Neurotrophic factor neurotrophin-4 regulates ameloblastin expression via full-length TrkB

Neurotrophic factors play an important role in the development and maintenance of not only neural but also nonneural tissues. Several neurotrophic factors are expressed in dental tissues, but their role in tooth development is not clear. Here, we report that neurotrophic factor neurotrophin (NT)-4 promotes differentiation of dental epithelial cells and enhances the expression of enamel matrix genes. Dental epithelial cells from 3-day-old mice expressed NT-4 and three variants of TrkB receptors for neurotrophins (full-length TrkB-FL and truncated TrkB-T1 and -T2). Dental epithelial cell line HAT-7 expressed these genes, similar to those in dental epithelial cells. We found that NT-4 reduced HAT-7 cell proliferation and induced the expression of enamel matrix genes, such as ameloblastin (Ambn). Transfection of HAT-7 cells with the TrkB-FL expression construct enhanced the NT-4-mediated induction of Ambn expression. This enhancement was blocked by K252a, an inhibitor for Trk tyrosine kinases. Phosphorylation of ERK1/2, a downstream molecule of TrkB, was induced in HAT-7 cells upon NT-4 treatment. TrkB-FL but not TrkB-T1 transfection increased the phosphorylation level of ERK1/2 in NT-4-treated HAT-7 cells. These results suggest that NT-4 induced Ambn expression via the TrkB-MAPK pathway. The p75 inhibitor TAT-pep5 decreased NT-4-mediated induction of the expression of Ambn, TrkB-FL, and TrkB-T1, suggesting that both high affinity and low affinity neurotrophin receptors were required for NT-4 activity. We found that NT-4-null mice developed a thin enamel layer and had a decrease in Ambn expression. Our results suggest that NT-4 regulates proliferation and differentiation of the dental epithelium and promotes production of the enamel matrix.     

Structural and thermodynamic analyses of solute-binding Protein from Bifidobacterium longum specific for core 1 disaccharide and lacto-N-biose I

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Galbeta1-3GlcNAc) and galacto-N-biose (GNB; Galbeta1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K(d) values of 0.087 and 0.010 microm, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.     

Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification

With an aim to develop a quick and simple method to survey pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the identification of Plasmodium-carrying mosquitoes, specifically a Plasmodium-transmitting experimental model using rodent malaria parasite (Plasmodium berghei) and anopheline mosquitoes (Anopheles stephensi). The detection sensitivity limit of the LAMP reaction amplifying the SPECT2 gene was determined to be 1 × 10² purified Plasmodium parasites, estimated to be sufficient for reliable identification of infectious mosquitoes. The robustness of the LAMP reaction was revealed by its ability to detect both Plasmodium oocysts and sporozoites from an “all-in-one” template using whole mosquito bodies. Moreover, LAMP successfully identified an infectious mosquito carrying just a single oocyst in its midgut, a level that can be easily overlooked in conventional microscopic analysis. These observations suggest that LAMP is more reliable and useful for routine diagnosis of vector mosquitoes in regions where vector-borne diseases such as malaria are endemic.     

Role of subsite +1 residues in pH dependence and catalytic activity of the glycoside hydrolase family 1 beta-glucosidase BGL1A from the basidiomycete Phanerochaete chrysosporium

The basidiomycete Phanerochaete chrysosporium produces two glycoside hydrolase family 1 intracellular beta-glucosidases, BGL1A and BGL1B, during the course of cellulose degradation. In order to clarify the catalytic difference between two enzymes, in spite of their high similarity in amino acid sequences (65%), five amino acids around the catalytic site of BGL1A were individually mutated to those of BGL1B (V173C, M177L, D229N, H231D, and K253A), and the effects of the mutations on cellobiose hydrolysis were evaluated. When the kinetic parameters (K(m) and k(cat)) were compared at the optimum pH for the wild-type enzyme, the kinetic efficiency was decreased in the cases of D229N, H231D, and K253A, but not V173C or M177L. The pH dependence of cellobiose hydrolysis showed a significantly more acidic pH profile for the D229N mutant, compared with the wild-type enzyme. Since D229 is located between K253 and the putative acid/base catalyst E170, we prepared the double mutant D229N/K253A, and found that its hydrolytic activity at neutral pH was restored to that of the wild-type enzyme. Our results indicate that the interaction between D229 and K253 is critical for the pH dependence and catalytic activity of BGL1A. Biotechnol. Bioeng.     

Structure-activity relationship study and NMR analysis of fluorobenzoyl pentapeptide GPR54 agonists

GPR54 is a Gq-protein coupled receptor involved in cancer metastasis and regulation of the endocrine system. GPR54 activation by endogenous ligands attenuates the mobility of carcinomas and stimulates the secretion of gonadotropin-releasing hormone. GPR54 agonists are, therefore, potential therapeutic candidates for cancer metastasis and hormonal diseases. Pentapeptide derivatives of kisspeptin C-terminus were identified as potent GPR54 agonists in our previous studies. In the present study, we investigated the structure-activity relationship of a variety of pentapeptides having various fluorine-substituted benzoyl groups at the N-terminus. Among these, a 4-fluorobenzoyl derivative was the most potent agonist. On the other hand, the derivatives having multiple fluoro-substituting groups showed less binding affinity. NMR analysis of these peptides and their N-terminal partial structures suggested that fluorine substituents affect the benzoyl conformation. o-Monofluorobenzoyl is likely to be in a coplanar conformation due to the intramolecular CF--HN hydrogen bonding between o-fluorine and amide hydrogen; the o,o-difluorobenzoyl moiety exists in a distorted conformation probably due to the steric hindrance and/or electrostatic repulsion between two o-fluorine atoms and carbonyl oxygen.     

Phosphorylation of claudin-4 is required for tight junction formation in a human keratinocyte cell line

Extensive studies have identified a large number of the molecular components of cellular tight junctions (TJ), including the claudins, occludin and ZO-1/2, and also many of the physical interactions between these molecules. However, the regulatory mechanisms of TJ formation are as yet poorly understood. In HaCaT, a human epidermal keratinocyte cell line, TJ were newly formed when cells were cultured in the presence of SP600125, a JNK inhibitor. Moreover, claudin-4 was newly phosphorylated during this process. We found that claudin-4 contains a sequence which is phosphorylated by atypical PKC (aPKC). Kinase assay demonstrated that the 195th serine (serine195) of mouse claudin-4 was phosphorylated by aPKC in vitro. The 194th serine (serine194) of human claudin-4 corresponding to serine195 of mouse claudin-4 was phosphorylated in HaCaT cells when TJ were formed, and the phosphorylated claudin-4 co-localized with ZO-1 at TJ. aPKC activity was required for both the claudin-4 phosphorylation and TJ formation in HaCaT. Furthermore, overexpression of mutant claudin-4 protein S195A, which was not phosphorylated by aPKC, perturbed the TJ formation mediated by SP600125. These findings suggest that aPKC regulates TJ formation through the phosphorylation of claudin-4.     

沈阳市郊耕地土壤交换性Ca含量的空间异质性特征

利用地统计学和地理信息系统相结合的方法研究了沈阳市郊耕地土壤(0～20crn)交换性钙含量的空间异质性特征,绘制了交换性钙含量的空间分布图。结果表明,土壤交换性钙含量经对数转换后基本符合正态分布,变异函数的最佳理论模型为球状模型,交换性钙含量具有很强的空间相关性,空间自相关距离为15．8km,其空间异质性主要受成土母质和土壤类型等结构性因子的影响。     

Dietary nucleotides increase the mucosal IgA response and the secretion of transforming growth factor β from intestinal epithelial cells in mice

We have investigated the influence of dietary nucleotides on the intestinal immune system in ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice (OVA-TCR Tg mice). When mice were supplied with water supplemented with 2% OVA ad libitum, the faecal OVA-specific immunoglobulin A (IgA) level significantly increased in those fed a nucleotide-supplemented diet (NT(+) diet) compared with those fed a nucleotide-free control diet (NT(–) diet). In the NT(+) diet-fed mice, secretion of transforming growth factor β (TGF-β), which is an isotype-specific switch factor for IgA, from intestinal epithelial cells (IECs) was significantly increased. Furthermore, an increased proportion of intestinal intraepithelial lymphocytes (IELs) bearing γδ TCR (TCRγδ⁺ IELs) and increased secretion from IECs of interleukin 7 (IL-7), which is essential for the development of TCRγδ⁺ IELs, were also observed in OVA-TCR-Tg mice fed the NT(+) diet, as we previously demonstrated using BALB/c mice (Nagafuchi et al., Biosci. Biotechnol. Biochem. 64: 1459-65 (2000)). Considering that TCRγδ⁺ T cells and TGF-β are important for an induction of the mucosal IgA response, our results suggest that dietary nucleotides augment the mucosal OVA-specific IgA response by increasing the secretion of TGF-β from IECs and the proportion of TCRγδ⁺ IELs. This revised version was published online in August 2006 with corrections to the Cover Date.