1. The effect of docosahexaenoic acid (DHA) on N-methyl-D-aspartic acid (NMDA) responses in the presence of glycine was investigated in pyramidal neurones acutely dissociated from rat cerebral cortex in whole-cell and single channel configurations. 2. DHA potentiated the NMDA-induced response but reduced the non-NMDA (kainateinduced) response in a concentration-dependent manner at a holding potential of -60 mV under voltage-clamp conditions. 3. Arachidonic acid (AA) also potentiated the NMDA-induced response in a manner similar to DHA. Oleic acid caused a slight potentiation. However, other polyunsaturated and saturated fatty acids had no such effects. 4. The facilitatory action of DHA on the NMDA-induced response was not affected by adding inhibitors of cyclo-oxygenase, lipoxygenase or phospholipase A2, suggesting that DHA may exert its facilitatory effect directly on the NMDA receptor. 5. The facilitatory action of DHA was observed in the presence of a saturating dose of NMDA. Moreover, a detailed analysis of the NMDA receptor-operated single channel currents revealed that, in the presence of DHA, the open probability of the channel increased without changing the conductance, indicating that DHA may act by binding directly to a novel site on the NMDA receptor or by altering the lipid environment of the NMDA receptor and thereby potentiating the response to NMDA. 6. The results are discussed in terms of the possibility that DHA may play an important role in the genesis of long-term potentiation, at least that involving the activation of NMDA receptors.

Lysophosphatidylinositol (LPI) is an endogenous ligand for GPR55, a putative novel type of cannabinoid receptor. In this study, we first examined the effects of LPI on p38 mitogen-activated protein kinase in HEK293 cells expressing GPR55. LPI induced the rapid phosphorylation of p38 mitogen-activated protein kinase in GPR55-expressing cells. No apparent effect was observed in the vector-transfected cells. The exposure of GPR55-expressing cells to LPI also triggered the phosphorylation of activating transcription factor 2 downstream of the p38 mitogen-activated protein kinase. Treatment of the cells with Y-27632 [a Rho-associated kinase (ROCK) inhibitor] blocked the LPI-induced phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor 2, suggesting that the Rho-ROCK pathway is involved in these cellular responses. Notably, GPR55 was found to be abundantly expressed in lymphoid organs such as the spleen and thymus. We obtained evidence that rapid phosphorylation of p38 mitogen-activated protein kinase and activating transcription factor 2 also takes place in IM-9 lymphoblastoid cells, which naturally express GPR55, after stimulation with LPI. These results suggest that GPR55 and its endogenous ligand LPI play essential roles in the homoeostatic responses to stress signals in several mammalian tissues and cells including certain types of immune cells.

We previously reported that G␤␥ signaling regulates cell spreading or cell shape change through activation of a Rho family small GTPase, suggesting the existence of a G␤␥-regulated Rho guanine-nucleotide exchange factor (RhoGEF). In this study we examined various RhoGEF clones, found FLJ00018 to be a G␤␥-activated RhoGEF, and investigated the molecular mechanism of G␤␥-induced activation of Rho family GTPases. Co-expression of the genes for FLJ00018 and G␤␥ enhanced serum response element-mediated gene transcription in HEK-293 cells. Combined expression of G␤␥ and FLJ00018 significantly induced activation of Rac and Cdc42 but not RhoA. FLJ00018 also enhanced gene transcription induced by carbachol-stimulated m2 muscarinic acetylcholine receptor, and this enhancement was blocked by pertussis toxin. Furthermore, we demonstrated G␤␥ to interact directly with the N-terminal region of FLJ00018 and the N-terminal fragment of this molecule to inhibit serum response element-dependent transcription induced by G␤␥/FLJ00018 and carbachol. In NIH3T3 cells, FLJ00018 enhanced lysophosphatidic acid-induced cell spreading, which was also blocked by the N-terminal fragment of FLJ00018. These results provide evidence for a signaling pathway by which G i -coupled receptor specifically induces Rac and Cdc42 activation through direct interaction of G␤␥ with FLJ00018.Rho family small GTPases belong to the Ras superfamily, comprise more than 20 distinct proteins, and control a wide variety of cellular processes. First identified as regulators of the actin cytoskeleton rearrangements, RhoA, Rac1, and Cdc42 induce stress fibers, lamellipodia, and filopodia formation, respectively, and it is now clear that Rho family proteins play pivotal roles in cell migration, outgrowth, extension and pathfinding of neuritis, and gene transcription. Like other small GTPases, Rho GTPases cycle between an inactive GDP-bound state and an active GTP-bound state (1, 2). This cycling of Rho GTPases is controlled by three distinct classes of regulatory proteins, namely (i) guanine-nucleotide dissociation inhibitors, which stabilize the inactive form (3), (ii) guanine-nucleotide exchange factors (GEFs), 2 which catalyze GDP/GTP exchange (4, 5), and (iii) GTPases-activating proteins, which stimulate low, intrinsic GTPase activity of Rho GTPases (6). In particular, 60 different GEFs for Rho family members (RhoGEFs) have been described so far (4). A common feature of RhoGEFs is the Dbl homology (DH) domain responsible for exchange activity followed by a pleckstrin homology (PH) domain considered to be involved in subcellular localization. Besides this tandem motif, RhoGEFs often contain one or more additional signal transduction domains, such as PDZ, Src homology (SH) 2, SH3, and RGS (regulator of G protein signaling), which can function as molecular bridges between different signal transduction pathways.It is well established that a large variety of G protein-coupled receptors (GPCRs), particularly those coupling to the G 12/13 type of heterotrimeric G proteins...

Responses of insulin-like growth factor (IGF)- 12University, 2-9-1 Sakura, Nanae, Kameda-gun, Hokkaido 041-1105, Japan. 32Fasting/re-feeding also affected their mRNA levels in the liver. These results suggest that 33 circulating IGF-I and IGFBP-1b could serve as positive and negative indices of growth, 34respectively, in masu salmon. Different sensitivities of IGBP-1a and IGFBP-1b may be useful to 35assess a broad range of catabolic conditions when they are combined.

Although tetraploidy is considered to be important and useful for the production of sterile triploids and fertile allotetraploids in aquaculture, in most cases the resultant embryos exhibit extremely low survival. In this study, we aimed to clarify the cause of the inviability of tetraploids in masu salmon. Firstly, we compared developmental rates of the first cell cycle among 9 single pairs produced by all possible matings between 3 females and 3 males. The results showed that the fertilized eggs developed almost synchronized among individuals from each pair but asynchronous among pairs and this asynchronous development was more apparent among pairs from different female than those from different male. Secondly, we induced both tetraploidy (4N) and gynogenetic diploidy (G2N) , 7 min duration) for the first cleavage inhibition using single-pair mating. This experiment was designed to verify whether the cause of mortality of induced tetraploidy was the elevated ploidy itself. Eggs from one female were divided into 2 groups and fertilized with normal or UV-irradiated sperm from one male, respectively. Then each group of eggs was subdivided into 6 groups: one was an intact control and the other 5 groups were treated with PS at every 30 min from 5 to 7 hpf at 10 °C. As a result, the treated eggs of 4N and G2N showed the highest survival (90.8% and 60.6%, respectively) and embryogenesis rate (both 100% relative to surviving eggs) at 33 dpf, only when PS treatment was performed at late prometaphase (6 hpf and 6.5 hpf, respectively). The other PS groups showed extremely low survival (1.5-52.8% and 3.1-14.8%), ceased morphogenetic development and developed into only undifferentiated cell masses. This experiment was repeated 2 more times using other single-pair mating and the results showed the same tendency. The embryonic bodies were confirmed by flow cytometry to have approximately objective ploidy, tetraploid, hypo-or hyper-tetraploid in 4N and diploid or hypo diploid in G2N.However, all tetraploid embryos, even those with normal morphology, began to die 3 simultaneously around the hatching period (34 dpf), while G2N embryos showed normal morphology and survived beyond 50 dpf (55.6%). Most tetraploid embryos showed malformation and had very poor vascular systems even in normal-looking ones. These results suggest that the mortality of induced tetraploids depends not only on the side effects of the PS treatment but also strongly depends on the induced tetraploidy itself. 5