Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA₄. Here we report that p2y5 is a novel LPA receptor coupling to the G₁₃-Rho signaling pathway. “LPA receptor-null” RH7777 and B103 cells exogenously expressing p2y5 showed [³H]LPA binding, LPA-induced [³⁵S]guanosine 5′-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and G_s/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA₆.     

Clock genes regulate neurogenic transcription factors,including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.     

Involvement of Rho-kinase in tumor necrosis factor-α-induced interleukin-6 release from C6 glioma cells

Tumor necrosis factor (TNF)-α stimulated interleukin (IL)-6 release and induced the phosphorylation of myosin phosphatase targeting subunit (MYPT)-1, a Rho-kinase substrate. The IL-6 release was significantly suppressed by Y-27632 and fasudil, Rho-kinase inhibitors. Although IκB inhibitor suppressed the TNF-α-induced IL-6 release, the Rho-kinase inhibitors did not affect the TNF-α-induced IκB phosphorylation. TNF-α induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p44/p42 MAP kinase. The TNF-α-induced IL-6 release was suppressed by SB203580, a p38 MAPK inhibitor, or SP600125, a SAPK/JNK inhibitor, but not by PD98059, a MAP kinase/extracellular signal-regulated kinase kinase inhibitor. The Rho-kinase inhibitors attenuated the TNF-α-induced phosphorylation of both p38 MAP kinase and SAPK/JNK.Rho-kinase, which has been used for the clinical treatment of cerebral vasospasms, may be involved in other central nervous system (CNS) disorders such as traumatic injury, stroke, neurodegenerative disease and neuropathic pain. TNF-α, a proinflammatory cytokine that affects the CNS through cytokines, such as IL-6, release from neurons, astrocytes and microglia. Therefore, we investigated the involvement of Rho-kinase in the TNF-α-induced IL-6 release from rat C6 glioma cells.These results strongly suggest that Rho-kinase regulates the TNF-α-induced IL-6 release at a point upstream from p38 MAPK and SAPK/JNK in C6 glioma cells. Therefore, Rho-kinase inhibitor may be considered to be a new clinical candidate for the treatment of CNS disorders in addition to cerebral vasospasms.     

Semiaquatic Heteroptera locomotion: coral treaders (Hermatobates weddi, Hermatobatidae), sea skaters (Halovelia septentrionalis, Veliidae), and water striders (Metrocoris histrio, Gerridae). Usual and unusual gaits

Using high-speed video recordings, we carried out an analysis of the locomotion gaits of the following aquatic Heteroptera: coral treaders Hermatobates weddi (Hermatobatidae), sea striders Halovelia septentrionalis (Veliidae), and water striders Metrocoris histrio (Gerridae), in the Island of Amami Oshima, Kagoshima Prefecture, Japan. Most insects use an alternating double tripod gait for walking, whereas species of Gerridae and some Veliidae use a synchronous rowing gait. We found that H. weddi used a peculiar locomotion gait, a modification of the double tripod gait. In this special gait, two alternating dipods (mid and hind legs) are used, while the forelegs remained inactive. Contralateral mid and hind stroked simultaneously. The mid leg recovered immediately after the stroke; however, the hind leg was delayed and remained extended after the stroke. Next, the following bipod stroked, and when that mid leg finished the stroke, both ipsilateral mid and hind (the one which did not recover after the stroke) legs recovered together. Turning is also unique in H. weddi because the body axis rotation and the course turning (deflection) were clearly separated in two phases. We compared the kinematics of H. weddi pattern with the synchronous rowing pattern found in H. septentrionalis and M. histrio and discussed some biomechanical consequences. We also analyzed phylogenetic implications of this gait, and we posit that the modified double dipod gait is a uniquely derived character of the family Hermatobatidae. The synchronous rowing gait would be an autapomorphy for the clade Gerridae + Veliidae. The modified thorax, with the meso and metacoxae horizontally directed, would be a synapomorphy for the superfamily Gerroidea (Hermatobatidae, Gerridae, and Veliidae). Handling editor: Koen Martens     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

Miscibility and phase behavior of DPPG and perfluorocarboxylic acids at the air-water interface

The miscibility and phase behavior of two components of phospholipids and perfluorocarboxylic acids [FCn; perfluorododecanoic acid (FC12), perfluorotetradecanoic acid (FC14), perfluorohexadecanoic acid (FC16), and perfluorooctadecanoic acid (FC18)] have been systematically investigated using Langmuir monolayer technique. Dipalmitoylphosphatidylglycerol (DPPG) is utilized as a phospholipid component in biomembranes. Surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms have been measured for the DPPG/FCn systems on 0.15 M NaCl (pH 2.0) at 298.2 K. From the isotherm results, two-dimensional phase diagrams are constructed and classified into miscible and immiscible patterns. Furthermore, the phase behavior of the DPPG/FCn systems has been morphologically examined using fluorescence microscopy (FM) and atomic force microscopy (AFM). These images indicate different phases among the four systems. In particular, specific phase morphology is observed in the middle molar fraction range for the DPPG/FC14 system; FC14 is selectively excluded from mixed DPPG-FC14 monolayers to be concentrated in the phase boundary as surface pressure increases. Then DPPG is refined as a patched film. Moreover, the data obtained here are compared to those in the previous systems in which different kinds of phospholipids were treated. Through a series of the miscibility investigations, it is proposed that combinations of hydrophobic chain lengths and of polar headgroups contribute to the monolayer miscibility between phospholipids and perfluorocarboxylic acids.     

RNA interference targeting against S100A4 suppresses cell growth and motility and induces apoptosis in human pancreatic cancer cells

S100A4 protein belongs to the S100 subfamily, which has grown to be one of the large subfamilies of the EF-hand Ca²⁺-binding proteins, and overexpression of S100A4 is suggested to associate with cell proliferation, invasion, and metastasis. We observed frequent overexpression of S100A4 in pancreatic cancer cell lines and further analyzed RNAi-mediated knockdown to address the possibility of its use as a therapeutic target for pancreatic cancer. The specific knockdown of S100A4 strongly suppressed cell growth, induced G2 arrest and eventual apoptosis, and decreased cell migration. Furthermore, microarray analyses revealed that knockdown of S100A4 induced expression of the tumor suppressor genes PRDM2 and VASH1. Our present results suggest the possibility that the inhibition of S100A4 can be utilized in antitumor applications for patients with pancreatic cancer.     

Reconstitution of blue fluorescent protein from recombinant apoaequorin and synthetic coelenteramide

Blue fluorescent protein of aequorin (BFP) is a complex of Ca²⁺-bound apoaequorin with coelenteramide and is a bifunctional protein, which shows blue fluorescence and the luminescence activity like a luciferase. To reconstitute synthetic BFP (syn-BFP) from apoaequorin and coelenteramide, we established new synthetic route of coelenteramide and prepared highly purified recombinant aequorin using the histidine-tagged secretion system in Escherichia coli cells. As a result, we succeeded in reconstituting syn-BFP quantitatively and the fluorescence and luminescence properties of syn-BFP were identical to that of BFP obtained from aequorin.     

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Vitamin D₃ (VD₃) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD₃ into its physiologically active forms, namely, 25(OH)VD₃ or 1α,25(OH)₂VD₃. In this study, we isolated and characterized Vdh (vitamin D₃ hydroxylase), which hydroxylates VD₃ from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V_max values for VD₃ 25-hydroxylation and 25(OH)VD₃ 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD₃. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD₃ 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD₃ into 25(OH)VD₃ was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD₃ by a single cytochrome P450.