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Organic/inorganic nanohybrid materials composed of terthiophenes and imogolite were prepared by means of specific surface interaction between a phosphonic acid group in terthiophene derivatives and...

We have demonstrated the preparation of hybrid films of aluminosilicate nanofiber (imogolite) and water-soluble poly( p -phenylene) (WS-PPP), which has sulfonate groups. The imogolite/WS-PPP hybrid gel could be prepared by mixing a solution of these two materials and subsequent centrifugation. The aluminol (Al OH) groups on the surface of imogolite would be protonated under acidic conditions to afford Al OH 2 + groups that can interact with sulfonate groups (SO 3 − ) of WS-PPP. Based on this ionic interaction, a layer-by-layer (LBL) assembly was applied to fabricate the hybrid films of imogolite nanofibers and WS-PPP. The deposited amounts of WS-PPP and imogolite were measured by quartz crystal microbalance (QCM) measurements and scanning electron microscopy (SEM) observation. Atomic force microscopy (AFM) observation revealed that imogolite nanofibers were well networked in the LBL hybrid film.

A novel gene, dfrG , encoding a trimethoprim (TMP)-resistant dihydrofolate reductase (DHFR, designated S3DHFR) was cloned from a clinical isolate of methicillin-resistant Staphylococcus aureus. Escherichia coli expressing dfrG was highly resistant to TMP. Recombinant S3DHFR exhibited DHFR activity that was not inhibited by TMP.

Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7-HS interaction is required for BMP-7 signaling.

Altered peptide ligands (APL) induce T cell responses different from those induced by the original agonistic peptide. As shown for CD4(+) T cells, partial agonists induce partial T cell activation without proliferation because of lower affinities and higher off rates to TCR than those of agonists. To determine whether overexpression of partially agonistic TCR ligands on antigen-presenting cells provides high-avidity TCR ligands, we generated L cell transfectants expressing various numbers of HLA-DR4 covalently linked with APL derived from a streptococcal peptide and observed responses of the cognate T cells. Some overexpressed HLA-DR4/partially agonistic APL complexes induced T cell proliferation in a density-dependent manner. However, tyrosine phosphorylation of zeta-associated protein-70 (ZAP-70) and linker for activation of T cells and kinase activity of ZAP-70 were not detectable. T cell proliferation stimulated with L cell transfectants was sensitive to the PKC inhibitor Gö6976, but to a lesser extent to Gö6983, suggesting the involvement of mu isotype of PKC (PKCmu). In vitro kinase assays revealed that PKCmu activity was up-regulated only in T cells stimulated with L cell transfectants that induced T cell proliferation. Our data suggest the presence of a unique signaling pathway coupling TCR ligation with T cell proliferation associated with PKCmu activation and impaired ZAP-70 activation.

Heparan sulfate (HS), a structurally diverse molecule comprising distinct sequences of sulfated disaccharide units, is abundant in the developing brain and binds to axon guidance molecules. Addition of HS to the developing Xenopus optic pathway causes severe targeting errors yet it is not known how the structural diversity of this molecule relates to its role in axon guidance. We have used an in vivo brain assay to identify the structural characteristics of HS that induce aberrant axon targeting. Inhibiting sulfation of endogenous HS with chlorate causes axons to bypass their target, the tectum, and treatment with chemically modified heparins reveals that 2-O- and 6-O-sulfate groups have potent bypass-inducing activity. Experiments with purified heparin saccharides show that bypass-inducing activity correlates with distinct structures, particularly those containing a combination of 2-O- and 6-O-sulfate groups. Taken together the results indicate that specific sequences, rather than gross structural composition, are critical for activity. In situ hybridisation revealed that HS 6-O-sulfotransferase is regionally expressed along the border of the dorsal optic tract whereas 2-O-sulfotransferase is expressed broadly. Our results demonstrate that specific HS sequences are essential for regulating retinotectal axon targeting and suggest that regionalised biosynthesis of specific HS structures is important for guiding axons into the tectum.

In super-finishing by cubic boron nitride (CBN) stones, it is considered that chips are formed by crossing of grain loci. Thus, crossing angles of grain loci are paid attention to as an important parameter. In this paper, the new method for prediction of cutting performance is proposed using average crossing angle of grain loci. On the other hand, chip formation process is classified into two types. Therefore, all crossing angles are calculated after considering these types of crossing of grain loci. Average crossing angle is defined as the mean value of those. Moreover, the relation between these average crossing angle and cutting performance is investigated by various calculations and experiments. As results, average crossing angle is one of the most important parameters to estimate the cutting performance on super-finishing using CBN stones.

N-Glycolylneuraminic acid (NeuGc) is abundantly expressed in most mammals, but it is not detectable in humans. The expression of NeuGc is controlled by cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase activity. We previously cloned a cDNA for mouse CMP-NeuAc hydroxylase and found that the human genome contains a homologue. We report here the molecular basis for the absence of NeuGc in humans. We cloned a cDNA for human CMP-NeuAc hydroxylase from a HeLa cell cDNA library. The cDNA encodes a 486-amino acid protein, and its deduced amino acid sequence lacks a domain corresponding to the N-terminal 104 amino acids of the mouse CMP-NeuAc hydroxylase protein, although the human protein is highly identical (93%) to the rest of the mouse hydroxylase protein. The N-terminal truncation of the human hydroxylase is caused by deletion of a 92-base pair-long exon in human genomic DNA. The human hydroxylase expressed in COS-7 cells exhibited no enzymatic activity, and a mouse hydroxylase mutant, which lacks the N-terminal domain, was also inactive. A chimera composed of the human hydroxylase and the N-terminal domain of the mouse hydroxylase displayed the enzyme activity. These results indicate that the human homologue of CMP-NeuAc hydroxylase is inactive because it lacks an N-terminal domain that is essential for enzyme activity. The absence of NeuGc in human glycoconjugates is due to a partial deletion in the gene that encodes CMP-NeuAc hydroxylase.

Integrin alphaIIbbeta3 plays a critical role in platelet aggregation through its interaction with fibrinogen. Elucidation of the mechanisms of alphaIIbbeta3-fibrinogen interaction is critical to understanding hemostasis and thrombosis. Here we report that mutations of Gly-184, Tyr-189, Tyr-190, Phe-191, and Gly-193 within the predicted turn structure of the third amino-terminal repeat of alphaIIb significantly block binding of alphaIIbbeta3 to soluble fibrinogen. These mutations also block binding of alphaIIbbeta3 to ligand-mimetic monoclonal antibodies PAC-1, OP-G2, LJ-CP3, which have an RGD-related RYD sequence in their antigen-binding sites. These mutations do not significantly affect the expression of alphaIIbbeta3, in contrast to most of the natural alphaIIb mutations occurring in Glanzmann's thrombasthenic patients. The data suggest that these residues are critically involved in alphaIIbbeta3-ligand interactions.

Neutral glycosphingolipids were purified from non-cataractous lenses of Sprague-Dawley rats by a combination of solvent extraction, Folch's partition, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major GSLs from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis and glycosidase digestion. Structural relationships among the six neutral glycosphingolipids revealed metabolic pathways leading to the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide (IV3Gal alpha nLc4), instead of a Lewis(x) glycolipid (Gal beta 1- 4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide, III3FucnLc4), from neolactotetraosylceramide (nLc4), together with isoglobotriaosylceramide (iGb3). The alpha-galactosyl epitope, Gal alpha 1-3Gal-R, is evolutionarily conserved in many types of cells of non-primate mammals, prosimians and New World monkeys, but not in those of Old World monkeys or humans. This evolution-related difference in carbohydrate epitopes suggests different cell-to-cell attachments, which may be mediated through cell surface glycosphingolipids, between rat and human lenses.

Bone is a typical nanocomposite of inorganic and organic substances mainly composed of nanocrystals of hydroxyapatite (Ca10(PO4)6(OH)2, HAp) with nonstoichiometry 20-40 nm in length and type-I collagen molecules 300 nm in length. In microscopic observation of bone, collagen molecules are covered with HAp nanocrystals, and they form nanocomposite fibers in which HAp c-axes are approximately aligned along with collagen molecules (Bacon et al., 1979, Sasaki & Sudoh, 1997). Bone has two important roles in vertebrates; one is as a structural material to maintain vertebrates body structure and to guard important internal organs, such as brain, heart and lung, and another is as an organ to control calcium ion homeostasis by resorbing bone mineral according to deficiency of calcium ion. These two roles are kind of antinomical properties as industrial materials, i.e., TOUGH to endure to external forces for whole life and BREAKABLE to allow calcium release by bone cell functions on demand. However, change our viewpoint about toughness of material, solution of the antinomy is concluded to one property. That is, every materials fatigue by long time use; thus, periodic renewal of material is necessary to maintain enough toughness of bone for whole life. Thus, bone has to be renewed easily by cell functions. That is almost the same meaning as a breakable property of bone. Accordingly, requirement of bone can be translated as follows; bone has to be decomposed by cell functions but be stable without them. Fortunately, our ancestors needed to preserve calcium and phosphate ions in their body when they left from sea. Calcium and phosphate easily form insoluble compound in aqueous solution, brushite (CaHPO4•2H2O), octacalcium phoshate (Ca8H2(PO4)6•5H2O) and HAp, and all calcium phosphates are changed into HAp in aqueous or moistured condition, because it is most stable compound in these conditions. Further, HAp formed in regular

We recently identified four isoforms of bovine prostaglandin E receptor EP3 subtype, which are coupled to different signaling pathways; EP3A is coupled to inhibition of adenylate cyclase, while EP3B and EP3C are coupled to its stimulation and EP3D is coupled to phosphatidylinositol turnover, in addition to the adenylate cyclase system (Namba, T., Sugimoto, Y., Negishi, M., Irie, A., Ushikubi, F., Kakizuka, Ito, S., A., Ichikawa, A., and Narumiya, S. (1993) Nature 365, 166-170). We examined here the identity of coupled G proteins and their regulation by one of the isoforms, EP3C, in the membranes of EP3C cDNA-transfected Chinese hamster ovary cells. M&B 28767, an EP3 agonist, stimulated the GTPase activity in the pertussis toxin (PT)-treated cell membrane, but inhibited it in the cholera toxin (CT)-treated cell membrane, while the agonist neither stimulated nor inhibited it in the both PT- and CT-treated cell membrane. In the PT- and CT-treated cell membrane reconstituted with various G proteins, M&B 28767 inhibited the GTPase activity of G(o), but stimulated that of Gs. On the other hand, M&B 28767 did not affect the GTPase activity of Gi1, Gi2, or Gi3. M&B 28767 increased the apparent affinity of G(o) for GDP without any change in that for GTP, as assessed by displacement of [35S]GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to G(o). In contrast, M&B 28767 increased the apparent affinity of Gs for GTP but decreased that for GDP. These results demonstrated that the EP3 receptor isoform is coupled to two different G proteins, and oppositely regulates their activities, inhibition of G(o), and stimulation of Gs.

Severe acute respiratory syndrome (SARS) spread during the winter of 2003, and attempt have been made to develop vaccines against SARS corona virus (SARS-CoV). The present study provides a strategy to rapidly identify SARS-CoV-derived antigenic peptides recognized by HLA-A2-restricted cytotoxic T lymphocytes (CTLs). Forty-three candidate peptides having HLA-A2-binding motifs were selected in silico and HLA-A2/Db chimeric MHC class I-transgenic mice were immunized with these peptides and a new derivative of muramyl dipeptide that can induce upregulation of HLA-DR, CD80, CD86, and CD40 in human CD14+ antigen presenting cells, was administered as an adjuvant. Six HLA-A2-restricted mouse CTL epitopes were identified, including two new epitopes which have never been reported before. One of the novel peptides was naturally processed and successfully induced HLA-A2-restricted specific CTLs in both HLA transgenic mice and healthy donors. The method was useful, convenient and efficient for rapid identification of CTL epitopes derived from SARS-CoV proteins and will be possibly applicable for other pathogens to develop a peptide-based vaccine.

A novel fucose-containing neutral glycosphingolipid (GL-5) was purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. The chemical structure was determined to be Fuc alpha 1-3GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-4Glc beta 1-1Cer by methylation analysis, partial acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The unique characteristics of GL-5 are that: the reducing terminal disaccharide portion is not Gal beta 1-4Glc but GlcNAc beta 1-4Glc; it includes a GalNAc beta 1-4GlcNAc sequence and a Fuc-GalNAc linkage; the defucosylated core is a novel trisaccharide chain; and the sugar structure is one of the smallest ever characterized for a difucosylated glycolipid. The major fatty acids were 22:1 and 22h:1, and about 30% of the total acids was 2-hydroxylated. All the long-chain bases were phytosphingosines, of which about 90% was n-t18:0. The similarity of the ceramide moiety to that of glucosylceramide from the same eggs [Kubo, H. et al. (1992) J. Biochem. 111, 726-731] suggests a close biosynthetic relationship between GL-5 and the glucosylceramide.

Glucosylceramide (Glc beta 1-1Cer) and a novel ceramide trihexoside (Gal beta 1-6Gal beta 1-6Glc beta 1-1Cer) were purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, chromic acid oxidation, enzymatic hydrolysis, enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ceramide trihexoside has a novel carbohydrate structure, and its core structure, Gal beta 1-6Glc, is also novel. The ceramide moieties of these glycolipids are almost identical. Two fatty acids, 22:1 and 22h:1, constitute more than 80% of the total acids. Long-chain bases are all phytosphingosine, approximately 90% of which is n-t18:0. The finding of melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) from the eggs of another sea urchin species [Kubo, H. et al. (1988) J. Biochem. 104, 755-760] and the present finding of the novel ceramide trihexoside suggest that there are a variety of unique sugar structures in sea urchin glycosphingolipids.

Ceramide dihexoside was obtained from the spermatozoa of the starfish, Asterias amurensis. Gas-liquid chromatography of the methanolysate to determine the sugar composition of the lipid demonstrated an unequal ratio of glucose and galactose, implying that two or more glycolipids are present. They were separated by thin-layer chromatography on a borate-impregnated plate into two bands. From the results of methylation analysis and chromic acid oxidation, one was determined to be lactosylceramide, while the other was suggested to be a mixture of two diglucosylceramides: gentiobiosylceramide (Glc beta 1-6Glc beta 1-1Cer) and cellobiosylceramide (Glc beta 1-4Glc beta 1-1Cer). The molar ratio of gentiobiosyl-, cellobiosyl-, and lactosylceramide was estimated to be 0.7 : 0.3 : 1.0. Ceramide dihexosides obtained from another batch of the spermatozoa, collected at the same place in a different year, consisted almost exclusively of gentiobiosylceramide as confirmed by proton-nuclear magnetic resonance spectroscopy and fast-atom bombardment mass-spectrometry. The fatty acid compositions of these glycolipids were similar and the main acids were 14h:0, 15h:0, 16h:0, 18h:0, and 24h:1 (constituting more than 80% of the total acids). The long-chain base compositions were qualitatively similar and the major constituents were commonly d18:2, d18:3, d19:3, d22:2, and t22:1. Lactosylceramide was rich in t22:1, while diglucosylceramides were rich in d22:2.

NeuGc alpha 2-6Glc beta 1-1Cer (M5 ganglioside) and HSO3-8NeuGc alpha 2-6Glc beta 1-1Cer (T1 ganglioside) were purified by column chromatographies with DEAE-Sephadex A-25 and silicic acid from the eggs of the sea urchin, Anthocidaris crassispina. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Long-chain base compositions of both gangliosides were almost identical: all the long-chain bases were phytosphingosines, and C18-phytosphingosine accounted for more than 95% of them. Fatty acid compositions were also very similar: the main fatty acids were 22:1, 23:1, 24:1, and their 2-hydroxylated forms, and the 2-hydroxy fatty acids amounted to 65.3 and 74.3% of the fatty acids in M5 and T1 gangliosides, respectively. Proton nuclear magnetic resonance spectroscopic study revealed a downfield-shifted H8 proton signal of NeuGc residue in T1 ganglioside, in agreement with the presence of sulfate ester at the C8 position.

Glucosylceramide (Glc beta 1-1Cer) was isolated from the spermatozoa of the starfish, Asterias amurensis. The long-chain bases of the glycolipid consisted of dihydroxy (d18:2, d18:3, d19:3, and d22:2), and trihydroxy (t22:1) types. Long-chain aldehydes derived from them were analyzed mainly by proton nuclear-magnetic resonance to determine the detailed structures. Two of the tri-unsaturated bases were identified as (4E,8E,10E)-2-amino-4,8,10-octadecatriene-1,3-di ol (d18:3) and (4E,8E,10E)-2-amino-9-methyl-4,8,10-octadecatriene+ ++-1,3-diol (d19:3), which is a novel base. Both d22:2 and t22:1 had a cis double bond at the C9 or C13 position. All fatty acids were 2-hydroxylated (C14-C25): Most of them were saturated and unbranched. About 10% was mono-unsaturated and unbranched (C22-C25), while saturated but branched (iso- and anteiso-types) C15-C18 acids were found as minor components. The main fatty acids, which summed up to more than 93% of the fatty acids in the glucosylceramide, were n-14h:0, n-15h:0, n-16h:0, n-17h:0, n-18h:0, and n-24h:1.

Little is known about the cues that guide retinal axons across the diencephalon en route to their midbrain target, the optic tectum. Here we show that chondroitin sulfate proteoglycans are differentially expressed within the diencephalon at a time when retinal axons are growing within the optic tract. Using exposed brain preparations, we show that the addition of exogenous chondroitin sulfate results in retinal pathfinding errors. Retinal axons disperse widely from their normal trajectory within the optic tract and extend aberrantly into inappropriate regions of the forebrain. Time-lapse analysis of retinal growth cone dynamics in vivo shows that addition of exogenous chondroitin sulfate causes intermittent stalling and increases growth cone complexity. These results suggest that chondroitin sulfate may modulate the guidance of retinal axons as they grow through the diencephalon towards the optic tectum. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 330–342, 2002

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified three isoforms which are produced through alternative splicing. In Chinese hamster ovary cells expressing each EP3 isoform, PGE2 induced an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i) due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. This increase was abolished by prior treatment with pertussis toxin (PT). PGE2 also stimulated an accumulation of inositol trisphosphate (IP3) in a PT-sensitive manner. Both the PGE2-induced increase in [Ca2+]i and accumulation of IP3 were blocked by the phospholipase C inhibitor U-73122. Thus, EP3 is linked to phospholipase C activation via Gi, and this activation leads to Ca2+ mobilization from internal stores and influx from the extracellular medium.

A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost ⪢ carbacyclin ⪢ prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF2α did not inhibit it. Iloprost in a concentration-dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.

Preparation of novel organic/inorganic nanofiber hybrid composed of one-dimensional P3HT nanofiber and aluminosilicate imogolite was demonstrated. To achieve better compatibility with hydrophobic P3HT, Al-OH group on imogolite surface was modified with terthiophene derivative, 2-[5-hexyl-2,2':5',2-terthiophene]-5-ethyl)-phosphonic acid (H3TP). Dissolving the mixture of P3HT and surface modified imogolite in poor solvent of anisole at elevated temperature (> 70?C) followed by subsequent slow cooling (25?C/h) to room temperature induced the formation of P3HT crystallite that winds around the surface modified imogolite core. The thickness of the nanofiber hybrid estimated from DFM is larger than the parent P3HT nanofiber and imogolite. UV-vis spectroscopy spectra indicate that addition of surface modified imogolite can influence the molecular aggregation state of P3HT via van der Waals interaction. Through GIWAXD experiments, it was found that P3HT molecular chains are strongly oriented and lying on surface modified imogolite core.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

PEPTIDE hormones, neurotransmitters, and autacoids activate a family of seven-transmembrane-domain receptors1. Each of these receptors specifically couples to one of several G proteins, Gs, Gi, Go and Gp, to activate a specific second messenger system2. Cell surface receptors for prostanoids have been characterized pharmacologically3 and the complementary DNAs for thrombox-ane A2 receptor4,5 and the EP3 subtype of the prostaglandin (PG)E receptor6 reveal that they belong to the seven-transmembrane-domain receptor family. The EP3 receptor mediates the diverse physiological actions of PGE2 (ref. 3). Although most of them occur through coupling of the EP3 receptor to Gi and inhibition of adenylyl cyclase, the EP3-mediated contraction of uterine muscle can only occur by activation of another second messenger pathway7. In chromaffin cells, two different second messenger pathways are activated by PGE2 binding to an apparently single EP3 receptor class8. Here we show that at least four isoforms of the EP3 receptor, which differ only at their C-terminal tails and are produced by alternative splicing, couple to different G proteins to activate different second messenger systems.

Neutral glycosphingolipids were purified from human senile cataractous lenses by a combination of solvent extraction, Folch's partition, acetylation, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major glycosphingolipids (A-F) from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis, secondary ion-mass spectrometry, glycosidase digestion, and chromium trioxide oxidation. Their structures suggested that they were closely related in their metabolism: their sugar chains were in sequence and their ceramide moieties were similarly composed, namely C16:0 and C24:1 constituted most of the fatty acids, and long-chain base components were mostly C18-dihydrosphingosine with a small portion of C18-sphingosine. The sugar chains implied two pathways branching from lactosylceramide: one to globotriaosylceramide and the other to lactotriaosylceramide, which leads to the production of Le(x) glycolipid via neolacto type 2 core chain.

We recently identified two isoforms of mouse prostaglandin (PG) E receptor EP3 subtype, EP3 alpha and EP 3 beta, which are produced by alternative splicing and different only in the carboxyl-terminal domain (Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, Y., T., Honda, A., Watabe, A., Hirata, M., Narumiya, S., and Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718). We examined here agonist-induced desensitization of the two isoforms using Chinese hamster ovary cells stably expressing these isoforms. Exposure of the EP3 alpha isoform to PGE2 for 30 min did not change maximal response but increased PGE2 concentration needed to inhibit forskolin-induced cAMP accumulation in the cells. Further exposure of this isoform to PGE2 suppressed the maximal response as well as sensitivity to PGE2 in a time-dependent manner; after 24-h exposure, it elicited only 50% of the maximal response of the control cells. Consistent with these results, short term exposure sequestered the EP3 alpha isoform away from the cell surface and long term incubation decreased the total receptor number in the cells. In contrast, exposure of the EP3 beta isoform to PGE2 did not affect its dose-response curve for PGE2, and no sequestration or decrease in the receptor number was observed in this isoform. Thus, alternative splicing produced the two isoforms with different carboxyl-terminal domains, which are different in sensitivity to agonist-induced desensitization.

A glycosphingolipid that reacted positively to anti-stage-specific embryonic antigen-1 (SSEA-1) antiserum accumulated in human lens in association with aging and senile cataract formation. Since this antiserum recognizes Lewis(x) (Le(x)) structure, Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, which is a typical tumor-associated and differentiation-related saccharide chain, the lens glycolipid was predicted to be a Lex antigen. The glycolipid purified from cataractous lens tissues was indeed a Lex glycolipid, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1- 4Glc beta 1-1 ceramide. Enhanced expression of the Lex glycolipid may affect the organization of lens plasma membranes through Le(x)-Le(x) interactions, as suggested for compaction in mouse preimplantation embryos and embryonic teratocarcinomas, resulting in lens opacification, namely cataract.

Glycyrrhetinic acid bound specifically to a particulate fraction of rat liver. The binding was dependent on time, temperature and pH, equilibrium being reached after 10 min at 37°C. The equilibrium dissociation constant and the maximal concentration of the binding site, as determined by Scatchard plot analysis, were 31 nM and 43 pmol/mg protein, respectively, indicating a single binding site entity. The binding site was highly specific for glycyrrhetinic acid, glycyrrhizin, various steroids, various fatty acids and retinoids showing no or only very low affinity. The binding was inhibited by boiling or treatment with trypsin or phospholipases. The specific activity of glycyrrhetinic acid binding was the highest in the liver, followed by in the kidney. The results suggest that glycyrrhetinic acid plays a significant role in the rat liver through its specific binding protein.

A functional cDNA clone for mouse prostaglandin (PG) F receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of cloned prostanoid receptors, and cross-hybridization screening. The mouse PGF receptor consists of 366 amino acid residues with putative seven transmembrane domains. The sequence revealed the highest homology to the EP1 subtype of PGE receptor and thromboxane (TX) A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGF2 alpha binding. The binding was displaced with unlabeled PGs in the order of PGF2 alpha = 9 alpha, 11 beta PGF2 > PGF 1 alpha > PGD2 > STA2 (a stable TXA2 agonist) > PGE2 > iloprost (a stable PGI2 agonist). PGF2 alpha increased inositol trisphosphate formation in a concentration-dependent manner in COS cells expressing PGF receptor. RNA blot and in situ hybridization analyses demonstrated that the PGF receptor transcripts are abundantly expressed in luteal cells of corpus luteum and in a lesser amount in kidney, heart, stomach, and lung.

Melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) was found as the sole ceramide dihexoside from the eggs of the sea urchin, Anthocidaris crassispina. Ceramide monohexoside of the eggs consisted only of glucosylceramide (Glc beta 1-1Cer). These lipids were purified by successive column chromatographies on DEAE-Sephadex A-25, silicic acid and Florisil, and identified by gas-liquid chromatography, negative-ion fast atom bombardment mass spectrometry and proton nuclear magnetic resonance spectroscopy as well as methylation analysis. Long-chain base compositions of both lipids were almost identical and comprised n-C18-phytosphingosine and small amounts of its homologs (C17-C19). Fatty acid compositions were qualitatively very similar, but the glucosylceramide contained more 2-hydroxy fatty acid than the melibiosylceramide. Although the chain length of fatty acids was distributed over a wide range, six major fatty acids, namely 22:1, 23:1, 24:1, 22h:1, 23h:1 and 24h:1, constituted more than 92% of the fatty acid content in these lipids.