Reiko Nakayama

LPT1 encodes a membrane-bound O-acyltransferase involved in the acylation of lysophospholipids in the yeast Saccharomyces cerevisiae.

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156–22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Δlpt1 mutant strain. Although the Δlpt1 mutant strain did not show other detectable defects, the Δlpt1 Δslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

Effect of 17β-estradiol on PAF and prostaglandin levels in oophorectomized rat uterus

The effects of 17 beta-estradiol on the levels of platelet-activating factor (PAF) and prostaglandins and their precursor phospholipid in the uterus of oophorectomized rats were studied. Oophorectomy results in the decrease in the uterine PAF level to one-third of that in natural estrus. This level was recovered by subcutaneous administration of 17 beta-estradiol. The level of uterine phospholipids, which are rich in arachidonic acid, was significantly decreased by estradiol treatment. More arachidonate-PC was depleted than arachidonate-PE. The molecular structure was confirmed by gas chromatography-mass spectrometry. The amount of PGF2 alpha in the oophorectomized uterine tissue was 10-times that of PAF, but like the latter, increased 3-4 times on estradiol treatment. The chemical structures of PAF and PGF2 alpha formed on estradiol treatment were confirmed by mass spectrometry. The present data strongly suggest a correlation between the formations of PAF and PGF2 alpha, and indicate that estradiol may regulate the physiological formations of PAF and PGs in non-pregnant rat uterus.

1-0-Hexadec-1′-enyl-2-acetyl-sn-glycero-3-phosphocholine and its biological activity

1-0-Alk-1′-enyl analog of platelet-activating factor (PAF, 1-0-alkyl-2-acetyl- sn -glycero-3-phosphocholine, alkylacetyl-GPC) was prepared semi-synthetically from choline plasmalogens of beef heart muscle. The main compound was identified mass spectrometrically as 1-hexadec-1′-enyl-2-acetyl- sn -glycero-3-phosphocholine (16:0 alk-1′-enylacetyl-GPC, 16:0 vinyl form of PAF) and its platelet aggregation activity was about one-fifth of that of the corresponding 16:0 alkylacetyl-GPC. The irreversible platelet aggregation activity induced by 5×10−10 M 16:0 alk-1′-enylacetyl-GPC was completely inhibited by 5×10−7 M CV-3988 and 1×10−7 M L-652,731, specific PAF antagonists, and more than 99% of the activity was also lost by acid treatment. The hydrogenated product, alkylacetyl analog, showed quite same activity as that of authentic 16:0 alkylacetyl-GPC. The platelets desensitized with 16:0 alkylacetyl-GPC and with 16:0 alk-1′-enylacetyl-GPC were not aggregated with 5×10−10 M 16:0 alk-1′-enylacetyl-GPC, suggesting that alk-1′-enylacetyl-GPC occupied the same receptor site of alkylacetyl-GPC.

Evidence for production of platelet-activating factor by yeast Saccharomyces cerevisiae cells

Various yeast strains were screened for platelet-activating factor (PAF) production. High PAF production was found mainly in the strains of Saccharomyces genus. Yeast PAF showed a typical platelet aggregation pattern, which was inhibited by specific PAF antagonists, such as CV-3988, CV-6209 and L-652731. The main molecular species of yeast PAF were identified as 1-palmitoleoyl-, -palmitoyl-, -oleoyl- and -stearoyl-2-acetyl-sn-glycero-3-phosphocholines (16:1, 16:0, 18:1 and 18:0 acylPAFs) and 1-hexadecenyl- and hexadecyl-2-acetyl-sn-glycero-3-phosphocholines (16:1 and 16:0 PAFs), by mass spectrometry. PAF formation in yeast cells increased at the middle stationary phase of growth.

Occurrence of Anserine as an Antioxidative Dipeptide in a Red Alga, Porphyra yezoensis.

To examine the antioxidative compounds of nonprotein amino acids in a red alga, Porphyra yezoensis, an ethanol extract of the cultured thalli was fractionated with Dowex columns. The basic fraction V showed strong antioxidative capacities with ferric thiocyanate and TBARS measurements. In this basic fraction, histidine, 3-methylhistidine, carnosine, and anserine were detected beside ornithine, lysine, and arginine by amino acid analysis. The occurrence of carnosine and anserine suggests that the histidine-related compounds also contribute to the antioxidative reactions in P. yezoensis.

Generation of acetyl glyceryl ether phosphorylcholine from the rat skin and muscle tissues stimulated by moxibustion

Platelet-activating factor was obtained from the rat skin and muscle tissues which were stimulated by moxibustion. It showed a typical aggregation pattern on interaction with washed rabbit platelets but when it was treated with phospholipases A2 and C, and CV 3988 the aggregation activity was lost. Platelet-activating factor was hydrolysed with phospholipase C and the resulting lipid product was converted to the tert-butyldimethylsilyl derivative. After purification by thin layer chromatography, the ether type of derivative was analysed by a selected ion monitoring technique of gas chromatography-mass spectrometry. 1-0-Hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine only was identified.

Physiological Action of PAF in Yeast Saccharomyces Cerevisiae

In mammalian cells, PAF (platelet-activating factor) is involved in allergy and inflammation1–3). It is now apparent that PAF also plays a role in a number of normal physiological processes, including reproduction and the lowering of blood pressure4,5). We have been reported PAF is generated not only by inflammatory cells but also by many normal tissues, such as those of the uterus, heart and stomach6).