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1.
Nat Commun ; 12(1): 1900, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772000

RESUMO

The computations performed by a neural circuit depend on how it integrates its input signals into an output of its own. In the retina, ganglion cells integrate visual information over time, space, and chromatic channels. Unlike the former two, chromatic integration is largely unexplored. Analogous to classical studies of spatial integration, we here study chromatic integration in mouse retina by identifying chromatic stimuli for which activation from the green or UV color channel is maximally balanced by deactivation through the other color channel. This reveals nonlinear chromatic integration in subsets of On, Off, and On-Off ganglion cells. Unlike the latter two, nonlinear On cells display response suppression rather than activation under balanced chromatic stimulation. Furthermore, nonlinear chromatic integration occurs independently of nonlinear spatial integration, depends on contributions from the rod pathway and on surround inhibition, and may provide information about chromatic boundaries, such as the skyline in natural scenes.


Assuntos
Potenciais de Ação/fisiologia , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Campos Visuais/fisiologia , Vias Visuais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Algoritmos , Animais , Cor , Feminino , HEPES/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Dinâmica não Linear , Estimulação Luminosa/métodos , Piridazinas/farmacologia , Retina/citologia , Estricnina/farmacologia
2.
J Cosmet Dermatol ; 20(6): 1766-1773, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33617668

RESUMO

BACKGROUND: Acne is a chronic disease that affects the pilosebaceous follicle and is characterized by the presence of non-inflammatory and/or inflammatory lesions, affecting both adolescents and adults. Inflammatory acne lesions are capable to increase their melanin production and promote a post-inflammatory hyperchromia. AIMS: To assess the efficacy of a serum containing dioic acid, glycolic acid, salicylic acid, LHA, citric acid, and HEPES in treating post-inflammatory hyperpigmentation and controlling skin oiliness in Brazilian patients with acne vulgaris. PATIENTS/METHODS: A single-center, prospective, open-label clinical study included 42 subjects, from both genders, presenting acne (grade I or II), oily skin and a clinical diagnosis of acne post-inflammatory hyperpigmentation. The study was conducted for 56 days, with clinical (skin quality and the number of post-inflammatory hyperchromic lesions) and instrumental (Sebumetry) evaluations after 7, 28, and 56 days of treatment. Standardized pictures were obtained using a VISIA-6® device. RESULTS: A significant decrease in the grade of post-inflammatory hyperchromic lesions was observed after 28 and 56 days, while the number of lesions decreases by 29.4% after 56 days (p < 0.001). Sebumetry values showed a significant decrease of 30.7% in the oiliness after 7 days of treatment, and then stable during the study conduction period of 56 days (p < 0.001 for all measurements). CONCLUSIONS: The daily treatment using the investigational product showed an interesting decrease both in the grade and the number of post-inflammatory hyperchromia acne lesions after 56 days, and in the oiliness after 7 days, being stable for all study period.


Assuntos
Acne Vulgar , Ácido Salicílico , Acne Vulgar/tratamento farmacológico , Adolescente , Adulto , Brasil , Ácido Cítrico , Feminino , HEPES , Humanos , Masculino , Estudos Prospectivos , Ácido Salicílico/uso terapêutico , Resultado do Tratamento
3.
Artigo em Inglês | MEDLINE | ID: mdl-32971367

RESUMO

The successful application of monoclonal antibodies (mAb) in oncology and autoimmune diseases paved the way for the development of therapeutic antibodies with a wider range of structural and physico-chemical properties. A pH-gradient combining 2-(N-morpholino)ethanesulfonic (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers and mediated with potassium chloride was developed to sufficiently retain acidic mAbs (pI < 7) in cation exchange chromatography (CEX), while keeping suitable separation performance for basic mAbs (pI > 7). Firstly, the MES and HEPES buffers were individually evaluated in their useful pH range by applying a salt gradient. The performance of a salt-mediated pH gradient combining the MES and HEPES buffers was then compared to a commercial pH gradient kit. The developed conditions were found superior to the salt-gradient approaches and provided a useful alternative to commercial pH gradient kits. In this study, the developed conditions were applied to separate a bispecific antibody (BsAb) from its two parental mAbs.


Assuntos
Anticorpos Monoclonais , Cromatografia por Troca Iônica/métodos , Cloreto de Sódio/química , Ácidos Alcanossulfônicos/química , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , HEPES/química , Concentração de Íons de Hidrogênio , Morfolinas/química
4.
Biotechnol Bioeng ; 117(6): 1649-1660, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32129469

RESUMO

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L-1 ·day-1 , exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.


Assuntos
Técnicas de Cultura de Células/economia , Meios de Cultura/economia , Microbiologia Industrial/economia , Synechocystis/crescimento & desenvolvimento , Tampões (Química) , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , HEPES/economia , HEPES/metabolismo , Microbiologia Industrial/métodos , Synechocystis/metabolismo
5.
Analyst ; 145(4): 1396-1407, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32016204

RESUMO

The evolution of Raman spectroscopy into a useful analytical technique has been due, in part, to the development of inexpensive, compact instrumentation and advancements in methodologies that enhance Raman intensities. Surface enhanced Raman scattering (SERS) is a primary methodology for quantitative and low detection limit measurements. While a broad array of applications using solid SERS substrates have been demonstrated, in-solution SERS measurements are not as widely pursued. This work seeks to optimize the synthesis of gold nanostars (AuNS) as a colloidal SERS substrate for in-solution measurements using handheld instrumentation. The types and concentrations of two buffers typically used for AuNS synthesis are examined to optimize the SERS intensity of a chemisorbed Raman probe. The observed SERS intensity primarily depends on conditions that allow higher surface coverage of the probe. Conditions that result in AuNS aggregates are found to be most optimal for SERS, similar to other nanoparticle shapes. A method to quantitate methimazole, an anti-hormone pharmaceutical, in urine is developed and reported. The primary impact of this work is the demonstration of the combination of water dispersible substrates and handheld instrumentation for rapid and sensitive analytical measurements.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Antitireóideos/urina , Tampões (Química) , Coloides , HEPES/química , Humanos , Limite de Detecção , Metimazol/urina , Tamanho da Partícula , Piperazinas/química , Soluções , Propriedades de Superfície
6.
Analyst ; 145(5): 1810-1816, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-31951229

RESUMO

An electrochemiluminescence (ECL) sensor based on a benzo[3]uril-modified glassy carbon electrode with sensitized luminescence, with the coexistence of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as the coreactant, was successfully constructed. The sensitization mechanism was proposed by analyzing the results of the control experiments for establishing the relationship of the luminescence effect with the concentration of HEPES. Under the optimized conditions, the fabricated sensor system was applied for the detection of Fe3+ in an aqueous solution with good sensitivity and selectivity. A low detection limit of 0.41 nM was achieved, indicating superior sensor performance over the previous analytical methods. The ECL sensor system was employed for the detection of Fe3+ in human serum samples to produce excellent recoveries ranging from 96.17% to 101.81%.


Assuntos
Benzimidazóis/química , Técnicas Eletroquímicas/métodos , HEPES/química , Ferro/sangue , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Humanos , Ferro/química , Limite de Detecção , Oxirredução
7.
Biointerphases ; 14(6): 061004, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830792

RESUMO

Copper surfaces are well known for their antibacterial effects due to the release of copper ions. This benefit has been shown in many antibacterial efficiency tests, however, without considering the corrosion behaviors of copper in the physiological solutions, which could play an indispensable role in ion release from the metallic surface. This study compared the ground copper surface and sputtered cuprous oxide (Cu2O) coating in two common physiological buffers: phosphate-buffered saline (PBS) and Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Na-HEPES). The growth of the cuprous oxide (Cu2O) layer was found on copper in pure PBS, inhibiting further copper ion release. In contrast, a continuous release of copper ions was recorded in Na-HEPES for 3 h, where no oxide formation was observed. The antibacterial efficiency of copper (against E. coli) was measured and discussed with the ion release kinetics in the presence of E. coli. Similar results were obtained from Cu2O coating, ruling out its assisting role in showing the antibacterial property from copper surfaces, but they did indicate the importance of taking environmental parameters into consideration in interpreting the antibacterial efficiency of copper surfaces.


Assuntos
Antibacterianos/química , Cobre/química , HEPES/química , Íons/química , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Propriedades de Superfície
8.
Cryobiology ; 91: 128-136, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31526802

RESUMO

This study aims at the thermal analysis of marginal conditions leading to cryopreservation by vitrification, which appears to be the only alternative for indefinite preservation of large-size tissues and organs. The term "marginal conditions" here refers to cooling rates in close range with the so-called critical cooling rate, above which crystallization is avoided. The analysis of thermal effects associated with partial crystallization during vitrification is associated with the coupled phenomena of heat transfer and kinetics of crystallization. This study takes a practical, semi-empirical approach, where heat transfer is analyzed based on its underlying theoretical principles, while the thermal effects associated with partial crystallization are taken into account by means of empirical correlations. This study presents a computation framework to solve the coupled problem, while presenting a proof-of-concept for DP6 as a representative cryoprotective agent. The thermal effects associated with crystallization at various relevant cooling rates are measured in this study by means of differential scanning calorimetry. Results of this study demonstrate that, due to the thermal effects associated with partial crystallization, the cooling rate at the center of a large organ may lag behind the cooling rate in its surroundings under some scenarios, but may also exceed the surroundings cooling rate in other scenarios, leading to counter-intuitive effects associated with partial crystallization.


Assuntos
Varredura Diferencial de Calorimetria/métodos , Criopreservação/métodos , Crioprotetores/farmacologia , Análise Diferencial Térmica/métodos , Dimetil Sulfóxido/farmacologia , HEPES/farmacologia , Preservação de Órgãos/métodos , Propilenoglicóis/farmacologia , Temperatura Baixa , Crioprotetores/química , Cristalização , Temperatura Alta , Transição de Fase , Vitrificação
9.
Commun Biol ; 2: 144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044169

RESUMO

A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid-base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3 - and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Animais , Bicarbonatos/química , Tampões (Química) , Células CACO-2 , Proliferação de Células , HEPES/química , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Laboratórios , Mamíferos , Pesquisadores/educação , Cloreto de Sódio/química
10.
FEBS J ; 286(10): 1925-1940, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30761759

RESUMO

Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l-malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X-ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221 , with unit-cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C-terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES-binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.


Assuntos
Fumarato Hidratase/química , Fumarato Hidratase/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Fumarato Hidratase/genética , HEPES/química , HEPES/metabolismo , Humanos , Cinética , Lisina/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 211: 299-305, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30562703

RESUMO

A novel fluorescence probe L2 based on coumarin has been designed and synthesized. The probe L2 can be used for relay recognition of metal ions Al3+ and anion F- in the aqueous HEPES buffer (0.05 M, pH = 7.4), and build a OFF-ON-OFF detection system. The probe showed high selectivity and sensitivity to target ions in the process of relay recognition, and the corresponding detection limit could be as low as 0.014 µM (Al3+) and 0.03 µM (F-). Besides, the geometry optimizations of probe L2 and [L2 + Al3+] complex were carried out using the Gaussian 16 program based on DFT, and the identification mechanism of the probe was also discussed by the mass spectrometry and theoretical calculations. Moreover, the probe has also been successfully applied to detection of target ions in living cells.


Assuntos
Alumínio/análise , Corantes Fluorescentes/química , Fluoretos/análise , Imagem Molecular/métodos , Alumínio/metabolismo , Linhagem Celular Tumoral , Cumarínicos/química , Teoria da Densidade Funcional , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , HEPES , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Estrutura Molecular , Distribuição Normal , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Chá/química , Cremes Dentais/análise
12.
Anal Bioanal Chem ; 411(4): 797-802, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30506504

RESUMO

HEPES is commonly used in cell culture media as a buffering substance. Compared to the bicarbonate/CO2 buffer system, it does not require a CO2 atmosphere, thereby ensuring stable pH values during handling of cell culture media outside of an incubator. Due to its intrinsic charge, HEPES is considered not to be taken up by cells, which was a prerequisite during buffer development for cell culture by Good and colleagues. However, during the last years, evidence has emerged that HEPES seems to be taken up into cells and that it has major effects on cellular functions. Investigating three different cell lines (MCF-7, U2OS, HeLa) showed that all of them accommodated HEPES-containing medium, i.e., they survive and proliferate in the presence of HEPES. Determination of intracellular metabolites revealed the presence of HEPES for all cell lines. Further analysis of MCF-7 cells showed that even 48 h after medium exchange from HEPES-containing medium to HEPES-free medium, intracellular HEPES could still be detected. Thus, contrary to the common view, HEPES is taken up by cells which should be taken into consideration for studies of specific cellular functions. Graphical abstract ᅟ.


Assuntos
Citoplasma/metabolismo , HEPES/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/métodos , Transporte Biológico , Tampões (Química) , Linhagem Celular Tumoral , Meios de Cultura , Humanos
13.
Sci Adv ; 4(9): eaas9365, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30255140

RESUMO

LmrA is a bacterial ATP-binding cassette (ABC) multidrug exporter that uses metabolic energy to transport ions, cytotoxic drugs, and lipids. Voltage clamping in a Port-a-Patch was used to monitor electrical currents associated with the transport of monovalent cationic HEPES+ by single-LmrA transporters and ensembles of transporters. In these experiments, one proton and one chloride ion are effluxed together with each HEPES+ ion out of the inner compartment, whereas two sodium ions are transported into this compartment. Consequently, the sodium-motive force (interior negative and low) can drive this electrogenic ion exchange mechanism in cells under physiological conditions. The same mechanism is also relevant for the efflux of monovalent cationic ethidium, a typical multidrug transporter substrate. Studies in the presence of Mg-ATP (adenosine 5'-triphosphate) show that ion-coupled HEPES+ transport is associated with ATP-bound LmrA, whereas ion-coupled ethidium transport requires ATP binding and hydrolysis. HEPES+ is highly soluble in a water-based environment, whereas ethidium has a strong preference for residence in the water-repelling plasma membrane. We conclude that the mechanism of the ABC transporter LmrA is fundamentally related to that of an ion antiporter that uses extra steps (ATP binding and hydrolysis) to retrieve and transport membrane-soluble substrates from the phospholipid bilayer.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Farmacorresistência Bacteriana , Etídio/farmacocinética , HEPES/farmacocinética , Concentração de Íons de Hidrogênio , Lactobacillus/efeitos dos fármacos , Lactobacillus/metabolismo , Bicamadas Lipídicas/metabolismo , Magnésio/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Técnicas de Patch-Clamp , Fosfolipídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sódio/metabolismo
14.
Ann Biomed Eng ; 46(11): 1857-1869, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29922954

RESUMO

Arteries with 1-mm thick walls can be successfully vitrified by loading cryoprotective agents (CPAs) such as VS55 (8.4 M) or less concentrated DP6 (6 M) and cooling at or beyond their critical cooling rates of 2.5 and 40 °C/min, respectively. Successful warming from this vitrified state, however, can be challenging. For example, convective warming by simple warm-bath immersion achieves 70 °C/min, which is faster than VS55's critical warming rate of 55 °C/min, but remains far below that of DP6 (185 °C/min). Here we present a new method that can dramatically increase the warming rates within either a solution or tissue by inductively warming commercially available metal components placed within solutions or in proximity to tissues with non-invasive radiofrequency fields (360 kHz, 20 kA/m). Directly measured warming rates within solutions exceeded 1000 °C/min with specific absorption rates (W/g) of 100, 450 and 1000 for copper foam, aluminum foil, and nitinol mesh, respectively. As proof of principle, a carotid artery diffusively loaded with VS55 and DP6 CPA was successfully warmed with high viability using aluminum foil, while standard convection failed for the DP6 loaded tissue. Modeling suggests this approach can improve warming in tissues up to 4-mm thick where diffusive loading of CPA may be incomplete. Finally, this technology is not dependent on the size of the system and should therefore scale up where convection cannot.


Assuntos
Artérias/química , Materiais Biocompatíveis/química , Crioprotetores/química , Dimetil Sulfóxido/química , Formamidas/química , HEPES/química , Temperatura Alta , Metais/química , Propilenoglicóis/química , Ondas de Rádio
15.
Biopreserv Biobank ; 16(4): 270-277, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29958001

RESUMO

Cryopreservation represents one if not the only long-term option for tissue and perhaps future organ banking. In one particular approach, cryopreservation is achieved by completely avoiding ice formation (or crystallization) through a process called vitrification. This "ice-free" approach to tissue banking requires a combination of high-concentration cryoprotective additives such as M22 (9.4 M), VS55 (8.4 M), or DP6 (6 M) and sufficiently fast rates of cooling and warming to avoid crystallization. In this article, we report the temperature-dependent specific heat capacity of the above-mentioned cryoprotective additives in small volumes (10 mg sample pans) at rates of 5°C/min and 10°C/min using a commercially available differential scanning calorimetry (TA Instruments Q1000), in the temperature range of -150°C to 30°C. This data can be utilized in heat-transfer models to predict thermal histories in a cryopreservation protocol. More specifically, the effects of temperature dependence of specific heat due to the presence of three different phases (liquid, ice, and vitreous phase) can dramatically impact the thermal history and therefore the outcome of the cryopreservation procedure. The crystallization potential of these cryoprotectants was also investigated by studying cases of maximal and minimal crystallization in VS55 and DP6, where M22 did not crystallize under any rates tested. To further reduce crystallization in VS55 and DP6, a stabilizing sugar (sucrose) was added in varying concentrations (0.15 M and 0.6 M) and was shown to further reduce crystallization, particularly in VS55, at modest rates of cooling (1°C/min, 5°C/min, and 10°C/min).


Assuntos
Criopreservação/métodos , Crioprotetores/química , Cristalização/métodos , Dimetil Sulfóxido/química , Formamidas/química , HEPES/química , Temperatura Alta , Propilenoglicóis/química , Sacarose/química
16.
Cryobiology ; 82: 70-77, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29660316

RESUMO

Vitrification tendency and stability of the amorphous state were analyzed by means of differential scanning calorimetry (DSC) for the vitrification solution DP6, with and without additional solutes to enhance ice suppression. This study is a part of an ongoing research effort to characterize the thermophysical and mechanical properties of DP6 and its derivatives, and their qualities as cryoprotective solutions. DP6 was determined to have a critical cooling rate necessary to ensure vitrification of 2.7 °C/min. The following additional solutions were tested: DP6 + 6% (2R, 3R) 2,3-butanediol, DP6 + 6% 1,3-cyclohexanediol, DP6 + 6% (0.175M) sucrose, DP6 + 12% PEG 400, and DP6 + 17.1% (0.5 M) sucrose. The additives decreased the critical cooling rate of the DP6 solution to rates below 1 °C/min that were not quantifiable by the DSC techniques used. The following critical warming rates necessary to avoid devitrification were identified for DP6 and the modified solutions, respectively: 189 °C/min, 5 °C/min, ≈ 1 °C/min, 15 °C/min, <1 °C/min, and <1 °C/min. Glass transition temperatures and melting temperatures were also measured. Sucrose was the least effective additive on a per mass basis, with 1,3-cyclohexanediol appearing to be the most effective additive for suppressing ice formation in DP6.


Assuntos
Butileno Glicóis/química , Criopreservação/métodos , Crioprotetores/química , Cicloexanóis/química , Dimetil Sulfóxido/química , HEPES/química , Polietilenoglicóis/química , Propilenoglicóis/química , Sacarose/química , Vitrificação , Animais , Varredura Diferencial de Calorimetria , Temperatura Baixa , Transição de Fase , Temperatura de Transição
17.
Autophagy ; 14(3): 437-449, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29455584

RESUMO

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in cell culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of MTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in cell culture media due to their potentially confounding effects on experimental results.


Assuntos
Autofagia/fisiologia , Redes Reguladoras de Genes/genética , HEPES/metabolismo , Lisossomos/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Humanos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
18.
Food Res Int ; 105: 556-562, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29433247

RESUMO

This study systematically explored the effect of HEPES, Tris and sodium phosphate (PB) buffers on the xanthine oxidase (XO) inhibitory activity of tuna protein hydrolysate (TPH, containing over 90% of constituents with molecular weight below 5kDa). The greatest XO inhibition by TPH was observed in HEPES buffer. The optimal HEPES concentration was 100mmol/L. Tryptophan fluorescence and circular dichroism measurements revealed the comparable stability of XO and TPH in the three buffers. The buffers did not alter the majority of XO or TPH structure but induced slight modifications to specific domains (e.g. Trp residues on α-helices) and certain rearrangements (e.g. XO unfolding or refolding). HEPES buffer exerted stronger interactions with XO or TPH, causing a lower α-helical content in XO and consequently a lower XO catalytic activity but greater XO inhibition, compared to Tris and PB buffers.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas de Peixes da Dieta/farmacologia , Manipulação de Alimentos/métodos , HEPES/química , Fosfatos/química , Hidrolisados de Proteína/farmacologia , Alimentos Marinhos , Trometamina/química , Atum , Xantina Oxidase/antagonistas & inibidores , Animais , Tampões (Química) , Dicroísmo Circular , Inibidores Enzimáticos/isolamento & purificação , Estabilidade Enzimática , Proteínas de Peixes da Dieta/isolamento & purificação , Concentração de Íons de Hidrogênio , Dobramento de Proteína , Hidrolisados de Proteína/isolamento & purificação , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Xantina Oxidase/química , Xantina Oxidase/metabolismo
19.
Phys Chem Chem Phys ; 20(19): 13263-13270, 2018 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-29423470

RESUMO

A simple fluorophore bearing a diethylaminocoumarin donor and a pyridinium acceptor was synthesized and utilized for the ultra-sensitive detection of heparin. The synthesized dicationic push-pull coumarin derivative emits strongly in the red-region (665 nm) and detects nanomolar concentrations (14.8 nM to 148 nM) of heparin in HEPES buffer and FBS serum solutions. The dication exhibits excellent fluorescence selectivity and sensitivity towards heparin over its analogues such as chondroitin 4-sulfate (CS), hyaluronic acid (HA) and dextran. This fluorescence assay is a convenient, sensitive method for monitoring heparin levels in biological samples. These findings were confirmed using coarse-grained Monte Carlo simulations, which provide us with a rationale for the selective binding of heparin.


Assuntos
Corantes Fluorescentes/química , Heparina/análise , Espectrometria de Fluorescência/métodos , Sítios de Ligação , Técnicas Biossensoriais/métodos , Sulfatos de Condroitina/química , Simulação por Computador , Dextranos/química , HEPES/química , Ácido Hialurônico/química , Limite de Detecção , Espectroscopia de Ressonância Magnética/métodos , Ligação Proteica , Sensibilidade e Especificidade
20.
Mol Pharmacol ; 93(3): 208-215, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29326243

RESUMO

The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folate-specific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-buffered medium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na+/H+ exchanger(s); it was eliminated by inhibitors of Na+/H+ exchanger (s) or Na+/K+ ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.


Assuntos
Antagonistas do Ácido Fólico/farmacocinética , Metotrexato/farmacocinética , Transportador de Folato Acoplado a Próton/metabolismo , Transporte Biológico Ativo , Tampões (Química) , HEPES/farmacologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Ácido Poliglutâmico/metabolismo , Microambiente Tumoral
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