Toxicity mechanisms of amphotericin B and its neutralization by conjugation with arabinogalactan.

Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity.

A safety and tolerability study of differently-charged nanoparticles for local pulmonary drug delivery.

Nanoparticle (NP) based drug delivery systems provide promising opportunities in the treatment of lung diseases. Here we examined the safety and tolerability of pulmonary delivered NPs consisting of PEG-PLA as a function of particle surface charge. The rationale for such a comparison should be attributed to the differential pulmonary toxicity of positively and negatively charged PEG-PLA NP.

Nanoparticle-cell interactions: drug delivery implications.

Design strategies to enhance drug delivery at specific cellular organelle by taking advantage of the endocytotic pathways are still in the early stages of development. This review provides a summary of the endocytosis machineries and pathways, as well as their involvement in nanoparticle internalization processes into either polarized epithelial cells or nonpolarized cells, in view of the marked differences in endocytic processes occurring within those cell types. The relevance of the physicochemical properties of nanoparticles upon their entry into the cells, as well as the experimental tools used to investigate the entry of nanoparticles into cells, is also addressed.

Alpha-synuclein and polyunsaturated fatty acids promote clathrin-mediated endocytosis and synaptic vesicle recycling.

Alpha-synuclein (alphaS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, alphaS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in alphaS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity.

The mammalian Nek1 kinase is involved in primary cilium formation.

Recent studies implicate primary cilium (PC) proteins in the etiologies of various polycystic kidney diseases (PKD). NIMA-related kinases (NRKs) are conserved serine/threonine kinases, which are usually defined as 'mitotic kinases'. Murine mutants for the NRKs, nek1 (kat mice) suffer from PKD, suggesting that it may be involved in cilium control.

Analysis of membrane traffic in polarized epithelial cells.

Spatial asymmetry is fundamental to the structure and function of most eukaryotic cells. A basic aspect of this polarity is that the cell's plasma membrane is divided into discrete domains. The best studied and simplest example of this occurs in epithelial cells, which line exposed body surfaces.

Surface charge of nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells.

A major challenge in drug delivery is the internalization through the apical plasma membrane of the polarized epithelial cells lining organs facing the external environment, e.g., lungs and the gastrointestinal tract.

Beta-tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure.

In epithelial cells, the apical junctional complex (AJC), composed of tight junctions (TJs) and adherens junctions (AJs), maintains cell-surface polarity by forming a fence that prevents lateral movement and diffusion of proteins and lipids between the apical and basolateral PM and holds the epithelial monolayer intact through cell-cell contacts. Disassembly of this complex is a prime event in development and cell transformation. Maintenance of the AJC has been shown to involve mainly the actin cytoskeleton.

Caveolin 2 regulates endocytosis and trafficking of the M1 muscarinic receptor in MDCK epithelial cells.

Clathrin and caveolins are known for their involvement in the internalization of numerous receptors. Here we show that in polarized epithelial Madin-Darby canine kidney cells, both the clathrin machinery and caveolins are involved in the endocytosis and delivery to the plasma membrane (PM) of the M1 muscarinic acetylcholine receptor (mAChR). We initially localized this receptor to the lateral membrane, where it accumulates proximal to the tight junctions.

Targeting of nanoparticles to the clathrin-mediated endocytic pathway.

Nanoparticles (NPs) are considered attractive carriers for gene therapy and drug delivery owing to their minor toxic effect and their ability to associate and internalize into mammalian cells. In this study, we compared the endocytosis into HeLa cells of NPs exposing either a negative or positive charge on their surface. The exposed charge significantly affected their ability to internalize as well as the cellular endocytosis mechanism utilized.

EFA6 regulates endosomal trafficking and affects early endosomes in polarized MDCK cells.

The small-GTPase family of ADP ribosylation factors (ARFs) recruit coat proteins to promote vesicle budding. ARFs are activated by an association with sec7-containing exchange factors which load them with GTP. In epithelial cells, the small GTPase ARF6 operates within the endocytic system and has been shown to associate with ARNO to promote apical endocytosis and early to late endosomal trafficking.

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis.

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein.

ARNO through its coiled-coil domain regulates endocytosis at the apical surface of polarized epithelial cells.

ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis.

Actin is required for endocytosis at the apical surface of Madin-Darby canine kidney cells where ARF6 and clathrin regulate the actin cytoskeleton.

In epithelial cell lines, apical but not basolateral clathrin-mediated endocytosis has been shown to be affected by actin-disrupting drugs. Using electron and fluorescence microscopy, as well as biochemical assays, we show that the amount of actin dedicated to endocytosis is limiting at the apical surface of epithelia. In part, this contributes to the low basal rate of clathrin-dependent endocytosis observed at this epithelial surface.

pH-specific sequestration of phosphoglucose isomerase/autocrine motility factor by fibronectin and heparan sulphate.

Phosphoglucose isomerase (PGI) is a glycolytic enzyme that moonlights as a cytokine under the aliases autocrine motility factor (AMF), neuroleukin and maturation factor. The cytokine function of PGI/AMF targets multiple cell types however mechanisms that regulate and sequester this ubiquitous, circulating cytokine remain largely unidentified. PGI/AMF is shown here to exhibit fibronectin (FN)-dependent cell surface association at both neutral and acid pH.

The activity of antiepileptic drugs as histone deacetylase inhibitors.

Purpose: Valproic acid (VPA), one of the widely used antiepileptic drugs (AEDs), was recently found to inhibit histone deacetylases (HDACs). HDAC inhibitors of a wide range of structures, such as hydroxamic acids, carboxylic acids, and cyclic tetrapeptides, have various effects on transformed and nontransformed cells, including neuromodulation and neuroprotection. The aim of this study was to assess comparatively the activity of traditional and newer AEDs as HDAC inhibitors.

The apical compartment: trafficking pathways, regulators and scaffolding proteins.

Defects in the trafficking of apical membrane proteins in polarized epithelial cells are often associated with diseases, including cystic fibrosis, Liddle's syndrome, nephrogenic diabetes insipidus and Dubin-Johnson syndrome. In recent years, we have learned much about the specialized apical trafficking pathways in polarized cells. Many laboratories have identified signals that direct proteins within these pathways and have defined protein interactions that mediate specific steps in the sorting and stabilization of these proteins.

Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.

Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae.