Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H₂O₂) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H₂O₂ compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.     

Determination of phenolic compounds and diterpenes in roots of Salvia miltiorrhiza and Salvia przewalskii by two LC–MS tools: Multi-stage and high resolution tandem mass spectrometry with assessment of antioxidant capacity

Comparative phytochemical analyses of hydroalcoholic (50% EtOH) extracts from roots of S. miltiorrhiza (SM) and S. przewalskii (SP) were performed using two complementary LC–MS systems: the first system HPLC-DAD-MSⁿ an ion trap mass spectrometer and the second system consisted high resolution MS/MS Orbitrap mass spectrometer. The individual compounds were identified using a previously published approach via comparison of the exact molecular masses, mass spectra and retention times to those of standard compounds, online available databases and literature data. Moreover, the determination of antioxidative activities of extracts by DPPH and FRAP methods was carried out. Analysis allowed to identify 39 chemical compounds in extracts from both species. Extract from root of SP differs from SM in the presence of several metabolites such as: przewalskinic acid and their derivatives, przewaquinone C, przewaquinonate A, glycosides of rosmarinic acid, methyltanshinonate, whereas tanshinones, salvianolic acids and lithospermic acids occurred in both species. Moreover, it was shown that hydroalcoholic extract from roots of SM exerted stronger antioxidant properties in a FRAP test (max. 323.92 μM Fe²⁺/L) and in DPPH test (max. 78.64 nM TE) in comparison with SP extract.     

Production of Bio-Energy from Pig Manure: A Focus on the Dynamics Change of Four Parameters under Sunlight-Dark Conditions

This study investigated the effect of sunlight-dark conditions on volatile fatty acids (VFAs), total ammonium nitrogen (TAN), total alkalinity (TA) and pH during pig manure (PM) digestion and then the subsequent influence on biogas yield of PM. PM₁ and PM₂ were performed in a transparent reactor and a non-transparent reactor, respectively. Two sets of experiments were conducted with a temperature of 35.0±2.0 °C and a total solid concentration of 8.0% to the digestion material. The dynamic change of the four parameters in response to sunlight-dark conditions resulted in variations of the physiological properties in the digester and affected the cumulative biogas production (CBP). PM₁ obtained higher CBP (15020.0 mL) with a more stable pH and a lower TAN concentration (1414.5 mg/L) compared to PM₂ (2675.0 mL and 1670.0 mg/L, respectively). The direct path coefficients and indirect path coefficients between the four parameters and CBP were also analyzed.     

Geochemical baseline determination and pollution assessment of heavy metals in urban soils of Karachi,Pakistan

Karachi is one of the most populated urban agglomerations in the world. No categorical study has yet discussed the geochemical baseline concentrations of metals in the urban soil of Karachi. The main objectives of this study were to establish geochemical baseline values and to assess the pollution status of different heavy metals. Geochemical baseline concentrations of heavy metals were estimated using the cumulative frequency distribution (CDF) curves. The estimated baseline concentrations of Pb, Cr, Cu, Zn and Fe were 56.23, 12.9, 36.31, 123.03 and 11,776 mg kg⁻¹, respectively. The pollution status of heavy metals in urban soils was evaluated using different quantitative indices (enrichment factor–EF, Geo-accumulation Index–I_geo, and pollution index–PI). Enrichments factors of the selected heavy metals determined by using Fe as a normalizer showed that metal contamination was the product of anthropogenic activities. The urban soils of Karachi were found to have a moderate to moderately severe enrichment with Pb, whereas Cr and Cu has moderate and Zn has minor enrichment. I_geo results indicated moderate soil contamination by Pb at some of the sampling locations. PI for Pb, Cr, Cu and Zn was found in the range of 0.04–3.42, 0.19–1.55, 0.27–2.45 and 0.32–1.57, respectively. Large variations in PI values of Pb revealed that soil in those areas of the city which are influenced by intensive anthropogenic activities have exceptionally high concentrations of Pb. The findings of this study would contribute to the environmental database of the soil of the region and would also facilitate both at the local and the international scales, in a more accurate global environmental monitoring, which will eventually facilitate the development of management and remediation strategies for heavy metal contaminated urban soil.     

A four-dimensional computed tomography comparison of healthy and asthmatic human lungs

The purpose of this study was to explore new insights in non-linearity, hysteresis and ventilation heterogeneity of asthmatic human lungs using four-dimensional computed tomography (4D-CT) image data acquired during tidal breathing. Volumetric image data were acquired for 5 non-severe and one severe asthmatic volunteers. Besides 4D-CT image data, function residual capacity and total lung capacity image data during breath-hold were acquired for comparison with dynamic scans. Quantitative results were compared with the previously reported analysis of five healthy human lungs. Using an image registration technique, local variables such as regional ventilation and anisotropic deformation index (ADI) were estimated. Regional ventilation characteristics of non-severe asthmatic subjects were similar to those of healthy subjects, but different from the severe asthmatic subject. Lobar airflow fractions were also well correlated between static and dynamic scans (R² > 0.84). However, local ventilation heterogeneity significantly increased during tidal breathing in both healthy and asthmatic subjects relative to that of breath-hold perhaps because of airway resistance present only in dynamic breathing. ADI was used to quantify non-linearity and hysteresis of lung motion during tidal breathing. Non-linearity was greater on inhalation than exhalation among all subjects. However, exhalation non-linearity among asthmatic subjects was greater than healthy subjects and the difference diminished during inhalation. An increase of non-linearity during exhalation in asthmatic subjects accounted for lower hysteresis relative to that of healthy ones. Thus, assessment of non-linearity differences between healthy and asthmatic lungs during exhalation may provide quantitative metrics for subject identification and outcome assessment of new interventions.     

RssB-mediated σS Activation is Regulated by a Two-Tier Mechanism via Phosphorylation and Adaptor Protein – IraD

Regulation of bacterial stress responding σ^S is a sophisticated process and mediated by multiple interacting partners. Controlled proteolysis of σ^S is regulated by RssB which maintains minimal level of σ^S during exponential growth but then elevates σ^S level while facing stresses. Bacteria developed different strategies to regulate activity of RssB, including phosphorylation of itself and production of anti-adaptors. However, the function of phosphorylation is controversial and the mechanism of anti-adaptors preventing RssB-σ^S interaction remains elusive. Here, we demonstrated the impact of phosphorylation on the activity of RssB and built the RssB-σ^S complex model. Importantly, we showed that the phosphorylation site - D58 is at the interface of RssB-σ^S complex. Hence, mutation or phosphorylation of D58 would weaken the interaction of RssB with σ^S. We found that the anti-adaptor protein IraD has higher affinity than σ^S to RssB and its binding interface on RssB overlaps with that for σ^S. And IraD-RssB complex is preferred over RssB-σ^S in solution, regardless of the phosphorylation state of RssB. Our study suggests that RssB possesses a two-tier mechanism for regulating σ^S. First, phosphorylation of RssB provides a moderate and reversible tempering of its activity, followed by a specific and robust inhibition via the anti-adaptor interaction.