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Biomedical subjects

M T Wilson

Publications and source records attributed to M T Wilson.

At least 55 records · Page 3Linked to original sources
PubMed1998

Reactivity of nitric oxide with cytochrome c oxidase: interactions with the binuclear centre and mechanism of inhibition.

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the Ki being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized CuB leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from CuB to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.

Animals↗
PubMed1998

Ultrastructure of the frog retina after high-pressure freezing and freeze substitution.

In many types of tissue, high-pressure freezing (HPF), followed by freeze substitution, can produce excellent ultrastructural preservation at depths over 10 times that obtained by other cryofixation techniques. However, in the case of neural tissue, the benefits of HPF have not been realized. In the present study, isolated frog (Rana pipiens) retina was sliced at a thickness of 150 or 350 microns, rapidly frozen in a Balzers HPM 010 high-pressure freezer, and freeze substituted with 1% OsO4 and 0.1% tannic acid in acetone. Specially designed HPF chambers and specific freezing media (35% high-MW dextran for 150-micron slices or 15% low-MW dextran for 350-micron slices) were required for adequate freezing. The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-micron slices, compared with chemically fixed slices. Specifically, HPF resulted in better preserved cellular, mitochondrial and nuclear membranes in all retinal layers. This is the first study to successfully cryofix all of the layers of the retina. The increased depths of adequate freezing achieved by HPF should facilitate various ultrastructural studies of retina, as well as of other CNS tissues, where preservation approaching that of the 'native' state is required.

Animals↗
PubMed1998

Functional mapping of human sensorimotor cortex with electrocorticographic spectral analysis. I. Alpha and beta event-related desynchronization.

Human scalp EEG studies have shown that event-related desynchronization (ERD) in the alpha (8-13 Hz) and beta (15-25 Hz) bands may be used to detect functional activation of sensorimotor cortex. However, in most previous studies somatotopy has not been examined in detail and brief, self-paced movements, focusing on the planning of motor output, have been used. We recorded electrocorticographic (ECoG) signals in five clinical subjects during a visual-motor decision task that was designed to activate the representations of different body parts in sensorimotor cortex. To focus more on execution of motor output than on its planning, subjects were instructed to make sustained isometric muscle contractions in different body parts (tongue protrusion, fist-clenching or foot dorsiflexion) in response to randomized visual stimuli depicting each action. ECoG spectral analysis utilized a mixed-effects analysis of variance model in which within-trial temporal dependencies were taken into account, and the magnitude and statistical significance of alpha and beta ERDs were mapped onto a surface rendering of each subject's brain MRI. Cortical electrical stimulation was performed in all subjects for clinical purposes, and the resulting maps of sensorimotor function were compared with those generated by ECoG spectral analysis. During the early phases of the motor responses, alpha ERD commonly occurred in a diffuse spatial pattern that was not somatotopically specific. During the late phases, the spatial pattern of alpha ERD usually became more focused and somatotopically specific. Maps of alpha ERD were closer to cortical stimulation maps when alpha ERD was sustained throughout the late phases of the motor responses. Thus, the topography of alpha ERD more resembled traditional somatotopy when its temporal profile approximated that of the motor response. The topography of beta ERD was often more discrete and somatotopically specific than that of alpha ERD, but beta ERD was often transient and sometimes absent. Sometimes, unilateral limb movement produced sustained alpha and beta ERD over bilateral sensorimotor cortices, with overlapping patterns for different body parts. The topographical spread of alpha ERD beyond expected functional-anatomical boundaries during early (and sometimes late) phases of motor responses invites a re-examination of traditional assumptions about sensorimotor functional neuroanatomy, as well as the role of alpha ERD as an index of cortical activation. We agree with others that the somatotopic representations of different body parts overlap more than previously thought. Also, unilateral limb movements may be associated with both contralateral and ipsilateral activation of sensorimotor cortex. We conjecture that alpha ERD may reflect activity within a broad synaptic network with distributed cortical representations.

Adult↗
PubMed1998

Comparative analysis of pulsed (activated) and resting chicken cytochrome c oxidases with sodium chloride dependence of the enzyme.

Comparative investigation of pulsed (activated) chicken cytochrome c oxidase and resting enzyme are compared with other species and pH dependent properties are also monitored Sodium chloride dependence of chicken oxidase is analysed and compared with that of beef cytochrome c oxidase. Carbon monoxides binding and spectral changes during this process are also discussed. S values are also determined at different pH of the enzyme ultra centrifugation.

Journal Article↗
PubMed1997

Nitric oxide ejects electrons from the binuclear centre of cytochrome c oxidase by reacting with oxidised copper: a general mechanism for the interaction of copper proteins with nitric oxide?

Small increases in NO concentration can inhibit mitochondrial oxygen consumption by reacting at the binuclear haem a3/CuB oxygen reduction site of cytochrome c oxidase. Here we demonstrate that under normal turnover conditions NO reacts initially with the oxidised CuB rather than the haem a3. We propose that hydration of an initial Cu+/NO+ complex forms nitrite, a proton and CuB+; the latter ejects an electron from the binuclear centre and results in the observed (100 s(-1)) reduction of other electron transfer centres in the enzyme (haem a and CuA). These reactions may have implications for the interactions of NO with other copper proteins.

Binding Sites↗
PubMed1997

Photochemical electron injection into redox-active proteins.

A new method is presented that makes it possible to inject electrons rapidly into redox-active proteins by means of a short light flash. Reduced carboxymethylated cytochrome c (CmCyt c) with carbon monoxide bound to the heme iron is mixed with the oxidized acceptor protein. Upon rapid photodissociation of CO the apparent redox potential of CmCyt c drops, resulting in electron transfer to the electron acceptor. In this study we have used mitochondrial cytochrome c oxidase as the acceptor protein, but the method also can be used to investigate electron transfer to other proteins that can interact with cytochrome c. In principle, it can be used with any redox protein into which a CO binding site at the heme iron can be engineered.

Animals↗
PubMed1997

The inhibition of cytochrome c oxidase by nitric oxide using S-nitrosoglutathione.

The effect of the free radical nitric oxide (NO) on the activity of isolated cytochrome sigma oxidase was investigated by using ferrocytochrome sigma as an electron donor, and the system SNOG/DTT, which produces a steady-state NO concentration similar to that expected to be found in vivo. The initial electron entry into the heme a/Cu a center and the initial rate of the electron transfer between the two hemes were not affected by the presence of NO. Under our conditions, the rate of inhibition of cytochrome c oxidase was found to be dependent both on the SNOG (NO concentration) and on the ferrocytochrome c concentration (electron entry rate). The data confirm that NO binds exclusively at the binuclear center, and that the NO binding in these conditions requires the presence of an intermediate populated only in turnover. Accordingly, we found that the rate of inhibition is directly related to the electron entry rate. In addition, a residual activity seems to be present in cytochrome c oxidase in the presence of nitric oxide, suggesting that NO can act as an electron acceptor to cytochrome c oxidase in the presence of oxygen.

Cytochrome c Group↗
PubMed1997

The globin-based free radical of ferryl hemoglobin is detected in normal human blood.

Normal human venous blood was studied by electron paramagnetic resonance (EPR) spectroscopy at -196 degrees C. The EPR signal of free radicals in frozen blood is shown to have the same radiospectroscopic parameters and properties as the signal of the globin based free radical, .Hb(Fe(IV)=O), formed in the reaction of purified methemoglobin (metHb) with H2O2 and therefore has been assigned as such. The globin-based radicals and metHb exhibited significant variation (fluctuations) in different frozen samples taken from the same liquid blood sample. In any given sample a high concentration of free radicals was associated with a low concentration of metHb and vice versa, i.e. the fluctuations were always of opposite sense. No such fluctuations were observed in the concentration of two other paramagnetic components of blood, transferrin and ceruloplasmin. The time course of free radical formation and decay upon the addition of H2O2 to purified metHb was studied at three different molar ratios H2O2/metHb. This kinetic study together with the results of an annealing experiment allow us to propose a mechanism for the formation and decay of the globin-based radical in blood. Within this mechanism, the source of H2O2 in blood is considered to be dismutation of O-2 radicals produced via autoxidation of Hb. We postulate that the dismutation is intensified on the phase separation surfaces during cooling and freezing of a blood sample. The fluctuations are explained within this hypothesis.

Electron Spin Resonance Spectroscopy↗
PubMed1997

Reduction of Cu(II) by lipid hydroperoxides: implications for the copper-dependent oxidation of low-density lipoprotein.

The Cu(II)-promoted oxidation of lipids is a lipid hydroperoxide (LOOH)-dependent process that has been used routinely to assess the oxidizability of low-density lipoprotein (LDL) in human subjects. Metal-dependent redox reactions, including those mediated by copper, have been implicated in the pathogenesis ofatherosclerosis. Despite its widespread use and possible biological significance, key elements of the mechanism are not clear. For example, although it is evident that copper acts as a catalyst, which implies a redox cycle between the Cu(II) and Cu(I) redox states, the reductants remain uncertain. In LDL these could include alpha-tocopherol, amino acid residues on the protein and LOOH. However, both alpha-tocopherol and amino acid residues are probably consumed before the most rapid phase of lipid peroxidation occurs, suggesting that another reductant must be donating electrons to Cu(II), the most likely candidate being LOOH. This role has been disputed, since LDLs nominally devoid of LOOH are still capable of reducing Cu(II) to Cu(I) and thermodynamic calculations for this reaction are not favourable. Direct investigation of the role of LOOH as reductant has not been reported and in the present study, using simple lipid systems and LDL, we have re-examined this issue using the Cu(I) chelator bathocuproine. We have shown that Cu(II) may promote lipid peroxidation in liposomes, which do not contain either protein or alpha-tocopherol, and that this is associated with reduction to Cu(I). The data also indicate that an equilibrium between free Cu(II) and LOOH exists, which only in the presence of an oxidizable substrate, i.e. unsaturated fatty acids, is shifted towards formation of Cu(I) and lipid-derived peroxyl radicals. We propose that reduction of Cu(II) by LOOH is a necessary component in sustaining the propagation of lipid peroxidation and that the formation of peroxyl radicals and their products in a lipid environment is sufficient to overcome thermodynamic barriers to the reaction.

Copper↗
PubMed1997

Spectroscopic studies of cobalt(II) binding to Escherichia coli bacterioferritin.

The iron storage protein bacterioferritin (BFR) consists of 24 identical subunits, each containing a dinuclear metal binding site called the ferroxidase center, which is essential for fast iron core formation. Cobalt(II) binding to wild-type and site-directed variants of Escherichia coli BFR was studied by optical and magnetic techniques. Data from absorption spectroscopy demonstrate the binding of two cobalt(II) ions per subunit of wild-type and heme-free BFR, each with a pseudotetrahedral or pentacoordinate geometry, and EPR studies show that the two cobalt(II) ions are weakly magnetically coupled. Studies of variants of BFR in which a single glutamic acid residue at the ferroxidase center is replaced by alanine confirm that this is the site of cobalt(II) binding, since the altered centers bind only one cobalt(II) ion. This work shows that the electroneutrality of the ferroxidase center is preserved on binding a pair of divalent metal ions. Optical and EPR data show that cobalt(II) binding to BFR exhibits positive cooperativity, with an average Kd of approximately 1 x 10(-5) M. The favored filling of the ferroxidase center with pairs of metal ions may have mechanistic implications for the iron(II) binding process. Discrimination against oxidation of single iron(II) ions avoids odd electron reduction products of oxygen.

Bacterial Proteins↗