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Electron beams are versatile tools for nanoscale fabrication processes, however, the underlying e-beam chemistry remains in its infancy. Through operando transmission electron microscopy investigations, we elucidate a redox-driven cargo release of individual metal atoms triggered by electron beams. The chosen organic delivery molecule, tetraphenylporphyrin (TPP), proves highly versatile, forming complexes with nearly all metals from the periodic table and being easily processed in solution. A comprehensive cinematographic analysis of the dynamics of single metal atoms confirms the nearly instantaneous ejection of complexed metal atoms under an 80 kV electron beam, underscoring the system's broad versatility. Providing mechanistic insights, we employ density functional theory to support the proposed reductive demetallation pathway facilitated by secondary electrons, contributing novel perspectives to electron beam-mediated chemical reaction mechanisms. Lastly, our findings demonstrate that all seven metals investigated form nanoclusters once ejected from TPP, highlighting the method's potential for studying and developing sustainable single-atom and nanocluster catalysts.
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The rising prospects of mechanochemically assisted syntheses hold promise for both academia and industry, yet they face challenges in understanding and, therefore, anticipating respective reaction kinetics. Particularly, dependencies based on variations in milling equipment remain little understood and globally overlooked. This study aims to address this issue by identifying critical parameters through kinematic models, facilitating the reproducibility of mechanochemical reactions across the most prominent mills in laboratory settings, namely planetary and mixer mills. Through a series of selected experiments replicating major classes of organic, organometallic, transition metal-catalyzed, and inorganic reactions from literature, we rationalize the independence of kinematic parameters on reaction kinetics when the accumulated energy criterion is met. As a step forward and to facilitate the practicability of our findings, we provide a freely accessible online tool that allows the calculation of respective energy parameters for different planetary and mixer mills. Our work advances the current understanding of mechanochemistry and lays the foundation for future rational exploration in this rapidly evolving field.
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Excessive stimulation of the quinolinic acid induces neuronal cell death and is implicated in developing several neurodegenerative diseases. This study investigated whether a Wnt5a antagonist plays a neuroprotective role by regulating the Wnt pathway, activating cellular signaling mechanisms, including MAP kinase and ERK, and acting on the antiapoptotic and the proapoptotic genes in N18D3 neural cells. The cells were pretreated with a Wnt5a antagonist Box5, for one hour and then exposed to quinolinic acid (QUIN), an NMDA receptor agonist for 24 hours. An MTT assay and DAPI staining were used to evaluate cell viability and apoptosis, respectively, demonstrating that Box5 protected the cells from apoptotic death. In addition, a gene expression analysis revealed that Box5 prevented the QUIN-induced expression of the pro-apoptotic genes, BAD and BAX, and increased that of the anti-apoptotic genes, Bcl-xL, BCL2, and BCLW. Further examination of potential cell signaling candidates involved in this neuroprotective effect showed that the immunoreactivity of ERK was significantly increased in the cells treated with Box5. These results suggest that the neuroprotective mechanism of Box5 against QUIN-induced excitotoxic cell death involves the regulation of ERK and modulation of cell survival and death genes through decreasing the Wnt pathway, specifically Wnt5a.
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Fármacos Neuroprotetores , Via de Sinalização Wnt , Apoptose , Morte Celular , Fármacos Neuroprotetores/farmacologia , Ácido Quinolínico/toxicidade , Animais , Camundongos , Linhagem Celular , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K(+) channel blocker). However, neither N (G)-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K(+) channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.
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Pim kinases play a key role in the regulation of signaling pathways including proliferation, migration, and metabolism and are a potential target for cancer therapy. A series of 5-benzylidenethiazolidine-2,4-diones were synthesized as pim kinase inhibitors. The structure-activity relationships (SAR) of the analogues in inhibiting in vitro pim kinase activity as well as the proliferation of leukemia cell lines were examined. SAR studies indicated that a hydroxyl group at the 2-position of the benzene ring of 5-benzylidenethiazolidine-2,4-dione plays an important role in the inhibitory activity against all three pim kinases and replacement with a pyrazinyl group at the 5-position of the benzene ring of 5-benzylidenethiazolidine-2,4-dione improved activity significantly. The compounds exerted anti-proliferative activity against the three leukemia cell lines we tested. The most potent compound, 5i, had an EC50 value of 0.8 µM in the MV4-11 cell line. The result of kinase profiling indicated that compound 5i was highly selective for pim-kinases.
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Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Tiazolidinas/síntese química , Tiazolidinas/farmacologia , Linhagem Celular Tumoral , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
Machines found in nature and human-made machines share common components, such as an engine, and an output element, such as a rotor, linked by a clutch. This clutch, as seen in biological structures such as dynein, myosin or bacterial flagellar motors, allows for temporary disengagement of the moving parts from the running engine. However, such sophistication is still challenging to achieve in artificial nanomachines. Here we present a spherical rotary nanomotor with a reversible clutch system based on precise molecular recognition of built-in DNA strands. The clutch couples and decouples the engine from the machine's rotor in response to encoded inputs such as DNA or RNA. The nanomotor comprises a porous nanocage as a spherical rotor to confine the magnetic engine particle within the nanospace (â¼0.004 µm3) of the cage. Thus, the entropically driven irreversible disintegration of the magnetic engine and the spherical rotor during the disengagement process is eliminated, and an exchange of microenvironmental inputs is possible through the nanopores. Our motor is only 200 nm in size and the clutch-mediated force transmission powered by an embedded ferromagnetic nanocrystal is high enough (â¼15.5 pN at 50 mT) for the in vitro mechanical activation of Notch and integrin receptors, demonstrating its potential as nano-bio machinery.
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DNA , Nanotecnologia , DNA/química , Nanotecnologia/métodos , Nanoporos , MagnetismoRESUMO
Background: We previously demonstrated the validity of a regression model that included ethnicity as a novel predictor for predicting normative brain volumes in old age. The model was optimized using brain volumes measured with a standard tool FreeSurfer. Objective: Here we further verified the prediction model using newly estimated brain volumes from Neuro I, a quantitative brain analysis system developed for Korean populations. Methods: Lobar and subcortical volumes were estimated from MRI images of 1,629 normal Korean and 786 Caucasian subjects (age range 59-89) and were predicted in linear regression from ethnicity, age, sex, intracranial volume, magnetic field strength, and scanner manufacturers. Results: In the regression model predicting the new volumes, ethnicity was again a substantial predictor in most regions. Additionally, the model-based z-scores of regions were calculated for 428 AD patients and the matched controls, and then employed for diagnostic classification. When the AD classifier adopted the z-scores adjusted for ethnicity, the diagnostic accuracy has noticeably improved (AUCâ=â0.85, ΔAUCâ=â+â0.04, Dâ=â4.10, pâ<â0.001). Conclusions: Our results suggest that the prediction model remains robust across different measurement tool, and ethnicity significantly contributes to the establishment of norms for brain volumes and the development of a diagnostic system for neurodegenerative diseases.
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Doença de Alzheimer , Encéfalo , Imageamento por Ressonância Magnética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Alzheimer/etnologia , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Doença de Alzheimer/diagnóstico , Povo Asiático , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Tamanho do Órgão , População Branca , População do Leste AsiáticoRESUMO
Interstitial cells of Cajal (ICCs) from the urinary bladder regulate detrusor smooth muscle activities. We cultured ICCs from the urinary bladder of mice and performed patch clamp and intracellular Ca(2+) ([Ca(2+)]i) imaging to investigate whether cultured ICCs can be a valuable tool for cellular functional studies. The cultured ICCs displayed two types of spontaneous electrical activities which are similar to those recorded in intact bladder tissues. Spontaneous electrical activities of cultured ICCs were nifedipine-sensitive. Carbachol and ATP, both excitatory neurotransmitters in the urinary bladder, depolarized the membrane and increased the frequency of spike potentials. Carbachol increased [Ca(2+)]i oscillations and basal Ca(2+) levels, which were blocked by atropine. These results suggest that cultured ICCs from the urinary bladder retain rhythmic phenotypes similar to the spontaneous electrical activities recorded from the intact urinary bladder. Therefore, we suggest that cultured ICCs from the urinary bladder may be useful for cellular and molecular studies of ICCs.
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To understand how the molecular machinery of synapses works, it is essential to determine an inventory of synaptic proteins at a subsynaptic resolution. Nevertheless, synaptic proteins are difficult to localize because of the low expression levels and limited access to immunostaining epitopes. Here, we report on the exTEM (epitope-exposed by expansion-transmission electron microscopy) method that enables the imaging of synaptic proteins in situ. This method combines TEM with nanoscale resolution and expandable tissue-hydrogel hybrids for enhanced immunolabeling with better epitope accessibility via molecular decrowding, allowing successful probing of the distribution of various synapse-organizing proteins. We propose that exTEM can be employed for studying the mechanisms underlying the regulation of synaptic architecture and function by providing nanoscale molecular distribution of synaptic proteins in situ. We also envision that exTEM is widely applicable for investigating protein nanostructures located in densely packed environments by immunostaining of commercially available antibodies at nanometer resolution.
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Sinapses , Expansão de Tecido , Sinapses/fisiologiaRESUMO
Although bismuth vanadate (BiVO4) has been promising as photoanode material for photoelectrochemical water splitting, its charge recombination issue by short charge diffusion length has led to various studies about heterostructure photoanodes. As a hole blocking layer of BiVO4, titanium dioxide (TiO2) has been considered unsuitable because of its relatively positive valence band edge and low electrical conductivity. Herein, a crystal facet engineering of TiO2 nanostructures is proposed to control band structures for the hole blocking layer of BiVO4 nanodots. We design two types of TiO2 nanostructures, which are nanorods (NRs) and nanoflowers (NFs) with different (001) and (110) crystal facets, respectively, and fabricate BiVO4/TiO2 heterostructure photoanodes. The BiVO4/TiO2 NFs showed 4.8 times higher photocurrent density than the BiVO4/TiO2 NRs. Transient decay time analysis and time-resolved photoluminescence reveal the enhancement is attributed to the reduced charge recombination, which is originated from the formation of type II band alignment between BiVO4 nanodots and TiO2 NFs. This work provides not only new insights into the interplay between crystal facets and band structures but also important steps for the design of highly efficient photoelectrodes.
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Dedications to achieve the highly efficient metal oxide semiconductor for the photoelectrochemical water splitting system have been persisted to utilize the TiO2 as the promising photoanode material. Herein, we report notable progress for nanostructured TiO2 photoanodes using facile sequential one-pot hydrothermal synthesis and annealing in hydrogen. A photocurrent density of 3.04 mA·cm-2 at 1.23 V vs. reversible hydrogen electrode was achieved in TiO2 nanorod arrays annealed in hydrogen ambient, which is approximately 4.25 times higher than that of pristine TiO2 annealed in ambient air. 79.2% of incident photon-to-current efficiency at 380 nm wavelength demonstrates the prominence of the material at the near-UV spectral range region and 100 h chronoamperometric test exhibits the stability of the photoanode. Detailed studies regarding crystallinity, bandgap, and elemental analysis provide the importance of the optimized annealing condition for the TiO2-based photoanodes. Water contact angle measurement displays the effect of hydrogen annealing on the hydrophilicity of the material. This study clearly demonstrates the marked improvement using the optimized hydrogen annealing, providing the promising methodologies for eco-friendly mass production of water splitting photoelectrodes.
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The aim of this study was to determine the effects of transplanted neural differentiated human mesenchymal stem cells (hMSCs) in a guinea pig model of auditory neuropathy. In this study, hMSCs were pretreated with a neural-induction protocol and transplanted into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. A control model was made by injection of Hanks balanced salt solution alone into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. We established the auditory neuropathy guinea pig model using 1 mM ouabain application to the round window niche. After application of ouabain to the round window niche, degeneration of most spiral ganglion neurons (SGNs) without the loss of hair cells within the organ of Corti and increasing the auditory brain responses (ABR) threshold were found. After transplantation of neural differentiated hMSCs, the number of SGNs was increased, and some of the SGNs expressed immunoreactivity with human nuclear antibody under confocal laser scanning microscopy. ABR results showed mild hearing recovery after transplantation. Based on an auditory neuropathy animal model, these findings suggest that it may be possible to replace degenerated SGNs by grafting stem cells into the scala tympani.
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Perda Auditiva Central/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Animais , Cardiotônicos/toxicidade , Cóclea/efeitos dos fármacos , Cóclea/patologia , Modelos Animais de Doenças , Feminino , Cobaias , Perda Auditiva Central/induzido quimicamente , Perda Auditiva Central/patologia , Humanos , Neurogênese , Ouabaína/toxicidade , Gânglio Espiral da Cóclea/patologia , Transplante HeterólogoRESUMO
Hydrogen peroxide (H(2)O(2)) is involved in intestinal motility through changes of smooth muscle activity. However, there is no report as to the modulatory effects of H(2)O(2) on interstitial cells of Cajal (ICC). We investigated the H(2)O(2) effects and signal transductions to determine whether the intestinal motility can be modulated through ICC. We performed whole-cell patch clamp in cultured ICC from murine intestine and molecular analyses. H(2)O(2) hyperpolarized the membrane and inhibited pacemaker currents. These effects were inhibited by glibenclamide, an inhibitor of ATP-sensitive K+ (K(ATP)) channels. The free-radical scavenger catalase inhibited the H(2)O(2)-induced effects. MAFP and AACOCF3 (a cytosolic phospholipase A2 inhibitors) or SC-560 and NS-398 (a selective COX-1 and 2 inhibitor) or AH6809 (an EP2 receptor antagonist) inhibited the H(2)O(2)-induced effects. PD98059 (a mitogen activated/ERK-activating protein kinase inhibitor) inhibited the H(2)O(2)-induced effects, though SB-203580 (a p38 MAPK inhibitor) or a JNK inhibitor did not affect. H(2)O(2)-induced effects could not be inhibited by LY-294002 (an inhibitor of PI3-kinases), calphostin C (a protein kinase C inhibitor) or SQ-22536 (an adenylate cyclase inhibitor). Adenoviral infection analysis revealed H2O2 stimulated tyrosine kinase activity and AG 1478 (an antagonist of epidermal growth factor receptor tyrosine kinase) inhibited the H(2)O(2)-induced effects. These results suggest H(2)O(2) can modulate ICC pacemaker activity and this occur by the activation of K(ATP) channels through PGE(2) production via receptor tyrosine kinase-dependent MAP kinase activation.
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Peróxido de Hidrogênio/farmacologia , Células Intersticiais de Cajal/efeitos dos fármacos , Células Intersticiais de Cajal/enzimologia , Intestinos/citologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxidantes/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/fisiologia , Catalase/metabolismo , Células Cultivadas , Inibidores Enzimáticos/metabolismo , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Células Intersticiais de Cajal/citologia , Intestinos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/genética , Técnicas de Patch-Clamp , Fosfolipases A2/metabolismo , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Adult mesenchymal stem cells (MSCs) derived from adipose tissue have the capacity to differentiate into mesenchymal as well as endodermal and ectodermal cell lineage in vitro. We characterized the multipotent ability of human adipose tissue-derived stem cells (hADSCs) as MSCs and investigated the neural differentiation potential of these cells. RESULTS: Human ADSCs from earlobe fat maintained self-renewing capacity and differentiated into adipocytes, osteoblasts, or chondrocytes under specific culture conditions. Following neural induction with bFGF and forskolin, hADSCs were differentiated into various types of neural cells including neurons and glia in vitro. In neural differentiated-hADSCs (NI-hADSCs), the immunoreactivities for neural stem cell marker (nestin), neuronal markers (Tuj1, MAP2, NFL, NFM, NFH, NSE, and NeuN), astrocyte marker (GFAP), and oligodendrocyte marker (CNPase) were significantly increased than in the primary hADSCs. RT-PCR analysis demonstrated that the mRNA levels encoding for ABCG2, nestin, Tuj1, MAP2, NFL, NFM, NSE, GAP43, SNAP25, GFAP, and CNPase were also highly increased in NI-hADSCs. Moreover, NI-hADSCs acquired neuron-like functions characterized by the display of voltage-dependent tetrodotoxin (TTX)-sensitive sodium currents, outward potassium currents, and prominent negative resting membrane potentials under whole-cell patch clamp recordings. Further examination by RT-PCR showed that NI-hADSCs expressed high level of ionic channel genes for sodium (SCN5A), potassium (MaxiK, Kv4.2, and EAG2), and calcium channels (CACNA1C and CACNA1G), which were expressed constitutively in the primary hADSCs. In addition, we demonstrated that Kv4.3 and Eag1, potassium channel genes, and NE-Na, a TTX-sensitive sodium channel gene, were highly induced following neural differentiation. CONCLUSIONS: These combined results indicate that hADSCs have the same self-renewing capacity and multipotency as stem cells, and can be differentiated into functional neurons using bFGF and forskolin.
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Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Colforsina/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Neurônios/citologia , Células-Tronco/citologia , Adolescente , Separação Celular , Células Cultivadas , Criança , Pré-Escolar , Orelha Externa/citologia , Humanos , Células-Tronco Pluripotentes/citologia , Adulto JovemRESUMO
BACKGROUND: There are conflicting evidences on endothelial function in lacunar infarction. This may be attributed to the effects of risk factors on the vascular smooth muscle. To test endothelial function only in patients with lacunar infarction, we evaluated the endothelium-dependent and -independent vasodilatation of the brachial artery. METHODS: We enrolled consecutive patients with lacunar infarction defined by clinical characteristics and MRI findings. The control group included age- and sex-matched patients with hypertension who do not have any history of clinical stroke, coronary artery disease or peripheral vascular disease. Endothelial function was evaluated using flow-mediated dilatation (FMD) and nitrogen-mediated dilatation (NMD) of the brachial artery. FMD and NMD were examined by an experienced vascular sonographer using a high-resolution ultrasound. Intracranial stenosis was defined as flow gap or >50% reduction in vessel diameter on MRA. RESULTS: FMD was 6.6 +/- 4.5% in the lacunar infarction group and 12.2 +/- 4.6% in the control group (p = 0.000). NMD was 14.3 +/- 4.9% in the lacunar infarction group and 13.8 +/- 4.9% in the control group (p = 0.37). FMD in patients with lacunar infarction and intracranial arterial stenosis was 6.4 +/- 3.9%, and FMD in patients with lacunar infarction was only 6.9 +/- 5.5%. In the control group, it was 12.2 +/- 4.6%. CONCLUSION: FMD was low in patients with lacunar infarction. NMD was similar between the lacunar infarction group and the control group. These results are suggestive of pure endothelial dysfunction in lacunar infarction. Endothelial dysfunction was as severe in lacunar infarction as in intracranial arterial stenosis.
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Infarto Cerebral/fisiopatologia , Endotélio Vascular/fisiopatologia , Arteriosclerose Intracraniana/fisiopatologia , Vasodilatação , Idoso , Artéria Braquial/diagnóstico por imagem , Artéria Braquial/fisiopatologia , Estudos de Casos e Controles , Infarto Cerebral/diagnóstico , Constrição Patológica , Feminino , Humanos , Hiperemia/fisiopatologia , Arteriosclerose Intracraniana/diagnóstico , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/fisiopatologia , Nitroglicerina , Fluxo Sanguíneo Regional , Ultrassonografia , VasodilatadoresRESUMO
The benefit of introducing gold nanoparticles is due to the plasmon relaxation process. The plasmon decay induces various phenomena such as near-field enhancement, hot electron injection, and resonance energy transfer. Shape-controlled octahedral gold nanoparticles can maximize the efficiency of these processes. For practical purposes, a high-coverage decoration method, comparable to physical vapor deposition on a metal oxide semiconductor nanostructure, is indispensable. However, the ligand exchange reaction to attach octahedral gold nanoparticles is limited in aqueous solution due to the inactivity of the gold (111) surface as a result of a densely-packed cetyltrimethylammonium bilayer structure. Herein, we report a controllable high-coverage surface decoration method of octahedral gold nanoparticles on the targeted semiconductor nanostructures via phase transfer by an organic medium with thiolated-polyethylene glycol. Our results deliver an innovative platform for future plasmonic gold nanoparticle applications.
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Despite many studies on the biological and pharmacological properties of (-)-epigallocatechin-3-gallate (EGCG), an active component of green tea, information on neuronal modulation by EGCG is limited. This study was designed to investigate the effects of EGCG on the electrical activity of rat substantia nigra dopaminergic neurons using whole-cell patch clamp recordings. The spike frequency was increased to 6.33+/-0.23 (p<0.05) and 7.15+/-0.29 (p<0.05) by 5 and 10 microM EGCG, respectively, from the control level of 5.49+/-0.19 spikes/second, respectively (n=18). The resting membrane potential of the cells was decreased to -45.66+/-0.45 and -43.99+/-0.87 (p<0.05), by 5 and 10 microM EGCG, respectively, from -47.82+/-0.57 mV. The amplitude of afterhyperpolarization was decreased to 12.73+/-0.45 (p<0.05) and 11.60+/-0.57 (p<0.05) by 5 and 10 microM EGCG, respectively, from 13.80+/-0.31 mV. The neuronal activity of dopaminergic neurons is closely linked to dopamine release. When neurons switch from a single-spike firing to bursts of action potentials, the release of dopamine increases. The above experimental results suggest that EGCG increases the neuronal activity via inhibition of calcium-dependent potassium currents underlying the afterhyperpolarization, and it could act as a facilitating factor that elicits NMDA-dependent bursts of action potentials like apamin or bicuculline methiodide.
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Antioxidantes/farmacologia , Catequina/análogos & derivados , Dopamina/metabolismo , Neurônios/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Animais , Catequina/farmacologia , Imuno-Histoquímica , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Substância Negra/citologia , Substância Negra/metabolismoRESUMO
Bone marrow-derived mesenchymal stem cells (BM-MSCs), which are characterized by multipotency and self-renewal, are responsible for tissue regeneration and repair. We have previously reported in adipose tissue-derived MSCs that only Wnt5a is enhanced at neurogenic differentiation, and the mechanism of differentiation is dependent on the Wnt5a/JNK pathway; however, the role of Wnt/MAPK pathway is yet to be investigated in neurogenic differentiation in BM-MSCs. We compared the transcriptional expression of Wnt in neurogenic induced-hBM-MSCs (NI-hBM-MSCs) with that in primary hBM-MSCs, using RT-PCR, qPCR, and western blotting. Although the expression of Wnt1 and Wnt2 was unchanged, the expression of Wnt4, Wnt5a, and Wnt11 increased after neurogenic differentiation. In addition, only the expression of frizzled class receptor (Fzd) 3 gene was increased, but not of most of the Fzds and Wnt ligands in NI-hBM-MSCs. Interestingly, Wnt4, Wnt5a, and Wnt11 gene expressions significantly increased in NI-hBM-MSCs by qPCR. In addition, the protein expression level of Wnt4 and Wnt5a, but not Wnt3, increased after neurogenic induction. Furthermore, the expressions of phosphorylated-GSK-3ß, ERK1/2, and PKC decreased; however, JNK was activated after neurogenic differentiation. Thus, non-canonical Wnts, i.e., Wnt4, Wnt5a, and Wnt11, regulate neurogenic differentiation through Fzd3 activation and the increase in downstream targets of JNK, which is one of the non-canonical pathways, in hBM-MSCs.
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Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Neurônios/metabolismo , Proteínas Wnt/metabolismo , Células Cultivadas , Receptores Frizzled/metabolismo , Expressão Gênica , Humanos , Via de Sinalização Wnt , Proteína Wnt-5a/metabolismo , Proteína Wnt4/metabolismoRESUMO
Excessive noise, ototoxic drugs, infections, autoimmune diseases, and aging can cause loss of spiral ganglion neurons, leading to permanent sensorineural hearing loss in mammals. Stem cells have been confirmed to be able to differentiate into spiral ganglion neurons. Little has been reported on adipose tissue-derived stem cells (ADSCs) for repair of injured spiral ganglion neurons. In this study, we hypothesized that transplantation of neural induced-human ADSCs (NI-hADSCs) can repair the injured spiral ganglion neurons in guinea pigs with neomycin-induced sensorineural hearing loss. NI-hADSCs were induced with culture medium containing basic fibroblast growth factor and forskolin and then injected to the injured cochleae. Guinea pigs that received injection of Hanks' balanced salt solution into the cochleae were used as controls. Hematoxylin-eosin staining showed that at 8 weeks after cell transplantation, the number of surviving spiral ganglion neurons in the cell transplantation group was significantly increased than that in the control group. Also at 8 weeks after cell transplantation, immunohistochemical staining showed that a greater number of NI-hADSCs in the spiral ganglions were detected in the cell transplantation group than in the control group, and these NI-hADSCs expressed neuronal markers neurofilament protein and microtubule-associated protein 2. Within 8 weeks after cell transplantation, the guinea pigs in the cell transplantation group had a gradually decreased auditory brainstem response threshold, while those in the control group had almost no response to 80 dB of clicks or pure tone burst. These findings suggest that a large amount of NI-hADSCs migrated to the spiral ganglions, survived for a period of time, repaired the injured spiral ganglion cells, and thereby contributed to the recovery of sensorineural hearing loss in guinea pigs.
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This study aims to investigate whether (-)-epigallocatechin-3-gallate (EGCG) affects neuronal activity of acutely isolated rat medial vestibular nuclear neurons in whole-cell configuration patch-clamp experiments. EGCG (0.5 and 1 muM) lowered the spontaneous firing rate and hyperpolarized the membrane potential of medial vestibular nuclear neurons. However, it did not change the amplitude of afterhyperpolarization or the spike width of the action potential. A second application of EGCG with the same concentration elicited lesser responses. These results suggest that EGCG decreases neuronal activity by affecting potassium currents which are responsible for membrane potentials.