Pluripotency and nuclear reprogramming

Embryonic stem cells are promising donor cell sources for cell transplantation therapy, which may in the future be used to treat various diseases and injuries. However, as is the case for organ transplantation, immune rejection after transplantation is a potential problem with this type of therapy. Moreover, the use of human embryos presents serious ethical difficulties. These issues may be overcome if pluripotent stem cells are generated from patients' somatic cells. Here, we review the molecular mechanisms underlying pluripotency and the currently known methods of inducing pluripotency in somatic cells.     

Role of Tyr residues on the protein surface of cationic cell-wall-peroxidase (CWPO-C) from poplar: potential oxidation sites for oxidative polymerization of lignin

It was previously reported that an unique peroxidase isoenzyme, cationic cell-wall-bound peroxidase (CWPO-C), from poplar callus oxidizes sinapyl alcohol, ferrocytochrome c and synthetic lignin polymers, unlike other plant peroxidases. Here, the catalytic mechanism of CWPO-C was investigated using chemical modification and homology modeling. The simulated CWPO-C structure predicts that the entrance to the heme pocket of CWPO-C is the same size as those of other plant peroxidases, suggesting that ferrocytochrome c and synthetic lignin polymers cannot interact with the heme of CWPO-C. Since Trp and Tyr residues are redox-active, such residues located on the protein surface were predicted to be active sites for CWPO-C. Modification of CWPO-C Trp residues did not suppress its oxidation activities toward guaiacol and syringaldazine. On the other hand, modification of CWPO-C Tyr residues using tetranitromethane strongly suppressed its oxidation activities toward syringaldazine and 2,6-dimethoxyphenol by 90%, respectively, and also suppressed its guaiacol oxidation activity to a lesser extent. Ferrocytochrome c was not oxidized by Tyr-modified CWPO-C. These results indicate that the Tyr residues in CWPO-C mediate its oxidation of syringyl compounds and high-molecular-weight substrates. Homology modeling indicates that Tyr-177 and Tyr-74 are located near the heme and exposed on the protein surface of CWPO-C. These results suggest that Tyr residues on the protein surface are considered to be important for the oxidation activities of CWPO-C with a wide range of substrates, and potentially unique oxidation sites for the plant peroxidase family.     

Absence of Ku70 gene obliterates X-ray-induced lacZ mutagenesis of small deletions in mouse tissues

With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.     

Invention and modification of a new tool use behavior: ant-fishing in trees by a wild chimpanzee (Pan troglodytes verus) at Bossou, Guinea

Wild chimpanzees are known to have a different repertoire of tool use unique to each community. For example, "ant-dipping" is a tool use behavior known in several chimpanzee communities across Africa targeted at driver ants (Dorylus spp.) on the ground, whereas "ant-fishing," which is aimed at carpenter ants (Camponotus spp.) in trees, has primarily been observed among the chimpanzees of Mahale in Tanzania. Although the evidence for differences between field sites is accumulating, we have little knowledge on how these tool use behaviors appear at each site and on how these are modified over time. This study reports two"ant-fishing" sessions which occurred 2 years apart by a young male chimpanzee at Bossou, Guinea. Ant-fishing had never been observed before in this community over the past 27 years. During the first session, at the age of 5, he employed wands of similar length when ant-fishing in trees to those used for ant-dipping on the ground, which is a customary tool use behavior of this community. Two years later, at the age of 7, his tools for ant-fishing were shorter and more suitable for capturing carpenter ants. This observation is a rare example of innovation in the wild and does provide insights into problem-solving and learning processes in chimpanzees.     

Mutational analysis of predicted intracellular loop domains of human motilin receptor

Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin- and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63-66, 135-137, and 296-301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.     

Establishment of canine RNA polymerase I-driven reverse genetics for influenza A virus: its application for H5N1 vaccine production

In the event of a new influenza pandemic, vaccines whose antigenicities match those of circulating strains must be rapidly produced. Here, we established an alternative reverse genetics system for influenza virus using the canine polymerase I (PolI) promoter sequence that works efficiently in the Madin-Darby canine kidney cell line, a cell line approved for human vaccine production. Using this system, we were able to generate H5N1 vaccine seed viruses more efficiently than can be achieved with the current system that uses the human PolI promoter in African green monkey Vero cells, thus improving pandemic vaccine production.     

Developmental expression of GLUT2 in the rat retina

We previously demonstrated that GLUT2, a facilitated-diffusion glucose transporter isoform known to play critical roles in the regulation of systemic blood glucose level, is present at the apical ends of Müller cells in the rat retina. As a means of elucidating the ontogeny and possible role(s) of GLUT2 in the developing retina, this study examined its expression at various stages of retinal development by immunofluorescence staining using GLUT2-specific antibody. Evidence of GLUT2 expression first appeared at embryonic day 14 (E14) as linear staining along the boundary between the inner and outer layers of the optic cup, with this staining pattern being present throughout subsequent embryonic and neonatal stages. After the development of photoreceptor cell inner and outer segments (i.e., photoreceptor layer), GLUT2 immunoreactivity was localized along the boundary between the outer nuclear layer and photoreceptor layer. Localization of GLUT2 expression and the timing of its appearance, which coincided with the formation of choriocapillaries, together suggest that GLUT2 is involved in the anterior transport of glucose supplied by choroidal circulation from the early stages of retinal development.     

Inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma

Although matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components in vitro, the role of MMPs in the in vivo accumulation of the cells to the site of inflammation in bronchial asthma is still obscure. In this study, we investigated the role of MMPs in the pathogenesis of bronchial asthma, using a murine model of allergic asthma. In this model, we observed the increase of the release of MMP-2 and MMP-9 in bronchoalveolar lavage fluids after Ag inhalation in the mice sensitized with OVA, which was accompanied by the infiltration of lymphocytes and eosinophils. Administration of tissue inhibitor of metalloproteinase-2 to airways inhibited the Ag-induced infiltration of lymphocytes and eosinophils to airway wall and lumen, reduced Ag-induced airway hyperresponsiveness, and increased the numbers of eosinophils and lymphocytes in peripheral blood. The inhibition of cellular infiltration to airway lumen was observed also with tissue inhibitor of metalloproteinase-1 and a synthetic matrix metalloproteinase inhibitor. These data suggest that MMPs, especially MMP-2 and MMP-9, are crucial for the infiltration of inflammatory cells and the induction of airway hyperresponsiveness, which are pathophysiologic features of bronchial asthma, and further raise the possibility of the inhibition of MMPs as a therapeutic strategy of bronchial asthma.     

Protective effects of steroid saponins from Paris polyphylla var. yunnanensis on ethanol- or indomethacin-induced gastric mucosal lesions in rats: structural requirement for activity and mode of action

The methanolic extract from the rhizomes of Paris polyphylla SM. var. yunnanensis (FR.) H-M. was found to potently inhibit ethanol-induced gastric lesions in rats. Through bioassay-guided separation, four known spirostanol-type steroid saponins, pennogenin 3-O-alpha-L-rhamnopyranosyl(1-->2)-[alpha-L-arabinofuranosyl(1-->4)]-beta-D-glucopyranoside (1), pennogenin 3-O-alpha-L-rhamnopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (2), diosgenin 3-O-alpha-L-rhamnopyranosyl(1-->2)-[alpha-L-arabinofuranosyl(1-->4)]-beta-D-glucopyranoside (3), and diosgenin 3-O-alpha-L-rhamnopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (4), and a new furostanol-type steroid saponin, parisaponin I (5), together with two known furostanol-type steroid saponins, trigofoenoside A (6) and protogracillin (7), were isolated from the active fraction. Compounds 1-4 (1.25-10 mg/kg, po) strongly inhibited gastric lesions induced by ethanol and indomethacin. With regard to structural requirement of steroid saponins, the 3-O-glycoside moiety and spirostanol structure were found to be essential for the activity and the 17-hydroxyl group in the aglycon part enhanced the protective effects against ethanol-induced gastric lesions. The protective effects of 1 and 3 against ethanol-induced gastric lesions were attenuated by pretreatment with indomethacin and N-ethylmaleimide. Compounds 1 and 3 weakly inhibited acid secretions in pylorus-ligated rats. These findings suggested that endogenous prostaglandins and sulfhydryl compounds were involved in the protective activity.