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C Pidgeon

Publications and source records attributed to C Pidgeon.

At least 37 records · Page 2Linked to original sources
PubMed1994

Membrane properties of antiviral phospholipids containing heteroatoms in the acyl chains.

Phospholipids containing heteroatoms in the lipid acyl chains, e.g., 1,2-bis(12-methoxydodecanoyl)-sn-3-phosphocholine (L-AC2), exhibit potent anti-HIV activity [Pidgeon, C., Markovich, R. J., Liu, M. D., Holzer, T., Novak, R., & Keyer, K. (1993) J. Biol. Chem. 268, 7773-7778]. AC2 is a synthetic chemical analog of the long-chain phospholipid, dimyristoylphosphatidylcholine (DMPC). Sonicated AC2 lipid dispersions would not entrap either Dextran-4000 or Mn2+ used as aqueous space markers. The lack of entrapment of aqueous space markers indicates that the AC2 structures do not contain an aqueous core that is the characteristic morphology of conventional lipid vesicles formed by sonication. Transmission electron microscopy (TEM) showed that sonicated AC2 lipid dispersions are small homogeneous particles approximately 70-100 A in diameter. 1H NMR experiments using Mn2+ as a broadening reagent indicated that Mn2+ was accessible to all of the AC2 phospholipid headgroups in the AC2 lipid particles formed by sonication. The temperature dependence of 1H spin-lattice (T1) relaxation time measurements revealed that the motional activation energies increased from the choline headgroup to the end of the acyl chains of AC2 molecules in the AC2 lipid particles formed by sonication. Collectively these results demonstrate that AC2 forms micelles. NOESY experiments showed that the AC2 molecules forming the micelle structures have hindered motion compared to conventional short-chain phosphatidylcholine micelles. 31P NMR spectroscopy and TEM showed that the AC2 micelles extensively fuse into giant bilayer liposomes (single-layered) when the temperature is reduced from above to below the main phase transition temperature of AC2. This micelle-to-liposome transition is an irreversible process; increasing the temperature above the Tm does not cause the formation of micelles. Thus, a main finding is that AC2 micelles formed by sonication are not thermodynamically stable because they fuse into large unilamellar vesicles that are stable to further changes in temperature. These unusual membrane properties of sonicated AC2 dispersions may be important for the antiviral activity and metabolism of the phospholipids.

Antiviral Agents↗
PubMed1993

Antiviral phospholipids. Anti-HIV drugs conjugated to the glycerobackbone of phospholipids.

Heteroatom fatty acid analogs of myristic acid containing oxygen or sulfur substituted for the alkyl methylene groups inhibit replication of the human immunodeficiency virus (HIV) in infected cells by acting as alternative substrates during the viral protein myristoylation event. In this class of compounds, 12-methoxydodecanoic acid is the most potent compound but is approximately 10(3)-fold less active than azidothymidine. The antiviral activity of 12-methoxydodecanoic acid can be enhanced > 40-fold by preparing L-alpha-phosphatidylethanolamine containing 12-methoxydodecanoic acid in both alkyl chains. In addition, the diacylated L-alpha-phosphatidylcholine analog containing 12-methoxydodecanoic acid in both alkyl chains (i) has a 15-fold better antiviral selectivity, (ii) is 7-fold more potent, and (iii) is 10-100-fold more synergistic with azidothymidine than 12-methoxydodecanoic acid. Because of potent synergism, the antiviral selectivity of the diacylated L-alpha-phosphatidylcholine analog is > 10(4) when coadministered with azidothymidine. Phospholipid conjugates are chiral at the C-2 carbon of the glycerol backbone and most interesting is the observation that both the D- and L-isomers of phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, and phosphatidylserine have approximately equal antiviral activity. Phospholipase A2 stereospecifically hydrolyzes only the L isomer of phospholipids and similar activity for both the D- and L- phospholipid isomers suggests that phospholipase A2 is not the rate-limiting enzyme for release of the drugs in vivo.

Antiviral Agents↗
PubMed1993

Magnetically induced orientation of phosphatidylcholine membranes.

Lipid bilayers prepared from natural phospholipids orient in magnetic fields with the long axis of the lipid molecules perpendicular to the magnetic field. This magnetically induced orientation was studied at high (11.7 Tesla (T)), mid (9.36 T), and low (4.68 T) magnetic field strengths using lipid aggregates prepared from natural and synthetic phosphatidylcholine analogs. Phosphatidylcholine analogs containing saturated diacylated chains (12 to 16 carbons/chain) exhibited extensive orientation of the lipid when bilayer formation occurred by gentle hydration conditions. Gentle hydration involved incubating dried phosphatidylcholine C above the main phase transition (Tm); brief shaking or swirling by hand was occasionally needed to completely disperse the lipids. The method of bilayer formation significantly influenced the amount of lipid that orients in magnetic fields. Thus the supramolecular structures (and % orientation) above Tm in an 11.7 T field of dimyristoylphosphatidylcholine (DMPC) bilayers are SUV (0%), LUV (approximately 15%), SPLV (approximately 40%), vortexed-MLV (approximately 60%) and non-vortexed MLV (approximately 90%). Single layered vesicles prepared by the REV method exhibited orientation at 11.7 T similar to LUV prepared by freeze thaw cycles. Aqueous dispersions of eggPC prepared by gentle hydration exhibit approximately 40% orientation at 11.7 T which decreased to approximately 30% orientation if 30% cholesterol is added to the membrane. Magnetic orientation of bilayers thus appears to be a general phenomenon for both saturated and unsaturated natural phospholipids either with or without cholesterol in the membrane.

Lipid Bilayers↗
PubMed1992

Nelson's syndrome following partial pituitary microadenomectomy and pregnancy.

We report for the first time the development of Nelson's syndrome in a patient who had previously undergone unsuccessful pituitary microadenomectomy and subsequently bilateral adrenalectomy. The removal of a 3-mm portion of a microadenoma did not protect against the development of Nelson's syndrome within 3 years which was closely related to pregnancy, a previously noted association. This report suggests that the development of Nelson's syndrome depends not on the size of the initial pituitary tumour but rather on its individual potential for proliferating once free of the inhibitory effects of glucocorticoid excess.

Adenoma, Chromophobe↗
PubMed1991

Silica subsurface amine effect on the chemical stability and chromatographic properties of end-capped immobilized artificial membrane surfaces.

The silica surface of immobilized artificial membranes containing phosphatidylcholine (IAM.PC) has approximately two aminopropyl groups per immobilized phosphatidylcholine molecule. Primary amines near the silica subsurface adsorb biomolecules and also decrease the chemical stability of IAM.PC surfaces. Consequently, subsurface amines were end-capped by several methods including silylating reagents, acetyl analogues, glycidol, methyl glycolate, short-chain anhydrides (3-6 carbons/anhydride chain), and long-chain anhydrides (10-12 carbons/anhydride chain). All end-capping reactions resulted in loss of the initially immobilized phosphatidylcholine molecule. However, the amount of PC loss during end capping was very low (for alkyl anhydride end-capping reactions) to very high (for silylation end-capping reactions). After end capping, IAM.PC showed increased chemical stability compared to non end-capped IAM.PC surfaces. The chemical stability of IAM packing material was monitored by phospholipid leaching from IAM surfaces exposed to organic and aqueous solvents using thin-layer chromatography, 1H NMR spectroscopy, infrared spectroscopy, and mass spectrometry. IAM.PC packing material end capped with long-chain anhydrides exhibited the greatest chemical stability, i.e., little or no detectable phospholipid leaching when challenged with aqueous and/or organic solvents. The chromatography of acidic and basic compounds on end-capped and non-end-capped IAM.PC surfaces was studied. Compared to non-end-capped IAM.PC HPLC columns, the chromatographic retention times of acidic compounds (deoxynucleotides) decreased after end capping. In contrast, the retention times of basic compounds (amphetamine analogues) increased on end-capped IAM.PC HPLC columns relative to non-end-capped IAM.PC HPLC columns. This indicates that these solutes have access to the silica subsurface amines during chromatography.

Amines↗
PubMed1991

Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization.

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.

Animals↗
PubMed1991

Immobilized artificial membrane chromatography: rapid purification of functional membrane proteins.

A solid-phase membrane mimetic system, denoted as immobilized artificial membranes (IAM), has been developed and utilized as a novel high-performance liquid chromatography (HPLC) matrix for the first step in the rapid purification of functional membrane proteins. IAM phases consist of monolayers of amphiphilic membrane lipid molecules covalently bonded to a rigid silica particle. These monolayers of lipids have proved remarkably effective for the chromatography of biomolecules. Several cytochrome P450 isozymes, an extremely important family of hydrophobic membrane proteins with a labile heme catalytic center, have been partially purified in functional conformations from rat liver, kidney, and adrenal microsomes on IAM supports. Functionality of purified P450 and P450 reductase has been demonstrated by optical difference spectroscopy, by carbon monoxide binding, and by reconstitution of enzymatic activity in vitro. Other membrane proteins, including rat liver plasma membrane NADH oxidase and ferricyanide oxidoreductase have also been partially purified by IAM HPLC. The methods for purification of these proteins are described.

Adrenal Glands↗
PubMed1991

Introduction to Fourier transform infrared spectroscopy and applications in the pharmaceutical sciences.

The applications of infrared spectroscopy to pharmaceutical sciences is small compared to the applications of infrared spectroscopy to the fields of chemistry, biology, and biochemistry. This is unfortunate because modern routine infrared spectrometers are excellent research tools that provide very high signal-to-noise, high resolution, and extensive data-manipulation computer software packages. This review summarizes basic principles of infrared spectrometers and the use of Fourier self-deconvolution.

Fourier Analysis↗
PubMed1991

A novel adrenocorticotropin-inducible cytochrome P450 from rat adrenal microsomes catalyzes polycyclic aromatic hydrocarbon metabolism.

7,12-Dimethylbenz(a)anthracene (DMBA) causes massive ACTH-dependent necrosis of the rat adrenal cortex. This may be related to an ACTH-inducible adrenal microsomal cytochrome P450 that metabolizes polycyclic aromatic hydrocarbons (PAH). The proportions of major monooxygenated products of rat adrenal microsomal DMBA metabolism (DMBA-8,9-diol, DMBA-3,4-diol, and DMBA-phenols) differ significantly from that of P450IA1, the most active PAH-metabolizing P450 in rat liver microsomes. After hypophysectomy, both DMBA metabolic activity and a 57K protein which is distinct from P450XXI disappear from rat adrenal microsomes. ACTH restores both 57K protein and DMBA metabolic activity in hypophysectomized rats almost to the levels in intact untreated rats, but not to levels in ACTH-induced intact rats. The 57K protein has been partially purified from solubilized microsomes in a single step, using detergent elution from a new HPLC matrix consisting of monolayers of phosphatidylcholine covalently bound to a silica support. The resulting P450 preparation contains a single major (57K) band, constituting approximately 70% of the total protein (specific content, 2 nmol P450/mg protein; turnover number, 1.5 nmol DMBA min-1. A rabbit polyclonal antibody raised against this preparation also recognizes a single ACTH-inducible 57K rat adrenal microsomal protein on immunoblots and dose-dependently inhibits DMBA metabolism in solubilized reconstituted rat adrenal microsomes. This 57K P450 is immunochemically distinct from rat P450s of the I, II, III, XVII, and XXI families, but it is immunochemically closely related to a 55K benz(a)anthracene-inducible P450 in the 10T1/2 mouse embryo fibroblast cell line.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Formation of the antiplanar-antiplanar phosphate conformation of dilauroylphosphatidylcholine bilayers.

Infrared spectroscopy was used to investigate lipid conformational changes that occur in dilauroylphosphatidylcholine (diC12PC) bilayers with and without fatty-acid-amino-acids as guest molecules in the membrane. Incorporating 2.5 mole% N-decanoylglycine (decgly) into diC12PC liposomes caused formation of the antiplanar-antiplanar (ap-ap) phosphodiester conformation which was stable in room temperature IR spectra. Several other fatty-acid-amino-acids incorporated into diC12PC bilayers were found to also elicit the ap-ap phosphodiester conformation. Unlike these diC12PC/fatty-acid-amino-acid mixed bilayers, pure diC12PC bilayers would form the ap-ap phosphodiester conformation only under low temperature incubation conditions. Dry diC12PC films incubated at 5 degrees C for 0.5 h (brief incubation) or 16 h (prolonged incubation), and then rapidly hydrated (i.e., vortexed at 25 degrees C in D2O), caused the ap-ap phosphodiester conformation to persist in the diC12PC liposomes equilibrated to room temperature. Slow hydration for 16 h at 5 degrees C in both buffered and non-buffered D2O of diC12PC lipid films also produced the ap-ap phosphodiester conformation. In contrast, slow hydration for 16 h at 5 degrees C in PBS/D2O of diC12PC/decgly mixed films caused the greatest number of ap-ap phosphodiester conformers. Using pure diC12PC bilayers, infrared data indicate that incubation of diC12PC films causes the headgroup phosphodiester conformation to change from gauche-gauche (g-g) conformation to the ap-ap conformation. Under all liposome formation conditions examined, no changes in hydration of either the phosphate group or the carbonyl ester group were detected and in addition, no trans/gauche conformational changes in the acyl chain were observed.

Lipid Bilayers↗
PubMed1990

Solid phase membrane mimetics: immobilized artificial membranes.

The studies discussed demonstrate the importance of developing rapid methods to purify membrane proteins and also quantitate binding events between cell membranes and biomolecules. Traditional equilibrium methods are experimentally very difficult because of long equilibration times, peptide aggregation, and the need to make several measurements to obtain a single binding constant. We are developing chromatographic methods to measure binding constants between membranes and biomolecules by using solid phase membrane mimetics. Solid phase binding assays are well established for reactions that typically occur in solution, whereas for reactions that require a membrane environment, no solid phase assay exists. Solid phase membrane mimetics have the potential of filling this gap.

Membrane Fusion↗
PubMed1989

Fourier transform infrared assay of membrane lipids immobilized to silica: leaching and stability of immobilized artificial membrane-bonded phases.

A nondestructive, sensitive assay to monitor the hydrocarbon content of silica-based chromatography particles has been developed. The assay requires a microscope accessory interfaced with a Fourier transform infrared (FTIR) spectrometer. For determining hydrocarbon content, undiluted alkyl-silica-bonded phases were pressed into a thin wafer. Hydrocarbon content was quantitated using the integrated hydrocarbon band intensity between 2995 and 2825 cm-1 [i.e., band area C-H] and the integrated silica oxide band intensity between 1945 and 1780 cm-1 [i.e., band area Si-O]. Plotting the [band area C-H]/[band area Si-O] ratio vs the carbon content determined by elemental analysis gave a correlation coefficient of r = 0.997. The FTIR assay was validated on 5-, 7-, and 12-microns silica particles using three different immobilized artificial membrane (IAM) silica-bonded phases. The utility of the FTIR assay in determining hydrocarbon content was demonstrated by evaluating hydrocarbon leaching from IAM phases exposed to mobile-phase solvents. The ability of organic solvents to leach hydrocarbon from IAM phases containing phosphatidylcholine (PC) as the immobilized ligand was chloroform greater than ethanol approximately methanol greater than ethyl acetate greater than methylene chloride greater than acetonitrile greater than acetone. Acetone and acetonitrile cause very little hydrocarbon leaching from HPLC-IAM.PC columns. When challenged with different mobile phases, IAM.PC columns perfused with mobile phase are more stable than IAM.PC-bonded phases stirred in mobile phases. IAM.PC contains lecithin linked to silica by amide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1989

Fourier transform infrared assay of liposomal lipids.

Quantitating the lipid content in organic lipid solutions and extracted membrane preparations is described. Fourier transform infrared analysis of thin films using perdeuterated nonadecane as an internal standard permitted quantitation with greater than 95% accuracy.

Alkanes↗
PubMed1989

Immobilized artificial membrane chromatography: supports composed of membrane lipids.

Cell membranes provide an environment for several types of molecular processes and we are attempting to mimic the cell membranes' environment on a chromatography solid support. Chromatography solid supports utilizing lecithin as the bonded phase were synthesized and the HPLC behavior of hydrophilic peptides evaluated. A diC14 lecithin containing a terminal carboxy group on the C2 fatty acid chain was amidated with the surface amines of Nucleosil-300 (7NH2) silica particles. Based on elemental analysis, lecithin was coupled to Nucleosil-300 (7NH2) at a surface density near that of lecithin found in biological membranes and this novel chromatographic support material is denoted as Nucleosil-lecithin, the prototype immobilized artificial membrane. Infrared difference spectra of Nucleosil-lecithin minus Nucleosil-300 (7NH2) clearly showed amide I (1653.1 cm-1) and amide II (1550.9 cm-1) bands, giving direct spectroscopic evidence for the amide linkage. Spectral deconvolution resolved two peaks for the amide I band, and three peaks for the amide II band. This demonstrates lecithin interchain amide hydrogen bonding and/or hydrogen bonds between the lecithin amide link and unreacted silica surface amines. Nucleosil-lecithin as a solid phase mimics membranes and can be used to study the interactions of biomolecules with membranes. Our primary objective is to develop HPLC methods for studying the interaction between cell membranes and peptide sequences found near the interfaces of cell membranes. A frequency distribution of amino acids bracketing approximately 400 transmembrane peptide sequences showed Cys to be the least frequently occurring amino acid at this putative interfacial membrane region. Hydrophilic peptide analogs bearing Cys were used as model compounds to test Nucleosil-lecithin solid supports. Small peptides, six to eight amino acids in length, containing Cys bind approximately 2X tighter to Nucleosil-lecithin compared to identical peptides without the Cys residue. Thus, Cys at the interface of cells may stabilize protein-lipid interactions.

Chromatography, High Pressure Liquid↗
PubMed1989

Amino acids bracketing the predicted transmembrane domains of membrane proteins.

The cell membrane is a complex mixture of several classes of biomolecules but amino acids and lipids are the main constituents. For this reason we are establishing a data base of transmembrane proteins with the intent of using the data base to identify interfacial peptide sequences useful for studying protein-lipid interactions at membrane interfaces. Our present intention is to characterize transmembrane peptides and amino acids found near the membrane interface. A data base containing only signal peptides is available (G. von Heijne, Prot. Seq. Data Anal. 1:41-42, 1987).

Amino Acid Sequence↗
PubMed1988

Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins.

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.

Amino Acid Sequence↗
PubMed1987

Multilayered vesicles prepared by reverse-phase evaporation: liposome structure and optimum solute entrapment.

Liposome structure and solute entrapment in multilayered vesicles (MLVs) prepared by reverse-phase evaporation (REV) were studied. MLV-REV vesicles prepared from ether/water emulsions have high entrapment. Entrapment depends on drug, drug concentration, lipid, lipid concentration, and the container used to prepare the vesicles. By use of 300 microL of aqueous phase and 100 mg of phosphatidylcholine (PC), vesicles prepared in a test tube 25 mm X 175 mm have higher entrapment than vesicles prepared in a 100-mL round-bottom or pear-shaped flask. By use of a test tube, 100 mg of PC, and 300 microL of aqueous phase containing sucrose (1-50 mg/mL), greater than 90% sucrose entrapment was obtained. Increasing lipid content to 150 mg of PC decreased entrapment to approximately 80%. Neutral PC MLV-REV vesicles have optimum entrapment. Mixing negatively charged lipids or cholesterol (CH) with PC to make MLV-REV vesicles results in decreased entrapment compared to using only PC. Preparing vesicles with the solid lipid dipalmitoylphosphatidylcholine (DPPC) or DPPC/CH mixtures (0 less than or equal to mol % CH less than or equal to 50) results in approximately 30-40% entrapment when diethyl ether is used to make the MLV-REV emulsion. Substituting diisopropyl ether for diethyl ether and heating the MLV-REV emulsion during vesicle formation generate DPPC/CH vesicles that entrap 60% of added solutes. The high entrapment found for MLV vesicles prepared from water/organic solvent emulsions depends on maintaining a core during the process of liposome formation. A method to calculate the fraction of water residing in the liposomes' core is presented and used to compare multilayered vesicles prepared by different processes. X-ray diffraction data demonstrate that a heterogeneous distribution of lipid may exist in multilayered vesicles prepared by the REV process.

Chemical Phenomena↗