JNK1-Dependent Phosphorylation of GAP-43 Serine 142 is a Novel Molecular Marker for Axonal Growth

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.

     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.     

Structure-based screening for functional non-coding RNAs in fission yeast identifies a factor repressing untimely initiation of sexual differentiation

Non-coding RNAs (ncRNAs) ubiquitously exist in normal and cancer cells. Despite their prevalent distribution, the functions of most long ncRNAs remain uncharacterized. The fission yeast Schizosaccharomyces pombe expresses >1800 ncRNAs annotated to date, but most unconventional ncRNAs (excluding tRNA, rRNA, snRNA and snoRNA) remain uncharacterized. To discover the functional ncRNAs, here we performed a combinatory screening of computational and biological tests. First, all S. pombe ncRNAs were screened in silico for those showing conservation in sequence as well as in secondary structure with ncRNAs in closely related species. Almost a half of the 151 selected conserved ncRNA genes were uncharacterized. Twelve ncRNA genes that did not overlap with protein-coding sequences were next chosen for biological screening that examines defects in growth or sexual differentiation, as well as sensitivities to drugs and stresses. Finally, we highlighted an ncRNA transcribed from SPNCRNA.1669, which inhibited untimely initiation of sexual differentiation. A domain that was predicted as conserved secondary structure by the computational operations was essential for the ncRNA to function. Thus, this study demonstrates that in silico selection focusing on conservation of the secondary structure over species is a powerful method to pinpoint novel functional ncRNAs.     

New entry to the enantioselective formation of substituted cyclohexenes bearing an all-carbon quaternary stereogenic center

Enantioselective formation of cyclohexene derivatives bearing an all-carbon quaternary stereogenic center is described. The racemic cyclohexenes are readily transformed to chiral substituted cyclohexenes in good yield with excellent enantioselectivity and diastereoselectivity by a palladium-mediated deracemization. The resulting products are promising synthetic intermediates of biologically active natural products. This protocol provides us with a new entry to the concise and scalable synthesis of multifunctionalized compounds.     

Protective Effect Against 17β-Estradiol on Neuronal Apoptosis in Hippocampus Tissue Following Transient Ischemia/Recirculation in Mongolian Gerbils via Down-Regulation of Tissue Transglutaminase Activity

We analyzed the protective effect of 17β-estradiol (17β-ED) injection against delayed neuronal death in the hippocampus tissue of the brain in Mongolian gerbils after transient ischemia/recirculation treatment, especially in relation with bcl-2 gene expression and enzymatic activity changes of caspase-3 and tissue transglutaminase (tTGase). Daily intraperitoneal injection of 17β-ED to the animal after the ischemia stimulated the expression of an apoptosis suppressor gene, bcl-2, in the hippocampal tissue for a week. The gradually increasing apoptotic enzyme activity of caspase-3 and increased number of TUNEL positive fragmented neuronal nuclei caused by ischemic attack in the gerbil brain were clearly suppressed by 17β-ED administration. The reduced activity and enzyme protein of tTGase, a neurodegenerative marker of apoptosis in the hippocampus after ischemia, were also restored to nearly normal levels by 17β-ED injection. These results suggest that daily 17β-ED administration to the gerbil after transient ischemic insult with progressing neuronal deteriorative changes in hippocampus tissue can effectively prevent apoptotic changes through a molecular cascade involving gene expression regulation.     

31P-NMR STUDIES ON INORGANIC POLYPHOSPHATES IN MICROALGAE1

Using “P nuclear magnetic resonance analysis, total inorganic polyphosphate in algae could be quantitatively estimated, For this purpose the algal suspension, which had been kept in cold trichloroacetic acid, was further treated with 6 mM EDTA, or the cells were kept in 2 N KOH containing 100 mM EDTA for 18 h at 37°C. These simple methods avoid hydrolysis of cellular inorganic polyphosphate and, therefore, are useful for the study of phosphorus metabolism in algae. The effects of these treatments on visualization of the signal for inorganic polyphosphate in nuclear magnetic resonance spectra were discussed in comparison with in vivo, ‘P nuclear magnetic resonance spectra of algae.     

A novel L-glutamate transporter inhibitor reveals endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

We previously reported for the first time that D-aspartate (D-Asp) is biosynthesized by cultured mammalian cells such as pheochromocytoma (PC)12 cells and its subclone MPT1 (FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92). We speculated that D-Asp levels in the intra- and extracellular spaces of the cultured cells are maintained in a dynamic state of homeostasis. To test this here, we utilized a novel and potent L-Glu transporter inhibitor, TFB-TBOA. This inhibitor proved to be a genuine nontransportable blocker of the transporter even during long periods of culture. Use of this inhibitor with MPT1 cells confirmed that D-Asp levels are in a dynamic steady state where it is constantly released into the extracellular space by a yet undefined mechanism as well as being constantly and intensively taken up by the cells via the L-Glu transporter. We estimated the rate with which D-Asp is constitutively released from MPT1 cells is approx. 3.8 pmol/h/1x10(5) cells.     

Cell differentiation inducers derived from thalidomide

5-Hydroxy- and 4-amino-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33 and 4APP-33, respectively) have been shown to possess cell differentiation-inducing activity toward human leukemia cell line HL-60.     

Design and synthesis of phthalimide-type histone deacetylase inhibitors

Several hydroxamic acid derivatives with a substituted phthalimide group as a linker and/or cap structure, prepared during structural development studies based on thalidomide, were found to have histone deacetylase (HDAC)-inhibitory activity. Structure-activity relationship studies indicated that nature of the substituent introduced at the phthalimide nitrogen atom, introduction of a hydroxamic acid structure, and distance between the N-hydroxyl group and the cap structure are important for HDAC-inhibitory activity.