Consequences of Sphaeropsis tip blight disease for the phytohormone profile and antioxidative metabolism of its pine host

Phytopathogenic fungi infections induce plant defence responses that mediate changes in metabolic and signalling processes with severe consequences for plant growth and development. Sphaeropsis tip blight, induced by the endophytic fungus Sphaeropsis sapinea that spreads from stem tissues to the needles, is the most widespread disease of conifer forests causing dramatic economic losses. However, metabolic consequences of this disease on bark and wood tissues of its host are largely unexplored. Here, we show that diseased host pines experience tissue dehydration in both bark and wood. Increased cytokinin and declined indole‐3‐acetic acid levels were observed in both tissues and increased jasmonic acid and abscisic acid levels exclusively in the wood. Increased lignin contents at the expense of holo‐cellulose with declined structural biomass of the wood reflect cell wall fortification by S. sapinea infection. These changes are consistent with H₂O₂ accumulation in the wood, required for lignin polymerization. Accumulation of H₂O₂ was associated with more oxidized redox states of glutathione and ascorbate pools. These findings indicate that S. sapinea affects both phytohormone signalling and the antioxidative defence system in stem tissues of its pine host during the infection process.     

Mapping Sites Where Replication Initiates in Mammalian Cells Using DNA Fibers

DNA replication in mammalian chromosomes takes place as a unit of replicon clusters. Here we show a powerful method to detect replication origins and fork movement on DNA fibers from mammalian cells. Cells were loaded with nucleotide analogs, DNA fibers were prepared, and replicated DNA was detected. Using this approach, we could detect origins as close as 10 kb apart and found that the average size of replicon is smaller ( approximately 46 kb) than previously estimated. In addition, the procedure visualizes the complex structure of replicon clusters, e.g. sequential activation of origins in a cluster and flexible initiation sites in different cell cycles. Combined with fluorescence in situ hybridization, replication origins can be mapped in genomic loci including repetitive DNA and a single-copy gene.     

Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells

We previously reported that serotonin (5-HT) increased glial cell line-derived neurotrophic factor (GDNF) release in a 5-HT₂ receptor (5-HT₂R) and mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK)-dependent manner in rat C6 glioma cells (C6 cells), a model of astrocytes. We herein found that 5-HT-induced rapid ERK phosphorylation was blocked by 5-HT₂R antagonists in C6 cells. We therefore examined 5-HT-induced ERK phosphorylation to reveal the mechanism of 5-HT-induced GDNF mRNA expression. As 5-HT-induced ERK phosphorylation was blocked by inhibitors for Gα_q/11 and fibroblast growth factor receptor (FGFR), but not for second messengers downstream of Gα_q/11, 5-HT₂R-mediated FGFR transactivation was suggested to be involved in the ERK phosphorylation. Although FGFR1 and 2 were functionally expressed in C6 cells, 5-HT selectively phosphorylated FGFR2. Indeed, small interfering RNA for FGFR2, but not for FGFR1, blocked 5-HT-induced ERK phosphorylation. As Src family tyrosine kinase inhibitors and microtubule depolymerizing agents blocked 5-HT-induced FGFR2 phosphorylation, Src family tyrosine kinase and stabilized microtubules were suggested to act upstream of FGFR2. Finally, 5-HT-induced GDNF mRNA expression was also inhibited by the blockade of 5-HT₂R, FGFR, and Src family tyrosine kinase. In conclusion, our findings suggest that 5-HT induces GDNF mRNA expression via 5-HT₂R-mediated FGFR2 transactivation in C6 cells.     

Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-alpha in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-gamma-producing Th cells depending on their capacity to residually produce IFN-alpha. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-alpha-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.