Search

Milk fat depression (MFD) is a naturally occurring condition in dairy cows where milk fat synthesis is inhibited by intermediates of ruminal biohydrogenation. One of these bioactive fatty acids (FA), trans-10, cis-12 conjugated linoleic acid (CLA), decreases milk fat synthesis through transcriptional downregulation of genes involved in mammary lipid synthesis. Energy partitioning during MFD is not well characterized because of the complexity of observing energy metabolism in ruminant animals. To investigate energy partitioning during MFD, adipose tissue biopsies were taken from 4 cows arranged in a switchback design. Treatments were control and 4-d abomasal infusion of trans-10, cis-12 CLA (7.5 g/d). CLA decreased milk fat yield by 38 % and milk fat content by 34%, but yields of milk and other milk components were unchanged. In contrast to reported changes in mammary tissue, adipose tissue expression of lipid synthesis enzymes, including lipoprotein lipase, FA synthase, stearoyl-CoA desaturase, and FA binding protein 4, was increased. Expression of regulators of lipid synthesis, including sterol-response element binding protein 1, thyroid hormone responsive spot 14, and PPAR-[gamma], also increased in adipose tissue. Thus, a CLA dose resulting in near maximal inhibition of mammary lipid synthesis resulted in increased expression of lipid synthesis-related genes in adipose tissue. A meta-analysis of intake response during CLA infusion was conducted to extend the investigation of energy metabolism during MFD. Voluntary intake decreased (P< 0.001) by 1.5 kg/d during CLA-induced MFD in the 14 studies analyzed, but the reduction in intake only partially accounts for the energy spared from reduced milk fat synthesis. Results are consistent with energy spared from the reduction in milk fat synthesis being partitioned toward adipose tissue fat stores during short-term MFD.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.01.024 Byline: Jan-Michael Kugler, Jarred Chicoine, Paul Lasko Keywords: Ribonucleoprotein particles; Endoplasmic reticulum; Trafficking; Epidermal growth factor signaling; Pattern formation Abstract: Bicaudal-C (Bic-C) is a multiple KH-domain RNA-binding protein required for Drosophila oogenesis and, maternally, for embryonic patterning. In early oogenesis, Bic-C negatively regulates target mRNAs, including Bic-C, by recruiting the CCR4 deadenylase through a direct association with its NOT3 subunit. Here, we identify a novel function for Bic-C in secretion of the TGF-[alpha] homolog Gurken (Grk). In Bic-C mutant egg chambers, Grk is sequestered within actin-coated structures during mid-oogenesis. As a consequence, Egfr signalling is not efficiently activated in the dorsal-anterior follicle cells. This phenotype is strikingly similar to that of trailer hitch (tral) mutants. Consistent with the idea that Bic-C and Tral act together in Grk secretion, Bic-C co-localizes with Tral within cytoplasmic granules, and can be co-purified with multiple protein components of a Tral mRNP complex. Taken together, our results implicate translational regulation by Bic-C and Tral in the secretory pathway. Author Affiliation: Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, Canada H3A 1B1 Article History: Received 2 June 2008; Revised 5 January 2009; Accepted 19 January 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.12.025 Byline: Kota Saito (1), Mei Chen (3), Fred Bard (4), Shenghong Chen (5), Huilin Zhou (5), David Woodley (3), Roman Polischuk (6), Randy Schekman (7), Vivek Malhotra (1)(2) Keywords: CELLBIO; SIGNALING Abstract: A genome-wide screen revealed previously unidentified components required for transport and Golgi organization (TANGO). We now provide evidence that one of these proteins, TANGO1, is an integral membrane protein localized to endoplasmic reticulum (ER) exit sites, with a luminal SH3 domain and a cytoplasmic proline-rich domain (PRD). Knockdown of TANGO1 inhibits export of bulky collagen VII from the ER. The SH3 domain of TANGO1 binds to collagen VII; the PRD binds to the COPII coat subunits, Sec23/24. In this scenario, PRD binding to Sec23/24 subunits could stall COPII carrier biogenesis to permit the luminal domain of TANGO1 to guide SH3-bound cargo into a growing carrier. All cells except those of hematopoietic origin express TANGO1. We propose that TANGO1 exports other cargoes in cells that do not secrete collagen VII. However, TANGO1 does not enter the budding carrier, which represents a unique mechanism to load cargo into COPII carriers. Author Affiliation: (1) Department of Cell and Developmental Biology, CRG-Centre de Regulacio Genomica, Dr. Aiguader, 88 08003 Barcelona, Spain (2) Institutcio Catalana de Recerca I Estudis Avancats, Dr. Aiguader, 88 08003 Barcelona, Spain (3) Department of Dermatology, USC/Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA (4) Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673 Proteos, Singapore (5) Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA (6) Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy (7) Department of Molecular and Cellular Biology, HHMI, University of California, Berkeley, Berkeley, CA 94720, USA Article History: Received 20 June 2008; Revised 8 October 2008; Accepted 12 December 2008 Article Note: (miscellaneous) Published: March 5, 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.02.014 Byline: Chan Young Park (1), Paul J. Hoover (2), Franklin M. Mullins (2)(3), Priti Bachhawat (2)(4), Elizabeth D. Covington (2), Stefan Raunser (5), Thomas Walz (5)(6), K. Christopher Garcia (2)(4), Ricardo E. Dolmetsch (1), Richard S. Lewis (2) Keywords: CELLBIO Abstract: Store-operated Ca.sup.2+ channels activated by the depletion of Ca.sup.2+ from the endoplasmic reticulum (ER) are a major Ca.sup.2+ entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca.sup.2+ sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca.sup.2+ entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions. Author Affiliation: (1) Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA (2) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA (3) Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA (4) Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA (5) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA (6) Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA Article History: Received 24 September 2008; Revised 6 January 2009; Accepted 9 February 2009 Article Note: (miscellaneous) Published online: February 26, 2009

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided [approximately equal to] 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches. fatty acid elongation | S. cerevisiae | shotgun lipidomics

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.11.047 Byline: Catalina Ruiz-Canada (1), Daniel J. Kelleher (1), Reid Gilmore (1) Keywords: CELLBIO; PROTEINS Abstract: Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation. Author Affiliation: (1) Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School, Worcester, MA 01605, USA Article History: Received 23 June 2008; Revised 17 October 2008; Accepted 25 November 2008 Article Note: (miscellaneous) Published: January 22, 2009

The molecular details of how lipids influence final topological organization of membrane proteins are not well understood. Here, we present evidence that final topology is influenced by lipid--protein interactions most likely outside of the translocon. The N-terminal half of Escherichia coli lactose permease (LacY) is inverted with respect to the C-terminal half and the membrane bilayer when assembled in mutants lacking phosphatidylethanolamine and containing only negatively charged phospholipids. We demonstrate that inversion is dependent on interactions between the net charge of the cytoplasmic surface of the N-terminal bundle and the negative charge density of the membrane bilayer surface. A transmembrane domain, acting as a molecular hinge between the two halves of the protein, must also exit from the membrane for inversion to occur. Phosphatidylethanolamine dampens the translocation potential of negative residues in favor of the cytoplasmic retention potential of positive residues, thus explaining the dominance of positive over negative amino acids as co- or post-translational topological determinants.

It is well known that fatty acid biosynthesis in leaves is closely related to photosynthesis; although a residual synthesis is also detected at night. But less known is the effect of the day-night cycle in fatty acid biosynthesis in heterotrophic tissues such as developing seeds. Through the use of radioactive precursors, we detected variations in the metabolism of fatty acids and lipids during the day--night period in sunflower (Helianthus annuus L.) seeds. These oscillations have been observed both in the fatty acids bound to triacylglycerols and in polar lipids, indicating a fine regulation of the lipids biosynthetic activities in sunflower developing seeds. Additionally, in the triacylglycerol fraction, periodic variations were observed in the oleic and linoleic acids content as a consequence of qualitative variations in the biosynthetic machinery during the day--night period related to temperature changes. Analyzing the rate of oleic acid desaturation, we observed a significant increase in activity in the middle of the night, indicative of the importance of nocturnal desaturation. Similar variations were also observed in the stearoyl-acyl carrier protein (ACP) desaturase activity. This study has shown the variation in the lipid biosynthetic capacity in sunflower developing seeds during the day--night cycle--including control over individual enzymes such as the stearoyl-ACP desaturase--and the influence of diurnal and nocturnal temperatures in the quality (oleic/linoleic ratio) of the oil.

ABSTRACT. To determine whether nitrogen or light exerts the most control for the rates of carbon production of ice algae and phytoplankton under the ice, nitrogen addition ([NO.sub.3] or [NH.sub.4]) [...]

The goal of this study was to determine the magnitude by which acute consumption of fructose in a morning bolus would stimulate lipogenesis (measured by infusion of [sup.13][C.sub.1]-acetate and analysis by GC-MS) immediately and after a subsequent meal. Six healthy subjects [4 men and 2 women; aged (mean [+ or -] SD) 28 [+ or -] 8 y; BMI, 24.3 [+ or -] 2.8 kg/[m.sup.2]; and serum triacylglycerols (TG), 1.03 [+ or -] 0.32 mmol/L] consumed carbohydrate boluses of sugars (85 g each) in a random and blinded order, followed by a standardized lunch 4 h later. Subjects completed a control test of glucose (100:0) and a mixture of 50:50 glucose:fructose and one of 25:75 (wt:wt). Following the morning boluses, serum glucose and insulin after 100:0 were greater than both other treatments (P < 0.05) and this pattern occurred again after lunch. In the morning, fractional lipogenesis was stimulated when subjects ingested fructose and peaked at 15.9 [+ or -] 5.4% after the 50:50 treatment and at 16.9 [+ or -] 5.2% after the 25:75 treatment, values that were greater than after the 100:0 treatment (7.8 [+ or -] 5.7%; P < 0.02). When fructose was consumed, absolute lipogenesis was 2-fold greater than when it was absent (100:0). Postlunch, serum TG were 11-29% greater than 100:0 and TG-rich lipoprotein-TG concentrations were 76-200% greater after 50:50 and 25:75 were consumed (P< 0.05). The data demonstrate that an early stimulation of lipogenesis after fructose, consumed in a mixture of sugars, augments subsequent postprandial lipemia. The postlunch blood TG elevation was only partially due to carry-over from the morning. Acute intake of fructose stimulates lipogenesis and may create a metabolic milieu that enhances subsequent esterification of fatty acids flowing to the liver to elevate TG synthesis postprandially.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.02.049 Byline: Asvin K.K. Lakkaraju (1), Camille Mary (1), Anne Scherrer (1), Arthur E. Johnson (2), Katharina Strub (1) Keywords: CELLBIO; PROTEINS Abstract: SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted. Author Affiliation: (1) Departement de biologie cellulaire, Universite de Geneve, Sciences III, 1211 Geneva, Switzerland (2) Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA Article History: Received 18 October 2007; Revised 8 January 2008; Accepted 14 February 2008 Article Note: (miscellaneous) Published: May 1, 2008

Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the [beta]-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of [beta]-hydroxydecanoyl-[beta]-hydroxydecanoate from two molecules of [beta]-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily [beta]-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for [beta]-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

In contrast to other cereals, typical barley cultivars have caryopses with adhering hulls at maturity, known as covered (hulled) barley. However, a few barley cultivars are a free-threshing variant called naked (hulless) barley. The covered/naked caryopsis is controlled by a single locus (nud) on chromosome arm 7HL. On the basis of positional cloning, we concluded that an ethylene response factor (ERF) family transcription factor gene controls the covered/naked caryopsis phenotype. This conclusion was validated by (i) fixation of the 17-kb deletion harboring the ERF gene among all 100 naked cultivars studied; (ii) two x-ray-induced nud alleles with a DNA lesion at a different site, each affecting the putative functional motif; and (iii) gene expression strictly localized to the testa. Available results indicate the monophyletic origin of naked barley. The Nud gene has homology to the Arabidopsis WIN1/SHN1 transcription factor gene, whose deduced function is control of a lipid biosynthesis pathway. Staining with a lipophilic dye (Sudan black B) detected a lipid layer on the pericarp epidermis only in covered barley. We infer that, in covered barley, the contact of the caryopsis surface, overlaid with lipids to the inner side of the hull, generates organ adhesion. caryopsis | domestication epidermis | ethylene response factor | grass

The synthesis of a sulfolipid-I (SL-I) analogue bearing unnatural lipid substituents is described. The route developed for the model compound is extended for the synthesis of native SL-I as well as additional analogues for use examining the functions of SL-I.

The nonclassical biological activities of vitamin E are investigated. It is observed that vitamin E is involved in novel transcriptional activities that cause a pronounced inhibition of cholesterol biosynthesis.

The ability to store fat in the form of cytoplasmic triglyceride droplets is conserved from Saccharomyces cerevisiae to humans. Although much is known regarding the composition and catabolism of lipid droplets, the molecular components necessary for the biogenesis of lipid droplets have remained obscure. Here we report the characterization of a conserved gene family important for lipid droplet formation named fat-inducing transcript (FIT). FIT1 and FIT2 are endoplasmic reticulum resident membrane proteins that induce lipid droplet accumulation in cell culture and when expressed in mouse liver, shRNA silencing of FIT2 in 3T3-LI adipocytes prevents accumulation of lipid droplets, and depletion of FIT2 in zebrafish blocks diet-induced accumulation of lipid droplets in the intestine and liver, highlighting an important role for FIT2 in lipid droplet formation in vivo. Together these studies identify and characterize a conserved gene family that is important in the fundamental process of storing fat. adipocytes | diabetes | FIT | obesity | triglyceride

The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) that are annotated as lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes, olsA was previously shown to be an O-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of c-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining plsC316 and plsC3498 genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the Escherichia coli plsC, neither plsC316 nor plsC3498 was essential in R. capsulatus. In contrast, no plsC316 olsA double mutant could be isolated, indicating that an intact copy of either olsA or plsC316 was required for R. capsulatus growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no c-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an E. coli plsC(Ts) mutant of either R. capsulatus plsC316 or olsA gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that plsC316 encodes the main AGPAT required for glycerophospholipid synthesis in R. capsulatus, while olsA acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of R. capsulatus, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.

The liver produces plasma sex hormone-binding globulin (SHBG), which transports sex steroids and regulates their access to tissues. In overweight children and adults, low plasma SHBG levels are a biomarker of the metabolic syndrome and its associated pathologies. Here, we showed in transgenic mice and HepG2 hepatoblastoma cells that monosaccharides (glucose and fructose) reduce human SHBG production by hepatocytes. This occurred via a downregulation of hepatocyte nuclear factor-4[alpha] (HNF-4[alpha]) and replacement of HNF-4[alpha] by the chicken OVA upstream promoter-transcription factor 1 at a cis-element within the human SHBG promoter, coincident with repression of its transcriptional activity. The dose-dependent reduction of HNF-4[alpha] levels in HepG2 cells after treatment with glucose or fructose occurred in concert with parallel increases in cellular palmitate levels and could be mimicked by treatment with palmitoyl-CoA. Moreover, inhibition of lipogenesis prevented monosaccharide-induced downregulation of HNF-4[alpha] and reduced SHBG expression in HepG2 cells. Thus, monosaccharide-induced lipogenesis reduced hepatic HNF-4[alpha] levels, which in turn attenuated SHBG expression. This provides a biological explanation for why SHBG is a sensitive biomarker of the metabolic syndrome and the metabolic disturbances associated with increased fructose consumption., Introduction Body mass index is a major determinant of sex hormone-binding globulin (SHBG) concentrations in the blood of men and women (1-3). Low serum SHBG levels in overweight individuals are [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.10.025 Byline: R. Luke Wiseman (1)(2), Evan T. Powers (1)(2), Joel N. Buxbaum (3), Jeffery W. Kelly (1)(2), William E. Balch (4)(5) Keywords: CELLBIO; PROTEINS; HUMDISEASE Abstract: To provide an integrated view of endoplasmic reticulum (ER) function in protein export, we have described the interdependence of protein folding energetics and the adaptable biology of cellular protein folding and transport through the exocytic pathway. A simplified treatment of the protein homeostasis network and a formalism for how this network of competing pathways interprets protein folding kinetics and thermodynamics provides a framework for understanding cellular protein trafficking. We illustrate how folding and misfolding energetics, in concert with the adjustable biological capacities of the folding, degradation, and export pathways, collectively dictate an adaptable standard for protein export from the ER. A model of folding for export (FoldEx) establishes that no single feature dictates folding and transport efficiency. Instead, a network view provides insight into the basis for cellular diversity, disease origins, and protein homeostasis, and predicts strategies for restoring protein homeostasis in protein-misfolding diseases. Author Affiliation: (1) Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (2) The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (3) Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (4) Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (5) The Institute for Childhood and Neglected Diseases, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Article History: Received 9 March 2007; Revised 31 July 2007; Accepted 10 October 2007 Article Note: (miscellaneous) Published: November 15, 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.10.018 Byline: David Reczek (1), Michael Schwake (2), Jenny Schroder (2), Heather Hughes (1), Judith Blanz (2), Xiaoying Jin (1), William Brondyk (1), Scott Van Patten (1), Tim Edmunds (1), Paul Saftig (2) Keywords: CELLBIO; HUMDISEASE Abstract: [beta]-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of [beta]-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. [beta]-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of [beta]-glucocerebrosidase was secreted. Missorting of [beta]-glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of [beta]-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a [beta]-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for [beta]-glucocerebrosidase. Author Affiliation: (1) Genzyme Corporation, 1 Mountain Road, Framingham, MA 01701, USA (2) Biochemical Institute, Christians-Albrechts University Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany Article History: Received 15 May 2007; Revised 5 September 2007; Accepted 8 October 2007 Article Note: (miscellaneous) Published: November 15, 2007

The biogenesis of thylakoid membranes, an indispensable event for the photoautotrophic growth of plants, requires a significant increase in the level of the unique thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG), which constitutes the bulk of membrane lipids in chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. Here we report the identification and characterization of an Arabidopsis mutant showing a complete defect in MGDG synthase 1. The mutant seeds germinated as small albinos only in the presence of sucrose. The seedlings lacked galactolipids and had disrupted photosynthetic membranes, leading to the complete impairment of photosynthetic ability and photoautotrophic growth. Moreover, invagination of the inner envelope, which is not seen in mature WT chloroplasts, was observed in the mutant, supporting an old hypothesis that envelope invagination is a major event in early chloroplast biogenesis. In addition to the defective seedling phenotype, embryo development was arrested in the mutant, although seeds with impaired embryos could germinate heterotrophically. These results demonstrate the importance of galactolipids not only in photosynthetic growth but also in embryogenesis. glycosyltransferase | thylakoid membrane | monogalactosyldiacylglycerol | monogalactosyldiacylglycerol synthase

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the reversible transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein to the glucosamine 3-OH group of UDP-GlcNAc. Escherichia coli LpxA is highly selective for R-3-hydroxymyristate. The crystal structure of the E. coli LpxA homotrimer, determined previously in the absence of lipid substrates or products, revealed that LpxA contains an unusual, left-handed parallel [beta]-helix fold. We have now solved the crystal structures of E. coli LpxA with the bound product UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc at a resolution of 1.74 [Anstrom] and with bound UDP-3-O-(R-3-hydroxydecanoyl)GlcNAc at 1.85 [Angstrom]. The structures of these complexes are consistent with the catalytic mechanism deduced by mutagenesis and with a recent 3.0-[Angtrom] structure of LpxA with bound UDP-GlcNAc. Our structures show how LpxA selects for 14-carbon R-3-hydroxyacyl chains and reveal two modes of UDP binding. UDP-3-O-(R-3-hydroxymyristoyl)-acetylglucosamine | acyl carrier protein LpxA | Escherichia coli | x-ray crystallography

The accumulation of low-density lipoprotein (LDL) is recognized as one of the main contributors in atherogenesis. Mathematical models have been constructed to simulate mass transport in large arteries and the consequent lipid accumulation in the arterial wall. The objective of this study was to investigate the influences of wall shear stress and transmural pressure on LDL accumulation in the arterial wall by a multilayered, coupled lumen-wall model. The model employs the Navier-Stokes equations and Darcy's Law for fluid dynamics, convection-diffusion-reaction equations for mass balance, and Kedem-Katchalsky equations for interracial coupling. To determine physiologically realistic model parameters, an optimization approach that searches optimal parameters based on experimental data was developed. Two sets of model parameters corresponding to different transmural pressures were found by the optimization approach using experimental data in the literature. Furthermore, a shear-dependent hydraulic conductivity relation reported previously was adopted. The integrated multilayered model was applied to an axisymmetric stenosis simulating an idealized, mildly stenosed coronary artery. The results show that low wall shear stress leads to focal LDL accumulation by weakening the convective clearance effect of transmural flow, whereas high transmural pressure, associated with hypertension, leads to global elevation of LDL concentration in the arterial wall by facilitating the passage of LDL through wall layers. low-desity lipoprotein transport; lipid accumulation; atherosclerosis; hypertension doi:10.1152/ajpheart.01281.2006

Two experiments were conducted with lactating Angus x Gelbvieh beef cows to determine the effects of postpartum lipid supplementation, BCS at parturition, and day of lactation on fatty acid profiles in plasma, adipose tissue, and milk. In Exp. 1, 36 primiparous cows (488 [+ or -] 10 kg of initial BW; 5.5 [+ or -] 0.02 initial BCS) were given ad libitum access to hay and assigned randomly to a low-fat (control) supplement or supplements with cracked, high-linoleate safflower seeds (linoleate) or cracked, high-oleate safflower seeds (oleate) from d 3 to 90 of lactation. Diets were formulated to be isonitrogenous and isocaloric; safflower seed diets provided 5% of DMI as fat. Plasma and milk samples were collected on d 30, 60, and 90 of lactation. Adipose tissue biopsies were collected near the tailhead region of cows on d 45 and 90 of lactation. In Exp. 2, 3-yr-old cows achieving a BCS of 4 [+ or -] 0.07 (479 [+ or -] 36 kg of BW) or 6 [+ or -] 0.07 (580 [+ or -] 53 kg of BW) at parturition were used in a 2-yr experiment (n = 36/yr). Beginning 3 d postpartum through d 61 of lactation, cows were fed diets similar to those of Exp. 1. Adipose tissue and milk samples were collected on d 30 and 60, and plasma was collected on d 31 and 61 of lactation. Responses to postpartum dietary treatment were comparable in both experiments. Cows fed linoleate and oleate had greater (P [less than or equal to] 0.001) total fatty acid concentrations in plasma than cows fed control. Except for 15:1, milk fatty acids with Key words: adipose tissue, beef cow, body condition score, fatty acid, lipid supplementation, milk

The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.09.043 Byline: Xiaodong Wang (1), John Venable (1), Paul LaPointe (1), Darren M. Hutt (1), Atanas V. Koulov (1), Judith Coppinger (1), Cemal Gurkan (1), Wendy Kellner (1), Jeanne Matteson (1), Helen Plutner (1), John R. Riordan (5), Jeffery W. Kelly (2)(3), John R. Yates (1), William E. Balch (1)(4) Abstract: The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, [DELTA]F508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of [DELTA]F508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the 'chaperome') is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease. Author Affiliation: (1) Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (2) Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (3) The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (4) The Institute for Childhood and Neglected Disease, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA (5) Department of Biochemistry and Biophysics, 5011 Thurston-Bowles Building, University of North Carolina, Chapel Hill, NC, 27510, USA Article History: Received 8 April 2006; Revised 8 July 2006; Accepted 11 September 2006 Article Note: (miscellaneous) Published: November 16, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.08.004 Byline: Ramanujan S. Hegde (1), Harris D. Bernstein (2) Abstract: Most secreted and many membrane proteins contain cleavable N-terminal signal sequences that mediate their targeting to and translocation across the endoplasmic reticulum or bacterial cytoplasmic membrane. Recent studies have identified many exceptions to the widely held view that signal sequences are simple, degenerate and interchangeable. Growing evidence indicates that signal sequences contain information that specifies the choice of targeting pathway, the efficiency of translocation, the timing of cleavage and even postcleavage functions. As a consequence, signal sequences can have important roles in modulating protein biogenesis. Based on a synthesis of studies in numerous experimental systems, we propose that substrate-specific sequence elements embedded in a conserved domain structure impart unique and physiologically important functionality to signal sequences. Author Affiliation: (1) Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA (2) Genetics and Biochemistry Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.06.059 Byline: Huiping Tu (1), Omar Nelson (1), Arseny Bezprozvanny (1), Zhengnan Wang (1), Sheu-Fen Lee (2), Yi-Heng Hao (2), Lutgarde Serneels (3), Bart De Strooper (3), Gang Yu (2), Ilya Bezprozvanny (1) Abstract: Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for [approximately equal to]40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca.sup.2+) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for [approximately equal to]80% of passive Ca.sup.2+ leak from the endoplasmic reticulum. Deficient Ca.sup.2+ signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca.sup.2+ leak function of presenilins is independent of their [gamma]-secretase activity. Our data suggest a Ca.sup.2+ signaling function for presenilins and provide support for the 'Ca.sup.2+ hypothesis of AD.' Author Affiliation: (1) Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, TX, 75390, USA (2) Center for Basic Neuroscience, UT Southwestern Medical Center at Dallas, Dallas, TX, 75390, USA (3) Neuronal Cell Biology and Gene Transfer, Center for Human Genetics, Flanders Interuniversity Institute for Biotechnology (VIB4) and KU Leuven, 3000 Leuven, Belgium Article History: Received 1 June 2005; Revised 9 April 2006; Accepted 30 June 2006 Article Note: (miscellaneous) Published: September 7, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.06.045 Byline: Soo Hee Lee (1), Jennifer L. Stephens (1), Kimberly S. Paul (1), Paul T. Englund (1) Abstract: All eukaryotic and prokaryotic organisms are thought to synthesize fatty acids using a type I or type II synthase. In addition, eukaryotes extend pre-existing long chain fatty acids using microsomal elongases (ELOs). We have found that Trypanosoma brucei, a eukaryotic human parasite that causes sleeping sickness, uses three elongases instead of type I or type II synthases for the synthesis of nearly all its fatty acids. Trypanosomes encounter diverse environments during their life cycle with different fatty acid requirements. The tsetse vector form requires synthesis of stearate (C18), whereas the bloodstream form needs myristate (C14). We find that trypanosome fatty acid synthesis is modular, with ELO1 converting C4 to C10, ELO2 extending C10 to C14, and ELO3 elongating C14 to C18. In blood, ELO3 downregulation favors myristate synthesis, whereas low concentrations of exogenous fatty acids in cultured parasites cause upregulation of the entire pathway, allowing the parasite to adapt to different environments. Author Affiliation: (1) Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, MD 21205, USA Article History: Received 17 April 2006; Revised 15 May 2006; Accepted 12 June 2006 Article Note: (miscellaneous) Published: August 24, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.06.001 Byline: Christian W. Gruber, MaA a AemaA3/4ar, BegoA[+ or -]a Heras, Jennifer L. Martin, David J. Craik Abstract: Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell. Author Affiliation: Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, QLD 4072, Australia

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.05.043 Byline: Pedro Carvalho (1), Veit Goder (1), Tom A. Rapoport (1) Abstract: Many misfolded endoplasmic reticulum (ER) proteins are eliminated by ERAD, a process in which substrates are polyubiquitylated and moved into the cytosol for proteasomal degradation. We have identified in S. cerevisiae distinct ubiquitin-ligase complexes that define different ERAD pathways. Proteins with misfolded ER-luminal domains use the ERAD-L pathway, in which the Hrd1p/Hrd3p ligase forms a near stoichiometric membrane core complex by binding to Der1p via the linker protein Usa1p. This core complex associates through Hrd3p with Yos9p, a substrate recognition protein in the ER lumen. Substrates with misfolded intramembrane domains define a pathway (ERAD-M) that differs from ERAD-L by being independent of Usa1p and Der1p. Membrane proteins with misfolded cytosolic domains use the ERAD-C pathway and are directly targeted to the Doa10p ubiquitin ligase. All three pathways converge at the Cdc48p ATPase complex. These results lead to a unifying concept for ERAD that may also apply to mammalian cells. Author Affiliation: (1) Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA Article History: Received 17 February 2006; Revised 30 April 2006; Accepted 18 May 2006 Article Note: (miscellaneous) Published: July 27, 2006

Sterol regulatory element-binding protein (SREBP)-1a and -1c are key transcription factors that regulate lipid biosynthesis in cells. We identified that Ser338 located at the N[H.sub.2] terminus of SREBP-1a is a PKA phosphorylation site in vitro and in HepG2 cells. PKA phosphorylation of this site attenuated DNA occupancy, as revealed by chromatin immunoprecipitation assay, and the ensuing transactivation. In contrast, replacing Ser with Ala [SREBP-1a(N)-S338A] increased transactivation. Although it forms heterodimers with the wild-type SREBP-1a(N) or $338A but not a homodimer with itself, SREBP-1a(N)-S338D (replacing Ser with Asp) decreased DNA binding. Ser314 of SREBP1c, the counterpart of SREBP-1a Ser338, was also phosphorylated by PKA. Accordingly, the adenovirus-mediated expression of SREBP-1c(N)-S314D in HepG2 cells retarded lipogenesis. Our results indicate that the cAMP-PKA pathway, by phosphorylating SREBP-1, may modulate lipid metabolism in liver cell lines. metabolism; glucagon; insulin; lipogenesis

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.11.040 Byline: Kezhong Zhang (1), Xiaohua Shen (2), Jun Wu (1), Kenjiro Sakaki (1), Thomas Saunders (3), D. Thomas Rutkowski (2), Sung Hoon Back (2), Randal J. Kaufman (1)(2) Abstract: Regulated intramembrane proteolysis (RIP) of endoplasmic reticulum (ER) membrane-anchored transcription factors is known to maintain sterol homeostasis and to mediate the unfolded protein response (UPR). Here, we identified CREBH as a RIP-regulated liver-specific transcription factor that is cleaved upon ER stress and required to activate expression of acute phase response (APR) genes. Proinflammatory cytokines increase expression of ER membrane-anchored CREBH. In response to ER stress, CREBH is cleaved by site-1 and site-2 proteases to liberate an amino-terminal fragment that transits to the nucleus to activate transcription of the genes encoding serum amyloid P-component (SAP) and C-reactive protein (CRP). Proinflammatory cytokines and lipopolysaccharide activate the UPR and induce cleavage of CREBH in the liver in vivo. Together, our studies delineate a molecular mechanism for activation of an ER-localized transcription factor, CREBH, and reveal an unprecedented link by which ER stress initiates an acute inflammatory response. Author Affiliation: (1) Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, MI 48109, USA (2) Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, MI 48109, USA (3) Transgenic Animal Model Core, University of Michigan Medical Center, Ann Arbor, MI 48109, USA Article History: Received 9 May 2005; Revised 8 September 2005; Accepted 11 November 2005 Article Note: (miscellaneous) Published: February 9, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.11.047 Byline: Gia K. Voeltz (1), William A. Prinz (2), Yoko Shibata (1), Julia M. Rist (1), Tom A. Rapoport (1) Abstract: How is the characteristic shape of a membrane bound organelle achieved? We have used an in vitro system to address the mechanism by which the tubular network of the endoplasmic reticulum (ER) is generated and maintained. Based on the inhibitory effect of sulfhydryl reagents and antibodies, network formation in vitro requires the integral membrane protein Rtn4a/NogoA, a member of the ubiquitous reticulon family. Both in yeast and mammalian cells, the reticulons are largely restricted to the tubular ER and are excluded from the continuous sheets of the nuclear envelope and peripheral ER. Upon overexpression, the reticulons form tubular membrane structures. The reticulons interact with DP1/Yop1p, a conserved integral membrane protein that also localizes to the tubular ER. These proteins share an unusual hairpin topology in the membrane. The simultaneous absence of the reticulons and Yop1p in S. cerevisiae results in disrupted tubular ER. We propose that these 'morphogenic' proteins partition into and stabilize highly curved ER membrane tubules. Author Affiliation: (1) Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA (2) Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA Article History: Received 26 September 2005; Revised 31 October 2005; Accepted 30 November 2005 Article Note: (miscellaneous) Published: February 9, 2006

Model biomembranes composed of poly-DATT/DGS/ POPA and poly-DATY/DGS/CL were separately prepared on gold electrodes. A monolayer of 1,2-dioleoyl-sn-glycero-3-succinate (DGS) was covalently bonded onto electrochemically grown poly-(3,4-diamiono-2,2:5,2-terthiophene) (DATI) layers (thickness of ~300 nm; particle size of ~50 to 70 nm). The numbers of unit molecules of the poly-DATT layer and of the DGS immobilized onto the poly-DATT layers were 1.53 x [10.sup.-7] and 1.56 x [10.sup.-9] mol [cm.sup.-2], respectively, using a quartz crystal microbalance technique. The lipid bilayers of 1-palmitoyl-2-oleoyl-snglycero-3-phosphate (POPA) and cardiolipin (CL) were formed onto the poly-DATr/DGS layer using the Langmuir Blodgett technique. The surface characterizations of each step were investigated by SEM, AFM, and XPS analyses. Cytochrome c (cyt c) was immobilized onto these model biomembranes through the charge interaction between the positive charges of cyt c and the negative charges of phosphate groups in CL or POPA lipids. At the POPA- and CL-modified biomembranes, the formal potentials of the redox couple of the immobilized cyt c were 0.22 and 0.23 V (vs Ag/AgCl), respectively. The redox reaction of the immobilized cyt c at the POPA- and CL-modified biomembranes was quasireversible, and the electron-transfer rate constants were 0.121 [s.sup.-1] and 0.133 [s.sup.-1], respectively. The applicability of these cyt c immobilized bioimitation membranes as the biosensors was tested for the determination of superoxide.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2005.09.002 Byline: Francis P. Kuhajda (a)(b)(c), Leslie E. Landree (d), Gabriele V. Ronnett (d)(e) Abstract: Obesity and its attendant disorders, such as Type II diabetes, have reached epidemic proportions in the USA, and their prevalence is increasing globally. C75 is a small-molecule inhibitor of fatty acid synthase (FAS) and a stimulator of carnitine palmitoyl 1 activity, which causes profound weight loss in mice. Although C75 is not a compound that is destined for human drug development, it has provided two potential pathways to target in obesity therapy: fatty acid synthesis and fatty acid oxidation. In this article, we discuss the latest data challenging the relationship between fatty acid synthase inhibition and C75-induced anorexia. Author Affiliation: (a) Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA (b) Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA (c) Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA (d) Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA (e) Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.08.018 Byline: Nicolas Touret (1), Paul Paroutis (1)(3), Mauricio Terebiznik (2), Rene E. Harrison (4), Sergio Trombetta (5), Marc Pypaert (5), Amy Chow (5), Aimin Jiang (5), James Shaw (1)(3), Christopher Yip (3), Hsiao-Ping Moore (6), Nicole van der Wel (7), Diane Houben (7), Peter J. Peters (7), Chantal de Chastellier (8), Ira Mellman (5), Sergio Grinstein (1)(3) Abstract: Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles. Author Affiliation: (1) Programme in Cell Biology, University of Toronto, Toronto, Ontario, M5G 1X8, Canada (2) Gastroenterology, Hepatology, and Nutrition Department, Hospital for Sick Children, University of Toronto, Toronto, Ontario, M5G 1X8, Canada (3) Department of Biochemistry and, University of Toronto, Toronto, Ontario, M5G 1X8, Canada (4) Life Sciences, University of Toronto, Toronto, Ontario, M5G 1X8, Canada (5) Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520 (6) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720 (7) Netherlands Cancer Institute, Tumor Biology - H4, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands (8) Centre d'Immunologie de Marseille-Luminy, INSERM-CNRS, Universite de la Mediterranee, 13288 Marseille Cedex 09, France Article History: Received 8 April 2005; Revised 29 June 2005; Accepted 1 August 2005 Article Note: (miscellaneous) Published: October 6, 2005

The results of long-term in vitro incubations with (super 13)CH(sub 4) of a microbial mat from the Black Sea with high anaerobic oxidation of methane (AOM) activity are described. It is observed that there were differences in (super 13)C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.

Apolipoproteins (lipid-free) are lipid-binding proteins that circulate in the plasma of human blood and are responsible for the clearance of lipoproteins. Apolipoprotein E (ApoE) is one of the several classes of this protein family. It acts as a ligand for the low-density lipid (LDL) receptors and is important for the clearance of very low-density lipid (VLDL) and chylomicron remnants. The APOE gene locus is polymorphic, with three major known alleles, APOE*3, *4, and *2. We investigated the distribution of the allele frequency of the APOE gene locus and describe here the genetic variation in four Kuwaiti subpopulations: Arab origin (Arabian peninsula), Arab Bedouin tribes, Iranian origin, and the heterogeneous population. We also describe the use of Spreadex gels in resolving the amplified and digested products of the APOE gene locus. DNA was extracted from whole blood and subjected to PCR and then to RFLP analysis. Allele and genotype frequencies were estimated for the total population and for each subpopulation. Statistical analysis showed no difference in the allele frequencies between the four groups. The frequency of APOE*3 in the Kuwaiti population was highest (88.4%) followed by the frequency of APOE*4 (6.5%) and APOE*2 (5.1%). The genotype and allele frequencies obtained for the Kuwaiti population fell within the reported worldwide distribution for the APOE gene locus. Moreover, the results obtained in this study showed no statistical difference (p > 0.05) between the APOE allele and genotype frequencies between the subgroups for all six genotypes and three alleles, supporting the assumption of admixture in the Kuwaiti population and that the obtained frequencies were in Hardy-Weinberg equilibrium. Finally, we found that the distribution of the APOE alleles in Kuwait differs somewhat from those reported in other Arab populations, suggesting that the Arabs originating from the Arabian peninsula are different from those of Lebanon, Morocco, and Sudan. KEY WORDS: APOE, ALLELE FREQUENCIES, POLYMORPHISMS, KUWAIT, ARABS, LIPOPROTEINS, SPREADEX GELS., Apolipoproteins are lipid-binding proteins that circulate in the bloodstream of all vertebrates. The apolipoproteins form a structural part of the lipoprotein particles. Their primary function is signaling and targeting lipoproteins [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.04.025 Byline: Dominic Hoepfner (1)(2), Danny Schildknegt (1), Ineke Braakman (1), Peter Philippsen (2), Henk F. Tabak (1)(3)(4) Abstract: How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we followed the trafficking route of newly made YFP-tagged Pex3 and Pex19 proteins by real-time fluorescence microscopy in Saccharomyces cerevisiae. Remarkably, Pex3 (an integral membrane protein) could first be observed in the endoplasmic reticulum (ER), where it concentrates in foci that then bud off in a Pex19-dependent manner and mature into fully functional peroxisomes. Pex19 (a farnesylated, mostly cytosolic protein) enriches first at the Pex3 foci on the ER and then on the maturing peroxisomes. This trafficking route of Pex3-YFP is the same in wild-type cells. These results demonstrate that peroxisomes are generated from domains in the ER. Author Affiliation: (1) Department of Cellular Protein Chemistry, University of Utrecht, Padualaan 8, NL-3548 CH Utrecht, The Netherlands (2) Lehrstuhl fur Angewandte Mikrobiologie, Biozentrum, Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland (3) Laboratory of Cell Biology, University Medical Center Utrecht, Heidelberglaan 100, NL-3584 CX Utrecht, The Netherlands (4) Academic Biomedical Centre, University of Utrecht, Yalelaan 1, NL-3548 CL Utrecht, The Netherlands Article History: Received 13 December 2004; Revised 15 March 2005; Accepted 25 April 2005 Article Note: (miscellaneous) Published: July 14, 2005

The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, L-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32% to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8.3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [[sup.13]C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [[sup.13]C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.

The hexosamine signaling pathway has been shown to serve a nutrient-sensing function. We have previously shown that overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine-fructose-6-phosphate amidotransferase) in adipose tissue of transgenic mice results in skeletal muscle insulin resistance and altered regulation of leptin and adiponectin. To dissect the pathways by which the hexosamine pathway affects fuel storage and energy homeostasis, we have examined the characteristics of adipocytes from these animals. After 3 mo of age, epididymal fat pads from adult transgenic animals are 42% heavier (P = 0.003) and individual adipocytes are 23% larger in diameter (P < 0.05) than those from littermate wild-type controls. Isolated adipocytes from transgenic mice are insulin resistant, with a 2.5-fold increase in the E[D.sub.50] for stimulation of 2-deoxy-D-glucose uptake. However, maximal insulin-stimulated glucose uptake is increased in transgenic adipocytes by 39% (P < 0.05). This upregulation of glucose uptake was associated with a 41% increase in the expression of GLUT4 mRNA and a 28% increase in GLUT4 protein in transgenics compared with controls (P < 0.05). GLUT1 mRNA and protein did not significantly differ between fasted control and transgenics. Total lipid synthesis was also increased in epididymal adipocytes from transgenic animals by 206% compared with controls (P < 0.05). Fatty acid oxidation was increased 1.6-fold in the transgenic adipocytes (P < 0.05). We conclude that the hexosamine signaling pathway upregulates fat storage in adipocytes in states of carbohydrate excess, in part by increasing GLUT4 and glucose uptake and by augmenting fatty acid synthesis. hexosamine; insulin resistance; adipocyte

A report on an investigation on the aggregation of 1,2-dilauroyl-sn-glycero-3-phosphatidyluridine (DLPU), a surfactant molecule that merges the self-assembling properties of lecithins with the molecular recognition characteristics of nucleic bases in a phosphate-buffered aqueous solution at physiological pH and as a functin of lipid concentration is presented.

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [[sup.14]C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P < 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [[sup.14]C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [[sup.14]C]oleic acid as a precursor, butyrate significantly (P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [[sup.35]S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations. lipid esterification; cholesterol synthesis; phospholipid; chylomicron; very low-density lipoprotein; apolipoprotein A-I; apolipoprotein B

Results demonstrate that a lipid transfer component pocket of the apolipoprotein B, is formed by the hydrophobic association of the N-terminal first 1000 residues of apolipoprotein B and the microsomal triglyceride transfer protein along with a nascent lipid nucleus.

Animal cells exert exquisite control over the physical and chemical properties of their membranes, but the mechanisms are obscure. We show that phosphatidylethanolamine, the major phospholipid in Drosophila, controls the release of sterol regulatory element-binding protein (SREBP) from Drosophila cell membranes, exerting feedback control on the synthesis of fatty acids and phospholipids. The finding that SREBP processing is controlled by different lipids in mammals and flies (sterols and phosphatidylethanolamine, respectively) suggests that an essential function of SREBP is to monitor cell membrane composition and to adjust lipid synthesis accordingly., The lipid composition of membranes in animal cells is maintained within strict limits, primarily by feedback regulation of lipid biosynthesis. The mechanism for this homeostasis is beginning to be understood. [...]