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1.
Proc Natl Acad Sci U S A ; 121(14): e2315264121, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38551837

RESUMO

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.


Assuntos
Corantes Fluorescentes , Potenciais da Membrana , Potenciais de Ação , Membrana Celular , Microscopia de Fluorescência/métodos
2.
RSC Chem Biol ; 5(3): 209-215, 2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38456036

RESUMO

PHD fingers are a type of chromatin reader that primarily recognize chromatin as a function of lysine methylation state. Dysregulated PHD fingers are implicated in various human diseases, including acute myeloid leukemia. Targeting PHD fingers with small molecules is considered challenging as their histone tail binding pockets are often shallow and surface-exposed. The KDM5A PHD1 finger regulates the catalytic activity of KDM5A, an epigenetic enzyme often misregulated in cancers. To identify ligands that disrupt the PHD1-histone peptide interaction, we conducted a high-throughput screen and validated hits by orthogonal methods. We further elucidated structure-activity relationships in two classes of compounds to identify features important for binding. Our investigation offers a starting point for further optimization of small molecule PHD1 ligands.

3.
Heliyon ; 10(4): e25620, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38380000

RESUMO

The cooking process is fundamental for bean consumption and to increase the bioavailability of its nutritional components. The study aimed to determine the effect of cooking on bean seed coat through morphological analyses with different microscopy techniques and image analyses. The chemical composition and physical properties of raw black bean (RBB) and cooked black bean (CBB) seeds were determined. The surface and cross-sectional samples were studied by Optical microscopy (OM), environmental scanning electron microscopy (ESEM), atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). The composition of samples showed significant differences after the cooking process. OM images and gray level co-occurrence matrix algorithm (GLCM) analysis indicated that cuticle-deposited minerals significantly influence texture parameters. Seed coat surface ESEM images showed cluster cracking. Texture fractal dimension and lacunarity parameters were effective in quantitatively assessing cracks on CBB. AFM results showed arithmetic average roughness (Ra) (121.67 nm) and quadratic average roughness (Rq) (149.94 nm). The cross-sectional ESEM images showed a decrease in seed coat thickness. The CLSM results showed an increased availability of lipids along the different multilayer tissues in CBB. The results generated from this research work offer a valuable potential to carry out a strict control of bean seed cooking at industrial level, since the structural changes and biochemical components (cell wall, lipids and protein bodies) that occur in the different tissues of the seed are able to migrate from the inside to the outside through the cracks generated in the multilayer structure that are evidenced by the microscopic techniques used.

4.
Foods ; 13(2)2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38254478

RESUMO

During vanilla bean curing, the cell arrangement derived from the killing technique applied to start bean ripening is essential to obtain the characteristic aroma and flavor of vanilla. Hence, killing is an important step to release the enzymes and compounds required for vanillin production. In this work, high hydrostatic pressure (HHP) at 100-400 MPa for 5 min, using water at 7 °C as the pressure-transmitting medium, was applied as the killing method, and its effect on the microstructural changes in vanilla beans during different curing cycles (C0-C20) was evaluated and compared with that observed after scalding by using water at 100 °C for 8 s. Microstructural changes in the cross-sectioned beans were analyzed using a stereomicroscope (SM), confocal laser scanning microscopy (CLSM), and environmental scanning electron microscopy (ESEM). The vanilla beans were cross-sectioned and three main sectors were analyzed: the total, annular, and core. The morphometric descriptors, namely, area, Feret's diameter, and circularity, were quantified via digital image analysis (DIA), from which a shrinkage ratio was calculated. The results show that the total area in the beans presented a maximum decrease in the C16 of curing. The core area was most affected by the HHP treatment, mainly at 400 MPa, rather than scalding. CSLM observations revealed the autofluorescence of the compounds inside the beans. In conclusion, the use of microscopy techniques and DIA allowed us to determine the microstructural changes in the HHP-treated pods, which were found to be more numerous than those found in the scalded beans.

5.
bioRxiv ; 2023 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-37873283

RESUMO

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes, but also measure values of membrane potentials. This study discloses a new fluorescent indicator which can address both. We describe the synthesis of a new sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials with greater signal-to-noise than state-of-the-art BeRST (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of action potentials in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

6.
Curr Opin Chem Biol ; 74: 102286, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36948085

RESUMO

Reader domains that recognize methylated lysine and arginine residues on histones play a role in the recruitment, stabilization, and regulation of chromatin regulatory proteins. Targeting reader proteins with small molecule and peptidomimetic inhibitors has enabled the elucidation of the structure and function of specific domains and uncovered their role in diseases. Recent progress towards chemical probes that target readers of lysine methylation, including the Royal family and plant homeodomains (PHD), is discussed here. We highlight recently developed covalent cyclic peptide inhibitors of a plant homeodomain. Additionally, inhibitors targeting previously untargeted Tudor domains and chromodomains are discussed.


Assuntos
Histonas , Lisina , Cromatina , Histonas/química , Lisina/metabolismo , Metilação , Ligação Proteica
7.
Science ; 379(6639): eabm5658, 2023 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-36996219

RESUMO

It is known that interactions between nociceptors and dendritic cells (DCs) can modulate immune responses in barrier tissues. However, our understanding of the underlying communication frameworks remains rudimentary. Here, we show that nociceptors control DCs in three molecularly distinct ways. First, nociceptors release the calcitonin gene-related peptide that imparts a distinct transcriptional profile on steady-state DCs characterized by expression of pro-interleukin-1ß and other genes implicated in DC sentinel functions. Second, nociceptor activation induces contact-dependent calcium fluxes and membrane depolarization in DCs and enhances their production of proinflammatory cytokines when stimulated. Finally, nociceptor-derived chemokine CCL2 contributes to the orchestration of DC-dependent local inflammation and the induction of adaptive responses against skin-acquired antigens. Thus, the combined actions of nociceptor-derived chemokines, neuropeptides, and electrical activity fine-tune DC responses in barrier tissues.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Quimiocina CCL2 , Células Dendríticas , Interleucina-1beta , Neuroimunomodulação , Nociceptores , Pele , Quimiocina CCL2/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Nociceptores/metabolismo , Transdução de Sinais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Cálcio/metabolismo , Masculino , Feminino , Animais , Camundongos , Camundongos Endogâmicos C57BL , Pele/imunologia , Pele/microbiologia , Inflamação/imunologia , Inflamação/microbiologia
8.
Pharmaceutics ; 14(10)2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36297649

RESUMO

The objective of this study was to assess the effectiveness of liposomes loaded with soybean lunasin and amaranth unsaponifiable matter (UM + LunLip) as a source of squalene in the prevention of melanoma skin cancer in an allograft mice model. Tumors were induced by transplanting melanoma B16-F10 cells into the mice. The most effective treatments were those including UM + LunLip, with no difference between the lunasin concentrations (15 or 30 mg/kg body weight); however, these treatments were statistically different from the tumor-bearing untreated control (G3) (p < 0.05). The groups treated with topical application showed significant inhibition (68%, p < 0.05) compared to G3. The groups treated with subcutaneous injections showed significant inhibition (up to 99%, p < 0.05) in G3. During tumor development, UM + LunLip treatments under-expressed Ki-67 (0.2-fold compared to G3), glycogen synthase kinase-3ß (0.1-fold compared to G3), and overexpressed caspase-3 (30-fold compared to G3). In addition, larger tumors showed larger necrotic areas (38% with respect to the total tumor) (p < 0.0001). In conclusion, the UM + LunLip treatment was effective when applied either subcutaneously or topically in the melanoma tumor-developing groups, as it slowed down cell proliferation and activated apoptosis.

9.
Artigo em Inglês | MEDLINE | ID: mdl-36141952

RESUMO

Inflammation is a normal response in defense to agents that may cause damage to the human body. When inflammation becomes chronic, reactive oxygen species (ROS) are produced; which could lead to diseases such as cancer. The aim was to assess liposomes' antioxidant and anti-inflammatory capacity loaded with amaranth unsaponifiable matter and soybean lunasin (UM + LunLip) in an in vitro model using fibroblasts and macrophages. To evaluate ROS production, fibroblasts CHON-002 ABAP were added to promote ROS production; and the cells were treated with UM + LunLip. For inflammation markers production, lipopolysaccharides (LPS)-stimulated RAW 264.7 and peritoneal macrophages were treated with empty liposomes (EmLip), liposomes loaded with unsaponifiable matter (UMLip), liposomes loaded with lunasin (LunLip), and UM + LunLip. ROS production was significantly decreased by 77% (p < 0.05) when fibroblasts were treated with UM + LunLip at 2 mg lunasin/mL compared with the control treated with ABAP. Treatment with UMLip was the most effective in reducing tumor necrosis factor-α (71-90%) and interleukin-6 (43-55%, p < 0.001). Both liposomes containing unsaponifiable matter (UMLip and UM + LunLip) were more effective than EmLip or LunLip. In conclusion, amaranth unsaponifiable matter-loaded liposomes are effective in decreasing pro-inflammatory cytokine production.


Assuntos
Glycine max , Lipopolissacarídeos , Amidinas , Anti-Inflamatórios , Antioxidantes/farmacologia , Fibroblastos , Humanos , Inflamação , Interleucina-6 , Lipossomos , Macrófagos , Espécies Reativas de Oxigênio , Fator de Necrose Tumoral alfa
10.
J Med Chem ; 65(13): 9281-9294, 2022 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-35776775

RESUMO

A series of new (tricarbonyl)rhenium(I) complexes were synthesized using chiral bidentate ligands (+)/(-)-iminopyridines (LR/LS). The reaction yielded a mixture of mononuclear Re(I) diastereoisomers, formulated as fac-[Br(CO)3Re(S/R)L(S/R)]. Each single diastereoisomer was isolated and fully characterized. X-ray crystallography and circular dichroism spectra verified their enantiomeric nature. The cytotoxicity of each complex was evaluated against six cancer cell lines. The effect of the two complexes on viability, proliferation, and migration was analyzed on glioblastoma cell lines (U251 and LN229). Changes in the expression of histones, apoptotic, and key signaling proteins, as well as alterations in DNA structure, were also observed. These experiments showed that the chirality associated with both metal and ligand has a strong influence on cytotoxicity.


Assuntos
Glioblastoma , Rênio , Cristalografia por Raios X , Glioblastoma/tratamento farmacológico , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Rênio/química
11.
Food Chem ; 395: 133638, 2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-35816985

RESUMO

Hard to cook phenomenon results from inadequate post-harvest storage of the bean associated with the microstructure and changes in seed color and texture. The aim of this study was to evaluate the physical and chemical properties, identify the phenolic compounds and their relationship with the black bean seed coat microstructure during 270 days at 30 °C and 70% r. h. The water absorption capacity decrease to 12.19% that induced changes in seed texture observed by increasing the hardness from 5.42 to 19.96 N. A total of 37 compounds were identified by UPLC-ESI-MS and the changes in phenolic profile during storage period contribute to the seed coat color saturation. The identification of flavonoids, hydroxybenzoic and hydroxycinnamic acids, as well as distribution of condensed tannins in the seed coat, the changes in physical properties evidenced by seed darkening and hardening contribute to the seed coat impermeability.


Assuntos
Fabaceae , Phaseolus , Proantocianidinas , Flavonoides/análise , Phaseolus/química , Fenóis/análise , Proantocianidinas/análise , Sementes/química
12.
Chem Sci ; 13(22): 6599-6609, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35756531

RESUMO

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3-a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3-histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic 'groove' of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains.

13.
Food Chem X ; 14: 100284, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35345793

RESUMO

In Mexico, local ripened cheeses such as Chihuahua, Ranchero, and Cotija are produced, being consumed in great quantities together with imported cheeses. Proteolysis that takes place during ripening generates bioactive peptides; in this way the cheese acquires potential as a functional food. The ripening process of Gouda cheese was studied based on its bromatological and sensorial properties, bioactivity, and peptide profile. Ripened cheese met bromatological standard parameters and showed higher overall acceptability. After 90 days, bioactivity reached maximum values for radical scavenging (6.6%), ferric reducing power (11.2%), metal chelating effect (49%), and angiotensin I-converting enzyme inhibitory activity (66.2%). Eight peptides were identified, four from αS1-casein, f(1-9, 1-13, 1-14, and 25-36), and four from ß-casein, f(11-28, 60-63, 193-209, and 197-205). Ripening of Gouda cheese results in a product with functional potential due to the presence of peptides with biological activity. Additionally, the methodology proposed in this work could be used by the dairy industry to monitor the manufacturing process and ripening of other types of cheeses.

14.
Entramado ; 17(2): 208-225, jul.-dic. 2021. tab, graf
Artigo em Espanhol | LILACS-Express | LILACS | ID: biblio-1360423

RESUMO

RESUMEN Se presentan resultados del proyecto de investigación, mapa de conocimiento del uso de medios de comunicación en instituciones educativas de básica y media realizado en Cartago, Colombia en la I.E. (Institución educativa) GABO, la I.E Ciudad Cartago y la I.E Antonio Holguín Garcés. La metodología aplicada fue cualitativa y se utiliza la Investigación Acción Participativa para la construcción de biografías mediáticas a modo de síntesis de resultados. Se identifica una desarticulación entre las políticas públicas e iniciativas del estado a nivel nacional con las dinámicas regionales, un interés particular de la Secretaria de Educación municipal en las acciones por la calidad educativa a nivel local y una postura positiva, crítica y significativa en el papel de las TIC y los medios de comunicación en los procesos de aprendizaje en los estudiantes.


ABSTRACT Results of the research project are presented, map of knowledge of the use of media in basic and medium educational institutions carried out in Cartago, Colombia, in I.E GABO, I.E Ciudad Cartago and I.E Antonio Holguín Garcés. The methodology applied was qualitative and Participatory Action Research is used for the construction of Media Biographies as a synthesis of results. A disarticulation is identified between public policies and initiatives of the state at the national level with regional dynamics, a particular interest of the municipal education secretary in actions for educational quality at the local level and a positive, critical posture and significant in the role of ICT and the media in student learning processes.


RESUMO São apresentados os resultados do projecto de investigação, mapa de conhecimento do uso dos media nas instituições de ensino básico e secundário realizado em Cartago, Colômbia, na I.E. (Instituição Educativa) GABO, na I.E. Ciudad Cartago e na I.E. Antonio Holguín Garcés. A metodologia aplicada foi qualitativa e utilizou a Investigação de Acção Participativa para a construção de biografias dos meios de comunicação como síntese de resultados. É identificada uma desarticulação entre políticas públicas e iniciativas estatais a nível nacional com dinâmica regional, um interesse particular da Secretaria Municipal de Educação nas acções para a qualidade educativa a nível local e uma posição positiva, crítica e significativa sobre o papel das TIC e dos media nos processos de aprendizagem dos estudantes.

15.
Nanomaterials (Basel) ; 11(8)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34443791

RESUMO

Amaranthus hypochondriacus is a source of molecules with reported health benefits such as antioxidant activity and cancer prevention. The objective of this research was to optimize the conditions for preparing a liposome formulation using amaranth unsaponifiable matter as a source of squalene in order to minimize the particle size and to maximize the encapsulation efficiency of liposomes for carrying and delivering soybean lunasin into melanoma cell lines. Amaranth oil was extracted using supercritical dioxide carbon extraction (55.2 MPa pressure, 80 °C temperature, solvent (CO2)-to-feed (oil) ratio of 20). The extracted oil from amaranth was used to obtain the unsaponifiable enriched content of squalene, which was incorporated into liposomes. A Box-Behnken response surface methodology design was used to optimize the liposome formulation containing the unsaponifiable matter, once liposomes were optimized. Soybean lunasin was loaded into the liposomes and tested on A-375 and B16-F10 melanoma cells. The squalene concentration in the extracted oil was 36.64 ± 0.64 g/ 100 g of oil. The particle size in liposomes was between 115.8 and 163.1 nm; the squalene encapsulation efficiency ranged from 33.14% to 76.08%. The optimized liposome formulation contained 15.27 mg of phospholipids and 1.1 mg of unsaponifiable matter. Cell viability was affected by the liposome formulation with a half-maximum inhibitory concentration (IC50) equivalent to 225 µM in B16-F10 and 215 µM in A-375. The liposomes formulated with lunasin achieved 82.14 ± 3.34% lunasin encapsulation efficiency and improved efficacy by decreasing lunasin IC50 by 31.81% in B16-F10 and by 41.89% in A-375 compared with unencapsulated lunasin.

16.
Food Sci Nutr ; 9(7): 4007-4017, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34262754

RESUMO

The nopal cactus is an essential part of the Mexican diet and culture. The per capita consumption of young cladodes averages annually to 6.4 kg across the nation. In addition to contributing to the country's food culture, the nopal is considered a food with functional characteristics since, in addition to providing fiber, an important group of polyphenolic compounds is present, which has given cladodes to be considered a healthy food, for what they have been incorporated into the diet of Mexican people and many other countries worldwide. Research suggests that polyphenols from cladodes act as antioxidants and antidiabetics. This review studies the main phenolic components in cladodes and summarizes both conventional and novel methods to identify them.

17.
J Food Sci ; 86(3): 643-655, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33586793

RESUMO

The common bean (Phaseolus vulgaris L.) represents one of the main crops for human consumption, due to its nutritional and functional qualities. Phenolic compounds have beneficial health effects, and beans are an essential source of these molecules, being found mainly in the seed coat and its color depends on the concentration and type of phenolic compounds present. The bean during storage and processing, such as cooking, germination, extrusion, and fermentation, undergoes physical, chemical, and structural changes that affect the bioavailability of its nutrients; these changes are related to the interactions between phenolic compounds and other components of the food matrix. This review provides information about the identification and quantification of phenolic compounds present in beans and the changes they undergo during processing. It also includes information on the interactions between the phenolic compounds and the components of the bean's cell wall and the analytical methods used to identify the interactions of phenolic compounds with macromolecules.


Assuntos
Manipulação de Alimentos , Phaseolus/química , Fenóis/química , Humanos , Substâncias Macromoleculares/química
18.
J Biol Chem ; 296: 100302, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465376

RESUMO

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 µM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 µM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-µM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-µM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-µM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.


Assuntos
Amifampridina/farmacologia , Fármacos Neuromusculares/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Canais de Potássio Shaw/metabolismo , Acetilcolina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Masculino , Camundongos , Microeletrodos , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Rana pipiens , Canais de Potássio Shaw/antagonistas & inibidores , Canais de Potássio Shaw/genética , Técnicas de Cultura de Tecidos
19.
RSC Chem Biol ; 2(6): 1594-1599, 2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34977574

RESUMO

We describe the design, synthesis, and application of voltage-sensitive silicon rhodamines. Based on the Berkeley Red Sensor of Transmembrane potential, or BeRST, scaffold, the new dyes possess an isomeric molecular wire for improved alignment in the plasma membrane and 2' carboxylic acids for ready functionalization. The new isoBeRST dyes have a voltage sensitivity of 24% ΔF/F per 100 mV. Combined with a flexible polyethyleneglycol (PEG) linker and a chloroalkane HaloTag ligand, isoBeRST dyes enable voltage imaging from genetically defined cells and neurons and provide improved labeling over previous, rhodamine-based hybrid strategies. isoBeRST-Halo hybrid indicators achieve single-trial voltage imaging of membrane potential dynamics from cultured hippocampal neurons or cortical neurons in brain slices. With far-red/near infrared excitation and emission, turn-on response to action potentials, and effective cell labeling in thick tissue, the new isoBeRST-Halo derivatives provide an important complement to voltage imaging in neurobiology.

20.
Int J Nanomedicine ; 15: 7627-7650, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116492

RESUMO

The skin is the largest organ in the human body, providing a barrier to the external environment. It is composed of three layers: epidermis, dermis and hypodermis. The most external epidermis is exposed to stress factors that may lead to skin conditions such as photo-aging and skin cancer. Some treatments for skin disease utilize the incorporation of drugs or bioactive compounds into nanocarriers known as liposomes. Liposomes are membranes whose sizes range from nano to micrometers and are composed mostly of phospholipids and cholesterol, forming similar structures to cell membranes. Thus, skin treatments with liposomes have lower toxicity in comparison to traditional treatment routes such as parenteral and oral. Furthermore, addition of edge activators to the liposomes decreases the rigidity of the bilayer structure making it deformable, thereby improving skin permeability. Liposomes are composed of an aqueous core and a lipidic bilayer, which confers their amphiphilic property. Thus, they can carry hydrophobic and hydrophilic compounds, even simultaneously. Current applications of these nanocarriers are mainly in the cosmetic and pharmaceutic industries. Nevertheless, new research has revealed promising results regarding the effectiveness of liposomes for transporting bioactive compounds through the skin. Liposomes have been well studied; however, additional research is needed on the efficacy of liposomes loaded with bioactive peptides for skin delivery. The objective of this review is to provide an up-to-date description of existing techniques for the development of liposomes and their use as transporters of bioactive compounds in skin conditions such as melanoma and skin inflammation. Furthermore, to gain an understanding of the behavior of liposomes during the process of skin delivery of bioactive compounds into skin cells.


Assuntos
Inflamação/patologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Pele/patologia , Transporte Biológico , Humanos , Lipossomos/ultraestrutura , Absorção Cutânea
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