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Biomedical subjects

P Gale

Publications and source records attributed to P Gale.

26 records · Page 2Linked to original sources
PubMed1993

Defining the statistical distribution of vesicle diameters facilitates quantitative assessment of spectral narrowing from small vesicles in protein/lipid interaction studies by 2H-NMR.

The diameters of vesicles comprising complexes of an integral membrane protein and 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the choline gamma-methyl groups, exhibited a lognormal distribution defined by the geometric mean (GM) and the logarithmic standard deviation (LSD). The range of vesicle sizes within each complex was very large (e.g., 99% of vesicles measured between 60 nm and 4,000 nm in one complex). This complicated assessment of spectral narrowing artefacts from small vesicles in broad line 2H-NMR results. Artefacts (e.g., decreases in quadrupole splitting and generation of two component spectra) resembled results attributed to membrane proteins in other 2H-NMR studies for which spectral narrowing was dismissed by demonstration of some vesicles > 400 nm in diameter. This was inadequate since 2H-NMR spectra from complexes with GM < 350 nm exhibited spectral narrowing artefacts despite the presence of some vesicles 1,200 nm in diameter. The nature and degree of artefact were determined by both GM and LSD. Quantifying vesicle sizes by GM and LSD accommodated the large size variation within a complex and facilitated quantitative assessment of artefacts through comparison of different complexes.

Bacteriorhodopsins↗
PubMed1992

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.

Animals↗
PubMed1992

Mechanism of inhibition of human leukocyte elastase by two cephalosporin derivatives.

The cephalosporin derivatives L 658758 [1-[[3-(acetoxymethyl)-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-2-yl]carbonyl]proline S,S-dioxide] and L 659286 [1-[[7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo- 1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-aza-(6R)-bicyclo[4.2.0]-o ct-2-en-2-yl]carbonyl]pyrrolidine S,S-dioxide] are mechanism based inhibitors of human leukocyte elastase (HLE). The mechanism involves initial formation of a Michaelis complex followed by acylation of the active site serine. The group on the 3'-methylene is liberated during the course of these reactions, followed by partitioning of an intermediate between hydrolysis to regenerate active enzyme and further modification to produce a stable HLE-inhibitor complex. The partition ratio of 2.0 obtained for the reaction with L 658758 approaches that of an optimal inhibitor. These compounds are functionally irreversible inhibitors as the recovery of activity after inactivation is slow. The half-lives at 37 degrees C of the L 658758 and L 659286 derived HLE-I complexes were 9 and 6.5 h, respectively. The complexes produced by both inhibitors are similar chemically since the thermodynamic parameters for activation to regenerate active enzyme are essentially identical. The free energy of activation for this process is dominated primarily by the enthalpy term. The stability of the final complexes likely arises from Michael addition on the active site histidine to the 3'-methylene.

Catalysis↗
PubMed1992

Effect of bacteriorhodopsin on the orientation of the headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in bilayers: a 31P- and 2H-NMR study.

Bacteriorhodopsin (BR), purified from the halophilic bacterium, Halobacterium halobium, has been separated from the endogenous purple membrane lipids and reconstituted by detergent dialysis into bilayers of the zwitterionic phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), which was selectively deuterated at the headgroup in the choline alpha- and beta-methylene segments and the choline gamma-methyl groups. Complexes of DMPC/BR contents from 67:1 to 222:1 (mol/mol) were produced under conditions to promote formation of large vesicles (mean diameters 600-700 nm). The magnitudes of the 2H-NMR quadrupole splittings recorded from the deuterium-labelled headgroup segments, and the 31P-NMR chemical shift anisotropy (CSA) of the phosphate group appeared to vary linearly with the BR content in the complexes over the range of DMPC/BR ratios studied. On increasing the proportion of BR in the DMPC-BR complexes, the 2H-NMR quadrupole splittings measured from the choline gamma-methyl groups and the beta-methylene segments and the 31P-NMR CSA increased in magnitude, while the 2H-NMR quadrupole splitting from the alpha-methylene segment decreased. Such opposing changes in the choline alpha- and beta-methylene segment quadrupole splittings are similar to those reported on increasing the proportion of positively charged amphiphile at the bilayer surface (Seelig et al. (1987) Biochemistry 26, 7535-7541). It is suggested that BR presents a net positive charge to the phosphocholine headgroups at the protein/lipid interface.

Bacteriorhodopsins↗
PubMed1991

Characterization of phospholipid compositions and physical properties of DMPC/bacteriorhodopsin vesicles produced by a detergent-free method.

Homogeneous complexes of bacteriorhodopsin (BR) from Halobacterium halobium purple membrane (PM) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) have been produced by a detergent-free process in which bovine liver non-specific phospholipid transfer protein (nsTP) promotes net transfer of DMPC from small unilamellar vesicles directly into PM. The number of DMPC molecules incorporated per BR monomer follows a close to linear dependence with the relative proportions of DMPC and PM added to the initial mixture over the ranges studied. The resulting complexes, with total lipid phosphate/BR contents of between 31:1 and 152:1 (mole/mole), were purified free from any remaining unincorporated DMPC by sucrose density gradient centrifugation. Broad line 31P-NMR spectra and partitioning studies with the nitroxide spin label, Tempo, confirm that the BR and DMPC coexist in bilayer complexes. Quantitative analysis of high resolution 31P-NMR spectra from complexes after solubilization in 4% SDS revealed 74-84% of the major PM phospholipid to be retained in the complexes.

Animals↗
PubMed1984

Kaolin pneumoconiosis. Radiologic, pathologic, and mineralogic findings.

Pulmonary tissue obtained at thoracotomy or autopsy from 5 kaolin workers with complicated pneumoconiosis was studied by optical light and scanning electron microscopy. Premortem or preoperative chest roentgenograms demonstrated small irregular shadows and large opacities typical of kaolin pneumoconiosis. On gross examination, there were firm, grey-brown nodules and masses in the parenchyma and in the hilar lymph nodes. Histologically, there was extensive pulmonary kaolinite deposition associated with formation of peribronchiolar macules and nodules. The latter were comprised of kaolinite aggregates traversed by bands of fibrous tissue rather than dense whorled collagen, as seen in silicosis. Crystallographic studies confirmed the presence of kaolinite in the lungs, but silica was not demonstrable by either analytical scanning electron microscopy or X-ray diffractometry. These findings illustrate the pathology of human kaolin pneumoconiosis, confirm the fibrogenic potential of kaolinite, and emphasize differences in pulmonary responses to kaolinite and to silica.

Adult↗
PubMed1976

Studies of a glycoprotein in the oocysts of Eimeria tenella.

A glycoprotein unique to the cytoplasm of the unsporulated oocyst of Eimeria tenella has been purified and partially characterized. The protein has a molecular weight of 30,000, of which approximately 40% is carbohydrate. The carbohydrate portion of the molecule consists of glucose, galactose, mannose, xylose, glucosamine, and galactosamine, with no detectable sialic acid. The protein portion contains approximately 141 residues, being rich in hydrophilic amino acids with very few aromatic amino acids and no cystine. The protein comprises 14% of the total soluble protein of the unsporulated oocyst but has not been identified in the cytoplasm of any other developmental stage of the organism. Using polyacrylamide gel electrophoresis and a radioimmunoassay specific for the protein, it has been shown to disappear from the cytoplasm between the 15th and 20th hour of the 20-hour sporulation process. Subsequent immunofluorescence experiments have shown a reactive material as a component of the sporozoite membrane. These results indicate that the glycoprotein is a structural protein of the sporozoite membrane, apparently synthesized by the unsporulated oocyst and incorporated into the sporozoite membrane as one of the last steps involved in the sporulation process.

Amino Acids↗