The Combination of Cholecystokinin and Stress Amplifies an Inhibition of Appetite,Gastric Emptying,and an Increase in c-Fos Expression in Neurons of the Hypothalamus and the Medulla Oblongata

Cholecystokinin (CCK) had been the first gastrointestinal hormone known to exert anorexic effects. CCK had been inferred to contribute to the onset of functional dyspepsia (FD) symptoms. To understand the pathophysiology of FD, the roles of stress have to be clarified. In this study, we aimed to clarify the influence of stress on the action of cholecystokinin (CCK) on appetite and gastric emptying. Using rats, stress was simulated by giving restraint stress or intraperitoneal injection of the stress-related peptide hormone urocortin 1 (UCN1). The effects of CCK and restraint stress, alone or in combination, on food intake and gastric motility were examined, and c-Fos expression in the neurons of appetite control network in the central nervous system was assessed by immunohistochemical staining. CCK inhibited food intake and gastric emptying in a dose-dependent manner. Food intake for 1 h was significantly lower with UCN1 (2 nmol/kg) than with the saline control. Restraint stress amplified the suppressive effects of CCK on food intake for 1 h and on gastric emptying. With regard to brain function, the CCK induced c-Fos expression in the neurons of the nucleus tractus solitarius and paraventricular nucleus of the hypothalamus was markedly and significantly amplified by the addition of restraint stress with CCK. The results suggested that stress might amplify the anorexic effects of CCK through activation of the nuclei that comprise the brain neuronal network for satiation; this might play a role in the pathogenesis of the postprandial distress syndromes of functional dyspepsia.

     

Production of haploids and doubled haploids from unfertilized ovule culture of various wild species of gentians (Gentiana spp.)

The production of haploids and doubled haploids (DHs) on unfertilized ovule culture was examined in 19 wild species of gentians (Gentiana ssp.) classified into eight sections. Of the 19 species including 35 strains, embryo-like structures (ELSs) were obtained from unfertilized ovule culture in 15 species, and regenerated plants were produced in 11 species. ELS production has varied greatly among the15 species, i.e., 0.5–79.2% frequency of responding flower buds and 0.01–1.99 ELSs per flower bud. Of the ELS-producing species, almost all were classified into the sects. Pneumonanthe or Cruciata. Species in sect. Pneumonanthe showed higher responses than those in sect. Cruciata. In examining the effect of flower bud stage on ELS production, more than twice as many ELSs were observed at the anther-dehiscent stage than that at the anther-indehiscent stage. Ploidy level was determined in 117 randomly selected regenerated plantlets, which suggests that most were diploid (46.2%) and haploid (32.5%). When 12 diploid plants were examined using simple sequence repeat (SSR) markers, 8 (66.7%) were DH. This study revealed that unfertilized ovule culture can be applied not only on cultivated gentian species but also on a number of wild species. The production of haploids and DHs in wild gentians provides novel prospects for ornamental and/or medicinal gentian breeding.

     

Structure of RecJ Exonuclease Defines Its Specificity for Single-stranded DNA

RecJ is a single-stranded DNA (ssDNA)-specific 5′-3′ exonuclease that plays an important role in DNA repair and recombination. To elucidate how RecJ achieves its high specificity for ssDNA, we determined the entire structures of RecJ both in a ligand-free form and in a complex with Mn²⁺ or Mg²⁺ by x-ray crystallography. The entire RecJ consists of four domains that form a molecule with an O-like structure. One of two newly identified domains had structural similarities to an oligonucleotide/oligosaccharide-binding (OB) fold. The OB fold domain alone could bind to DNA, indicating that this domain is a novel member of the OB fold superfamily. The truncated RecJ containing only the core domain exhibited much lower affinity for the ssDNA substrate compared with intact RecJ. These results support the hypothesis that these structural features allow specific binding of RecJ to ssDNA. In addition, the structure of the RecJ-Mn²⁺ complex suggests that the hydrolysis reaction catalyzed by RecJ proceeds through a two-metal ion mechanism.     

Interleukin-1beta induces sialyl Lewis X on hepatocellular carcinoma HuH-7 cells via enhanced expression of ST3Gal IV and FUT VI gene

We previously demonstrated that human hepatocellular carcinoma-derived HuH-7 cells stimulated with interleukin-1beta (IL-1beta) produce alpha(1)-acid glycoprotein (AGP) with increased amounts of sialyl Lewis X (sLeX) antigen, although the mechanism remained obscure. Here, we report our investigation of the mechanism. sLeX expression on HuH-7 cells was induced 2.5 times more after 48 h stimulation with 100 U/mL IL-1 beta compared with control, as indicated by anti-sLeX antibody binding. Furthermore, expression of 2,3-sialylated N-acetyllactosamine increased gradually up to 48 h after IL-1 beta stimulation; this preceded the increase in sLeX expression. Increases in alpha 2,3-sialyltransferase activity also preceded increases in alpha1,3-fucosyltransferase activity. Furthermore, mRNA levels of ST3Gal IV, FUT IV and VI in HuH-7 cells stimulated with IL- 1beta were increased at 2-4 h, while increases in FUT VI mRNA level occurred gradually after 24 h. IL-1 beta-induced sLeX expression on HuH-7 cells was suppressed by transfection of gene-specific small interference RNAs against FUT VI and ST3Gal IV but not against FUT IV and ST3Gal III. These data results that IL-1 beta induces expression of sLeX on HuH-7 cells by enhanced expression of FUT VI and ST3Gal IV gene.     

Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.     

Transglutaminase-mediated internal protein labeling with a designed peptide loop

Post-translational internal protein labeling was explored through the insertion of a 13-mer peptidyl loop specifically recognized by microbial transglutaminase (MTG). The peptidyl loop included one lysine residue (abbreviated as the K-loop), and was designed and inserted into two different regions of the protein bacterial alkaline phosphatase (BAP). MTG-mediated selective labeling of a lysine residue in the K-loop was achieved with a functional Gln-donor substrate. Internal protein labeling in the vicinity of the active site of BAP (residues 91–93) markedly decreased the activity of the enzyme. Conversely, insertion of the K-loop at a site distal from the active site (residues 219–221) afforded site-specific and covalent internal protein labeling without impairing the activity of the enzyme.