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Biomedical subjects

D G Greene

Publications and source records attributed to D G Greene.

At least 19 recordsLinked to original sources
PubMed2000

Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation.

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.

Adult↗
PubMed1996

Comparison of acceptor and donor substrates in the CoA-independent transacylase reaction in human neutrophils.

In human neutrophils (PMN) the ethanolamine-containing phosphoglyceride fraction (PE), principally plasmalogen-linked PE (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine), is the major store of arachidonic acid (AA). Exogenous AA is initially incorporated into 1-acyl-linked phosphoglycerides and is believed to be transferred into the 1-ether-linked phosphoglycerides via the action of a CoA-independent transacylase (CoA-IT). We have investigated the selectivity for both the "acceptor' lysophospholipids and "donor' AA-containing phospholipid substrates in the CoA-IT reaction. Evidence suggests CoA-IT may also participate in the synthesis of platelet activating factor. The transfer of [3H]AA from endogenously labeled choline-containing phosphoglycerides (PC) to exogenously added alkenyl-lyso-PE (0-50 microM) was examined in saponin-permeabilized PMN. In these "donor' studies, we observed that [3H]AA was transferred from both alkyl- and diacyl-linked PC in a proportional manner. More detailed molecular species analysis showed that [3H]AA was deacylated from all the major AA-containing molecular species in both the alkyl and diacyl subclasses with no selectivity for either subclass. To investigate the "acceptor' selectivity, membrane fractions prelabeled with either [3H]alkyl-arachidonoyl-PE or -PC were utilized as donor substrates. Various unlabeled lysophospholipids (10 microM) were added and the generation of [3H]lyso-PE or -PC was monitored as a measure of CoA-IT activity. Significant subclass preference was observed upon addition of lyso-PE species (1-alkenyl > 1-alkyl > 1-acyl) however, little selectivity was seen with the corresponding lyso-PC species. On the other hand, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid all served as poor acceptor substrates in the reaction. These data from PMN are consistent with other evidence that the CoA-IT plays a pivotal role in the enrichment of AA into plasmalogen-linked PE.

Acylation↗
PubMed1991

Evidence that hydrolysis of ethanolamine plasmalogens triggers synthesis of platelet-activating factor via a transacylation reaction.

Addition of 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (alkenyl-lyso-GPE) to human neutrophil membrane preparations containing 1-O-[3H]hexadecyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (1-O-[3H]alkyl-2-arachidonoyl-GPC) resulted in rapid deacylation of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-platelet-activating factor, lyso-PAF). When acetyl-CoA was included in the incubation mixture, the [3H]lyso-PAF was converted to [3H]PAF. Studies of [3H]arachidonate-labeled neutrophils permeabilized with Staphlococcus aureus alpha-toxin revealed a major shift of labeled [3H]arachidonate from the choline to the ethanolamine-containing phosphoglycerides upon addition of alkenyl-lyso-GPE. The studies indicated that lyso-PAF is formed in the system by the transfer of arachidonate from 1-O-alkyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE by a CoA-independent transacylase reaction. Mass measurements revealed a rapid loss of arachidonate from 1-radyl-2-acyl-GPE and a concomitant increase in alkenyl-lyso-GPE upon stimulation of the neutrophils by ionophore A23187. Based on these and other findings, a pathway is proposed that may play a significant, if not obligatory, role in the synthesis of PAF in intact stimulated neutrophils. It has been widely accepted that phospholipase A2 acts directly on 1-O-alkyl-2-arachidonoyl-GPC as the first step in the synthesis of PAF via formation of lyso-PAF. In the proposed scheme, phospholipase A2, upon stimulation, acts rapidly on ethanolamine plasmalogen selectively releasing arachidonic acid and generating alkenyl-lyso-GPE. The CoA-independent transacylase then selectively transfers arachidonate from 1-radyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE generating lyso-PAF, which is then acetylated to form PAF. The interactions outlined can account for the synthesis of 1-acyl-2-acetyl-GPC, 1-O-alk-1'-enyl-2-acetyl-GPE, and eicosanoids, in parallel with PAF.

Acylation↗
PubMed1990

Selective deacylation of arachidonate-containing ethanolamine-linked phosphoglycerides in stimulated human neutrophils.

The involvement of the ethanolamine-linked phosphoglyceride fraction (PE) in neutrophil signal transduction is suggested by the stimulus-induced release of arachidonic acid from PE (Chilton, F. H., and Connell, T. R. (1988) J. Biol. Chem. 263, 5260-5265) and by the synthesis of acetylated PE species, predominantly 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alkenylacetyl-GPE; Tessner, T. G., and Wykle, R. L. (1987) J. Biol. Chem. 262, 12660-12664) in stimulated cells. In the studies reported here, we investigated the relationship between arachidonic acid release from PE and generation of the lysophospholipid precursor required in the biosynthesis of alkenylacetyl-GPE. In order to follow these reactions, we prelabeled neutrophils with 1-O-[3H]alk-1'-enyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (alkenyl-acyl-GPE). We also followed the hydrolysis of endogenous PE by analysis as the dinitrophenyl derivative using a high pressure liquid chromatography method we developed. Our results coupled with those of Chilton et al. (Chilton, F. H., Ellis, J. M., Olson, S. C., and Wykle, R. L. (1984) J. Biol. Chem. 259, 12014-12019) indicate that in human neutrophils the metabolism of alkenylacyl-GPE and alkylacyl-sn-glycero-3-phosphocholine (GPC) are strikingly similar with regard to arachidonate metabolism. When added to neutrophils, both 1-O-[3H]alkenyl-2-lyso-GPE and 1-O-[3H]alkyl-2-lyso-GPC are acylated predominantly with arachidonic acid, and the resulting arachidonoyl-containing phospholipids are extensively deacylated upon stimulation. However, hydrolysis of PE in the neutrophil differs from hydrolysis of choline-containing phosphoglycerides in that stimulation leads to a greater accumulation of the ethanolamine-linked lysophospholipid. A comparison of the molecular species of endogenous PE (based on molar concentrations measured as the dinitrophenyl derivative) from resting and stimulated neutrophils indicated that only those species which contain arachidonate are significantly hydrolyzed.

Arachidonic Acids↗
PubMed1990

Ether-linked phosphoglyceride content of human leukemia cells.

The glycerolipids of most cells are characterized by a specific proportion of ether linkages at the sn-1 position of the glycerol backbone. A number of tumors are known to have altered concentrations of ether-linked lipids compared to normal tissues. However, no through examination of the ether-lipid content of human leukemia cells has been reported despite the importance of these lipids in normal leukocyte function. In the present study samples were obtained from adults with acute myelogenous leukemia (AML), chronic granulocytic leukemia in blast crisis, and acute lymphocytic leukemia and from healthy human donors. The cellular lipids were extracted, the individual phospholipid classes were isolated, lipid phosphorus content was determined, and the lipids were converted to diglyceride benzoate derivatives for separation and quantitation of the subclasses by high performance liquid chromatography. The data indicate that all the leukemic cells analyzed have an altered phospholipid composition compared to their respective normal leukocytes. Furthermore, among the AML patients both the percentage of the choline-containing phosphoglyceride fraction (PC) which is alkyl linked and the nmoles alkyl-PC/10(6) cells differ significantly by FAB subtype. A positive correlation between the levels of alkyl-PC and the degree of cellular differentiation is observed. Although no differences are observed between chronic granulocytic leukemia in blast crisis and AML lipids, the leukemic cells contain dramatically lower levels of alkyl-linked PC than do normal polymorphonuclear leukocytes. In contrast, no differences are observed between the alkyl-PC content of normal and leukemic lymphocytes. In light of the relations among ether-lipids, protein kinase C, and cell differentiation, these data suggest the ether-linked lipids are important in myeloid cell function and differentiation.

Alkylation↗
PubMed1984

Reductions in coronary flow under resting conditions in collateral-dependent myocardium of patients with complete occlusion of the left anterior descending coronary artery.

Flow per unit weight in collateral-dependent myocardium was quantified selectively in seven patients with complete occlusion of the proximal left anterior descending artery and prominent distal collateralization from the right coronary artery by infusing dissolved hydrogen into the right coronary artery for 10 to 15 minutes and monitoring hydrogen desaturation in the great cardiac vein. Coronary flow per unit weight in all myocardium draining into the great cardiac vein was quantified simultaneously by having the patient breathe helium and by monitoring arterial and great cardiac vein helium desaturation. Flow per unit weight in collateral-dependent myocardium averaged 38 +/- 8 (standard deviation) ml/min per 100 g and was in each case below the 95% confidence limit for normal individuals with the same rate-pressure product. Flow per unit weight in all myocardium draining into the great cardiac vein was systematically higher (51 +/- 8 ml/min per 100 g); because arteries other than the anterior descending had no stenoses greater than 30% in diameter, these values presumably reflect mixtures of subnormally perfused collateralized myocardium and adjacent normally perfused tissue. The findings suggest that coronary flow per unit weight is not maintained at usual basal values in densely collaterlized myocardium that is entirely collateral-dependent. The reductions in flow are presumably associated with marked reductions in local arterial pressure and raise the possibility of a chronic reduction in local myocardial metabolic demand.

Adult↗
PubMed1984

1-O-alkyl-linked glycerophospholipids of human neutrophils: distribution of arachidonate and other acyl residues in the ether-linked and diacyl species.

This study was done to determine whether human neutrophils contain sufficient 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine to support the synthesis of platelet activating factor by a deacylation-reacylation mechanism, and to examine the relative distribution of arachidonate among the 1,2-diacyl, 1-O-alkyl-2-acyl, and the 1-O-alk-1'-enyl-2-acyl classes of choline- and ethanolamine-containing phospholipids. The predominant phospholipid species of human neutrophils were choline-containing glycerophospholipids (41%), ethanolamine-containing glycerophospholipids (39%), and sphingomyelin (14%), with smaller quantities of phosphatidylserine (4%) and phosphatidylinositol (1%). The choline-linked fraction contained high amounts of 1-O-alkyl-2-acyl-X (44%) and 1,2-diacyl-sn-glycero-3-phosphocholine (47%), and a lesser amount of 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine (9%). In contrast, the ethanolamine-linked fraction contained a large amount of 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (66%), and lower levels of the 1,2-diacyl (24%) and 1-O-alkyl-2-acyl (10%) species. The major 1-O-alkyl and 1-O-alk-1'-enyl ether chains found in the choline and ethanolamine phospholipid pools were 16:0, 18:0, 18:1, and 20:0. The predominant fatty acyl residues found in the 1,2-diacyl and the sn-2 position of the 1-O-alkyl-2-acyl and 1-O-alk-1'-enyl-2-acyl choline and ethanolamine glycerophospholipids were 16:0, 18:0, 18:1, 18:2, and 20:4.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1981

Long-term survival after coronary bypass surgery. Comparison of various subsets of patients with general population.

Life-table analysis consecutive cases of isolated coronary bypass surgery at the Buffalo Hospital between 1973 and 1977 showed an estimated survival of 94 per cent at five years, equal to that of an age- and sex-matched group of the US population. Subsets of these patients divided according to sex, age, number of vessels narrowed, number of segments grafted, history of myocardial infarction, ejection fraction, and presence of unstable angina have estimated survivals not statistically less in any of these subsets than that of matched cohorts of the general population.

Adult↗
PubMed1979

Study of the atherogenic dyslipoproteinemia induced by dietary cholesterol in rhesus monekys (Macaca mulatta).

Hypercholesterolemia was induced in adult male rhesus monkeys with a high-fat diet containing an elevated cholesterol level (0.5%). Plasma lipoproteins were chromatographically separated into four size populations (regions) that were subdivided by density until fractions with single electrophoretic mobilities were obtained. The region III lipoproteins (LDL) contained 80% of plasma cholesterol and were present in the highest concentration of all fractions. Their molecular weight was increased over that of controls so that each particle averaged 1.8 times the number of cholesteryl ester molecules as did control LDL. Region II lipoproteins, a heterogeneous group, were present in next highest concentration. Most were cholesteryl ester-rich, beta-migrating lipoproteins that overlapped the VLDL and LDL density ranges; apoB was the predominant apoprotein. One region II subfraction had pre beta 2 migration and the density range. 1.050 less than d less than 1.10. Another subfraction, cholesteryl ester-rich VLDL including only about 1% of plasma cholesterol, had pre beta 1 migration and apoB and apoC as the predominant apoproteins with no apoprotein E. Region I lipoproteins were larger sized, slow beta-migrating cholesteryl ester-rich VLDL that included 5% of plasma cholesterol. ApoB and apoE were the predominant apoproteins. Region IV lipoproteins (HDL) contained 4% of the plasma cholesterol; their concentration was decreased to about 1/3 of the control level. Atherogenic features of the diet-induced dyslipoproteinemia included the increased plasma concentrations and cholesteryl ester contents of the region I, II, and III lipoproteins in addition to the decreased HDL concentration.

Animals↗
PubMed1978

Coronary artery fistulas emptying into left heart chambers.

We present three cases of coronary artery fistulas entering into the left heart chambers. Coronary arteriography in one showed aneurysmal dilatation of the main left coronary artery and a fistulous communication with a large left atrium. Exploration during repair revealed an anomalous branch of the left circumflex emptying into the left atrium. In the second case the proximal left circumflex gave rise to a branch supplying a hemangioma which emptied into the left atrium. Coronary arteriograms of the third patient showed an enlarged left anterior descending artery with an anomalous branch emptying into the left ventricle. Shunt flow was estimated with hydrogen as a tracer in the last two cases and was two thirds and one third of the left coronary inflow, respectively. Review of the literature shows 32 previously reported cases of a fistula draining into the left side of the heart.

Adult↗
PubMed1977

Separation and characterization of plasma lipoproteins of rhesus monkeys (Macaca mulatta).

A group of 14 adult male rhesus monkeys was maintained on a low cholesterol-high fat diet. Periodically, animals were fasted and blood samples were taken for characterization of the plasma lipoproteins. Complete separation of individual plasma lipoprotein classes was not achieved by traditional sequential ultracentrifugation techniques. Rather, initial separation of lipoprotein classes according to size was effected and density centrifugation was used subsequently for further separation. At least six lipoprotein fractions were identified, each of which was unique as defined by the properties of size, density (d), and electrophoretic mobility. These lipoprotein fractions were characterized by determination of chemical compositions and apoprotein patterns. The lipoproteins present in highest concentration in these monkeys were designated as region IV lipoproteins. This fraction had alpha-migration on agarose electrophoresis, 1.063 < d < 1.225, and the size, composition, and apoprotein pattern characteristic of HDL. No fewer than three fractions were identified with densities that overlapped the 1.019 < d < 1.063 range. Of these, the fraction designated as region III lipoproteins was present in highest concentration, had beta-migration by agarose electrophoresis, a predominant B apoprotein, and a chemical composition and size characteristic of LDL. Two larger subfractions, identified as region II lipoproteins, were separated from each other at a density of 1.050 g/ml. Agarose electrophoresis showed that the fraction with d < 1.050 had a migration intermediate between beta and pre-beta. The chemical composition and apoprotein pattern were consistent with the possibility that these lipoproteins were remnants of VLDL catabolism. The fraction with d > 1.050, had pre-beta mobility and a size and composition similar to the Lp(a) lipoprotein in plasma of human beings. At least two VLDL subfractions, identified as region I and IIa lipoproteins, were found although both were present in very low concentrations. Region I lipoproteins were larger and contained relatively more cholesteryl ester and more of the apoproteins that migrated with the mobility of apo-B and arg-rich apoprotein in SDS-polyacrylamide gel electrophoresis. Some of the region I lipoproteins were beta-migrating by agarose electrophoresis. These results suggested the possibility that a beta-migrating VLDL was present in these normal animals.

Animals↗
PubMed1975

Lordotic right posterior oblique projection of the left coronary artery: a speical view for special anatomy.

The lordotic right posterior oblique projection of the left coronary artery is obtained by combining cranial angulation of the X-ray beam with rotation of the patient into the right posterior oblique position. This projection is helpful for separation of the main left coronary artery and the proximal portions of the left anterior descending and circumflex divisions, especially in patients in whom the left anterior descending artery is directed cephalad early in its course. The obtaining of an image from the lordotic right posterior oblique projection adds less than two minutes to the procedure and improves arteriographic assessment of the left coronary artery.

Coronary Angiography↗