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Biomedical subjects

G A Thompson

Publications and source records attributed to G A Thompson.

At least 109 records · Page 6Linked to original sources
PubMed1988

Protein acylation in Tetrahymena.

Examination of exhaustively delipidated Tetrahymena mimbres cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of several protein bands containing covalently linked fatty acids. Palmitic (16:0) and stearic (18:0) acids together accounted for approximately 90% of the protein-linked acyl chains, with myristic acid (14:0) comprising most of the remainder. Each of these three fatty acids was present mainly in alkali-stable linkage, indicating that unlike most other systems examined, fatty acids are attached to proteins of Tetrahymena principally by amide bonds. Smaller proportions of the acyl chains were susceptible to release by hydroxylaminolysis or by alkaline hydrolysis as would be expected from an ester linkage. The protein-bound acyl chains accounted for 0.3% of the cells' total fatty acids. They closely resembled in composition the highly saturated free fatty acid pool but not the vast pool of glycerolipid-associated fatty acids, which were mainly unsaturated. Cells subjected to thermal stress by rapid chilling from 39 to 15 degrees C responded by sharply increasing the ratio of palmitate to stearate in covalent association with proteins.

Acylation↗
PubMed1988

Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock.

The inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) comprise 14.8, 1.2, and 0.3 mol %, respectively, of Dunaliella salina phospholipids. In isolated plasma membrane fractions, PIP and PIP2 are highly concentrated, together comprising 9.5 mol % of plasmalemma phospholipids. The metabolism of these inositol phospholipids and phosphatidic acid (PA) is very rapid under normal growth conditions. Within 5 min after introduction of 32Pi into the growth medium, over 75% of lipid-bound label was found in these quantitatively minor phospholipids. Within 2 min after a sudden hypoosmotic shock, the levels of PIP2 and PIP dropped to 65 and 79%, respectively, of controls. Within the same time frame, PA rose to 141% of control values. These data suggest that a rapid breakdown of the polyphosphoinositides may mediate the profound morphological and physiological changes which allow this organism to survive drastic hypoosmotic stress. In contrast to hypoosmotic shock, hyperosmotic shock induced a rise in PIP2 levels to 131% of control values, whereas the level of PA dropped to 56% of controls after 4 min. These two different types of osmotic stress affect inositol phospholipid metabolism in a fundamentally opposite manner, with only hypoosmotic shock inducing a net decrease in polyphosphoinositides.

Cell Membrane↗
PubMed1988

Recovery of Dunaliella salina cells following hydrogenation of lipids in specific membranes by a homogeneous palladium catalyst.

Unsaturated fatty acyl chains of Dunaliella salina membrane lipids can be catalytically reduced by the homogeneous hydrogenation catalyst palladium di(sodium alizarine monosulphonate), Pd(QS)2, under conditions permitting full recovery of the cells within 24 h. The hydrogenation is accomplished by incubation of cells with the hydride form of Pd(QS)2 under 1 atmosphere of H2 and for 2 min or less. Following this protocol, hydrogenation reduces only those fatty acids located in the plasma membrane and other membranes located near the cell surface. The limited reactivity in vivo is due to the fact the Pd(QS)2 permeates into the living cells more slowly than it does into liposomes prepared from extracted Dunaliella membrane lipids. While Dunaliella is completely unaffected by exposure to the oxygenated, inactive catalyst, hydrogenated cells cease growth for approximately 12 h, during which time the hydrogenated acyl chains are being enzymatically retroconverted to their original unsaturated form. When the lipid composition approaches its prehydrogenation values, growth resumes, presumably due to the restoration of normal membrane functions. The system shows promise for studying the metabolic regulation of membrane microviscosity.

Chlorophyta↗
PubMed1988

Human serum and plasma protein binding of enoximone and its sulfoxide metabolite.

Experimental factors and determinants of the protein binding of enoximone (a new cardiotonic agent) were investigated in human serum from healthy, drug-free subjects using a rapid ultrafiltration method; these factors and determinants included nonspecific binding to the apparatus, ultrafiltrate volume, temperature, serum pH, enoximone serum concentration, and enoximone sulfoxide (metabolite) concentration. It was demonstrated from mass balance experiments that nonspecific binding to the apparatus did not occur. Within the range investigated, ultrafiltrate volume did not affect the binding result. However, serum pH and temperature were critical variables. At pH 7.4 and 37 degrees C, enoximone serum binding occurred to the extent of approximately 70%; over the therapeutic serum concentration range, this binding was concentration independent. Experiments with purified albumin solutions indicated that much of the serum binding could be accounted for by albumin. At concentrations exceeding those observed clinically, enoximone sulfoxide did not affect enoximone serum binding. In another experiment, enoximone binding to serum was compared with that from plasma containing either heparin or disodium EDTA. There were essentially no differences. Enoximone sulfoxide serum protein binding was also investigated in serum from healthy, drug-free human subjects; binding occurred to the extent of approximately 5%.

Animals↗
PubMed1988

Concurrent changes in Dunaliella salina ultrastructure and membrane phospholipid metabolism after hyperosmotic shock.

Hyperosmotic shock, induced by raising the NaCl concentration of Dunaliella salina medium from 1.71 to 3.42 M, elicited a rapid decrease of nearly one-third in whole cell volume and in the volume of intracellular organelles. The decrease in cell volume was accompanied by plasmalemma infolding without overall loss of surface area. This contrasts with the dramatic increase in plasmalemma surface area after hypoosmotic shock (Maeda, M., and G. A. Thompson. 1986. J. Cell Biol. 102:289-297). Although plasmalemma surface area remained constant after hyperosmotic shock, the nucleus, chloroplast, and mitochondria lost membrane surface area, apparently through membrane fusion with the endoplasmic reticulum. Thus the endoplasmic reticulum serves as a reservoir for excess membrane during hyperosmotic stress, reversing its role as membrane donor to the same organelles during hypoosmotically induced cell expansion. Hyperosmotic shock also induced rapid changes in phospholipid metabolism. The mass of phosphatidic acid dropped to 56% of control and that of phosphatidylinositol 4,5-bisphosphate rose to 130% of control within 4 min. Further analysis demonstrated that within 10 min after hyperosmotic shock, there was 2.5-fold increase in phosphatidylcholine turnover, a twofold increase in lysophosphatidylcholine mass, a four-fold increase in lysophosphatidate mass, and an elevation in free fatty acids to 124% of control, all observations suggesting activation of phospholipase A. The observed biophysical and biochemical phenomena are likely to be causally interrelated in providing mechanisms for successful accommodation to such severe osmotic extremes.

Cell Membrane↗
PubMed1987

The hydrogenation of phospholipid-bound unsaturated fatty acids by a homogeneous, water-soluble, palladium catalyst.

The hydrogenation of unsaturated phospholipids by palladium di(sodium alizarine monosulphonate) activated for 5 min under H2 proceeded rapidly at 20 degrees C and 1 atm. H2. Multibilayer liposomes of dioleoyl- and dilinolenoylphosphatidylcholine were hydrogenated at similar rates while dilinoleoyl- and 1-palmitoyl-2-oleoylphosphatidylcholine were hydrogenated at slightly slower rates. The reduction of polyunsaturated fatty acids gave rise to a variety of natural and unnatural positional cis and trans isomers which were largely reduced further to saturated fatty acids as the hydrogenation continued. Dioleoylphosphatidylethanolamine was attacked by the catalyst more slowly at 20 degrees C than was the equivalent phosphatidylcholine molecular species. Experiments conducted using mixtures of phosphatidylethanolamine and phosphatidylcholine in varying proportions also suggested that phospholipids are slightly more susceptible to catalytic hydrogenation in the bilayer phase than in the hexagonalII phase. Understanding the sequence of hydrogenation reactions involving these one and two component lipid preparations is useful in interpreting the action of the palladium catalyst on living cells under the same mild conditions.

Catalysis↗
PubMed1987

Biotransformation and pharmacokinetic overview of enoximone and its sulfoxide metabolite.

Enoximone possesses both positive inotropic and vasodilatory activities and may be useful in the treatment of patients with congestive heart failure (CHF). In all animal species investigated (rat, dog, monkey and man), the major urinary metabolite is the sulfide oxidation product (sulfoxide); very little unchanged drug appears in urine. Both in vitro and in vivo animal studies indicate reversibility of the sulfoxidation reaction; therefore, it is presumed that sulfoxidation is reversible in man. In normal healthy subjects, no difference in extent of absorption due to dietary state is observed. In patients with New York Heart Association class III to IV CHF, median terminal disposition half-lives for enoximone and its sulfoxide metabolite are 6.2 to 7.6 hours, respectively. Enoximone and sulfoxide plasma concentrations from high dose intravenous infusion studies in patients with class III to IV CHF were also investigated. The collective data suggest nonlinearity in one or more pharmacokinetic processes, of which one may be saturation of sulfoxidation. No direct relation between enoximone and/or the sulfoxide metabolite plasma concentration and pharmacologic effect has been established.

Absorption↗
PubMed1987

On the metabolic relationships between monogalactosyldiacylglycerol and digalactosyldiacylglycerol molecular species in Dunaliella salina.

Dunaliella salina cells were pulse-labeled for 2 min with [14C]palmitic acid, [14C]oleic acid, or [14C]lauric acid in order to trace the pathway of galactolipid biosynthesis and desaturation. Through the use of high performance liquid chromatography it was possible to follow the movement of radioactivity through many individual molecular species of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) for periods of 24 h and, in some cases, as much as 120 h. Analysis of the fatty acid fluxes permitted us to refine current views regarding biosynthesis of the predominantly "prokaryotic" galactolipids. The initial D. salina MGDG molecular species, containing paired oleate and palmitate (18:1/16:0), can follow two metabolic routes. If the palmitoyl chain is desaturated to 16:1, the resulting 18:1/16:1 MGDG is subject to rapid further desaturation to varying degrees, and a part of these products is subsequently galactosylated to DGDG. Contrary to widely held opinions, these DGDG molecular species can themselves be further desaturated toward a 18:3/16:4 final product. In a separate series of reactions, a smaller portion of the nascent 18:1/16:0 MGDG is directly galactosylated to 18:1/16:0 DGDG. This molecular species can then be sequentially desaturated to 18:2/16:0 DGDG and 18:3/16:0 DGDG. However, there is only very limited desaturation of the palmitoyl group attached to these molecular species.

Carbon Radioisotopes↗
PubMed1987

Alterations of the composition and size of the free fatty acid pool of Tetrahymena responding to low-temperature stress.

Cells of Tetrahymena mimbres (formerly T. pyriformis NT-1) in midlogarithmic growth under isothermal conditions (at 39 degrees C) contained a very small, compositionally discrete pool of free fatty acids, principally (60.6% of the total free fatty acid mass) palmitic and stearic acids. The composition, degree of unsaturation, and size of this free fatty acid pool were rapidly (15 min or less) altered in response to chilling. During the acclimation period following chilling to 15 degrees C, the size of the free fatty acid pool increased from a mean value of 15.5 nmol free fatty acid/mumol lipid phosphorus in 39 degrees C cells to 24.2 nmol free fatty acid/mumol lipid phosphorus. The degree of free fatty acid saturation rapidly increased over the initial hour following the onset of hypothermal conditions, but by 24 h the unsaturated free fatty acid/saturated free fatty acid ratio was 0.35 (equivalent to a 2.7-fold increase in unsaturation relative to 39 degrees C controls (unsaturated/saturated ratio = 0.13) and 4.4-fold greater than cells acclimated for 1 h (unsaturated/saturated ratio = 0.08)). By 24 h the percentage of palmitic and stearic acids had decreased to 45.6%. Similar, and in some instances more pronounced, changes were observed to occur in triacylglycerol-bound fatty acids. Modulation of steady-state free fatty acid composition could also be achieved by the addition of exogenous fatty acids to the growth medium. The ability to manipulate the level of intracellular free fatty acids should prove to be a valuable experimental tool in determining how specific fatty acids regulate various lipid-modifying enzymes.

Acclimatization↗
PubMed1987

Metabolism of exogenous long-chain fatty acids by spinach leaves.

When applied in liquid paraffin to the upper surface of expanding spinach leaves, [1-14C]palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol. A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact. Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-[14C]hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h. There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with [1-14C]palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position. Exogenous [1-14C]stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to [14C]linolenate. Exogenous [1-14C]oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol. There, desaturation of linoleate to linolenate took place. Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-[14C]linolenoyl molecular species of phosphatidylcholine as those species with two [14C]linoleoyl residues were drawn off for monogalactolipid synthesis. Glycerolipids synthesised from exogenous [1-14C]acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions. These results are interpreted as providing strong support for the two-pathway scheme of glycerolipid synthesis in plants.

Acetates↗
PubMed1986

On the mechanism of rapid plasma membrane and chloroplast envelope expansion in Dunaliella salina exposed to hypoosmotic shock.

Dunaliella salina cells rapidly diluted from their normal 1.71 M NaCl-containing growth medium into medium containing 0.86 M NaCl swelled within 2--4 min to an average volume 1.76 X larger and a surface area 1.53 X larger than found in control cells. Morphometric analysis of thin section electron micrographs revealed that certain organelles, including the chloroplast, nucleus, and some types of vacuoles, also expanded in surface area as much or more than did the entire cell. It is likely that glycerol, the most important osmotically active intracellular solute, was present in high concentration within these organelles as well as in the cytoplasm itself. Thin section and freeze-fracture electron microscopy were utilized to trace the origin of membrane material whose addition permitted the large increase in plasma membrane surface area and the equally large growth of the chloroplast outer envelope. The findings indicated that the plasma membrane's expansion resulted from its selective fusion with numerous small (less than or equal to 0.25 micron diam) vesicles prevalent throughout the cytoplasm. In contrast, new membrane added to the chloroplast outer envelope was drawn from an entirely different source, namely, elements of the endoplasmic reticulum.

Cell Membrane↗
PubMed1986

Effects of Orthophosphate and Adenosine 5'-Phosphate on Threonine Synthase and Cystathionine gamma-Synthate of Lemna paucicostata Hegelm. 6746.

Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine gamma-synthase less strongly. AMP is an extremely potent and structurally specific inhibitor of threonine synthase. Each inhibition progressively decreases with increasing concentrations of O-phosphohomoserine (OPH). To study the in vivo effects of these inhibitions, Lemna was grown with a range of Pi concentrations. A 25,000-fold increase in Pi concentration in the culture medium caused an increase of only 6-fold in total phosphorus of the plants. This is explained by the fact that a high affinity Pi uptake system is selectively down-regulated during growth with high concentrations of Pi. Pi and AMP in plants grown with various Pi concentrations were determined, and concentrations estimated for chloroplasts, the organelle containing threonine synthase and cystathionine gamma-synthase. Calculations indicated that for growth at standard external Pi (0.4 millimolar) or above, if total OPH were uniformly distributed within the plants, activities of the two enzymes in question would be severely inhibited, and each would fall two orders of magnitude below the amount required to provide threonine (plus isoleucine) or methionine adequate for growth. If OPH were restricted to chloroplasts, these inhibitions would be much less severe, resulting in enzyme activities approaching the required physiological amounts. Evidence is presented that even up to 50 millimolar external Pi, this ion does not limit production of threonine or methionine sufficiently to retard growth, consistent with the postulated localization of OPH within chloroplasts.

Journal Article↗
PubMed1986

Spinach Leaves Desaturate Exogenous [C]Palmitate to Hexadecatrienoate : Evidence that de Novo Glycerolipid Synthesis in Chloroplasts Can Utilize Free Fatty Acids Imported from Other Cellular Compartments.

Long-chain (14)C-fatty acids applied to the surface of expanding spinach leaves were incorporated into all major lipid classes. When applied in diethyleneglycol monomethyl ether solution, as done by previous workers, [(14)C]palmitic acid uptake was much lower than that of [(14)C] oleic acid. However, when applied in a thin film of liquid paraffin the rate of [(14)C] palmitic acid metabolism was rapid and virtually complete. Considerable radioactivity from [(14)C]palmitate incorporated into lipids following either application method gradually appeared in polyunsaturated C(16) fatty acids esterified to those molecular species of galactolipids previously thought to be made using only fatty acids synthesized and retained within the chloroplast. Evidence for the incorporation of radioactivity from exogenous [(14)C]oleate into those same molecular species of galactolipids was less compelling. The unexpected availability of fatty acids bound to extrachloroplastidal lipids for incorporation into galactolipids characteristically assembled entirely within the chloroplast emphasizes the need to reassess interrelations between the "prokaryotic" and "eukaryotic" pathways of galactolipid formation.

Journal Article↗
PubMed1986

Metabolism and control of lipid structure modification.

The lipid composition characteristic of a particular cellular membrane can become significantly altered, sometimes quite suddenly, when the cell is placed under environmental stress. In the majority of cases examined, the alterations seem to return the membrane's physical state towards that existing prior to imposition of the stress. The compositional changes are often diverse in their nature and also in their site of origin within the cell. Certain modifications, such as changes in the degree of phospholipid acyl chain unsaturation and in the reordering of fatty acid pairing on specific phospholipids, are now recognized as crucial first responses to stress, while others (e.g., fluctuations in relative proportions of different phospholipid classes and in the sterol:phospholipid ratio) develop more slowly and may represent secondary adjustments to the initial lipid changes. The factors directly responsible for modifying membrane lipid composition are generally unknown at the molecular level, but recent advances provide new clues favoring involvement, in some cases, of the ubiquitous mediator Ca2+. In other cases, the physical state of a membrane may directly modulate the activity of lipid-metabolizing enzymes embedded therein.

Cell Membrane↗
PubMed1986

The repeated one-point method for predicting dose sizes with irregular dosing intervals.

To initiate drug therapy a preliminary dosage regimen is often calculated using literature values for pharmacokinetic parameters. However, in some cases literature data are not available or indicate large interindividual variations, or in certain cases an altered volume of distribution (such as in acute congestive heart failure, edema, obesity, etc.) and changes in renal and nonrenal elimination are expected. For such cases the repeated one-point method (ROPM) was suggested. The ROPM like the other methods for dosage regimens assumes equal maintenance doses at equal dosing intervals. Yet, in practice and in most hospitals, doses administered 3 or 4 times daily are often not administered at equal intervals. A modification of the ROPM is suggested for unequal dosing intervals. The following 4 situations are presented: different dose sizes to maintain Cssmin constant, different dose sizes to maintain Cssmax constant, equal dose sizes where the highest peak does not exceed a certain maximum concentration and equal dose sizes where the lowest trough does not fall below a certain minimum concentration.

Circadian Rhythm↗