RESUMEN
In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFß signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32WT/eGFP-AMP-LacZ). The addition of dopamine to MSNs either increased, decreased or had no effect on intracellular calcium, indicating the presence of multiple dopamine receptor subtypes. In summary, we demonstrate greater control over early fate decisions using activin-A, Wnt and SHH to direct differentiation into MSNs. We also generate a DARPP32 reporter line that enables deeper pharmacological profiling and interrogation of complex receptor interactions in human MSNs.
Asunto(s)
Diferenciación Celular/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Fosfoproteína 32 Regulada por Dopamina y AMPc/fisiología , Genes Reporteros/fisiología , Células Madre Embrionarias Humanas/fisiología , Neuronas/fisiología , Línea Celular , Proteínas Hedgehog/fisiología , Humanos , Vía de Señalización Wnt/fisiologíaRESUMEN
Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10µM), thapsigargin (1µM) and ZD7288 (10µM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1µM). Human cell survival was sensitive to the addition of trastuzumab and doxorubicin, while the switch from a low to a high oxygen environment affected oscillation frequency. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.
Asunto(s)
Miocitos Cardíacos/fisiología , Células Madre Pluripotentes/fisiología , Animales , Relojes Biológicos/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Oxígeno/metabolismo , Células Madre Pluripotentes/metabolismoRESUMEN
Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca(2+) imaging and [(3)H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), L-glutamate (30 µM), noradrenaline (30 µM), acetylcholine (30 µM) and dopamine (30 µM), with similar elevations of intracellular Ca(2+)([Ca(2+)](i)). The presence of GABA(A) and GABA(B) antagonists, bicuculline (30 µM) and CGP55845 (1 µM), increased the elevation of [Ca(2+)](i) in response to dopamine (30 µM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [(3)H]-GABA release from forebrain and midbrain cultures. Dopamine (30 µM) did not stimulate significant [(3)H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA(+) neurons contain NPY, and show similar agonist-induced elevations of [Ca(2+)](i) and [(3)H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates.
Asunto(s)
Tipificación del Cuerpo , Mesencéfalo/embriología , Neuronas/fisiología , Neuropéptido Y/fisiología , Prosencéfalo/embriología , Ácido gamma-Aminobutírico/fisiología , Animales , Secuencia de Bases , Diferenciación Celular , Cartilla de ADN , Dopamina/farmacología , Inmunohistoquímica , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Ratones , Neuronas/citología , Prosencéfalo/citología , Prosencéfalo/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have applications in understanding cardiac disease pathophysiology, pharmacology, and toxicology. Comprehensive characterization of their basic physiological and pharmacological properties is critical in determining the suitability of ESC-CMs as models of cardiac activity. In this study we use video microscopy and quantitative PCR to investigate the responses of mouse ESC-CMs to adrenoceptor, muscarinic, angiotensin II (Ang II), and endothelin-1 (ET-1) receptor activation. Isoprenaline (10 nM-10 µM) increased beating rate and contraction amplitude in all beating bodies (BBs), whereas carbachol (up to 1 µM) and the I(f) channel blocker ZD-7288 (10 µM) decreased contraction frequency. ET-1 (0.01-100 nM) reduced contraction amplitude in all BBs and increased contraction frequency in 50% of BBs; these effects were blocked by the ET(A) receptor antagonist BQ123 (250 nM). Ang II (0.01 nM-1 µM) increased both contraction amplitude (all BBs) and frequency (in 50% of BBs), effects blocked, respectively, by losartan (100 nM) and PD123,319 (200 nM). These results indicate the presence of functional ET(A) and both AT1 and AT2 receptors in murine ESC-CMs, but their expression and or activity appears to be evident only in a limited set of BBs.
Asunto(s)
Angiotensina II/metabolismo , Células Madre Embrionarias/metabolismo , Endotelina-1/metabolismo , Miocitos Cardíacos/metabolismo , Angiotensina II/genética , Animales , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Endotelina-1/genética , Ratones , Contracción Miocárdica , Miocitos Cardíacos/citologíaRESUMEN
BACKGROUND AND PURPOSE: Neurons derived from mouse embryonic stem cells (mESCs) are a valuable resource for basic pharmacological research. With the exception of cardiomyocytes, there is relatively little understanding of the pharmacology of stem cell-derived differentiated cells. In this study we investigate P2 receptor agonist effects on GABAergic neurons derived from mESCs. EXPERIMENTAL APPROACH: mESCs were differentiated into GABAergic neurons in the presence of N2B27 culture medium. At day 24 of differentiation GABAergic neuronal responsiveness to purinergic agonists was investigated using calcium imaging and [3H]-GABA release studies. KEY RESULTS: Sub-populations of GABAergic neurons responded to some or all of the adenine and uracil nucleotides ATP, ADP, UTP and UDP (all 100 microM) with elevations of intracellular Ca2+ ([Ca2+]i). The number of neurons responding to ATP was reduced by suramin (100 microM), PPADS (10 microM) and MRS2179 (10 microM), but not by NF023 (10 microM). The response to ATP was modulated by extracellular Zn2+ and pH. Neurons also responded to ATP (100 microM) with the release of [3H]-GABA, an effect completely inhibited by tetrodotoxin (100 nM). Ap4A and 2-methylthioATP both elicited significant [3H]-GABA release. Reverse transcriptase PCR showed the presence of P2X1,2,3,4,5,6 and P2X7, and P2Y1,2 and P2Y6 receptors. mESCs expressed P2X2,5 and P2X7 and P2Y1,2 and P2Y6 receptors. CONCLUSIONS AND IMPLICATIONS: GABAergic neurons derived from stem cells elevate [Ca2+]i predominantly via the activation of P2X2, P2X4 and P2Y1 receptors. This study shows that mESCs generate good models of neuronal function for in vitro pharmacological investigation.
Asunto(s)
Calcio/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Difosfato/administración & dosificación , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/administración & dosificación , Adenosina Trifosfato/metabolismo , Animales , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Neuronas/metabolismo , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2Y1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Uridina Difosfato/administración & dosificación , Uridina Difosfato/metabolismo , Uridina Trifosfato/administración & dosificación , Uridina Trifosfato/metabolismo , Zinc/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Insoluble, pure protein particles could be advantageous as single-entity vaccines or as carriers for small peptide epitopes. Dense gas anti-solvent precipitation was employed to produce pure protein particles which were found to be insoluble in water. As particulate and multimerized antigens are more immunogenic and hence more advantageous for vaccination, particles were produced via this method using ovalbumin as a model antigen. The particles produced had a mean diameter of approximately 300nm, and remained as discrete particles at low pH. At neutral pH or in the presence of electrolyte, the particles exhibited predictable flocculation behaviour to produce aggregates 1-5microm in diameter. Immunisation of mice with these flocculates elicited specific ovalbumin antibody production, T-cell proliferation and a cytotoxic T-cell response, all in the absence of adjuvant. Thus, dense gas processing could be used as a generic method to produce pure protein particulate vaccines.
Asunto(s)
Adyuvantes Inmunológicos , Formación de Anticuerpos/inmunología , Antígenos/inmunología , Inmunidad Celular/inmunología , Material Particulado/inmunología , Vacunas/inmunología , Animales , Antígenos/química , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Química Farmacéutica , Pollos , Inmunización , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Muramidasa/inmunología , Ovalbúmina/inmunología , Tamaño de la Partícula , Linfocitos T/inmunología , Vacunas/químicaAsunto(s)
Glicoproteínas de Membrana/fisiología , Receptores Virales/fisiología , Virosis/virología , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/fisiología , Hepacivirus/fisiología , Humanos , Estructura Terciaria de Proteína , Retroviridae/fisiología , Tetraspanina 28 , Virosis/tratamiento farmacológico , Replicación ViralRESUMEN
Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post-plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na(+) and tetraethylammonium (TEA)-sensitive K(+) channels. Neurons reached their maximal cell capacitance after D6-7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca(2+)-free, 2.5 mm Mg(2+) saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca(2+) levels within ES cell-derived neurons increased in response to glutamate receptor agonists l-glutamate, AMPA, N-methyl-d-aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl(-)-selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons.
Asunto(s)
Potenciales de Acción/fisiología , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Neurotransmisores/farmacología , Células Madre/citología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Ácido Glutámico/farmacología , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Factores de Transcripción SOXB1 , Células Madre/efectos de los fármacos , Células Madre/fisiologíaRESUMEN
Inefficient transport of DNA from the cytoplasm into the nucleus remains a limiting step in the development of non-viral gene delivery systems. This is particularly acute in non-dividing cells, where entry to the nucleus is thought to occur only through the nuclear pore complex. Active import of physiological proteins is mediated by nuclear localization sequences (NLSs) within cargo proteins such as transcription factors. Here we review current knowledge of this import machinery and consider its exploitation by mammalian viruses. Significant research effort has been directed at incorporating NLSs into synthetic gene delivery systems to take advantage of this physiological pathway. Both non-covalent and covalent methods of conjugation are evaluated, with NLS linkage to both DNA and carrier, and compared with activities of simple cationic polymers. Finally, progress in the field of DNA sequence-specific nuclear import is examined and the current state of the technology assessed.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Técnicas de Transferencia de Gen , Transgenes , Adenoviridae/fisiología , ADN/genética , ADN/metabolismo , Dependovirus/fisiología , Vectores Genéticos , Herpesviridae/fisiología , Humanos , Señales de Localización Nuclear/metabolismoRESUMEN
The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that influence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efficiency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future.
Asunto(s)
Terapia Genética/métodos , Animales , Portadores de Fármacos , Marcación de Gen , HumanosRESUMEN
'Lipid' formulations for oral administration of drugs generally consist of a drug dissolved in a blend of two or more excipients, which may be triglyceride oils, partial glycerides, surfactants or co-surfactants. The primary mechanism of action which leads to improved bioavailability is usually avoidance, or partial avoidance, of the slow dissolution process which limits the bioavailability of hydrophobic drugs from solid dosage forms. Ideally the formulation allows the drug to remain in a dissolved state throughout its transit through the gastrointestinal tract. The availability of the drug for absorption can be enhanced by presentation of the drug as a solubilizate within a colloidal dispersion. This objective can be achieved by formulation of the drug in a self-emulsifying system or alternatively by taking advantage of the natural process of triglyceride digestion. In practice 'lipid' formulations range from pure oils, at one extreme, to blends which contain a substantial proportion of hydrophilic surfactants or cosolvents. Knowledge of the efficiency of emulsification of these formulations, the nature of the colloidal system formed by dispersion, their susceptibility to digestion, and the subsequent fate of the drug is desirable for formulation. Yet the literature on this subject is limited, so this article represents part review and part commentary on current status of lipid formulations. A simple classification system for lipid formulations, based on the polarity of the blend and reviewed here, will help comparison of data between laboratories. Priorities for future work are discussed. More data is needed on the solubility of drugs in various types of formulations, and in particular, on the relationship between the physical chemistry of the drug and its fate, subsequent to dilution and digestion of the formulation in the lumen of the gastrointestinal tract. The mechanisms of action and practical uses of each type of lipid formulation are discussed.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Excipientes/química , Lípidos/química , Tensoactivos/química , Administración Oral , Animales , Precipitación Química , Excipientes/farmacología , Humanos , Lípidos/farmacología , Lipólisis/efectos de los fármacos , Lipólisis/fisiología , Tamaño de la Partícula , Solubilidad/efectos de los fármacos , Tensoactivos/farmacologíaRESUMEN
Improved non-viral vector systems are needed for efficient delivery of DNA to target cell nuclei in gene therapy. A series of linear polyamine poly(ethylene glycol) (PEG) constructs has been synthesised by reaction of appropriately Boc-protected thermine derivatives with omega-methoxyPEG oxiranylmethyl ethers. Constructs carrying 1-3 MeOPEG units and 0, 2 or 4 N-methyl groups have been prepared by this method. H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NHBoc was prepared efficiently by mono-trifluoroacetylation of thermine, attachment of Boc and removal of the trifluoroacetyl group in one pot. A similar process gave H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2. BocMeN(CH2)3NHMe was alkylated by 1,3-dibromopropane to give BocMeN(CH2)3NMe(CH2)3NMe(CH2)3NMeBoc. A cyanoethylation/reduction sequence extended H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NH2 to give H2N(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2)3NBoc(CH2) 3NH2, which was converted to its mono- and di-MeOPEG550 derivatives. Deprotection gave the linear polyamine MeOPEG constructs. A branched triamine-poly(ethylene glycol) construct was prepared by acylation of (BocHN(CH2)3)2N(CH2)3NH2 with omega-methoxyPEG 550 chloroformate, followed by deprotection. A cyanoethylation/reduction/protection sequence from (H2N(CH2)3)2 N(CH2)3NHBoc gave a protected pentamine. Alkylation with Br(CH2)5CONH(CH2)2NHBoc, deprotection, acylation with MeOPEG chloroformate and deprotection gave a pentamine MeOPEG construct in which the MeOPEG is attached through a linker to the central amine. The linear hexamine construct carrying MeOPEG550 at only one terminus was the most effective DNA-interactive member of the two series in an ethidium displacement assay and was effective in delivering a reporter gene to RIF-1 tumours.
Asunto(s)
ADN/metabolismo , Sistemas de Liberación de Medicamentos , Poliaminas/metabolismo , Polietilenglicoles/metabolismo , Animales , Unión Competitiva , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN/administración & dosificación , ADN/ultraestructura , Composición de Medicamentos , Etidio/metabolismo , Femenino , Genes Reporteros/genética , Humanos , Ratones , Ratones Endogámicos C3H , Poliaminas/síntesis química , Polietilenglicoles/síntesis química , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Tensoactivos/síntesis química , Tensoactivos/química , Tensoactivos/metabolismo , Transfección/métodosRESUMEN
A novel peptide containing the lysosomally degradable sequence GlyPheLeuGly and a sequence-inverting unit has been prepared. This peptide presents the methylamino groups of sarcosine (N-methylglycine) at both termini for co-polymerisation with alpha, omega-bis(oxiranylmethoxy)poly(ethylene glycol) of mean M(w) 1650. Gel permeation chromatographic analysis showed the presence of a mixture of oligomers. Preliminary degradation studies showed that these oligomers are cleaved by cathepsin B, an important lysosomal enzyme.
Asunto(s)
Sistemas de Liberación de Medicamentos , Péptidos/síntesis química , Polietilenglicoles/síntesis química , Catepsina B/química , Espectroscopía de Resonancia Magnética , Péptidos/administración & dosificación , Péptidos/química , Polietilenglicoles/administración & dosificación , Polietilenglicoles/químicaRESUMEN
153N-6 (H-[Met5,Pro6,D-Phe7,D-Trp9,Phe10]-MSH(5-13)) has emerged as the most potent antagonist of alpha-MSH activity on Xenopus laevis melanophores, from a library of 32 360 peptides based on alpha-MSH(5-13) [22]. A recent report has confirmed our observation that 153N-6 also binds to mammalian melanocortin receptors. Here we report the receptor-binding affinities and biologic activities of 153N-6 and 17 selected alpha-MSH analogues at the native MCI receptor expressed by murine B16 melanoma cells. Our intention is to determine the structural requirements for agonism and competitive antagonism of melanocortin activity at the MC1-R and to discover more potent antagonists. 153N-6 was able to inhibit the action of native alpha-MSH and the potent synthetic agonist, [Nle4,D-Phe7]alpha-MSH, at the murine MC1-R. However, the Ki of 153N-6 was 439 times higher than that of alpha-MSH and 4475 times higher than that of [Nle4,D-Phe7]alpha-MSH; too high to allow 153N-6 to be considered as a practical antagonist for use in vivo (Ki of 153N-6 = 9.0 X 10(-6) M). Because Met4 is an important component of alpha-MSH binding at the MC1-R, we investigated alpha-MSH(1-13) and alpha-MSH(4-13) analogues to produce compounds with higher MC1-R-binding affinity than 153N-6. The binding affinity of 153N-6 was not significantly different from alpha-MSH(5-13), but it was 232 times lower than alpha-MSH(4-13). Coupling of H-Nle (as an isosteric replacement for Met) or acetyl-Nle to the N-terminus of 153N-6 raised the binding affinity by a factor of 46, but this and all full-length alpha-MSH analogues with Met or Nle in position 4 were full agonists of the MC1-R. A full-length alpha-MSH(1-13) derivative of 153N-6 with Ala4 did not exhibit significantly greater binding affinity than 153N-6 and appeared to be a partial agonist at the MC1-R in the cAMP assay. These data suggest that Met4 is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at the MCI-R. Pro6 and Phe10 (with respect to alpha-MSH) were found to be the most influential substitutions that determined the antagonist activity of 153N-6.
Asunto(s)
Oligopéptidos/química , Receptores de Corticotropina/química , Secuencia de Aminoácidos , Animales , Ratones , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Receptores de Corticotropina/metabolismo , Receptores de Melanocortina , Relación Estructura-Actividad , alfa-MSH/antagonistas & inhibidores , alfa-MSH/química , alfa-MSH/metabolismoRESUMEN
Physicochemical properties of polyplexes formed between pRSVlacZ and poly(amino acid)s were investigated as a paradigm of more complex, synthetic virus-like, DNA delivery systems, that are of interest to many gene delivery laboratories. We observed the interaction between polymer and DNA using ethidium exclusion, and determined the size distributions and the zeta potentials of polyplexes. We correlated these properties with their fundamental interactions with cultured B16 murine melanoma cells, and the resulting efficiency of transfection. A variety of poly(amino acid)s each condensed DNA to produce particles with mean hydrodynamic diameters of approximately 100 nm (a typical span of a population was 80-120nm). Poly(amino acid) polyplexes were unstable in electrolyte solutions such as cell culture media. The apparent particle size increased in electrolyte, depending on the charge ratio, to diameters up to 700 nm. This was thought to be due to aggregation, since neutral particles were most sensitive. When the charge ratio (+/-) exceeded unity polyplexes had positive zeta potentials (which peaked at approximately +30 mV), bound non-specifically to cells, were internalised and in the presence of an endosomolytic agent were able to transfect cells. Though all cationic poly(amino acid)s investigated formed polyplexes with similar physical properties, their biological properties were significantly different. Polyplexes prepared with poly-L-ornithine were the most effective transfection agents, but poly(lys-co-ala, 1: 1) systems appeared to be inactive. This may reflect the differences in uncoupling of DNA and polymer, which is expected to be necessary for passage through the nuclear pore. Uncoupling of polycation and DNA was investigated by exposing the complexes to dextran sulphate. Release of DNA was detected by increased fluorescence at 600 nm in the presence of ethidium. Release of DNA was incomplete from polyplexes formed with high molecular weight polylysine. This may explain the lower levels of transfection observed with high molecular weight polylysine. The significance of these observations for design of advanced non-viral gene delivery systems is discussed.
Asunto(s)
Aminoácidos , ADN/administración & dosificación , Técnicas de Transferencia de Gen , Polímeros , Animales , Cationes , Etidio , Melanoma/genética , Ratones , Transfección , Células Tumorales CultivadasRESUMEN
PURPOSE: The use of rapidly dividing in vitro cell culture systems to assess the efficiency of gene delivery is now recognised as a poor indicator of in vivo success. We investigated whether differentiated Caco-2 cell filter-cultures would make a more suitable model for studying gene transfer to an epithelium. METHODS: Caco-2 cells were cultured on semi-permeable membrane filters into differentiated polarised monolayers. Monolayer differentiation was assessed by monitoring the transport of taurocholic acid. Cells at different stages of differentiation were transfected with DNA/DOTAP lipoplexes and later analysed for reporter gene activity. The uptake of radiolabled DNA was also evaluated at various stages of differentiation. RESULTS: Caco-2 cultures developed a resistance to lipoplex-mediated transfection as early as three days, when some cells were still dividing and undifferentiated. As cultures matured, expression of reporter gene progressively decreased partly due to reduced internalisation of DNA. The resistance to transfection could be overcome in part by pre-treatment of monolayers with calcium chelating agents or surfactants. However, transgene expression in treated monolayers was still significantly lower than that in dividing cultures. CONCLUSIONS: Differentiated Caco-2 cells are a more appropriate model for gene-transfer studies to the intestinal epithelium because they demonstrate a resistance to transfection similar to that observed in vivo.
Asunto(s)
Portadores de Fármacos , Células Epiteliales/fisiología , Lípidos/química , Transfección/métodos , Células CACO-2 , Cationes , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , ADN/genética , Filtración , Humanos , Liposomas , Tamaño de la Partícula , Plásmidos/genética , Ácido Taurocólico/metabolismoRESUMEN
DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.
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ADN/administración & dosificación , Lípidos/farmacología , Péptidos/farmacología , Poliaminas/química , Transfección , Animales , Células COS , Línea Celular Transformada , ADN/química , Terapia Genética , Humanos , Lípidos/administración & dosificación , Lípidos/química , Ratones , Péptidos/administración & dosificación , Plásmidos , Polielectrolitos , Células Tumorales CultivadasRESUMEN
Reaction of alkane-1, omega-diamines with 6-chloro-1,3-dimethylpyrimidine-2,4-dione under carefully controlled conditions gives 6-(omega-aminoalkylamino)-1,3-dimethylpyrimidine-2,4-diones, which can be readily separated from traces of products of disubstitution after benzyloxycarbonyl protection. A sequence of nitrosation at the pyrimidine 5-position, thermal cyclization, and deprotection affords 8-(omega-aminoalkyl) derivatives of theophylline, an important drug in the treatment of asthma and related diseases. These 8-(omega-aminoalkyl)theophyllines can be coupled to fluorescein-5-isothiocyanate and to dansyl chloride, giving fluorescent derivatives of theophylline with applications in automated immunoassay of the drug in biofluids using the fluorescence capillary fill device.
Asunto(s)
Colorantes Fluorescentes/química , Inmunoensayo , Teofilina/análogos & derivados , Isotiocianatos/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ácidos Sulfínicos/química , Teofilina/químicaRESUMEN
Five subtypes of melanocortin receptors have to date been identified, but to date little is known about the different structural requirements for binding and biological activity at these receptors. In this study, the role of C-terminal melanocortin peptide residues in imparting selectivity for the receptor subtypes was examined. C-terminally modified analogues of alpha-MSH and gamma-MSH were synthesized and their interaction with MC1 and MC3 melanocortin receptors was investigated. This study provides further evidence for an important role of proline 12 (numbering with respect to alpha-MSH) for binding and activity at the MC1 receptor. Although the influence of C-terminal amino acids on binding and activity at MC3-R was less marked, some of them were nevertheless observed to be beneficial for the interaction with this receptor subtype.