103 research outputs found
Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli
Cytochrome bd is a tri-heme (b558, b595, d) respiratory oxygen reductase that is found in many bacteria including pathogenic
species. It couples the electron transfer from quinol to O2 with generation of an electrochemical proton gradient. We
examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in oneelectron
reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm
excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast
(t,1.5 ms) electron transfer from heme d to heme b595 in a small fraction of the protein, not reported earlier. Then the
electron migrates to heme b558 (t,16 ms). It returns from the b-hemes to heme d with t,180 ms. Unlike cytochrome bd in
the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is
small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an
internal ligand (L) at the opposite side of the heme. CO recombines with heme d (t,16 ms) yielding a transient
hexacoordinate state (CO-Fe2+
-L). Then the ligand slowly (t,30 ms) dissociates from heme d. Recombination of CO with a
reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to
heme d (t,20 ms), some heme b558 (t,0.2–3 ms), and finally migrates from heme d to heme b595 (t,24 ms) in ,5% of the
enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the
membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in
excitation and other experimental conditions
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Automated Segmentation of HeLa Nuclear Envelope from Electron Microscopy Images
This paper describes an image-processing pipeline for the automatic segmentation of the nuclear envelope of HeLcells observed through Electron Microscopy. The pipeline was applied to a 3D stack of 300 images. The intermediate results of neighbouring slices are further combined to improve the final results. Comparison with a handsegmented ground truth reported Jaccard similarity values between 94-98% on the central slices with a decrease towards the edges of the cell where the structure was considerably more complex. The processing is unsupervised and each 2D slice is processed in about 5-10 seconds running on a MacBook Pro. No systematic attempt to make the code faster was made. These encouraging results could be further used to provide data for more complex segmentation techniques like Deep Learning, which require a considerable amount of data to train architectures like Convolutional Neural Networks. The code is freely available from https://github.com/reyesaldasoro/HeLa-Cell-Segmentatio
Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.
Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Ă… structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Ă… structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I
A Model of Brain Circulation and Metabolism: NIRS Signal Changes during Physiological Challenges
We construct a model of brain circulation and energy metabolism. The model is
designed to explain experimental data and predict the response of the
circulation and metabolism to a variety of stimuli, in particular, changes in
arterial blood pressure, CO2 levels, O2 levels, and
functional activation. Significant model outputs are predictions about blood
flow, metabolic rate, and quantities measurable noninvasively using
near-infrared spectroscopy (NIRS), including cerebral blood volume and
oxygenation and the redox state of the CuA centre in cytochrome
c oxidase. These quantities are now frequently measured in
clinical settings; however the relationship between the measurements and the
underlying physiological events is in general complex. We anticipate that the
model will play an important role in helping to understand the NIRS signals, in
particular, the cytochrome signal, which has been hard to interpret. A range of
model simulations are presented, and model outputs are compared to published
data obtained from both in vivo and in vitro
settings. The comparisons are encouraging, showing that the model is able to
reproduce observed behaviour in response to various stimuli
Three dimensional electron microscopy reveals changing axonal and myelin morphology along normal and partially injured optic nerves
Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma
Mammalian NADH:ubiquinone oxidoreductase (Complex I) and nicotinamide nucleotide transhydrogenase (Nnt) together regulate the mitochondrial production of H2O2—Implications for their role in disease, especially cancer
Influence of heat treatment on the electrophysical properties of thin films of copper-indium diselenide
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