The significance of the activity of dissolved oxygen, and other gases, enhanced by high hydrostatic pressure

The partial pressure of oxygen and other gases dissolved in water and subjected to high hydrostatic pressure is increased. Although this was established many years ago it remains a problematical phenomenon. The review deals with some of the underlying theoretical difficulties and discusses the kinetic and environmental implications of the pressure-enhanced partial pressures.     

An amyloid organelle, solid-state NMR evidence for cross-β assembly of gas vesicles

Functional amyloids have been identified in a wide range of organisms, taking on a variety of biological roles and being controlled by remarkable mechanisms of directed assembly. Here, we report that amyloid fibrils constitute the ribs of the buoyancy organelles of Anabaena flos-aquae. The walls of these gas-filled vesicles are known to comprise a single protein, GvpA, arranged in a low pitch helix. However, the tertiary and quaternary structures have been elusive. Using solid-state NMR correlation spectroscopy we find detailed evidence for an extended cross-β structure. This amyloid assembly helps to account for the strength and amphiphilic properties of the vesicle wall. Buoyancy organelles thus dramatically extend the scope of known functional amyloids.     

Site-specific protein dynamics in communication pathway from sensor to signaling domain of oxygen sensor protein, HemAT-Bs: Time-resolved Ultraviolet Resonance Raman Study

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ～50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ～50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.     

Detection of Pseudomonas aeruginosa in sputum headspace through volatile organic compound analysis

Introduction

Chronic pulmonary infection is the hallmark of Cystic Fibrosis lung disease. Searching for faster and easier screening may lead to faster diagnosis and treatment of Pseudomonas aeruginosa (P. aeruginosa). Our aim was to analyze and build a model to predict the presence of P. aeruginosa in sputa.

Methods

Sputa from 28 bronchiectatic patients were used for bacterial culturing and analysis of volatile compounds by gas chromatography–mass spectrometry. Data analysis and model building were done by Partial Least Squares Regression Discriminant analysis (PLS-DA). Two analysis were performed: one comparing P. aeruginosa positive with negative cultures at study visit (PA model) and one comparing chronic colonization according to the Leeds criteria with P. aeruginosa negative patients (PACC model).

Results

The PA model prediction of P. aeruginosa presence was rather poor, with a high number of false positives and false negatives. On the other hand, the PACC model was stable and explained chronic P. aeruginosa presence for 95% with 4 PLS-DA factors, with a sensitivity of 100%, a positive predictive value of 86% and a negative predictive value of 100%.

Conclusion

Our study shows the potential for building a prediction model for the presence of chronic P. aeruginosa based on volatiles from sputum.     

Measurement of hepatic protein fractional synthetic rate with stable isotope labeling technique in thapsigargin stressed HepG2 cells

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97 ± 0.02 and 0.99 ± 0.05%/hr calculated from 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68 ± 0.03 and 0.60 ± 0.06%/hr in the TG treatment group (p<0.05, vs. control). TG-induced ER stress inhibited hepatic protein synthesis. The stable isotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis.     

气相色谱法测定食品中的抗氧化剂BHA，BHT，TBHQ

本文利用甲醇和石油醚直接提取油脂和含油脂食品中的抗氧化剂丁基羟基茴香醚(BHA),二丁基羟基甲苯(BHT),特丁基对苯二酚(TBHQ),建立同时测定这三种抗氧化剂的气相色谱方法。采用安捷伦HP-5毛细管柱(i.d.0.25μm×30.0m),氢火焰离子化检测器,外标法定量测量,经实验,样品加标回收率为95%-104%,检出限分别为BHA:0.22 mg/L,BHT:0.23 mg/L,TBHQ:0.33 mg/L。该方法具有前处理操作简便,灵敏度高,重现性好等优点。     

Grafting of cucumber as a means to minimize copper toxicity

The aim of the current work was to determine whether grafting could improve copper (Cu) tolerance of cucumber, and to study the changes induced by the rootstock in the shoot growth at agronomical and physiological levels. A greenhouse experiment was carried out to determine yield, growth, fruit quality, leaf gas exchange, chlorophyll and carotenoids contents, electrolyte leakage, mineral composition and assimilate partitioning of cucumber plants (Cucumis sativus L. cv. ‘Akito’), either ungrafted or grafted onto the commercial rootstock ‘Shintoza’ (Cucurbita maxima Duchesne × Cucurbita moschata Duchesne) and cultured in nutrient film technique (NFT). Plants were supplied with nutrient solutions having three levels of Cu concentration [0.3 (control), 47, or 94 μM]. Significant depression of yield, shoot and root biomass production, and fruit quality (low fruit pH, and high Cu content) in Cu treated cucumber plants was observed, and this effect varied as a function of Cu concentration in NFT solution. The relative yield of cucumber decreased by ≈3.4% for each unit of increase in leaf tissue Cu concentrations above the threshold value (7.8 μg g⁻¹). At the two higher Cu concentrations (47 and 94 μM Cu), the percentages of yield, shoot and root biomass weight reductions were significantly lower in grafted plants in comparison to those of the ungrafted plants. Excessive Cu, especially at 94 μM Cu, inhibited photosynthesis, pigment synthesis, and membrane integrity. The Cu-related reductions in net assimilation, stomatal conductance, chlorophyll and carotenoid content were more severe in ungrafted plants in comparison with those grafted on 'Shintoza’. The percentage of electrolyte leakage was significantly higher in ungrafted plants especially those with severe Cu toxicity (94 μM Cu). The accumulation of Cu in leaf tissue at 47 and 94 μM Cu, with respect to control, were significantly lower in grafted plants (138 and 181%, respectively) in comparison to that of ungrafted plants (about 235 and 392%, respectively). Significant reduction of macro- (N, K, Ca and Mg) and micro-elements (Fe, Mn and Zn) in cucumber leaf tissue was found under moderate and severe Cu stresses especially on ungrafted plants. The improved crop performance of grafted plants was attributed to their strong capacity to inhibit Cu accumulation in the aerial parts and to maintain a better plant nutritional status.     

Phylogenetic characterization of bacterial consortia obtained of corroding gas pipelines in Mexico

Characterization of the microbial populations formed in gas pipelines is essential to understand the metallic surface-microbe interaction, their role in metal corrosion, and to implement efficient monitoring and control strategies. Microbial community analysis in a corroded gas pipeline in a petroleum-producing facility in the Southeast region in Mexico was performed by traditional cultivation techniques and identification based on 16S rRNA gene sequence. In all samples, thin bacterial biofilms were observed and pitting corrosion was reveled after removing the biofilms. Six pure or mixed cultures of anaerobic bacteria were obtained and their 16S rRNA libraries were constructed, respectively. At least two members of each RFLP profile were sequenced and the phylogenetic affiliations of cloned bacterial 16S rRNA genes indicated that native biofilms were mainly colonized by Desulfovibrio vulgaris and Desulfovibrio desulfuricans, sulfate-reducing bacteria members; Citrobacter freundii, an Enterobacteriaceae member; Clostridium celerecrescens and Clostridium sporogenes, spore-forming anaerobic species and Cetobacterium somerae, a microaerotolerant, non-spore-forming fusobacteria. Some of these species have been observed consistently in other steel pipelines previously, but Cetobacterium members and C. celerecrescens are described for the fist time in this corroded gas pipeline. The potential role of each species in biofilm formation and steel corrosion is discussed.     

Capture and reconstitution of G protein-coupled receptors on a biosensor surface

Surface plasmon resonance (SPR) biosensors offer a unique opportunity to study the binding activity of G protein-coupled receptors (GPCRs) in real time with minimal sample preparation. Using two chemokine receptors (CXCR4 and CCR5) as model systems, we captured the proteins from crude cell preparations onto the biosensor surface and reconstituted a lipid environment to maintain receptor activity. The conformational states of the receptors were probed using conformationally dependent antibodies, and by characterizing the binding properties of a native chemokine ligand (stromal cell-derived factor 1alpha). The results suggest that the detergent-solubilized receptors are active for ligand binding in the presence and absence of a reconstituted bilayer. There are three advantages to using this receptor-capturing approach: (1) there is no need to purify the receptor prior to immobilization on the biosensor surface, (2) the receptors are homogeneously immobilized through the capturing step, and (3) the receptors can be captured at high enough densities to allow the study of relatively low-molecular-mass ligands (2000-4000Da). We also demonstrated that the receptors are sensitive to the solubilizing conditions, which illustrates the potential for using SPR biosensors to rapidly screen solublization conditions for GPCRs.     

Identification of carbonyl sulfide and sulfur dioxide in porcine coronary artery by gas chromatography/mass spectrometry, possible relevance to EDHF

Incubation of porcine coronary artery rings and cardiac muscle tissue in Krebs buffer followed by GC/MS analysis of the headspace gas revealed two gases, carbonyl sulfide (COS) and sulfur dioxide (SO(2)). The gases were identified by characteristic ions obtained by electron ionization, and by comparison of the retention time on a chromatographic column (GS GasPro) with standards of these gases. Stimulation of the arterial rings with acetylcholine and calcium ionophore A23187 increased the levels of SO(2) and COS in the vascular tissue. We also provide evidence that SO(2) could originate from disproportionation of a very unstable gas, sulfur monoxide (S=O). We suggest potential origins of these gases and discuss their relevance to endothelium-derived hyperpolarizing factor.