RESUMO
Although predictions from the past about the future have been of major interest to current neuroscience, how past and present behavioral experience interacts at the level of a single neuron remains largely unknown. Using the pond snail Lymnaea stagnalis we found that recent experience of terrestrial locomotion (exercise) results in a long-term increase in the firing rate of serotonergic pedal (PeA) neurons. Isolation from the CNS preserved the "memory" about previous motor activity in the neurons even after the animals rested for two hours in deep water after the exercise. In contrast, in the CNS, no difference in the firing rate between the control and "exercise-rested" (ER) neurons was seen. ER snails, when placed again on a surface to exercise, nevertheless showed faster locomotor arousal. The difference in the firing rate between the control and ER isolated neurons disappeared when the neurons were placed in the microenvironment of their home ganglia. It is likely that an increased content of dopamine in the CNS masks an increased excitation of PeA neurons after rest: the dopamine receptor antagonist sulpiride produced sustained excitation in PeA neurons from ER snails but not in the control. Therefore, our data suggest the involvement of two mechanisms in the interplay of past and present experiences at the cellular level: intrinsic neuronal changes in the biophysical properties of the cell membrane and extrinsic modulatory environment of the ganglia.
Assuntos
Lymnaea/fisiologia , Neurônios Serotoninérgicos/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Dopamina/metabolismo , Locomoção , Lymnaea/citologia , Memória , Atividade Motora , Neurônios Serotoninérgicos/citologia , Análise de Célula ÚnicaRESUMO
Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.
Assuntos
Coriocarcinoma/genética , Coriocarcinoma/patologia , Espectroscopia Dielétrica , Perfilação da Expressão Gênica , Modelos Biológicos , Placenta/citologia , Placenta/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , GravidezRESUMO
Extracellular microRNA are one of the indicators of the functional state of cells. Culturing of Caco-2 cells under the conditions of microcirculation in a Homunculus microfluidic device allows better simulating natural environment of the body in comparison with static culturing. Impedance spectroscopy (BioClinicum Research Center) was used for non-invasive estimation of the monolayer quality and changes in the cell apical membrane due to the formation of microvilli. Under static conditions, Caco-2 cells release more microRNA from the apical membrane than under microcirculation conditions, while secretion of miR-320a, miR-24-3p, and miR-221-3p microRNA under static conditions can indicate stress of the cells and activation of inflammatory response. Under microcirculation conditions, the expression of laminin-α1 (LAMA1) was lower than under static conditions, which indicates deeper differentiation of cells.
Assuntos
Membrana Celular/metabolismo , Dispositivos Lab-On-A-Chip , MicroRNAs/metabolismo , Microcirculação/fisiologia , Células CACO-2 , Diferenciação Celular/fisiologia , Espectroscopia Dielétrica , HumanosRESUMO
We compared available methods for monitoring the integrity of in vitro models of barrier tissues and studied the possibility of using impedance spectroscopy to solve this problem. It was demonstrated (theoretically and experimentally) that TEER measurements are not sufficiently sensitive to detect small defects in the cell barrier that significantly affect its permeability. For obtaining reliable results, it is necessary to set a sufficiently high threshold TEER, which leads to the loss of many intact samples. At the same time, impedance spectroscopy has all advantages of the classical method of measuring TEER (it is rapid and non-invasive method), while its application in combination with the methods of machine learning allows reliable detection of defects in the cell barrier.
Assuntos
Espectroscopia Dielétrica/métodos , Mucosa Intestinal/metabolismo , Células CACO-2 , Impedância Elétrica , HumanosRESUMO
External magnetic field is characterized by low toxicity and existence of magnetic properties, which contributes to an interest in the development of products from ferromagnetic nanoparticles (FNP) for antitumor therapy. Previously we synthesized a conjugate of ferromagnetic magnetite nanoparticles and viscumin (mistletoe lectin I, MLI), which exhibits the antitumor activity. Studying the pharmacological properties of this conjugate (FNP-MLI) was directed to the evaluation of FNP-MLI elimination after intratumor injection in mice. The elimination rate of FNP-MLI was much lower than that of native plant MLI. The presence of FNP-MLI was not accompanied by undesired changes in the tumor tissue. The use of a FNP-MLI conjugate allowed us to prolong the time of MLI presence in tissues without increasing the dose of exogenous lectin. These features contribute to the prolongation of an immunomodulatory effect of MLI.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/administração & dosagem , Nanopartículas de Magnetita/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 2/farmacocinética , Toxinas Biológicas/farmacocinética , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Portadores de Fármacos/farmacocinética , Feminino , Humanos , Injeções Intralesionais , Injeções Subcutâneas , Imageamento por Ressonância Magnética , Camundongos , Camundongos SCID , Proteínas Inativadoras de Ribossomos Tipo 2/farmacologia , Toxinas Biológicas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We studied the possibility of using viscumin lectin (MLI) for targeted delivery of antitumor drugs. Affinity of MLI for more than 600 oligosaccharide structures was determined and the glycosylation profiles of cell surface in various mouse tissues were analyzed. It was found that biodistribution of MLI was determined by not only expression of oligosaccharides specifically recognized by the lectin in tissue cells, but also by the structure of glycan in general.
Assuntos
Lectinas de Plantas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 2/metabolismo , Toxinas Biológicas/metabolismo , Animais , Feminino , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismoRESUMO
We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.
Assuntos
Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Dispositivos Lab-On-A-Chip , Bupropiona/metabolismo , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2B6/análise , Citocromo P-450 CYP2C19/análise , Citocromo P-450 CYP2C9/análise , Citocromo P-450 CYP3A/análise , Inibidores das Enzimas do Citocromo P-450/farmacologia , Humanos , Cetoconazol/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Espectrometria de Massas , Mefenitoína/análogos & derivados , Mefenitoína/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Omeprazol/metabolismo , Fenciclidina/análogos & derivados , Fenciclidina/farmacologia , Especificidade por Substrato , Sulfafenazol/farmacologia , Testosterona/metabolismo , Tolbutamida/metabolismoRESUMO
We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.
Assuntos
Fígado/citologia , Reatores Biológicos , Sobrevivência Celular/fisiologia , Hepatócitos/citologia , Humanos , Microcirculação/fisiologiaRESUMO
We studied the effects of DMSO and fibroblasts during HepaRG cell spheroid formation and conditions of their subsequent culturing on the levels of mRNA of the major cytochromes P450. A protocol of spheroid formation from differentiated HepaRG cells and their culturing in serum- and DMSO-free medium is developed.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Hepatócitos/enzimologia , Esferoides Celulares/enzimologia , Linhagem Celular , Meios de Cultura/química , Sistema Enzimático do Citocromo P-450/metabolismo , Dimetil Sulfóxido/química , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The behavior of magnetite nanoparticles was studied in the cell chip microcapillaries. No aggregation of magnetite nanoparticles under conditions of long-term circulation was noted. Biodistribution and toxicity of magnetite nanoparticles (14 nm) and aminated magnetite after their intragastric administration to mice were studied in vivo. According to mass spectrometry and microscopy data, accumulation of nanoparticles occurred mainly in the liver cells.
Assuntos
Nanopartículas de Magnetita/química , Animais , Animais não Endogâmicos , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Nanopartículas de Magnetita/toxicidade , Masculino , Camundongos , Microcirculação , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Soluções , Distribuição TecidualRESUMO
We analyzed hepatotoxicity of three drugs: acetaminophen, metformin, and isoniazid. Spheroids of differentiated HepaRG cells cultured under microfluidic conditions were used as the model. Acute toxicity of substances was assessed by analyzing cell viability, while lactate concentration in the culture medium was used as the potential marker for evaluation of chronic exposure and non-lethal side effects of xenobiotics. The results were compared with mitochondrial activity and DNA fragmentation data. The efficiency and possibility of applying the integrated approach for assessment of drug hepatotoxicity are discussed.
Assuntos
Hepatócitos/efeitos dos fármacos , Microfluídica/métodos , Xenobióticos/toxicidade , Linhagem Celular , Fragmentação do DNA/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Testes de Toxicidade/métodosRESUMO
3D cell cultures are extensively used to study in vitro toxic effect of xenobiotics. When using multicellular spheroids, the question about their optimal size should be solved: small spheroids are difficult to manipulate, while large size of spheroids impairs the transport of nutrients and oxygen into the center. Mathematical models describing the distribution of substances in multicellular spheroids numerical procedure for solving differential equation system, which complicates their use in laboratory practice. We proposed and experimentally evaluated a new mathematical model describing oxygen distribution in HepaRG cell spheroids. Markers of functional activity were studied in spheroids of different size. The maximum size of spheroids that can be maintained in culture for 9 days without necrosis was determined.
Assuntos
Oxigênio/metabolismo , Esferoides Celulares/metabolismo , Linhagem Celular , Difusão , Hepatócitos/metabolismo , Humanos , Modelos Biológicos , Consumo de OxigênioRESUMO
Acetaminophen in a concentration of 5 mM increased the expression of JNK, HIF1A (hypoxiainduced factor), and CASP3, which indicated development of oxidative stress and apoptotic cell death. Acetaminophen in a concentration of 10 mM did not induce expression of HIF1A and CASP3, but reduced expression of chaperone HSP90, which attested to activation of a caspase-3-independent mechanism of cell death. The methods of preventing acetaminophen intoxication are discussed.
Assuntos
Acetaminofen/farmacologia , Analgésicos não Narcóticos/farmacologia , Caspase 3/metabolismo , Neurônios/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Estresse OxidativoRESUMO
Magnetite particles modified by polyethylene glycol with a molecular weight of 3 kDa and hydrodynamic diameter of ~60 nm were used. Plant lectin viscumin covalently immobilized on these nanoparticles retained its binding activity. Immunochemical characteristics of conjugated viscumin were evaluated using monoclonal antibodies. The resultant conjugate with a hydrodynamic diameter of 70 nm was used for studies of binding and internalization by target cells. Binding of viscumin and its conjugate was determined by receptors containing terminal galactose, while intracellular distribution varied. The model system presented in this study can be used for creation of drugs for target therapy.
Assuntos
Nanopartículas de Magnetita/química , Nanoconjugados/química , Proteínas Inativadoras de Ribossomos Tipo 2/química , Toxinas Biológicas/química , Linhagem Celular Tumoral , Glioblastoma , Humanos , Tamanho da Partícula , Proteínas Inativadoras de Ribossomos Tipo 2/metabolismo , Toxinas Biológicas/metabolismoRESUMO
The central pattern generator (CPG) is defined as a set of neurons involved in joint production of patterned motor output. The roundtable discussion on the CPG was a part of the 5th All-Russian Conference on Animal Behavior (Moscow, Nov. 21, 2012). The discussion centred on three core themes: 1) the mechanisms of the organization and reconfiguration of pattern generating neuronal ensembles, 2) extrapolations that extend the CPG concept beyond the motor systems, and 3) evolutionary and developmental aspects of CPG.
Assuntos
Geradores de Padrão Central/fisiologia , Animais , Evolução Biológica , Geradores de Padrão Central/crescimento & desenvolvimento , HumanosRESUMO
There is a growing recognition that Central Pattern Generators (CPGs) play the fundamental role in the production of motor commands. A CPG is defined as a set of neurons involved in joint production of patterned sequenced output. It is generally believed that the sequence arises from the pattern of synaptic connections among these neurons. Alternatively, it was suggested (Sakharov, 1985) that the orderly organization of pattern-generating units might be due to the chemical diversity of their constituent neurons representing different phenotypes. Recent researches demonstrate that a given CPG can be reorganized to produce a different pattern of output activity. This multifunctionality can hardly be explained in terms of the anatomical (= synaptic) organization. The heterochemical approach appears to be more flexible. We hypothesize that dynamic fluctuations of the local extracellular milieu determine the physiological properties and receptor profile of individual neurons, and thus the self-organisation of the latter into a CPG.
Assuntos
Encéfalo/fisiologia , Geradores de Padrão Central/fisiologia , Atividade Motora/fisiologia , Neurônios Motores/fisiologia , Animais , HumanosAssuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologiaAssuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Procedimentos Analíticos em Microchip/métodos , Paclitaxel/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50RESUMO
The analysis of the protein composition of exhaled breath to diagnose diseases of the respiratory system raises a problem of differentiation proteins of expressed in the tissues of the lungs and respiratory tract (endogenous) and got in the respiratory system from the ambient air in the process of respiration (exogenous). In this work an attempt was made to estimate a set of exhaled exogenic proteins by mass spectrometry coupled with nanoflow HPLC. Six-month isolation of healthy donors indoors with air cleaned of dust leads to removal from the spectrum of exhaled proteins of some keratins that are considered therefore to be exogenic. Non-keratin proteins may also circulate between the ambient air and human respiratory ways, but their concentration appears to be significantly lower the keratin concentrations (especially epidermis keratin). Among non-keratins dermcidin seems to be the most significant exogenic protein of exhaled air. The conclusion of the diagnostic value of exhaled proteins can be done only after careful comparison of the results of quantitative and qualitative analysis of their composition in norm and pathology for a statistically significant sample of donors.
Assuntos
Proteínas/análise , Adulto , Poluição do Ar em Ambientes Fechados , Sequência de Aminoácidos , Testes Respiratórios , Cromatografia Líquida de Alta Pressão , Expiração , Humanos , Queratinas/análise , Masculino , Espectrometria de Massas , Mapeamento de Peptídeos , Fatores de TempoRESUMO
Extracellular concentration of heat shock protein (Hsp) with a molecular weight of 70 kDa (Hsp70) rapidly increases in the serum in response to stress and returns to the basal level during recovery. Further regulation of its blood concentration is unclear. A possible regulator is HspBP1, a protein binding Hsp70. Binding to ATPase domain of Hsp70, HspBP1 inactivates it, thus acting as a factor of nucleotide exchange. Blood sera from athletes were examined at the beginning and end of the last mesocycle of the training period by two-staged immunoaffinity test system. The concentration of HspBP1 increased with decreasing Hsp70 concentration under conditions of long-term training. Presumably, the dynamics of Hsp70 and HspBP1 concentrations can serve as the test for evaluating the adaptation potential.