Nanocarrier-assisted sub-cellular targeting to the site of mitochondria improves the pro-apoptotic activity of paclitaxel.

Many drug molecules exert their biological action on intracellular molecular targets present on or inside various cellular organelles. Consequently, it has become more evident that the efficiency and efficacy of drug action is dependent largely on how well an unaided drug molecule is able to reach its intracellular target. We hypothesized that the biological action of such drug molecules might be improved by specific delivery to the appropriate sub-cellular site by a pharmaceutical carrier designed for the purpose. To test our hypothesis, we used paclitaxel, a molecule that has recently been shown to have pro-apoptotic biological targets on the mitochondria but has a quantitative structure-activity relationship-predicted cytosolic accumulation and no affinity for mitochondria. Using a mitochondria-specific nanocarrier system (DQAsomes) prepared from the amphiphilic quinolinium derivative dequalinium chloride to deliver paclitaxel to mitochondria in cells, we report that it is possible to improve the pro-apoptotic action of paclitaxel.

Liposomes and liposome-like vesicles for drug and DNA delivery to mitochondria.

Mitochondrial research is presently one of the fastest growing disciplines in biomedicine. Since the early 1990s, it has become increasingly evident that mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Most remarkably, mitochondria, the "power house" of the cell, have also become accepted as the "motor of cell death" reflecting their recognized key role during apoptosis. Based on these recent exciting developments in mitochondrial research, increasing pharmacological efforts have been made leading to the emergence of "Mitochondrial Medicine" as a whole new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, colloidal vectors, i.e., delivery systems, need to be developed able to selectively transport biologically active molecules to and into mitochondria within living human cells. Here we review ongoing efforts in our laboratory directed toward the development of different phospholipid- and non-phospholipid-based mitochondriotropic drug carrier systems.

Localization and loss-of-function implicates ciliary proteins in early, cytoplasmic roles in left-right asymmetry.

Left-right asymmetry is a crucial feature of the vertebrate body plan. While much molecular detail of this patterning pathway has been uncovered, the embryonic mechanisms of the initiation of asymmetry, and their evolutionary conservation among species, are still not understood. A popular recent model based on data from mouse embryos suggests extracellular movement of determinants by ciliary motion at the gastrulating node as the initial step. An alternative model, driven by findings in the frog and chick embryo, focuses instead on cytoplasmic roles of motor proteins. To begin to test the latter hypothesis, we analyzed the very early embryonic localization of ciliary targets implicated in mouse LR asymmetry. Immunohistochemistry was performed on frog and chick embryos using antibodies that have (KIF3B, Polaris, Polycystin-2, acetylated alpha-tubulin) or have not (LRD, INV, detyrosinated alpha-tubulin) been shown to detect in frog embryos only the target that they detect in mammalian tissue. Immunohistochemistry revealed localization signals for all targets in the cytoplasm of cleavage-stage Xenopus embryos, and in the base of the primitive streak in chick embryos at streak initiation. Importantly, several left-right asymmetries were detected in both species, and the localization signals were dependent on microtubule and actin cytoskeletal organization. Moreover, loss-of-function experiments implicated very early intracellular microtubule-dependent motor protein function as an obligate aspect of oriented LR asymmetry in Xenopus embryos. These data are consistent with cytoplasmic roles for motor proteins in patterning the left-right axis that do not involve ciliary motion.

Mitochondrial pharmaceutics.

Since the end of the 1980s, key discoveries have been made which have significantly revived the scientific interest in a cell organelle, which has been studied continuously and with steady success for the last 100 years. It has become increasingly evident that mitochondrial dysfunction contributes to a variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Moreover, since the middle of the 1990s, mitochondria, the 'power house' of the cell, have also become accepted as the cell's 'arsenals' reflecting their increasingly acknowledged key role during apoptosis. Based on these recent developments in mitochondrial research, increased pharmacological and pharmaceutical efforts have lead to the emergence of 'Mitochondrial Medicine' as a whole new field of biomedical research. Targeting of biologically active molecules to mitochondria in living cells will open up avenues for manipulating mitochondrial functions, which may result in the selective protection, repair or eradication of cells. This review gives a brief synopsis over current strategies of mitochondrial targeting and their possible therapeutic applications.

DQAsome-mediated delivery of plasmid DNA toward mitochondria in living cells.

DQAsomes are mitochondriotropic cationic 'bola-lipid'-based vesicles, which have been developed by us for the transport of drugs and DNA to mitochondria in living cells. This has made direct mitochondrial gene therapy feasible for the very first time. Our strategy for the delivery of DNA into the matrix of mitochondria is based upon the DQAsomal transport of a DNA-signal peptide conjugate to mitochondria, the selective liberation of this conjugate from DQAsomes at the mitochondrial membrane followed by DNA uptake via the mitochondrial protein import machinery. Using membrane-mimicking liposomes and isolated rat liver mitochondria we have shown earlier that DQAsome-DNA complexes (DQAplexes) selectively release pDNA when in contact with mitochondria-like membranes. Employing a newly developed protocol for selectively staining free pDNA in the cytosol of living cells and based on confocal fluorescence microscopic imaging we demonstrate here that DQAplexes appear to be able to escape from endosomes without loosing their pDNA load and transport the pDNA to the site of mitochondria at which at least a portion of the pDNA is released from its DQAsomal carrier. Free pDNA could not be detected anywhere else inside the cytosol of transfected cells demonstrating the target-selectivity of DQAsome-mediated DNA delivery to mitochondria.

Myeloid expression of cytochrome P450 4F3 is determined by a lineage-specific alternative promoter.

The cytochrome P450 4F3 (CYP4F3) gene encodes two functionally distinct enzymes that differ only by the selection of exon 4 (CYP4F3A) or exon 3 (CYP4F3B). CYP4F3A inactivates leukotriene B4, a reaction that has significance for controlling inflammation. CYP4F3B converts arachidonic acid to 20-hydroxyeicosatetraenoic acid, a potent activator of protein kinase C. We have previously shown that mRNAs coding for CYP4F3A and CYP4F3B are generated from distinct transcription start sites in neutrophils and liver. We therefore investigated mechanisms that regulate the cell-specific expression of these two isoforms. Initially, we analyzed the distribution of CYP4F3 in human leukocytes and determined a lineage-specific pattern of isoform expression. CYP4F3A is expressed in myeloid cells and is coordinate with myeloid differentiation markers such as CD11b and myeloperoxidase during development in the bone marrow. In contrast, CYP4F3B expression is restricted to a small population of CD3+ T lymphocytes. We identified distinct transcriptional features in myeloid, lymphoid, and hepatic cells that indicate the presence of multiple promoters in the CYP4F3 gene. The hepatic promoter depends on a cluster of hepatocyte nuclear factor sites 123-155 bp upstream of the initiator ATG codon. The myeloid promoter spans 400 bp in a region 468-872 bp upstream of the ATG codon; it is associated with clusters of CACCT sites and can be activated by ZEB-2, a factor primarily characterized as a transcriptional repressor in cells that include lymphocytes. ZEB-2 interacts with C-terminal binding protein and Smads, and this would provide opportunities for integrating environmental signals in myelopoiesis and inflammation.

Early embryonic expression of ion channels and pumps in chick and Xenopus development.

An extensive body of literature implicates endogenous ion currents and standing voltage potential differences in the control of events during embryonic morphogenesis. Although the expression of ion channel and pump genes, which are responsible for ion flux, has been investigated in detail in nervous tissues, little data are available on the distribution and function of specific channels and pumps in early embryogenesis. To provide a necessary basis for the molecular understanding of the role of ion flux in development, we surveyed the expression of ion channel and pump mRNAs, as well as other genes that help to regulate membrane potential. Analysis in two species, chick and Xenopus, shows that several ion channel and pump mRNAs are present in specific and dynamic expression patterns in early embryos, well before the appearance of neurons. Examination of the distribution of maternal mRNAs reveals complex spatiotemporal subcellular localization patterns of transcripts in early blastomeres in Xenopus. Taken together, these data are consistent with an important role for ion flux in early embryonic morphogenesis; this survey characterizes candidate genes and provides information on likely embryonic contexts for their function, setting the stage for functional studies of the morphogenetic roles of ion transport.

K(ATP) channel activity is required for hatching in Xenopus embryos.

A growing body of work suggests that the activity of ion channels and pumps is an important regulatory factor in embryonic development. We are beginning to identify functional roles for proteins suggested by a survey of expression of ion channel and pump genes in Xenopus and chick embryos (Rutenberg et al. [2002] Dev Dyn 225, this issue). Here, we report that the ATP-sensitive K(+) channel protein is present in the hatching gland of Xenopus embryos; moreover, we show that its activity is necessary for hatching in Xenopus. Pharmacologic inhibition of K(ATP) channels not only specifically prevents the hatching process but also greatly reduces the endogenous expression of Connexin-30 in the hatching gland. Based on recent work which showed that gap-junctional communication mediated by Cx30 in the hatching gland was required for secretion of the hatching enzyme, we propose that K(ATP) channel activity is upstream of Cx30 expression and represents a necessary endogenous step in the hatching of the Xenopus embryo.