Genome-Guided Discovery of Antifungal Filipins from a Deep-Sea-Derived Streptomyces antibioticus.

Nine new (1-3, 5-8, 11, and 12; named filipins VI-XIV) and three known (4, 9, and 10) filipin-type polyene macrolides were isolated from the deep-sea-derived Streptomyces antibioticus OUCT16-23 using a genome-guided strategy coupled with bioassay. Their structures were elucidated based on the extensive MS and NMR spectroscopic analyses together with ECD calculations. In an antifungal assay, compounds 4, 5, and 7-10 showed different degrees of growth inhibition against Candida albicans with minimum inhibitory concentrations (MICs) of 1.56-12.5 µg/mL, by which the alkyl side-chain substitution affecting the activity was preliminarily studied. A biosynthetic pathway to 1-12 in S. antibioticus OUCT16-23 is also proposed.

High-content screen for modifiers of Niemann-Pick type C disease in patient cells.

Niemann-Pick type C (NPC) disease is a rare lysosomal storage disease caused primarily by mutations in NPC1. NPC1 encodes the lysosomal cholesterol transport protein NPC1. The most common NPC1 mutation is a missense mutation (NPC1I1061T) that causes misfolding and rapid degradation of mutant protein in the endoplasmic reticulum. Cholesterol accumulates in enlarged lysosomes as a result of decreased levels of lysosomal NPC1I1061T protein in patient cells. There is currently no cure or FDA-approved treatment for patients. We sought to identify novel compounds that decrease lysosomal cholesterol storage in NPC1I1061T/I1061T patient fibroblasts using a high-content screen with the cholesterol dye, filipin and the lysosomal marker, LAMP1. A total of 3532 compounds were screened, including 2013 FDA-approved drugs, 327 kinase inhibitors and 760 serum metabolites. Twenty-three hits were identified that decreased both filipin and LAMP1 signals. The majority of hits (16/21) were histone deacetylase (HDAC) inhibitors, a previously described class of modifiers of NPC cholesterol storage. Of the remaining hits, the antimicrobial compound, alexidine dihydrochloride had the most potent lysosomal cholesterol-reducing activity. Subsequent analyses showed that alexidine specifically increased levels of NPC1 transcript and mature protein in both control and NPC patient cells. Although unsuitable for systemic therapy, alexidine represents a unique tool compound for further NPC studies and as a potent inducer of NPC1. Together, these findings confirm the utility of high-content image-based compound screens of NPC1 patient cells and support extending the approach into larger compound collections.

The Streptomyces filipinensis Gamma-Butyrolactone System Reveals Novel Clues for Understanding the Control of Secondary Metabolism.

Streptomyces γ-butyrolactones (GBLs) are quorum sensing communication signals triggering antibiotic production. The GBL system of Streptomyces filipinensis, the producer of the antifungal agent filipin, has been investigated. Inactivation of sfbR (for S. filipinensis γ-butyrolactone receptor), a GBL receptor, resulted in a strong decrease in production of filipin, and deletion of sfbR2, a pseudo-receptor, boosted it, in agreement with lower and higher levels of transcription of filipin biosynthetic genes, respectively. It is noteworthy that none of the mutations affected growth or morphological development. While no ARE (autoregulatory element)-like sequences were found in the promoters of filipin genes, suggesting indirect control of production, five ARE sequences were found in five genes of the GBL cluster, whose transcription has been shown to be controlled by both S. filipinensis SfbR and SfbR2. In vitro binding of recombinant SfbR and SfbR2 to such sequences indicated that such control is direct. Transcription start points were identified by 5' rapid amplification of cDNA ends, and precise binding regions were investigated by the use of DNase I protection studies. Binding of both regulators took place in the promoter of target genes and at the same sites. Information content analysis of protected sequences in target promoters yielded an 18-nucleotide consensus ARE sequence. Quantitative transcriptional analyses revealed that both regulators are self-regulated and that each represses the transcription of the other as well as that of the remaining target genes. Unlike other GBL receptor homologues, SfbR activates its own transcription whereas SfbR2 has a canonical autorepression profile. Additionally, SfbR2 was found here to bind the antifungal antimycin A as a way to modulate its DNA-binding activity.IMPORTANCEStreptomyces GBLs are important signaling molecules that trigger antibiotic production in a quorum sensing-dependent manner. We have characterized the GBL system from S. filipinensis, finding that two key players of this system, the GBL receptor and the pseudo-receptor, each counteracts the transcription of the other for the modulation of filipin production and that such control over antifungal production involves an indirect effect on the transcription of filipin biosynthetic genes. Additionally, the two regulators bind the same sites, are self-regulated, and repress the transcription of three other genes of the GBL cluster, including that encoding the GBL synthase. In contrast to all the GBL receptors known, SfbR activates its own synthesis. Moreover, the pseudo-receptor was identified as the receptor of antimycin A, thus extending the range of examples supporting the idea of signaling effects of antibiotics in Streptomyces The intricate regulatory network depicted here should provide important clues for understanding the regulatory mechanism governing secondary metabolism.

Primary cilium alterations and expression changes of Patched1 proteins in niemann-pick type C disease.

Niemann-Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease-causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3-ß-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A), a cholesterol transport-inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A-treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A-treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A-treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.

Influence of membrane cholesterol in the molecular evolution and functional regulation of TRPV4.

TRPV4 is involved in several physiological and sensory functions as well as with several diseases and genetic disorders, though the molecular mechanisms for these are unclear. In this work we have analyzed molecular evolution and structure-function relationship of TRPV4 using sequences from different species. TRPV4 has evolved during early vertebrate origin (450million years). Synteny analysis confirms that TRPV4 has coevolved with two enzymes involved in sterol biosynthesis, namely MVK and GLTP. Cholesterol-recognizing motifs are present within highly conserved TM4-Loop4-TM5 region of TRPV4. TRPV4 is present in lipid raft where it co-localizes with Caveolin1 and Filipin. TM4-Loop4-TM5 region as well as Loop4 alone can physically interact with cholesterol, its precursor mevalonate and derivatives such as stigmasterol and aldosterone. Mobility of TRPV4-GFP depends on membrane cholesterol level. Molecular evolution of TRPV4 shared striking parallelism with the cholesterol bio-synthesis pathways at the genetic, molecular and metabolic levels. We conclude that interaction with sterols and cholesterol-dependent membrane dynamics have influence on TRPV4 function. These results may have importance on TRPV4-medaited cellular functions and pathophysiology.

Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-ß-cyclodextrin (MßC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MßC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MßC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

Antifungal activity of azole compounds CPA18 and CPA109 against azole-susceptible and -resistant strains of Candida albicans.

OBJECTIVES: In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. METHODS: Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. RESULTS: Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. CONCLUSIONS: Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

Synthesis of 2-hydroxypropyl-ß-cyclodextrin/pluronic-based polyrotaxanes via heterogeneous reaction as potential Niemann-Pick type C therapeutics.

Five polyrotaxanes were synthesized by threading 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) onto a variety of α,ω-ditriethylenediamino-N-carbamoyl-poly-(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronic) triblock copolymers using a two-pot strategy under heterogeneous, nonaqueous conditions. The threaded HP-ß-CD units were retained on the pseudopolyrotaxane precursors by end-capping the branched diamine termini with sodium 2,4,6-trinitrobenzene sulfonate. Inclusion of the Pluronic copolymers within the HP-ß-CD cavities was more favorable in nonpolar solvents, such as diethyl ether and n-hexane, both of which gave better coverage ratios than polar solvents. (1)H NMR and MALDI-TOF were used to estimate the average molecular weights of the purified polyrotaxane products. A globular morphology of aggregated polyrotaxanes was observed by tapping-mode AFM imaging of dried samples. Treatment of Niemann-Pick C (NPC) type 2-deficient fibroblasts with the polyrotaxane derivatives produced substantial reductions in sterol accumulation, as seen by diminished filipin staining in these cells, suggesting that Pluronic-based polyrotaxanes may be promising vehicles for delivery of HP-ß-CD to cells with abnormal cholesterol accumulation.

Erythrocyte Plasma Membrane Lipid Composition Mirrors That of Neurons and Glial Cells in Murine Experimental In Vitro and In Vivo Inflammation.

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly.

Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder.

Niemann-Pick type C disease is a fatal lysosomal storage disorder caused by loss of NPC1 function. The disorder severely affects multiple body systems, particularly the nervous system. To test whether rescue of NPC1 activity in neurons, astrocytes, or other cell types can correct the neurological defects, a Tet-inducible Npc1-YFP transgene was introduced into Npc1(-/-) mice for the cell type-specific rescue of NPC1 loss. NPC1-YFP produced in neurons prevented neuron degeneration, slowed reactive glial activity, and ameliorated the disease. NPC1-YFP produced in astrocytes or in cells of visceral tissue did not. These results suggest that loss of NPC1 activity from neurons is the primary cause of the neuropathology and that rescue of NPC1 function in neurons is sufficient to mitigate the disease. The ability of neurons to survive and function in a cell-autonomous fashion allowed the use of this newly engineered rescue system to further define the brain regions or neuron populations required to ameliorate a neurological symptom. NPC1-YFP produced specifically in cerebellar Purkinje neurons reduced ataxia, increased weight, and prolonged life, but it did not prevent the eventual decline and premature death of Npc1(-/-) mice. Significant increase in lifespan correlated with sustained reduction of inflammation in the thalamus. Neuron rescue of other forebrain areas provided little benefit. Future work targeting increasingly discrete neuronal networks should reveal which CNS areas are critical for survival. This work may have broad implications for understanding the anatomical and cellular basis of neurological signs and symptoms of other neurodegenerative and lysosomal disorders.

Brain region-specific immunolocalization of the lipolysis-stimulated lipoprotein receptor (LSR) and altered cholesterol distribution in aged LSR+/- mice.

Brain lipid homeostasis is important for maintenance of brain cell function and synaptic communications, and is intimately linked to age-related cognitive decline. Because of the blood-brain barrier's limiting nature, this tissue relies on a complex system for the synthesis and receptor-mediated uptake of lipids between the different networks of neurons and glial cells. Using immunofluorescence, we describe the region-specific expression of the lipolysis-stimulated lipoprotein receptor (LSR), in the mouse hippocampus, cerebellum Purkinje cells, the ependymal cell interface between brain parenchyma and cerebrospinal fluid, and the choroid plexus. Colocalization with cell-specific markers revealed that LSR was expressed in neurons, but not astrocytes. Latency in arms of the Y-maze exhibited by young heterozygote LSR(+/-) mice was significantly different as compared to control LSR(+/+), and increased in older LSR(+/-) mice. Filipin and Nile red staining revealed membrane cholesterol content accumulation accompanied by significantly altered distribution of LSR in the membrane, and decreased intracellular lipid droplets in the cerebellum and hippocampus of old LSR(+/-) mice, as compared to control littermates as well as young LSR(+/-) animals. These data therefore suggest a potential role of LSR in brain cholesterol distribution, which is particularly important in preserving neuronal integrity and thereby cognitive functions during aging.

Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain.

Filipin is an antibiotic polyene widely used as a histochemical marker for cholesterol. We previously reported cholesterol/filipin-positive staining in brain of ß-galactosidase (ß-gal) knockout ((-/-)) mice (GM1 gangliosidosis). The content and distribution of cholesterol and gangliosides was analyzed in plasma membrane (PM) and microsomal (MS) fractions from whole-brain tissue of 15 week-old control (ß-gal(+/-)) and GM1 gangliosidosis (ß-gal(-/-)) mice. Total ganglioside content (µg sialic acid/mg protein) was 3-fold and 7-fold greater in the PM and MS fractions, respectively, in ßgal(-/-) mice than in ßgal(+/-) mice. GM1 content was 30-fold and 50-fold greater in the PM and MS fractions, respectively. In contrast, unesterified cholesterol content (µg/mg protein) was similar in the PM and the MS fractions of the ßgal(-/-) and ßgal(+/-) mice. Filipin is known to bind to various sterol derivatives and phospholipids on thin-layer chromatograms. Biochemical evidence is presented showing that filipin also binds to GM1 with an affinity similar to that for cholesterol, with a corresponding fluorescent reaction. Our data suggest that the GM1 storage seen in the ß-gal(-/-) mouse contributes to the filipin ultraviolet fluorescence observed in GM1 gangliosidosis brain. The data indicate that in addition to cholesterol, filipin can also be useful for detecting GM1.

Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution.

An automated fluorescence microscopy assay using a nontoxic cholesterol binding protein, toxin domain 4, (D4), was developed in order to identify chemical compounds modifying intracellular cholesterol metabolism and distribution. Using this method, we screened a library of 1,056 compounds and identified 35 compounds that decreased D4 binding to the cell surface. Among them, 8 compounds were already reported to alter the biosynthesis or the intracellular distribution of cholesterol. The remaining 27 hit compounds were further analyzed biochemically and histochemically. Cell staining with another fluorescent cholesterol probe, filipin, revealed that 17 compounds accumulated cholesterol in the late endosomes. Five compounds decreased cholesterol biosynthesis, and two compounds inhibited the binding of D4 to the membrane. This visual screening method, based on the cholesterol-specific probe D4 in combination with biochemical analyses, is a cell-based, sensitive technique for identifying new chemical compounds and modifying cholesterol distribution and metabolism. Furthermore, it is suitable for high-throughput analysis for drug discovery.

T cell signal regulation by the actin cytoskeleton.

T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase Lck. Conversely, the membrane phosphatase CD45, which activates Src family kinases, is excluded, and this is necessary to avoid quenching of T cell receptor phosphosignals. Data suggest that this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B, which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following the addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells.

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.

Isolation of Lipid Rafts (Detergent-Resistant Microdomains) and Comparison to Extracellular Vesicles (Exosomes).

Lipid rafts (LRs) represent cellular microdomains enriched in sphingolipids and cholesterol which may fuse to form platforms in which signaling molecules can be organized and regulated (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62-75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 190:1549-1550, 1999) the LR has a well-ordered central core composed mainly of cholesterol and sphingolipids that is surrounded by a zone of decreasing lipid order. Detergents such as Triton X-100 can solubilize the core (and a significant amount of phosphoglyceride), but the LRs will be insoluble at 4 °C and be enriched in a well-characterized set of biomarkers. Model 2 proposes that the LRs are homogeneous, but there is selectivity in the lipids (and proteins) extracted by the 1% Triton X-100. Model 3 proposes LRs with distinct lipid compositions are highly structured and can be destroyed by binding molecules such as beta-methylcyclodextrin or filipin. These may be Caveolin in some cell types but not in brain. Since it is unlikely that two LR preparations will be exactly the same this review will concentrate on LRs defined as "small (50 nm) membranous particles which are insoluble in 1% Triton X-100 at 4 °C and have a low buoyant density (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1):62-75, 2003; Testai et al., J Neurochem 89:636-644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1-6].

Lipid microdomain polarization is required for NADPH oxidase-dependent ROS signaling in Picea meyeri pollen tube tip growth.

The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca(2+) gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.

Dynamin- and lipid raft-dependent entry of decay-accelerating factor (DAF)-binding and non-DAF-binding coxsackieviruses into nonpolarized cells.

Group B coxsackieviruses (CVB) use the CVB and adenovirus receptor (CAR) to enter and infect cells. Some CVB also bind to decay-accelerating factor (DAF), but that interaction alone is insufficient for infection. We previously found that CVB3 entry into polarized human intestinal cells (Caco-2) occurs by a caveolin-dependent but dynamin-independent mechanism that requires DAF-mediated tyrosine kinase signals. In this study, we examined how CVB enter and infect nonpolarized HeLa cells and how DAF binding affects these processes. Using immunofluorescence microscopy and a combination of dominant-negative proteins, small interfering RNAs, and drugs targeting specific endocytic pathways, we found that both DAF-binding and non-DAF-binding virus isolates require dynamin and lipid rafts to enter and infect cells. Unlike what we observed in Caco-2 cells, CVB3 entered HeLa cells with CAR. We found no role for clathrin, endosomal acidification, or caveolin. Inhibition of tyrosine kinases blocked an early event in infection but did not prevent entry of virus into the cell. These results indicate that CVB3 entry into nonpolarized HeLa cells differs significantly from entry into polarized Caco-2 cells and is not influenced by virus binding to DAF.

Post-ictal psychosis in adolescent Niemann-Pick disease type C.

We describe the presentation of an adolescent with juvenile-onset Niemann-Pick disease type C (NPC) who presented with post-ictal psychosis in the context of a developing seizure disorder. After demonstrating mild gait disturbance beginning at the age of 4 years, he was diagnosed with NPC at age 12 on the basis of 95% of cultured fibroblasts staining positive for filipin and a reduced fibroblast cholesterol esterification rate. He then developed a seizure disorder at age 15, where clusters of seizures produced typical psychotic symptoms, including hallucinations and delusions. His seizure disorder responded to valproate, which resulted in a settling of his psychotic symptoms. Whilst post-ictal psychosis is rarely reported prior to the age of 16, NPC in adolescents and adults is particularly psychotogenic and may increase the risk for post-ictal psychosis in the pediatric population.

n-3 and n-6 Polyunsaturated fatty acids suppress sterol regulatory element binding protein activity and increase flow of non-esterified cholesterol in HepG2 cells.

The plasma lipid-lowering effect of PUFA, one of their main beneficial effects, is considered to be related to the regulation of lipid biosynthesis through transcription factors including sterol regulatory element binding proteins (SREBP). In the present study, we compared the effect of different PUFA on SREBP activity in HepG2 cells, using a sterol regulatory element-luciferase reporter construct as a probe. Supplementation with different fatty acids reduced SREBP activity in the order 20 : 5n-3 = 18 : 2n-6 = 20 : 4n-6 " 18 : 3n-3 = 22 : 6n-3 = 22 : 5n-6 " 18 : 1n-9. The suppression of SREBP activity greatly depended on the degree of incorporation of the supplemented PUFA into cellular lipids, and correlated positively with the unsaturation index (r 0.831; P < 0.01) of total cell lipids. Supplemented PUFA were also metabolised to longer and more unsaturated species. These processing activities were higher for n-3 than n-6 PUFA (P < 0.01). We studied the effect of PUFA on the intracellular distribution of non-esterified cholesterol, using filipin staining and fluorescence microscopy with or without the cholesterol traffic blocker U18666A. The data show that the incorporation of PUFA increases non-esterified cholesterol flow from the plasma membrane to intracellular membranes. We conclude that suppression of SREBP activity by PUFA depends on the degree of incorporation into cellular lipids, and is associated with increased flow of non-esterified cholesterol between the plasma membrane and intracellular membranes.