of our knowledge, this is the first study provide evidence that upregulated P2X3R expression exists in both epileptic humans and rats and may aggravate the epileptic state in vitro. Thus, P2X3Rs may represent a novel therapeutic target for antiepileptic drugs.


Assuntos
Modelos Animais de Doenças , Epilepsia do Lobo Temporal/metabolismo , Receptores Purinérgicos P2X3/biossíntese , Regulação para Cima/fisiologia , Potenciais de Ação/fisiologia , Adolescente , Adulto , Animais , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Pilocarpina/toxicidade , Ratos , Ratos Sprague-Dawley , Adulto Jovem
17.
Am J Physiol Cell Physiol ; 308(8): C665-72, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25652453

RESUMO

Hydrogen peroxide (H2O2) causes cell damage via oxidative stress. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that can protect cardiomyocytes against oxidative stress. In this study, we investigated whether the heme precursor 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) could protect cardiomyocytes from H2O2-induced hypertrophy via modulation of HO-1 expression. HL-1 cells pretreated with/without 5-ALA and SFC were exposed to H2O2 to induce a cardiomyocyte hypertrophy model. Hypertrophy was evaluated by planar morphometry, (3)H-leucine incorporation, and RT-PCR analysis of hypertrophy-related gene expressions. Reactive oxygen species (ROS) production was assessed by 5/6-chloromethyl-2',7'-ichlorodihydrofluorescein diacetate acetylester. HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expressions were analyzed by Western blot. In our experiments, HL-1 cells were transfected with Nrf2 siRNA or treated with a signal pathway inhibitor. We found several results. 1) ROS production, cell surface area, protein synthesis, and expressions of hypertrophic marker genes, including atrial natriuretic peptide, brain natriuretic peptide, atrial natriuretic factor, and ß-myosin heavy chain, were decreased in HL-1 cells pretreated with 5-ALA and SFC. 2) 5-ALA and SFC increased HO-1 expression in a dose- and time-dependent manner, associated with upregulation of Nrf2. Notably, Nrf2 siRNA dramatically reduced HO-1 expression in HL-1 cells. 3) ERK1/2, p38, and SAPK/JNK signaling pathways were activated and modulate 5-ALA- and SFC-enhanced HO-1 expression. SB203580 (p38 kinase), PD98059 (ERK), or SP600125 (JNK) inhibitors significantly reduced this effect. In conclusion, our data suggest that 5-ALA and SFC protect HL-1 cells from H2O2-induced cardiac hypertrophy via activation of the MAPK/Nrf2/HO-1 signaling pathway.


Assuntos
Ácido Aminolevulínico/farmacologia , Antioxidantes/farmacologia , Cardiomegalia/tratamento farmacológico , Compostos Ferrosos/farmacologia , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/genética , Animais , Fator Natriurético Atrial/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Estresse Oxidativo , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Miosinas Ventriculares/metabolismo
18.
Nanoscale Res Lett ; 9(1): 172, 2014 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-24717037

RESUMO

A method for situ preparing a hybrid material consisting of silica nanoparticles (SiO2) attached onto the surface of functionalized graphene nanoplatelets (f-GNPs) is proposed. Firstly, polyacrylic acid (PAA) was grafted to the surface of f-GNPs to increase reacting sites, and then 3-aminopropyltriethoxysilane (APTES) KH550 reacted with abovementioned product PAA-GNPs to obtain siloxane-GNPs, thus providing reaction sites for the growth of SiO2 on the surface of GNPs. Finally, the SiO2/graphene nanoplatelets (SiO2/GNPs) hybrid material is obtained through introducing siloxane-GNPs into a solution of tetraethyl orthosilicate, ammonia and ethanol for hours' reaction. The results from Fourier transform infrared spectroscopy (FTIR) showed that SiO2 particles have situ grown on the surface of GNPs through chemical bonds as Si-O-Si. And the nanostructure of hybrid materials was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). All the images indicated that SiO2 particles with similar sizes were grafted on the surface of graphene nanoplatelets successfully. And TEM images also showed the whole growth process of SiO2 particles on the surface of graphene as time grows. Moreover, TGA traces suggested the SiO2/GNPs hybrid material had stable thermal stability. And at 900°C, the residual weight fraction of polymer on siloxane-GNPs was about 94.2% and that of SiO2 particles on hybrid materials was about 75.0%. However, the result of Raman spectroscopy showed that carbon atoms of graphene nanoplatelets became much more disordered, due to the destroyed carbon domains during the process of chemical drafting. Through orthogonal experiments, hybrid materials with various sizes of SiO2 particles were prepared, thus achieving the particle sizes controllable. And the factors' level of significance is as follows: the quantity of ammonia > the quantity of tetraethyl orthosilicate (TEOS) > the reaction time.

19.
Apoptosis ; 19(6): 946-57, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24664781

RESUMO

Diabetes increases the risk of cardiovascular diseases. Berberine (BBR), an isoquinoline alkaloid used in Chinese medicine, exerts anti-diabetic effect by lowering blood glucose and regulating lipid metabolism. It has been reported that BBR decreases mortality in patients with chronic congestive heart failure. However, the molecular mechanisms of these beneficial effects are incompletely understood. In the present study, we sought to determine whether BBR exerts cardioprotective effect against ischemia/reperfusion (I/R) injury in diabetic rats and the underlying mechanisms. Male Sprague-Dawley rats were injected with low dose streptozotocin and fed with a high-fat diet for 12 weeks to induce diabetes. The diabetic rats were intragastrically administered with saline or BBR (100, 200 and 400 mg/kg/d) starting from week 9 to 12. At the end of week 12, all rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. BBR significantly improved the recovery of cardiac systolic/diastolic function and reduced myocardial apoptosis in diabetic rats subjected to myocardial I/R. Furthermore, in cultured neonatal rat cardiomyocytes, BBR (50 µmol/L) reduced hypoxia/reoxygenation-induced myocardial apoptosis, increased Bcl-2/Bax ratio and decreased caspase-3 expression, together with enhanced activation of PI3K-Akt and increased adenosine monophosphate-activated protein kinase (AMPK) and eNOS phosphorylation. Pretreatment with either PI3K/Akt inhibitor wortmannin or AMPK inhibitor Compound C blunted the anti-apoptotic effect of BBR. Our findings demonstrate that BBR exerts anti-apoptotic effect and improves cardiac functional recovery following myocardial I/R via activating AMPK and PI3K-Akt-eNOS signaling in diabetic rats.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Berberina/uso terapêutico , Diabetes Mellitus Experimental/patologia , Hipoglicemiantes/uso terapêutico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Berberina/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Feminino , Coração/efeitos dos fármacos , Coração/fisiopatologia , Hipoglicemiantes/farmacologia , Antagonistas da Insulina/farmacologia , Masculino , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Estreptozocina , Wortmanina
20.
J Biol Chem ; 288(19): 13728-40, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23530048

RESUMO

BACKGROUND: The origin of eukaryotic histone modification enzymes still remains obscure. RESULTS: Prototypic KMT4/Dot1 from Archaea targets chromatin proteins (Sul7d and Cren7) and shows increased activity on Sul7d, but not Cren7, in the presence of DNA. CONCLUSION: Promiscuous aKMT4 could be regulated by chromatin environment. SIGNIFICANCE: This study supports the prokaryotic origin model of eukaryotic histone methyltransferases and sheds light on chromatin dynamics in Archaea. Histone methylation is one of the major epigenetic modifications even in early diverging unicellular eukaryotes. We show that a widespread lysine methyltransferase from Archaea (aKMT4), bears striking structural and functional resemblance to the core of distantly related eukaryotic KMT4/Dot1. aKMT4 methylates a set of various proteins, including the chromatin proteins Sul7d and Cren7, and RNA exosome components. Csl4- and Rrp4-exosome complexes are methylated in different patterns. aKMT4 can self-methylate intramolecularly and compete with other proteins for the methyl group. Automethylation is inhibited by suitable substrates or DNA in a concentration-dependent manner. The automethylated enzyme shows relatively compromised activity. aKMT4-8A mutant with abrogated automethylation shows a more than 150% increase in methylation of substrates, suggesting a possible mechanism to regulate methyltransferase activity. More interestingly, methylation of Sul7d, but not Cren7, by aKMT4 is significantly enhanced by DNA. MS/MS and kinetic analysis further suggest that aKMT4 methylates Sul7d in the chromatin context. These data provide a clue to the possible regulation of aKMT4 activity by the local chromatin environment, albeit as a promiscuous enzyme required for extensive and variegated lysine methylation in Sulfolobus. This study supports the prokaryotic origin model of eukaryotic histone modification enzymes and sheds light on regulation of archaeal chromatin.


Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Metiltransferases de Proteína/química , Processamento de Proteína Pós-Traducional , Sulfolobus/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas Arqueais/genética , Cromatina/química , Sequência Conservada , DNA Arqueal/química , Metilação , Dados de Sequência Molecular , Metiltransferases de Proteína/genética , Especificidade por Substrato
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