Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.     

Novel and orally bioavailable inducible nitric oxide synthase inhibitors: synthesis and evaluation of optically active 4,5-dialkyl-2-iminoselenazolidine derivatives

We have previously reported that (4R,5R)-5-ethyl-2-imino-4-methylthiazolidine (3) strongly inhibits inducible nitric oxide synthase (iNOS). In a successive search for strong and selective iNOS inhibitors, we, herein, describe the synthesis of the selenium analogue of 3 (4: ES-2133) and its related optically active compounds and examine their in vitro and in vivo inhibitory activity against iNOS. In addition, an alternative synthetic method to the selected compound 4 and its pharmacokinetic profile is also reported.     

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.     

Nonuniform concerted evolution and chloroplast capture: heterogeneity of observed introgression patterns in three molecular data partition phylogenies of Asian Mitella (saxifragaceae)

Interspecific hybridization is one of the major factors leading to phylogenetic incongruence among loci, but the knowledge is still limited about the potential of each locus to introgress between species. By directly sequencing three DNA regions: chloroplast DNAs (matK gene and trnL-F noncoding region), the nuclear ribosomal external transcribed spacer (ETS) region, and internal transcribed spacer (ITS) regions, we construct three phylogenetic trees of Asian species of Mitella (Saxifragaceae), a genus of perennials in which natural hybrids are commonly observed. Within this genus, there is a significant topological conflict between chloroplast and nuclear phylogenies and also between the ETS and the ITS, which can be attributed to frequent hybridization within the lineage. Chloroplast DNAs show the most extensive introgression pattern, ITS regions show a moderate pattern, and the ETS region shows no evidence of introgression. Nonuniform concerted evolution best explains the difference in the introgression patterns between the ETS region and ITS regions, as the sequence heterogeneity of the ITS region within an individual genome is estimated to be twice that of an ETS in this lineage. Significant gene conversion patterns between two hybridizing taxa were observed in contiguous arrays of cloned ETS-ITS sequences, further confirming that only ITS regions have introgressed bidirectionally. The relatively slow concerted evolution in the ITS regions probably allows the coexistence of multiple alleles within a genome, whereas the strong concerted evolution in the ETS region rapidly eliminates heterogeneous alleles derived from other species, resulting in species delimitations highly concordant with those based on morphology. This finding indicates that the use of multiple molecular tools has the potential to reveal detailed organismal evolution processes involving interspecific hybridization, as an individual locus varies greatly in its potential to introgress between species.     

Precursor structure of egg proteins in the coral Galaxea fascicularis

In the egg of the reef coral Galaxea fascicularis, four proteins (named GfEP-1 to -4) are stored in high abundance. In the present study, a cDNA containing a full-length open reading frame for GfEP-1 was cloned, and the translated protein sequence was compared to the N-terminal sequences of GfEP-2, -3, and -4. GfEP-1 and -2 were shown to be generated by processing of a precursor of 1439 amino acids, and GfEP-3 turned out to be a partial fragment of GfEP-2. The precursor protein contained regions which exhibited similarities to vitellogenins (Vgs) in bilaterian animals (oviparous vertebrates and invertebrates including nematodes, arthropods, and molluscs). This study reports the first cloning and characterization of a full-length cDNA encoding a Vg in a non-bilaterian animal, and argues that the emergence of Vg as a precursor of egg yolk proteins predated the divergence of the cnidarian and bilaterian lineages.     

Activation of Notch1 signaling in cardiogenic mesoderm induces abnormal heart morphogenesis in mouse

Notch signaling is implicated in many developmental processes. In our current study, we have employed a transgenic strategy to investigate the role of Notch signaling during cardiac development in the mouse. Cre recombinase-mediated Notch1 (NICD1) activation in the mesodermal cell lineage leads to abnormal heart morphogenesis, which is characterized by deformities of the ventricles and atrioventricular (AV) canal. The major defects observed include impaired ventricular myocardial differentiation, the ectopic appearance of cell masses in the AV cushion, the right-shifted interventricular septum (IVS) and impaired myocardium of the AV canal. However, the fates of the endocardium and myocardium were not disrupted in NICD1-activated hearts. One of the Notch target genes, Hesr1, was found to be strongly induced in both the ventricle and the AV canal of NICD1-activated hearts. However, a knockout of the Hesr1 gene from NICD-activated hearts rescues only the abnormality of the AV myocardium. We searched for additional possible targets of NICD1 activation by GeneChip analysis and found that Wnt2, Bmp6, jagged 1 and Tnni2 are strongly upregulated in NICD1-activated hearts, and that the activation of these genes was also observed in the absence of Hesr1. Our present study thus indicates that the Notch1 signaling pathway plays a suppressive role both in AV myocardial differentiation and the maturation of the ventricular myocardium.     

Type I IFN-mediated enhancement of anti-leukemic cytotoxicity of gammadelta T cells expanded from peripheral blood cells by stimulation with zoledronate

BACKGROUND: In order to establish efficient gammadelta T-cell based tumor immunotherapy, we explored a method to enhance the cytotoxicity of gammadelta T cells against leukemia cells by stimulating gammadelta T cells with type I IFN. METHODS: Gammadelta T cells were expanded from normal PBMC by culturing with zoledronate and a low concentration of IL-2 for 2 weeks. For the activation of gammadelta T cells, gammadelta T cells were cultured with type I IFN (HLBI, IFN-alpha2b and IFN-beta) for 1-3 days. The cytotoxicity of HLBI-activated gammadelta T cells against leukemia cell lines and fresh leukemia cells was evaluated by 51Cr-release assay. RESULTS: Gammadelta T cells, which were expanded and purified with magnetic beads using an anti-gammadelta TCR MAb, were demonstrated to be cytotoxic against leukemia cell lines of both lymphoid and myeloid origin and fresh myeloid leukemia cells. By culturing expanded gammadelta T cells with type I IFN, the expression of the activation marker CD69 was increased and the cytometric bead array showed an elevated production of IFN-gamma by gammadelta T cells. In addition, the cytotoxicity of gammadelta T cells against leukemia cells was definitely enhanced by culturing gammadelta T cells with HLBI. DISCUSSION: The present study has demonstrated that type I IFN could enhance the anti-leukemic cytotoxicity of expanded gammadelta T cells, which implies that in vitro bisphosphonate (such as zoledronate)-expanded and type I IFN-activated gammadelta T cells could be applied to immunotherapy for hematologic malignancies such as leukemia and lymphoma.     

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.