LIPID arrays: new tools in the understanding of membrane dynamics and lipid signaling

Phospholipids are the structural building blocks of the membrane bilayer, which retains and regulates intra-cellular content. In addition to creating a protective barrier around the cell, lipids modulate membrane trafficking and are themselves precursors of important intracellular signaling molecules. Identification and quantification of these molecular species is essential for a more complete understanding of cell signaling pathways, and more reliable and sensitive methods are needed for determining membrane phospholipid content. Recent improvements in electrospray ionization mass spectrometry have made possible the direct identification of more than 400 phospholipid species from biological extracts of a single cell type. Changes in the cellular concentration of diverse lipids can be determined by analysis of the mass spectra by statistical algorithms. In the future, lipid arrays will be integrated with other high-throughput profiling technologies, and computational lipidomics will expand our understanding of the molecular basis of cellular processes and diseases.     

Cloning, expression, and characterization of the acyl-CoA-binding protein in African trypanosomes

African trypanosomes are shielded from their hosts' defenses by a coat of variant surface glycoprotein molecules, each of which is attached to the plasma membrane by a glycosylphosphatidylinositol anchor. During the later stages of glycosylphosphatidylinositol biosynthesis, myristic acid is incorporated into the anchor from the donor myristoyl-CoA by a series of unique fatty acid remodeling and exchange reactions. We have cloned and expressed a recombinant trypanosome acyl-CoA-binding protein that has a preference for binding relatively short chain acyl-CoAs and that has a high affinity for binding myristoyl-CoA (K(d) = 3.5 x 10(-10) M). This protein enhances fatty acid remodeling of glycosylphosphatidylinositol precursors in the trypanosome cell-free system. We speculate that the trypanosome acyl-CoA-binding protein plays an active role in supplying myristoyl-CoA to the fatty acid remodeling machinery in the parasite.     

Ultrastructural demonstration of cell coat on the cell surfaces of normal human oesophageal epithelium

Synopsis The cell coat in human oesophageal biopsies was studied with Alcian Blue, Ruthenium Red, Safranin O, colloidal iron and the ferrocyanide-osmium tetroxide techniques. Alcianophilic material was found on the cell surface of the basal, prickle cell and functional layers, being most abundant on the superficial cells where it appeared as a continuous coat. In the deeper layers, it tended to have a particulate distribution. Some membrane-coating granules were alcianophilic. Ruthenium Red had a particulate distribution over all cell surfaces. Intercellular debris was also stained. Safranin O produced no staining. Colloidal iron stained the cell coat in a particulate manner. The ferrocyanide-osmium technique showed a uniform filamentous cell coat. The oesophageal epithelial cell coats are, in part, acid mucosubstances which, on the surface cells, may have a protective function.     

Formation of F-ring isoprostane-like compounds (F3-isoprostanes) in vivo from eicosapentaenoic acid

Eicosapentaenoic acid (EPA, C20:5, omega-3) is the most abundant polyunsaturated fatty acid (PUFA) in fish oil. Recent studies suggest that the beneficial effects of fish oil are due, in part, to the generation of various free radical-generated non-enzymatic bioactive oxidation products from omega-3 PUFAs, although the specific molecular species responsible for these effects have not been identified. Our research group has previously reported that pro-inflammatory prostaglandin F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether F2-IsoP-like compounds (F3-IsoPs) are formed from the oxidation of EPA in vivo. Oxidation of EPA in vitro yielded a series of compounds that were structurally established to be F3-IsoPs using a number of chemical and mass spectrometric approaches. The amounts formed were extremely large (up to 8.7 + 1.0 microg/mg EPA) and greater than levels of F2-IsoPs generated from arachidonic acid. We then examined the formation of F3-IsoPs in vivo in mice. Levels of F3-IsoPs in tissues such as heart are virtually undetectable at baseline, but supplementation of animals with EPA markedly increases quantities up to 27.4 + 5.6 ng/g of heart. Interestingly, EPA supplementation also markedly reduced levels of pro-inflammatory arachidonate-derived F2-IsoPs by up to 64% (p < 0.05). Our studies provide the first evidence that identify F3-IsoPs as novel oxidation products of EPA that are generated in vivo. Further understanding of the biological consequences of F3-IsoP formation may provide valuable insights into the cardioprotective mechanism of EPA.     

Glycoprotein D receptor-dependent, low-pH-independent endocytic entry of herpes simplex virus type 1

Two herpes simplex virus type 1 (HSV-1) entry pathways have been described: direct fusion between the virion envelope and the plasma membrane, as seen on Vero cells, and low-pH-dependent endocytosis, as seen on CHO nectin-1 and HeLa cells. In this paper, we studied HSV entry into C10 murine melanoma cells and identified a third entry pathway for this virus. During entry into C10 cells, virion envelope glycoproteins rapidly became protected from the membrane-impermeable chemical cross-linker BS3 and from proteinase K. Protection was gD receptor dependent, and the time taken to detect protected protein was proportional to the rate of virus entry. Ultrastructural examination revealed that virions attached to the surface of C10 cells were localized to membrane invaginations, whereas those on the surface of receptor-negative B78 cells were peripherally attached. Virus entry into C10 cells was energy dependent, and intracellular enveloped virions were seen within membrane-bound vesicles consistent with endocytic entry. Entry was not inhibited by bafilomycin A1 or ammonium chloride, showing that passage of the virion through a low-pH environment was not required for infection. Resistance to similar reagents should therefore not be taken as proof of HSV entry by a nonendosomal pathway. These data define a novel gD receptor-dependent acid-independent endocytic entry pathway for HSV.     

Higher Thermal Acclimation Potential of Respiration but Not Photosynthesis in Two Alpine Picea Taxa in Contrast to Two Lowland Congeners

The members of the genus Picea form a dominant component in many alpine and boreal forests which are the major sink for atmospheric CO₂. However, little is known about the growth response and acclimation of CO₂ exchange characteristics to high temperature stress in Picea taxa from different altitudes. Gas exchange parameters and growth characteristics were recorded from four year old seedlings of two alpine (Picea likiangensis vars. rubescens and linzhiensis) and two lowland (P. koraiensis and P. meyeri) taxa. Seedlings were grown at moderate (25°C/15°C) and high (35°C/25°C) day/night temperatures, for four months. The approximated biomass increment (ΔD²H) for all taxa decreased under high temperature stress, associated with decreased photosynthesis and increased respiration. However, the two alpine taxa exhibited lower photosynthetic acclimation and higher respiratory acclimation than either lowland taxon. Moreover, higher leaf dry mass per unit area (LMA) and leaf nitrogen content per unit area (N_area), and a smaller change in the nitrogen use efficiency of photosynthesis (PNUE) for lowland taxa indicated that these maintained higher homeostasis of photosynthesis than alpine taxa. The higher respiration rates produced more energy for repair and maintenance biomass, especially for higher photosynthetic activity for lowland taxa, which causes lower respiratory acclimation. Thus, the changes of ΔD²H for alpine spruces were larger than that for lowland spruces. These results indicate that long term heat stress negatively impact on the growth of Picea seedlings, and alpine taxa are more affected than low altitude ones by high temperature stress. Hence the altitude ranges of Picea taxa should be taken into account when predicting changes to carbon fluxes in warmer conditions.     

Endocytotic pathways across the human gall-bladder mucosa: permeability studies using horseradish peroxidase

Summary Human gall-bladder epithelium obtained straight from the operating theatre was incubated in an Ussing chamber with the fluid phase marker, horseradish peroxidase (HRP), for up to 60 min. When the marker was presented on the apical surface, within 30 min it had moved readily across the apical cytoplasm in transport vesicles to receptosomes and into the lateral intercellular space, extending across the basement membrane into the lamina propria. When HRP was presented at the basal aspect, within 30 min it had moved through the lamina propria, across the basement membrane and into the lateral intercellular space. By 60 min, only small amounts had been taken up by the epithelial cells and transported to receptosomes. These data indicate a rapid transmucosal endocytotic pathway for blood-or bile-borne macromolecules.     

Fish oil and indomethacin in combination potently reduce dyslipidemia and hepatic steatosis in LDLR -/- mice

Fish oil (FO) is a potent anti-inflammatory and lipid-lowering agent. Because inflammation can modulate lipid metabolism and vice versa, we hypothesized that combining FO with cyclooxygenase inhibitors (COXIBs), well-known anti-inflammatory drugs, can enhance the anti-inflammatory and lipid-lowering effect of FO. LDLR (-/-) mice were fed a high-fat diet supplemented with 6% olive oil or FO for 12 wk in the presence or absence of indomethacin (Indo, 6 mg/l drinking water). FO reduced plasma total cholesterol by 30% but, in combination with Indo, exerted a greater decrease (44%). The reduction of liver cholesterol ester (CE) and triglycerides (TG) by FO (63% and 41%, respectively) was enhanced by Indo (80% in CE and 64% in TG). FO + Indo greatly increased the expression of genes modulating lipid metabolism and reduced the expression of inflammatory genes compared with control. The mRNA and/or protein expression of pregnane X receptor (PXR) and cytochrome P450 isoforms that alter inflammation and/or lipid metabolism are increased to a greater extent in mice that received FO + Indo. Moroever, the nuclear level of PXR is significantly increased in FO + Indo group. Combining FO with COXIBs may exert their beneficial effects on inflammation and lipid metabolism via PXR and cytochrome P450.