Biomedical subjects

C Pidgeon

Publications and source records attributed to C Pidgeon.

At least 19 recordsLinked to original sources

PubMed2000

Alkylation of cysteine-containing peptides to mimic palmitoylation.

Numerous proteins that are involved in cell signaling and viral replication require post-translational modification by palmitoylation to function properly. The molecular details by which this palmitoyl modification affects protein function remain poorly understood. To facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated C16 groups at cysteine residues and demonstrated using peptides derived from the palmitoylated region of Sindbis virus E2 glycoprotein. The synthetic approach takes advantage of disulfide chemistry to specifically modify only the cysteine residues within peptides and covalently links C16 groups via disulfide bridges using a new thioalkylating reagent, hexyldexyldithiopyridine. The chemistry presented here takes place in solution under mild conditions without the need for protection of the peptide functional groups. A method for purifying these modified peptides is also described. This protocol can be of general use to investigators studying the role of palmitoylation in biological systems.

Alkylation↗

PubMed1999

Contributions of residues of pancreatic phospholipase A2 to interfacial binding, catalysis, and activation.

Primary rate and equilibrium parameters for 60 site-directed mutants of bovine pancreatic phospholipase A2 (PLA2) are analyzed so incremental contributions of the substitution of specific residues can be evaluated. The magnitude of the change is evaluated so a functional role in the context of the N- and C-domains of PLA2 can be assigned, and their relationship to the catalytic residues and to the i-face that makes contact with the interface. The effect of substitutions and interfacial charge is characterized by the equilibrium dissociation constant for dissociation of the bound enzyme from the interface (Kd), the dissociation constant for dissociation of a substrate mimic from the active site of the bound enzyme (KL), and the interfacial Michaelis constants, KM and kcat. Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decrease in kcat is seen with the substitution of F5, I9, D99, A102, or F106, which form the substrate binding pocket. Certain residues, which are not part of the catalytic site or the substrate binding pocket, also modulate kcat. Interfacial anionic charge lowers Kd, and induces kcat activation through K56, K53, K119, or K120. Significant changes in KL are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in KM [=(k2+k-1)/k1] are attributed to kcat (=k2) and KL (=k-1/k1). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on KS, and the effect of the interfacial anionic charge on kcat. Interpreted in the context of the overall structure, results provide insights into the role of segments and domains in the microscopic events of catalytic turnover and processivity, and their allosteric regulation. We suggest that the interfacial recognition region (i-face) of PLA2, due to the plasticity of certain segments and domains, exercises an allosteric control on the substrate binding and chemical step.

Animals↗

PubMed1999

Intestinal peptide transport systems and oral drug availability.

The intestinal peptide transport system has broad substrate specificities. In addition to its physiological function of absorbing di- and tripeptides resulting from the digestion of dietary proteins, this transport system also absorbs some orally administered peptidomimetic drugs, including beta-lactam antibiotics, angiotensin converting enzyme inhibitors, renin inhibitors, bestatin, thrombin inhibitors, and thyrotropin-releasing hormone and its analogues. There have been several studies on the mechanism and substrate structure-affinity relationship for this transport system. Rapid progress has been made recently in studies on the molecular basis of the intestinal peptide transport system. A protein apparently involved in peptide transport has been isolated from rabbit small intestines, and genes for human intestinal peptide transporters have been cloned, sequenced and functionally expressed. This review summarizes these studies and addresses the pharmaceutical potential of the intestinal peptide transport system.

Administration, Oral↗

PubMed1999

The sitting position in neurosurgery: a critical appraisal.

The potential for serious complications after venous air embolism and successful malpractice liability claims are the principle reasons for the dramatic decline in the use of the sitting position in neurosurgical practice. Although there have been several studies substantiating the relative safety compared with the prone or park bench positions, its use will continue to decline as neurosurgeons abandon its application and trainees in neurosurgery are not exposed to its relative merits. How can individual surgeons continue to use this position? Will individual, difficult surgical access cases be denied the obvious technical advantages of the sitting position? Limited use of the sitting position should remain in the neurosurgeon's armamentarium. However, several caveats must be emphasized. Assessment of the relative risk-benefit, based on the individual patient's physical status and surgical implications for the particular intracranial pathology, is of paramount importance. The patient should be informed of the specific risks of venous air embolism, quadriparesis and peripheral nerve palsies. Appropriate charting of patient information provided and special consent issues are essential. An anaesthetic input into the decision to use the sitting position is a sine qua non. The presence of a patient foramen ovale is an absolute contraindication. Preoperative contrast echocardiography should be used as a screening technique to detect the population at risk of paradoxical air embolism caused by the presence of a patent foramen ovale. The technique involves i.v. injection of saline agitated with air and a Valsalva manoeuvre is applied and released. Use of this position necessitates supplementary monitoring to promptly detect and treat venous air embolism. Doppler ultrasonography is the most sensitive of the generally available monitors to detect intracardiac air. The use of a central venous catheter is recommended, with the tip positioned close to the superior vena cava junction with the right atrium, to aspirate intravascular gas. Measures to minimize hypotension associated with the sitting position include a slow, staged positioning over 5-10 min and use of the 'G suit' inflated with compressed air applied to the lower extremities and pelvis. Use of the sitting or upright position for patients undergoing posterior fossa and cervical spine surgery presents unique challenges for the anaesthetist. With appropriate patient selection and preparation, and using prudent intraoperative monitoring and anaesthetic techniques, selected patients should still benefit from the optimum access to mid-line lesions, improved cerebral venous decompression, lower intracranial pressure and enhanced gravity drainage of blood and CSF associated with the sitting position.

Anesthesia↗

PubMed1998

Synthesis and immobilization of ceramide analogs on silica particles.

Ceramides are the major lipid components of the stratum corneum, the major permeability barrier of the skin. Here we report a chemical synthesis of ceramide analogs covalently bonded on the silica particles, that can be used to predict the skin permeability of chemicals via HPLC methods.

Ceramides↗

PubMed1997

Single step purification of rat liver aldolase using immobilized artificial membrane chromatography.

Aldolase B is a peripheral membrane protein. Immobilized artificial membrane (IAM) surfaces were used to purify rat liver aldolase B in a single chromatographic step. Selective elution required dipalmitoylphosphatidylcholine (DPPC) to be included in the mobile phase. Selective elution of aldolase from the IAM column when DPPC (0.2 mM) was added to the mobile phase indicates that DPPC was an affinity displacing ligand for this membrane associated protein. Since tissue preparation involved only homogenization and centrifugation, the single step purification of aldolase B using IAM chromatography is a very convenient method. The IAM stationary phase (1.5 g) has a loading capacity of at least 4.39 mg total protein from rat liver homogenates and typically approximately 17.7 microg of pure aldolase in a single step from approximately 60 mg wet weight rat liver cytosol can be obtained.

Amino Acid Sequence↗

PubMed1996

Affinity purification of phospholipase A2 on immobilized artificial membranes containing and lacking the glycerol backbone.

Immobilized artificial membranes (IAMs) are chromatography surfaces containing monolayers of phospholipid ligands. etherIAM.PCC10/C3 contains the glycerol backbone whereas delta GIAM.PCC10/C3 lacks the glycerol backbone. Affinity purification of PLA2 on these IAM surfaces demonstrated that the surface structural differences were not important for phospholipase A2 (PLA2) binding. This suggests that the chromatographically important binding event involves the PLA2 surface and the monolayer of polar choline headgroups on the IAM surface. After sample loading, short-chain alkylsulfonates were used as low eluotropic strength detergents to remove contaminating proteins, and PLA2 were eluted with CH3CN (30%). Octyllysophosphatidylcholine (0.5%) can replace CH3CN to elute PLA2 from IAM surfaces. The PLA2 purity after IAM chromatography depends on the protein loading; analytical-scale loadings (0.8 mg protein/g IAM) resulted in a PLA2 purity of ca. 70% based on densitometric scans of proteins in polyacrylamide gels after electrophoresis. Preparative loadings of 3.21 mg protein/g IAM resulted in 48% PLA2 purity. Purification of PLA2 to electrophoretic homogeneity was achieved using an IAM column followed by a strong anion-exchange column. These results suggests that IAMs may be used to develop purification methods for PLA2 enzymes obtained from diverse biological specimens.

Chromatography, Affinity↗

PubMed1996

Immobilized-artificial-membrane chromatography: measurements of membrane partition coefficient and predicting drug membrane permeability.

Immobilized artificial membranes (IAMs) are chromatographic surfaces prepared by covalently immobilizing cell membrane phospholipids to solid surfaces at monolayer densities. IAM surfaces mimic fluid cell membranes. For 23 structurally unrelated compounds, solute capacity factors [log (k'IAM)] measured on IAM columns correlate very well with the solute equilibrium partition coefficients [log (Km)] measured in fluid liposome systems (r = 0.907). This indicates that solute partitioning between the IAM bonded phase and the aqueous mobile phase is similar to the solute partitioning between liposomes and the aqueous phase. IAMs also predicted oral drug absorption in mice and drug permeability through Caco-2 cells. IAM chromatography is experimentally simple and large volume screening of experimental compounds for drug absorption is possible. Solute retention on IAMs was found to be dominated by a partitioning mechanism. The structural requirements for HPLC bonded phases to predict solute-membrane partitioning are briefly discussed.

Adsorption↗

PubMed1996

Mobile phase effects on membrane protein elution during immobilized artificial membrane chromatography.

The eluotropic strength of different mobile phases for eluting membrane proteins from immobilized artificial membrane (IAM) chromatography surfaces was studied. Two protein mixtures containing bovine pancreatic PLA2 were used in this study. Protein mixture I was PLA2 obtained from Sigma which contained approximately 5-10 major protein bands in electrophoretic gels. Protein mixture II was obtained from flesh bovine pancreatic tissue and contained > 100 proteins including the target protein, PLA2. After adsorbing Sigma PLA2 to IAM columns, the elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA2. Elution conditions tested were (i) detergent gradients, (ii) salt gradients used during ion-exchange chromatography, (iii) salt conditions used during hydrophobic interaction chromatography, (iv) acetonitrile gradients used during reversed-phase chromatography, and (v) a two-step gradient consisting of first a detergent gradient followed by an acetonitrile gradient. Based on silver-stained electrophoretic protein gels. PLA2 from protein mixture I was purified to electrophoretic homogeneity with 417-fold increase in specific activity in one step using elution condition (v), and PLA2 from protein mixture II was purified in one step (660-fold increase in specific activity) using elution condition (iv). Total protein recovery from IAM columns is 70-100%.

Animals↗

PubMed1995

Predicting drug-membrane interactions by HPLC: structural requirements of chromatographic surfaces.

Drug-membrane interactions have recently been studied by immobilized artificial membrane (IAM) chromatography (Pidgeon, C.; et al. J. Med. Chem. 1995, 38, 590-595. Ong, S.; et al. Anal. Chem. 1995, 67, 755-762), and the molecular recognition properties of IAM surfaces toward drug binding/partitioning appear to be remarkably close to the molecular recognition properties of fluid membranes. The structural requirements of chromatography surfaces to emulate biological partitioning are unknown. To begin to elucidate the surface structural requirements needed to predict drug partitioning into membranes, three bonded phases were prepared. The chromatography bonded phases were prepared by immobilizing (i) a single-chain analog containing the phosphocholine (PC) headgroup (IAM.PC.DD), (ii) a long-chain alcohol containing polar OH groups protruding from the surface (12-OH-silica), and (iii) a long-chain fatty acid containing OCH3 groups protruding from the surface (12-MO-silica). The 12-OH-silica surface can be considered as an immobilized "octanol" phase with OH groups protruding from the surface and is therefore a solid phase model of octanol/water partitioning systems. As expected, improved capability of predicting solute-membrane interactions as found for the chromatographic surface containing the PC polar head-group because the PC headgroup is also found in natural cell membranes. For instance, the IAM.PC.DD column predicted drug partitioning into dimyristoylphosphatidylcholine liposomes (r = 0.864) better than 12-OH-silica (r = 0.812), and 12-MO-silica (r = 0.817). IAM. PC.DD columns also predicted intestinal drug absorption (r = 0.788) better than 12-OH-silica (r = 0.590) and 12-MO-silica (r = 0.681); reversed phase octadecylsilica (ODS) columns could not predict intestinal absorption (r = 0.10). Collectively, these results suggest that chromatographic surfaces containing interfacial polar groups, i.e., PC, OH, and OCH3, model drug-membrane interactions, but surfaces lacking interfacial polar functional groups (e.g., ODS surface) are poor models. Most interestingly, drug partitioning into octanol/water systems does not correlate with drug binding to the immobilized octanol phase. However, drug partitioning into immobilized octanol correlates with drug partitioning into liposomes (r = 0.812).

Chromatography, High Pressure Liquid↗

PubMed1995

Thermodynamics of solute partitioning into immobilized artificial membranes.

The solute retention mechanism on immobilized artificial membranes (IAMs) was studied using three different IAM.PC phases. IAMs were prepared by immobilizing either single-chain or double-chain phosphatidylcholine (PC) ligands. Solute affinity for the single-chain IAM.PC columns (with a ligand density of 127 mumol of PC/g of IAM) was 3-fold lower compared to solute affinity on the double-chain IAM.PC column (with a ligand density of 98 mumol of PC/g of IAM). This suggests that the solute retention on IAMs is dominated by a solute partitioning mechanism. Temperature-dependent studies indicated that the thermodynamics of solute partitioning is similar on both the single-chain and double-chain IAM.PC surfaces. For a set of phenol derivatives, the partitioning into IAM.PC surfaces is both enthalpy and entropy driven. For beta-blockers, the partitioning into IAM.PC surfaces is entropy driven. The free energy of solute partitioning into IAMs correlates very well with the free energy of solute partitioning into liposomes.

Hydrocarbons↗

PubMed1995

Rapid purification of cotton seed membrane-bound N-acylphosphatidylethanolamine synthase by immobilized artificial membrane chromatography.

N-Acylphosphatidylethanolamine synthase (NAPES) is a membrane-bound enzyme present in cotton seedlings at a concentration of < or = 0.02% of the total protein. NAPES was purified to electrophoretic homogeneity in a single chromatographic step using immobilized artificial membrane (IAM) chromatography. The IAM column used for NAPES purification was etherIAM.PEC10/C3 and this surface contains a monolayer of immobilized phosphatidyl-ethanolamine (PE). Since PE is an analogue of the natural substrate for NAPES, etherIAM.PEC10/C3 columns function as an affinity column for this enzyme. Detergent-solubilized microsomal proteins from cotton were loaded on to the etherIAM.PEC10/C3 column and eluted with buffered mobile phases containing 0.2 mM dimyristoylphosphatidylethanolamine (DMPE) and 2 mM dodecylmaltoside. Little NAPES functional activity eluted if DMPE was removed from the mobile phase. Mobile phase DMPE is also a substrate for NAPES, both the mobile phase and IAM surface contains NAPES substrates. Mobile phase DMPE may function as both a surfactant-type affinity displacing ligand effecting protein elution and also a stabilizing factor of NAPES functional activity. The loading capacity on semi-preparative etherIAM.PEC10/C3 (6.5 x 1.0 cm) columns was ca. 5 mg of total detergent solubilized microsomal proteins, and protein recovery was quantitative. This one-step IAM purification of NAPES resulted in a single band on silver-stained polyacrylamide gels, and 3940 fold increase in NAPES specific activity. The molecular mass of the purified NAPES protein is 64,000. 125I labeled [12-(4-azidosalicyl)amino]dodecanoic acid is a photoreactive fatty acid substrate of NAPES that was used to confirm protein purity.

Acyltransferases↗

PubMed1995

IAM chromatography: an in vitro screen for predicting drug membrane permeability.

Fluid cell membranes are the main barrier to drug absorption when diffusion limits uptake. Immobilized artificial membranes (IAMs) are solid phase models of fluid membranes that predicted oral drug absorption in mice for a homologous set of cephalosporins. IAMs also predicted drug permeability through Caco-2 cells. Since drug permeability in Caco-2 cells is known to correlate with the oral absorption of drugs in humans, IAMs may also model drug absorption in humans. IAM analysis is experimentally simple, and large-volume screening of experimental compounds for drug absorption is possible.

Animals↗

PubMed1995

Membrane partition coefficients chromatographically measured using immobilized artificial membrane surfaces.

Immobilized artificial membranes (IAMs) are chromatographic surfaces prepared by covalently immobilizing cell membrane phospholipids. IAM surfaces mimic fluid cell membranes. Solute capacity factors (k'IAM) measured on IAM columns correlate very well with solute equilibrium partition coefficients (Km') measured in fluid liposome systems. For 23 structurally unrelated compounds, log-(k'IAM) correlates with log(Km') with a linear correlation coefficient r = 0.907. This indicates that solute partitioning between the IAM bonded phase and the aqueous mobile phase is similar to the solute partitioning between liposomes and the aqueous phase. Although both IAM chromatography and liposome partitioning can be used as in vitro methods to predict solute partitioning into cell membranes, IAM chromatography is experimentally convenient compared to liposome systems. To study the effect of lipid structure on drug binding to IAMs, IAMs were prepared from three different phosphatidylcholine ligands: (i) a diacylated phosphatidylcholine ligand, (ii) a single chain ether phosphatidylcholine ligand, and (iii) a single chain phosphatidylcholine ligand that lacks a glycerol backbone. Solute retention data were identical for all of these IAMs, and consequently, predictions of solute binding to fluid membranes were also identical. This indicates that the structure of the phosphatidylcholine ligand that is immobilized is not critical for the binding of solutes. Since the structure is not important, the binding of solutes to membranes is a bulk phase property, i.e., it is the interface created by the ligands that determines the solute binding properties, not the ligands themselves. Solute partitioning using octanol/water systems does not correlate with k'IAM unless a homologous series of hydrophobic solutes is being evaluated.

Binding Sites↗

PubMed1994

Preparation of mixed ligand immobilized artificial membranes for predicting drug binding to membranes.

Mixed ligand immobilized artificial membranes (IAMs) are surfaces that contain at least two immobilized membrane phospholipids which are designated as either the primary phospholipid or the secondary phospholipid. The primary immobilized phospholipid refers to the immobilized phospholipid that has the highest surface density. For this work, the primary immobilized phospholipid was a single-chain ether phosphatidylcholine (PC) analog. Four mixed-ligand IAMs were prepared by use of immobilized PC as the primary immobilized phospholipid. The secondary immobilized phospholipid ligand was either phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. All of these secondary phospholipids are bonded at approximately 6-10 mol % relative to the molar amount of immobilized PC. Each secondary phospholipid contains functional groups in the polar head group region that require protecting groups during the immobilization process. The four-step synthetic strategy to prepare mixed-ligand IAMs involves (i) immobilization of the PC analog at high density to silica propylamine (SPA), (ii) immobilization of the second phospholipid (PL) analog at low density, (iii) end capping residual amines with a long-chain anhydride followed by end capping with a short-chain anhydride, and (iv) deprotection of the polar head group protecting groups. The surface density of the mixed PLs bonded to the silica support was approximately 130 mumol of PLs/g of SPA. High-performance liquid chromatography using these mixed lipid IAMs can be exploited to rapidly predict the membrane binding properties of drugs.

Adrenergic alpha-Agonists↗

PubMed1994

Phospholipid immobilization on solid surfaces.

Single chain ether phospholipids (PLs) containing omega-carboxyl groups in the alkyl chain were immobilized on silica propylamine (SPA) to form IAM chromatography packing material. The PL ligands are analogs of phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA). All of these PLs contain polar functional groups in the lipid head group that require protection prior to PL immobilization and then deprotection after immobilization. The IAM surface was prepared in four steps: (i) the omega-carboxyl group was activated with carbonyldiimidazole, (ii) the activated PL-imidazolide ligand was bonded to SPA, (iii) the surface was end capped with a long chain anhydride and then end-capped with a short chain anhydride, and (iv) protecting groups were removed to form the IAM surface. The extent of deblocking the protecting groups was typically > or = 90%. This immobilization strategy generated a phospholipid surface that was stable when solvated with all organic solvents and aqueous buffers between pH 2 and 8. Both FT-IR spectroscopy and elemental analysis indicated that the bonding densities were 64-83 mg of PL/g of SPA, which corresponds to an area per molecule of 66-104 A2. These bonding densities for the immobilized PLs are very close to the area per molecule of mobile phospholipids comprising liposome membrane. The similar areas per molecule of immobilized PLs and mobile phospholipid in liposomes indicate that the lipid environments are similar.

Liposomes↗

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↗