Sphingosine kinase-1 (SPHK1) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, sphingosine 1-phosphate, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK1. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK1 was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G 1 /S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK1, where SPHK1 was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.Sphingosine 1-phosphate (SPP) 1 is a bioactive lipid that regulates diverse biological processes such as calcium mobilization, cell growth, differentiation, survival, motility, and cytoskeletal reorganization, acting both inside and outside the cells (1, 2). Recently, SPP was identified as the ligand for a family of G protein-coupled receptors known as the endothelial differentiation gene-1 family, now collectively renamed SPP receptors (3-6), supporting a role for SPP as an extracellular ligand. However, the intracellular targets of SPP have not yet been identified.Sphingosine kinase (SPHK), the enzyme that catalyzes the phosphorylation of sphingosine, regulates the intracellular levels of SPP. Two isoforms of mammalian SPHK (SPHK1 and SPHK2) have been cloned and characterized (7,8). SPHK1 predominantly localizes in the cytosol, and its overexpression induces cell proliferation by promoting the G 1 to S transition of the cell cycle as well as by inhibiting the apoptotic response to serum deprivation or ceramide treatment (9). Several cellular proteins have recently been identified as SPHK1-interacting molecules, namely TRAF2 (10), RPK118 (11), and AKAP-related protein (12), which should help facilitate the understanding of the regulation and intracellular site of action of SPHK1.In contrast to SPHK1, little is known about the cellular actions of the other isozyme, SPHK2. In the present studies, we investigated the biological features of SPHK2. We have discovered that SPHK2 localizes in the nuclei of cells through its novel nuclear localization signal (NLS) sequence, depending on cell type and cell density. We have also demonstrated that nuclear localization of SPHK2 causes inhibition of DNA synthesis in various cell types.

Corneas of tadpole, mouse, rat, guinea pig, rabbit, cat, cattle, and human were examined by TEM and SEM in a comparative study. The differences between species were noted mainly by using TEM. Bowman's layer showed a tendency to be well developed in higher mammals. Tadpoles lack a Bowman's layer, lower mammals have a thin Bowman's layer, and higher mammals have a thick Bowman's layer. The boundary between the substantia propria and Descemet's membrane was distinct in higher mammals. On the other hand, there are no differences in thickness of the collagen fibrils that constitute Bowman's layer and those of the substantia propria. NaOH digestion was utilized for SEM preparation. SEM imaging revealed a textured appearance of the epithelial side of Bowman's layer. In Descemet's membrane, fibrous long spacing (FLS) fiber-like structures, which are arranged in parallel to the endothelium, were observed by both TEM and SEM. To our knowledge, this is the first report of SEM observations of FLS fiber-like structures on the endothelial surface of Descemet's membrane. SEM at a plane normal to the plane of the cornea showed that Descemet's membrane has a piled laminar structure. Descemet's membrane is closely associated with the collagen layer of the substantia propria. Collagen fibrils invading from the substantia propria into Descemet's membrane were observed with both TEM and SEM.

Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell. Sphingosine kinase (SPHK)1 is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events including cell growth, survival, motility, cytoskeletal changes, and the release of calcium from intracellular stores (1, 2) by acting both as an extracellular agonist and an intracellular messenger (3). The extracellular effects of SPP are mediated by the recently identified endothelial differentiation gene (EDG) receptors, novel members of the G-proteincoupled heptahelical receptor family (4). For example, the binding of SPP to HEK293 cells stably expressing EDG-1 induced the inhibition of cAMP accumulation and the activation of extracellular signal-regulated kinase (ERK) in a pertussis toxin-sensitive manner (5). On the other hand, the following findings were important clues to a specific intracellular action of SPP. First, the activation of various plasma membrane receptors such as the platelet-derived growth factor (6, 7) and the Fc⑀RI (8) was found to rapidly increase intracellular SPP production through the stimulation of SPHK. Second, microinjected SPP mobilized Ca 2ϩ from the internal stores of cells that had been pretreated with pertussis toxin to inactivate G i -or G o -coupled receptor signaling (9). Third, the manipulation of intracellular SPP content in yeast cells, which lack a cell surface receptor for SPP, by the overexpression or deletion of genes that encode SPHK has revealed an important role for SPP in yeast survival and proliferation during exposure to heat or nutrient-deprivation stress. However, the intracellular site of action of SPP remains unknown.The present studies were designed to determine the intracellular site of action of SPP by identifying molecules interacting with sphingosine kinase-1 (SPHK1) using a yeast...

Galactocerebroside and sulfatide are two major glycolipids in myelin; however, their independent functions are not fully understood. The absence of these glycolipids causes disruption of paranodal junctions, which separate voltage-gated Na(+) and Shaker-type K(+) channels in the node and juxtaparanode, respectively. In contrast to glial cells in the central nervous system (CNS), myelinating Schwann cells in the peripheral nervous system (PNS) possess characteristic structures, including microvilli and Schmidt-Lanterman incisures, in addition to paranodal loops. All of these regions are involved in axo-glial interactions. In the present study, we examined cerebroside sulfotransferase-deficient mice to determine whether sulfatide is essential for axo-glial interactions in these PNS regions. Interestingly, marked axonal protrusions were observed in some of the nodal segments, which often contained abnormally enlarged vesicles, like degenerated mitochondria. Moreover, many transversely cut ends of microvilli surrounded the mutant nodes, suggesting that alignments of the microvilli were disordered. The mutant PNS showed mild elongation of nodal Na(+) channel clusters. Even though Caspr and NF155 were completely absent in half of the paranodes, short clusters of these molecules remained in the rest of the paranodal regions. Ultrastructural analysis indicated the presence of transverse bands in some paranodal regions and detachment of the outermost several loops. Furthermore, the numbers of incisures were remarkably increased in the mutant internode. Therefore, these results indicate that sulfatide may play an important role in the PNS, especially in the regions where myelin-axon interactions occur.

Gas-phase photoelectron spectroscopy( PES) was conducted on [XAg 24 (SPhMe 2 ) 18 ] À (X = Ag, Au) and [YAg 24 -(SPhMe 2 ) 18 ] 2À (Y = Pd, Pt), which have aformal superatomic core (X@Ag 12 ) 5+ or (Y@Ag 12 ) 4+ with icosahedral symmetry. PES results show that superatomic orbitals in the (Au@Ag 12 ) 5+ core remain unshifted with respect to those in the (Ag@Ag 12 ) 5+ core,whereas the orbitals in the (Y@Ag 12 ) 4+ (Y = Pd, Pt) core shift up in energy by about 1.4 eV.T he remarkable doping effect of asingle Yatom (Y = Pd, Pt) on the electronic structure of the chemically modified (Ag@Ag 12 ) 5+ superatom was reproduced by theoretical calculations on simplified model systems and was ascribed to 1) the weaker binding of valence electrons in Y@(Ag + ) 12 compared to Ag + @(Ag + ) 12 due to the reduction in formal charge of the core potential, and 2) the upward shift of the apparent vacuum level due to the presence of ar epulsive Coulomb barrier between [YAg 24 (SPhMe 2 ) 18 ] À and electron.Thiolate (RS)-or dithiolate (RS 2 )-protected silver clusters, such as [Ag 25 (SR) 18 ] À , [1] [Ag 29 (S 2 R) 12 ] 3À , [2] and [Ag 44 -(SR) 30 ] 4À , [3][4][5] are an emerging class of nanomaterials.S inglecrystal X-ray diffraction (SCXRD) analysis showed that [Ag 25 (SR) 18 ] À and [Ag 29 (S 2 R) 12 ] 3À have an icosahedral Ag 13 core (Scheme 1), [1,2] whereas [Ag 44 (SR) 30 ] 4À has at wo-shell Keplerate Ag 32 core. [5] TheA g 13 and Ag 32 cores form the closed-shell electronic configurations (1S) 2 (1P) 6 and (1S) 2 -(1P) 6 (1D) 10 ,r espectively:1 S, 1P,a nd 1D represent superatomic orbitals with angular momenta of 0, 1, and 2, respectively. [6] Structural similarities to gold analogues indicate that the thiolate-protected Ag clusters represent another family of chemically modified superatoms. [7][8][9] Thiolate-protected Ag clusters have attracted researchers due to specific properties such as photoluminescence [10] although they are generally less stable than the gold analogues.Doping with heteroatoms is apromising approach to enhance the stability and further improve the properties of the Ag clusters.S tate-of-the-art synthesis based on coreduction [11,12] and galvanic replacement [13,14] allowed us to precisely define the number, element, and location of the heteroatom(s) introduced into the Ag clusters.F or example, as ingle Ma tom (M = Au,P d, Pt) can be integrated exclusively at the central position of an icosahedral Ag 13 core of [Ag 25 (SPhMe 2 ) 18 ] À[1] to form M@Ag 12 cores in [AuAg 24 (SPhMe 2 ) 18 ] À[13] and [MAg 24 (SPhCl 2 ) 18 ] 2À (M = Pd, Pt) [11] (Scheme 1). Both the undoped Ag 13 and doped M@Ag 12 cores form ac losed electron configuration, (1S) 2 -(1P) 6 .These atomically defined bimetallic clusters provide an ideal platform to study the effect of single-atom doping on their properties.O ptical spectroscopy (Figure 1a)a nd voltammetry [15] showed that the doping slightly modulates the HOMO-LUMO gap of [Ag 25 (SPhMe 2 ) 18 ] À .T he stability [13,15] and photoluminescence quantum yield (PLQY) [10] of [A...

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A novel series of rhodanin (RH) and thiohydantoin (TH) derivatives were designed and synthesized for detecting tau pathology in the brains of patients with Alzheimer’s disease (AD). In experiments in vitro using tau and β-amyloid (Aβ) aggregates, the TH derivative, TH2, showed high specific binding to tau aggregates. In hippocampal sections obtained from AD patients, TH2 intensely stained neurofibrillary tangles. In experiments using normal mice, [125I]TH2 showed good uptake (1.54%ID/g, 2 min postinjection) into and a rapid washout (0.25%ID/g, 60 min postinjection) from the brain. [123I]TH2 should be further investigated as a potential imaging agent for detecting tau pathology.

Brønsted and Lewis base catalysis of hexametalate clusters of group 5 metals [M­(V)6O19]8– (M­(V) = Ta, Nb) and group 6 metals [M­(VI)6O19]2– (M­(VI) = Mo, W) was studied using Knoevenagel condensation and CO2 fixation reaction, respectively, as test reactions. It was found from mass spectrometry and elemental analysis that tetrabutylammonium salts of [M­(V)6O19]8– were partially protonated to form [H4M­(V)6O19]4– under ambient conditions, whereas those of [M­(VI)6O19]2– were not. Base catalytic activity increased in the order of [Mo6O19]2–, [W6O19]2– ≪ [H4Nb6O19]4– < [H4Ta6O19]4–, which is consistent with the order of the average NBO charges on the surface O atoms. This trend suggests that the highest activity of [H4Ta6O19]4– is due to the large amount of negative charges on surface O atoms. Theoretical calculations on [H n Ta6O19](8–n)– (n = 1–4) demonstrated that protonation of the O atoms at the edges is energetically favorable regardless of n and that the NBO charges on the remaining unprotonated O atoms in [H4Ta6O19]4– are still more negative than those of pristine [Nb6O19]8–, [W6O19]2–, and [Mo6O19]2–. Theoretical calculations also predicted that CO2 can be reductively activated at all of the surface O atoms of [H4Ta6O19]4– regardless of their locations. This work demonstrates that group 5 polyoxometalates are promising candidates for active base catalysts.