Gene expression and sensitivity in response to copper stress in rice leaves

Gene expression in response to Cu stress in rice leaves was quantified using DNA microarray (Agilent 22K Rice Oligo Microarray) and real-time PCR technology. Rice plants were grown in hydroponic solutions containing 0.3 (control), 10, 45, or 130 microM of CuCl(2), and Cu accumulation and photosynthesis inhibition were observed in leaves within 1 d of the start of treatment. Microarray analysis flagged 305 Cu-responsive genes, and their expression profile showed that a large proportion of general and defence stress response genes are up-regulated under excess Cu conditions, whereas photosynthesis and transport-related genes are down-regulated. The Cu sensitivity of each Cu-responsive gene was estimated by the median effective concentration value (EC50) and the range of fold-changes (F) under the highest (130 microM) Cu conditions (|log(2)F|(130)). Our results indicate that defence-related genes involved in phytoalexin and lignin biosynthesis were the most sensitive to Cu, and that plant management of abiotic and pathogen stresses has overlapping components, possibly including signal transduction.     

Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.     

Small GTP-binding protein Rho-mediated signaling promotes proliferation of rheumatoid synovial fibroblasts

Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Gα13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Gα13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Gα13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells.     

Titanium dioxide nanoparticles impart protection from ultraviolet irradiation to fermenting yeast cells

The photocatalytic activity of titanium dioxide is widely utilized in science and technology. In the biological field, titanium dioxide is believed to be a disinfectant because it produces reactive oxygen species (ROS). However, there are multiple types of ROS such as hydroxyl radicals, superoxide anions, singlet oxygen, and hydrogen peroxide. In this study, we attempted to characterize the various mechanisms and roles of ROS in disinfection. Surprisingly, we found that titanium dioxide protected yeast cells from ultraviolet irradiation. We characterized the ROS produced under these conditions. The production of hydroxyl radicals and superoxide anions was confirmed; however, glucose in the yeast medium scavenged hydroxyl radicals. The photocatalytic activity of titanium dioxide produced oxidative products and reductive products, as oxidation and reduction occurred simultaneously. Once hydroxyl radicals are scavenged, the photocatalytic activity of titanium dioxide produces a reductive environment for fermenting yeast cells and protects them from oxidative stress by ultraviolet irradiation.     

Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or Pyrocathechol

Reaction conditions for the synthesis of L-tyrosine or L-dopa from DL-serine and phenol or pyrocatechol were studied with intact cells of Erwinia herbicola (ATCC 21434) containing high tyrosine phenol lyase activity. The optimum pH for this reaction was around 8.0, and the optimum temperature range was between 37~40°C for the synthesis of L-tyrosine and between 15~25°C for that of L-dopa. Sodium sulfite and EDTA were added to protect the synthesized L-dopa from decomposition. As high concentrations of phenol or pyrocatechol denatured the enzyme, each substrate was fed to maintain the optimum concentration during incubation.

The reaction mixture (100 ml) containing 4.0 g of DL-serine, 1.0 g of phenol or 0.7 g of pyrocatechol, 0.5 g of ammonium acetate and the cells, was incubated. During incubation, phenol or pyrocatechol was fed at intervals to maintain the substrate at the initial concentration. 5.35 g of L-tyrosine or 5.10 g of L-dopa was synthesized in 100 ml of the reaction mixture.     

Enhancement of tolerance to heavy metals and oxidative stress in Dunaliella tertiolecta by Zn-induced phytochelatin synthesis

The synthesis of phytochelatins (PCs) in a marine alga, Dunalliela tertiolecta, is strongly induced by Zn. Pretreatment of the cells with Zn enhances the tolerance toward toxic heavy metals such as Cd, Hg, Cu, Pb, and arsenate. Moreover, the pretreatment also increases the tolerance toward oxidative stress caused by hydrogen peroxide or paraquat. In vitro analysis shows that PC is a stronger scavenger of hydrogen peroxide and superoxide radical than glutathione. These results suggest that PCs inducibly synthesized by Zn treatment could play a role not only in detoxification of heavy metals but also in mitigation of oxidative stress.     

Cause of mortality in insects under severe stress.

Mortality in the host armyworm larvae Pseudaletia separata parasitized by the parasitic wasp Cotesia kariyai was dramatically increased when they were simultaneously infected by the entomopathogen Serratia marcescens. Previous studies have shown that this strong insecticidal effect is due to a metalloprotease-like insecticide (MPLI) released from S. marcescens enterobacter. This study was conducted to elucidate the exact cause of the mortality resulting from MPLI. Injection of MPLI caused a sharp increase in hemolymph dopamine concentration followed by elevated levels of brain dopamine in armyworm larvae. [3H]Dopamine injected into the hemocoel, was incorporated into the brains of MPLI-injected larvae to a level eight times greater than in BSA-injected control larvae. Transmission electron microscopy showed an obvious decrease in thickness and density of the brain sheath in insects injected with MPLI. This was probably due to the MPLI-induced elevation of hemocyte metalloprotease activities. Further, electron microscopic and TUNEL staining analyses showed a significant increase in apoptotic cells in the brain 12 h after the injection. Injection of 3-iodotyrosine (a tyrosine hydroxylase inhibitor) before MPLI completely prevented the increase in hemolymph dopamine in test larvae and their following death. From these observations, we conclude that MPLI-injected larvae may have suffered mortal damage through increased apoptosis of brain cells caused by an influx of dopamine from the hemolymph.