Acclimation to irradiance of leaf photosynthesis and associated nitrogen reallocation in photosynthetic apparatus in the year following thinning of a young stand of Chamaecyparis obtusa

In order to quantify the effects of thinning on photosynthetic parameters and associated change in leaf nitrogen (N) contents, half of the trees in a 10-year-old Chamaecyparis obtusa (Sieb. et Zucc.) Endl. stand (36° 3′N, 140°7′E) were removed, giving a final density of 1 500 trees ha⁻¹, in May 2004. Photosynthetic photon flux density (PPFD) and leaf N and carbon (C) contents in the lower (L), middle (M), and upper (U) crowns were monitored one, three, and five months after thinning in both the thinned stand and a non-thinned control stand. In addition, leaves’ photosynthetic responses to CO₂ concentration were simultaneously measured in situ to estimate the maximum rates of carboxylation (V_cmax) and electron transport (J_max). Thinning increased PPFD in the L and M crowns but not in the U crown. V_cmax in both the L and M crowns of the thinned stand increased significantly in comparison with the same crown position of the control stand in the three and five months following thinning. In addition, the thinned stand exhibited an increase in N partitioned to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in the L and M crowns relative to the control stand three and five months after thinning, indicating that N had been redistributed within the photosynthetic machinery. Thinning did not affect N per unit area at any of the crown positions, but significantly increased the content of N as a fraction of the total leaf dry mass in the L and M crowns three and five months after thinning. This was a consequence of a decrease in leaf dry mass due to rapid shoot growth. Thus thinning did not cause a redistribution of N between leaves. Thinning improved irradiance in the L and M crowns of C. obtusa, leading to photosynthetic acclimation. Photosynthetic acclimation in the first year mainly occurred via redistribution of N within but not between leaves.     

Heparanase regulates esophageal keratinocyte differentiation through nuclear translocation and heparan sulfate cleavage

Heparanase is an endo-beta-glucuronidase that specifically cleaves heparan sulfate (HS) chains. Heparanase is involved in the process of metastasis and angiogenesis through the degradation of HS chains of the extracellular matrix and cell surface. Recently, we demonstrated that heparanase was localized in the cell nucleus of normal esophageal epithelium and esophageal cancer, and that its expression was correlated with cell differentiation. However, the nuclear function of heparanase remains unknown. To elucidate the role of heparanase in esophageal epithelial differentiation, primary human esophageal cells were grown in monolayer as well as organotypic cultures, and cell differentiation was induced. Expression of heparanase, HS, involucrin, and p27 was determined by immunostaining and Western blotting. SF4, a novel pharmacological inhibitor, was used to specifically inhibit heparanase activity. Upon esophageal cell differentiation, heparanase was translocated from the cytoplasm to the nucleus. Such translocation of heparanase appeared to be associated with the degradation of HS chains in the nucleus and changes in the expression of keratinocyte differentiation markers such as p27 and involucrin, whose induction was inhibited by SF4. Furthermore, these in vitro observations agreed with the expression pattern of heparanase, HS, involucrin, cytokeratin 13, and p27 in normal esophageal epithelium. Nuclear translocation of heparanase and its catalytic cleavage of HS may play a critical role in the differentiation of esophageal epithelial cells. Our study provides a novel insight into the role of heparanase in an essential differentiation process.     

Fibroblast growth factor 2 facilitates the differentiation of transplanted bone marrow cells into hepatocytes

We have developed an in vivo mouse model, the green fluorescent protein (GFP)/carbon tetrachloride (CCl₄) model, and have previously reported that transplanted GFP-positive bone marrow cells (BMCs) differentiate into hepatocytes via hepatoblast intermediates. Here, we have investigated the growth factors that are closely related to the differentiation of transplanted BMCs into hepatocytes, and the way that a specific growth factor affects the differentiation process in the GFP/CCl₄ model. We performed immunohistochemical analysis to identify an important growth factor in our model, viz., fibroblast growth factor (FGF). In liver samples, the expression of FGF1 and FGF2 and of FGF receptors (FGFRs; FGFR1, FGFR2) was significantly elevated with time after bone marrow transplantation (BMT) compared with other factors, and co-expression of GFP and FGFs or FGFRs could be detected. We then analyzed the effect and molecular mechanism of FGF signaling on the enhancement of BMC differentiation into hepatocytes by immunohistochemistry, immunoblotting, and microarray analysis. Treatment with recombinant FGF (rFGF), especially rFGF2, elevated the repopulation rate of GFP-positive cells in the liver and significantly increased the expression of both Liv2 (hepatoblast marker) and albumin (hepatocyte marker). Administration of rFGF2 at BMT also raised serum albumin levels and improved the survival rate. Transplantation of BMCs with rFGF2 specifically activated tumor necrosis factor-alpha (TNF-α) signaling. Thus, FGF2 facilitates the differentiation of transplanted BMCs into albumin-producing hepatocytes via Liv2-positive hepatoblast intermediates through the activation of TNF-α signaling. Administration of FGF2 in combination with BMT improves the liver function and prognosis of mice with CCl₄-induced liver damage. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 13470121, 13770262, 15790348, 16390211, and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5).     

Control of isoperoxidases involved in chlorophyll degradation of stored broccoli (Brassica oleracea) florets by heat treatment

Changes in isoperoxidases involved in chlorophyll (Chl) degradation of stored broccoli (Brassica oleracea L.) florets and their control by heat treatment (HT) were determined. Chl a and b contents in non-heat-treated broccoli florets decreased greatly after 2 days at 15 degrees C, whereas the contents in heat-treated florets (50 degrees C for 2 h) showed almost no change. Three isoperoxidases involved in Chl degradation were detected by means of molecular exclusion chromatography and the molecular weights of those isoperoxidases were about 95 (Type I), 67 (Type II) and 56 (Type III) kDa, respectively. Only Type I was detected in broccoli florets immediately after harvest, and its activity in non-heat-treated broccoli increased greatly during storage. Both Type II and Type III were present in non-heat-treated broccoli with floret senescence. HT suppressed the enhancement of all of the isoperoxidase activities. Cycloheximide treatment also effectively retarded the increase in Types I, II and III isoperoxidase activities concomitant with the suppression of floret yellowing. The K(m) values corresponding to Chl a of Type II and Type III were lower than Type I, and the V(max)/K(m) values corresponding to Chl a of Type II and Type III were higher than Type I. This suggests that both Types II and III could be closely associated with Chl degradation in broccoli florets and that HT might inhibit floret senescence by suppression of isoperoxidase activities.     

A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11

Phosphorylation often regulates protein-protein interactions to control biological reactions. The Sld2 and Dpb11 proteins of budding yeast form a phosphorylation-dependent complex that is essential for chromosomal DNA replication. The Sld2 protein has a cluster of 11 cyclin-dependent kinase (CDK) phosphorylation motifs (Ser/Thr-Pro), six of which match the canonical sequences Ser/Thr-Pro-X-Lys/Arg, Lys/Arg-Ser/Thr-Pro and Ser/Thr-Pro-Lys/Arg. Simultaneous alanine substitution for serine or threonine in all the canonical CDK-phosphorylation motifs severely reduces complex formation between Sld2 and Dpb11, and inhibits DNA replication. Here we show that phosphorylation of these canonical motifs does not play a direct role in complex formation, but rather regulates phosphorylation of another residue, Thr84. This constitutes a non-canonical CDK-phosphorylation motif within a 28-amino-acid sequence that is responsible, after phosphorylation, for binding of Sld2-Dpb11. We further suggest that CDK-catalysed phosphorylation of sites other than Thr84 renders Thr84 accessible to CDK. Finally, we argue that this novel mechanism sets a threshold of CDK activity for formation of the essential Sld2 to Dpb11 complex and therefore prevents premature DNA replication.     

Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Methamphetamine Reward in Mice as Assessed by Conditioned Place Preference Test with Supermex® Sensors: Effect of Subchronic Clorgyline Pretreatment

Recent studies in our laboratory have shown that methamphetamine (METH)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. In this study, the effect of clorgyline pretreatment on METH-induced rewarding effect was assessed by a conditioned place preference (CPP) test, using an apparatus developed with Supermex sensors (infrared pyroelectric sensors). Although intact male ICR mice showed significant CPP for METH (0.5 mg/kg, i.p.), pretreatment with subchronic clorgyline (0.1 and 10 mg/kg, s.c.) did not affect the magnitude of CPP. At a dose of 1 mg/kg, pretreatment of the mice with clorgyline showed a similar CPP index in both saline/saline and METH/saline pairing groups. During the conditioning session, the mice did not express behavioral sensitization to METH. Pretreatment with clorgyline (0.1, 1, and 10 mg/kg) decreased striatal apparent monoamine turnover in a dose-dependent manner. These results indicated that clorgyline pretreatment (0.1 and 10 mg/kg) did not influence the METH-induced rewarding effect in mice, although pretreatment of the mice with clorgyline at a dose of 1 mg/kg appeared to influence the CPP for METH.     

Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication

Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.