Identification and characterisation of the E3 ligase, RAP1, in Arabidopsis

Changes in cellular redox status are implicated in the regulation of developmental and defence-related responses. The absence of S-nitrosoglutathione reductase (GSNOR) function in Arabidopsis leads to an accumulation of cellular S-nitrosoglutathione (GSNO), a mobile reservoir of nitric oxide (NO) which impacts the cellular redox tone. Consequently, the GSNOR knockout mutant, atgsnor1-3 displays defects in growth, time to flowering and pathogen resistance. Although it is now well established that GSNO is a key redox signalling molecule, the molecular mechanisms that underpin GSNO function remains largely unknown. RAP1 (REDOX-ASSOCIATED PROTEIN 1) was identified based on its dynamic changes of expression in atgsnor1-3 and sid2 plants upon avirulent Pseudomonas syringae pv. tomato (Pst) DC3000 (avrB) challenge. Pathogen-induced RAP1 expression was shown to be independent of the plant hormones salicylic acid, jasmonic acid, abscisic acid and ethylene. Recombinant RAP1 protein was shown to exhibit E3 ligase activity in vitro. Application of the NO donors (GSNO and Cysteine-NO (CysNO)) reduced the E3 ligase activity of RAP1 significantly. Biotinswitch analysis showed that RAP1 was S-nitrosylated and site-directed mutagenesis of RAP1 suggested that the S-nitrosylated site is the cysteine residue C325. The rap1 line does not show obvious developmental phenotypes, however, overexpressing RAP1 enhanced lateral root branching in young seedlings. Overexpression of a truncated RAP1 (RAP1ΔRING) led to a loss of apical dominance. In addition, rap1/rap2 double mutants showed delayed flowering, suggesting RAP1 might be involved in the regulation of plant growth and development. RAP1 may also be involved in plant defence, as rap1, rap2 and rap1/rap2 mutants exhibited increased susceptibility to PstDC3000 and Arabidopsis powdery mildew. Interestingly, rap1 plants showed enhanced resistance to methyl viologen (MV), which is in line with the phenotype of atgsnor mutants. Also, expression of RAP1 was rapidly inducible by ultraviolet-B (UV-B) light. As RAP1 expression and RAP1 E3 ligase activity are redox-related, it is speculated that RAP1 may be involved in redoxmediated regulation of a broad range of physiological responses

Topological analysis of a haloacid permease of a Burkholderia sp. bacterium with a PhoA-LacZ reporter

<p>Abstract</p> <p>Background</p> <p>2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium <it>Burkholderia </it>sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system.</p> <p>Results</p> <p>Thirty-six Deh4p-reporter recombinants were constructed and expressed in <it>E. coli</it>. Both PhoA and LacZ activities were determined in these cells. Strength indices were calculated to determine the locations of the reporters. The results mainly agree with the predicted model. However, two of the TMS were not verified. This lack of confirmation of the TMS, using a reporter, has been reported previously. Further comparative analysis of Deh4p has assigned it to the Metabolite:H⁺Symporter (MHS) 2.A.1.6 family with twelve TMS. Deh4p exhibits many common features of the MHS family proteins. Deh4p is apparently a member of the MFS but with some atypical features.</p> <p>Conclusion</p> <p>The PhoA-LacZ reporter system is convenient for analysis of the topology of membrane proteins. However, due to the limitation of the biological system, verification of some of the TMS of the protein was not successful. The present study also makes use of bioinformatic analysis to verify that the haloacid permease Deh4p of <it>Burkholderia </it>sp. MBA4 is a MFS protein but with atypical features.</p

Changes in physico-chemical parameters during forced-aeration static-pile co-composting of cattle manure with calcium cyanamide

The goal of this research was to determine the effect of CaCN2 addition into manure mixed with sawdust on the composting process under forced-aeration static condition, especially on nitrogen (N). The changes in the physical and chemical parameters over the entire composting period were evaluated. The profile of temperature, pH, and NO3 –-N was improved in the piles mixed with 2%, 3% and 4% CaCN2 (the test piles). The NH4 +-N met the limit value accepted for mature compost. Finally, the additive contents of not less than 2% but not more than 3% in CaCN2 were recommended for this system based upon a comprehensive evaluation of the measured parameters

Reduction of foodborne pathogens during cattle manure composting with addition of calcium cyanamide

Inactivation of four species of foodborne pathogens (E. coli, Salmonella, E. faecalis, and S. aureus) was investigated during laboratory-scale composting of fresh cow manure with addition of calcium cyanamide (CaCN2) at constant temperature conditions, and the effects of temperature and additive content of CaCN2 on the efficacy of inactivation were evaluated. At different composting temperatures (20, 30, 37 and 50 °C), a significant inhibition or quick inactivation of pathogens were observed during 10 d composting with addition of 2.0% CaCN2, and the effect was more obvious at mesophilic temperatures compared to thermophilic temperature. Therefore, the ideal additive content of CaCN2 was determined at 30 °C through mixing 2.0%, 2.5%, and 3.0% CaCN2 with manure. With increase in additive content of CaCN2, the efficacies of pathogen inactivation also increased. However, the result indicates that, no less than 2.5% CaCN2 should be mixed with the manure to entirely eliminate the pathogens during composting

Spatial variation in prostate cancer survival in the Northern and Yorkshire region of England using Bayesian relative survival smoothing

Primary Care Trust (PCT) estimates of survival lack robustness as there are small numbers of deaths per year in each area, even when incidence is high. We assess PCT-level spatial variation in prostate cancer survival using Bayesian spatial models of excess mortality. We extracted data on men diagnosed with prostate cancer between 1990 and 1999 from the Northern and Yorkshire Cancer Registry and Information Service database. Models were adjusted for age at diagnosis, period of diagnosis and deprivation. All covariates had a significant association with excess mortality; men from more deprived areas, older age at diagnosis and diagnosed in 1990–1994 had higher excess mortality. The unadjusted relative excess risks (RER) of death by PCT ranged from 0.75 to 1.66. After adjustment, areas of high and low excess mortality were smoothed towards the mean, and the RERs ranged from 0.74 to 1.49. Using Bayesian smoothing techniques to model cancer survival by geographic area offers many advantages over traditional methods; estimates in areas with small populations or low incidence rates are stabilised and shrunk towards local and global risk estimates improving reliability and precision, complex models are easily handled and adjustment for covariates can be made

C IV black hole mass measurements with the Australian Dark Energy Survey (OzDES)

Black hole mass measurements outside the local Universe are critically important to derive the growth of supermassive black holes over cosmic time, and to study the interplay between black hole growth and galaxy evolution. In this paper, we present two measurements of supermassive black hole masses from reverberation mapping (RM) of the broad C IV emission line. These measurements are based on multiyear photometry and spectroscopy from the Dark Energy Survey Supernova Program (DES-SN) and the Australian Dark Energy Survey (OzDES), which together constitute the OzDES RM Program. The observed reverberation lag between the DES continuum photometry and the OzDES emission line fluxes is measured to be 358+126−123 and 343+58−84 d for two quasars at redshifts of 1.905 and 2.593, respectively. The corresponding masses of the two supermassive black holes are 4.4 × 109 and 3.3 × 109 M⊙, which are among the highest redshift and highest mass black holes measured to date with RM studies. We use these new measurements to better determine the C IV radius−luminosity relationship for high-luminosity quasars, which is fundamental to many quasar black hole mass estimates and demographic studies.This research was funded partially by the Australian Government through the Australian Research Council through project DP160100930. PM and ZY were supported in part by the United States National Science Foundation under grant no. 161553

Protective Effects of a Rhodiola Crenulata Extract and Salidroside on Hippocampal Neurogenesis against Streptozotocin-Induced Neural Injury in the Rat

Previously we have demonstrated that a Rhodiola crenulata extract (RCE), containing a potent antioxidant salidroside, promotes neurogenesis in the hippocampus of depressive rats. The current study was designed to further investigate the protective effect of the RCE on neurogenesis in a rat model of Alzheimer's disease (AD) induced by an intracerebroventricular injection of streptozotocin (STZ), and to determine whether this neuroprotective effect is induced by the antioxidative activity of salidroside. Our results showed that pretreatment with the RCE significantly improved the impaired neurogenesis and simultaneously reduced the oxidative stress in the hippocampus of AD rats. In vitro studies revealed that (1) exposure of neural stem cells (NSCs) from the hippocampus to STZ strikingly increased intracellular reactive oxygen species (ROS) levels, induced cell death and perturbed cell proliferation and differentiation, (2) hydrogen peroxide induced similar cellular activities as STZ, (3) pre-incubation of STZ-treated NSCs with catalase, an antioxidant, suppressed all these cellular activities induced by STZ, and (4) likewise, pre-incubation of STZ-treated NSCs with salidroside, also an antioxidant, suppressed all these activities as catalase: reduction of ROS levels and NSC death with simultaneous increases in proliferation and differentiation. Our findings indicated that the RCE improved the impaired hippocampal neurogenesis in the rat model of AD through protecting NSCs by its main ingredient salidroside which scavenged intracellular ROS

ROS release by PPARβ/δ-null fibroblasts reduces tumor load through epithelial antioxidant response.

Tumor stroma has an active role in the initiation, growth, and propagation of many tumor types by secreting growth factors and modulating redox status of the microenvironment. Although PPARβ/δ in fibroblasts was shown to modulate oxidative stress in the wound microenvironment, there has been no evidence of a similar effect in the tumor stroma. Here, we present evidence of oxidative stress modulation by intestinal stromal PPARβ/δ, using a FSPCre-Pparb/d &lt;sup&gt;-/-&lt;/sup&gt; mouse model and validated it with immortalized cell lines. The FSPCre-Pparb/d &lt;sup&gt;-/-&lt;/sup&gt; mice developed fewer intestinal polyps and survived longer when compared with Pparb/d &lt;sup&gt;fl/fl&lt;/sup&gt; mice. The pre-treatment of FSPCre-Pparb/d &lt;sup&gt;-/-&lt;/sup&gt; and Pparb/d &lt;sup&gt;fl/fl&lt;/sup&gt; with antioxidant N-acetyl-cysteine prior DSS-induced tumorigenesis resulted in lower tumor load. Gene expression analyses implicated an altered oxidative stress processes. Indeed, the FSPCre-Pparb/d &lt;sup&gt;-/-&lt;/sup&gt; intestinal tumors have reduced oxidative stress than Pparb/d &lt;sup&gt;fl/fl&lt;/sup&gt; tumors. Similarly, the colorectal cancer cells and human colon epithelial cells also experienced lower oxidative stress when co-cultured with fibroblasts depleted of PPARβ/δ expression. Therefore, our results establish a role for fibroblast PPARβ/δ in epithelial-mesenchymal communication for ROS homeostasis