A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

Identification of archaeobotanical Pistacia L. fruit remains: implications for our knowledge on past distribution and use in prehistoric Cyprus

Vegetation History and Archaeobotany - Pistacia spp. remains are common finds among archaeobotanical assemblages in prehistoric sites in Southwest Asia, both in the form of endocarps and charcoal...     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a(3) as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK(a) values during the pumping process are derived for comparison to experiments.     

Molecular rearrangement in POR macrodomains as a reason for the blue shift of chlorophyllide fluorescence observed after phototransformation

The activation energy and activation volume of the spectral blue shift subsequent to protochlorophyllide phototransformation (called Shibata shift in intact leaves) were studied in prolamellar body (PLB) and prothylakoid-(PT)-enriched membrane fractions prepared from dark-grown wheat (Triticum aestivum, L.) leaves. The measurements were done at 20, 30 and 40 degrees C and at various pressure values. The activation energy values were 181+/-8 kJ mol(-1) and 188+/-6 kJ mol(-1) for the PLBs and the PTs, respectively. The pressure stabilized the structure of the NADPH:protochlorophyllide oxidoreductase (POR) macrodomains; it prevented or slowed down the blue shift. There were no significant differences between the activation volumes of PLBs and PTs at 30 or 40 degrees C giving values around 100-125 ml mol(-1) which correspond to changes in the tertiary structure of proteins but also resemble the volume changes occurring during the disaggregation of protein dimers or oligomers, or during dissociation of peripheral membrane proteins from membranes. The small differences in the activation parameters of PLBs and PTs indicate that molecular rearrangements inside the POR macrodomains are the primary reasons of the fluorescence blue shift; however, their lipid microenvironment must be also important in the initialization of the shift.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a₃ as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK_a values during the pumping process are derived for comparison to experiments.     

Automatic fermentation control based on a real-time in situ SIRE biosensor regulated glucose feed

Monitoring and regulation of fermentations is of a paramount industrial and academic importance in order to keep conditions optimal during the entire process. Established techniques employed today include HPLC and spectrophotometry, which both have the disadvantage that broth samples have to be drawn from the fermentor and that they often require sample pre-treatment. The objectives of this study was to design and evaluate a software controlled automatic real-time SIRE biosensor connected to a glucose feed solution pump for in situ based monitoring and regulation of the glucose concentration during a yeast fermentation process. The maximal frequency for the measuring-regulation cycles was 30/h. A 10 mM mean glucose concentration level was successfully maintained within +/-0.013 mM during 60 min fermentations at various concentrations of yeast (10, 20, 40 and 80g/l). The on/off-regulator used caused some expected fluctuations (oscillations) of the glucose concentration around the mean value (+/-0.12 mM at 10 g/l, +/-0.26 mM at 20 g/l, +/-0.51 mM at 40 g/l, and +/-0.99 mM at 80 g/l). A 7-h fermentation process (10 mM glucose and 20 g/l yeast) was successfully monitored and regulated. The obtained measuring data were found to be 8.5-22.9% lower than data obtained with a commercially available spectrophotometric kit. The difference increased linearly (-0.26 mM/h), during the fermentation process and indicated that some clogging of the in situ positioned probe occurred. The speed and the automatisation adaptability of the presented device suggest advantages compared to established techniques.     

Exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle RNA

Background

Exosomes are small extracellular nanovesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling functional RNA from one cell to another. Exosomes are released by many cells including mast cells, dendritic cells, macrophages, epithelial cells and tumour cells. Exosomes differ compared to their donor cells, not only in size, but also in their RNA, protein and lipid composition.

Methodology/Principal Findings

In this study, we show that exosomes, released by mouse mast cells exposed to oxidative stress, differ in their mRNA content. Also, we show that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Furthermore, Affymetrix microarray analysis revealed that the exosomal mRNA content not only differs between exosomes and donor cells, but also between exosomes derived from cells grown under different conditions; oxidative stress and normal conditions. Finally, we also show that exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress.

Conclusions/Significance

These results argue that the exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stress stimulus.