To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A 2 (PLA 2 ), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca 2؉ -independent group IVC PLA 2 isozyme (cPLA 2 ␥), a paralogue of cytosolic PLA 2 ␣ (cPLA 2 ␣), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA 2 ␥ increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H 2 O 2 and other hydroperoxides induced arachidonic acid release in cPLA 2 ␥-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA 2 ␥ is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.

Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, MspI and HpaII, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with MspI and HpaII. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C(5m)CGG sites. Further analyses revealed an additional methylated site of NotI. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.

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We evaluated the pharmacological profiles of FMS586 [3-(5,6,7,8-tetrahydro-9-isopropyl-carbazol-3-yl)-1-methyl-1-(2-pyridin-4-yl-ethyl)-urea hydrochloride], a novel tetrahydrocarbazole derivative as a neuropeptide Y (NPY) Y5 receptor antagonist. This compound showed a highly selective in vitro affinity for Y5 (IC 50 ϭ 4.3 Ϯ 0.4 nM) relative to other NPY receptor subtypes like Y1 or Y2. Its binding to Y5 was found to be fully antagonistic from cyclic AMP accumulation assays in human embryonic kidney 293 cells. Pharmacokinetic analysis revealed sufficient oral availability and brain permeability of this compound accompanied with clear dose relation. We attempted to assess the selectivity of FMS586 and, thereby, to infer the physiological role of Y5 in the following feeding experiments in normal rats. An intracerebroventricular injection of NPY and Y5-selective agonist peptide induced acute and robust feeding responses in satiated rats, and prior administration of FMS586 at the doses from 25 to 100 mg/kg clearly inhibited these responses by approximately 55 and 90%, respectively. This compound also showed dose-dependent but transient suppression in natural feeding models of both overnight fasting-induced hyperphagia and spontaneous daily intake. FMS586 did not modulate food intake induced by the topical injection of norepinephrine, galanin, or ␥-aminobutyric acid receptor agonist muscimol to the paraventricular nucleus. In addition, we confirmed the Y5-specific activity profile of FMS586 by immunohistochemical analysis. Taken together, we propose not only that our compound potentially expresses specific blockade of central Y5 signals but also that Y5 receptor would certainly contribute to physiological regulation of food intake in normal rats, as suggested from its origin.

We have established a series of monoclonal antibodies that bind to phosphatidylserine (PS). One mAb, PS4A7, showed a strict specificity for PS and distinguished the stereospecific configuration of its serine moiety. We determined the amino acid sequences of the heavy and light chain variable regions of PS4A7, and examined the reactivity of the synthetic peptides corresponding to the complementarity determining region (CDR) of the mAb with phospholipids. We found that a 12-amino acid synthetic peptide corresponding to the third CDR of the heavy chain (amino acid residues 93-102, referred to as CDR3-H) bound specifically to PS. Although the affinity of the peptide to PS was markedly lower, the peptide was shown to bind to 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS), but not to 1,2-diacyl-sn-glycero-3-phospho-D-serine, showing a similar specificity to that of PS4A7. The specific binding of the CDR3-H peptide to PS was confirmed by ELISA and TLC-immunostaining assay. The interaction between the CDR3-H peptide and water-soluble PS-derivatives was investigated by inhibition of the ELISA. PS effectively inhibited the binding and phosphoserine showed a weak but significant inhibition, but no appreciable inhibition was observed with serine. These observations suggest that the CDR3-H peptide plays a major role in the interaction of PS4A7 with the phosphoserine residue of the PS molecule.

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We analyzed the inheritance of DNA methylation in the first filial generation(F1) hybrid of Oryza sativa L. ("Nipponbare"x"Kasalath") by restriction landmark genome scanning (RLGS). Most parental RLGS spots were found in the F1, but eight spots (4%) showed abnormal inheritance: seven of the eight spots were missing in the F1, and one was newly detected in the F1. Here we show demethylation at restriction enzyme sites in the F1. We also found a candidate site of stable heterozygous methylation in the genome. These results show the applicability of the RLGS method for analysis of the inheritance and alteration of methylation in F1 hybrid plants.