RESUMO
Precise measurement of mechanical and electrical properties of single cells can yield useful information on the physiological and pathological state of cells. In this work, we develop a differential multiconstriction microfluidic device with self-aligned 3D electrodes to simultaneously characterize the deformability, electrical impedance and relaxation index of single cells at a high throughput manner (>430 cell/min). Cells are pressure-driven to flow through a series of sequential microfluidic constrictions, during which deformability, electrical impedance and relaxation index of single cells are extracted simultaneously from impedance spectroscopy measurements. Mechanical and electrical phenotyping of untreated, Cytochalasin B treated and N-Ethylmaleimide treated MCF-7 breast cancer cells demonstrate the ability of our system to distinguish different cell populations purely based on these biophysical properties. In addition, we quantify the classification of different cell types using a back propagation neural network. The trained neural network yields the classification accuracy of 87.8% (electrical impedance), 70.1% (deformability), 42.7% (relaxation index) and 93.3% (combination of electrical impedance, deformability and relaxation index) with high sensitivity (93.3%) and specificity (93.3%) for the test group. Furthermore, we have demonstrated the cell classification of a cell mixture using the presented biophysical phenotyping technique with the trained neural network, which is in quantitative agreement with the flow cytometric analysis using fluorescent labels. The developed concurrent electrical and mechanical phenotyping provide great potential for high-throughput and label-free single cell analysis.
Assuntos
Fenômenos Biofísicos , Técnicas Biossensoriais , Dispositivos Lab-On-A-Chip , Análise de Célula Única , Citocalasina B/química , Espectroscopia Dielétrica , Eletrodos , Etilmaleimida/química , Citometria de Fluxo , Humanos , Células MCF-7 , MicrofluídicaRESUMO
The targeted delivery of therapeutics to sites of rheumatoid arthritis (RA) has been a long-standing challenge. Inspired by the intrinsic inflammation-targeting capacity of macrophages, a macrophage-derived microvesicle (MMV)-coated nanoparticle (MNP) was developed for targeting RA. The MMV was efficiently produced through a novel method. Cytochalasin B (CB) was applied to relax the interaction between the cytoskeleton and membrane of macrophages, thus stimulating MMV secretion. The proteomic profile of the MMV was analyzed by iTRAQ (isobaric tags for relative and absolute quantitation). The MMV membrane proteins were similar to those of macrophages, indicating that the MMV could exhibit bioactivity similar to that of RA-targeting macrophages. A poly(lactic- co-glycolic acid) (PLGA) nanoparticle was subsequently coated with MMV, and the inflammation-mediated targeting capacity of the MNP was evaluated both in vitro and in vivo. The in vitro binding of MNP to inflamed HUVECs was significantly stronger than that of the red blood cell membrane-coated nanoparticle (RNP). Compared with bare NP and RNP, MNP showed a significantly enhanced targeting effect in vivo in a collagen-induced arthritis (CIA) mouse model. The targeting mechanism was subsequently revealed according to the proteomic analysis, indicating that Mac-1 and CD44 contributed to the outstanding targeting effect of the MNP. A model drug, tacrolimus, was encapsulated in MNP (T-RNP) and significantly suppressed the progression of RA in mice. The present study demonstrates MMV as a promising and rich material, with which to mimic macrophages, and demonstrates that MNP is an efficient biomimetic vehicle for RA targeting and treatment.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Nanopartículas/administração & dosagem , Proteômica , Animais , Artrite Reumatoide/patologia , Citocalasina B/química , Modelos Animais de Doenças , Eritrócitos/química , Eritrócitos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Receptores de Hialuronatos/genética , Antígeno de Macrófago 1/genética , Macrófagos/química , Camundongos , Nanopartículas/química , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , TacrolimoRESUMO
Skeletal muscle-associated toxicity is an underresearched area in the field of high-throughput toxicity screening; hence, the potential adverse effects of drugs and chemicals on skeletal muscle are largely unknown. Novel organotypic microphysiological in vitro models are being developed to replicate the contractile function of skeletal muscle; however, the throughput and a need for specialized equipment may limit the utility of these tissue chip models for screening. In addition, recent developments in stem cell biology have resulted in the generation of induced pluripotent stem cell (iPSC)-derived skeletal myoblasts that enable high-throughput in vitro screening. This study set out to develop a high-throughput multiplexed assay using iPSC-derived skeletal myoblasts that can be used as a first-pass screen to assess the potential for chemicals to affect skeletal muscle. We found that cytotoxicity and cytoskeletal integrity are most useful and reproducible assays for the skeletal myoblasts when evaluating overall cellular health or gauging disruptions in actin polymerization following 24 h of exposure. Both assays are based on high-content imaging and quantitative image processing to derive quantitative phenotypes. Both assays showed good to excellent assay robustness and reproducibility measured by interplate and interday replicability, coefficients of variation of negative controls, and Z'-factors for positive control chemicals. Concentration response assessment of muscle-related toxicants showed specificity of the observed effects compared to the general cytotoxicity. Overall, this study establishes a high-throughput multiplexed assay using skeletal myoblasts that may be used for screening and prioritization of chemicals for more complex tissue chip-based and in vivo evaluation.
Assuntos
Citocalasina B/farmacologia , Doxorrubicina/farmacologia , Ensaios de Triagem em Larga Escala , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Células Cultivadas , Citocalasina B/efeitos adversos , Citocalasina B/química , Relação Dose-Resposta a Droga , Doxorrubicina/efeitos adversos , Doxorrubicina/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Compostos de Amônio Quaternário/efeitos adversos , Compostos de Amônio Quaternário/química , Relação Estrutura-AtividadeRESUMO
Endophytic fungi are fungi that colonize internal tissues of plants; several biologically active compounds have been isolated from these fungi. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, this study aimed the isolation and structural identification of ergosterol (1), ergosterol peroxide (2), mevalonolactone (3), cytochalasin B (4) and cytochalasin H (5) from Aspergillus sp. EJC 04, an endophytic fungus from Bauhinia guianensis. The cytochalasin B (4) and the diacetate derivative of cytochalasin B (4a) showed high lethality in the brine shrimp assay. This is the first occurrence of cytochalasins in Amazonian endophytic fungi from B. guianensis.
Assuntos
Artemia/efeitos dos fármacos , Aspergillus/química , Citocalasina B/toxicidade , Citocalasinas/toxicidade , Endófitos/química , Ergosterol/análogos & derivados , Fabaceae/microbiologia , Ácido Mevalônico/análogos & derivados , Acetilação , Animais , Argentina , Aspergillus/isolamento & purificação , Citocalasina B/química , Citocalasina B/isolamento & purificação , Citocalasinas/química , Citocalasinas/isolamento & purificação , Endófitos/isolamento & purificação , Ergosterol/química , Ergosterol/isolamento & purificação , Ergosterol/toxicidade , Dose Letal Mediana , Ácido Mevalônico/química , Ácido Mevalônico/isolamento & purificação , Ácido Mevalônico/toxicidade , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
Due to their enhanced metabolic needs many cancers need a sufficient supply of glucose, and novel inhibitors of glucose import are in high demand. Cytochalasin B (CB) is a potent natural glucose import inhibitor which also impairs the actin cytoskeleton leading to undesired toxicity. With a view to identifying selective glucose import inhibitors we have developed an enantioselective trienamine catalyzed synthesis of a CB-inspired compound collection. Biological analysis revealed that indeed actin impairment can be distinguished from glucose import inhibition and led to the identification of the first selective glucose import inhibitor based on the basic structural architecture of cytochalasin B.
Assuntos
Alcenos/química , Aminas/química , Citocalasina B/química , Citocalasina B/farmacologia , Glucose/antagonistas & inibidores , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Catálise , Glucose/metabolismo , Células HCT116 , Células HeLa , Humanos , Estrutura Molecular , EstereoisomerismoRESUMO
The dose-responses of micronuclei (MN) in binucleated (BN) and mononucleated (MONO) lymphocytes cultivated with cytochalasin B (CBMN-assay) were studied. Irradiation of lymphocytes was performed in vitro (donor A) at the single dose of 1 and 2 Gy of (60)Co y-rays, or in vivo, during whole-body exposure of a cancer patient (donor B) to (60)Co γ-rays each day at a single dose of 0.115 Gy up to a total dose of 1.15 Gy. The linear dose-response for MN was determined in both BN and MONO lymphocytes of donor B. It means that when CBMN assay is applied, the MN in MONO cells represent those preexisted in vivo before each exposure. On the contrary, in lymphocytes of donor A irradiated in vitro an essential elevated MN yield with an - increased dose was observed only in BN lymphocytes. A slight dose dependent elevation of MN in MONO cells seems to be due to either their division before cytochalasin was introduced in the culture medium or their insensitivity to the CB block of cytokinesis.
Assuntos
Relação Dose-Resposta à Radiação , Linfócitos/efeitos da radiação , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Neoplasias/radioterapia , Adulto , Radioisótopos de Cobalto/efeitos adversos , Citocalasina B/química , Raios gama/efeitos adversos , Humanos , Masculino , Neoplasias/sangueRESUMO
Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Embrião não Mamífero/metabolismo , Oócitos/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Ciclina B1/genética , Citocalasina B/química , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Oócitos/citologia , Oogênese , Regiões Promotoras Genéticas , Testículo/metabolismo , Peixe-Zebra , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Although the actin cytoskeleton is vital for carcinogenesis and subsequent pathology, no microfilament-directed agent has been approved for cancer chemotherapy. One of the most studied classes of microfilament-directed agents has been the cytochalasins, mycotoxins known to disrupt the formation of actin polymers. In the present study, we sought to determine the effects of cytochalasin congeners toward human drug sensitive and multidrug resistant cell lines. METHODS: SKOV3 human ovarian carcinoma and several multidrug resistant derivatives were tested for sensitivity against a panel of nine cytochalasin congeners, as well as three clinically approved chemotherapeutic agents (doxorubicin, paclitaxel, and vinblastine). In addition, verapamil, a calcium ion channel blocker known to reverse P-glycoprotein (P-gp) mediated drug resistance, was used in combination with multiple cytochalasin congeners to determine whether drug sensitivity could be increased. RESULTS: While multidrug resistant SKVLB1 had increased drug tolerance (was more resistant) to most cytochalasin congeners in comparison to drug sensitive SKOV3, the level of resistance was 10 to 1000-fold less for the cytochalasins than for any of the clinically approved agents. While cytochalasins did not appear to alter the expression of ATP binding cassette (ABC) transporters, several cytochalasins appeared to inhibit the activity of ABC transporter-mediated efflux of rhodamine 123 (Rh123), suggesting that these congeners do have affinity for drug efflux pumps. Cytochalasins also appeared to significantly decrease the F/G-actin ratio in both drug sensitive and drug resistant cells, indicative of marked microfilament inhibition. The cytotoxicity of most cytochalasin congeners could be increased with the addition of verapamil, and the drug sensitivity of resistant SKVLB1 to the clinically approved antineoplastic agents could be increased with the addition of cytochalasins. As assessed by isobolographic analysis and Chou-Talalay statistics, cytochalasin B and 21,22-dihydrocytochalasin B (DiHCB) demonstrated notable synergy with doxorubicin and paclitaxel, warranting further investigation in a tumor-bearing mammalian model. CONCLUSION: Cytochalasins appear to inhibit the activity of P-gp and potentially other ABC transporters, and may have novel activity against multidrug resistant neoplastic cells that overexpress drug efflux proteins.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma/tratamento farmacológico , Citocalasina B/farmacologia , Citocalasinas/farmacologia , Doxorrubicina/farmacologia , Microtúbulos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Linhagem Celular Tumoral , Citocalasina B/química , Citocalasinas/química , Avaliação Pré-Clínica de Medicamentos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Feminino , HumanosRESUMO
Olfactory receptor (OR) genes are considered to be the largest superfamily of the mammalian genome, and in the case of humans, approximately 390 kinds of functional ORs play a role in perceiving odors. In spite of their significance in olfaction, the function of all ORs has not yet been fully revealed. In order to efficiently identify specific ligands of orphan ORs, methods that can generate olfactory signals in a reliable manner and that can convert the cellular signals into measurable responses are required. Here, we describe an OR screening assay method using olfactory sensors that are based on cell-derived nanovesicles combined with single-walled carbon nanotube field-effect transistors (SWNT-FETs). The nanovesicles contain ORs on their surface membrane and induce influx of calcium ions similar to olfactory signal transduction. This ion influx causes an electrical current change along the carbon nanotube, and then this change is measured by the SWNT-FET sensor. This technique facilitates the simple and rapid screening of OR functions.
Assuntos
Técnicas Biossensoriais , Ensaios de Triagem em Larga Escala , Nanotubos de Carbono/química , Receptores Odorantes/genética , Proteínas Recombinantes de Fusão/genética , Rodopsina/genética , Animais , Bovinos , Citocalasina B/química , Expressão Gênica , Células HEK293 , Humanos , Microtecnologia , Plasmídeos/química , Plasmídeos/metabolismo , Receptores Odorantes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/metabolismo , Silício/química , Transfecção , Transistores EletrônicosRESUMO
As a consequence of the increased use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics to prevent fungal and bacterial growth, there is a need for validated rapid screening methods to assess the safety of nanoparticle exposure. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential genotoxicity of 20-nm nanosilver. The average silver nanoparticle size as determined by transmission electron microscopy (TEM) was 20.4 nm. Dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The silver concentration in a 20-nm nanosilver solution determined by the inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Analysis by ICP-MS and TEM demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Genotoxicity was determined by the cytochalasin B-blocked micronucleus assay with acridine orange staining and fluorescence microscopy. Concentration- and time-dependent increases in the frequency of binucleated cells with micronuclei induced by the nanosilver was observed in the concentration range of 0.5 to 15 µg ml(-1) in both HepG2 and Caco2 cells compared with the control. Our results indicated that HepG2 cells were more sensitive than Caco2 cells in terms of micronuclei formation induced by nanosilver exposure. In summary, the results of this study indicate that the widely used in vitro models, HepG2 and Caco2 cells in culture, represent potential screening models for prediction of genotoxicity of silver nanoparticles by in vitro micronucleus assay.
Assuntos
Dano ao DNA/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Células CACO-2 , Colo/citologia , Colo/efeitos dos fármacos , Citocalasina B/química , Células Hep G2 , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Testes para Micronúcleos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Tamanho da PartículaRESUMO
The cytokinesis-block micronucleus cytome (CBMN) assay, introduced by Fenech, was used to demonstrate different types of DNA damage in MOLT-3 human lymphoblastoid cells exposed to 10 µM zidovudine (AZT). In addition, we explored the cytoprotective potential of two antioxidants, WR-1065 and Tempol, to decrease AZT-induced genotoxicity. Binucleated cells, arrested by Cytochalasin B (Cyt B), were evaluated for micronuclei (MN), caused by DNA damage or chromosomal loss, and chromatin nucleoplasmic bridges (NPBs), caused by telomere attrition. Additionally, nuclear buds (NBUDs), caused by amplified DNA, and apoptotic and necrotic (A/N) cells were scored. We hypothesized that AZT exposure would increase the frequency of genotoxic end points, and that the antioxidants Tempol and WR-1065 would protect against AZT-induced genotoxicity. MOLT-3 cells were exposed to 0 or 10 µM AZT for a total of 76 hr. After the first 24 hr, 0 or 5 µM WR-1065 and/or 0 or 200 µM Tempol were added for the remainder of the experiment. For the last 28 hr (of 76 hr), Cyt B was added to arrest replication after one cell division, leaving a predominance of binucleated cells. The nuclear division index (NDI) was similar for all treatment groups, indicating that the exposures did not alter cell viability. MOLT-3 cells exposed to AZT alone had significant (P < 0.05) increases in MN and NBs, compared to unexposed cells. Both Tempol and WR-1065 protected against AZT-induced MN formation (P < 0.003 for both), and WR-1065, but not Tempol, reduced the levels of A/N (P = 0.041). In cells exposed to AZT/Tempol there were significantly reduced levels of NBUDs, compared to cells exposed to AZT alone (P = 0.015). Cells exposed to AZT/WR-1065 showed reduced levels of NPBs, compared to cells exposed to AZT alone (P = 0.037). Thus WR-1065 and Tempol protected MOLT-3 cells against specific types of AZT-induced DNA damage.
Assuntos
Antioxidantes/química , Cromatina/química , Óxidos N-Cíclicos/química , Dano ao DNA , Mercaptoetilaminas/química , Zidovudina/química , Apoptose , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Cromossomos/ultraestrutura , Citocalasina B/química , Humanos , Testes para Micronúcleos , Mutagênicos/química , Necrose , Protetores contra Radiação/química , Marcadores de SpinRESUMO
Electric cell-substrate impedance sensing (ECIS) is a powerful instrument for quantifying cell behavior in tissue culture. As cells attach and spread on the sensing electrode, they restrict the current flow and hence cause the increase of electrical impedance. Furthermore, cell motion may reveal itself as electrical fluctuations, which are always associated with living cells and continue even when the cells become fully confluent. The impedance fluctuation is attributed to incessant changes in the size of the cell-substrate space as cells persistently rearrange their cell-substrate adhesion sites. The magnitude of this sort of vertical motion detected by ECIS is of the order of nanometers and referred to as micromotion. In this study, Hilbert-Huang Transform was used as a micromotion analysis tool to distinguish the in vitro cytotoxicity of human umbilical vein endothelial cells (HUVECs) exposed to low levels of cytochalasin B. Hilbert-Huang Transform consists of two procedures: the empirical mode decomposition (EMD) and the Hilbert Transform. The measured impedance fluctuations due to cell micromotion were decomposed into several intrinsic mode functions (IMFs) by EMD, and then these IMFs were transferred to instantaneous frequencies by Hilbert Transform. Both amplitude and phase of instantaneous frequencies were expressed as a time-frequency spectrum, called Hilbert spectrum, which displayed different distribution pattern in response to different cytochalasin B concentration. The total instantaneous energy (IE) of each IMF was also calculated to quantify the spectral difference. In addition to the observation of a dose-dependent relationship, the IE value of the first IMF at 0.1 µM decreased to about 48% of the control value and significantly distinguished the cytotoxic effect of 0.1 µM of cytochalasin B (P<0.05).
Assuntos
Impedância Elétrica , Algoritmos , Adesão Celular , Citocalasina B/química , Eletrodos , Células Endoteliais da Veia Umbilical Humana , Humanos , Movimento (Física) , Processamento de Sinais Assistido por Computador , Técnicas de Cultura de TecidosRESUMO
Emerging evidence suggests that cytoplasmic streaming can regulate the plasma-membrane H(+) transport and photosynthetic electron flow. Microfluorometric and surface pH measurements on Chara corallina internodes revealed the transmission of photoinduced signals by the cytoplasmic flow for a distance of few millimeters from the site of stimulus application. When a 30-s pulse of bright light was locally applied, the downstream cell regions responded with either release or enhancement of non-photochemical quenching of chlorophyll fluorescence, depending on the background irradiance of the analyzed cell area. Under dim background irradiance (<20 µmol m(-2) s(-1)), the arrival of the distant signal from the brightly illuminated 400-µm-wide zone elevated the maximal fluorescence F m (') in the analyzed downstream area, whereas at higher background irradiances it induced strong quenching of F m (') . At intermediate irradiances the increase and decrease in F m (') appeared as two successive waves. The transition between the F m (') responses of opposite polarities occurred at a narrow threshold range of irradiances. This indicates that inevitable slight variations in irradiance at the bottom chloroplast layer combined with the cyclosis-transmitted signals may contribute to the formation of a photosynthetic activity pattern. The rapid cyclosis-mediated release of non-photochemical quenching, unlike the delayed response of opposite polarity, was associated with opening of H(+) (OH(-))-conducting plasma membrane channels, as evidenced by the concurrent alkaline pH shift on the cell surface. It is proposed that the initial increase in F m (') after application of a distant photostimulus is determined, among other factors, by the wave of alkaline cytoplasmic pH.
Assuntos
Chara/metabolismo , Cloroplastos/química , Citoplasma/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Clorofila/química , Citocalasina B/química , Fluorometria/métodos , Temperatura Alta , Concentração de Íons de Hidrogênio , Luz , Microscopia de Fluorescência/métodos , Fótons , PrótonsRESUMO
Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experiment, we investigated the microtubule and microfilament alterations in bovine oocytes after demecolcine and/or cytochalasin B (CB) treatments by immunocytochemical staining. We also examined mechanical enucleation of demecolcine-treated oocytes in cytochalasin-free medium. The results showed that demecolcine-treatment disrupts the balance between microtubule/microfilament interactions primarily by deleting microtubules and with little effect on the microfilaments that we believe accounts for the membrane protrusion. The CB treatment reduced the amount of microfilaments but had little effect on the microtubules. Most demecolcine-induced membrane protrusions disappeared when exposed to CB. Western blotting showed that CB treatment increases G-actin, which indicates a decrease in the microfilaments. High oocyte enucleation, survival, and developmental rates occurred when demecolcine-assisted enucleation was carried out in a CB-free solution. Higher blastocyst development rates and blastocyst cell numbers were achieved compared to control, indicating that CB is not necessary in the enucleation procedure of bovine oocytes. This study provides a clearer understanding of the mechanism for the demecolcine-induced oocyte membrane protrusion, and substantiates the practical use of demecolcine-assisted enucleation in a CB-free environment.
Assuntos
Núcleo Celular , Demecolcina/química , Técnicas de Transferência Nuclear , Oócitos , Moduladores de Tubulina/química , Citoesqueleto de Actina , Animais , Bovinos , Membrana Celular , Sobrevivência Celular , Citocalasina B/química , Citocalasina B/farmacologia , Demecolcina/farmacologia , Feminino , Microtúbulos , Moduladores de Tubulina/farmacologiaRESUMO
GLUT4 is a 12 transmembrane (TM) protein belonging to the Class I facilitated glucose transporter family that transports glucose into the cells in an insulin regulated manner. GLUT4 plays a key role in the maintenance of blood glucose homeostasis and inhibition of glucose transporter activity may lead to insulin resistance, hallmark of type 2 diabetes. No crystal structure data is available for any members of the facilitated glucose transporter family. Here, in this paper, we have generated a homology model of GLUT4 based on experimental data available on GLUT1, a Class I facilitated glucose transporter and the crystal structure data obtained from the Glycerol 3-phosphate transporter. The model identified regions in GLUT4 that form a channel for the transport of glucose along with the substrate interacting residues. Docking and electrostatic potential data analysis of GLUT4 model has mapped an ATP binding region close to the binding site of cytochalasin B and genistein, two GLUT4 inhibitors, and this may explain the mechanism by which these inhibitors could potentially affect the GLUT4 function.
Assuntos
Trifosfato de Adenosina/química , Citocalasina B/química , Genisteína/química , Transportador de Glucose Tipo 4/química , Simulação de Acoplamento Molecular , Sequência de Aminoácidos , Sítios de Ligação , Glucose/química , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 4/antagonistas & inibidores , Humanos , Proteínas de Membrana Transportadoras/química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
Five novel epoxide derivatives of cytochalasin B were synthesized. Reaction of cytochalasin B with t-BHP and BuLi led to selective epoxidation of the C-21/22 double bond to give a single monoepoxide, while reaction with m-CPBA yielded two diepoxides. Reaction of the monoepoxide with m-CPBA yielded two triepoxides. The relative configurations of the epoxides were elucidated by analogy with the natural product by means of spectroscopic methods; full assignment of NMR signals was achieved, and the absolute configuration was confirmed by X-ray crystallography.
Assuntos
Citocalasina B/química , Compostos de Epóxi/síntese química , Espectroscopia de Ressonância Magnética , Isótopos de Carbono , Cristalografia por Raios X , Compostos de Epóxi/química , Modelos Moleculares , Estrutura Molecular , PrótonsRESUMO
Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.
Assuntos
Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Proteínas de Transporte de Monossacarídeos/química , 3-O-Metilglucose/antagonistas & inibidores , 3-O-Metilglucose/sangue , Sítios de Ligação , Transporte Biológico Ativo/fisiologia , Citocalasina B/sangue , Citocalasina B/química , Membrana Eritrocítica/fisiologia , Líquido Extracelular/química , Líquido Extracelular/metabolismo , Transportador de Glucose Tipo 1 , Hemólise/fisiologia , Humanos , Soluções Hipotônicas , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Maltose/sangue , Maltose/química , Modelos Biológicos , Modelos Químicos , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Proteínas de Transporte de Monossacarídeos/sangue , Floretina/sangue , Floretina/química , Temperatura , Fatores de Tempo , TrítioRESUMO
In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.
Assuntos
Criopreservação/métodos , Técnicas de Cultura/métodos , Oócitos/patologia , Injeções de Esperma Intracitoplásmicas/métodos , Animais , Blastocisto/metabolismo , Crioprotetores/farmacologia , Citocalasina B/química , Feminino , Oócitos/metabolismo , Ovário/metabolismo , Suínos , TemperaturaRESUMO
The cytochalasins are structurally complex natural products with a broad range of apparently unrelated effects in different biological systems. Different members of the family have variously demonstrated inhibitory activity toward the formation of actin filaments, toward the functioning of HIV protease, and toward the process of angiogenesis. The structural series is defined by a largely conserved, rigid bicyclic isoindolone core that is fused to a macrocyclic appendage. The latter structural component varies widely within the cytochalasins and seems to play an important role in the determination of biological activity. In this work, we describe the development of a convergent and enantioselective synthetic route to the cytochalasins that allows for the late-stage introduction of macrocyclic appendages of different sizes and constitutions. We illustrate the route with the synthesis of the 14-membered macrolactone cytochalasin B (1, an inhibitor of the formation of actin filaments) and the 11-membered macrocarbocyclic cytochalasin L-696,474 (2, an inhibitor of HIV protease) by using common precursors.
Assuntos
Citocalasina B/síntese química , Citocalasinas/síntese química , Química Orgânica/métodos , Citocalasina B/química , Citocalasinas/química , Isoindóis , Estrutura Molecular , EstereoisomerismoRESUMO
Evidence indicates that a large portion of the facilitative glucose transporter isoform GLUT1 in certain animal cells is kept inactive and activated in response to acute metabolic stresses. A reversible interaction of a certain inhibitor molecule with GLUT1 protein has been implicated in this process. In an effort to identify this putative GLUT1 inhibitor molecule, we studied here the effects of adenosine and adenosine triphosphate (ATP) on the binding of D-glucose to GLUT1 by assessing their abilities to displace cytochalasin B (CB), using purified GLUT1 in vesicles. At pH 7.4, adenosine competitively inhibited CB binding to GLUT1 and also reduced the substrate binding affinity by more than an order of magnitude, both with an apparent dissociation constant (K(D)) of 3.0 mM. ATP had no effect on CB and D-glucose binding to GLUT1, but reduced adenosine binding affinity to GLUT1 by 2-fold with a K(D) of 30 mM. At pH 3.6, however, ATP inhibited the CB binding nearly competitively, and increased the substrate binding affinity by 4--5-fold, both with an apparent K(D) of 1.22 mM. These findings clearly demonstrate that adenosine and ATP interact with GLUT1 in vitro and modulate its substrate binding affinity. They also suggest that adenosine and ATP may regulate GLUT1 intrinsic activity in certain cells where adenosine reduces the substrate-binding affinity while ATP increases the substrate-binding affinity by interfering with the adenosine effect and/or by enhancing the substrate-binding affinity at an acidic compartment.