Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance.     

High-level expression of naked DNA delivered to rat liver via tail vein injection

Background

High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.

Methods

We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS‐Epo.

Results

We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose‐response relationship between serum Epo levels and the amount of injected DNA up to 800 µg. Using quantitative real‐time PCR, the vector‐derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, β‐galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose‐dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.

Conclusions

These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright © 2002 John Wiley & Sons, Ltd.

     

N-terminal mutational analysis of the interaction between growth-blocking peptide (GBP) and receptor of insect immune cells

GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.     

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al

Human plasmain vitro inhibits the growth of coagulase negative staphylococci,S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity againstS. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated withS. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation.To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition.These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.     

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.     

Caenorhabditis elegans geminin homologue participates in cell cycle regulation and germ line development

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins.     

Increased binding and chemotactic capacities of PDGF-BB on fibroblasts in radiation pneumonitis

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of (125)I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that (125)I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.     

Tissue tracking imaging for identifying the origin of idiopathic ventricular arrhythmias: a new role of cardiac ultrasound in electrophysiology

Several strategies for mapping ventricular outflow tract tachycardia have been reported as useful indices for differentiating between those originating from the right and the left side. Recently, tissue tracking imaging (TTI) has been demonstrated as a novel non-invasive modality for identifying the origin of outflow tract tachycardias. Tissue tracking imaging is an ultrasonographic technique that measures the myocardial motion amplitude towards the transducer in each region during systole, identifying regional myocardial displacement on the basis of myocardial velocities using color Doppler myocardial imaging principles. In this technique, the origin of the arrhythmia could be recognized as the site where the earliest color-coded signal (ECCS) appeared on the myocardium at the onset of the systole. In preliminary studies this modality was found to be useful in differentiating outflow tract ventricular tachycardias. ECCS was always found below or at the level of the pulmonary valve in all arrhythmias which could be ablated from the right ventricular outflow tract, while in those where the ECCS was above and close to the pulmonary valve it could be ablated from the left sinus of valsalva. These results indicate that TTI can provide detailed and accurate information on the arrhythmia origin of outflow tract tachycardia and may be useful for differentiating between an outflow tract tachycardia originating from the LV epicardium remote from the LSV and that from the LSV. Newer advances in echocardiographic technologies like high resolution, high frame rate real time three dimensional echocardiography with speckle tracking may further improve the precise localization of arrhythmias in the future.