RESUMEN
Bacterial intercellular signaling mediated by small molecules, also called autoinducers (AIs), enables synchronized behavior in response to environmental conditions, and in many bacterial pathogens, intercellular signaling controls virulence gene expression. However, in the intestinal pathogen Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), although three signals, named AI-1, AI-2 and AI-3, have been described, their roles in virulence remain elusive. AI-3 is the 3,6- isomer of a previously described Vibrio cholerae signaling molecule; 3,5-dimethylpyrazin-2-ol (3,5-DPO). To elucidate the role of AI-3/DPO in S. Typhimurium, we have mapped the global transcriptomic responses to 3,5- and 3,6-DPO isomers in S. Typhimurium. Our studies showed that DPO affects expression of almost 8% of all genes. Specifically, expression of several genes involved in gut-colonization respond to DPO. Interestingly, most of the affected genes are similarly regulated by 3,5-DPO and 3,6-DPO, respectively, indicating that the two isomers have overlapping roles in S. Typhimurium.
Asunto(s)
Transcriptoma , Vibrio cholerae , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Lactonas/metabolismo , Perfilación de la Expresión Génica , Vibrio cholerae/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
In this communication we describe the construction of four succinic-based cationic lipids, their formulation with plasmid DNA (pDNA), and an evaluation of their in vitro gene delivery into Chinese hamster ovarian (CHO-K1) cells. The cationic lipids employed in this work possess either a dimethylamine or trimethylamine headgroup, and a macrocyclic or an acyclic hydrophobic domain composed of, or derived from two 16-atom, succinic-based acyl chains. The synthesized lipids and a co-lipid of neutral charge, either cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were formulated in an overall 3:2 cationic-to-neutral lipid molar ratio, then complexed with plasmid DNA (pDNA). The relative transfection performance was evaluated via a comparison between matched versus mismatched formulations defined by the rigidity relationship between the lipids employed. Gel electrophoresis was used to characterize the binding of the lipid formulations with plasmid DNA and the relative degree of plasmid degradation using a DNase I degradation assay. Small angle X-ray diffraction (SAXD) was employed to characterize the packing morphology of the lipid-DNA complexes. In general, the succinic unit embedded within the hydrophobic domain of the cationic lipids was found to improve lipid hydration. The transfection assays revealed a general trend in which mismatched formulations that employed a rigid lipid combined with a non-rigid (or flexible) lipid, outperformed the matched formulations. The results from this work suggest that the design of the cationic lipid structure and the composition of the lipoplex formulation play key roles in governing the transfection performance of nonviral gene delivery agents.
Asunto(s)
ADN/metabolismo , Lípidos/química , Succinatos/química , Transfección/métodos , Animales , Células CHO , Cationes/química , Cricetinae , Cricetulus , ADN/química , Técnicas de Transferencia de Gen/normas , Hidrocarburos Acíclicos , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos Macrocíclicos , Plásmidos , Succinatos/metabolismo , Transfección/normasRESUMEN
The delivery of nucleic acids into cells remains an important laboratory cell culture technique and potential clinical therapy, based upon the initial cellular uptake, then translation into protein (in the case of DNA), or gene deletion by RNA interference (RNAi). Although viral delivery vectors are more efficient, the high production costs, limited cargo capacity, and the potential for clinical adverse events make nonviral strategies attractive. Cationic lipids are the most widely applied and studied nonviral vectors; however, much remains to be solved to overcome limitations of these systems. Advances in the field of cationic lipid-based nucleic acid (lipoplex) delivery rely upon the development of robust and reproducible lipoplex formulations, together with the use of cell culture assays. This chapter provides detailed protocols towards the formulation, delivery, and assessment of in vitro cationic lipid-based delivery of DNA.
Asunto(s)
Cationes/química , Lípidos/química , Ácidos Nucleicos/genética , Animales , Células CHO , Cricetulus , Liposomas/química , Liposomas/farmacología , Ácidos Nucleicos/química , Tamaño de la Partícula , TransfecciónRESUMEN
Previously we reported the synthesis and in vitro evaluation of four novel, short-chain cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic hydrophobic regions composed of, or derived from, two 7-carbon chains. Herein we describe a revised synthesis of an expanded library of related cationic lipids to include extended chain analogues, their formulation with plasmid DNA (pDNA) and in vitro delivery into Chinese hamster ovarian (CHO-K1) cells. The formulations were evaluated against each other based on structural differences in the hydrophobic domain and headgroup. Structurally the library is divided into four sets based on lipids derived from two 7- or two 11-carbon hydrophobic chains, C7 and C11 respectively, which possess either a dimethylamine or a trimethylamine derived headgroup. Each set includes four cationic lipids based on an acyclic or macrocyclic, saturated or unsaturated hydrophobic domain. All lipids were co-formulated with the commercial cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) in a 1:1 molar ratio, along with one of two distinct neutral co-lipids, cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an overall cationic-to-neutral lipid molar ratio of 3:2. Binding of lipid formulations with DNA, and packing morphology associated with the individual lipid-DNA complexes were characterized by gel electrophoresis and small angle X-ray diffraction (SAXD), respectively. As a general trend, lipoplex formulations based on mismatched binary cationic lipids, composed of a shorter C7 lipid and the longer lipid EPC (C14), were generally associated with higher transfection efficiency and lower cytotoxicity than their more closely matched C11/EPC binary lipid formulation counterparts. Furthermore, the cyclic lipids gave transfection levels as high as or greater than their acyclic counterparts, and formulations with cholesterol exhibited higher transfection and lower cytotoxicity than those formulated with DOPE. A number of the lipid formulations with cholesterol as co-lipid performed as well as, or better than Lipofectamine 2000™ and EPC, the two positive controls employed in these studies. These results suggest that our novel cyclic and acyclic cationic lipid vectors are effective nonviral gene transfer agents that warrant further investigation.
Asunto(s)
Lípidos/química , Transfección , Animales , Células CHO , Cationes/química , Cricetinae , Cricetulus , Dimiristoilfosfatidilcolina/análogos & derivados , Dimiristoilfosfatidilcolina/química , Lípidos/síntesis química , Liposomas/síntesis química , Liposomas/química , Liposomas/metabolismo , Fosfatidiletanolaminas/química , Plásmidos/genética , Plásmidos/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).
Asunto(s)
Lípidos/síntesis química , Compuestos Macrocíclicos/síntesis química , Animales , Células CHO , Cationes/química , Supervivencia Celular/efectos de los fármacos , Cricetinae , Lípidos/farmacología , Compuestos Macrocíclicos/farmacología , Relación Estructura-Actividad , TransfecciónRESUMEN
A description of the structurally unusual "phomactin" family of platelet activating factor antagonists recently found in the marine fungus Phoma sp. is presented. The phomactins show an interesting structural and biosynthetic relationship with the more familiar taxane group of antitumor compounds isolated from yew trees. The Account highlights and discusses this unique relationship and also presents a cogent picture of plausible biogenetic interrelationships within the family of phomactins. Complementary synthetic endeavors with the phomactins are interwoven in the discussions, alongside contemporaneous biosynthetic studies with both the phomactins and the taxanes.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Factor de Activación Plaquetaria/antagonistas & inhibidores , Animales , Hidrocarburos Aromáticos con Puentes/química , Compuestos Epoxi/química , Compuestos Epoxi/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Humanos , Estructura Molecular , Taxoides/químicaRESUMEN
A range of natural and unnatural phomactins, recently synthesised in our laboratory, were found to exhibit PAF antagonism with pIC(50) values in the range 5.6-6.2. The variation in structural and stereochemical features between the phomactins was found to have only a modest effect on the inhibition of binding of PAF to its human platelet receptors.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Factor de Activación Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/química , Receptores Acoplados a Proteínas G/química , Plaquetas , Membrana Celular/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Concentración 50 Inhibidora , Ligandos , Glicoproteínas de Membrana Plaquetaria/aislamiento & purificación , Receptores Acoplados a Proteínas G/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
A total synthesis of phomactin G (), which is a central intermediate in the biosynthesis of phomactin A () in Phoma sp. is described. The synthesis is based on a Cr(ii)/Ni(ii) macrocyclisation from the aldehyde vinyl iodide, leading to, followed by sequential conversion of into the [small beta]-epoxide and the ketone which, on deprotection, led to (+/-)-phomactin G. Phomactin G () shares an interesting structural homology with phomactin D (), the most potent PAF-antagonist metabolite in Phoma sp. It is most likely converted into phomactin A (), by initial allylic oxidation to the transient [small alpha]-alcohol 'phomactin' structure, known as Sch 49028, followed by spontaneous pyran ring formation.
Asunto(s)
Ascomicetos/química , Compuestos Epoxi/síntesis química , Factor de Activación Plaquetaria/antagonistas & inhibidores , Estructura MolecularRESUMEN
A total synthesis of the PAF antagonist phomactin A (1), isolated from the marine fungus Phoma sp. is described. The synthesis is based on a Cr(II)/Ni(II) macrocyclisation from the aldehyde vinyl iodide 14, leading to the key phomactatrienol intermediate 16a, followed by elaboration of 16a to the epoxyketone 21, which undergoes spontaneous pyran and hemiacetal ring formation to 1 on deprotection with DDQ.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Aldehídos/química , Ascomicetos , Benzoquinonas/química , Cromo/química , Cetonas/química , Modelos Químicos , Níquel/química , Factor de Activación Plaquetaria/química , Piranos/química , EstereoisomerismoRESUMEN
The total syntheses of the epoxy cyclic hemiacetal structures 8 and 9, which are isomeric with the structure 6 proposed for the phomactin known as Sch 49028 isolated from the marine fungus Phoma sp. are described. Neither of these structures showed spectroscopic data consistent with those reported for the purported natural product, adding credibility to the proposal that the structure Sch 49028 does not exist in nature and that its NMR spectroscopic data should have been assigned as phomactin A (1).
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
A total synthesis of phomactin A (1) based on a Cr(II)/Ni(II) macrocyclisation from the aldehyde vinyl iodide 11, leading to 12, followed by elaboration of the epoxyketone 16, which then undergoes spontaneous pyran-hemiacetal formation on deprotection, is described.