Anionic lipid domains: correlation with functional topography in a mammalian cell membrane.

Polymyxin B was used to explore distribution of anionic phospholipids in sperm plasma membranes by electron microscopy of freeze-fracture replicas. After exposure to Hepes/Tris-buffered polymyxin at 4 mM, phosphatidylcholine liposomes showed no perturbations nor did they fluoresce with dansylated incubation. When phosphatidylethanolamine was included in the liposomes, they became perturbed and fluoresced. Plasma membranes of Drosophila larval cells, containing or lacking cholesterol, were also disrupted by polymyxin. The cell membranes of guinea pig sperm were likewise disrupted but in specific functional areas. Fusional membrane domains showed protrusions; the stable membrane of the flagellum revealed diffuse bubbling. Regions of well-defined particle arrays and the postacrosomal segment maintained smooth contours. By fluorescence microscopy, we detected the same heterogeneous binding of the polymyxin dansyl derivative.     

HCV and the hepatic lipid pathway as a potential treatment target

Atherosclerosis has been described as a liver disease of the heart [1]. The liver is the central regulatory organ of lipid pathways but since dyslipidaemias are major contributors to cardiovascular disease and type 2 diabetes rather than liver disease, research in this area has not been a major focus for hepatologists. Virus-host interaction is a continuous co-evolutionary process [2] involving the host immune system and viral escape mechanisms [3]. One of the strategies HCV has adopted to escape immune clearance and establish persistent infection is to make use of hepatic lipid pathways. This review aims to: ? update the hepatologist on lipid metabolism ? review the evidence that HCV exploits hepatic lipid pathways to its advantage ? discuss approaches to targeting host lipid pathways as adjunctive therapy.     

Antibodies bound to lipid haptens in model membranes diffuse as rapidly as the lipids themselves.

A pattern photobleaching method has been used to measure the rates of lateral diffusion of fluorescent-labeled specific anti-nitroxide IgG bound with both combining sites to nitroxide-containing phospholipids in liposomal membranes composed of dimyristoyl phosphatidylcholine at 28 degrees C ("fluid"), dipalmitoyl phosphatidylcholine at 32 degrees C ("Solid"), and dipalmitoyl phosphatidylcholine containing 15 or 25 mol% cholesterol ("solid" or "fluid," respectively, at 32 degrees C). The diffusion coefficients of the bound immunoglobulin were found to be the same as those of fluorescent-labeled phospholipids in each case even though these diffusion coefficients range from 10(-11) to 10(-8) cm2/sec. Hapten-containing liposomal membranes of the type studied here have previously been shown to elicit a number of antibody-dependent immune responses. Therefore, this work indicates that membrane-bound but otherwise freely diffusing antibodies are sufficient for these reponses.     

Intrinsic curvature hypothesis for biomembrane lipid composition: a role for nonbilayer lipids.

A rationale is presented for the mix of "bilayer" and "nonbilayer" lipids, which occurs in biomembranes. A theory for the L alpha-HII phase transition and experimental tests of the theory are reviewed. It is suggested that the phase behavior is largely the result of a competition between the tendency for certain lipid monolayers to curl and the hydrocarbon packing strains that result. The tendency to curl is quantitatively given by the intrinsic radius of curvature, Ro, which minimizes the bending energy of a lipid monolayer. When bilayer (large Ro) and nonbilayer (small Ro) lipids are properly mixed, the resulting layer has a value of Ro that is at the critical edge of bilayer stability. In this case, bilayers may be destabilized by the protein-mediated introduction of hydrophobic molecules, such as dolichol. An x-ray diffraction investigation of the effect of dolichol on such a lipid mixture is described. This leads to the hypothesis that biomembranes homeostatically adjust their intrinsic curvatures to fall into an optimum range. Experimental strategies for testing the hypothesis are outlined.     

Mechanics of surface area regulation in cells examined with confined lipid membranes

Cells are wrapped in inelastic membranes, yet they can sustain large mechanical strains by regulating their area. The area regulation in cells is achieved either by membrane folding or by membrane exo- and endocytosis. These processes involve complex morphological transformations of the cell membrane, i.e., invagination, vesicle fusion, and fission, whose precise mechanisms are still under debate. Here we provide mechanistic insights into the area regulation of cell membranes, based on the previously neglected role of membrane confinement, as well as on the strain-induced membrane tension. Commonly, the membranes of mammalian and plant cells are not isolated, but rather they are adhered to an extracellular matrix, the cytoskeleton, and to other cell membranes. Using a lipid bilayer, coupled to an elastic sheet, we are able to demonstrate that, upon straining, the confined membrane is able to regulate passively its area. In particular, by stretching the elastic support, the bilayer laterally expands without rupture by fusing adhered lipid vesicles; upon compression, lipid tubes grow out of the membrane plane, thus reducing its area. These transformations are reversible, as we show using cycles of expansion and compression, and closely reproduce membrane processes found in cells during area regulation. Moreover, we demonstrate a new mechanism for the formation of lipid tubes in cells, which is driven by the membrane lateral compression and may therefore explain the various membrane tubules observed in shrinking cells.     

Characterization of lipid domains in erythrocyte membranes.

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.     

Protonophores induce plastoquinol oxidation and quench chloroplast fluorescence: Evidence for a cyclic, proton-conducting pathway in oxygenic photosynthesis

The photosynthetic apparatus converts light into chemical energy by a series of reactions that give rise to a coupled flow of electrons and protons that generate reducing power and ATP, respectively. A key intermediate in these reactions is plastoquinone (PQ), the most abundant electron and proton (hydrogen) carrier in photosynthetic membranes (thylakoids). PQ ultimately transfers electrons to a terminal electron acceptor by way of the Rieske Fe-S center of the cytochrome bf complex. In the absence of a terminal acceptor, electrons accumulate in the PQ pool, which is reduced to plastoquinol (PQH₂), and also on a specialized PQ, Q_A, which is reduced to an unprotonated semiquinone anion (Q_A^-). The accumulation of Q_A^- is measured by a rise in fluorescence yield and the accumulation of PQH₂ is measured by absorption difference spectrometry. We have found that in the absence of a terminal electron acceptor, two chemically diverse proton-conducting ionophores (protonophores), 2,6-di-t-butyl-4-(2′,2′-dicyanovinyl)phenol (SF 6847) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), induced oxidation of PQH₂ and quenching of chloroplast fluorescence, signifying oxidation of Q_A^-. The two protonophores produced the same effects even when the only recognized pathway of PQH₂ oxidation by way of the cytochrome bf complex was inhibited by dibromothymoquinone. Two other uncouplers, gramicidin and nigericin, which are not protonophores but facilitate proton movement across membranes by other mechanisms, were ineffective. These findings are consistent with the operation in the oxygen-generating photosystem (photosystem II) of a cyclic, proton-conducting pathway.     

Lipid advanced glycosylation: pathway for lipid oxidation in vivo.

To address potential mechanisms for oxidative modification of lipids in vivo, we investigated the possibility that phospholipids react directly with glucose to form advanced glycosylation end products (AGEs) that then initiate lipid oxidation. Phospholipid-linked AGEs formed readily in vitro, mimicking the absorbance, fluorescence, and immunochemical properties of AGEs that result from advanced glycosylation of proteins. Oxidation of unsaturated fatty acid residues, as assessed by reactive aldehyde formation, occurred at a rate that paralleled the rate of lipid advanced glycosylation. Aminoguanidine, an agent that prevents protein advanced glycosylation, inhibited both lipid advanced glycosylation and oxidative modification. Incubation of low density lipoprotein (LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components. Oxidized LDL formed concomitantly with AGE-modified LDL. Of significance, AGE ELISA analysis of LDL specimens isolated from diabetic individuals revealed increased levels of both apoprotein- and lipid-linked AGEs when compared to specimens obtained from normal, nondiabetic controls. Circulating levels of oxidized LDL were elevated in diabetic patients and correlated significantly with lipid AGE levels. These data support the concept that AGE oxidation plays an important and perhaps primary role in initiating lipid oxidation in vivo.     

Interaction of lymphocytes with lipid bilayer membranes: a model for lymphocyte-mediated lysis of target cells.

Horizontal lipid bilayer membranes were used as a model system to study lymphocyte-mediated killing of target cells. Dinitrophenylated lipid bilayers can physically support dozens of lymphocytes for periods of over one hour without breakage or increasing the electrical conductance of the membrane. However, in the presence of antibody against Dnp, human lymphocytes rapidly induced increases in membrane conductance of several orders of magnitude without membrane breakage. Such ionic permeability increases occurred only when the membrane voluage was positive on the lymphocyte side, as would be the case with a target cell membrane. The lymphocyte and antibody dependence of this conductance increase parallels that observed for lymphocyte killing of antibody-coated target cells. The results are interpreted as evidence that the primary event in lymphocyte killing of antibody-coated target cells is the creation of ion-conducting channels in the target membrane.     

Trypanosome variant surface glycoprotein transfer to target membranes: a model for the pathogenesis of trypanosomiasis.

The variant surface glycoprotein (VSG) of trypanosomes is attached to the cell surface by means of a phosphatidylinositol-containing glycolipid membrane anchor. The studies presented in this paper support the hypothesis that the transfer of VSG from trypanosomes to erythrocytes could lead to one of the pathological features associated with trypanosome infection--i.e., anemia. Migration of trypanosome VSG from live trypanosomes to target cells (sheep erythrocytes) could be shown by preincubating erythrocytes with trypanosomes and subsequently testing the washed erythrocytes for insertion of VSG by their susceptibility to lysis by complement in the presence of an anti-VSG antibody. Complement-mediated lysis was found to depend on the strain-specific anti-VSG antibody used. Extent of erythrocyte lysis increased with time of cell exposure to trypanosomes and with trypanosome concentration. No erythrocyte lysis was observed when trypanosomes were preincubated with anti-VSG antibody before adding erythrocytes. Purified membrane-form VSG (which retains the glycolipid anchor), but not soluble VSG (which no longer has the terminal diacylglycerol moiety), could sensitize erythrocytes to anti-VSG antibody-mediated complement lysis. The intermembrane transfer of VSG from trypanosomes to cells of the infected host could provide a molecular mechanism for the pathogenesis of trypanosomiasis.     

Interferon induction: a conformational hypothesis.

The ability of polynucleotides or polynucleotide duplexes such as poly(I).poly(C) to induce interferon production is proposed to depend on the existence of certain stable glycosidic orientations. It appears that a slight increase in instability of 1--3 kcal/mole (1 cal = 4.184 J) in the conformational regions near 20 degrees, 80 degrees, and 160 degrees leads to a loss of potency with respect to interferon induction. Thus, it is proposed that, for a polynucleotide to exist in the overall conformation necessary for interferon induction, stability of glycosidic orientations near 20 degrees, 80 degrees, and 160 degrees may be necessary to confer flexibility and activity on polynucleotide structures. This proposed conformational triad of stable conformational regions essential to interferon induction is based on the results of conformational energy calculations of the glycoside rotational profiles of adenosine, 7-deazaadenosine, inosine, and 7-deazainosine, as well as the conformational properties of other purine nucleoside analogs, and on inferences derived from calculations about the conformational effect in polynucleotides of removing the 2'-OH group.     

Evidence for conduction of protons along the interface between water and a polar lipid monolayer.

Movements of H+ along the polar heads of phospholipids spread in monolayers were compared to movements of H+ in the aqueous subphase. The probe for detecting H+ movement along the monolayer was a pH-sensitive fluorescein chromophore covalently bound to the head group of phosphatidylethanolamine. The behavior of this probe was not affected by the electrical properties of the lipid/water interface. Lateral diffusion of H+ along the phospholipid/water interface was then studied by acid-jump experiments in which advantage was taken of the large size of the monolayer. H+ was injected a few centimeters away from the probe observation area. The time needed for H+ diffusion to the probe was monitored by the change in the fluorescence signal, fluorescein being nonfluorescent in an acid medium. Diffusion of H+ in the bulk phase was monitored by the fluorescence change of water-soluble fluorescein isothiocyanate. Diffusion along the lipid monolayer was found to be 20 times faster than in the bulk water phase and required a structured monolayer in order to occur, as revealed by variation of the molecular area occupied by the lipid molecules. The molecular basis of rapid H+ transfer along the lipid monolayer may be the existence of a hydrogen-bond network along the polar heads, capable of supporting a rapid "hop and turn" of H+.     

Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes

Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3'-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of approximately 3 x 10(-10) cm(2)/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.     

Cronkhite Canada syndrome: a new hypothesis.

The occurrence of Cronkhite Canada syndrome in a 78 year old man is described. The presence of total gastrointestinal mucosal atrophy with nail loss is reported for the first time. It is suggested that the polyps represent residues of aged cells with no absorptive function and that the condition results from the loss of normal proliferative stimuli or acquired resistance to them. The primary biochemical abnormality may be in the affected epithelia but the changes here could alternatively be secondary to failure of synthesis or release of growth factors.     

The cytoskeleton and the regulation of gluconeogenesis: a hypothesis.

Previous data from our laboratory indicated a role for the cytoskeleton in the hormonal stimulation of gluconeogenesis. To gain further insight into the role of the cytoskeleton in the regulation of gluconeogenesis, we performed further experiments to examine the possibility that the cytoskeleton is involved in the glucagon-mediated changes in calcium distribution. Perfused livers or isolated liver cells were pretreated with either cytochalasin B or colchicine, agents that disrupt microfilaments and microtubules, respectively. Pretreatment with either agent significantly decreased the glucagon-evoked efflux of labeled calcium. Pretreatment with colchicine was more effective in blocking the influx of calcium into the cells than pretreatment with cytochalasin B. These drugs also prevented the characteristic increase in O(2) uptake, which is usually observed after glucagon administration. Thus, an intact cytoskeleton seems to be a prerequisite for the glucagon-evoked changes in calcium distribution to occur. These changes in calcium fluxes were shown previously to be an essential link in the chain of events leading to the metabolic effects of glucagon. Based on these results and on data obtained by others, a hypothesis is presented here. The hypothesis presumes that the cytoskeleton plays a crucial role in the regulation of gluconeogenesis. The hypothesis further assumes that the cytoskeleton influences gluconeogenesis in 3 ways: (1) by influencing the process of calcium signaling; (2) by changing the rate of enzymatic reactions through association and dissociation of enzymes with the cytoskeleton; and (3) by altering the position of intracellular organelles and the movements of molecules. Each of these points is discussed separately. It is known that the intracellular environment exists as a dense mesh in dynamic motion. According to our hypothesis, hormonal stimulation changes this environment by affecting the ionic composition in the cytosol and the structure of the cytoskeleton. Motion and conformational changes by the cytoskeleton play crucial regulatory functions, influencing metabolic processes.