Phenolics with PPAR-gamma ligand-binding activity obtained from licorice (Glycyrrhiza uralensis roots) and ameliorative effects of glycyrin on genetically diabetic KK-A(y) mice

The EtOAc extract of licorice (Glycyrrhiza uralensis roots) exhibited considerable PPAR-gamma ligand-binding activity. Bioassay-guided fractionation of the extract using a GAL-4-PPAR-gamma chimera assay method resulted in the isolation of two isoflavenes, one of which is a new compound named dehydroglyasperin D, an isoflavan, two 3-arylcoumarins, and an isoflavanone as the PPAR-gamma ligand-binding active ingredients of licorice. The isoprenyl group at C-6 and the C-2' hydroxyl group in the aromatic ring-C part in the isoflavan, isoflavene, or arylcoumarin skeleton were found to be the structural requirements for PPAR-gamma ligand-binding activity. Glycyrin, one of the main PPAR-gamma ligands of licorice, significantly decreased the blood glucose levels of genetically diabetic KK-A(y) mice.     

Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae

The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprosequence was analyzed by an expression system using Saccharomyces cerevisiae. When the mature portion of ROL (mROL) fused to the pre-alpha-factor leader sequence was expressed, secretion of active mROL was not observed. However, when mROL was synthesized together with the prosequence in trans (individually and coincidentally), secretion of active mROL was observed. The results indicate that the prosequence of ROL helped correct folding of mROL and its subsequent secretion from the yeast cells, and that physical linkage (cis) of the prosequence to the mature region was not prerequisite. From the expression of the ROL mutants with deletions at the N-terminal end of the prosequence together with mROL in trans, the residues from 20 to 37 in the prosequence were essential for the secretion, and those from 38 to 57 were essential for the formation of the active ROL and might play a role as an intramolecular chaperone. The results using the fragment of the prosequence confirmed that these residues (20-57) were significant for in vivo folding and secretion of active mROL.     

Expression of placental lactogen and cytokeratin in bovine placental binucleate cells in culture

Binucleate cells are present in ruminant placenta and play an endocrine role in the production of many hormones during pregnancy. We isolated and cultured binucleate cells from bovine placenta at middle to late gestation and characterized these cells using immunofluorescence techniques. Enriched preparations of binucleate cells were obtained using Percoll density gradient centrifugation following collagenase digestion. Binucleate cells in culture preferentially attached to collagen-coated dishes rather than to noncoated plastic dishes. The cells gradually extended their edges on collagen substrata, and finally assumed a flattened morphology. Antibodies to placental lactogen (PL) and pregnancy-associated glycoprotein-1 (PAG-1) specifically stained the majority of round binucleate cells, but not the flat cells. We found that PL-positive binucleate cells were consistently devoid of cytokeratin. In contrast, cytokeratin was expressed in PL-negative binucleate cells as well as mononuclear epithelial cells. Furthermore, the PL-negative flat binucleate cells also developed intense cytokeratin networks in the cytoplasm. These results indicate that cytokeratin expression is inversely proportionate to that of PL in cultured binucleate cells. We conclude that downregulation of cytokeratin in binucleate cells is a function of the state of cellular differentiation.     

Neonatal presentation of adult-onset type II citrullinemia

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific argininosuccinate synthetase deficiency caused by a deficiency of the citrin protein encoded by the SLC25A13 gene. Until now, however, no SLC25A13 mutations have been reported in children with liver diseases. We described three infants who presented as neonates with intrahepatic cholestasis associated with hypermethioninemia or hypergalactosemia detected by neonatal mass screening. DNA analyses of SLC25A13 revealed that one patient was a compound heterozygote for the 851de14 and IVS11+IG-->A mutations and two patients (siblings) were homozygotes for the IVS11+lG-->A mutation. These results suggested that there may be a variety of liver diseases related to CTLN2 in children.     

TNF-alpha induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes

The activation of phosphatidylinositol (PI) 3-kinase and Akt/protein kinase B (PKB) by tumor necrosis factor (TNF)-alpha and their roles on stimulation of protein synthesis were investigated in cultured neonatal rat cardiac myocytes. Treatment of cells with TNF-alpha resulted in enlargement of cell surface area and stimulation of protein synthesis without affecting myocyte viability. TNF-alpha induced marked activation of PI3-kinase and Akt/PKB, and the activation of PI3-kinase and Akt/PKB was rapid (maximal at 10 and 15 min, respectively) and concentration dependent. Akt/PKB activation by TNF-alpha was inhibited by a PI3-kinase-specific inhibitor LY-294002 and adenovirus-mediated expression of a dominant negative mutant of PI3-kinase, indicating that TNF-alpha activates Akt/PKB through PI3-kinase activation. Furthermore, TNF-alpha-induced protein synthesis was inhibited by pretreatment with LY-294002 and expression of a dominant negative mutant of PI3-kinase or Akt/PKB. These results indicate that activation of the PI3-kinase-Akt/PKB pathway plays an essential role in protein synthesis induced by TNF-alpha in cardiac myocytes.     

Role of the furrow of the proximal colon in the production of soft and hard feces in nutrias, Myocastor coypus

The bacterial level of soft feces is higher than that of hard feces in nutrias. This suggests the heterogeneity of bacterial density in the large intestine. To show the heterogeneity of bacteria in the contents of the large intestine in nutrias, we divided the contents of the large intestine into 12 regions, then measured the nitrogen (N), total amino acids (TAA) and diaminopimelic acid (DAP), a bacterial marker, of these regions. Levels of N, TAA and DAP varied along the cross section of the proximal colon. The greater curvature of the main lumen and furrow had higher N, TAA and DAP concentrations than the lesser curvature. We also examined the involvement of the furrow in producing two types of feces differing in bacterial nitrogen content by surgically preventing the flow of the furrow contents. We compared the concentrations of N, TAA and DAP between soft and hard feces among operated, sham-operated and intact animals. Surgical closure of the furrow abolished the difference in levels of N, TAA and DAP between soft and hard feces, suggesting that the furrow of the proximal colon is responsible for making the bacterial density higher in soft feces than in hard feces. Accepted: 13 July 2000     

Production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1, by cultured human adrenocortical carcinoma cells

The production and secretion of peptides by adrenocortical tumors have not been well studied. We therefore studied the production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1 in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreactive-adrenomedullin and immunoreactive-endothelin were detected in the culture medium of SW-13 cells (27.7 +/- 1.6 fmol/10 (5) cells/24 h and 11.0 +/- 0.8 fmol/10 (5) cells/24 h, respectively, mean +/- SEM, n = 6). Northern blot analysis showed the expression of adrenomedullin mRNA and endothelin-1 mRNA in SW-13 cells. On the other hand, no significant amount of calcitonin gene-related peptide, corticotropin-releasing hormone, neuropeptide Y, or urocortin was secreted by SW-13 cells. Treatment with ACTH (10(-9)-10(-7) mol/l), angiotensin II (10(-9)-10(-7) mol/l), or dexamethasone (10(-8)-10(-6) mol/l) for 24 h had no significant effects on immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium of SW-13. Treatment with tumor necrosis factor (TNF)-alpha (20 ng/ml) increased significantly both immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium. Interferon-gamma (100 U/ml) increased the immunoreactive-endothelin levels, but not immunoreactive-adrenomedullin levels, whereas interleukin-1 (IL-1)beta (10 ng/ml) increased immunoreactive-adrenomedullin levels, but not immunoreactive-endothelin levels. These findings indicate that SW-13 human adrenocortical carcinoma cells produce and secrete two vasoactive peptides, adrenomedullin, and endothelin-1 and that the secretion of these two peptides is modulated differently by cytokines.     

ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase

Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis: ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase. Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon-induced allele suggests a non-cell-autonomous function for the ACL5 gene product. We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock-dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis: We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.     

Identification and characterization of TMEFF2, a novel survival factor for hippocampal and mesencephalic neurons

We have identified a novel mammalian gene, TMEFF2, that encodes a putative transmembrane protein containing two follistatin-like domains and one epidermal growth factor (EGF)-like domain. The TMEFF2 gene is predominantly expressed in the brain. In situ hybridization analysis revealed that TMEFF2 is widely expressed in the brain, including hippocampal cornu ammonis, dentate gyrus, and substantia nigra pars compacta. We evaluated the survival effect of TMEFF2 using primary cultured neurons from several regions of fetal rat brain following treatment with a recombinant TMEFF2 protein fragment consisting of the putative extracellular domain. TMEFF2 increased survival of neurons from the hippocampus and midbrain, but not from the cerebral cortex, indicating that the survival effects of TMEFF2 are specific to certain cell types. Recombinant TMEFF2 also promoted survival of mesencephalic dopaminergic neurons. Together, these findings suggest that TMEFF2 may be a novel survival factor for hippocampal and mesencephalic, but not for cortical, neurons.