ABSTRACT
In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Drug Delivery Systems/methods , Electroporation/methods , Poloxamer/administration & dosage , Animals , Bleomycin/pharmacokinetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival , Cisplatin/pharmacokinetics , Flow Cytometry , HeLa Cells , Hemolysis/drug effects , Humans , Hydrophobic and Hydrophilic Interactions , Jurkat Cells , Luciferases/genetics , Luciferases/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Nude , Porosity/drug effects , Propidium , Spectrometry, Fluorescence , Whole Body Imaging , Xenograft Model Antitumor Assays , Red Fluorescent ProteinABSTRACT
The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.
Subject(s)
Gene Knockdown Techniques/methods , MicroRNAs/metabolism , RNA Interference , Transcription, Genetic/drug effects , Animals , Cell Line , Doxycycline/pharmacology , HeLa Cells , Humans , Kinetics , Mice , Mice, Nude , MicroRNAs/chemistry , MicroRNAs/genetics , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA Polymerase III/metabolism , RNA, Messenger/metabolismABSTRACT
The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and the mitochondria of most eukaryotes. In general, the bacterial complex consists of 14 different subunits. In addition to the homologues of these subunits, the mitochondrial complex contains approximately 31 additional proteins. While it was shown that the mitochondrial complex is assembled from distinct intermediates, nothing is known about the assembly of the bacterial complex. We used Escherichia coli mutants, in which the nuo-genes coding the subunits of complex I were individually disrupted by an insertion of a resistance cartridge to determine whether they are required for the assembly of a functional complex I. No complex I-mediated enzyme activity was detectable in the mutant membranes and it was not possible to extract a structurally intact complex I from the mutant membranes. However, the subunits and the cofactors of the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of some of the nuo-mutants. It is discussed whether this fragment represents an assembly intermediate. In addition, a membrane-bound fragment exhibiting NADH/ferricyanide oxidoreductase activity and containing the iron-sulfur cluster N2 was detected in one mutant.
Subject(s)
Electron Transport Complex I/genetics , Escherichia coli/enzymology , Centrifugation, Density Gradient , Cytoplasm/enzymology , Electron Spin Resonance Spectroscopy , Electron Transport Complex I/chemistry , Electron Transport Complex I/isolation & purification , Electron Transport Complex I/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Genes, Bacterial , Kinetics , MutationABSTRACT
OBJECTIVE: The authors conducted an investigation of the association between air pollution and arrhythmia. METHODS: A prospective panel study (October 2000-April 2001) was conducted in Erfurt, Germany. Fifty-seven men with coronary heart disease were subjected to six 24-hour electrocardiogram recordings. Runs of supraventricular and ventricular tachycardia were associated with continuous ultrafine particle counts (UFP), accumulation mode particle counts (ACP), PM2.5, and gaseous pollutants. Poisson and linear regression models were applied adjusting for trend, weekday, and meteorologic data. RESULTS: Elevated concentrations of UFP, ACP, PM2.5, and nitrogen dioxide increased the risk for supraventricular runs and the number of ventricular runs at almost all lags. Statistically significant associations were found predominantly in the previous 24 to 71 hours and with the 5-day moving average. CONCLUSION: Elevated concentrations of fine and ultrafine particle increased the risk of arrhythmia in men with coronary heart disease.
