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1.
Front Aging Neurosci ; 14: 848380, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35250550

RESUMO

OBJECTIVE: Cortical electrical stimulation (CES) can modulate cortical excitability through a plasticity-like mechanism and is considered to have therapeutic potentials in Parkinson's disease (PD). However, the precise therapeutic value of such approach for PD remains unclear. Accordingly, we adopted a PD rat model to determine the therapeutic effects of CES. The current study was thus designed to identify the therapeutic potential of CES in PD rats. METHODS: A hemiparkinsonian rat model, in which lesions were induced using unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, was applied to identify the therapeutic effects of long-term (4-week) CES with intermittent theta-burst stimulation (iTBS) protocol (starting 24 h after PD lesion observation, 1 session/day, 5 days/week) on motor function and neuroprotection. After the CES intervention, detailed functional behavioral tests including gait analysis, akinesia, open-field locomotor activity, apomorphine-induced rotation as well as degeneration level of dopaminergic neurons were performed weekly up to postlesion week 4. RESULTS: After the CES treatment, we found that the 4-week CES intervention ameliorated the motor deficits in gait pattern, akinesia, locomotor activity, and apomorphine-induced rotation. Immunohistochemistry and tyrosine hydroxylase staining analysis demonstrated that the number of dopamine neurons was significantly greater in the CES intervention group than in the sham treatment group. CONCLUSION: This study suggests that early and long-term CES intervention could reduce the aggravation of motor dysfunction and exert neuroprotective effects in a rat model of PD. Further, this preclinical model of CES may increase the scope for the potential use of CES and serve as a link between animal and PD human studies to further identify the therapeutic mechanism of CES for PD or other neurological disorders.

2.
Circ Res ; 130(1): 112-129, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34816743

RESUMO

BACKGROUND: Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RBM24 (RNA-binding motif protein 24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim is to study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner. METHODS: We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. RESULTS: Although RBM24-/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (eg, ACTN2 [α-actinin 2], TTN [titin], and MYH10 [non-muscle myosin IIB]) were misspliced. Consequently, MYH6 (muscle myosin II) cannot replace nonmuscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the ABD (actin-binding domain; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24-/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity. CONCLUSIONS: RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.


Assuntos
Processamento Alternativo , Células-Tronco Embrionárias Humanas/metabolismo , Desenvolvimento Muscular , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Actinina/genética , Actinina/metabolismo , Diferenciação Celular , Linhagem Celular , Conectina/genética , Conectina/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Proteínas de Ligação a RNA/genética
3.
Biomedicines ; 9(8)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34440230

RESUMO

Mutated channelopathy could play important roles in the pathogenesis of aldosterone-producing adenoma (APA). In this study, we identified a somatic mutation, KCNJ5 157-159delITE, and reported its immunohistological, pathophysiological and pharmacological characteristics. We conducted patch-clamp experiments on HEK293T cells and experiments on expression of aldosterone synthase (CYP11B2) and aldosterone secretion in HAC15 cells to evaluate electrophysiological and functional properties of this mutated KCNJ5. Immunohistochemistry was conducted to identify expressions of several steroidogenic enzymes. Macrolide antibiotics and a calcium channel blocker were administrated to evaluate the functional attenuation of mutated KCNJ5 channel in transfected HAC15 cells. The interaction between macrolides and KCNJ5 protein was evaluated via molecular docking and molecular dynamics simulation analysis. The immunohistochemistry analysis showed strong CYP11B2 immunoreactivity in the APA harboring KCNJ5 157-159delITE mutation. Whole-cell patch-clamp data revealed that mutated KCNJ5 157-159delITE channel exhibited loss of potassium ion selectivity. The mutant-transfected HAC15 cells increased the expression of CYP11B2 and aldosterone secretion, which was partially suppressed by clarithromycin and nifedipine but not roxithromycin treatment. The docking analysis and molecular dynamics simulation disclosed that roxithromycin had strong interaction with KCNJ5 L168R mutant channel but not with this KCNJ5 157-159delITE mutant channel. We showed comprehensive evaluations of the KCNJ5 157-159delITE mutation which revealed that it disrupted potassium channel selectivity and aggravated autonomous aldosterone production. We further demonstrated that macrolide antibiotics, roxithromycin, could not interfere the aberrant electrophysiological properties and gain-of-function aldosterone secretion induced by KCNJ5 157-159delITE mutation.

4.
J Mol Endocrinol ; 67(4): 203-215, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34463641

RESUMO

Somatic mutation in the KCNJ5 gene is a common driver of autonomous aldosterone overproduction in aldosterone-producing adenomas (APA). KCNJ5 mutations contribute to a loss of potassium selectivity, and an inward Na+ current could be detected in cells transfected with mutated KCNJ5. Among 223 unilateral primary aldosteronism (uPA) individuals with a KCNJ5 mutation, we identified 6 adenomas with a KCNJ5 p.Gly387Arg (G387R) mutation, previously unreported in uPA patients. The six uPA patients harboring mutant KCNJ5-G387R were older, had a longer hypertensive history, and had milder elevated preoperative plasma aldosterone levels than those APA patients with more frequently detected KCNJ5 mutations. CYP11B2 immunohistochemical staining was only positive in three adenomas, while the other three had co-existing multiple aldosterone-producing micronodules. The bioinformatics analysis predicted that function of the KCNJ5-G387R mutant channel could be pathological. However, the electrophysiological experiment demonstrated that transfected G387R mutant cells did not have an aberrantly stimulated ion current, with lower CYP11B2 synthesis and aldosterone production, when compared to that of the more frequently detected mutant KCNJ5-L168R transfected cells. In conclusion, mutant KCNJ5-G387R is not a functional KCNJ5 mutation in unilateral PA. Compared with other KCNJ5 mutations, the observed mildly elevated aldosterone expression actually hindered the clinical identification of clinical unilateral PA. The KCNJ5-G387R mutation needs to be distinguished from functional KCNJ5 mutations during genomic analysis in APA evaluation because of its functional silence.


Assuntos
Alelos , Substituição de Aminoácidos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Mutação , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Idoso , Sequência de Aminoácidos , Biomarcadores , Linhagem Celular , Análise Mutacional de DNA , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/terapia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Relação Estrutura-Atividade
5.
Front Neural Circuits ; 15: 693073, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34194304

RESUMO

Objective: Individuals with different severities of traumatic brain injury (TBI) often suffer long-lasting motor, sensory, neurological, or cognitive disturbances. To date, no neuromodulation-based therapies have been used to manage the functional deficits associated with TBI. Cortical electrical stimulation (CES) has been increasingly developed for modulating brain plasticity and is considered to have therapeutic potential in TBI. However, the therapeutic value of such a technique for TBI is still unclear. Accordingly, an animal model of this disease would be helpful for mechanistic insight into using CES as a novel treatment approach in TBI. The current study aims to apply a novel CES scheme with a theta-burst stimulation (TBS) protocol to identify the therapeutic potential of CES in a weight drop-induced rat model of TBI. Methods: TBI rats were divided into the sham CES treatment group and CES treatment group. Following early and long-term CES intervention (starting 24 h after TBI, 1 session/day, 5 days/week) in awake TBI animals for a total of 4 weeks, the effects of CES on the modified neurological severity score (mNSS), sensorimotor and cognitive behaviors and neuroinflammatory changes were identified. Results: We found that the 4-week CES intervention significantly alleviated the TBI-induced neurological, sensorimotor, and cognitive deficits in locomotor activity, sensory and recognition memory. Immunohistochemically, we found that CES mitigated the glial fibrillary acidic protein (GFAP) activation in the hippocampus. Conclusion: These findings suggest that CES has significant benefits in alleviating TBI-related symptoms and represents a promising treatment for TBI.


Assuntos
Lesões Encefálicas Traumáticas , Transtornos Cognitivos , Disfunção Cognitiva , Animais , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/terapia , Cognição , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/terapia , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/terapia , Modelos Animais de Doenças , Estimulação Elétrica , Ratos
6.
Microsc Microanal ; 27(2): 420-424, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33487212

RESUMO

Lysosomes are integration hubs for several signaling pathways, such as autophagy and endocytosis, and also crucial stores of ions, including Zn2+. Lysosomal dysfunction caused by changes in their morphology by fusion and fission processes can result in several pathological disorders. However, the role of Zn2+ in modulating the morphology of lysosomes is unclear. The resolution of conventional epifluorescence microscopy restricts accurate observation of morphological changes of subcellular fluorescence punctum. In this study, we used a modified epifluorescence microscopy to identify the center of a punctum from a series of z-stack images and calculate the morphological changes. We stained primary cultured rat embryonic cortical neurons with FluoZin3, a Zn2+-sensitive fluorescent dye, and Lysotracker, a lysosome-specific marker, to visualize the distribution of Zn2+-enriched vesicles and lysosomes, respectively. Our results revealed that treating neurons with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, a cell-permeable Zn2+ chelator, shrank Zn2+-enriched vesicles and lysosomes by up to 25% in an hour. Pretreating the neurons with YM201636, a blocker of lysosome fission, could suppress this shrinkage. These results demonstrate the usefulness of the modified epifluorescence microscopy for investigating the homeostasis of intracellular organelles and related disorders.


Assuntos
Lisossomos , Neurônios , Animais , Autofagia , Células Cultivadas , Ratos , Zinco
7.
ACS Appl Bio Mater ; 4(9): 6865-6873, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-35006986

RESUMO

Recording ion fluctuations surrounding biological cells with a nanoelectronic device offers seamless integration of nanotechnology into living organisms and is essential for understanding cellular activities. The concentration of potassium ion in the extracellular fluid (CK+ex) is a critical determinant of cell membrane potential and must be maintained within an appropriate range. Alteration in CK+ex can affect neuronal excitability, induce heart arrhythmias, and even trigger seizure-like reactions in the brain. Therefore, monitoring local fluctuations in real time provides an early diagnosis of the occurrence of the K+-induced pathophysiological responses. Here, we modified the surface of a silicon nanowire field-effect transistor (SiNW-FET) with K+-specific DNA-aptamers (AptK+) to monitor the real-time variations of CK+ex in primary cultured rat embryonic cortical neurons or human embryonic stem cell-derived cardiomyocytes. The binding affinity of AptK+ to K+, determined by measuring the dissociation constant of the AptK+-K+ complex (Kd = 10.1 ± 0.9 mM), is at least 38-fold higher than other ions (e.g., Na+, Ca2+, and Mg2+). By placing cultured cortical neurons over an AptK+/SiNW-FET device, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation raised the CK+ex dose-dependently to 16 mM when AMPA concentration was >10 µM; this elevation could be significantly suppressed by an AMPA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. Likewise, the stimulation of isoproterenol to cardiomyocytes raised the CK+ex to 6-8 mM, with a concomitant increase in the beating rate. This study utilizing a robust nanobiosensor to detect real-time ion fluctuations surrounding excitable cells underlies the importance of ion homeostasis and offers the feasibility of developing an implant device for real-time monitoring.


Assuntos
Nanofios , Animais , Íons , Nanofios/química , Potássio/metabolismo , Ratos , Silício/química , Transistores Eletrônicos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
8.
PLoS One ; 15(9): e0232729, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915786

RESUMO

Zinc ions (Zn2+) are important messenger molecules involved in various physiological functions. To maintain the homeostasis of cytosolic Zn2+ concentration ([Zn2+]c), Zrt/Irt-related proteins (ZIPs) and Zn2+ transporters (ZnTs) are the two families of proteins responsible for decreasing and increasing the [Zn2+]c, respectively, by fluxing Zn2+ across the membranes of the cell and intracellular compartments in opposite directions. Most studies focus on the cytotoxicity incurred by a high concentration of [Zn2+]c and less investigate the [Zn2+]c at physiological levels. Zinc oxide-nanoparticle (ZnO-NP) is blood brain barrier-permeable and elevates the [Zn2+]c to different levels according to the concentrations of ZnO-NP applied. In this study, we mildly elevated the [Zn2+]c by ZnO-NP at concentrations below 1 µg/ml, which had little cytotoxicity, in cultured human neuroblastoma SH-SY5Y cells and characterized the importance of Zn2+ transporters in 6-hydroxy dopamine (6-OHDA)-induced cell death. The results show that ZnO-NP at low concentrations elevated the [Zn2+]c transiently in 6 hr, then declined gradually to a basal level in 24 hr. Knocking down the expression levels of ZnT1 (located mostly at the plasma membrane) and ZIP8 (present in endosomes and lysosomes) increased and decreased the ZnO-NP-induced elevation of [Zn2+]c, respectively. ZnO-NP treatment reduced the basal levels of reactive oxygen species and Bax/Bcl-2 mRNA ratios; in addition, ZnO-NP decreased the 6-OHDA-induced ROS production, p53 expression, and cell death. These results show that ZnO-NP-induced mild elevation in [Zn2+]c activates beneficial effects in reducing the 6-OHDA-induced cytotoxic effects. Therefore, brain-delivery of ZnO-NP can be regarded as a potential therapy for neurodegenerative diseases.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Nanopartículas Metálicas , Óxido de Zinco/farmacologia , Zinco/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Expressão Gênica/efeitos dos fármacos , Humanos , Oxidopamina/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Small ; 14(24): e1704439, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29770576

RESUMO

The Zn2+ stored in the secretory vesicles of glutamatergic neurons is coreleased with glutamate upon stimulation, resulting in the elevation of extracellular Zn2+ concentration (CZn2+ex). This elevation of CZn2+ex regulates the neurotransmission and facilitates the fibrilization of amyloid-ß (Aß). However, the exact CZn2+ex surrounding neurons under (patho)physiological conditions is not clear and the connection between CZn2+ex and the Aß fibrilization remains obscure. Here, a silicon nanowire field-effect transistor (SiNW-FET) with the Zn2+ -sensitive fluorophore, FluoZin-3 (FZ-3), to quantify the CZn2+ex in real time is modified. This FZ-3/SiNW-FET device has a dissociation constant of ≈12 × 10-9 m against Zn2+ . By placing a coverslip seeded with cultured embryonic cortical neurons atop an FZ-3/SiNW-FET, the CZn2+ex elevated to ≈110 × 10-9 m upon stimulation with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Blockers against the AMPA receptor or exocytosis greatly suppress this elevation, indicating that the Zn2+ stored in the synaptic vesicles is the major source responsible for this elevation of CZn2+ex. In addition, a SiNW-FET modified with Aß could bind Zn2+ with a dissociation constant of ≈633 × 10-9 m and respond to the Zn2+ released from AMPA-stimulated neurons. Therefore, the CZn2+ex can reach a level high enough to bind Aß and the Zn2+ homeostasis can be a therapeutic strategy to prevent neurodegeneration.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Espaço Extracelular/química , Nanofios/química , Neurônios/metabolismo , Transistores Eletrônicos , Zinco/farmacologia , Animais , Feminino , Íons , Neurônios/efeitos dos fármacos , Neurotransmissores/metabolismo , Ratos Sprague-Dawley , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
10.
ACS Sens ; 2(1): 69-79, 2017 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-28722429

RESUMO

The concentration gradient of K+ across the cell membrane of a neuron determines its resting potential and cell excitability. During neurotransmission, the efflux of K+ from the cell via various channels will not only decrease the intracellular K+ content but also elevate the extracellular K+ concentration. However, it is not clear to what extent this change could be. In this study, we developed a multiple-parallel-connected silicon nanowire field-effect transistor (SiNW-FET) modified with K+-specific DNA-aptamers (aptamer/SiNW-FET) for the real-time detection of the K+ efflux from cultured cortical neurons. The aptamer/SiNW-FET showed an association constant of (2.18 ± 0.44) × 106 M-1 against K+ and an either less or negligible response to other alkali metal ions. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation induced an outward current and hyperpolarized the membrane potential in a whole-cell patched neuron under a Na+/K+-free buffer. When neurons were placed atop the aptamer/SiNW-FET in a Na+/K+-free buffer, AMPA (13 µM) stimulation elevated the extracellular K+ concentration to ∼800 nM, which is greatly reduced by 6,7-dinitroquinoxaline-2,3-dione, an AMPA receptor antagonist. The EC50 of AMPA in elevating the extracellular K+ concentration was 10.3 µM. By stimulating the neurons with AMPA under a normal physiological buffer, the K+ concentration in the isolated cytosolic fraction was decreased by 75%. These experiments demonstrate that the aptamer/SiNW-FET is sensitive for detecting cations and the K+ concentrations inside and outside the neurons could be greatly changed to modulate the neuron excitability.

11.
Nanoscale ; 9(27): 9457-9466, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28660935

RESUMO

Optogenetics is an innovative technology now widely adopted by researchers in different fields of biological sciences. However, most light-sensitive proteins adopted in optogenetics are excited by ultraviolet or visible light which has a weak tissue penetration capability. Upconversion nanoparticles (UCNPs), which absorb near-infrared (NIR) light to emit shorter wavelength light, can help address this issue. In this report, we demonstrated the target selectivity by specifically conjugating the UCNPs with channelrhodopsin-2 (ChR2). We tagged the V5 epitope to the extracellular N-terminal of ChR2 (V5-ChR2m) and functionalized the surface of UCNPs with NeutrAvidin (NAv-UCNPs). After the binding of the biotinylated antibody against V5 onto the V5-ChR2m expressed in the plasma membrane of live HEK293T cells, our results showed that the NAv-UCNPs were specifically bound to the membrane of cells expressing V5-ChR2m. Without the V5 epitope or NAv modification, no binding of UCNPs onto the cell membrane was observed. For the cells expressing V5-ChR2m and bound with NAv-UCNPs, both 488 nm illumination and the upconverted blue emission from UCNPs by 980 nm excitation induced an inward current and elevated the intracellular Ca2+ concentration. Our design reduces the distance between UCNPs and light-sensitive proteins to the molecular level, which not only minimizes the NIR energy required but also provides a way to guide the specific binding for optogenetics applications.

12.
Mol Cell Neurosci ; 82: 35-45, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28427888

RESUMO

Zinc ion (Zn2+), the second most abundant transition metal after iron in the body, is essential for neuronal activity and also induces toxicity if the concentration is abnormally high. Our previous results show that exposure of cultured cortical neurons to dopamine elevates intracellular Zn2+ concentrations ([Zn2+]i) and induces autophagosome formation but the mechanism is not clear. In this study, we characterized the signaling pathway responsible for the dopamine-induced elevation of [Zn2+]i and the effect of [Zn2+]i in modulating the autophagy in cultured rat embryonic cortical neurons. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a membrane-permeable Zn2+ chelator, could rescue the cell death and suppress the autophagosome puncta number induced by dopamine. Dopamine treatment increased the lipidation level of the endogenous microtubule-associated protein 1A/1B-light chain 3 (LC3 II), an autophagosome marker. TPEN added 1h before, but not after, dopamine treatment suppressed the dopamine-induced elevation of LC3 II level. Inhibitors of the dopamine D1-like receptor, protein kinase A (PKA), and NOS suppressed the dopamine-induced elevation of [Zn2+]i. PKA activators and NO generators directly increased [Zn2+]i in cultured neurons. Through cell fractionation, proteins with m.w. values between 5 and 10kD were found to release Zn2+ following NO stimulation. In addition, TPEN pretreatment and an inhibitor against PKA could suppress the LC3 II level increased by NO and dopamine, respectively. Therefore, our results demonstrate that dopamine-induced elevation of [Zn2+]i is mediated by the D1-like receptor-PKA-NO pathway and is important in modulating the cell death and autophagy.


Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Zinco/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Células Cultivadas , Quelantes/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Etilenodiaminas/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
13.
IBRO Rep ; 2: 63-71, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30135934

RESUMO

Ca2+ influx through voltage-gated Ca2+ channels (CaVs) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca2+ concentration ([Ca2+]i), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca2+-dependent inactivation (CDI) of several types of CaVs; however, little is known about how CaM modulates CaV2.2. Here, we expressed CaV2.2 with CaM or CaM mutants with a Ca2+-binding deficiency in HEK293T cells and measured the currents to characterize the CDI. The results showed that CaV2.2 displayed a fast inactivation with Ca2+ but not Ba2+ as the charge carrier; when CaV2.2 was co-expressed with CaM mutants with a Ca2+-binding deficiency, the level of inactivation decreased. Using glutathione S-transferase-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of CaV2.2 in the presence or absence of Ca2+. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. CaV2.2 with mutations in the CaM-binding site showed a greatly reduced current that could be rescued by CaM12 (Ca2+-binding deficiency at the N-lobe) overexpression; in addition, CaM12 enhanced the total expression level of CaV2.2, but the ratio of CaV2.2 present in the membrane to the total fraction remained unchanged. Together, our data suggest that CaM, with different Ca2+-binding abilities, modulates not only the inactivation of CaV2.2 but also its expression to regulate Ca2+-related physiological activities.

14.
Biomed Opt Express ; 7(11): 4416-4423, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27895984

RESUMO

Optogenetics has emerged as a revolutionary technology especially for neuroscience and has advanced continuously over the past decade. Conventional approaches for patterned in vivo optical illumination have a limitation on the implanted device size and achievable spatio-temporal resolution. In this work, we developed a fabrication process for a microfiber array platform. Arrayed poly(methyl methacrylate) (PMMA) microfibers were drawn from a polymer solution and packaged with polydimethylsiloxane (PDMS). The exposed end face of a packaged microfiber was tuned to have a size corresponding to a single cell. To demonstrate its capability for single cell optogenetics, HEK293T cells expressing channelrhodopsin-2 (ChR2) were cultured on the platform and excited with UV laser. We could then observe an elevation in the intracellular Ca2+ concentrations due to the influx of Ca2+ through the activated ChR2 into the cytosol. The statistical and simulation results indicate that the proposed microfiber array platform can be used for single cell optogenetic applications.

15.
Small ; 12(40): 5524-5529, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27551968

RESUMO

Silicon nanowire field-effect transistors modified with specific aptamers can directly detect the minute dopamine and neuropeptide Y released from cells. The binding of these molecules to the aptamers results in a conductance change of the transistor biosensor and illustrates the differential releasing mechanisms of these molecules stored in various vesicle pools.


Assuntos
Aptâmeros de Peptídeos/química , Dopamina/análise , Histamina/farmacologia , Nanofios/química , Neuropeptídeo Y/análise , Transistores Eletrônicos , Animais , Células PC12 , Ratos , Silício/química
16.
Curr Cancer Drug Targets ; 16(3): 275-86, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26882030

RESUMO

Anti-angiogenesis therapy is one major approach of cancer therapies nowadays. Unfortunately, anti-angiogenesis therapy targeting VEGF-A was recently stumbled by the drugresistance that results from adaptive mechanisms, such as intratumor hypoxia. To obtain a more efficient therapeutic response, we created and identified a novel chimeric fusion of VEGF121 and VEGF165, which was connected by Fc region of human IgG1 to enhance dimerization. We found that the treatment of VEGF121-VEGF165 chimeric protein reduces proliferation, migration, invasion, and tube formation in endothelial and/or cancer cells through competing VEGF165 homodimer in a paracrine and an autocrine manner. Furthermore, the fusion protein attenuated autocrine VEGFR2-HIF-1α-VEGF165/Lon signaling through PI3KAKT- mTOR pathway in cancer cells. In conclusion, our data demonstrated that the chimeric VEGF121-VEGF165 arrests the tube formation of endothelial cells and interferes with tumor cell growth, migration and invasion, suggesting that it could be a potential drug as an angiogenesis antagonist in cancer therapy. The VEGF121-VEGF165 targets not only paracrine angiogenic cascade of endothelial cells but also autocrine PI3K-AKT-mTOR-mediated VEGFR2-HIF-1α- VEGF165/Lon signaling that drives drug resistance in tumor cells. Our study will open up the patient opportunities to combat drug resistance to antiangiogenic therapy.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neovascularização Patológica/prevenção & controle , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética
17.
Sci Rep ; 5: 17375, 2015 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-26616332

RESUMO

Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.


Assuntos
Inativação Gênica , MicroRNAs/genética , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Interferência de RNA , Processamento Pós-Transcricional do RNA , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , MicroRNAs/química , Nanofios , Conformação de Ácido Nucleico , Silício , Transistores Eletrônicos
18.
PLoS One ; 10(9): e0138856, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26421717

RESUMO

Changes in intracellular Ca2+ concentrations ([Ca2+]i) are an important signal for various physiological activities. The Na+/Ca2+ exchangers (NCX) at the plasma membrane transport Ca2+ into or out of the cell according to the electrochemical gradients of Na+ and Ca2+ to modulate [Ca2+]i homeostasis. Calmodulin (CaM) senses [Ca2+]i changes and relays Ca2+ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca2+ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM1234 (a Ca2+-binding deficient mutant) in HEK293T cells and measured the increase in [Ca2+]i contributed by the influx of Ca2+ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM1234. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3rd or 4th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2nd and 3rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca2+]i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Processamento Alternativo/fisiologia , Calmodulina/genética , Membrana Celular/genética , Células HEK293 , Humanos , Mutação Puntual , Estrutura Secundária de Proteína , Trocador de Sódio e Cálcio/genética
19.
Neuropharmacology ; 83: 54-61, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24721626

RESUMO

Phenytoin is an effective anti-epileptic drug that inhibits Na(+) channel activities; however, how phenytoin modulates synaptic transmission to soothe epileptic symptoms is not clear. To characterize the effects of phenytoin regulation on neurotransmission, we studied the electrophysical properties of cultured embryonic cortical neurons. Phenytoin inhibited the inward Na(+) current in a dose-dependent manner with an IC50 of 16.8 µM, and at 100 µM, the inhibitory effect of phenytoin on the Na(+) current was proportional to the frequency applied. In cultured neurons, phenytoin significantly decreased the action potential firing rate and the peak potential. To study the effect of phenytoin in neurotransmission, we measured the Ca(2+) responses from stimulated target neurons and their neighboring neurons. Phenytoin significantly suppressed the Ca(2+) responses evoked by strong stimulations in the target and neighboring neurons, and exerted a decreased inhibitory effect under moderate stimulation. Picrotoxin, a GABAA receptor antagonist, enhanced the recorded spontaneous excitatory postsynaptic current activities. After picrotoxin-induced enhancement, phenytoin had a more pronounced effect on the suppression of the spontaneous hyper-exciting excitatory postsynaptic current (>100 pA), but it only mildly inhibited the general excitatory postsynaptic current. Our results demonstrate that phenytoin suppresses the efficacy of neurotransmission especially for the high-frequency stimulation by reducing the Na(+) channel activity and can potentially alleviate epileptiform activity.


Assuntos
Anticonvulsivantes/farmacologia , Córtex Cerebral/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fenitoína/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Células Cultivadas , Córtex Cerebral/embriologia , Potenciais da Membrana/efeitos dos fármacos , Ratos Sprague-Dawley
20.
Cell Biol Toxicol ; 29(6): 415-29, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24077806

RESUMO

Dopamine oxidation and divalent cations have been reported to induce neuronal cell death. Although autophagy is involved in neuronal cell death, it has also been suggested to facilitate cell survival. We sought to investigate the role of autophagy in PC12 cells and cultured neurons treated with dopamine and Zn2+. Cells expressing EGFP-LC3 were treated with high concentrations of dopamine and Zn2+, and the formation of EGFP-LC3 fluorescence aggregates was monitored. Our results showed a significant increase in the number of fluorescent puncta in the cytosol of PC12 cells treated with these chemicals. These treatments enhanced LC3 lipidation levels in PC12 cells. Decreasing the ATG7 protein level using specific small interference RNA (siRNA) and pretreating with phosphatidylinositol 3-phosphate kinase blockers, wortmannin and LY294002, inhibited puncta formation. Dopamine or Zn2+ treatment significantly elevated the intracellular Zn2+ concentration ([Zn2+] i ); however, inhibiting the [Zn2+] i elevation in dopamine-treated cells suppressed the puncta formation. LY294002 or siRNA-directed members of the autophagy pathway increased the fraction of phosphatidylserine present on the outer membrane leaflet in PC12 cells treated with dopamine or Zn2+, suggesting an increase in apoptosis. Primary embryonic midbrain neurons expressing EGFP-LC3 also displayed a significant increase in the number of fluorescent aggregates in cells upon treatment with dopamine or Zn2+. Dopamine or Zn2+ treatment significantly elevated the [Zn2+] i in neurons and caused neuronal death. Our results indicate that treating cells with dopamine and Zn2+ results in the activation of the autophagy pathway in an effort to enhance cell survival.


Assuntos
Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromonas/farmacologia , Dopamina/farmacologia , Proteínas de Fluorescência Verde , Morfolinas/farmacologia , Células PC12 , Fagossomos/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/metabolismo , RNA Interferente Pequeno , Ratos , Transdução de Sinais/efeitos dos fármacos , Zinco/farmacologia
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