Enhancement of In Vivo Antioxidant Ability in the Brain of Rats Fed Tannin

The effect of the oral administration of mimosa tannin (MMT) on the rat intra-hippocampal antioxidant ability was examined. Wistar rats at the age of 6 weeks were reared for 8 weeks with the rodent diet (RD) consisting of 0.1 g/kg of MMT (RD–MMT). The antioxidant ability of rat brain was evaluated from the decay of a brain–blood-barrier permeable stable nitroxide, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) measured by the microdialysis-electron spin resonance system under a freely moving state. The decay rate of PCAM in the brain of rats fed RD–MMT was significantly larger than that of rats fed control rodent diet, which indicates the increase of the antioxidant ability in the brain of rats fed RD–MMT. In vitro study showed that MMT did not reduce PCAM directly but enhanced the reduction of PCAM by ascorbic acid. These results indicate that MMT is a potent antioxidant in vitro and in vivo.     

Conformational Change of the L-Shaped tRNAPhe Molecule

Abstract

Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNA^Phe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNA^Phe was measured by a singlet-singlet energy transfer. Conformational changes of tRNA^Phe with binding of tRNA^Glu ₂, which has the anticodon UUC complementary to the anticodon GAA of tRNA^Phe, were investigated. The distance obtained at the ionic strength of 100 mM K⁺ and 10 mM Mg²⁺ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNA^Glu ₂ is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ₅₅ in the TΨC loop of tRNA^Phe, Stern-Volmer quenching experiments for the probe with or without added tRNA^Glu ₂were carried out. The results showed greater access of the probe to the quencher with added tRNA^Glu ₂. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA^Glu ₂ and some structural collapse occurs at the corner of the L-shaped structure.     

Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186–200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.     

EXTL2, a Member of the EXT Family of Tumor Suppressors,Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear. Here we show that EXTL2 transfers a GlcNAc residue to the tetrasaccharide linkage region that is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminates chain elongation. We isolated an oligosaccharide from the mouse liver, which was not detected in EXTL2 knock-out mice. Based on structural analysis by a combination of glycosidase digestion and 500-MHz ¹H NMR spectroscopy, the oligosaccharide was found to be GlcNAcα1-4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), which was considered to be a biosynthetic intermediate of an immature GAG chain. Indeed, EXTL2 specifically transferred a GlcNAc residue to a phosphorylated linkage tetrasaccharide, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate). Remarkably, the phosphorylated linkage pentasaccharide generated by EXTL2 was not used as an acceptor for heparan sulfate or chondroitin sulfate polymerases. Moreover, production of GAGs was significantly higher in EXTL2 knock-out mice than in wild-type mice. These results indicate that EXTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates GAG biosynthesis might be a “quality control system” for proteoglycans.     

Display of active beta-glucosidase on the surface of Schizosaccharomyces pombe cells using novel anchor proteins

Here, we demonstrate display of beta-glucosidase (BGL) on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. A total of four candidate anchor proteins (SPBC21D10.06c, SPBC947.04, SPBC19C7.05, and SPBC359.04c) were selected from among almost all of S. pombe membrane proteins. The C-terminus of each anchor protein was genetically fused to the N-terminus of BGL, and the fusion protein was expressed using S. pombe as a host. The highest cell surface-associated BGL activity (107 U/10⁵ cells was achieved with SPBC359.04c serving as the anchor, followed by SPBC947.04 (44 U/10⁵ cells) and SPBC21D10.06c (38 U/10⁵ cells). S. pombe displaying BGL with SPBC359.04c as an anchor showed the highest growth on 2 % cellobiose (10.7?×?10⁷ cells/mL after 41 h of cultivation from an initial density of 0.1?×?10⁷ cells/mL). Additionally, culturing BGL-displaying S. pombe in medium containing cellobiose as the sole carbon source did not affect protein expression, and ethanol fermentation from cellobiose was successfully demonstrated using BGL-displaying S. pombe. This is the first report describing a cell surface display system for the functionalization of S. pombe.     

Absence of intervening sequences and point mutations in the V domain within 23S rRNA in Campylobacter lari isolates

Although the absence of intervening sequences (IVSs) within the 23S rRNA genes in Campylobacter lari isolates has been described, there are apparently no reports regarding correlations between the nucleotide sequences of 23S rRNA genes and erythromycin (Ery) susceptibility in C. lari isolates. Here, we determined the minimum inhibitory concentrations of 35 C. lari isolates [n?=?19 for urease-positive thermophilic Campylobacter (UPTC); n?=?16 urease-negative (UN) C. lari] obtained from Asia, Europe, and North America. We found that the 18 isolates were resistant to the Ery (defined as ≧8 μg/mL), and three isolates, UPTC A1, UPTC 92251, and UPTC 504, showed increased resistance (16 μg/mL). No correlations between the IVSs in the helix 45 region within the 23S rRNA gene sequences and Ery resistance were identified in the C. lari isolates examined. In addition, no point mutations occurred at any expected or putative position within the V domain in the isolates. In conclusion, antibiotic resistance against the macrolide erythromycin is mediated through an alternative pathway to that described above.     

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M1 and M4 muscarinic acetylcholine receptors agonists

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M₁ and M₄ agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.