Initial disulfide formation steps in the folding of an omega-conotoxin

To determine whether the native disulfides of omega-conotoxins are preferentially stabilized early in the folding of these small proteins, the rates and equilibria for disulfide formation were measured for three analogues of omega-conotoxin MVIIA. In each analogue, one of the three pairs of disulfide-bonded Cys residues was replaced with Ala residues, leaving four Cys residues that can form six intermediates with one disulfide and three species with two disulfides. For each analogue, all of the disulfide-bonded species were identified, and the equilibrium constants for forming the individual species via exchange with oxidized and reduced glutathione were measured. These equilibrium constants represent effective concentrations of the Cys thiols and ranged from 0.01 to 0.4 M in the fully reduced protein. There was little or no preference for forming the native disulfides, and the equilibria for forming the first and second disulfides decreased only slightly upon the addition of 8 M urea. The data for the four-Cys analogues, together with equilibrium data for the six-Cys form, were also used to estimate effective concentrations for forming a third disulfide once two native disulfides are present. These effective concentrations were approximately 100 and 10 M in the presence of 0 and 8 M urea, respectively. The results indicate that there is little or no preferential formation of native interactions in the folding of these molecules until two disulfides have formed, after which there is a high degree of cooperativity among the native interactions.     

Overexpression of the cgtA (yhbZ,obgE) gene,coding for an essential GTP-binding protein,impairs the regulation of chromosomal functions in Escherichia coli

GTPases belonging to the Obg/Gtp1 subfamily are essential proteins in most bacterial species and are evolutionarily conservative from bacteria to humans. However, their specific functions in the regulation of cellular processes are largely unknown. Here we demonstrate that overproduction of a member of the Obg/Gtp1 subfamily, cgtA ( yhbZ, obgE) gene product, in Escherichia coli is deleterious for bacterial growth. However, syntheses of DNA, RNA, and proteins were not significantly affected under these conditions as measured by efficiency of incorporation of radioactive precursors. On the other hand, flow cytometry studies revealed that cgtA-overexpressing bacteria form enlarged cells with significantly changed distribution of chromosomal DNA. These results strongly suggest that overproduction of a GTP-binding protein from the Obg/Gtp1 subfamily impairs regulation of some chromosomal functions in E. coli, especially synchronization of DNA replication initiation and possibly also partitioning of daughter chromosomes after a replication round.     

An iTRAQ characterisation of the role of TolC during electron transfer from Shewanella oneidensis MR‐1

Anodophilic bacteria have the ability to generate electricity in microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigated the anode‐specific responses of Shewanella oneidensis MR‐1, an exoelectroactive Gammaproteobacterium, using for the first time iTRAQ and 2D‐LC MS/MS driven membrane proteomics to compare protein abundances in S. oneidensis when generating power in MFCs, and growing in a continuous culture. The regulated dataset produced was enriched in membrane proteins. Proteins shown to be more abundant in anaerobic electroactive anodic cells included efflux pump TolC and an uncharacterised tetratricopeptide repeat (TPR) protein, whilst the TonB2 system and associated uncharacterised proteins such as TtpC2 and DUF3450 were more abundant in microaerobic planktonic cells. In order to validate the iTRAQ data, the functional role for TolC was examined using a δTolC knockout mutant of S. oneidensis. Possible roles for the uncharacterised proteins were identified using comparative bioinformatics. We demonstrate that employing an insoluble extracellular electron acceptor requires multiple proteins involved in cell surface properties. All MS and processed data are available via ProteomeXchange with identifier PXD004090.     

Autonomic Predictors of Hospitalization Due to Heart Failure Decompensation in Patients with Left Ventricular Systolic Dysfunction

Introduction

Autonomic nervous system balance can be significantly deteriorated during heart failure exacerbation. However, it is still unknown whether these changes are only the consequence of heart failure decompensation or can also predict development thereof. Objectives were to verify if simple, non-invasive autonomic parameters, such as baroreflex sensitivity and short-term heart rate variability can provide independent of other well-known clinical parameters information on the risk of heart failure decompensation in patients with left ventricular systolic dysfunction.

Methods

In 142 stable patients with left ventricular ejection fraction ≤ 40%, baroreflex sensitivity and short-term heart rate variability, as well as other well-known clinical parameters, were analyzed. During 23 ± 9 months of follow-up 19 patients were hospitalized due to the heart failure decompensation (EVENT).

Results

Pre-specified cut-off values of baroreflex sensitivity (≤2.4 ms/mmHg) and low frequency power index of heart rate variability (≤19 ms²) were significantly associated with the EVENTs (hazard ratio 4.43, 95% confidence interval [CI] 1.35–14.54 and 5.41, 95% CI 1.87–15.65 respectively). EVENTs were also associated with other parameters, such as left ventricular ejection fraction, NYHA class, diuretic use, renal function, brain natriuretic peptide and hemoglobin level, left atrial size, left and right ventricular heart failure signs. After adjusting baroreflex sensitivity and low frequency power index for each of the abovementioned parameters, autonomic parameters were still significant predictors of hospitalization due to the heart failure decompensation.

Conclusion

Simple, noninvasive autonomic indices can be helpful in identifying individuals with increased risk of hospitalization due to the heart failure decompensation among clinically stable patients with left ventricular systolic dysfunction, even when adjusted for other well-known clinical parameters.     

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H₂O₂), H₂O₂ and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H₂O₂ or H₂O₂ and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24?h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.     

The resolution and biochemical characterization of subcomplexes of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II)

LHC II isolated from carnation leaves has been solubilized and resolved by a newly developed, vertical-bed non-denaturing isoelectric focusing in polyacrylamide slab gels to yield three trimeric subcomplexes focusing at pH 4.52, 4.42 and 4.37 (designated a, b and c, respectively), comprising approximately 38%, 24% and 38% of the chlorophyll. The spectroscopic data demonstrated a close similarity among LHC II subcomplexes concerning their chlorophyll content and organization. The most alkaline and the most acidic subcomplex contained the 27 kDa polypeptide of LHC II while the intermediate pI fraction contained both LHC II polypeptides, i.e. 27 kDa and 26 kDa ones associated at 2:1 stoichiometry. The 27 kDa polypeptide could be resolved by denaturing isoelectrofocusing into 10 pI molecular isoforms covering 5.90–4.20 pH range. Three of the isoforms were found in the subcomplexes a and b and eight in the subcomplex c. The 26 kDa polypeptide comprised the unique pI molecular isoform focusing at pH 5.61.Abbreviations CBB G-250 Coomassie Brilliant Blue G-250 - chl chlorophyll - DM n-dodecyl--d-maltoside - EDTA ethylendiaminotetraacetic acid - IEF isoelectric focusing - LHC II the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - LHCP II apoprotein of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - NP-40 polyethyleneglycol-p-isooctylphenyl ether - pI isoelectric point - OG octyl--d-glucopyranoside - PS II Photosystem II - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TCA trichlorooacetic acid