Overexpression of an ADP-ribose pyrophosphatase, AtNUDX2, confers enhanced tolerance to oxidative stress in Arabidopsis plants

Mutant pqr-216 from an Arabidopsis activation-tagged line showed a phenotype of increased tolerance to oxidative stress after treatment with 3 μ m paraquat (PQ). Based on the phenotype of transgenic plants overexpressing the genes flanking the T-DNA insert, it was clear that enhanced expression of a Nudix (nucleoside diphosphates linked to some moiety X) hydrolase gene, AtNUDX2 (At5g47650), was responsible for the tolerance. It has been reported that the AtNUDX2 protein has pyrophosphatase activities towards both ADP-ribose and NADH ( Ogawa et al ., 2005 ). Interestingly, the pyrophosphatase activity toward ADP-ribose, but not NADH, was increased in pqr-216 and Pro _35S :AtNUDX2 plants compared with control plants. The amount of free ADP-ribose was lower in the Pro _35S :AtNUDX2 plants, while the level of NADH was similar to those in control plants under both normal conditions and oxidative stress. Depletion of NAD⁺ and ATP resulting from activation of poly(ADP-ribosyl)ation under oxidative stress was observed in the control Arabidopsis plants. Such alterations in the levels of these molecules were significantly suppressed in the Pro _35S :AtNUDX2 plants. The results indicate that overexpression of AtNUDX2 , encoding ADP-ribose pyrophosphatase, confers enhanced tolerance of oxidative stress on Arabidopsis plants, resulting from maintenance of NAD⁺ and ATP levels by nucleotide recycling from free ADP-ribose molecules under stress conditions.     

Immunohistochemical localization of aldehyde and xanthine oxidase in rat tissues using polyclonal antibodies

Tissues from male Wistar rats, fixed with 4% paraformaldehyde and embedded in paraffin, were studied with immunoperoxidase techniques using polyclonal antibodies raised against aldehyde oxidase or xanthine oxidase purified from rat liver. Immunohistochemical studies demonstrated that aldehyde oxidase-bearing cells were strongly stained in renal tubules, esophageal, gastric, intestinal and bronchial epithelium as well as liver cytoplasm. Weak but positive immunoreactivity was observed on the pulmonary alveolar epithelial cells, gastric glands and intestinal goblet cells. In contrast, it was demonstrated that cells with xanthine oxidase were strongly stained in renal tubules, esophageal, gastric, and small and large intestinal and bronchial epithelia etc. Positive immunostaining was also found in adrenal gland, skeletal muscle, spleen and cerebral hippocampus. Immunoreactivity againt aldehyde oxidase was not found in adrenal gland, spleen, mesentery or aorta, while immunoreactivity against xanthine oxidase was not found in mesentery or aorta. Although the significance of this ubiquitous and similar localization of aldehyde and xanthine oxidase seems unclear at present, these results may provide a clue as to the full understanding of the pathophysiological role of these oxidases in tissues.     

Isolation and identification of potent allelopathic substances in rattail fescue

Aqueous methanol extracts of rattail fescue (Vulpia myuros) inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), timothy (Phleum pratense), Digitaria sanguinalis and Lolium multiflorum. Increasing the extract concentration increased the inhibition, suggesting that rattail fescue may have growth inhibitory substances and possess allelopathic potential. The aqueous methanol extract of rattail fescue was purified and two main inhibitory substances were isolated and identified by spectral data as (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol. Both substances inhibited root and shoot growth of cress at concentrations greater than 0.3 μM. The concentrations required for 50% growth inhibition on root and shoot growth of cress, lettuce, alfalfa, timothy, D. sanguinalis and L. multiflorum were 2.7–19.7 μM for (−)-3-hydroxy-β-ionone, and 2.1–34.5 μM for (+)-3-oxo-α-ionol. The concentration of (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol, respectively, in rattail fescue was 7.8 and 3.7 μg g⁻¹ fresh weight. Considering the endogenous level and the inhibitory activity, (−)-3-hydroxy-β-ionone and (+)-3-oxo-α-ionol may work as allelopathic substances in rattail fescue through the growth inhibition of neighboring plant species.     

Comparative Evaluation of Direct Thrombin and Factor Xa Inhibitors with Antiplatelet Agents under Flow and Static Conditions: An In Vitro Flow Chamber Model

Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-{"type":"entrez-nucleotide","attrs":{"text":"C66096","term_id":"2424801","term_text":"C66096"}}C66096, a P₂Y₁₂ antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s⁻¹. Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.     

Cloning and characterization of erythroid-specific DNase I-hypersensitive site in human rhesus-associated glycoprotein gene

Rhesus-associated glycoprotein is a critical co-factor in the expression of rhesus blood group antigens. We identified and cloned an erythroid-specific major DNase I-hypersensitive site located about 10 kilobases upstream from the translation start site of the RHAG gene. A short core enhancer sequence of 195 base pairs that corresponded with the major hypersensitive site and possessed position- and orientation-independent enhancer activity in K562 cells. In vitro DNase I footprint analysis revealed four protected regions in the core enhancer; two GATA motifs, an Ets-like motif and an unknown motif. The GATA motifs bound GATA-1 and mutagenesis analysis revealed that the proximal one is critical for the enhancing activity. Homology plot analysis using the 5' sequence of the mouse RHAG gene revealed four homologous stretches and multiple insertions of repetitive sequences among them; four LINE/L1 and four Alu in the human and as well as one LINE/L1 and one LTR/MaLR in the mouse gene. The highly conservative enhancer region was flanked by SINE and LINE/L1 in both species. These results suggest that the 5'-flanking sequence of RHAG gene is a preferable target sequence for retroviral transposition and that the enhancer was inserted in the same manner, resulting in the acquisition of erythroid dominant expression.     

Epizootiology of virus diseases in three lepidopterous insect species of alfalfa

Summary The incidence of virus infections in three lepidopterous insect species was studied from 1965 to 1968 in alfalfa fields in California. The insects were the alfalfa caterpillar,Colias eurytheme; the beet armyworm,Spodoptera exigua; and the alfalfa looper,Autographa californica. InC. eurytheme, the major virus was a nuclear polyhedrosis virus (NPV); inS. exigua, a granulosis virus (GV) and an NPV; inA. californica, a GV. Virus epizootics did not develop in very high densities ofC. eurytheme. Virus epizootics occurred in low host densities of the three insect species, especially in populations ofA. californica. The virus acted as a density-dependent factor in the regulation of the populations ofS. exigua andA. californica. Temperature, humidity and rainfall had no marked effect on the incidence of virus infections.     

Delta1 family members are involved in filopodial actin formation and neuronal cell migration independent of Notch signaling

Delta family proteins are transmembrane molecules that bind Notch receptors and activate downstream signaling events in neighboring cells. In addition to serving as Notch ligands, Notch-independent roles for Delta have been suggested but are not fully understood. Here, we demonstrate a previously unrecognized role for Delta in filopodial actin formation. Delta1 and Delta4, but not Delta3, exhibit filopodial protrusive activity, and this activity is independent of Notch signaling. The filopodial activity of Delta1 does not depend on the PDZ-binding domain at the C-terminus; however, the intracellular membrane-proximal region that is anchored to the plasma membrane plays an important role in filopodial activity. We further identified a Notch-independent role of DeltaD in neuronal cell migration in zebrafish. These findings suggest a possible functional link between Notch-independent filopodial activity of Delta and the control of cell motility.     

An arginine residue instead of a conserved leucine residue in the recognition helix of the finger 3 of Zif268 stabilizes the domain structure and mediates DNA binding

The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.