Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways

IL-11 inhibits the activation of NF-kappaB and induces the Th2 polarization of CD4+ T cells. The clinical utility of IL-11 is being investigated in Crohn's disease. However, physiological secretion of IL-11 in the intestine remains unclear. In this study, we investigated IL-11 secretion in human intestinal subepithelial myofibroblasts (SEMFs). Intestinal SEMFs were isolated from the human colonic mucosa. IL-11 secretion and mRNA expression were determined by ELISA and Northern blot analysis. The activating protein (AP)-1-DNA binding activity was evaluated by EMSA. IL-11 secretion was induced by IL-1beta and transforming growth factor (TGF)-beta1. These were also observed at the mRNA level. The EMSAs demonstrated that both IL-1beta and TGF-beta1 induced AP-1 activation within 2 h after stimulation, and a blockade of AP-1 activation by the recombinant adenovirus containing a dominant negative c-Jun markedly reduced the IL-1beta- and TGF-beta1-induced IL-11 mRNA expression. IL-1beta and TGF-beta1 induced an activation of ERK p42/44 and p38 MAP kinases, and the MAP kinase inhibitors (SB-202190, PD-98059, and U-0216) significantly reduced the IL-1beta- and TGF-beta1-induced IL-11 secretion. The upregulation of IL-11 mRNA by IL-1beta- and TGF-beta1 was also mediated by a p38 MAP kinase-mediated mRNA stabilization. The combination of IL-1beta and TGF-beta1 additively enhanced IL-11 secretion. Intestinal SEMFs secreted IL-11 in response to IL-1beta- and TGF-beta1. Mucosal IL-11 secretion might be important as an anti-inflammatory response in the pathogenesis of intestinal inflammation.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).     

Histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow (Capricornis crispus, Artiodactyla, Mammalia) with special reference to glandular structures

The histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow ( Capricornis crispus ) was studied by light microscopic histochemical methods, particularly lectin histochemistry. The eccrine glands present exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine, alpha-D-galactose and N-acetyl-neuraminic acid) especially in the cells of the secretory acini, the free surface of the collecting duct cells also showed distinct positive reactions with most of the histochemical methods. The thick epidermis of the nasolabial skin contained smaller amounts of glycoproteins. The results obtained are discussed with regard to possible functions of the glandular secretions. This substance mixture may particularly improve water retention on the skin surface, and protect against physical damage as well as microbial contamination. There seem to be no basic differences of muzzle functions between wild and domesticated bovine species.     

Cerebrosides in grapevine leaves: distinct composition of sphingoid bases among the grapevine species having different tolerances to freezing temperature

Cerebrosides from leaves of three grapevine species were analyzed in detail. The relative proportions of 8-E/Z isomers of 4-hydroxy-8-sphingenines [i.e. 8-E/Z t18:1(8E) and (8Z)] differed amongst the species in respect to freezing tolerance. This suggests that the occurrence of high levels of t18:1(8Z) in cerebrosides is correlated with freezing tolerance in these species.     

Induction of permanent mixed chimerism and skin allograft tolerance across fully MHC-mismatched barriers by the additional myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers in mice has been established. The components of the method are i.v. administration of 1 x 108 allogeneic spleen cells on day 0, i.p. injection of 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell-depleted 1 x 107 bone marrow cells from the same donor on day 3. Recipient B10 (H-2b; IE-) mice prepared with this conditioning developed donor-specific tolerance, and long-lasting survival of skin allografts was shown in almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive Vbeta11+ T cells were reduced in periphery as seen in untreated B10.D2 (H-2d; IE+) mice. The specific tolerant state was confirmed by the specific abrogation against donor Ag in the assays of CTL activity and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophosphamide-induced tolerance, which have been reported by us since 1984, can be overcome by the additional treatments with the myelosuppressive drug busulfan, followed by 1 x 107 T cell-depleted bone marrow cells. To our knowledge, this is the first report to induce allograft tolerance with a short course of the Ag plus immunosuppressive drug treatment without any kind of mAbs (pure drug-induced tolerance).     

Alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe operates both in nucleus and in mitochondria

The fission yeast, Schizosaccharomyces pombe, possesses a UV-damaged DNA endonuclease-dependent excision repair (UVER) pathway in addition to nucleotide excision repair pathway for UV-induced DNA damage. We examined cyclobutane pyrimidine dimer removal from the myo2 locus on the nuclear genome and the coI locus on the mitochondrial genome by the two repair pathways. While nucleotide excision repair repairs damage only on the nuclear genome, UVER efficiently removes cyclobutane pyrimidine dimers on both nuclear and mitochondrial genomes. The ectopically expressed wild type UV-damaged DNA endonuclease was localized to both nucleus and mitochondria, while modifications of N-terminal methionine codons restricted its localization to either of two organelles, suggesting an alternative usage of multiple translation initiation sites for targeting the protein to different organelles. By introducing the same mutations into the chromosomal copy of the uvde(+) gene, we selectively inactivated UVER in either the nucleus or the mitochondria. The results of UV survival experiments indicate that although UVER efficiently removes damage on the mitochondrial genome, UVER in the mitochondria hardly contributes to UV resistance of S. pombe cells. We suggest a possible UVER function in mitochondria as a backup system for other UV damage tolerance mechanisms.     

PEBP2alphaA/CBFA1 mutations in Japanese cleidocranial dysplasia patients

Cleidocranial dysplasia (CCD) is an autosomal dominant human bone disease whose genetic locus has been located on chromosome 6p21, where the PEBP2alphaA/CBFA1 gene essential for osteogenesis also maps. Previously, several heterozygous mutations in PEBP2alphaA/CBFA1 were found in CCD patients. In this study, we identified six different types of mutations in PEBP2alphaA/CBFA1 in Japanese CCD patients. Four cases were similar to those reported previously: two were nonsense mutations in the Runt domain, one was a hemizygous deletion, and the other was a missense mutation in the Runt domain which abolished the DNA-binding activity of Runx2/PEBP2alphaA/CBFA1. The remaining two mutations were novel: one had a heterozygous gt-to-tt mutation at the splice donor site (gt) between the exon3-intron junction, which resulted in abnormal exon3 skipping, and the other had a mutation in exon7, which led to the introduction of a translational stop codon in the middle of the transactivation domain. Thus, defects in either the DNA-binding domain or transactivation domain of Runx2/PEBP2alphaA/CBFA1 can cause CCD. The results not only provide a strong genetic evidence that mutations involving in PEBP2alphaA/CBFA1 contribute to CCD, but also provide a useful tool to study how Runx2/PEBP2alphaA/CBFA1 plays its pivotal role during osteoblastic differentiation.     

Metallokinetic analysis of disposition of vanadyl complexes as insulin-mimetics in rats using BCM-ESR method

Among vanadium's wide variety of biological functions, its insulin-mimetic effect is the most interesting and important. Recently, the vanadyl ion (+4 oxidation state of vanadium) and its complexes have been shown to normalize the blood glucose levels of streptozotocin-induced diabetic rats (STZ-rats). During our investigations to find more effective and less toxic vanadyl complexes, the vanadyl-methylpicolinate complex (VO-MPA) was found to exhibit higher insulin-mimetic activity and less toxicity than other complexes, as evaluated by both in vitro and in vivo experiments. Electron spin resonance (ESR) is capable of measuring the paramagnetic species in biological samples. We have developed the in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method to analyze the ESR signals due to stable organic radicals in real time. In the present investigation, we have applied this method to elucidate the relationship between the blood glucose normalizing effect of VO-MPA and the global disposition of paramagnetic vanadyl species. This paper describes the results of vanadyl species in the circulating blood of rats following intravenous administration of vanadyl compounds. ESR spectra due to the presence of vanadyl species were obtained in the circulating blood, and their pharmacokinetic parameters were estimated using compartment models. The results indicate that vanadyl species are distributed considerably to the peripheral tissues, as estimated by BCM-ESR, and eliminated from the body through the urine, as estimated by ESR at 77 K. The exposure of vanadyl species in the blood was found to be enhanced by VO-MPA treatment. Given these results, we concluded that the pharmacokinetic character of vanadyl species is closely related with the structure and antidiabetic activity of the vanadyl compounds.