Molecular binding behavior of pyridine-2,6-dicarboxamide-bridged bis(beta-cyclodextrin) with oligopeptides: switchable molecular binding mode

Bridged bis(beta-cyclodextrin) 1 with a pyridine-2,6-dicarboxamide linker was synthesized, and its inclusion complexation behavior with some aliphatic oligopeptides was investigated in aqueous buffer solution of pH 2.0 and 7.2 at 25 degrees C by means of circular dichroism, fluorescence, and 2D NMR techniques. The results show that the resulting inclusion complexes of 1 with oligopeptides adopt a cooperative "cyclodextrin-guest-cyclodextrin" sandwich binding mode in a neutral media, but a "guest-linker-cyclodextrin" coinclusion binding mode in an acidic media. These switchable binding modes consequently rationalize the binding ability of bis(beta-cyclodextrin) 1 at different pH values; that is, 1 shows the stronger association with oligopeptides in a neutral media. Because of the simultaneous contributions of hydrophobic, hydrogen bond, and electrostatic interactions, bis(beta-cyclodextrin) 1 affords length-selectivity up to 4.7 for the Gly-Gly/Gly-Gly-Gly pair at pH 2.0 and sequence-selectivity up to 4.2 for the Gly-Leu/Leu-Gly pair at pH 7.2. These phenomena are discussed from the viewpoint of the size-fit concept and the multipoint recognitions between host and guest.     

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.     

Phylogenetic relationships of Drosophila melanogaster species group deduced from spacer regions of histone gene H2A-H2B

Nucleotide sequences of the spacer region of the histone gene H2A-H2B from 36 species of Drosophila melanogaster species group were determined. The phylogenetic trees were reconstructed with maximum parsimony, maximum likelihood, and Bayesian methods by using Drosophila pseudoobscura as the out group. Our results show that the melanogaster species group clustered in three main lineages: (1). montium subgroup; (2). ananassae subgroup; and (3). the seven oriental subgroups, among which the montium subgroup diverged first. In the third main lineage, suzukii and takahashii subgroups formed a clade, while eugracilis, melanogaster, elegans, ficusphila, and rhopaloa subgroups formed another clade. The bootstrap values at subgroup levels are high. The phylogenetic relationships of these species subgroups derived from our data are very different from those based on some other DNA data and morphology data.     

CD1d function is regulated by microsomal triglyceride transfer protein

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.     

Adiponectin acts in the brain to decrease body weight

Adiponectin (ADP) is an adipocyte hormone involved in glucose and lipid metabolism. We detected a rise in ADP in cerebrospinal fluid after intravenous (i.v.) injection, consistent with brain transport. In contrast to leptin, intracerebroventricular (i.c.v.) administration of ADP decreased body weight mainly by stimulating energy expenditure. Full-length ADP, mutant ADP with Cys39 replaced with serine, and globular ADP were effective, whereas the collagenous tail fragment was not. Lep(ob/ob) mice were especially sensitive to i.c.v. and systemic ADP, which resulted in increased thermogenesis, weight loss and reduction in serum glucose and lipid levels. ADP also potentiated the effect of leptin on thermogenesis and lipid levels. While both hormones increased expression of hypothalamic corticotropin-releasing hormone (CRH), ADP had no substantial effect on other neuropeptide targets of leptin. In addition, ADP induced distinct Fos immunoreactivity. Agouti (A(y)/a) mice did not respond to ADP or leptin, indicating the melanocortin pathway may be a common target. These results show that ADP has unique central effects on energy homeostasis.     

Mutational analysis of BRAF and K-ras in gastric cancers: absence of BRAF mutations in gastric cancers

Recently, BRAF mutations were found in a variety of human cancers. Interestingly, the most common of BRAF mutation (V599E) has not been identified in tumors with K-ras mutations. Whereas the majority of human cancer types has been screened for BRAF mutations, no detailed studies on gastric cancers have been investigated. Thus, we decided to investigate the incidence of BRAF mutations in gastric cancers, and the relationship between BRAF and K-ras mutations in such cancers. Three non-pathogenic BRAF polymorphisms and seven K-ras missense mutations were found in 66 gastric cancers and 16 gastric cancer cell lines. Although only 9% of our gastric cancer panels had K-ras mutations, the incidence of BRAF mutations was not high. Thus, BRAF mutations, which are present in a variety of other human cancers, do not seem to be involved in gastric cancer development.     

Antioxidant Effect of Phenelzine on MPP+-Induced Cell Viability Loss in Differentiated PC12 Cells

Phenelzine, deprenyl, and antioxidants (SOD, catalase, ascorbate, or rutin) reduced the loss of cell viability in differentiated PC12 cells treated with 250 M MPP⁺, whereas N-acetylcysteine and dithiothreitol did not inhibit cell death. Phenelzine reduced the condensation and fragmentation of nuclei caused by MPP⁺ in PC12 cells. Phenelzine and deprenyl prevented the MPP⁺-induced decrease in mitochondrial membrane potential, cytochrome c release, formation of reactive oxygen species, and depletion of GSH in PC12 cells. Phenelzine revealed a scavenging action on hydrogen peroxide and reduced the hydrogen peroxide–induced cell death in PC12 cells, whereas deprenyl did not depress the cytotoxic effect of hydrogen peroxide. Both compounds reduced the iron and EDTA-mediated degradation of 2-deoxy-d-ribose degradation. The results suggest that phenelzine attenuates the MPP⁺-induced viability loss in PC12 cells by reducing the alteration of mitochondrial membrane permeability that seems to be mediated by oxidative stress.     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞