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Biomedical subjects

F Snyder

Publications and source records attributed to F Snyder.

At least 109 records · Page 6Linked to original sources
PubMed1984

Cytotoxicity of ether-linked phytanyl phospholipid analogs and related derivatives in human HL-60 leukemia cells and polymorphonuclear neutrophils.

A cytotoxic activity, highly selective for neoplastic cells, is expressed by 1-alkyl-2-methoxy-sn-glycero-3-phosphocholine and by other derivatives closely related to the chemical structure of platelet activating factor. The antineoplastic potencies of a new series of analogs tested in HL-60 human leukemia cells and human polymorphonuclear neutrophils are reported. The degree of cytotoxicity was documented according to the ability of each analog to 1) destroy leukemic or normal cells or 2) to release lactic acid dehydrogenase from these cells. An index of selectivity of the analogs for their cytotoxicity toward leukemia cells is presented. Substitution by the twenty carbon branched-chain phytanyl moiety in place of the straight chain alkyl ether-linked group at the sn-1 position of various phospholipid analogs resulted in a 3- to 10-fold reduction in their cytotoxic potency in HL-60 leukemia cells. The enantiomeric isomers (D-forms) of several of the analogs possessed slightly greater phospholipid analogs possessing the sn-2-acetyl (platelet activating factor) or sn-2- propionoyl substituents, both biologically active in their ability to aggregate platelets and to induce hypotension, were relatively innocuous in terms of the measured cytotoxic responses in both HL-60 cells and neutrophils.

Antineoplastic Agents↗
PubMed1983

Inactivation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by a plasma acetylhydrolase: higher activities in hypertensive rats.

We have partially characterized the properties of a specific acetylhydrolase in plasma from spontaneous hypertensive rats. This enzyme inactivates 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a lipid involved in platelet aggregating, hypotensive, and allergic responses) by removal of the acetate group. The extent of acetate hydrolysis was linear with both time and protein concentration, and the enzyme had an apparent Km of 2.5 microM and a Vmax of 2.6 nmol/min/mg protein. As with an intracellular acetylhydrolase previously characterized by us, the plasma activity was not affected by addition of phosphatidylcholine, EDTA, or Ca2+. However, in contrast to the acetylhydrolase activity in the rat kidney soluble fraction, the plasma activity was associated with a higher molecular weight protein resolved on a Sepharose 6B column and the plasma acetylhydrolase was not inhibited by treatment with trypsin, pronase, or subtilisin. We also compared the acetylhydrolase activity in plasma of age-matched spontaneous hypertensive rats and their normotensive controls, and found approximately 20% higher levels of activity in plasma from the hypertensive animals (P less than 0.01).

Animals↗
PubMed1983

The mechanism of alkyldihydroxyacetone-P synthase. Formation of [3H]H2O from acyl[1-R-3H]dihydroxyacetone-P by purified alkyldihydroxyacetone-P synthase in the absence of acylhydrolase activity.

Alkyldihydroxyacetone-P (alkyl-DHAP) synthase catalyzes the exchange of the fatty acid esterified to C-1 of the DHAP portion of acyl-DHAP for a fatty alcohol to form 1-O-alkyl-DHAP, the first ether-linked intermediate in ether lipid biosynthesis. Another characteristic of the reaction is the exchange of the pro-R hydrogen at C-1. We have investigated this hydrogen exchange using palmitoyl-[1-R-3H]DHAP and a 1000-fold purified preparation of alkyl-DHAP synthase. We found a small but significant pro-R hydrogen exchange in the absence of the co-substrate, fatty alcohol. When [14C]hexadecanol was added, the increase in pro-R 3H exchange was equal to the [14C]hexadecyl-DHAP formed. Addition of [14C]palmitic acid resulted in an increase in pro-R 3H exchange that matched the formation of [14C]palmitoyl-DHAP by the acyl exchange activity of alkyl-DHAP synthase. Furthermore, although whole microsomes contain at least two acyl hydrolases for acyl-DHAP, purified preparations of alkyl-DHAP synthase do not form DHAP from acyl-DHAP. These results are discussed with respect to data obtained from other laboratories using whole microsomes and in support of our proposed ping-pong mechanism for alkyl-DHAP synthase.

Acyltransferases↗
PubMed1983

Spontaneous and protein-catalyzed transfer of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) between phospholipid bilayers.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor or alkylacetyl-GPC), a bioactive phospholipid that possesses hypotensive, platelet-aggregating and inflammatory properties, is known to be secreted by a variety of cell types. The biological activity of alkylacetyl-GPC is related to a precise chemical structure that implicates interaction with proteins. We have studied the spontaneous and protein-catalyzed transfer of alkylacetyl-GPC between phospholipid vesicles and have demonstrated the following: 1. There are at least two transferable pools of alkylacetyl-GPC in sonicated phospholipid vesicles. 2. These two pools differ in the rate at which they dissociate from the vesicles; one pool equilibrates between donor and acceptor vesicles instantaneously while the other pool is transferred much more slowly. 3. Dialysis of alkylacetyl-GPC between phospholipid vesicles through the aqueous phase is slow. 4. A protein fraction derived from rat lung cytosol catalyzes the transfer of the nonequilibrating pool of alkylacetyl-GPC between phospholipid vesicles; this transfer is superimposed on the spontaneous transfer and is unchanged in experiments using vesicles from which the rapidly equilibrating pool has been removed.

Kinetics↗
PubMed1983

Antihypertensive effects of I-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on plasma renin activity and catecholamine responses in spontaneously hypertensive rats.

Fourteen 23 week old male spontaneously hypertensive rats (SHR) were randomly divided into saline control or phospholipid (I-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) treatment groups. Four weeks of baseline systolic blood pressure (SBP) and heart rate (HR) measurements were determined via tail plethysmography. On week 25 of the baseline period a 1.5 ml blood sample was taken by tail clip for analysis of norepinephrine (NE), epinephrine (E), and plasma renin activity (PRA). On the following week, a single injection of phospholipid (11 ug/kg, s.c.) was given to the experimental animals following baseline SBP and HR determinations. A similar procedure was employed for control subjects, except they received an injection of normal saline (0.5 ml, s.c.). Systolic BP and HR responses were monitored for 24 minutes following the injection. A 1.5 ml blood sample was taken at the end of the 4th minute for NE, E, and PRA assays. A significant drop in SBP (202 +/- 5 mmHg to 124 +/- 6 mmHg) and an increase in HR (431 +/- 17 bpm to 519 +/- 21 bpm) were observed for experimental animals, but not for control subjects. Plasma NE increased significantly (446 +/- 42 pg/ml to 1099 +/- 77 pg/ml), but E remained unchanged following treatment with the phospholipid. Plasma renin activity increased for both groups, but this change was only significant for the experimental group (18.1 +/- 5.7 ng Al/ml/hr to 34.3 +/- 3.6 ng Al/ml/hr). Thus, it appears that I-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine is a potent antihypertensive vasodilating agent which stimulates baroreceptor mediated sympathetic discharge to the heart and kidneys of the SHR.

Animals↗
PubMed1983

Biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine by rat alveolar macrophages. Phospholipase A2 and acetyltransferase activities during phagocytosis and ionophore stimulation.

1-Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) comprises 11% of the total phospholipids of rat alveolar macrophages. This endogenous pool of alkylacyl-GPC was prelabeled by incubating the macrophages with [1,2-3H]alkyllyso-GPC (54 Ci/mmol), which enters the cells and is acylated. The effect of various stimuli on the synthesis and release into the media of labeled alkylacetyl-GPC (platelet-activating factor) from the cells was used to establish the role of inactive alkylacyl-GPC as a precursor of the biologically active derivative. A phagocytic agent (zymosan, 100 micrograms/ml) and an ionophore (A23187, 2 microM) stimulated the release of both alkylacetyl-GPC and alkyllyso-GPC into the media at the expense of cellular alkylacyl-GPC. Phospholipase A2 activity (at pH 4.5 and in 1 mM EDTA) was also increased in the media. The stimulatory effect of zymosan and the ionophore on alkylacetyl-GPC release was prevented by mepacrine (0.1 mM), an agent that inhibits the release of fatty acids from phospholipids. These data indicate that phospholipase activity is required for the biosynthesis of alkylacetyl-GPC. However, since the inhibitory effect of mepacrine was not apparent when acetate was present, it appears that the acetylation step is rate limiting. Exposure of alveolar macrophages in culture to zymosan or A23187 stimulated acetyltransferase activity 250-300%. In contrast, phorbol myristate acetate (1.6 microM), which stimulated the accumulation of lysophospholipids but not the level of alkylacetyl-GPC in the media, did not substantially increase acetyltransferase activity. We conclude that alkylacyl-GPC serves as a precursor of alkylacetyl-GPC and that the production of this potent mediator by rat alveolar macrophages can be stimulated by agents that affect phospholipase A2 and acetyltransferase activities. The latter enzyme appears to have a regulatory function in the biosynthesis of alkylacetyl-GPC.

Acetyltransferases↗
PubMed1983

Quantitative analysis of ether-linked lipids as alkyl- and alk-1-enyl-glycerol benzoates by high-performance liquid chromatography.

Methodology for the quantitative and qualitative analyses of alkyl- and alk-l-enyl-glycerols derived by Vitride reduction of ether-linked glycerolipids in the presence of an internal standard was developed. The procedure involved preparation of benzoate derivatives that were subsequently analyzed by high-performance liquid chromatography with detection at 228 nm. Separation of the glycerol ether dibenzoates on a C18 reverse-phase column allowed for the simultaneous quantitation and the determination of chain length of both alkyl- and alk-l-enyl-glycerols in a single chromatographic run of less than 15 min. The method was accurate (less than 10% error), reproducible, and sensitive (less than 1 microgram per component). Application of the method to the analysis of phospholipids from L-M cells grown in the presence and absence of elaidic acid demonstrated that the cells incorporated a portion of the trans acid supplement (presumably via the fatty alcohol) into the side chains of both alkyl- and alk-l-enyl-glycerol-containing phosphatides.

Animals↗
PubMed1983

Stimulation of calcium uptake by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) in rabbit platelets: possible involvement of the lipoxygenase pathway.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) induces an increase of Ca2+ uptake in rabbit platelets. This process depends upon the extracellular concentration of Ca2+ with the maximum stimulation occurring at 1-3 mM; uptake under these conditions is blocked by verapamil, a calcium-entry blocker. Increase of calcium uptake by the bioactive phospholipid was independent of ADP-induced platelet responses and of metabolites of arachidonic acid metabolism formed through the cyclooxygenase pathway. However, mepacrine, p-bromophenacyl bromide, eicosatetraynoic acid, and nordihydroguaiaretic acid significantly or totally inhibited the stimulation of Ca2+ uptake by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine. When arachidonic acid was given sufficient time to be metabolized to other products by the platelets, stimulation of Ca2+ uptake also occurred. Arachidonic acid and platelet-activating factor did not produce an additive or synergistic effect. Our data suggest that a metabolite(s) generated from arachidonic acid through the lipoxygenase pathway may be the mediator(s) responsible for the action of platelet-activating factor in the induction of increased Ca2+ uptake in rabbit platelets.

Adenosine Diphosphate↗
PubMed1983

Peptide utilization by nitrogen-starved Neurospora crassa.

Peptides ranging in size from a mean number of 30 residues down to dipeptides supported growth of a leucine auxotroph when used as both a nitrogen and leucine source. Under nitrogen-limiting conditions, the peptides induced extracellular peptidohydrolytic activity, hydrolyzing peptides to monomer amino acids. Growth of a leu-2 mutant of Neurospora crassa on those peptides transportable by the oligopeptide transport system did not result in induction of hydrolytic activity, whereas growth of a leu-2; gltR mutant on these same peptides resulted in induction of peptidohydrolytic activity. The induced extracellular proteolytic activity was shown to be analogous to that inducible by growth on proteins, e.g., bovine serum albumin.

Leucine↗
PubMed1983

Cytotoxicity of alkyl-lysophospholipid derivatives and low-alkyl-cleavage enzyme activities in rat brain tumor cells.

Alkyl-lysophospholipids (ALP) and related derivatives inhibited the in vitro incorporation of [3H]thymidine into seven different permanent cell lines derived from rat brain tumors. The cytostatic effect of ALP was dependent on dosage and incubation time. Naturally occurring 2-lysophosphatidylcholine did not exhibit cytostatic effects; under these conditions, the incorporation rates of [3H]thymidine were generally more than 100% of the controls. The trypan blue dye exclusion test, which was used to assess severe cell damage, correlated with the extent that [3H]thymidine incorporation was inhibited by ALP. Preincubation of ALP (rac-1-octadecyl-lyso-glycero-3-phosphocholine) for more than 8 min with a tetrahydropteridine-dependent O-alkyl cleavage enzyme preparation from rat liver microsomes destroyed almost all of the cytotoxic properties of ALP when tested at a concentration that previously inhibited tumor growth by more than 50%. [3H]Thymidine incorporation rates were greater than 100% for astrocytoma cells incubated with ALP after exposure to the alkyl cleavage enzyme. Comparison of the microsomal activities of the tetrahydropteridine-dependent alkyl-cleavage enzyme present in astrocytoma 78-FR-G-299 cells and the pleomorphic glioma 78-FR-G-219/S4 cells to that found in normal skin fibroblasts and rat livers revealed a markedly reduced activity in the neoplastic cell lines. Moreover, those tumor cells that were more resistant to ALP cytotoxicity (pleomorphic glioma, 78-FR-G-219/S4) had a 3-fold higher tetrahydropteridine-dependent cleavage activity than a more cytotoxic sensitive line (astrocytoma cells, 78-FR-G-299). Our results indicate that the low-alkyl-cleavage enzyme activities in these neoplastic cells in comparison to normal cells might be a factor in explaining the relatively high cytotoxicity of ALP in tumor cells.

Animals↗
PubMed1982

Formation of alkylacyl- and diacylglycerophosphocholines via diradylglycerol cholinephosphotransferase in rat liver.

We found that diacylglycerol and alkylacylglycerol choline phosphotransferase activities exhibited similar pH optima, thermolabilities and inhibitions by Mn2+, and dithiothreitol. The Vmax of diacylglycerol cholinephosphotransferase was higher (approx. 1-2-fold) than the Vmax of alkylacylglycerol cholinephosphotransferase. The Km value for diacylglycerol was somewhat greater than for the alkylacylglycerol, but no differences were found for the Km of CDPcholine with either type of diradylglycerol as the other substrate. Endogenous levels of diacylglycerols in microsomes were 24.3 nmol/mg protein, whereas no detectable amount of alkylacylglycerols was observed. Results from this study indicate that a similar or identical enzyme catalyzes the formation of 1,2-diacyl- and 1-alkyl-2-acyl-sn-glycero-3-phosphocholine and that the availability of diradylglycerols and the turnover rate of ether-linked lipids would appear to be important factors in controlling the level (under 1%) of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine in rat liver.

Animals↗
PubMed1982

Increasing the levels of ether-linked lipids in L-M cells by glyceryl ether supplementation depresses growth and choline utilization.

To gain insight into the role of alkyl-linked lipids in biological systems, we added hexadecylglycerol (a precursor of complex ether-linked lipids) to medium required for the growth of L-M cells in culture. L-M fibroblasts cultured through several generations in the presence of hexadecylglycerol grow at a reduced rate. Experimental cells at their sixth passage, with 2 microgram supplement/ml, double at 50% the rate of control cell populations. Hexadecylglycerol (10 microgram/ml) added 1 day after cell passage does not retard growth; however, within 1 h it decreases the incorporation of choline into the choline glycerophosphatide fraction. Inhibition is specific for choline; ethanolamine incorporation is not affected. The inhibition of choline utilization by hexadecylglycerol-treated cells is dose-dependent and reaches a maximum 12 h after supplementation. Cellular uptake of choline is reduced (approx. 17%) but not as much as the incorporation of choline into the phospholipids (approx. 60% at 12 h). The assimilation of ether lipid precursor into cellular phospholipids was followed by incubating cells with [1-14C]hexadecylglycerol. Incorporation of radioactivity into cellular phospholipids begins to plateau after 24 h, whereas the interference of hexadecylglycerol with choline metabolism could be detected as early as 1 h. The majority of the radioactivity recovered from cells incubated with labeled hexadecylglycerol is localized in the microsomal fraction (56%), where the label was distributed as free hexadecyglycerol, alkylacyl-phospholipids and alkyldiacylglycerols. These results show that the supplementation of a glyceryl ether to L-M fibroblast growth media selectively inhibits the utilization of choline for choline glycerophospholipid biosynthesis and causes a reduction in cell growth rate when cells are continually passaged in the presence of the glyceryl ether.

Choline↗
PubMed1982

Alkyldihydroxyacetone-P synthase. Solubilization, partial purification, new assay method, and evidence for a ping-pong mechanism.

Alkyldihydroxyacetone-P (alkyl-DHAP) synthase, the enzyme that forms the ether linkage of alkyl and alk-1-enyl glycerolipids, has been solubilized from ehrlich ascites cell microsomes using Triton X-100 and acetone precipitation. the solubilized enzyme, which is stabilized by glycerol or ethylene glycol, was then purified further by chromatography on DEAE-cellulose, QAE-Sephadex, Matrex Red, and hydroxylapatite with the aid of a new rapid assay method using DEAE-cellulose disks. Four enzymes involved in the metabolism of acyl-DHAP and alkyl-DHAP (acyl-DHAP/alkyl-DHAP oxidoreductase, DHAP acyltransferase, alkyl-DHAP phosphohydrolase, and a dinitrofluorobenzene-insensitive acyl-DHAP acylhydrolase) are removed under these conditions along with endogenous fatty acids and fatty alcohols. Two other activities copurify with the alkyl-DHAP synthase forward reaction: an acyl exchange reaction, in which [1-14C]palmitic acid is incorporated into palmitoyl-DHAP, and an alkyl exchange reaction, in which [1-14C]hexadecanol is incorporated into hexadecyl-DHAP. Exchange reactions of this type are characteristic properties of a ping-pong mechanism but not a sequential mechanism. This is confirmed by documentation that palmitic acid is a competitive inhibitor with respect to hexadecanol. In addition, low levels of palmitoyl-DHAP (less than 100 microM) show competitive inhibition with respect to hexadecanol, possibly due to palmitic acid formed from palmitoyl-DHAP by alkyl-DHAP synthase under these conditions. Based on the observations presented here and previously, a molecular mechanism for alkyl-DHAP synthase is proposed.

Alkyl and Aryl Transferases↗