Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1

The respiratory chain in the inner mitochondrial membrane contains three large multi‐enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19‐Å 3D map of the 1.7‐MDa amphipol‐solubilized supercomplex I₁III₂IV₁ from bovine heart obtained by single‐particle electron cryo‐microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X‐ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol‐binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.     

The diagnostic accuracy of high-mobility group box 1 protein and twelve other markers in discriminating bacterial, viral and co-infected bronchial pneumonia in Han children

Pneumonia in children is common and can lead to grave consequences if not addressed in a proper and timely manner. In the management of pneumonia, early identification of the causative infective agent is of obvious importance for treatment, as it allows selection of the appropriate antibiotics. However, such identification requires laboratory test results, which may not be immediately available. The aim of this study was to evaluate the accuracy and usefulness of 13 markers in differentiating between viral and bacterial pneumonia in Han children (34 healthy controls and 78 patients). It was found that WBC counts were more accurate in diagnosis of the type of agent responsible for infection than was the degree of expression of HMGB1. Among the 13 markers investigated, HMGB1 was the best at discriminating between co-infected (bacterium and virus) and single-infected (bacterium or virus) children with bronchial pneumonia. HMGB1 expression of less than 1.0256, excluded most co-infections (the negative predictive value was greater than 89.7%). Diagnosed sole viral pneumonia clinically overlapped with bacterial pneumonia, but bacterial pneumonia was more often associated with higher white blood cell (WBC) counts (WBC ≥ 13,000 cells/mm(3)). When the two marker readouts--HMGB1 < 1.0256 and WBC ≥ 13,000 cells/mm(3)--were combined, the positive predictive value for bacterial pneumonia alone was 92.3%. These findings can help clinicians discriminate between bronchial pneumonia caused by virus, bacterium or both with a high specificity.     

Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve

In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8-7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.     

Extracellular-superoxide dismutase expression during monocytic differentiation of U937 cells

Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.     

Phosphoproteome profiling for cold temperature perception

Temperature sensation initiates from the activation of cellular receptors when the cell is exposed to a decrease in temperature. Here, we applied a phosphoproteome profiling approach to the human lung epithelial cell line BEAS‐2B to elucidate cellular cold‐responsive processes. The primary aim of this study was to determine which intracellular changes of phosphorylation are accompanied by cold sensation. Eighteen protein spots that exhibited differentially phosphorylated changes in cells were identified. Most of the proteins that were phosphorylated after 5 or 10 min were returned to control levels after 30 or 60 min. Identified proteins were mainly RNA‐related (i.e., they were involved in RNA binding and splicing). Temperature (18 and 10°C) stimuli showed homologies that were detected for time course changes in phosphoproteome. The data indicated a time‐shift between two temperatures. The phosphorylation of putative cold responsive markers, such as ribosomal protein large P0 and heterochromatin‐associated proteins 1, were verified by Western blotting in cells transfected with TRPM8 or TRPA1. J. Cell. Biochem. 112: 633–642, 2011. © 2010 Wiley‐Liss, Inc.     

Recombinant immunosuppressive protein from Pimpla hypochondrica venom (rVPr1) increases the susceptibility of Mamestra brassicae larvae to the fungal biological control agent, Beauveria bassiana

Although fungi are used to control a variety of insect pests, it is accepted that their usage could be increased if their efficacy was greater. The outcome of the interaction of a fungus and a pest insect may be influenced by a number of criteria, including the ability of the insect to mount effective immune responses against the pathogen. In view of this, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) can increase the susceptibility of larvae of the lepidopteran pest, Mamestra brassicae, to the fungal biological control agent, Beauveria bassiana. Bioassays indicated that when larvae were injected with 3.5 μl of rVPr1 and 100 B. bassiana conidia (combined injection assays), a significant reduction in survival of larvae occurred compared with each treatment on its own (P=0.006). Similar results were obtained when larvae were dipped in a solution containing 3 × 10(6) B. bassiana conidia per ml and then injected with 3.5 μl of rVPr1 2 days later (topical application assays), (P<0.001). These results indicate that rVPr1 can increase the efficacy of B. bassiana toward a lepidopteran pest, and are discussed within the context of insect immune responses and integrated pest management.     

α-Enolase binds to RNA

To detect proteins binding to CUG triplet repeats, we performed magnetic bead affinity assays and North-Western analysis using a (CUG)₁₀ ssRNA probe and either nuclear or total extracts from rat L6 myoblasts. We report the isolation and identification by mass spectrometry and immunodetection of α-enolase, as a novel (CUG)n triplet repeat binding protein. To confirm our findings, rat recombinant α-enolase was cloned, expressed and purified; the RNA binding activity was verified by electrophoretic mobility shift assays using radiolabeled RNA probes. Enolase may play other roles in addition to its well described function in glycolysis.     

Mechanistic insight of photo-induced aggregation of chicken egg white lysozyme: the interplay between hydrophobic interactions and formation of intermolecular disulfide bonds

Recently, it was reported that ultraviolet (UV) illumination could trigger the unfolding of proteins by disrupting the buried disulfide bonds. However, the consequence of such unfolding has not been adequately evaluated. Here, we report that unfolded chicken egg white lysozyme (CEWL) triggered by UV illumination can form uniform globular aggregates as confirmed by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The assembling process of such aggregates was also monitored by several other methods, such as circular dichroism, fluorescence spectroscopy, mass spectrometry based on chymotrypsin digestion, ANS-binding assay, Ellman essay, and SDS-PAGE. Our finding is that due to the dissociation of the native disulfide bonds by UV illumination, CEWL undergoes drastic conformational changes resulting in the exposure of some hydrophobic residues and free thiols. Subsequently, these partially unfolded molecules self-assemble into small granules driven by intermolecular hydrophobic interaction. With longer UV illumination or longer incubation time, these granules can further self-assemble into larger globular aggregates. The combined effects from both the hydrophobic interaction and the formation of intermolecular disulfide bonds dominate this process. Additionally, similar aggregation behavior can also be found in other three typical disulfide-bonded proteins, that is, α-lactalbumin, RNase A, and bovine serum albumin. Thus, we propose that such aggregation behavior might be a general mechanism for some disulfide-bonded proteins under UV irradiation.     

Predicted structures of agonist and antagonist bound complexes of adenosine A3 receptor

We used the GEnSeMBLE Monte Carlo method to predict ensemble of the 20 best packings (helix rotations and tilts) based on the neutral total energy (E) from a vast number (10 trillion) of potential packings for each of the four subtypes of the adenosine G protein-coupled receptors (GPCRs), which are involved in many cytoprotective functions. We then used the DarwinDock Monte Carlo methods to predict the binding pose for the human A(3) adenosine receptor (hAA(3)R) for subtype selective agonists and antagonists. We found that all four A(3) agonists stabilize the 15th lowest conformation of apo-hAA(3)R while also binding strongly to the 1st and 3rd. In contrast the four A(3) antagonists stabilize the 2nd or 3rd lowest conformation. These results show that different ligands can stabilize different GPCR conformations, which will likely affect function, complicating the design of functionally unique ligands. Interestingly all agonists lead to a trans χ1 angle for W6.48 that experiments on other GPCRs associate with G-protein activation while all 20 apo-AA(3)R conformations have a W6.48 gauche+ χ1 angle associated experimentally with inactive GPCRs for other systems. Thus docking calculations have identified critical ligand-GPCR structures involved with activation. We found that the predicted binding site for selective agonist Cl-IB-MECA to the predicted structure of hAA(3)R shows favorable interactions to three subtype variable residues, I253(6.58), V169(EL2), and Q167(EL2), while the predicted structure for hAA(2A)R shows weakened to the corresponding amino acids: T256(6.58), E169(EL2), and L167(EL2), explaining the observed subtype selectivity.