VEGFR1-Positive Macrophages Facilitate Liver Repair and Sinusoidal Reconstruction after Hepatic Ischemia/Reperfusion Injury

Liver repair after acute liver injury is characterized by hepatocyte proliferation, removal of necrotic tissue, and restoration of hepatocellular and hepatic microvascular architecture. Macrophage recruitment is essential for liver tissue repair and recovery from injury; however, the underlying mechanisms are unclear. Signaling through vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to play a role in macrophage migration and angiogenesis. The aim of the present study was to examine the role of VEGFR1 in liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion (I/R). VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK^-/- mice) and wild-type (WT) mice were subjected to hepatic warm I/R, and the processes of liver repair and sinusoidal reconstruction were examined. Compared with WT mice, VEGFR1 TK^-/- mice exhibited delayed liver repair after hepatic I/R. VEGFR1-expressing macrophages recruited to the injured liver showed reduced expression of epidermal growth factor (EGF). VEGFR1 TK^-/- mice also showed evidence of sustained sinusoidal functional and structural damage, and reduced expression of pro-angiogenic factors. Treatment of VEGFR1 TK^-/- mice with EGF attenuated hepatoceullar and sinusoidal injury during hepatic I/R. VEGFR1 TK^-/- bone marrow (BM) chimeric mice showed impaired liver repair and sinusoidal reconstruction, and reduced recruitment of VEGFR1-expressing macrophages to the injured liver. VEGFR1-macrophages recruited to the liver during hepatic I/R contribute to liver repair and sinusoidal reconstruction. VEGFR1 activation is a potential therapeutic strategy for promoting liver repair and sinusoidal restoration after acute liver injury.     

Rearrangements of the DNA in carbon ion-induced mutants of Arabidopsis thaliana

To elucidate the nature of structural alterations in plants, three carbon ion-induced mutations in Arabidopsis thaliana, gl1-3, tt4(C1), and ttg1-21, were analyzed. The gl1-3 mutation was found to be generated by an inversion of a fragment that contained GL1 and Atpk7 loci on chromosome 3. The size of the inverted fragment was a few hundred kilobase pairs. The inversion was found to accompany an insertion of a 107-bp fragment derived from chromosome 2. The tt4(C1) mutation was also found to be due to an inversion. The size of the intervening region between the breakpoints was also estimated to be a few hundred kilobase pairs. In the case of ttg1-21, it was found that a break occurred at the TTG1 locus on chromosome 5, and reciprocal translocation took place between it and chromosome 3. From the sequences flanking the breakpoints, the DNA strand breaks induced by carbon ions were found to be rejoined using, if present, only short homologous sequences. Small deletions were also observed around the breakpoints. These results suggest that the nonhomologous end-joining (NHEJ) pathway operates after plant cells are exposed to ion particles.     

Expression of the human atypical kinase ADCK3 rescues coenzyme Q biosynthesis and phosphorylation of Coq polypeptides in yeast coq8 mutants

Coenzyme Q (ubiquinone or Q) is a lipid electron and proton carrier in the electron transport chain. In yeast Saccharomyces cerevisiae eleven genes, designated COQ1 through COQ9, YAH1 and ARH1, have been identified as being required for Q biosynthesis. One of these genes, COQ8 (ABC1), encodes an atypical protein kinase, containing six (I, II, III, VIB, VII, and VIII) of the twelve motifs characteristically present in canonical protein kinases. Here we characterize seven distinct Q-less coq8 yeast mutants and show that unlike the coq8 null mutant, each maintained normal steady-state levels of the Coq8 polypeptide. The phosphorylation states of Coq polypeptides were determined with two-dimensional gel analyses. Coq3p, Coq5p, and Coq7p were phosphorylated in a Coq8p-dependent manner. Expression of a human homolog of Coq8p, ADCK3(CABC1) bearing an amino-terminal yeast mitochondrial leader sequence, rescued growth of yeast coq8 mutants on medium containing a nonfermentable carbon source and partially restored biosynthesis of Q(6). The phosphorylation state of several of the yeast Coq polypeptides was also rescued, indicating a profound conservation of yeast Coq8p and human ADCK3 protein kinase function in Q biosynthesis.     

Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1

E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. In this study, we examined the role of SMAP1, a clathrin-interacting GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (Arf6) GTPase, in E-cadherin endocytosis. Mardin-Darby canine kidney (MDCK) epithelial cells were used as a model, and SMAP1 localized in the cytoplasm and along the adherens junction where E-cadherin was present. Next, activity of SMAP1 was compared with that of other Arf6GAPs (and/or an effector of Arf6-GTP), namely GIT1 and AMAP2/DDEF2. Overexpression of SMAP1 but not GIT1 nor AMAP2/DDEF2 strongly inhibited basal, as well as phorbolester-induced, internalization of E-cadherin. Notably, AMAP2/DDEF2 rather enhanced the caveolae-mediated incorporation of a membrane protein other than E-cadherin. Thus, in MDCK cells, E-cadherin appeared to be endocytosed solely through SMAP1-regulated clathrin-coated vesicles. Furthermore, MDCK cells overexpressing SMAP1 showed a reduced degree of cell migration compared to untransfected cells, as assessed by wound healing and Transwell assays, and this reduction in migration appeared to be due to the accumulation of E-cadherin at the adherens junction in cells overexpressing SMAP1. Collectively, SMAP1 likely represents a key Arf6GAP in clathrin dependent endocytosis of E-cadherin in MDCK cells. This activity of SMAP1 in E-cadherin turnover may be involved in epithelial organization and/or epithelial-mesenchymal transition.     

Glyceraldehyde-3-phosphate dehydrogenase of horseshoe crab (Tachypleus tridentatus).

Glyceraldehyde-3-phosphate dehydrogenase [ED 1.2.1.12] was purified from the horseshoe crab, a living fossil, and its properties were examined. 1 The purified enzyme was homogeneous as judged by various tests. The enzyme, like enzymes from other sources, was a tetramer with a subunit molecular weight of 36,000. The kinetic parameters and pH optimum were also similar to those of other enzymes, though the enzyme was more stable against heat and pH denaturations. 2 Analysis of SH groups showed that there were 4 SH groups per subunit, one of which was essential for the enzyme activity and was highly reactive. 3. CD spectra of the enzyme suggested that the enzyme had a very high content of beta-structure (ca. 45 per cent). 4. The horseshoe crab enzyme could form a hybrid in vitro with the rabbit muscle enzymes in concentrated salt solution at acidic pH. 5. There results indicate that the enzyme has overall structural similarity to other enzymes and that the enzyme is highly conserved during a long period of evolution. Some discussions on the structure and activity of the horseshoe crab enzyme are made in comparison with the enzymes from other sources.     

Crystallization and preliminary X-ray analysis of plant class I chitinase from rice

We report here on crystallization and preliminary X-ray analysis of plant class I chitinase from rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained under two independent conditions. The crystals grown under these conditions diffracted up to 2.1 and 2.5 angstroms resolution, respectively, at a synchrotron beamline, and were found to belong to the tetragonal space group P4(3)2(1)2.     

Secretory surface phenomena in freeze-etched preparations of the adenohypophysical cells and neurosecretory fibers

Summary The freeze-etching technique was used in studies of cell surface phenomena during the release of secretory products from the adenohypophysis and from neurosecretory terminals of rats in which exocytosis had been stimulated by the administration of hypothalamic extracts (somatotrophs, thyrotrophs, gonadotrophs and mammotrophs) or severe hemorrhage (neurohypophysis). The observations suggest that secretory granules are extruded through an opening at the tip of a protrusion of the cellular surface. The protrusions seem to result from the abutting of secretory granules on the inner surface of the plasma membrane. These structural details revealed in freeze-etched preparations have not been seen previously in conventional micrographs of ultrathin sections and may provide a clue to the mechanism of secretion.Supported by grants from the Japanese Educational Ministry. — The technical assistance of Mr. Takeshi Fukunishi is acknowledged with gratitude.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.