Studies on molecular species of choline glycerophospholipids of developing rat brain.

The chronological changes in molecular species of choline glycerophospholipids were studied for cerebra of 17-, 19- and 21-day-old rat fetuses, and 3-, 6-, 12-, 24- and 90-day-old rats. The molecular species found by gas chromatography-mass spectrometry and selected ion retrieval technique were phosphatidylcholines of '30 : 0, 32 : 0, 32 : 1, 34 : 0, 34 : 1, 34 : 2, 36 : 0, 36 : 1, 36 : 2, 36 : 3, and 36 : 4' where the larger number indicates the sum of chain lengths on positions C-1 and C-2; the smaller number is the total number of double bonds. Of these molecular species, '32 : 0' (mainly 16 : 0/16 : 0, dipalmitoyl glycerophosphorylcholine), '34 : 1' (mainly 16 : 0/18 : 1, palmitoyloleoyl glycerophosphorylcholine), '34 : 0' (16 : 0/18 : 0, palmitoylstearoyl glycerophosphorylcholine), '32 : 1' (mainly 16 : 0/16 : 1, palmitoylpalmitoleoyl glycerophosphorylcholine and '30 : 0' (14 : 0/16 : 0, myristoylpalmitoyl glycerophosphorylcholine) were main species. The '32 : 0' species increased to about 44% at around the 10th day and thereafter remained nearly constant. '34 : 1' and '34 : 0' decreased to about 17 and 6% at that time and then increased to about 30 and 14%, respectively. '30 : 0' increased from last stage of gestation to the 6th day and then decreased. '32 : 1' was about 16% for 17-day-old fetus and decreased grandually. '36 : 1' (18 : 0/18 : 1, stearoyloleoyl glycerophosphorylcholine) increased at the latter part of development.     

Statistical analysis of the processes controlling choline and ethanolamine glycerophospholipid molecular species composition

The regulation and maintenance of the cellular lipidome through biosynthetic, remodeling, and catabolic mechanisms are critical for biological homeostasis during development, health and disease. These complex mechanisms control the architectures of lipid molecular species, which have diverse yet highly regulated fatty acid chains at both the sn1 and sn2 positions. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serve as the predominant biophysical scaffolds in membranes, acting as reservoirs for potent lipid signals and regulating numerous enzymatic processes. Here we report the first rigorous computational dissection of the mechanisms influencing PC and PE molecular architectures from high-throughput shotgun lipidomic data. Using novel statistical approaches, we have analyzed multidimensional mass spectrometry-based shotgun lipidomic data from developmental mouse heart and mature mouse heart, lung, brain, and liver tissues. We show that in PC and PE, sn1 and sn2 positions are largely independent, though for low abundance species regulatory processes may interact with both the sn1 and sn2 chain simultaneously, leading to cooperative effects. Chains with similar biochemical properties appear to be remodeled similarly. We also see that sn2 positions are more regulated than sn1, and that PC exhibits stronger cooperative effects than PE. A key aspect of our work is a novel statistically rigorous approach to determine cooperativity based on a modified Fisher's exact test using Markov Chain Monte Carlo sampling. This computational approach provides a novel tool for developing mechanistic insight into lipidomic regulation.     

Different metabolic rates for arachidonoyl molecular species of ethanolamine glycerophospholipids in rat brain

The ethanolamine glycerophospholipids (EGP) contain most of the arachidonate (20:4, n-6) and adrenate (22:4, n-6), potential precursors of biologically potent prostaglandins and related compounds. Much better methods utilizing high performance liquid chromatography (HPLC) techniques are now available for the study of the molecular species of all three classes, namely diacyl, alkenylacyl (plasmalogen), and alkylacyl. Different molecular species may have different functions. This possibility was studied by examining the rates of incorporation of [3H]arachidonic acid into the three major molecular species of each of the three classes of ethanolamine glycerophospholipids. After the intracerebral injection of [3H]20:4 into rat brain, it was rapidly converted to 22:4(n-6). Of the total radioactivity, 10-20% was located in 22:4 in alkenylacyl and diacyl-GPE. In the alkylacyl-GPE, labeled 22:4 was preferentially incorporated and accounted for 50-60% of the total radioactivity. The primary arachidonoyl molecular species of alkenylacyl, alkylacyl, and diacyl-GPE were the 18:1-20:4, 16:0-20:4, and 18:0-20:4 species. The alkylacyl class contained almost equal proportions of these three molecular species. On the other hand, the 20:4 in alkenylacyl and diacyl classes was combined largely with 18:0 groups at the sn-1 position. In particular, the 18:0-20:4 species comprised about 80% of arachidonoyl molecular species of the diacyl class. In all three classes, the highest specific radioactivities were found in the 18:1-20:4 species, whereas the 18:0-20:4 species had the lowest specific radioactivity. Over the period 60 min-24 hr, the diacyl 18:0-20:4 and all three arachidonoyl molecular species of the alkenylacyl class increased in specific radioactivity more rapidly than the other arachidonoyl molecular species.     

Fatty acid changes in liver choline and ethanolamine glycerophospholipids in aspirin-treated rats fed linoleate, gamma-linolenate and fish oil

The effects of dietary linoleic acid, gamma-linolenic acid and marine fatty acids on the development of aspirin-induced gastric hemorrhage and the distribution of liver glycerophospholipid fatty acids in fat-deficient growing rats were studied. Aspirin (100 mg/day)-treated and nontreated rats were fed for 7 days, a mixed diet of 2.5% safflower oil and 7.5% hydrogenated coconut oil (SFO/HCO) or 7.5% fish oil (SFO/FO), or 2.5% gamma-linolenate concentrate and 7.5% fish oil (GLA/FO). Gastric hemorrhage was induced in animals by aspirin treatment to various extents. It was not affected by FO feeding, but was significantly alleviated by GLA feeding. Aspirin treatment reduced the proportions of 20:4n-6 in liver phosphatidylcholine. FO feeding (in SFO/FO and GLA/FO rats) further reduced the 20:4n-6 level and replaced it by n-3 fatty acids. GLA feeding, on the other hand, elevated the proportion of 20:4n-6. As a result, the reduction of 20:4n-6 by fish oil feeding, was less significant in GLA/FO rats than in SFO/FO rats. The degree of gastric hemorrhage appeared to relate negatively to the levels of 20:4n-6 in liver phosphatidylcholine, and to the sum of 20:4n-6 and 20:5n-3 when FO was included in the diet. It is suggested that long-chain polyunsaturated fatty acids (20:4n-6 and 20:5n-3) per se in addition to being precursors of prostaglandins, may also affect the development of gastric hemorrhage, possibly by modulating the permeability of cell membranes in the gastric mucosa.     

Altered acyl chain compositions of alkylacyl, alkenylacyl, and diacyl subclasses of choline and ethanolamine glycerophospholipids in rat heart by dietary fish oil

The effects of dietary fish oil containing n - 3 polyunsaturated fatty acids on the fatty acid compositions of the alkylacyl and alkenylacyl species of choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) were studied in rat heart and compared with the corresponding diacylglycerophospholipids. After a 7 week feeding period, all phospholipid classes from the fish oil group exhibited much higher levels of the n - 3 polyunsaturated fatty acids including eicosapentaenoic acid (20:5(n - 30)), docosapentaenoic acid (22:5(n - 3)) and docosahexaenoic acid (22:6(n - 3)), as well as lower levels of the n - 6 series (18:2, 20:4, 22:4 and 22:5), relative to animals given sunflower seed oil-enriched in 18:2(n - 6). However, the docosahexaenoic acid rather than eicosapentaenoic acid provided a much greater contribution to the n - 3 accumulation (fish oil group) in the ether-containing CGP, as indicated by the 20:5(n - 3)/22:6(n - 3) molar ratios of 0.32, 0.26 and 0.56 in the alkylacyl, alkenylacyl and diacyl classes, respectively. In addition to accumulating very high levels of docosahexaenoic acid (e.g., 47.2 mol% of fatty acids in alkenylacylglycerophosphoethanolamine of fish oil group), both ether-linked classes of EGP exhibited significantly higher levels of docosapentaenoic acid than the diacylglycerophosphoethanolamine (GPE) and all classes of CGP. These findings may bear relevance to possible beneficial effects of dietary fish oil on pathophysiological states (including myocardial ischemia) in cardiac tissue and their mediation via platelet-activating factor, 1-alkyl-2-acetylglycerophosphocholine (PAF) and arachidonic acid (20:4(n - 6))-derived eicosanoids.     

Preferential synthesis of diacyl and alkenylacyl ethanolamine and choline glycerophospholipids in rabbit platelet membranes

In rabbit platelet membranes, the contents of alkenylacyl phospholipids (plasmalogen) were 56% of phosphatidylethanolamine and 3% of phosphatidylcholine. This uneven distribution of plasmalogens in each phospholipid class could be attributed to the different substrate specificity of ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2). The properties of the enzymes were studied, using endogenous diglycerides and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. The newly formed phospholipids were mainly diacyl and alkenylacyl and only rarely alkylacyl type. The ratios of the labeled alkenylacyl to diacyl type of phospholipids clearly varied with the concentrations of CDP-ethanolamine or CDP-choline. When 1, 10, and 30 microM CDP-[3H]ethanolamine were used, the labeled phospholipids contained 53, 37, and 27% of the alkenylacyl type, respectively. The apparent Km for CDP-ethanolamine to synthesize alkenylacyl and diacyl types were 2.2 and 8.1 microM. On the other hand, when 1, 10, and 30 microM CDP-[14C]choline were used, the labeled lipids contained 10, 17, and 24% alkenylacyl type, respectively. The apparent Km for CDP-choline to synthesize alkenylacyl and diacyl types were 24 and 4.3 microM. Further, the syntheses of diacyl type of phosphatidylethanolamine and the alkenylacyl type of phosphatidylcholine were markedly inhibited by unlabeled CDP-choline and CDP-ethanolamine, respectively. The two enzymes had opposite substrate specificities, and ethanolaminephosphotransferase showed a high preference to plasmalogen synthesis, especially in the presence of CDP-choline.     

Effect of fish oil and coconut oil diet on the LDL receptor activity of rat liver plasma membranes

The influence of 4 weeks treatment with fish oil and coconut oil enriched diets on the chemical composition of rat liver plasma membranes and LDL and on the binding of LDL to liver membranes was investigated. Rats fed fish oil diet showed a total, LDL and HDL plasma cholesterol concentration lower than the values observed in rats fed coconut oil and to a lesser extent lower than those of rats fed standard laboratory diet. LDL of rats on fish oil diet had a relative percentage of cholesterol and phospholipid lower, while that of triacylglycerol was greater. Furthermore, fish oil feeding was associated with a greater concentration of n - 3 fatty acids and a lower arachidonic and linoleic acid content in LDL. Liver plasma membranes isolated from fish oil rats showed a higher percentage of n - 3 fatty acids, while only a trace amount of these fatty acids was found in control and coconut oil fed animals. In binding experiments performed with LDL and liver membranes from fish oil fed rats and control rats, binding affinity (Kd = 3.47 +/- 0.93 and 4.56 +/- 1.27, respectively) was significantly higher (P less than 0.05) as compared to that found using membranes and lipoprotein from coconut oil fed rats (Kd = 6.82 +/- 2.69). In cross-binding experiments performed with fish oil LDL and coconut oil liver plasma membranes or coconut oil LDL and fish oil liver plasma membranes, the LDL binding affinity was comparable and similar to that found in fish oil fed animals. No difference was found in the Bmax among all the groups of binding experiments. Our data seem to indicate that during fish oil diet the higher binding affinity of LDL to liver plasma membranes might be partly responsible of the hypocholesterolemic action of marine oil rich diet as compared to saturated diet. Furthermore, the modifications of binding affinity induced by changes of LDL and membrane source, suggest that lipoprotein and liver plasma membrane composition may be an important variable in binding studies.     

Effect of age on the modification of rat plasma lipids by fish and soybean oil diets

The effects of sardine and soybean oils on plasma lipids have been studied in young and aged rats. Plasma cholesterol and bile acids of aged rats fed on a sardine oil diet decreased to a greater degree than those of young rats. Cholesterol, bile acids and phospholipids of the soybean oil diet group decreased only in aged rats. Increases in plasma eicosapentaenoic (sardine) and linoleic (soybean) acid levels of aged rats were observed to be greater than those of young rats. These results indicate that the age enhances the effects of fish and soybean oils on plasma lipids by suppressing their characteristic fatty acid metabolism.     

Relative degradation of different arachidonoyl molecular species of choline glycerophospholipids in opsonized zymosan-stimulated rabbit alveolar macrophages

The relative degradation of arachidonoyl molecular species of glycerophospholipids prelabeled with [3H]20:4 caused by opsonized zymosan was studied in rabbit alveolar macrophages using a recently developed high-performance liquid chromatographic method. The opsonized zymosan caused the release of [3H]20:4 only from choline glycerophospholipids, no significant changes being observed in the radioactivities of other glycerophospholipids and triacylglycerol. Choline glycerophospholipids were resolved into seven arachidonoyl molecular species, which differed as to the alkyl ether or acyl residue bound at the 1-position, by high-performance liquid chromatography. Arachidonate was predominantly located in the alkyl type having 16:0 at the 1-position which comprised more than half of the total arachidonoyl molecular species of choline glycerophospholipids. The radioactivities of all arachidonoyl molecular species of choline glycerophospholipids, except for the 18:2-20:4 and 18:1-20:4 species of diacylglycerophosphocholine, decreased to 80-85% of the control values as a result of the challenge with opsonized zymosan for 1 h. However, 50% of the released 20:4 came from the 16:0-20:4 species of alkylacylglycerophospholipids, which were the most predominant species of choline glycerophospholipids. The present results indicate that the 16:0-20:4 species of alkylacylglycerophosphocholine is a significant source of arachidonate and 1-O-alkyl-2-lysoglycerophosphocholine, the precursor of the platelet-activating factor, relative to other arachidonoyl species in activated alveolar macrophages.     

Metabolism of unique diarachidonoyl and linoleoylarachidonoyl species of ethanolamine and choline phosphoglycerides in rat testes

Selected molecular species of rat testicular 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines were quantitated as their diradylglycerobenzoate derivatives, using a recently developed high-performance liquid chromatographic method. Increased amounts of docosapentaenoic acid were found in glycerophospholipids containing ether moieties compared with the diacyl phospholipids (e.g., docosapentaenoate-containing species comprised more than 80% of the alkylacyl subclass of the ethanolamine phosholipids as opposed to 29.3% of the diacyl subclass). Within 2 h after intratesticular injections of [5,6,8,9,11,12,14,15-3H]arachidonic acid, the 20:4-20:4 and 18:2-20:4 molecular species of the diacyl subclass of both the choline and ethanolamine glycerophosphatides had the highest specific radioactivities. These unique molecular species (20:4-20:4 and 18:2-20:4) also exhibited the largest percentage decrease in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonic acid, which indicates these two species possess a high metabolic turnover. Two of the arachidonate-containing molecular species (18:1-20:4 and 18:0-20:4) in the ethanolamine plasmalogens showed only a small decrease in specific radioactivity, whereas a third species (16:0-20:4) actually had a 44% increase in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonate. These data indicate that the 20:4-20:4, 18:2-20:4 and 18:1-20:4 species of phosphatidylcholine and/or phosphatidylethanolamine are most rapidly labeled after administration of [3H]arachidonic acid and that they appear to serve as the source of the [3H]arachidonate that is ultimately transferred to ethanolamine plasmalogens.     

Distribution of newly formed palmitate and stearate among molecular species of choline and ethanolamine phosphatides.

The choline and ethanolamine phosphatides derived from isolated rat livers during perfusion with 75 percent deuterated water (Kuksis, A., Myher, J.J., Marai L., Yeung, S.K.F., Steiman, I. & Mookerjea, S. (1975) Can. J. Biochem. 53, 509-518) were resolved into molecular species by argentation thin-layer chromatography. The time course of percentage replacement of the newly synthesized fatty acids in each molecular species was determined by gas chromatography with mass spectrometry. The results confirmed the earlier postulated differential utilization of palmitic and stearic acids in glycerolipid biosynthesis as well as supported the hypothesis of a precursor-product relationship between the oligoenoic and tetranoic species of both phosphatides. Calculations of half-lives gave values of 14-19 h for palmitoyl oligoenes, 40-50 h for palmitoyl tetraenes, and 22-28 h for palmitoyl hexaenes of both choline and ethanolamine phosphatides. The corresponding stearoyl species had half-lives which ranged from 89 to 200 h. Evidence was obtained for a metabolic heterogeneity among subsets of molecular species recognized on the basis of combinations of new and old glycerol and fatty acids in the same glycerolipid molecule.     

Fatty acid composition of choline and ethanolamine glycerophospholipid subclasses in heart tissue of mammals and migratory and demersal fish

The distribution and fatty acid composition of cardiac choline and ethanolamine glycerophospholipids in both migratory and demersal fish and bovine and pig were determined. Phospholipid contents (mg/g heart) were 4.7-9.4 in demersal fish, 14.0-16.5 in migratory fish, and 16.8-20.6 in mammals. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the major components in the phospholipid fraction. Diacyl forms represented 50.2-88.1% of PC in all animals, while plasmalogens comprised 47.0% in bovine, 8.2% in pig and 6.2-7.2% in four species of fish. In PE, plasmalogens varied from 45.0% in bovine and 57.9% in pig to 26.1-29.7% in fish. This glycerophospholipid subclass was identified as containing higher proportions of polyunsaturated fatty acids (PUFAs; 20:4, 20:5, and 22:6) than found in alkylacyl- and diacyl-glycerophospholipids. Qualitative and quantitative differences were found in PE-plasmalogen between land mammals and fish, especially with regard to n-3 fatty acid composition, but no significant difference was noted between migratory and demersal fish.     

Effect of salt stress on the metabolism of ethanolamine and choline in leaves of the betaine-producing mangrove species Avicennia marina

Glycinebetaine synthesis from [methyl-14C]choline and [1,2-14C]ethanolamine in leaf disks of Avicennia marina, was increased by salt stress (250 and 500 mM NaCl). After 18 h incubation with [methyl-14C]choline, phosphocholine and CO(2) were found to be heavily labelled. Phosphocholine contained 39% of the total radioactivity taken up by non-salinised (control) leaf disks and 15% of the total for salinised leaf disks stressed with 500 mM NaCl. Eighteen and 49% of the radioactivity absorbed by control and salinised disks, respectively, were released as CO(2). Metabolic studies of [1,2-14C]ethanolamine revealed that the radioactivity taken up by the leaf disks was recovered as the following compounds after 18 h: phosphorylated compounds (mainly phosphoethanolamine, phosphodimethylethanolamine and phosphocholine) (40-50%); choline (1-2%); glycinebetaine (3-5%); lipids (20-28%); CO(2) (6-10%). Unlike glycinebetaine, incorporation into phosphorylated compounds and lipids were reduced by salt stress. Incorporation of [methyl-14C]S-adenosyl-L-methionine (SAM) into choline, phosphocholine and glycinebetaine in leaf disks was stimulated by salt stress. In vitro activities of adenosine kinase and adenosine nucleosidase, which are implicated in stimulating the SAM regeneration cycle, increased after the leaf disks were incubated with 250 and 500 mM NaCl for 18 h. Changes in metabolism involving choline and glycinebetaine due to salt stress are discussed.     

Effect of dietary supplementation with a fish oil concentrate on the alkenylacyl class of ethanolamine phospholipid in human platelets

It has been demonstrated that the alkenylacyl class of ethanolamine phospholipid (PE) represents one of the major forms of eicosapentaenoic acid (EPA)-containing phospholipid in the circulating platelets isolated from human subjects consuming a fish oil concentrate. Since the alkenylacyl PE from human platelets is enriched in the eicosanoid precursor arachidonic acid (AA) and the n-6 polyunsaturate adrenic acid (AdA), it was of interest to study changes in alkenylacyl PE fatty acid composition upon fish oil supplementation. Healthy volunteers were given 20 capsules of MaxEPA daily (3.6 g of EPA plus 2.4 g of docosahexaenoic acid, DHA) for 6 weeks followed by a 6-week recovery period. Washed platelet suspensions were prepared and the fatty acid compositions of the phospholipid components were evaluated by thin-layer and gas-liquid chromatography at weeks 0, 3, 6, 9, and 12. Fatty acid composition changes were more pronounced in the alkenylacyl PE than in other platelet phospholipids as a result of fish oil consumption. The alkenylacyl PE exhibited a greater drop (by 20.3 mol%, i.e., from 72.0 to 51.7 mol%) in AA than diacyl PE (by 1.6 mol%) or total (predominantly diacyl) choline phospholipids (PC) (by 4.5 mol%). In alkenylacyl PE, the predominant reservoir of AdA in human platelet phospholipid, a dramatic reduction in the level of AdA also resulted with MaxEPA supplementation (from 7.9 to 3.1 mol%); diacyl PE and total PC decreased by 0.6 and 0.3 mol%, respectively. With respect to the n-3 fatty acids, EPA rose by 12.5 mol% in alkenylacyl PE, compared to only 3.8 and 2.5 mol% in diacyl PE and total PC, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.     

Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney.

The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline-ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.     

Purification and characterization of choline/ethanolamine kinase from rat liver

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.     

Effect of fish oil diet on immune response and proteinuria in mice

In this study, we examined the immune response and proteinuria caused by dietary polyunsaturated fatty acids in normal NZW/N and autoimmune NZB/NZW mice. Mice were maintained more than one year on five dietary groups: normal (5% corn oil), calorie-restricted, high fat (20% corn oil), high fat (20% fish oil), and Purina laboratory rodent chow. Normal mice fed with the fish oil diet had a more reduced anti-sheep red blood cells (SRBC) plaque-forming cell (PFC) response and less interleukin-2 (IL-2) enhancement of PFC than did the group with the restricted diet and the young control group. The corn oil (5 and 20%) diet animals also showed reduced PFC response and IL-2 utilization. NZB/NZW mice fed with the fish oil diet showed similar reduced PFC response but had a significantly lower response to IL-2 than did those on the corn oil diets and the restricted diet. The IL-2 production by macrophages from NZW/N mice was reduced in both the fish oil and corn oil diet groups. However, mice fed with the fish oil diet had less proteinuria and good survival rates, similar to the group with the restricted diet. These results suggest that the beneficial effect of the fish oil diet in these animals may be attributed in part to the immunosuppression mechanism.     

Effect of the removal of cleaner fish on the abundance and species composition of reef fish

The ecological significance of cleaner fish on coral reefs was investigated. I removed all cleaner fish, Labroides dimidiatus, from eight small reefs, measured the subsequent effect on the abundance and species composition of all reef fish after 3 and 6 months, and compared it with eight control reefs with cleaner fish. The removal of cleaner fish had no detectable effect on the total abundance of fish on reefs and the total number of fish species at both times. Multivariate analysis by non-metric multidimensional scaling and ANOSIM pairwise tests based on 191 fish species revealed no effect of cleaners on the community structure of fish. Similar results were obtained using principal components analysis on subsets of the data using the 33 most common fish species and the 15 most abundant species (≥5 individuals per reef ) with both log10 (x + 1) transformed data and with fish numbers standardized for abundance. This study demonstrates that the removal of cleaner fish for 6 months did not result in fish suffering increased mortality nor in fish leaving reefs to seek cleaning elsewhere. Received: 28 October 1996 / Accepted: 7 February 1997