Priming in plant-pathogen interactions

Plants can acquire enhanced resistance to pathogens after treatment with necrotizing attackers, nonpathogenic root-colonizing pseudomonads, salicylic acid, beta-aminobutyric acid and many other natural or synthetic compounds. The induced resistance is often associated with an enhanced capacity to mobilize infection-induced cellular defence responses - a process called 'priming'. Although the phenomenon has been known for years, most progress in our understanding of priming has been made only recently. These studies show that priming often depends on the induced disease resistance key regulator NPR1 (also known as NIM1 or SAI1) and that priming has a major effect on the regulation of cellular plant defence responses.     

Direct evidence that toll-like receptor 9 (TLR9) functionally binds plasmid DNA by specific cytosine-phosphate-guanine motif recognition

Cytosine-phosphate-guanine (CpG) motifs in bacterial DNA are known to activate the mammalian immune system, and this activation is thought to depend on the Toll-like receptor 9 (TLR9) signaling pathway. Previous studies strongly suggested that TLR9 is involved as the specific receptor for CpG motifs but did not provide direct evidence of their interaction. In this study, we demonstrate for the first time that murine TLR9 binds an unmethylated CpG-containing plasmid. This interaction is sequence-specific and is influenced by the methylation status of the plasmid. Furthermore, we demonstrate that this interaction leads to the activation of the NF-kappaB pathway in mTLR9-expressing cells. Our results provide a molecular basis for the interaction between CpG-DNA and TLR9.     

Neuropeptide chronomics in clinically healthy young adults: circaoctohoran and circadian patterns

Endothelin-1 (ET-1) undergoes an about 8-h (circaoctohoran) rather than a circadian variation in clinical health. Herein, 24 h plasma concentrations of vasoactive intestinal peptide (VIP), substance P (SP), neuropeptide Y (NpY), and cortisol used as reference, were obtained from 20 healthy young adults starting at 07:00 or 19:00 h. Like ET-1, SP and NpY undergo a circaoctohoran variation, whereas VIP is circadian rhythmic, peaking during the night, some 8 h prior to the circadian acrophase of cortisol. Maps of circadian and extra-circadian patterns may serve for screening, diagnosis and a better understanding of mechanisms underlying the etiology of various diseases.     

Chemical characterization of Azadirachta indica grafted on Melia azedarach and analyses of azadirachtin by HPLC‐MS‐MS (SRM) and meliatoxins by MALDI‐MS

Introduction – Melia azedarach adapted to cool climates was selected as rootstocks for vegetative propagation of Azadirachta indica. Cleft grafting of A. indica on M. azedarach rootstock showed excellent survival. Little is known about the chemistry of grafting. Objective – The roots, stems, leaves and seeds of this graft were examined in order to verify if grafted A. indica would produce limonoids different from those found in non‐grafted plants. Intact matured fruits were also studied to verify if they were free of meliatoxins. Methodology – After successive chromatographic separations the extracts afforded several limonoids. HPLC‐MS/MS and MALDI‐MS were used to develop sensitive methods for detecting azadirachtin on all aerial parts of this graft and meliatoxins in fruits, respectively. Results – The stem afforded the limonoid salannin, which was previously found in the oil seeds of A. indica. Salannin is also found in the root bark of M. azedarach. Thus, the finding of salannin in this study suggests that it could have been translocated from the M. azedarach rootstock to the A. indica graft. HPLC‐MS/MS analyses showed that azadirachtin was present in all parts of the fruits, stem, flowers and root, but absent in the leaves. The results of MALDI‐MS analyses confirmed the absence of meliatoxins in graft fruits. Conclusion – This study showed that A. indica grafted onto M. azedarach rootstock produces azadirachtin, and also that its fruits are free of meliatoxins from rootstocks, confirming that this graft forms an excellent basis for breeding vigorous Neem trees in cooler regions. Copyright © 2010 John Wiley & Sons, Ltd.     

OCP3 is an important modulator of NPR1-mediated jasmonic acid-dependent induced defenses in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Upon appropriate stimulation, plants increase their level of resistance against future pathogen attack. This phenomenon, known as induced resistance, presents an adaptive advantage due to its reduced fitness costs and its systemic and broad-spectrum nature. In Arabidopsis, different types of induced resistance have been defined based on the signaling pathways involved, particularly those dependent on salicylic acid (SA) and/or jasmonic acid (JA).     

Selected contribution: circadian rhythm of tumor necrosis factor-alpha uptake into mouse spinal cord.

Circadian variations in the actions of tumor necrosis factor-alpha (TNF-alpha) have been observed. Because a saturable transport system at the blood-brain barrier mediates most of the influx of TNF-alpha from blood to the central nervous system (CNS), the circadian variation of the CNS effects of TNF-alpha could be related to changes in this transport system. Accordingly, we measured the uptake of intravenously injected TNF-alpha into various CNS regions at different times and compared these measurements with the uptake into a peripheral control (muscle). We found that the spinal cord, but not the brain, showed a circadian rhythm in the uptake of TNF-alpha. This pattern is similar to that of leptin but different from that of interleukin-1. The circadian rhythm of the influx of TNF-alpha into this region of the CNS suggests a functional role for the spinal cord in the physiological actions of TNF-alpha.     

Automated electron-density sampling reveals widespread conformational polymorphism in proteins

Although proteins populate large structural ensembles, X-ray diffraction data are traditionally interpreted using a single model. To search for evidence of alternate conformers, we developed a program, Ringer, which systematically samples electron density around the dihedral angles of protein side chains. In a diverse set of 402 structures, Ringer identified weak, nonrandom electron-density features that suggest of the presence of hidden, lowly populated conformations for >18% of uniquely modeled residues. Although these peaks occur at electron-density levels traditionally regarded as noise, statistically significant (P < 10⁻⁵) enrichment of peaks at successive rotameric χ angles validates the assignment of these features as unmodeled conformations. Weak electron density corresponding to alternate rotamers also was detected in an accurate electron density map free of model bias. Ringer analysis of the high-resolution structures of free and peptide-bound calmodulin identified shifts in ensembles and connected the alternate conformations to ligand recognition. These results show that the signal in high-resolution electron density maps extends below the traditional 1 σ cutoff, and crystalline proteins are more polymorphic than current crystallographic models. Ringer provides an objective, systematic method to identify previously undiscovered alternate conformations that can mediate protein folding and function.     

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.