PubMed Health⌕ Search

Biomedical subjects

L A Horrocks

Publications and source records attributed to L A Horrocks.

At least 73 records · Page 4Linked to original sources
PubMed1992

Phospholipid composition of cultured human endothelial cells.

Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay.

Cell Extracts↗
PubMed1992

Serine incorporation into phosphatidylserine in hypoxic rat brain cortex.

The effect of hypoxia on the incorporation of [14C]serine into serine glycerophospholipids was investigated in rat brain cortex. Brain slices were incubated, in the presence of the labeled precursor, in Krebs-Henseleit Ringer bicarbonate or Krebs Ringer phosphate, and hypoxia was induced by bubbling nitrogen in the medium. The lowering of oxygen caused an increase of the incorporation of the base into phosphatidylserine in slices incubated in both media, although the effect was greater in Krebs Ringer phosphate. Such an effect was also observed in the homogenate subjected to N2-treatment, with an increase in the incorporation similar to that obtained in slices incubated in Krebs-Henseleit Ringer bicarbonate. Phosphatidylserine is synthesized in mammalian tissues by a "base-exchange" enzyme, strictly Ca2+ dependent, and, moreover, is necessary for protein kinase C activity. We postulate that the increased synthesis of phosphatidylserine might affect signal transduction mechanisms and participate in the modification of lipid metabolism observed in hypoxia and/or ischemia.

Animals↗
PubMed1992

Quantitative association between altered plasma esterified omega-6 fatty acid proportions and psychological stress.

Medical students (MS) tested during the first year of medical school showed both greater stress on the Brief Symptom Inventory and lower plasma proportions of total esterified arachidonic acid (AA, C20:4n-6), and its omega-6 fatty acid (FA) precursor, linoleic acid (C18:2n-6) than control laboratory workers. This association suggests that omega-6 FA metabolism may be affected during stress. Low AA values might result from depletion of plasma stores for immunoregulatory prostenoids formation or from modification of metabolic pathways by cortisol or other cytokine compounds implicated in stress. Values for other major FA and the omega-3 neuronal metabolic substrate, docosahexaenoic acid (DHA, C22:6n-3) were similar between students and controls. The clear preservation of the omega-3 FA pathway suggests their programmed availability for neuronal function during stress. Since plasma FA proportions may affect immune cell membrane function(s), we suggest that altered values of plasma FAs may be an important component of the physiological effects of psychological stress.

Anxiety↗
PubMed1992

Cell swelling, blebbing, and death are dependent on ATP depletion and independent of calcium during chemical hypoxia in a glial cell line (ROC-1).

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.

Adenosine Triphosphate↗
PubMed1992

Identification and purification of calcium-independent phospholipase A2 from bovine brain cytosol.

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Stimulation of mono- and diacylglycerol lipase activities in ibotenate-induced lesions of nucleus basalis magnocellularis.

Ibotenic acid was injected into the nucleus basalis magnocellularis region of rat brain in order to study whether an elevation of lipase activities was associated with the degeneration of cholinergic neurons in this potential animal model of Alzheimer's disease. Two plasma membrane fractions were prepared from different regions of ibotenate injected (right hemisphere) and non-injected (left hemisphere) rat brain. One plasma membrane fraction was from synaptosomes (SPM) and the other from glial and neuronal cell bodies (PM). Activities of mono- and diacylglycerol lipases in these plasma membrane fractions were markedly increased (3- to 5-fold) in hippocampus, midbrain and frontal cortical regions of rat brain at 10 days after the injection of ibotenate. The activity of choline acetyltransferase was decreased in frontal cortex but unchanged in hippocampus and midbrain. Our results suggest that the increase in lipase activity is much more widespread and non-specific than is the decrease in cholinergic function.

Alzheimer Disease↗
PubMed1991

Stimulation of mono- and diacylglycerol lipase activities by gangliosides in chicken neuronal cultures.

Chicken neurons in culture display measurable activities of mono- and diacylglycerol lipases. Treatment of chicken neuronal cultures with gangliosides (10(-8)M to 10(-5)M) resulted in a time and dose dependent increase in monoacylglycerol lipase activity. The diacylglycerol lipase showed significant increase in specific activity before that of monoacylglycerol lipase. The increase was observed only up to 24 hours and no differences between diacylglycerol lipase activity of control and ganglioside treated cells were observed after 48 hours. The data indicate that the treatment of neurons with exogenous gangliosides affect the diglyceride metabolism in stimulating not only the enzymes catalyzing their production but also those involved in their catabolism.

Animals↗
PubMed1990

Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine.

We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.

Adrenal Gland Neoplasms↗
PubMed1990

Stimulation of mono- and diacylglycerol lipase activities by bradykinin in neural cultures.

Neural cultures of fetal mouse spinal cord, mouse neuroblastoma (N1E-115) and mixed primary glial cell cultures from neonatal rat brain display measurable activities of mono- and diacylglycerol lipases. Treatment of fetal mouse spinal cord cultures with bradykinin (10 nM) for 1-4 min resulted in a marked increase in specific activities of mono- and diacylglycerol lipases. This is the first direct demonstration that bradykinin can act through the lipase pathway. The increase in activities of lipases was dose and time dependent. The bradykinin response was blocked by [Thi5,8, D-Phe7]bradykinin, a bradykinin B-2 receptor antagonist, indicating that the bradykinin induced stimulation of lipase activities involves bradykinin receptors.

Animals↗
PubMed1990

Purification of plasmalogens using Rhizopus delemar lipase and Naja naja naja phospholipase A2.

Bovine heart ChoGpl (choline glycerophospholipid) and bovine brain EtnGpl (ethanolamine glycerophospholipid) contain diacyl, alkenylacyl and alkylacyl analogs. Purification of plasmalogens was achieved using R. delemar lipase and N. naja naja phospholipase A2 digestion. The R. delemar lipase hydrolyzes the acyl bond at the 1-position of 1,2-diacyl glycerophospholipids. The N. naja naja phospholipase A2 has greater activity with diacyl and alkylacyl than with alkenylacyl glycerophospholipids. These enzymes were mainly used to remove diacyl and alkylacyl analogs respectively. When the diacyl types were removed by double incubation with R. delemar lipase, the plasmalogen content was 94.2% +/- 0.21% (mean +/- S.E.M., n = 4) for PlsCho (plasmenylcholine) and 94.9% +/- 0.19% (mean +/- S.E.M., n = 3) for PlsEtn (plasmenylethanolamine). Recoveries were 74% and 88% respectively. These partially purified plasmalogens were treated with N. naja naja phospholipase A2. Finally, 97.7% +/- 0.24% (mean +/- S.E.M., n = 4) and 98.8% +/- 0.27% (mean +/- S.E.M., n = 3) pure plasmalogens were obtained for PlsCho and PlsEtn respectively. Plasmalogens were recovered in an overall yield of 7.7% +/- 0.7% (mean +/- S.E.M., n = 4) and 10.2% +/- 1.2% (mean +/- S.E.M., n = 3) for PlsCho and PlsEtn.

Animals↗
PubMed1989

Fluorimetric coupled enzyme assay for lysoplasmalogenase activity in liver.

We developed a continuous spectrofluorimetric assay of lysoplasmalogenase activity with the use of horse liver alcohol dehydrogenase as a coupling enzyme. In this method the disappearance of NADH is measured spectrofluorimetrically. The excitation and emission monochromators were set at 340 and 460 nm respectively. The assay is 10 times as sensitive as the previous u.v. spectrophotometric method. We could detect approx. 0.02 nmol of aldehyde produced/min per ml.

Alcohol Dehydrogenase↗
PubMed1989

Solubilization, purification and characterization of lysoplasmalogen alkenylhydrolase (lysoplasmalogenase) from rat liver microsomes.

Alkenylhydrolase (EC 3.3.2.2; EC 3.3.2.5) has been purified 200-fold to a specific activity of 8.0 mumol/min per mg from rat liver microsomes with 51% of the activity recovered. Purification was accomplished by solubilization of the membrane-associated enzyme with octylglucoside and chromatographic resolution on sequential DEAE cellulose and hydroxylapatite (HPLC) columns in the presence of octylglucoside. The partially purified enzyme, specific for the 2-deacylated plasmalogen, lysoplasmalogen (1-alk-1'-enyl-sn-glycero-3-phosphocholine or -ethanolamine), had no hydrolytic activity with intact plasmalogens or 1-acyl-sn-glycero-3-phosphoethanolamine. Kinetic analyses of enzymic activity demonstrated apparent Km values of 5.5 and 42 microM for 1-alk-1'-enyl-sn-glycero-3-phosphocholine and 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine, respectively. The Vmax values were 11.7 and 13.6 mumol/min per mg with the choline and ethanolamine substrates, respectively. The optimal pH range was between 6.6 and 7.1 with both substrates; the energy of activation for the purified enzyme was 15,200 cal. The enzyme required no cofactors and was unaffected by low millimolar concentrations of Ca2+, Mg2+, Mn2+ or EDTA. It was inhibited by the sulfhydryl-reacting reagent, p-chloromercuribenzoate. Mono- or diradylglycerophospholipids or sphingomyelin did not affect the enzymic activity at 37 degrees C. Activity of the purified enzyme, destroyed by freezing at -20 degrees C, was preserved if stored at this temperature in the presence of 300-600 microM diradylglycerophosphocholine or 50% glycerol. A continuous spectrophotometric assay, adapted in our laboratory for the assay of liver alkenylhydrolase, facilitated this purification. This is the first reported purification of alkenylhydrolase.

Animals↗
PubMed1989

Continuous spectrophotometric assay of phospholipase A2 activity hydrolyzing plasmalogens using coupling enzymes.

We developed a continuous spectrophotometric assay of the phospholipase A2 activity specific for choline plasmalogen using rat liver lysoplasmalogenase and horse liver alcohol dehydrogenase as coupling enzymes and Naja naja venom phospholipase A2 as a source of the phospholipase A2 activity. In these coupling reactions, choline lysoplasmalogen is hydrolyzed by lysoplasmalogenase to glycerophosphocholine and free aldehyde. The free aldehyde is quantitatively converted to alcohol by alcohol dehydrogenase with the oxidation of NADH. The disappearance of NADH is measured spectrophotometrically at 340 nm. The assay is sensitive to about 0.2 nmol aldehyde produced/ml/min and also is rapid, convenient, and continuous.

Animals↗
PubMed1989

Sources for brain arachidonic acid uptake and turnover in glycerophospholipids.

Brain arachidonic acid comes from linoleic acid and arachidonic acid in the blood. Part of the brain arachidonic acid is elongated to adrenic acid, 22:4 (n = 6), especially in higher animals. With labeled arachidonic acid injected into cerebral ventricles of mice, the highest specific radioactivity was in triacylglycerols. The highest labeling in PtdCho and PtdIns was found at 15 to 60 minutes. Labeling of PtdEtn was much less. The molecular species with 16:0 and 18:1 were labeled better than those with 18:0. Adrenic acid was preferred by alkylacyl-GroPEtn. The highest level of labeling by arachidonic acid was found in the choline plasmalogens and the alkylacyl-GroPCho at 24 hours after injection. The PtdCho arachidonic acid turned over several times within 24 hours. Part of this turnover probably represents the transfer of labeled arachidonic acid to unlabeled ether-linked choline glycerophospholipids, including 1-alkyl-2-lyso-GroPCho, also known as lyso platelet activating factor. The energy-independent transfer of arachidonic acid from PtdCho to ether-linked choline glycerophospholipids may follow removal of their arachidonic acid by phospholipase A2 due to receptor activation. The lack of pulse labeling of ether-linked choline glycerophospholipids complicates the study of their function.

Animals↗