RESUMEN
New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm (3) of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup.
RESUMEN
Microfluidic bioreactors are expected to impact cell therapy and biopharmaceutical production due to their ability to control cellular microenvironments. This work presents a novel approach for continuous cell culture in a microfluidic system. Microcarriers (i.e., microbeads) are used as growth support for anchorage-dependent mammalian cells. This approach eases the manipulation of cells within the system and enables harmless extraction of cells. Moreover, the microbioreactor uses a perfusion function based on the biocompatible integration of a porous membrane to continuously feed the cells. The perfusion rate is optimized through simulations to provide a stable biochemical environment. Thermal management is also addressed to ensure a homogeneous bioreactor temperature. Eventually, incubator-free cell cultures of Drosophila S2 and PC3 cells are achieved over the course of a week using this bioreactor. In future applications, a more efficient alternative to harvesting cells from microcarriers is also anticipated as suggested by our positive results from the microcarrier digestion experiments.
Asunto(s)
Reactores Biológicos , Técnicas de Cultivo de Célula , Técnicas Analíticas Microfluídicas , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Drosophila melanogaster , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodosRESUMEN
We report the fabrication of a thin silicon membrane with an array of micrometer and submicrometer pores that acts as a scaffold for suspending a lipid bilayer. We successfully deposited a lipid bilayer by the Langmuir-Blodgett method on a synthetic silicon membrane bearing arrays of pores with sizes of 1000, 650, and 300 nm. Topographic images obtained by AFM showed a suspended lipid film spanning the pores, whatever the pore size. Higher stability of bilayers supported on smaller pores was shown by AFM characterization. These results represent an important first step to creating a biomimetic environment to study cell membrane dynamics and/or in developing a biosensor.
Asunto(s)
Membrana Dobles de Lípidos , Imitación Molecular , Silicio/química , Microscopía de Fuerza AtómicaRESUMEN
K(ATP) channels incorporate a regulatory subunit of the ATP-binding cassette (ABC) transporter family, the sulfonylurea receptor (SUR), which defines their pharmacology. The therapeutically important K(+) channel openers (e.g. pinacidil, cromakalim, nicorandil) act specifically on the SUR2 muscle isoforms but, except for diazoxide, remain ineffective on the SUR1 neuronal/pancreatic isoform. This SUR1/2 dichotomy underpinned a chimeric strategy designed to identify the structural determinants of opener action, which led to a minimal set of two residues within the last transmembrane helix of SUR. Transfer of either residue from SUR2A to SUR1 conferred opener sensitivity to SUR1, while the reverse operation abolished SUR2A sensitivity. It is therefore likely that these residues form part of the site of interaction of openers with the channel. Thus, openers would target a region that, in other ABC transporters, is known to be tightly involved with the binding of substrates and other ligands. This first glimpse of the site of action of pharmacological openers should permit rapid progress towards understanding the structural determinants of their affinity and specificity.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Activación del Canal Iónico/efectos de los fármacos , Canales de Potasio de Rectificación Interna , Canales de Potasio/química , Canales de Potasio/fisiología , Receptores de Droga/química , Receptores de Droga/fisiología , Secuencia de Aminoácidos , Animales , Membrana Celular/fisiología , Cricetinae , Cromakalim/farmacología , Diazóxido/farmacología , Activación del Canal Iónico/fisiología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Músculo Esquelético/fisiología , Mutagénesis Sitio-Dirigida , Neuronas/fisiología , Nicorandil/farmacología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Páncreas/fisiología , Pinacidilo/farmacología , Canales de Potasio/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/fisiología , Estructura Secundaria de Proteína , Ratas , Receptores de Droga/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Receptores de SulfonilureasRESUMEN
The molecular mechanism of in vitro movement is assumed, by most investigators, to be identical to that of muscle contraction. We discuss this view, which raises various problems. We believe there are mechanisms for muscle contraction (in this case considerable forces are developed, with small displacements) and other mechanisms for in vitro movement (giving large displacements, without necessarily generating substantial forces). Hybrid models may explain muscle contraction. The traditional swinging-crossbridge model may explain in vitro movement. For muscle contraction, movement may result partly from the swinging-crossbridge mechanism and partly from other factors. Comparisons of different fibres at different moments of the Mg-ATPase cycle suggest that both the value of the isometric force in muscle and in vitro and that of the Mg-ATPase activity used in vitro need to be reconsidered. The recently reported dependence of the isometric active tension of smooth skinned fibres on temperature appears to be weaker than predicted by the swinging-crossbridge theory alone. This recent observation is compatible with the existence of other forces (electrostatic repulsions) decreasing with temperature as has been known for some years. From recent experimental data, we think the biochemistry of myosin and actomyosin should be reassessed, to try to find new details of the mechanisms of muscle contraction and in vitro motility.
Asunto(s)
Miosinas/fisiología , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Contracción IsométricaRESUMEN
Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Tampones (Química) , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Quimotripsina , Dimerización , Concentración de Iones de Hidrógeno , Matemática , Microscopía Electrónica , Modelos Moleculares , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/ultraestructura , Miosinas/síntesis química , Miosinas/química , Papaína , Conejos , TemperaturaRESUMEN
The pharmacological phenotype of ATP-sensitive potassium (K(ATP)) channels is defined by their tissue-specific regulatory subunit, the sulfonylurea receptor (SUR), which associates with the pore-forming channel core, Kir6.2. The potassium channel opener diazoxide has hyperglycemic and hypotensive properties that stem from its ability to open K(ATP) channels in pancreas and smooth muscle. Diazoxide is believed not to have any significant action on cardiac sarcolemmal K(ATP) channels. Yet, diazoxide can be cardioprotective in ischemia and has been found to bind to the presumed cardiac sarcolemmal K(ATP) channel-regulatory subunit, SUR2A. Here, in excised patches, diazoxide (300 microM) activated pancreatic SUR1/Kir6.2 currents and had little effect on native or recombinant cardiac SUR2A/Kir6.2 currents. However, in the presence of cytoplasmic ADP (100 microM), SUR2A/Kir6.2 channels became as sensitive to diazoxide as SUR1/Kir6. 2 channels. This effect involved specific interactions between MgADP and SUR, as it required Mg(2+), but not ATP, and was abolished by point mutations in the second nucleotide-binding domain of SUR, which impaired channel activation by MgADP. At the whole-cell level, in cardiomyocytes treated with oligomycin to block mitochondrial function, diazoxide could also activate K(ATP) currents only after cytosolic ADP had been raised by a creatine kinase inhibitor. Thus, ADP serves as a cofactor to define the responsiveness of cardiac K(ATP) channels toward diazoxide. The present demonstration of a pharmacological plasticity of K(ATP) channels identifies a mechanism for the control of channel activity in cardiac cells depending on the cellular ADP levels, which are elevated under ischemia.
Asunto(s)
Adenosina Difosfato/metabolismo , Diazóxido/farmacología , Canales de Potasio de Rectificación Interna , Canales de Potasio/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Cricetinae , Citoplasma/metabolismo , Cobayas/metabolismo , Ratones , Miocardio/metabolismo , Técnicas de Placa-Clamp , Mutación Puntual , Potasio/metabolismo , Canales de Potasio/genética , Ratas , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Xenopus/embriologíaRESUMEN
ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the sulfonylurea receptor (SUR), and an inward rectifier K(+) channel subunit, Kir6.2. The diverse pharmacological responsiveness of K(ATP) channels from various tissues are thought to arise from distinct SUR isoforms. Thus, when assembled with Kir6. 2, the pancreatic beta cell isoform SUR1 is activated by the hyperglycemic drug diazoxide but not by hypotensive drugs like cromakalim, whereas the cardiac muscle isoform SUR2A is activated by cromakalim and not by diazoxide. We exploited these differences between SUR1 and SUR2A to pursue a chimeric approach designed to identify the structural determinants of SUR involved in the pharmacological activation of K(ATP) channels. Wild-type and chimeric SUR were coexpressed with Kir6.2 in Xenopus oocytes, and we studied the resulting channels with the patch-clamp technique in the excised inside-out configuration. The third transmembrane domain of SUR is found to be an important determinant of the response to cromakalim, which possibly harbors at least part of its binding site. Contrary to expectations, diazoxide sensitivity could not be linked specifically to the carboxyl-terminal end (nucleotide-binding domain 2) of SUR but appeared to involve complex allosteric interactions between transmembrane and nucleotide-binding domains. In addition to providing direct evidence for the structure-function relationship governing K(ATP) channel activation by potassium channel-opening drugs, a family of drugs of the highest therapeutic interest, these findings delineate the determinants of ligand specificity within the modular ATP-binding cassette-transporter architecture of SUR.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de la Membrana/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Receptores de Droga/metabolismo , Animales , Cromanos/farmacología , Cricetinae , Diazóxido/farmacología , Magnesio/farmacología , Proteínas de la Membrana/genética , Ratones , Oocitos/metabolismo , Canales de Potasio/genética , Ingeniería de Proteínas , Isoformas de Proteínas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Receptores de Sulfonilureas , Xenopus laevisRESUMEN
In this review we consider available information on the cardio- and cytoprotective properties and mechanism of action of trimetazidine, an antianginal drug. Two points in particular are addressed: the advantages of trimetazidine over classic drugs and its possible use for the long-term treatment of hypertrophic cardiomyopathy, described as an ischemic disorder.
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Cardiomiopatía Hipertrófica/tratamiento farmacológico , Trimetazidina/uso terapéutico , Vasodilatadores/uso terapéutico , Animales , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/fisiopatología , Modelos Animales de Enfermedad , HumanosRESUMEN
We reinvestigated whether the native myosin LC2-free-subfragment 1 (S1) dimer exists by using viscometry, capillary electrophoresis, and laser light scattering. We found that the intrinsic viscosity of the monomer is [eta]m = 6.7 cm3/g and its translation diffusion coefficient is (c = 0) = 4.43 x 10(-)7 cm2/s. For the dimer, [eta]d = 19.8 cm3/g and (c = 0) = 2.54 x 10(-)7 cm2/s. Using the Svedberg equation and introducing the values of the sedimentation coefficients (5.05 S for the monomer and 6.05 S for the dimer), we find the following molecular weights: Mr,m = 108 000 Da and Mr,d = 213 000 Da, which agree well with previous determinations. Capillary electrophoresis successfully separated S1(A1) and S1(A2), in a monomer buffer, and S1(A1) and S1(A2) and a heterodimer S1(A1)-S1(A2), in a dimer buffer. An interesting feature of the monomer-dimer equilibrium is the presence of temperature transitions, whose positions and widths depend upon the buffer conditions. At low temperatures, a pure dimer was observed, whereas at high temperatures only the monomer was present. The dimerization site on both myosin and S1 is extremely labile.
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Músculo Esquelético/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Subfragmentos de Miosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Animales , Tampones (Química) , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Dimerización , Electroforesis Capilar , Modelos Químicos , Músculo Esquelético/química , Cadenas Ligeras de Miosina/química , Subfragmentos de Miosina/química , Conejos , ViscosidadRESUMEN
The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).
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Isoenzimas/química , Músculo Esquelético/química , Miosinas/química , Animales , Dimerización , Electroforesis Capilar/métodos , Isoenzimas/metabolismo , Magnesio/metabolismo , Músculo Esquelético/enzimología , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosinas/metabolismo , ConejosRESUMEN
1. In this study we investigated whether long-term trimetazidine (anti-ischaemic drug) therapy alters the ventricular myosin heavy chain (MHC) isoform composition in a model of cardiomyopathy. 2. MHC isoforms were analysed in the native state by electrophoresis in a pyrophosphate buffer. Myosin isoform patterns were studied in cardiac muscle from cardiomyopathic hamsters (CMH) of the BIO 14:6 strain during the time course of the disease and compared with those of healthy golden hamsters (F1B). The correlation between myosin profile and Ca2+-activated ATPase activity was determined from 220 days. 3. At the stage of insufficiency (350 days), CMH presented the most abnormal phenotype with 53% V1-24% V3 compared to 79% V1-7% V3 (P<0.001), in F1B. Trimetazidine was administered to cardiomyopathic hamsters from the early stage of active disease (30 days) to the congestive stages (220-350 days). Within 65 days, trimetazidine treatment, in CMH and F1B, reduced V1 to a low level (53% and 62%, respectively), which remained constant throughout the treatment. This level was similar to that in 220 and 350 days-old untreated-CMH. In sharp contrast, a standard calcium blocker, verapamil, administered to CMH in the same conditions resulted in a higher V1 (about 70%) and higher global myosin ATPase activity from 220 days. 4. Previous results in terms of hypertrophy and survival, compared to these results, suggest that verapamil and trimetazidine treatments reveal a dissociation between ventricular hypertrophy and isomyosin distribution. In addition, the shift in favour of V3 may not necessarily be an aggravating factor of the disease but an adaptative compensatory event.
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Bloqueadores de los Canales de Calcio/uso terapéutico , Cardiomiopatías/tratamiento farmacológico , Miosinas/genética , Trimetazidina/uso terapéutico , Vasodilatadores/uso terapéutico , Verapamilo/uso terapéutico , Animales , ATPasas Transportadoras de Calcio/metabolismo , Cardiomiopatías/enzimología , Cardiomiopatías/genética , Cricetinae , Electroforesis en Gel de Poliacrilamida , Mesocricetus , Miosinas/aislamiento & purificación , FenotipoRESUMEN
The cardiomyopathic Syrian hamster (CMH) of the strain BIO 14:6 is a model for both cardiac and skeletal muscle abnormalities. It has reduced longevity and noticeable hypertrophy of the heart and liver. At 220 days, CMHs display a total Ca2+ overload, 1.3-1.8-fold normal and a cytosolic Ca2+ concentration 2-4-fold higher than normal. Long-term oral treatment (18 mg/kg per day) with trimetazidine (anti-ischaemic drug), from age 30 to 350 days, was more efficient than the standard Ca2+ blocker verapamil. Trimetazidine increased the median survival time of CMH by 57% and the hypertrophy disappeared. The total Ca2+ level in CMHs reverted to that of normal Syrian hamsters (F1B). The cytosolic Ca2+ overload was limited to a factor of approximately 2. Therefore, trimetazidine possesses anti-Ca2+ properties and is effective in increasing survival and decreasing the heart and liver hypertrophy of CMH. This suggests that trimetazidine may be valuable in the prevention of congestive heart failure of similar aetiology.