Development of primary reference facilities and measurement comparison of standard artifacts for the bidirectional transmittance distribution function.

To determine the bidirectional transmittance distribution function (BTDF) of diffusely transmitting materials, two new primary facilities have been developed at the Physikalisch-Technische Bundesanstalt (PTB) and Aalto University (Aalto). A detailed description of both facilities and the different approaches used are presented in this paper. The performance of both facilities is compared by determining the BTDF of two different diffuser types in both in-plane and out-of-plane bidirectional geometries at four different wavelengths in the visible spectral range. Due to delayed completion of PTB's primary facility, the measured BTDF values are compared between Aalto's primary facility and another PTB setup, whose measurement scales are successfully transferred to PTB's primary facility by an internal comparison. A thorough analysis of the measurement uncertainty is presented, leading to a combined k = 1 standard uncertainty of 0.8%-1.2% for PTB's primary facility and 1.3%-1.7% for Aalto's primary facility. The BTDF results obtained agree well within their expanded k = 2 uncertainty. This indirect bilateral comparison shows that Aalto's and PTB's new facilities are suited as primary reference setups for the determination of the BTDF. These studies also reveal action points to improved measurement capabilities and for a reduction of the measurement uncertainty, depending on the type of diffuser under test.

Amiodarone disrupts cholesterol biosynthesis pathway and causes accumulation of circulating desmosterol by inhibiting 24-dehydrocholesterol reductase.

BACKGROUND: We have earlier reported that amiodarone, a potent and commonly used antiarrhythmic drug increases serum desmosterol, the last precursor of cholesterol, in 20 cardiac patients by an unknown mechanism. OBJECTIVE: Here, we extended our study to a large number of cardiac patients of heterogeneous diagnoses, evaluated the effects of combining amiodarone and statins (inhibitors of cholesterol synthesis at the rate-limiting step of hydroxy-methyl-glutaryl CoA reductase) on desmosterol levels and investigated the mechanism(s) by which amiodarone interferes with the metabolism of desmosterol using in vitro studies. METHODS AND RESULTS: We report in a clinical case-control setting of 236 cardiac patients (126 with and 110 without amiodarone treatment) that amiodarone medication is accompanied by a robust increase in serum desmosterol levels independently of gender, age, body mass index, cardiac and other diseases, and the use of statins. Lipid analyses in patient samples taken before and after initiation of amiodarone therapy showed a systematic increase of desmosterol upon drug administration, strongly arguing for a direct causal link between amiodarone and desmosterol accumulation. Mechanistically, we found that amiodarone resulted in desmosterol accumulation in cultured human cells and that the compound directly inhibited the 24-dehydrocholesterol reductase (DHCR24) enzyme activity. CONCLUSION: These novel findings demonstrate that amiodarone blocks the cholesterol synthesis pathway by inhibiting DHCR24, causing a robust accumulation of cellular desmosterol in cells and in the sera of amiodarone-treated patients. It is conceivable that the antiarrhythmic potential and side effects of amiodarone may in part result from inhibition of the cholesterol synthesis pathway.

Quantum interference of tunably indistinguishable photons from remote organic molecules.

We demonstrate two-photon interference using two remote single molecules as bright solid-state sources of indistinguishable photons. By varying the transition frequency and spectral width of one molecule, we tune and explore the effect of photon distinguishability. We discuss future improvements on the brightness of single-photon beams, their integration by large numbers on chips, and the extension of our experimental scheme to coupling and entanglement of distant molecules.

Respiratory disease in Niemann-Pick type C2 is caused by pulmonary alveolar proteinosis.

Niemann-Pick diseases are hereditary neurovisceral lysosomal lipid storage disorders, of which the rare type C2 almost uniformly presents with respiratory distress in early infancy. In the patient presented here, the NPC2 exon 4 frameshift mutation c.408_409delAA caused reduced NPC2 protein levels in serum and lung lavage fluid and the synthesis of an aberrant, larger sized protein of around 28 kDa. Protein expression was strongly reduced also in alveolar macrophages. The infant developed failure to thrive and tachypnea. Lung lavage, computer tomography, and histology showed typical signs of pulmonary alveolar proteinosis with an abnormal intraalveolar accumulation of surfactant as well as macrophages. An NPC2-hypomorph animal model also showed pulmonary alveolar proteinosis and accumulation of macrophages in the lung, liver, and spleen long before the mice died. Due to the elevation of cholesterol, the surfactant had an abnormal composition and function. Despite the removal of large amounts of surfactant from the lungs by therapeutic lung lavages, this treatment was only temporarily successful and the infant died of respiratory failure. Our data indicate that respiratory distress in NPC2 disease is associated with a loss of normal NPC2 protein expression in alveolar macrophages and the accumulation of functionally inactive surfactant rich in cholesterol.

Realization of two Fourier-limited solid-state single-photon sources.

We demonstrate two solid-state sources of indistinguishable single photons. High resolution laser spectroscopy and optical microscopy were combined at T = 1.4 K to identify individual molecules in two independent microscopes. The Stark effect was exploited to shift the transition frequency of a given molecule and thus obtain single photon sources with perfect spectral overlap. Our experimental arrangement sets the ground for the realization of various quantum interference and information processing experiments.

Endosomal cholesterol traffic: vesicular and non-vesicular mechanisms meet.

The endoplasmic reticulum is traditionally perceived as the key compartment for regulating intracellular cholesterol metabolism. Increasing evidence suggests that the endocytic pathway provides an additional regulatory level governing intracellular cholesterol trafficking and homoeostasis. Sterols can enter, and apparently also exit, endosomal compartments via both vesicular and non-vesicular mechanisms. A number of studies have focused on endosomal sterol removal as its defects lead to cholesterol storage diseases. So far, the bulk of evidence on endosomal sterol egress describes the involvement of membrane trafficking machineries. Interestingly, two late endosomal sterol-binding proteins were recently shown to regulate the movement of late endosomes along cytoskeletal tracks. These studies provide the first indications of how non-vesicular and vesicular mechanisms may co-operate in endosomal sterol trafficking.

Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages.

Liver X receptors (LXRs) are nuclear receptors that regulate macrophage cholesterol efflux by inducing ATP-binding cassette transporter A1 (ABCA1) and ABCG1/ABCG4 gene expression. The Niemann-Pick C (NPC) proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant governing its availability for efflux to extracellular acceptors. Here we investigated the influence of LXR activation on intracellular cholesterol trafficking in primary human macrophages. Synthetic LXR activators increase the amount of free cholesterol in the plasma membrane by inducing NPC1 and NPC2 gene expression. Moreover, ABCA1-dependent cholesterol efflux induced by LXR activators was drastically decreased in the presence of progesterone, which blocks postlysosomal cholesterol trafficking, and reduced when NPC1 and NPC2 mRNA expression was depleted using small interfering RNA. The stimulation of cholesterol mobilization to the plasma membrane by LXRs led to a decrease in cholesteryl ester formation and Acyl-coenzyme A cholesterol acyltransferase-1 activity. These data indicate that LXR activation enhances cholesterol trafficking to the plasma membrane, where it becomes available for efflux, at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.

Peroxisome proliferator-activated receptor alpha controls cellular cholesterol trafficking in macrophages.

The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant that governs its availability for efflux to extracellular acceptors. NPC1 and NPC2 are proteins localized in the late endosome and control cholesterol transport from the lysosome to the plasma membrane. Here, we report that NPC1 and NPC2 gene expression is induced by oxidized LDL (OxLDL) in human macrophages. Because OxLDLs contain natural activators of peroxisome proliferator-activated receptor alpha (PPARalpha), a fatty acid-activated nuclear receptor, the regulation of NPC1 and NPC2 by PPARalpha and the consequences on cholesterol trafficking were further studied. NPC1 and NPC2 expression is induced by synthetic PPARalpha ligands in human macrophages. Furthermore, PPARalpha activation leads to an enrichment of cholesterol in the plasma membrane. By contrast, incubation with progesterone, which blocks postlysosomal cholesterol trafficking, as well as NPC1 and NPC2 mRNA depletion using small interfering RNA, abolished ABCA1-dependent cholesterol efflux induced by PPARalpha activators. These observations identify a novel regulatory role for PPARalpha in the control of cholesterol availability for efflux that, associated with its ability to inhibit cholesterol esterification and to stimulate ABCA1 and scavenger receptor class B type I expression, may contribute to the stimulation of reverse cholesterol transport.

Caveolins and membrane cholesterol.

Caveolae (small plasma membrane invaginations) and their coat proteins, caveolins, have attracted the attention of researchers in diverse fields, including cell biology, cardiovascular and cancer research. The tight association between caveolin and cholesterol governs the biochemical behaviour of caveolae and is emerging as an important characteristic in a number of processes assigned to these multifunctional organelles. In this review, selected aspects of the caveolin-cholesterol association and its potential functional implications are discussed.

Mass spectrometric analysis reveals an increase in plasma membrane polyunsaturated phospholipid species upon cellular cholesterol loading.

Here we used electrospray ionization mass spectrometry for quantitative determination of lipid molecular species in human fibroblasts and their plasma membrane incorporated into enveloped viruses. Both influenza virus selecting ordered domains and vesicular stomatitis virus (VSV) depleted of such domains [Scheiffele, P., et al. (1999) J. Biol. Chem. 274, 2038-2044] were analyzed. The major difference between influenza and VSV was found to be a marked enrichment of glycosphingolipids in the former. The effect of chronic cholesterol loading on viral lipid composition was studied in Niemann-Pick type C (NPC) fibroblasts. Both NPC-derived influenza and VSV virions contained increased amounts of cholesterol. Furthermore, polyunsaturated phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were enriched in NPC-derived virions at the expense of the monounsaturated ones. When normal fibroblasts were acutely loaded with cholesterol using cyclodextrin complexes, an adjustment toward increasingly unsaturated phospholipid species was observed, most clearly for phosphatidylcholine and sphingomyelin. Our results provide evidence that (1) glycosphingolipids are enriched in domains through which influenza virus buds, (2) chronic cholesterol accumulation increases the cholesterol content of both glycosphingolipid-enriched and intervening plasma membrane domains, and (3) an increase in membrane cholesterol content is accompanied by an increased level of polyunsaturated species of the major membrane phospholipids. We suggest that remodeling of phospholipids toward higher unsaturation may serve as both an acute and a long-term adaptive mechanism in human cellular membranes against cholesterol excess.

The OSBP-related protein family in humans.

Oxysterols are oxygenated derivatives of cholesterol that have a number of biological effects and play a key role in the maintenance of the body cholesterol balance. In this study, we describe the cDNA sequences and genomic structures of the recently identified human oxysterol-binding protein (OSBP)-related protein (ORP) family (Laitinen, S. et al. 1999. J. Lipid Res. 40: 2204-2211). The family now includes 12 genes/proteins, which can be divided into six distinct subfamilies. The ORP have two major structural features: a highly conserved OSBP-type sterol-binding domain in the C-terminal half and a pleckstrin homology domain present in the N-terminal region of most family members. Several ORP genes are present in S. cerevisiae, D. melanogaster, and C. elegans, suggesting that the protein family has functions of fundamental importance in the eukaryotic kingdom. Analysis of ORP mRNA levels in unloaded or acetylated LDL-loaded human macrophages revealed that the expression of ORP genes was not significantly affected by the loading, with the exception of ORP6, which was up-regulated 2-fold. The present study summarizes the basic characteristics of the OSBP-related gene/protein family in humans, and provides tools for functional analysis of the encoded proteins.

Roles of lipid rafts in membrane transport.

Cholesterol-sphingolipid microdomains (lipid rafts) are part of the machinery ensuring correct intracellular trafficking of proteins and lipids. The most apparent roles of rafts are in sorting and vesicle formation, although their roles in vesicle movement and cytoskeletal connections as well as in vesicle docking and fusion are coming into focus. New evidence suggests that compositionally distinct lipid microdomains are assembled and may coexist within a given membrane. Important clues have also been uncovered about the mechanisms coupling raft-dependent signaling and endocytic uptake.

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance.

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV), specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

How cells handle cholesterol.

Cholesterol plays an indispensable role in regulating the properties of cell membranes in mammalian cells. Recent advances suggest that cholesterol exerts many of its actions mainly by maintaining sphingolipid rafts in a functional state. How rafts contribute to cholesterol metabolism and transport in the cell is still an open issue. It has long been known that cellular cholesterol levels are precisely controlled by biosynthesis, efflux from cells, and influx of lipoprotein cholesterol into cells. The regulation of cholesterol homeostasis is now receiving a new focus, and this changed perspective may throw light on diseases caused by cholesterol excess, the prime example being atherosclerosis.

Dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface.

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15 degrees C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.

Mobilization of late-endosomal cholesterol is inhibited by Rab guanine nucleotide dissociation inhibitor.

Cholesterol entering cells in low-density lipoproteins (LDL) via receptor-mediated endocytosis is transported to organelles of the late endocytic pathway for degradation of the lipoprotein particles. The fate of the free cholesterol released remains poorly understood, however. Recent observations suggest that late-endosomal cholesterol sequestration is regulated by the dynamics of lysobisphosphatidic acid (LBPA)-rich membranes [1]. Genetic studies have pinpointed a protein, Niemann-Pick C-1 (NPC-1), that is required for the mobilization of late-endosomal/lysosomal cholesterol by an unknown mechanism [2]. Here, we report the removal of accumulated cholesterol by overexpression of the NPC-1 protein in NPC-1-deficient fibroblasts from patients with Niemann-Pick disease, and in normal fibroblasts upon release of a progesterone-induced block of cholesterol transport. We show that late-endosomal/lysosomal cholesterol mobilization is specifically inhibited by microinjection of Rab GDP-dissociation inhibitor (Rab-GDI). Moreover, clearance of the cholesterol deposits by NPC-1 in patients' fibroblasts is accompanied by the redistribution of LBPA and of a lysosomal hydrolase that utilizes the mannose-6-phosphate receptor. Our results reveal, for the first time, the involvement of a specific molecular component of the membrane-trafficking machinery in cholesterol transport and the coupling of late-endosomal cholesterol egress to the trafficking of other lipid and protein cargo.

Caveolins and cellular cholesterol balance.

Caveolins are major integral membrane components of caveolae. Over the last few years, evidence has accumulated for a close link between caveolin, caveolae, and the regulation of cellular cholesterol levels. However, the exact role of caveolin in this process, the intracellular trafficking routes followed by caveolin/cholesterol complexes, and the relationship of caveolin-cholesterol to other caveolin-mediated processes such as signal transduction have remained unclear. Recent findings from a number of systems suggest that specific signaling pathways require precise regulation of cellular cholesterol. Here we review evidence for caveolin regulation of cholesterol transport and consider how this may relate to signal transduction.