INAUGURAL ARTICLE by a Recently Elected Academy Member:Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Synthetic lipid–oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5′-coupled conjugate and the other the 3′-coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane–membrane spacing on fusion, a series of conjugates was constructed by adding 2–24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.     

Sterols block binding of COPII proteins to SCAP,thereby controlling SCAP sorting in ER

Sterols inhibit their own synthesis in mammalian cells by blocking the vesicular endoplasmic reticulum-to-Golgi transport of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), a sterol-sensing protein that escorts SREBPs. Unable to reach the Golgi, SREBPs are not processed by Golgi-resident proteases, and they fail to activate genes required for cholesterol synthesis. The current studies were designed to reveal whether sterols block SCAP movement by inhibiting synthesis of special vesicles dedicated to SCAP, or whether sterols block SCAP incorporation into common coat protein (COP)II-coated vesicles. Through immunoisolation, we show that SCAP-containing vesicles, formed in vitro, also contain vesicular stomatitis virus glycoprotein (VSVG) protein, a classic marker of COPII-coated vesicles. Sterols selectively block incorporation of SCAP into these vesicles without blocking incorporation of VSVG protein. We show that the mammalian vesicular budding reaction can be reconstituted by recombinant yeast COPII proteins that support incorporation of SCAP as well as VSVG into vesicles. Sterols block SCAP incorporation into vesicles by blocking Sar1-dependent binding of the COPII proteins Sec 23/24 to SCAP. These studies demonstrate feedback control of a biosynthetic pathway by the regulated binding of COPII proteins to an endoplasmic reticulum-to-Golgi transport protein.     

Dynamic transport of SNARE proteins in the Golgi apparatus

Localization of a membrane protein in a subcellular compartment can be achieved by its retention in the compartment or by its continuous transport toward this compartment. Previous results have suggested that specific enzymes are localized in the Golgi apparatus at least in part by selective retention and exclusion from transport vesicles. However, the function of some Golgi SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins is not compatible with their exclusion from transport vesicles. To help understand the mechanism accounting for the localization of SNARE proteins in the Golgi apparatus, we analyzed their lateral distribution in the Golgi cisternae and their incorporation into transport vesicles. According to our results, all SNARE proteins are efficiently incorporated into transport vesicles, indicating that the localization of SNARE proteins in the Golgi apparatus is not based on a static retention mechanism. Detailed analysis suggested that incorporation into transport vesicles was more efficient for SNARE proteins restricted to the cis face of the Golgi as compared with SNAREs present at the trans face. Furthermore, overexpression of a cis-Golgi SNARE protein altered concomitantly its incorporation in transport vesicles and its intra-Golgi localization. These observations suggest that, contrary to resident Golgi enzymes, SNARE proteins are localized in the Golgi apparatus as the result of a dynamic transport equilibrium.     

内皮细胞脂质过氧化损伤与平滑肌细胞增殖的关系

用联胺作用于培养的人脐静脉内皮细胞引起其脂质过氧化损伤，用抗碱性纤维母细胞生长因子单克隆抗体和抗血小板源性生长因子ＢＢ多克隆抗体进行免疫组织化学染色，以检测内皮细胞暴露于联胺前后和ＰＤＧＦ－ＢＢ的表达，以及其条件培养基对平同细胞内ｂＦＧＦ表达的影响。用＾３Ｈ－ＴｄＲ掺入法观察内皮细胞暴露于联胺后其条件培养基对ＳＭＣ是否有增殖活性，结果显示，联胺引起内皮细胞脂质过氧化损伤后，其ＰＤＧＦ－ＢＢ和ｂＦＧ     

脂质过氧化对血管内皮细胞源性生长因子合成和基因表达的影响

本研究以氢过氧化枯烯作为脂质过氧化反应的引发剂，作用于培养的牛主动脉内皮细胞，制备内皮细胞条件培养液，测定基对Ｓｗｉｓｓ３Ｔ３细胞ＤＮＡ合成的影响；并以抗血小板源性生长因子抗体中和实验和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析测定了脂质过氧化作用对内皮细胞ＰＤＧＦ产生的影响。     

Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers.

Various methods to incorporate bacteriorhodopsin in black lipid membranes are reported. Both purple membrane patches and monomeric bacteriorhodopsin were used as starting material. The incorporation of bacteriorhodopsin into planar lipid bilayers was achieved by the following methods. (i) Purple membrane patches were transferred from water to solutions of lipids in n-alkanes. Black membranes were formed from such organic suspensions. (ii) Lipid layers containing solvent and purple membranes were spread on an air/water interface. These layers were used to form planar bilayers. (iii) Vesicles containing purple membranes or monomeric bacteriorhodopsin were spread on an air/water interface and, from the resulting layer, bilayers were formed. On illumination, steady-state photocurrents were observed in all three cases, indicating that these methods lead to functional transmembranous integration of the protein in the planar black lipid membrane. The influence of an applied electric field on the pumping process was studied on membranes formed by using method i. At approximately 200 mV, the photocurrent tends to zero. Furthermore, it was possible to make planar lipid bilayers photoelectrically active by adding vesicles containing monomeric bacteriorhodopsin to the bathing solution. Because, in this case, only transient photocurrents were observed, it can be concluded that the vesicles are attached to but not fused with the black lipid membrane.     

Stability of carbohydrate-modified vesicles in vivo: comparative effects of ceramide and cholesterol glycoconjugates.

The stability and tissue distribution of lipid vesicles modified at the surface by the incorporation of either a galactosyl ceramide (GalCer) or a galactosyl cholesterol (GalChol) glycoconjugate have been studied in mice by measuring the release of vesicle-entrapped 111In. Although the tissue distributions of both vesicle types were similar, the GalCer-containing vesicles were markedly less stable than those prepared with GalChol, whether administered orally or by intraperitoneal injection. Physical characterization of the vesicles in vitro suggests that the increased disruption rate for GalCer vesicles in vivo is related to structural instabilities induced by the cerebroside, which can then result in either an increased rate of vesicle uptake by tissues or a greater susceptibility to lysis. These studies demonstrate the importance of the nonpolar anchoring groups in determining the fate of surface-modified vesicles in vivo.     

Lung phospholipids during recovery from oxygen toxicity are altered by hydrocortisone

Corticosteroids affect type II alveolar epithelial cells, increase the lung's content of lipids associated with pulmonary surfactant, and have been used to treat the adult respiratory distress syndrome. We used hyperoxia to produce diffuse alveolar damage in rats and then determined the contents of lung lipids and the incorporation of precursors into these lipids while the rats recovered in air and received either hydrocortisone or saline injections. With saline injections (control) the total lipid content after hyperoxia and 48 hours recovery in air was nearly twice the lipid content of lungs never exposed to hyperoxia; many lipids, including phosphatidylcholine (PC), shared in this increase. With hydrocortisone injections the total lipid content, when expressed per lung, was not significantly greater than with saline injections but when expressed per mg DNA several lipid subgroups, including phosphatidylcholine, were significantly greater. Palmitate incorporation into most lipids of lung slices was not significantly different by 48 hours of recovery, and hydrocortisone had a modest effect after 96 hours. In contrast to the incorporation of palmitate, the incorporation of glycerol and of lysophosphatidylcholine (LPC) nearly doubled in most lipids during the recovery phase. Hydrocortisone injections were associated with greater glycerol incorporation after 96 hours of recovery in nearly all lipids and LPC incorporation was increased primarily into PC. We conclude that lung lipids increase markedly during recovery from severe oxygen toxicity and that hydrocortisone leads to additional increases of saturated and unsaturated PC.     

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.     

Distribution and Fate of Synthetic Lipid Vesicles in the Mouse: A Combined Radionuclide and Spin Label Study

Single compartmental spherules of various lipid constituents (vesicles), enclosing (99m)TcO(4) (-) as a radioactive marker, were injected intravenously into C(3)H mice, and the distribution of radioactivity was studied. About 25% of the administered radioactivity was present in the liver 5 min and 30 min after the injection of vesicles composed of phosphatidylcholine and gangliosides, which were sonicated for 5 min (standard preparation). About 10-20% of the radioactivity remained in the circulation. By use of a nonradioactive spin label (tempocholine) enclosed within vesicles, intact vesicles were demonstrated in the circulation for 46 min after intravenous injection. The distribution of radioactivity from (99m)TcO(4) (-) inside vesicles is very different from that of free (99m)TcO(4) (-) or of (99m)Tc sulfur colloid.Increase in the length of sonication or incorporation of cholesterol into the wall of the vesicles enhanced hepatic levels and reduced blood levels of radioactivity. These same manipulations also slowed the rate of transfer of (99m)TcO(4) (-) out of vesicles in dialysis experiments in vitro. Addition of phosphatidic acid, phosphatidylethanolamine, or phosphatidylserine to the standard constituents did not greatly alter the distribution of radioactivity in vivo but did increase the number and type of active coupling sites on the outside of the vesicle. The results indicate that vesicles might be valuable as carriers of diagnostic or therapeutic agents.     

Unilamellar vesicle formation and encapsulation by microfluidic jetting

Compartmentalization of biomolecules within lipid membranes is a fundamental requirement of living systems and an essential feature of many pharmaceutical therapies. However, applications of membrane-enclosed solutions of proteins, DNA, and other biologically active compounds have been limited by the difficulty of forming unilamellar vesicles with controlled contents in a repeatable manner. Here, we demonstrate a method for simultaneously creating and loading giant unilamellar vesicles (GUVs) using a pulsed microfluidic jet. Akin to blowing a bubble, the microfluidic jet deforms a planar lipid bilayer into a vesicle that is filled with solution from the jet and separates from the planar bilayer. In contrast with existing techniques, our method rapidly generates multiple monodisperse, unilamellar vesicles containing solutions of unrestricted composition and molecular weight. Using the microfluidic jetting technique, we demonstrate repeatable encapsulation of 500-nm particles into GUVs and show that functional pore proteins can be incorporated into the vesicle membrane to mediate transport. The ability of microfluidic jetting to controllably encapsulate solutions inside of GUVs creates new opportunities for the study and use of compartmentalized biomolecular systems in science, industry, and medicine.     

Direct activation of cGMP-dependent channels of retinal rods by the cGMP phosphodiesterase.

The cationic conductances of purified bovine retinal rod membranes were studied by incorporation of vesicles into planar lipid bilayers. When the membranes were stripped of all peripheral proteins [guanine nucleotide-binding protein (G protein) and cGMP phosphodiesterase (3',5'-cyclic-GMP 5'-nucleotidohydrolase), EC 3.1.4.35], sodium and calcium fluxes were almost only observed in the presence of cGMP. Reconstitution experiments in which purified cGMP phosphodiesterase alone or with G protein were reassociated to the vesicles in proportions similar to those found in the native rod provide evidence for a direct interaction between the cGMP-dependent channel protein and the phosphodiesterase. (i) In its inhibited state, phosphodiesterase markedly stimulates the activity of the channels in the presence of cGMP (situation in the dark-adapted rod) but is not capable of activating the channels in the absence of cGMP. (ii) In the absence of cGMP, activation of the phosphodiesterase by G protein with GTP bound (equivalent to photoexcitation) induces the opening of cation channels that have the same conductance for sodium ions as cGMP-activated channels (20-22 pS, with two sublevels of about 7 pS and 13 pS).     

Alcohol Causes a Fatty Pancreas. A Rat Model of Ethanol-Induced Pancreatic Steatosis

To develop an animal model of alcoholic pancreatic steatosis, female Wistar rats were pair fed liquid diets, containing ethanol as 36% of calories or an isocaloric amount of carbohydrate for 3 weeks. Electron microscopic examination showed lipid vesicles localized principally at the bases of pancreatic acinar cells in the ethanol-fed rats. Ethanol feeding significantly increased pancreatic content of cholesteryl ester without changing levels of other lipids. Ethanol feeding enhanced labeled acetate, palmitate, oleate, and lino-leate incorporation into cholesteryl ester. Therefore, increased esterification of cholesterol may, in part, explain the observed accumulation of cholesteryl ester.     

Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers

Membrane–membrane recognition and binding are crucial in many biological processes. We report an approach to studying the dynamics of such reactions by using DNA-tethered vesicles as a general scaffold for displaying membrane components. This system was used to characterize the docking reaction between two populations of tethered vesicles that display complementary DNA. Deposition of vesicles onto a supported lipid bilayer was performed by using a microfluidic device to prevent mixing of the vesicles in bulk during sample preparation. Once tethered onto the surface, vesicles mixed via two-dimensional diffusion. DNA-mediated docking of two reacting vesicles results in their colocalization after collision and their subsequent tandem motion. Individual docking events and population kinetics were observed via epifluorescence microscopy. A lattice-diffusion simulation was implemented to extract from experimental data the probability, P_dock, that a collision leads to docking. For individual vesicles displaying small numbers of docking DNA, P_dock shows a first-order relationship with copy number as well as a strong dependence on the DNA sequence. Both trends are explained by a model that includes both tethered vesicle diffusion on the supported bilayer and docking DNA diffusion over each vesicle's surface. These results provide the basis for the application of tethered vesicles to study other membrane reactions including protein-mediated docking and fusion.     

Vitamin E distribution and modulation of the physical state and function of pulmonary endothelial cell membranes

Vitamin E, a dietary antioxidant, is thought to incorporate into the lipid bilayer of biological membranes. We evaluated the lipid composition and distribution of [3H]-vitamin E in various membranes of pulmonary endothelial cells and determined whether vitamin E incorporation caused alterations in membrane structure and function in these cells. Following 6-, 12-, 18-, 24-, and 48-h incubation periods, vitamin E incorporation values were 3.0, 5.7, 6.9, 7.2, and 6.8 nmol/mg protein or 3.8, 7.3, 8.8, 9.2, and 8.7 nmol/mg phospholipid in mitochondrial membranes and 2.0, 4.4, 5.2, 5.3, and 5.0 nmol/mg protein or 3.5, 7.7, 9.1, 9.3, and 8.8 nmol/mg phospholipid in microsomal membranes, respectively. Vitamin E incorporation into the plasma membranes was greater than in mitochondrial and microsomal membranes after 12-, 24-, and 48-h incubations (18.9, 20.8, and 19.6 nmol/mg protein, respectively [P less than .001] versus mitochondria and microsomes or 12.2, 13.4, and 12.6 nmol/mg phospholipid, respectively [P less than .05] versus mitochondria and microsomes). The total phospholipid content, as well as the unsaturation index of the fatty acid content of these membranes, were in the same order, (i.e., plasma membrane greater than mitochondrial membranes and microsomal membranes). The physical state of the intact plasma membrane and the mitochondrial and microsomal membranes were measured by monitoring fluorescence anisotropies (rs) of the molecular probes, diphenylhexatriene (DPH) and trimethylamino-DPH (TMA-DPH). Vitamin E incorporation caused significant increases in rs for DPH (P less than .01) and TMA-DPH (P less than .01) in all three membranes compared to controls. Similar increases in rs values for DPH and TMA-DPH were observed in lipid vesicles prepared from these membranes. Following vitamin E incorporation, 5-hydroxytryptamine (5-HT) transport was measured as an index of plasma membrane function. Vitamin E incorporation resulted in an 18% reduction (P less than .05) in 5-HT uptake. These results indicate that vitamin E was distributed nonuniformly in endothelial cell membranes but resulted in comparable decreases in fluidity in all three membranes. In addition to its role as an antioxidant, vitamin E may alter the membrane physical state and modulate a variety of endothelial cell functions, including 5-HT transport.     

脂质过氧化对血管内皮细胞促增殖活性的影响

为探讨脂质过氧化对血管内皮细胞源生长因子的影响，以氢过氧化枯烯作用于培养的牛主动脉内皮细胞后，收集内皮细胞条件培养基，并用肝素亲和层析法处理，分组测定其对3T3细胞3H-胸腺嘧啶核苷掺入率的影响，并用放射免疫法测定各脂质过氧化作用组内皮细胞培养基中内皮素的含量。结果发现．氢过氧化枯烯使培养的内皮细胞的脂过氧化物蓄积增加，且内皮细胞条件培养基对3T3细胞的DNA合成有促进作用．对Swiss3T3细胞的促增殖活性主要存在于非肝素结合部分。氢过氧化枯烯作用后的内皮细胞的内皮素分泌水平比对照组增加。由此推测脂质过氧化作用引起的内皮细胞对平滑肌细胞的促增殖活性可能与内皮素等非肝素结合的分子有关。     

碱性纤维母细胞生长因子对血管平滑肌细胞增殖的影响

应用＾３Ｈ－ＲｄＲ掺入实验，ＲＮＡ印迹分析和反转录－多降酶链反应等方法观察碱性纤维母细胞生长因子对大鼠血管平滑肌细胞ＤＮＡ合成及细胞增殖相关基因表达的影响，结果发现，碱性纤维母细胞增殖相关基因表达的影响，结果发现，碱性纤维母细胞生长因子作用于血管平滑肌细胞６ｈ后，＾３Ｈ－ＴｄＲ掺入开始增加，２４ｈ达到高峰，在一定浓度范围内，碱性纤维母细胞生长因子以血管平滑肌细胞的促增殖效应与浓度呈正相关。碱性纤维     

脂质过氧化对血管内皮细胞促增殖活性的影响

为探讨脂质过氧化对血管内皮细胞源生长因子的影响，以氢过氧化枯烯作用于培养的牛主动脉内皮细胞后，收集内皮细胞条件培养基，并用肝素亲和层析法处理，分组测定其对３Ｔ３细胞＾３Ｈ－胸腺嘧啶核苷掺入率的影响，并用放射免疫法测定各脂质过氧化作用组内皮细胞培养基中内皮素的含量。结果发现，氢过氧化枯烯使培养的内皮细胞的脂过氧化物蓄积增加，且内皮细胞条件培养基对３Ｔ３细胞的ＤＮＡ合成有促进作用，对Ｓｗｉｓｓ３Ｔ３细     

Allogeneic cytolysis of reconstituted membrane vesicles.

The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described. Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 51Cr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets.