Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species

The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples. CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo. In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood. By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum. We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum. We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney. Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide. Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO⁻ and O₂^•−, as well as other not identified RONS, are detectable by CP-H in tissue. In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues. Increased formation of RONS in response to endotoxin challenge is organ specific.     

Skeletal muscles, heart, and lung are the main sources of oxygen radicals in old rats

The aim of this study was to compare rat tissues with respect to their reactive oxygen and nitrogen species (RONS) generating activities as a function of age. We quantified the RONS generation in vivo in young (6 months) and in old (30 months) male Sprague-Dawley rats using the recently developed spin trap 1-hydroxy-3-carboxy-pyrrolidine, applied intravenously. This spin trap reacts with superoxide radical and peroxynitrite yielding a stable spin adduct which is detectable by means of electron paramagnetic resonance (EPR) spectroscopy in frozen tissues. In old rats RONS generation was significantly increased compared to their young counterparts in the following order: blood相似文献   

Probabilistic Identification of Spin Systems and their Assignments including Coil–Helix Inference as Output (PISTACHIO)

We present a novel automated strategy (PISTACHIO) for the probabilistic assignment of backbone and sidechain chemical shifts in proteins. The algorithm uses peak lists derived from various NMR experiments as input and provides as output ranked lists of assignments for all signals recognized in the input data as constituting spin systems. PISTACHIO was evaluate00000000d by comparing its performance with raw peak-picked data from 15 proteins ranging from 54 to 300 residues; the results were compared with those achieved by experts analyzing the same datasets by hand. As scored against the best available independent assignments for these proteins, the first-ranked PISTACHIO assignments were 80–100% correct for backbone signals and 75–95% correct for sidechain signals. The independent assignments benefited, in a number of cases, from structural data (e.g. from NOESY spectra) that were unavailable to PISTACHIO. Any number of datasets in any combination can serve as input. Thus PISTACHIO can be used as datasets are collected to ascertain the current extent of secure assignments, to identify residues with low assignment probability, and to suggest the types of additional data needed to remove ambiguities. The current implementation of PISTACHIO, which is available from a server on the Internet, supports input data from 15 standard double- and triple-resonance experiments. The software can readily accommodate additional types of experiments, including data from selectively labeled samples. The assignment probabilities can be carried forward and refined in subsequent steps leading to a structure. The performance of PISTACHIO showed no direct dependence on protein size, but correlated instead with data quality (completeness and signal-to-noise). PISTACHIO represents one component of a comprehensive probabilistic approach we are developing for the collection and analysis of protein NMR data.Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Anti-Inflammatory Activity of S. Marianum and N. Sativa Extracts on Macrophages

Background:Nigella sativa (N. sativa) and Silybum marianum (S. marianum) are used to regulate macrophage polarization in lipopolysaccharide-induced RAW 264.7 cells and thioglycollate-elicited peritoneal inflammation.Methods:Cytotoxicity assays and acute toxicity tests were performed to investigate the safe dose and toxicity of the prepared extracts. Also, nitric oxide production was determined by Griess assay on RAW264.7 and peritoneal macrophage supernatants. After RNA extraction from macrophages, real-time PCR was performed to measure the relative gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β, and IL-10. Finally, regulatory T cells (Treg cells) were counted by flow cytometry.Results:S. marianum methanolic extract (SME), N. sativa ethanolic extract (NEE), and their mixture (SME+NEE) decreased NO levels significantly in RAW264.7 and peritoneal murine macrophages. N. sativa ethanolic extract significantly increased IL-10 gene expression and significantly decreased IL-6 and TNF-α expression in RAW264.7 cells. In mixture-treated peritoneal macrophages, IL-10 and TGF-β expression were significantly increased, while IL-6 and TNF-α were significantly decreased. Also, the percentage of Treg cells was significantly greater in the mixture-treated cells than in controls.Conclusion:These results suggest that an SME and NEE mixture has anti-inflammatory and immunomodulatory activities and may be useful in the treatment of diseases of immunopathologic origin characterized by macrophage hyperactivation.Key Words: Cytokine, Inflammation, Nigella sativa, Nitric oxide, Silybum marianum     

Pathogen-Triggered Ethylene Signaling Mediates Systemic-Induced Susceptibility to Herbivory in Arabidopsis

Multicellular eukaryotic organisms are attacked by numerous parasites from diverse phyla, often simultaneously or sequentially. An outstanding question in these interactions is how hosts integrate signals induced by the attack of different parasites. We used a model system comprised of the plant host Arabidopsis thaliana, the hemibiotrophic bacterial phytopathogen Pseudomonas syringae, and herbivorous larvae of the moth Trichoplusia ni (cabbage looper) to characterize mechanisms involved in systemic-induced susceptibility (SIS) to T. ni herbivory caused by prior infection by virulent P. syringae. We uncovered a complex multilayered induction mechanism for SIS to herbivory. In this mechanism, antiherbivore defenses that depend on signaling via (1) the jasmonic acid–isoleucine conjugate (JA-Ile) and (2) other octadecanoids are suppressed by microbe-associated molecular pattern–triggered salicylic acid (SA) signaling and infection-triggered ethylene signaling, respectively. SIS to herbivory is, in turn, counteracted by a combination of the bacterial JA-Ile mimic coronatine and type III virulence-associated effectors. Our results show that SIS to herbivory involves more than antagonistic signaling between SA and JA-Ile and provide insight into the unexpectedly complex mechanisms behind a seemingly simple trade-off in plant defense against multiple enemies.     

Spatial phylogenetics of the native woody plant species in Hainan,China

To better identify biodiversity hotspots for conservation on Hainan Island, a tropical island in southern China, we assessed spatial variation in phylogenetic diversity and species richness using 18,976 georeferenced specimen records and a newly reconstructed molecular phylogeny of 957 native woody plants. Within this framework, we delineated bioregions based on vegetation composition and mapped areas of neoendemism and paleoendemism to identify areas of priority for conservation. Our results reveal that the southwest of Hainan is the most important hot spot for endemism and plant diversity followed by the southeast area. The distribution of endemic species showed a scattered, rather than clustered, pattern on the island. Based on phylogenetic range‐weighted turnover metrics, we delineated three major vegetational zones in Hainan. These largely correspond to natural secondary growth and managed forests (e.g., rubber and timber forests) in central Hainan, old‐growth forests and natural secondary growth forest at the margins of Hainan, and nature reserves on the island (e.g., Jianfeng and Diaoluo National Nature Reserves). Our study helps to elucidate potential botanical conservation priorities for Hainan within an evolutionary, phylogenetic framework.