A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.     

Gene expression profiles of endothelial progenitor cells by oligonucleotide microarray analysis

Among the many tissue stem or progenitor cells recently being unveiled, endothelial progenitor cells (EPCs) have attracted particular attention, not only because of their cardinal role in vascular biology and embryology but also because of their potential use in the therapeutic development of a variety of postnatal diseases, including cardiovascular and peripheral vascular disorders and cancer. The aim of this study is to provide some basic and comprehensive information on gene expression of EPCs to characterize the cells in molecular terms. Here, we focus on EPCs derived from CD34-positive mononuclear cells of human umbilical cord blood. The EPCs were purified and expanded in culture and analyzed by a high-density oligonucleotide microarray and real-time RT-PCR analysis. We identified 169 up-regulated and 107 down-regulated genes in the EPCs compared with three differentiated endothelial cells of human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (LMEC) and human aortic endothelial cells (AoEC). It is expected that the obtained list include key genes which are critical for EPC function and survival and thus potential targets of EPC recognition in vivo and therapeutic modulation of vasculogenesis in cancer as well as other diseases, in which de novo vasculogenesis plays a crucial role. For instance, the list includes Syk and galectin-3, which encode protein tyrosine kinase and β-galactoside-binding protein, respectively, and are expressed higher in EPCs than the three control endothelial cells. In situ hybridization showed that the genes were expressed in isolated cells in the fetal liver at E11.5 and E14.5 of mouse development.     

The effect of temperature on the post-embryonic growth of Neomysis intermedia Czernlawsky (Crustacea, Mysidacea) under laboratory conditions

The effect of temperature on post-embryonic growth of Neomysisintermedia was investigated under unlimited food conditionsin the laboratory. The effect of temperature on the size ofnewly released animals was negligibly small, but body size wasinversely related to temperature in adults. This was mainlycaused by the difference in the number of molts before maturation.The specific growth rate of N. intermedia increased exponentiallywith a temperature coefficient, Q₁₀ of 4.6 from 0.018 d^–13C to 0.21 d^–1 at 20C in juveniles, and with a temperaturecoefficient of 2.7 from 0.006 d^–1 at 3C to 0.05 d^–1at 25C in adults. The rate in juveniles levelled off above20C, and dropped at 29C. Brood size and brood interval decreasedwith temperature increase, while the daily specific reproductionrate increased. The specific growth rate of gravid females,including production of egg matter, increased exponentiallywith a temperature coefficient of 3.3 from 0.015 d^–1 at10C to 0.093 d^–1 at 25C. The present laboratory experiments confirmed the temperaturecontrol on the growth of N. intermedia suggested in a hyper-eutrophiclake.     

ecNOS gene polymorphism is associated with endothelium-dependent vasodilation in Type 2 diabetes

The association between endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphism and vascular endothelial function has not been clarified. We investigated the impact of ecNOS gene polymorphism on endothelial function in 95 patients with Type 2 diabetes (ecNOS genotype: 4b/b, n = 62; 4b/a, n = 30; 4a/a, n = 3). Flow-mediated (endothelium dependent, FMD) and nitroglycerin-induced (endothelium independent, NTG) vasodilations of the right brachial artery were studied using a phase-locked echotracking system. There were no significant differences in clinical characteristics among the ecNOS genotypes. The FMD was significantly lower in the patients with ecNOS4a allele than in those without ecNOS4a allele (P < 0.05). Multiple regression analysis showed that ecNOS4a allele and mean blood pressure were significant independent determinants for reduced FMD in all patients (R(2) = 0.122, P = 0.0025). The ecNOS4a allele was an independent determinant for reduced FMD in smokers but not in nonsmokers. These results suggest that ecNOS4a allele is a genetic risk factor for endothelial dysfunction in diabetic patients, especially in smokers.     

Synthesis of 2′,3′-Dideoxypurinenucleosides via the Palladium Catalyzed Reduction of 9-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-d-xylofuranosyl)purine Derivatives

Abstract

Practical method to produce 2′,3′-dideoxypurinenucleosides from 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)purines (1) was developed. High ratio of 2′,3′-dideoxynucleoside to 3′-deoxyribonucleoside was obtained by selecting the reaction conditions (solvent, pH and/or base), or changing 2′-acyloxy leaving group. The reaction mechanism was studied by deuteration experiments of 1a and 1-(3,5-di-O-acety1-2-bromo-2-deoxy-β-D-ribofuranosyl)thymine (12).

     

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase.     

Knockout of the SREBP system increases production of the polyketide FR901512 in filamentous fungal sp. No. 14919 and lovastatin in Aspergillus terreus ATCC20542

Applied Microbiology and Biotechnology - In the production of useful microbial secondary metabolites, the breeding of strains is generally performed by random mutagenesis. However, because random...