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Biomedical subjects

David H Thompson

Publications and source records attributed to David H Thompson.

At least 19 recordsLinked to original sources
PubMed2006

Functional reconstitution of the integral membrane enzyme, isoprenylcysteine carboxyl methyltransferase, in synthetic bolalipid membrane vesicles.

Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (also known as bolalipids) have been prepared to determine (1) the influence of molecular structure on the physical properties of bolalipid membranes and (2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. Three bolalipids were synthesized: C20BAS, C32BAS, and C32phytBAS. These bolalipid structures differ in that the C20BAS derivative has a short sn-1 glyceryl diether C20H40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C32BAS and C32phytBAS derivatives have a longer sn-1 diether C32H64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C32BAS Tm > 85 degrees C; C32phytBAS Tm = 14 degrees C; and C20BAS Tm = 17 degrees C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 25 degrees C, were 1.5 x 10(-8) and 1.8 x 10(-9) cm2/s for C20BAS and C32phytBAS, respectively. The mobility of C32BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C20BAS/E. coli polar lipid, C20BAS/POPC, C32phytBAS/E. coli polar lipid, and C32phytBAS/POPC. Ste14p activity was lost in vesicles composed of 75-100 mol % C20BAS and 0-100 mol % C32BAS but retained in vesicles with 0-50 mol % C20BAS and 0-100 mol % C32phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol % C20BAS and 100 mol % C32phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C20BAS is only 32 A. Because Ste14p activity was not affected by either the gel-to-liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75-100 mol % C20BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p.

Cell Membrane↗
PubMed2006

Mechanism of the chiral SHG activity of bacteriorhodopsin films.

The nonlinear optical activity of bacteriorhodopsin (bR) Langmuir-Blodgett (LB) films were investigated computationally and experimentally. The second harmonic generation optical rotary dispersion (SHG-ORD) response was calculated directly from the known structure and orientation of the PSB retinal chromophore within bR with no adjustable parameters. The predicted results agree remarkably well in sign, magnitude, and trend with the experimental SHG-ORD measurements. The calculations indicated negligible chirality with the tensor for the PSB retinal chromophore, but significant chiral-specific activity for the thin film through a relatively simple orientational mechanism.

Bacteriorhodopsins↗
PubMed2006

pH-responsive movement of cucurbit[7]uril in a diblock polypseudorotaxane containing dimethyl beta-cyclodextrin and cucurbit[7]uril.

[Structure: see text] A polypseudorotaxane consisting of cucurbit[7]uril (CB[7])/N,N'-(3-phenylenebis(methylene)dipropargylamine (PMPA), [2]pseudorotaxane, and 2,6-O-dimethyl beta-cyclodextrin (DM-beta-CD)/alpha,omega-bisazidopropylene glycol 400 [2]pseudorotaxane was synthesized using the "click" reaction. The polypseudorotaxane structure was maintained in aqueous solution over a wide range of pH values with the DM-beta-CD units contributing to increased solubilization of the polypseudorotaxane without dethreading. The pH-responsive movement of the CB[7] units in the polypseudorotaxane was also observed.

Bridged-Ring Compounds↗
PubMed2006

Synthesis, characterization, and pH-triggered dethreading of alpha-cyclodextrin-poly(ethylene glycol) polyrotaxanes bearing cleavable endcaps.

The synthesis, characterization, and degradation kinetics of three alpha-cyclodextrin (alpha-CD)-poly(ethylene glycol) (PEG) polyrotaxanes with endcaps that were installed using Cu(I)-catalyzed Huisgen cyclization is reported. PEG1500, azidated with azidoacetic acid, was threaded with alpha-CD to form a pseudopolyrotaxane that was then capped in up to 82% yield with three different substituents to provide polyrotaxanes that were either acid-, base-, or fluoride-sensitive. NMR, GPC, XRD, and AFM methods were used to characterize the polyrotaxanes. Dethreading rates upon exposure to mild deprotection conditions were monitored by turbidity analysis. The vinyl ether-endcapped polyrotaxane is stable at pH 7 for 16 h but is solubilized at approximately 0.0211 min(-1) at pH 4. The ester-endcapped polyrotaxane is solubilized at 0.0122 min(-1) at pH 12.1. Our results show that pH-triggerable polyrotaxanes can be readily and efficiently prepared from pseudopolyrotaxanes in high yield by Huisgen cyclization of azido- and alkynyl-modified precursors in the presence of Cu(I).

Biocompatible Materials↗
PubMed2005

A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases.

We have developed a simple and sensitive fluorescence-based two-step coupled enzyme assay to report the activity of S-adenosylmethionine-dependent methyltransferases. This assay relies on a fluorescein-cystamine-methyl red (FL-S-S-MR) reporter molecule that can be activated by thiols. In the absence of thiols, fluorescence from the reporter is quenched through fluorescence resonance energy transfer between the two chromophores. In this report, we use catechol-O-methyltransferase with the addition of S-adenosylhomocysteine hydrolase to produce the thiol homocysteine. The presence of homocysteine leads to disulfide bond cleavage in the cystamine tether and fluorescence dequenching as the uncoupled chromophores are diluted into the surrounding media. The sensitivity and specificity of FL-S-S-MR to thiols enabled detection of <or=1 microM concentrations of homocysteine, suggesting that this assay is sensitive enough to detect biologically relevant amounts of homocysteine. We believe that this fluorescence reporter approach may be generalizable to all enzymatic or chemical assays that produce thiols.

Cystamine↗
PubMed2003

Acid-triggered release via dePEGylation of DOPE liposomes containing acid-labile vinyl ether PEG-lipids.

Four structurally related, acid-labile polyethylene glycol (PEG) conjugated vinyl ether lipids have been synthesized and used at low molar ratios to stabilize the nonlamellar, highly fusogenic lipid, dioleoylphosphatidyl ethanolamine, as unilamellar liposomes. Acid-catalyzed hydrolysis of the vinyl ether bond destabilized these liposomes by removal of the sterically-stabilizing PEG layer, thereby promoting contents release on the hours timescale at pH<5. Structure-property correlations of these compounds suggested that single vinyl ether linkages between the PEG headgroup and the lipid backbone produce faster leakage rates. These studies also suggested that the presence of a slight negative charge at the membrane surface can accelerate the acid-catalyzed leakage process.

Acids↗
PubMed2003

Direct synthesis of plasmenylcholine from allyl-substituted glycerols.

We report a new method for the facile preparation of plasmenylcholine via reaction of lithioalkoxy allyl intermediates with 1-iodoalkanes as the key step in the stereoselective formation of 1'-(Z)-alkenyl glyceryl ethers. The allyl anion intermediate is prepared by treating mono- or disiloxy-protected 1-allylglycerol precursors with s-BuLi at -65 to -80 degrees C. Subsequent addition of 1-iodoalkane solutions at low temperature gives moderate yields of gamma-coupled, Z-vinyl ethers as the major product and alpha-coupled product as the minor component. Several different preparative strategies for the total synthesis of plasmalogens are enabled by this simple transformation.

Glycerol↗
PubMed2003

Interactions between pH-sensitive liposomes and model membranes.

The structure and dynamics of two different pH-sensitive liposome systems were investigated by means of cryo-transmission electron microscopy and different photophysical techniques. Both systems consisted of dioleoylphosphatidylethanolamine (DOPE) and contained either oleic acid (OA) or a novel acid-labile polyethylene glycol-conjugated lipid (DHCho-MPEG5000) as stabiliser. Proton induced leakage, lipid mixing and structural changes were studied in the absence and presence of EPC liposomes, as well as in the presence of liposomes designed to model the endosome membrane. Neither DHCho-MPEG5000- nor OA-stabilised liposomes showed any tendency for fusion with pure EPC liposomes or endosome-like liposomes composed of EPC/DOPE/SM/Cho (40/20/6/34 mol.%). Our investigations showed, however, that incorporation of lipids from the pH-sensitive liposomes into the endosome membrane may lead to increased permeability and formation of non-lamellar structures. Taken together the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE, seems more plausible.

Cell Membrane↗
PubMed2003

Preparation of liposomes with glucose binding sites: liposomes containing di-branched amino acid derivatives.

Di-branched amino acid derivatives, such as dihexadecyl-glutamate-glutamine (DHD-glu-gln), dihexadecyl-glutamate-asparagine (DHD-glu-asn) and dihexadecyl-glutamate-glutamic acid (DHD-glu-glu), were synthesized, and then incorporated into lipid vesicles (liposomes) using dipalmitoylphosphatidylcholine (DPPC). To form binding sites toward glucose, the liposomes containing amino acid derivatives were mixed with glucose above the phase transition temperature (PTT) of DPPC and subsequently the temperature was lowered below the PTT. The glucose-binding affinity of liposomes containing amino acid derivative with or without glucose imprinting was evaluated by surface plasmon resonance (SPR) and equilibrium dialysis technique. SPR of liposomes containing each amino acid derivative or three amino acid derivatives revealed that only the liposomes containing all three amino acid derivatives had glucose-binding affinity and that the glucose-imprinting process was essential to fix the amino acid derivatives into a glucose binding site on the liposomes. Equilibrium dialysis studies of glucose-imprinted liposomes produced curvilinear Scatchard plots, indicating that the amino acid derivatives play a role in glucose binding. Di-branched amino acid derivatives synthesized in this study are promising agent for the development of biocompatible synthetic glucose binding materials.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

Optical imaging of metastatic tumors using a folate-targeted fluorescent probe.

We describe the use of a tumor targeting ligand, the vitamin folic acid, to deliver an attached fluorescent probe to both primary and metastatic tumors overexpressing the folate receptor. Upon laser excitation, derived images of normal tissues generally show little or no fluorescence, whereas images of folate receptor-expressing tumors display bright fluorescence that can be easily distinguished from adjacent normal tissue. Furthermore, metastatic tumor loci of submillimeter size can also be visualized without the aid of image processing or enhancement. The sharp distinction between tumor and normal tissues provided by this technique could find application in the localization and resection of tumor tissue during surgery or in the enhanced endoscopic detection and staging of cancers.

Animals↗
PubMed2003

Structural characterization of plasmenylcholine photooxidation products.

Oxidative damage to plasmenyl-type lipids contributes to decreased membrane barrier function, loss of membrane structure and formation of nonlamellar defects in membrane bilayers. Previous results from this laboratory have shown that membrane-soluble sensitizers (e.g. zinc phthalocyanine and bacteriochlorophyll a) mediate the photooxidation of palmitoyl plasmenylcholine (1-O-alk-1'-Z-enyl-2-palmitoyl-sn-glycero-3-phosphocholine; PPlsC) vesicles with the subsequent creation of lamellar defect structures, vesicle contents leakage and membrane-membrane fusion. Because plasmalogen lipids are significant components of sarcoplasma and myelin membranes, we sought to characterize the products of their photooxidation. This study focuses on the photooxidation of PPlsC vesicles in the presence of the water-soluble sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS4(4-)). Attack of photogenerated singlet oxygen on the 1-O-alkenyl ether linkage of PPlsC lipids was expected to generate dioxetane- and ene-type photoproducts. The products formed during continuous aerobic irradiation (28 mW/cm2, (610 nm) of PPlsC vesicles in the presence of AlPcS4(4-) were separated via reverse-phase high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) or evaporative light-scattering detection (ELSD). Photooxidized dipalmitoyl-phosphatidylcholine-cholesterol vesicles (control) were used to optimize the HPLC-ECD conditions, using 7 alpha-hydroperoxy-cholesterol as standard. HPLC-ECD was found to be most sensitive for PPlsC hydroperoxides, whereas HPLC-ELSD was more sensitive for nonhydroperoxide photoproducts. The three major photoproducts formed during vesicle irradiation were isolated via preparative HPLC and then characterized by 1H-nuclear magnetic resonance and mass spectrometry. 1-Formyl-2-palmitoyl-sn-glycero-3-phosphocholine and 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine were identified as dioxetane cleavage products that coeluted at approximately 3 min. The second fraction (retention time [RT] = 48 min) was identified as a PPlsC allylic hydroperoxide. The third photoproduct, eluting at RT = 64 min, is tentatively identified as an oxidation product arising from allylic hydroperoxide degradation via Hock rearrangement or free radical decomposition.

Chromatography, High Pressure Liquid↗
PubMed2002

Facile synthesis of plasmalogens via Barbier-type reactions of vinyl dioxanes and vinyl dioxolanes with alkyl halides in LiDBB solution.

Plasmalogens (i.e. plasmenylcholines or plasmenylethanolamines) are a biologically important class of glycerophospholipids that have been difficult to synthesize due to the presence of an acid and oxidatively labile (Z)-vinyl ether substituent at the sn-1 position and a base-labile sn-2 acyl substituent that easily migrates during silica gel purification. We report two facile synthetic methods for the preparation of racemic plasmenylcholines via a tandem reductive vinyl dioxane/dioxolane ring opening and alkyliodide coupling process that proceeds in a single pot reaction. The key step in the formation of (Z)-vinyl ether precursors for the production of plasmenylcholines is accomplished using LiDBB under Barbier-type conditions to give the corresponding TBDMS-protected 1-O-Z'-vinylglycerol intermediate in moderate yields. This pathway is the most direct synthetic route for the formation of plasmenylcholines to date, requiring a total of six transformations from acrolein and glycerol or solketal as inexpensive starting materials, to generate glycerophosphocholine-type plasmalogens in 4% overall yield.

Catalysis↗
PubMed2002

Size and structure of spontaneously forming liposomes in lipid/PEG-lipid mixtures.

The optimal size and structure of spontaneous liposomes formed from lipid/polymer-lipid mixtures was calculated using a molecular mean-field theory. The equilibrium properties of the aggregate are obtained by expanding the free energy of a symmetric planar bilayer up to fourth order in curvature and composition of lipid and polymer. The expansion coefficients are obtained from a molecular theory that explicitly accounts for the conformational degrees of freedom of the hydrophobic tails of the lipid and of the polymer chains. The polar headgroup interactions are treated using the opposing forces model. The onset of stability of the symmetric planar film is obtained from the expansion up to quadratic order. For unstable planar films the equilibrium size and structure of the spherical aggregates is obtained from the second- and fourth-order terms in curvature and composition of lipid and polymer. The driving force for the formation of spontaneous vesicles is the asymmetric distribution of polymers between the inner and outer monolayer. The composition asymmetry between the two monolayers in the aggregates is much larger for the polymer component than for the lipid, and it depends upon the size of the aggregate. The smaller the aggregate, the more asymmetric the distribution of polymer and lipid. The tendency of the polymer chains to be tethered on the outer surface of the aggregate is very strong, and it limits the range of polymer loading for which spherical liposomes are stable. A very small excess of polymer loading causes small spherical micelles to be the optimal aggregates. In these cases spontaneous liposomes can form as metastable aggregates, showing as a local minima in the free energy. Even for metastable aggregates the asymmetric distribution of polymers is very large. The elastic constants of the asymmetric bilayer in the spherical aggregate are found to be the same as those that are calculated from the planar symmetric film. Therefore, the stable structure of the aggregate is not needed to determine its mechanical properties. The range of stable liposomes is very narrow in the range of molecular weights studied, which include the experimental relevant domain of aggregates used in drug delivery. It is found that the stability of the spherical aggregates results from a very fine balance between the tendency of the polymer chains and lipid tails to pack in an asymmetric spherical aggregate and the tendency of the hydrophobic-water interface to keep the area per molecule fixed. The changes in free energy per molecules that are responsible for liposome formation are very small and are very sensitive to detailed molecular properties. The theoretical description of the aggregates requires a theory capable of incorporating these detailed molecular properties. The findings are discussed in the context of vesicle formation and liposome design for drug delivery.

Biophysical Phenomena↗
PubMed2002

Formation of plasmid-based transfection complexes with an acid-labile cationic lipid: characterization of in vitro and in vivo gene transfer.

PURPOSE: This study tests the hypothesis that gene transfer efficiency may be improved through the use of transiently stable transfection complexes that degrade within endosomal compartments and promote plasmid escape into the cytosol. METHOD: An acid labile cationic lipid, O-(2R-1,2-di-O-(1'Z, 9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), was designed, synthesized, and tested for enhanced gene transfer activity relative to non-labile controls. RESULTS: The O-alkenyl chains of BCAT were completely hydrolyzed after 4 h incubation in pH 4.5 buffer at 25 degrees C. Addition of BCAT to plasmid DNA in 40% ethanol followed by ethanol evaporation yielded transfection complexes that transfected several cell types in the presence of fetal calf serum and without the need of a helper lipid. Transfection complexes prepared from BCAT displayed higher luciferase expression than the corresponding DCAT complexes (an acid-insensitive derivative of BCAT) for all cell types tested. Uptake studies showed that this increase was not due to a difference in the amount of DNA being delivered. FAGS analysis for GFP expression showed that BCAT transfection complexes yielded 1.6 more transfected cells and 20% higher log mean fluorescence than DCAT transfection complexes. In vivo gene transfer was demonstrated in subcutaneous tumor-bearing mice by systemic administration of a 60 microg plasmid dose. Expression was observed in the lungs and in the tumor, with the highest activity being observed in the lungs. CONCLUSIONS: Our results show that increased transfection can be obtained by coupling the cationic headgroup to the hydrophobic amphiphilic tails via acid-labile bonds. Acid-catalyzed release of the alkyl chains should facilitate dissociation of the cationic lipid headgroup from the plasmid, thus accelerating one of the rate-limiting steps in cationic lipid mediated transfection.

3T3 Cells↗
PubMedDate not supplied

Formation of fibrinogen-based hydrogels using phototriggerable diplasmalogen liposomes.

We report the triggered release of Ca2+ from liposomal compartments to induce rapid gelation of protein-based hydrogels. Phototriggerable liposomes were designed by entrapping CaCl(2) within liposomes composed of 38:57:5 diplasmenylcholine (DPPlsC):disteroylphosphatidylcholine (DSPC):bacteriochlorophyll (Bchl). These liposomes release >80% of their entrapped Ca2+ within 15 min when irradiated at 800 nm (800 mW/cm2). A precursor solution, containing liposomes suspended in aqueous human fibrinogen and transglutaminase (TGase), remained fluid for several hours in the dark, but gelled rapidly when exposed to 800 nm excitation, as a result of photosensitized Ca2+ release and TG-induced fibrinogen cross-linking. TGase and hrFXIII activities, determined using a fluorimetric dansylcadaverine assay, were found to depend strongly on irradiation time and reaction temperature. SDS-PAGE of the photolyzed reaction mixture revealed that gelation arises from enzyme-catalyzed cross-linking of predominately the alpha and gamma chains of fibrinogen. This approach to the phototriggered formation of protein hydrogels creates new opportunities for biomaterials applications in drug delivery, tissue engineering, and wound healing.

Calcium↗
PubMedDate not supplied

Inhibition of membrane-associated methyltransferases by a cholesterol-based metal chelator.

We have designed, synthesized, and characterized a metal chelating compound that is based on the structure of cholesterol and contains the high affinity metal chelating group, lysine nitrilotriacetic acid (Lys-NTA). Using the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt) from yeast as a model integral membrane metalloenzyme, we find that this agent potently inhibits Icmt activity with an IC(50) value between 35 and 75 microM, which is at least 40 times more potent than the best known Icmt metal chelating inhibitor, Zincon. We propose that the rigid hydrophobic cholesterol moiety promotes partitioning into the membrane, enabling the metal-binding NTA group(s) to inactivate the enzyme by metal chelation. Because this compound is based on a naturally occurring membrane lipid and appears to chelate metals buried deeply within water insoluble environments, this agent may also be useful as a general tool for identifying previously unappreciated metal dependencies of other classes of membrane proteins.

Cell Membrane↗