Wayne H. Anderson

Distribution of arachidonic and eicosapentaenoic acids in the lipids of mosses.

Lipid classes from four species of mosses, Mnium cuspidatum, and Mnium medium from Minnesota, and Hylocomium splendens and Pleurozium schreberi from Alaska, were analyzed. The total lipids of all species contained 30-40% arachidonic and eicosapentaenoic acids. However, the lipids from the Alaskan mosses contained about 75% neutral lipids (triacylglycerols, steryl esters and wax esters) whereas the lipids of the other species contained only 20% or less of these neutral lipids. Consistently, monogalactosyldiacylglycerols and phosphatidylethanol-amines were enriched in arachidonic acid and the galactolipids in eicosapentaenoic acid. The distribution of these acids in the phospholipids shows some preference for position 2. Together, the highly unsaturated C20 acids represented 80% of acyl groups in steryl esters. In triacylglycerols they were at average levels, while they were much less in sulfolipids and phosphatidylglycerols. Wax esters contained very little of the highly unsaturated acids but appreciable amounts of phytol and phytenic acid were found as wax constituents.

Synthesis and analysis of phytyl and phytenoyl wax esters

An efficient procedure for preparing phytenic acid methyl ester, free of isomers, from phytol is reported. Phytyl phytenate and other isoprenoid wax esters were synthesized. Gas liquid chromatography of these wax esters and other compounds related to phytol and phytenic acid is described. The alkyl constituents of isoprenoid wax esters can be analyzed after alkaline methanolysis and the acyl constituents after acidic methanolysis. The applicability of these methods to natural mixtures was demonstrated with wax esters from mosses which contained both types of isoprenoids and with wax esters from healthy and frost damaged grass which contained phytol, but not phytenic acid.

ATP-dependent Choline Phosphate-induced Mitogenesis in Fibroblasts Involves Activation of pp70 S6 Kinase and Phosphatidylinositol 3′-Kinase through an Extracellular Site SYNERGISTIC MITOGENIC EFFECTS OF CHOLINE PHOSPHATE AND SPHINGOSINE 1-PHOSPHATE

In serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0.5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0.1-1 mM) both in the absence and presence of insulin; maximal potentiating effects required 50-100 μM ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5′-O-(3-thiotriphosphate), adenosine 5′-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10-100 μM) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3′-kinase (PI 3′-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3′-kinase or treatments with the PI 3′-kinase inhibitor wortmannin only partially (∼40-50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3′-kinase.

9,12,15-octadecatrien-6-ynoic acid, new acetylenic acid from mosses

Lipids of the moss,Ceratodon purpureus, yield up to 25% of an acetylenic acid which was identified as allcis-9,12,15-octadecatrien-6-ynoic acid. The methyl ester of this acid was isolated in 95% purity by gas liquid chromatography. Mass spectroscopy provided the mol wt and confirmed methyl stearate as product of hydrogenation. Ozonization indicated a triple bond in position 6 and a double bond in position 15. UV and IR spectra showedcis-double bonds, no conjugation, and notrans-double bonds. The Raman spectrum provided direct evidence for the triple bond and confirmed the presence of double bonds and the absence of conjugation. The ratio of intensities indicated 1 triple bond/3 double bonds. 9,12,15-Octadecatrien-6-ynoic acid has not previously been isolated from biological materials. It was found only in the triglycerides ofCeratodon purpureus and several other mosses. In contrast to arachidonic and eicosapentaenoic acids, the acetylenic acid seems to be restricted to few moss genera.

The bisindolylnialeimide GF 109203X, a selective inhibitor of protein kinase C, does not inhibit the potentiating effect of phorbol ester on ethanol-induced phospholipase C-mediated hydrolysis of phosphatidylethanolamine

In fibroblasts, the protein kinase C (PKC) activator phorbol 12-myristate (PMA) either inhibits or stimulates phospholipase C-mediated hydrolysis of phosphatidylethanolamine in the absence or presence of ethanol, respectively. Here, we demonstrate that the specific PKC inhibitor bisindolylmaleimide GF 109203X prevents only the inhibitory, but not the stimulatory, PMA effect.

Acetylenic acids from mosses

Two new acetylenic fatty acids, 9, 12-octadecadien-6-ynoic and 11,14-eicosadien-8-ynoic, were identified from lipids of the moss,Fontinalis antipyretica. They resemble the previously identified 9,12,15-octadecatrien-6-ynoic acid by having a methylene interrupted unsaturated system. The C20 acetylenic acid shows that the capability of mosses to synthesize polyolefinic acids of this chain length applies, in certain species, also to olefinic-acetylenic acids.

Highly Unsaturated Lipids of Mnium, Polytrichum, Marchantia, and Matteuccia

Arachidonic and 5,8,11,14,17-eicosapentaenoic acids are highly unsaturated fatty acids which occur in lipids of mosses, liverworts, and ferns, but not in seed plants. The lipids of Mnium cuspidatum, M. medium, Polytrichum juniperinum, Marchantia polymorpha, and Matteuccia struthiopteris were studied and the content of the highly unsaturated acids determined in several tissues of these species. Arachidonic acid was found in all tissues, mostly at levels of 10-30% of total fatty acids. Except for Mnium medium and the Marchantia, the pentaenoic acid was at a considerably lower level than arachidonic acid and very little of it was found in nonphotosynthesizing tissue. Arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid, abbreviated 20:4w6) and similar highly unsaturated fatty acids occur in all higher animals, but they are not synthesized by them except when appropriate precursors are provided in the food (Burr & Burr, 1929; Holman, 1970). These acids are important as components in the lipids of cell membranes and as precursors of prostaglandins. They have not been found in seed plants (Hilditch & Williams, 1964) but are present at significant levels in mosses, ferns, and other lower plants (Gellerman & Schlenk, 1964; Schlenk & Gellerman, 1965; Nichols, 1965; Wagner & Friedrich, 1965, 1969; Wolf et al., 1966; Haigh et al., 1969; Karunen, 1971). The widespread occurrence of arachidonic and 5,8,11,14,17-eicosapentaenoic (20: 5o3) acids in these plants poses the question of their function in relation to physiological characteristics which these plants have and which set them in contrast to seed plants, where arachidonic and eicosapentaenoic acids do not occur. In the work reported here, the lipids and the fatty acid compositions of several tissues from a variety of lower plants have been investigated. MATERIALS AND METHODS Plants.-Mnium cuspidatum Hedw., M. medium B.S.G., Marchantia polymorpha L., and Matteuccia struthiopteris (L.) Todaro were collected at or near the Jay C. Hormel Nature Center, east of Austin, Minnesota. All samples were collected between 8:00 and 10:00 A.M. and the plant surfaces were free of water. 1 This investigation was supported in part by U. S. Public Health Service Grant AM 05165 from the National Institutes of Health; U. S. Public Health Service Research Grant HE 08214 from the Program Project Branch, Extramural Programs, National Heart Institute; and by The Hormel Foundation. We thank Dr. C. H. Wetmore, Dr. D. M. J. Mueller, and Mr. D. G. Richardson for identifications and helpful discussions. 2 The Hormel Institute, University of Minnesota, Austin, Minnesota 55912. This content downloaded from 157.55.39.17 on Wed, 31 Aug 2016 04:49:42 UTC All use subject to http://about.jstor.org/terms 1972] GELLERMAN ET AL.: LIPIDS OF BRYOPHYTES AND MATTEUCCIA 551 The habitat of M. cuspidatum was a grass area partially shaded by elm trees. Mats of the moss were taken with a 3-4 cm layer of soil and were promptly brought to the laboratory. The plants were removed from extraneous material and separated into tissues as specified in Table 1. The immature sporophytes were brownish green and, one week later, brownish red after natural dispersal of spores. They were picked by hand into vials for weighing and storage in solvent. Gametophores harvested in May were bright-green new growth. Sporophytes were removed from them before weighing and extraction. The mats of rhizoids, without gametophores and sporophytes, were rinsed with water and then hand picked to remove extraneous material. The rhizoids were blotted and air dried and then stored for extraction. Mnium medium was collected from a stand of oak and basswood trees. The sampling of gametophores and rhizoids was carried out as described above. Polytrichum juniperinum was collected from a pine forest in July. The gametophores were darker green and drier than those of Mnium collected one or two months earlier. Some of the capsules were open and already emptied, but spores were left in others and were shaken, by means of a laboratory vibrator, into a small funnel for collection and separate analysis. Green, fully expanded thalli of Marchantia were picked from a shady, wet stream bank facing north. Matteuccia struthiopteris was dug from a moist, very shady area. The fronds were about 80 cm long, bright-green with the lower portion somewhat brownish. The buds were very light green, tightly coiled and the fronds inside appeared somewhat deeper green. Rhizomes and roots were hand picked and cleaned as described earlier. Storage and Extraction.-Most materials were extracted within 2 days after harvest, but comparative analyses showed that the tissues can be stored at -170C in chloroform + methanol (2:1, v/v) for several months without marked change in lipid composition. The lipids were extracted in a Sorvall Omni-mixer for 2 min. by homogenizing with the above solvent mixture. After filtration under suction, the fiber was extracted at least once more. Complete extraction was checked by alkaline saponification (see below) of the residue and isolation of the fatty acids that may have been liberated. The lipids of gametophores, which had a low water content, were resistant to complete extraction by solvent, but recovery of all lipids was achieved by soaking the tissue, or the ground fiber from the first extraction, for 5 min. in water. Lipids in the moss spores were bound even more tightly and only 2/3 of the lipid was extractable by the solvent mixture. The amount of the non-extractable lipids was estimated from that of the fatty acids obtained by saponification. The composition of these acids was similar to that found in the extracted lipids. The usual precautions against autoxidation were taken in handling the unsaturated lipids. For example, water was freed of oxygen by boiling and cooling while bubbling a stream of N, through it, and vacuum systems were filled with N2 before opening them. Moisture, Lipid, and Chlorophyll Content.-Fresh tissues were immediately weighed in a tared screw-cap vial. The samples were then thoroughly homogenized with the extracting solvent in the Omni-mixer. The volume of the slurry was measured and an aliquot air-dried to constant weight. The total dry weight of the tissue was calculated from the aliquot and the moisture content was determined by difference from fresh weight. The aliquots exposed to oxidation by this procedure were discarded. The main portion of the slurry was treated further for complete extraction of the lipids. Filtrates were combined and shaken with 0.2 volumes of 0.9% NaC1 solution to remove methanol and non-lipid contaminants. The lipids were recovered from the chloroform phase by evaporation of the solvent on a Rotovac at 400C. Residual water was removed by addition of methanol and repeated evaporation. The lipids were transferred in chloroform to tared teflon-lined screw-cap tubes. The solvent was removed under a stream of N2 at 400C to determine the lipid yield. For storage, the lipids were dissolved in chloroform + methanol, 2:1, and kept under N2 in the screw-cap tubes at -170C. Under such conditions, they can be stored for 3-4 weeks without deterioration. Relative amounts of chlorophyll were determined spectrophotometrically according to Arnon (1949) as modified by Bruinsma (1961). An aliquot of the lipid solution, containing exactly 1 mg lipid, was transferred to a 10 ml volumetric flask and brought to dryness by a stream of N2. The lipids were dissolved in 8 ml acetone and then 2 ml water was added. This content downloaded from 157.55.39.17 on Wed, 31 Aug 2016 04:49:42 UTC All use subject to http://about.jstor.org/terms 552 THE BRYOLOGIST [Volume 75 In some cases not all lipids dissolved and filtration was required. The absorbance was read at 652 my (Bausch & Lomb Spectronic 20). Although this method is not quantitative it permits sufficiently reliable relative assays of chlorophyll. Lipid Classes.-Separations were carried out by thin-layer chromatography (TLC) (Mangold, 1969) using the solvents A-D (see below). Authentic substances were purchased for comparison of Rf values, of color with the staining reagents, and of intensity of stains to estimate their amounts. The following reference compounds were obtained: glycerides and other neutral lipids, monogalactosyl diglyceride, and phosphatidyl inositol (Lipids Preparation Laboratory of The Hormel Institute, Austin, Minn.); phosphatidic acid, phosphatidyl ethanolamine, and digalactosyl diglyceride (Applied Science Laboratories, State College, Penn.); phosphatidyl glycerol and phosphatidyl choline (Supelco, Inc., Bellefonte, Penn.); and phosphatidyl serine (Calbiochem, Los Angeles, Calif.).

Resolution of binding sites in β-Lactoglobulin

β-Lactoglobulin has been considered to have two or three strong binding sites for organic anions. By means of calorimetry with a very strongly binding anion, dodecyl-sulfate, enthalpic titration indicates two binding sites available on the acid side of the pH 7.5 conformation transition. There remain two sites available on the alkaline side of the same transition. One of these sites was resolved as a single, rather strongly binding site by means of spectrofluorimetry of a fluorescent organic anion at concentrations of the order of 10−5m. The second site is a considerably weaker anionbinding site, but was not resolved as such. Instead, dialysis equilibrium-binding experiments using the same anion, at concentrations of the order of 10−4m were performed. The data, used in Scatchards method of plotting, yield the average of the two binding sites at this higher organic anion concentration. The molar association constants (which may be corrected further for statistical and electrostatic factors) for β-lactoglobulin at pH 8 with 0.02 m Cl− and a N-methylanilinonaphthalene sulfonate anion, are K1 = 3.4 ± 0.5 × 105 for n1 = 0.95 ± 0.10 sites, fully resolved, and K2 = 2.0 ± 0.2 × 104 for n2 = 2.0 ±0.2 sites which include the first strong binding site and the second weaker site. The K2 cited refers to an average binding constant for both sites. Probably Cl− competition for the same anion-binding sites, at this pH, is minimal. By using fluorescent compounds and adequate control of excitation wavelength, it is probable that binding constants for individual strong binding sites can be worked out for fluorescent compound binding to a variety of other proteins.

Arachidonic and eicosapentaenoic acids in developing gametophores and sporophytes of the moss,Mnium cuspidatum

The fatty acid composition of different parts of the moss,Mnium cuspidatum, which contains up to 35% arachidonic acid in its lipids, was studied through the annual cycle and especially during the period of rapid development of the reproductive parts. The content of 20∶4ω6 was highest in summer and lowest in winter; but for 20∶5ω3, the reverse was found. Levels of the acids, 20∶5ω3, 18∶3ω3 and 16∶3ω3 showed parallel fluctuations through the seasons of the year, and functionally they may substitute for each other. In contrast, 20∶5ω6 is at a high level when 18∶2ω6 is low. The latter acid accumulates in storage or dormant tissue and may be a reserve to form arachidonic acid for specific requirements in cell membranes when rapid growth resumes.

Carcinogens stimulate phosphorylation of ethanolamine derived from increased hydrolysis of phosphatidylethanolamine in, C3H/101/2 fibroblasts

Many human tumors contain high concentrations of ethanolamine phosphate (EtnP). An important question is whether increased formation of EtnP is merely the consequence of cell transformation, or is it associated with the process of carcinogenesis. Here we show that in C3H/10Tl/2 embryonic fibroblasts, an established cellular model for the study of carcinogenesis, the environmental carcinogens, 7,12‐dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) (0.1–1 concentration; 24 h treatment), stimulate phosphorylation of ethanolamine derived from increased hydrolysis of phosphatidylethanolamine. The results suggest that increased formation of EtnP is associated with the early stages of carcinogenesis. This observation may have prognostic value.