Generally, cationic vector-based intravenous delivery of at a ratio of 4 nitrogen equivalents per DNA phosphate. DNA is hindered by interactions of positively charged comLower levels of transfection were found in the heart, plexes with serum proteins. However, if optimally formuspleen, liver and kidney. Expression was dose-and timelated, cationic vectors can provide reasonable levels of dependent in all tissues examined. In the lung, -galactotransfection in the lung either by intravenous or intrapulsidase staining showed transgene expression in clusters monary routes. We investigated the in vivo transfection of 10 or more pulmonary cells including the alveolar endocapacity of a cationic polymer: linear, 22 kDa polyethylenithelium, squamous and great alveolar epithelial cells (type mine. PEI/DNA complexes were formulated in 5% glucose I and II pneumocytes) and septal cells. These findings indiand delivered into adult mice through the tail vein. Two cate that the complexes pass the capillary barrier in the marker genes were used, -galactosidase and luciferase.lung. Although the delivery mechanism requires eluciHigh levels of luciferase expression (10 7 RLU/mg protein) dation, linear PEI has promise as a vector for intravenous were found in the lung when DNA was complexed with PEI transfer of therapeutic genes.Keywords: cationic polymers; pneumocytes; plasmid DNA; nonviral; gene therapy A number of generations of cationic vectors have been synthetised and tested in a variety of in vivo models and some have been taken to clinical trials. Most of these vectors are either monocationic or polycationic lipids, but there has been more recent interest in cationic polymers. Indeed, we showed that the branched cationic polymer polyethylenimine (PEI) can provide high levels of transfection in vivo. [1][2][3] In particular the lowest molecular weight preparation then commercially available, the 25 kDa preparation from Aldrich, was shown to be a versatile and efficient vector in the mammalian brain. 2 In a more recent study, 3 we chose to examine the effect of formulation procedures (glucose or saline solutions) on the size and in vivo transfection activity of a mixture of linear polymers with a mean MW of 22 kDa (Exgene 500; Euromedex, Souffleweyersheim, France). We found that the complexes formed in glucose were an order of magnitude smaller than those formed in saline and these complexes provided high levels of gene transfer following dilution into a physiological medium, the cerebrospinal fluid. In the light of these findings we chose to examine the effects of injecting complexes of plasmid DNA formulated with 22 kDa PEI in 5% glucose directly into the blood system and to examine transgene expression in a variety of organs.PEI-DNA complexes with different ratios of PEI nitro- gen to DNA phosphate (N/P ratio) were prepared in 5% glucose using a CMV-Luc plasmid 4 and the 22 kDa linear PEI. This PEI is synthesised to a degree of polymerisation of 510 units. Earlier experiments carried out with the branched 25 kDa PEI (Aldri...
SummaryMultiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.
Feline leukemia virus subgroup C receptor 1 (FLVCR1) is a cell membrane heme exporter that maintains the balance between heme levels and globin synthesis in erythroid precursors. It was previously shown that Flvcr1-null mice died in utero due to a failure of erythropoiesis. Here, we identify Flvcr1b, a mitochondrial Flvcr1 isoform that promotes heme efflux into the cytoplasm. Flvcr1b overexpression promoted heme synthesis and in vitro erythroid differentiation, whereas silencing of Flvcr1b caused mitochondrial heme accumulation and termination of erythroid differentiation. Furthermore, mice lacking the plasma membrane isoform (Flvcr1a) but expressing Flvcr1b had normal erythropoiesis, but exhibited hemorrhages, edema, and skeletal abnormalities. Thus, FLVCR1b regulates erythropoiesis by controlling mitochondrial heme efflux, whereas FLVCR1a expression is required to prevent hemorrhages and edema. The aberrant expression of Flvcr1 isoforms may play a role in the pathogenesis of disorders characterized by an imbalance between heme and globin synthesis.
The human congenital syndromes ectrodactyly ectodermal dysplasia-cleft lip/palate syndrome, ankyloblepharon ectodermal dysplasia clefting, and split-hand/foot malformation are all characterized by ectodermal dysplasia, limb malformations, and cleft lip/palate. These phenotypic features are a result of an imbalance between the proliferation and differentiation of precursor cells during development of ectoderm-derived structures. Mutations in the p63 and interferon regulatory factor 6 (IRF6) genes have been found in human patients with these syndromes, consistent with phenotypes. Here, we used human and mouse primary keratinocytes and mouse models to investigate the role of p63 and IRF6 in proliferation and differentiation. We report that the ΔNp63 isoform of p63 activated transcription of IRF6, and this, in turn, induced proteasomemediated ΔNp63 degradation. This feedback regulatory loop allowed keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate. Importantly, mutations in either p63 or IRF6 resulted in disruption of this regulatory loop: p63 mutations causing ectodermal dysplasias were unable to activate IRF6 transcription, and mice with mutated or null p63 showed reduced Irf6 expression in their palate and ectoderm. These results identify what we believe to be a novel mechanism that regulates the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation and links the pathogenesis of 2 genetically different groups of ectodermal dysplasia syndromes into a common molecular pathway.
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