Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic EventsV⃞

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.

Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice.

We have studied the effects of endogenous and exogenous estrogen on atherosclerotic lesions in apolipoprotein E-deficient mice. Female mice ovariectomized (OVX) at weaning displayed increases (P < 0.01) in fatty streak lesions in the proximal aorta and aortic sinus compared with female mice with intact ovarian function. These differences between the OVX and sham controls were apparent in both chow- and "Western-type" diet-fed mice. Moreover, increases in lesion size following OVX occurred without changes in plasma cholesterol. Hormone replacement with subdermal 17-beta-estradiol pellets releasing either 6, 14, or 28 micrograms/day significantly decreased (P < 0.001) atherosclerotic lesion area in both male and OVX female mice. In contrast, neither 17-alpha-estradiol (28 micrograms/day) or tamoxifen (85 micrograms/day) affected lesion progression in OVX female mice. In the Western diet-fed group, exogenous estradiol markedly reduced plasma cholesterol and triglycerides, whereas, in animals fed the chow diet, exogenous estrogen and tamoxifen treatment only decreased plasma and very low density lipoprotein triglycerides. However, lesion area was only weakly correlated with plasma cholesterol and triglycerides, 0.35 and 0.44 tau values, respectively (P < 0.01). In summary, in the apolipoprotein E-deficient mouse 17-beta-estradiol protects against atherosclerotic lesion formation, and this can only be partially explained through effects on plasma lipoprotein levels.

Defective forebrain development in mice lacking gp330/megalin.

gp330/megalin, a member of the low density lipoprotein (LDL) receptor gene family, is expressed on the apical surfaces of epithelial tissues, including the neuroepithelium, where it mediates the endocytic uptake of diverse macromolecules, such as cholesterol-carrying lipoproteins, proteases, and antiproteinases. Megalin knockout mice manifest abnormalities in epithelial tissues including lung and kidney that normally express the protein and they die perinatally from respiratory insufficiency. In brain, impaired proliferation of neuroepithelium produces a holoprosencephalic syndrome, characterized by lack of olfactory bulbs, forebrain fusion, and a common ventricular system. Similar syndromes in humans and animals are caused by insufficient supply of cholesterol during development. Because megalin can bind lipoproteins, we propose that the receptor is part of the maternal-fetal lipoprotein transport system and mediates the endocytic uptake of essential nutrients in the postgastrulation stage.

Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.

Apolipoprotein (apo)-B is found in two forms in mammals: apo-B100, which is made in the liver and the yolk sac, and apo-B48, a truncated protein made in the intestine. To provide models for understanding the physiologic purpose for the two forms of apo-B, we used targeted mutagenesis of the apo-B gene to generate mice that synthesize exclusively apo-B48 (apo-B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100-only mice). Both the apo-B48-only mice and apo-B100-only mice developed normally, were healthy, and were fertile. Thus, apo-B48 synthesis was sufficient for normal embryonic development, and the synthesis of apo-B100 in the intestines of adult mice caused no readily apparent adverse effects on intestinal function or nutrition. Compared with wild-type mice fed a chow diet, the levels of low density lipoprotein (LDL)-cholesterol and very low density lipoprotein- and LDL-triacylglycerols were lower in apo-B48-only mice and higher in the apo-B100-only mice. In the setting of apo-E-deficiency, the apo-B100-only mutation lowered cholesterol levels, consistent with the fact that apo-B100-lipoproteins can be cleared from the plasma via the LDL receptor, whereas apo-B48-lipoproteins lacking apo-E cannot. The apo-B48-only and apo-B100-only mice should prove to be valuable models for experiments designed to understand the purpose for the two forms of apo-B in mammalian metabolism.

Purification and cDNA cloning of a second apoptosis-related cysteine protease that cleaves and activates sterol regulatory element binding proteins.

We have purified from hamster liver a second cysteine protease that cleaves and activates sterol regulatory element binding proteins (SREBPs). cDNA cloning revealed that this enzyme is the hamster equivalent of Mch3, a human enzyme that is related to the interleukin 1beta converting enzyme. We call this enzyme Mch3/SCA-2. It is 54% identical to hamster CPP32/SCA-1, a cysteine protease that was earlier shown to cleave SREBPs at a conserved Asp between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. This cleavage liberates an NH2-terminal fragment of approximately 460 amino acids that activates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. Mch3/SCA-2 and CPP32/SCA-I are synthesized as inactive 30-35 kDa precursors that are thought to be cleaved during apoptosis to generate active fragments of approximately 20 and approximately 10 kDa. The current data lend further support to the notion that SREBPs are cleaved and activated as part of the program in programmed cell death.

Myofibroblasts differentiate from fibroblasts when plated at low density.

Myofibroblasts, defined by their expression of smooth muscle alpha-actin, appear at corneal and dermal incisions and promote wound contraction. We report here that cultured fibroblasts differentiate into myofibroblasts by a cell density-dependent mechanism. Fibroblasts seeded at low density (5 cells per mm2) produced a cell culture population consisting of 70-80% myofibroblasts, 5-7 days after seeding. In contrast, fibroblasts seeded at high density (500 cells per mm2) produced cultures with only 5-10% myofibroblasts. When the myofibroblast-enriched cultures were subsequently passaged at high density, the smooth muscle alpha-actin phenotype was lost within 3 days. Furthermore, initially 60% of the low density-cultured cells incorporated BrdUrd compared to 30% of cells passaged at high density. Media from myofibroblast-enriched cultures had more latent and active transforming growth factor beta (TGF-beta) than did media from fibroblast-enriched cultures. Although there was a trend towards increased numbers of myofibroblasts after addition of exogenous TGF-beta, the results did not reach statistical significance. We conclude that myofibroblast differentiation can be induced in fibroblasts by plating at low density. We propose a cell density-dependent model of myofibroblast differentiation during wounding and healing in which at least two factors interact: loss of cell contact and the presence of TGF-beta.

Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid.

Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position of the SREBP sites relative to those for the ubiquitous auxiliary transcription factor Sp1 is reminiscent of that previously described for the sterol-regulated low density lipoprotein receptor promoter. The experiments provide molecular evidence that the metabolism of fatty acids and cholesterol, two different classes of essential cellular lipids, are coordinately regulated by cellular lipid levels.

Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.

Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.

Refinery Nonconventional Feedstocks: Influence of the Coprocessing of Vacuum Gas Oil and Low Density Polyethylene in Fluid Catalytic Cracking Unit on Full Range Gasoline Composition

Gasoline coming from refinery fluid catalytic cracking (FCC) unit is a major contributor to the total commercial grade gasoline pool. The contents of the FCC gasoline are primarily paraffins, naphthenes, olefins, aromatics, and undesirables such as sulfur and sulfur containing compounds in low quantities. The proportions of these components in the FCC gasoline invariable determine its quality as well as the performance of the associated downstream units. The increasing demand for cleaner and lighter fuels significantly influences the need not only for novel processing technologies but also for alternative refinery and petrochemical feedstocks. Current and future clean gasoline requirements include increased isoparaffins contents, reduced olefin contents, reduced aromatics, reduced benzene, and reduced sulfur contents. The present study is aimed at investigating the effect of processing an unconventional refinery feedstock, composed of blend of vacuum gas oil (VGO) and low density polyethylene (LDPE) on FCC full range gasoline yields and compositional spectrum including its paraffins, isoparaffins, olefins, napthenes, and aromatics contents distribution within a range of operating variables of temperature (500–700 °C) and catalyst-feed oil ratio (CFR 5–10) using spent equilibrium FCC Y-zeolite based catalyst in a FCC pilot plant operated at the University of Alicante’s Research Institute of Chemical Process Engineering (RICPE). The coprocessing of the oil-polymer blend led to the production of gasoline with very similar yields and compositions as those obtained from the base oil, albeit, in some cases, the contribution of the feed polymer content as well as the processing variables on the gasoline compositional spectrum were appreciated. Carbon content analysis showed a higher fraction of the C9–C12 compounds at all catalyst rates employed and for both feedstocks. The gasoline’s paraffinicity, olefinicity, and degrees of branching of the paraffins and olefins were also affected in various degrees by the scale of operating severity. In the majority of the cases, the gasoline aromatics tended toward the decrease as the reactor temperature was increased. While the paraffins and iso-paraffins gasoline contents were relatively stable at around 5 % wt, the olefin contents on the other hand generally increased with increase in the FCC reactor temperature.

Conversion of low density polyethylene into fuel through co-processing with vacuum gas oil in a fluid catalytic cracking riser reactor

In this work, mixtures of vacuum gas oil and low density polyethylene, a major component of common industrial and consumer household plastics, were pyrolytically co-processed in a fluid catalytic cracking (FCC) riser reactor as a viable alternative for the energy and petrochemical revalorisation of plastic wastes into valuable petrochemical feedstocks and fuel within an existing industrial technology. Using equilibrium FCC catalyst, the oil–polymer blends were catalytically cracked at different processing conditions of temperatures between 773 K and 973 K and catalyst feed ratios of 5:1, 7:1 and 10:1. The influence of each of these processing parameters on the cracking gas and liquid yield patterns were studied and presented. Further analysed and presented are the different compositional distributions of the obtained liquids and gaseous products. The analysis of the results obtained revealed that with very little modifications to existing process superstructure, yields and compositional distributions of products from the fluid catalytic cracking of the oil–polymer blend in many cases were very similar to those of the processed oil feedstock, bringing to manifest the viability of the feedstock co-processing without significant detriments to FCC product yields and quality.

Linear low-density polyethylene colored with a nanoclay-based pigment: Morphology and mechanical, thermal, and colorimetric properties

In this study, a novel kind of hybrid pigment based on nanoclays and dyes was synthesized and characterized. These nanoclay-based pigments (NCPs) were prepared at the laboratory with sodium montmorillonite nanoclay (NC) and methylene blue (MB). The cation-exchange capacity of NC exchanged with MB was varied to obtain a wide color gamut. The synthesized nanopigments were thoroughly characterized. The NCPs were melt-mixed with linear low-density polyethylene (PE) with an internal mixer. Furthermore, samples with conventional colorants were prepared in the same way. Then, the properties (mechanical, thermal, and colorimetric) of the mixtures were assessed. The PE–NCP samples developed better color properties than those containing conventional colorants and used as references, and their other properties were maintained or improved, even at lower contents of dye compared to that with the conventional colorants.

Production and Electrical Characterization of Low Density Polyethylene-based Micro- and Nano-dielectrics containing Graphene Oxide, Functionalized Graphene and Carbon Black additives

Oggigiorno la ricerca di nuovi materiali per gradatori di campo da impiegarsi in accessori di cavi ha iniziato a studiare alcuni materiali nano dielettrici con proprietà elettriche non lineari con la tensione ed aventi proprietà migliorate rispetto al materiale base. Per questo motivo in questo elaborato si sono studiati materiali nanostrutturati a base di polietilene a bassa densità (LDPE) contenenti nano polveri di grafene funzionalizzato (G*), ossido di grafene (GO) e carbon black (CB). Il primo obiettivo è stato quello di selezionare e ottimizzare i metodi di fabbricazione dei provini. La procedura di produzione è suddivisa in due parti. Nella prima parte è stata utilizzatala tecnica del ball-milling, mentre nella seconda un pressa termica (thermal pressing). Mediante la spettroscopia dielettrica a banda larga (BDS) si sono misurate le componenti reali e immaginarie della permettività e il modulo della conducibilità del materiale, in tensione alternata. Il miglioramento delle proprietà rispetto al provino di base composto dal solo polietilene si sono ottenute quando il quantitativo delle nanopolveri era maggiore. Le misure sono state effettuate sia a 3 V che a 1 kV. Attraverso misurazioni di termogravimetria (TGA) si è osservato l’aumento della resistenza termica di tutti i provini, soprattutto nel caso quando la % di nanopolveri è maggiore. Per i provini LDPE + 0.3 wt% GO e LDPE + 0.3 wt% G* si è misurata la resistenza alle scariche parziali attraverso la valutazione dell’erosione superficiale dei provini. Per il provino contenente G* è stato registrato una diminuzione del 22% del volume eroso, rispetto al materiale base, mentre per quello contenente GO non vi sono state variazioni significative. Infine si è ricercata la resistenza al breakdown di questi ultimi tre provini sopra citati. Per la caratterizzazione si è fatto uso della distribuzione di Weibull. Lo scale parameter α risulta aumentare solo per il provino LDPE + 0.3 wt% G*.

A theoretical comparison of fixed route bus and flexible route subscription bus feeder service in low density areas. Final report.

Transportation Department, Office of Research and Development Policy, Washington, D.C.

Scale model study of low-density concrete impact attenuators. Final report.

Ohio Department of Transportation, Columbus