Michael T. Wilson

The Simultaneous Generation of Superoxide and Nitric Oxide Can Initiate Lipid Peroxidation in Human Low Density Lipoprotein

Oxidation of low density lipoprotein (LDL) has been shown to occur in the artery wall of atherosclerotic lesions in both animal models and human arteries. The oxidant(s) responsible for initiating this process are under intensive investigation and 15-lipoxygenase has been suggested in this context. Another possibility is that nitric oxide and superoxide, generated by cells present in the artery wall, react together to form peroxynitrite which decomposes to form the highly reactive hydroxyl radical. In the present study we have modelled the simultaneous generation of superoxide and nitric oxide by using the sydnonimine, SIN-1 and have investigated its effects on LDL. SIN-1 liberates both superoxide and nitric oxide during autooxidation resulting in the formation of hydroxyl radicals. We have demonstrated that superoxide generated by SIN-1 is not available to take part in a dismutation reaction since it reacts preferentially with nitric oxide. It follows, therefore, that during the autooxidation of SIN-1 little or no superoxide, or perhydroxyl radical will be available to initiate lipid peroxidation. We have shown that SIN-1 is capable of initiating the peroxidation of LDL and also converts the lipoprotein to a more negatively charged form. The SIN-1-dependent peroxidation of LDL is completely inhibited by superoxide dismutase which scavenges superoxide. Neither sodium nitroprusside or S-nitroso-N-acetyl penicillamine, which only produce nitric oxide, are able to modify LDL. These results are consistent with the hypothesis that a product of superoxide and nitric oxide could oxidize lipoproteins in the artery wall and so contribute to the pathogenesis of atherosclerosis in vivo.

Discovery and characterization of a novel inhibitor of matrix metalloprotease-13 that reduces cartilage damage in vivo without joint fibroplasia side effects.

Matrix metalloproteinase-13 (MMP13) is a Zn2+-dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structure-based drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.

The oxidation of α-tocopherol in human low-density lipoprotein by the simultaneous generation of superoxide and nitric oxide

Peroxynitrite is the product of the reaction between nitric oxide and superoxide. It is an oxidant which can also decompose to form the hydroxyl radical and nitrogen dioxide. In this report we show that a powerful oxidant with reactivity similar to that of the hydroxyl radical is formed from the generation of superoxide from xanthine oxidase and nitric oxide from S‐nitroso‐n‐acetylpenicillamine (SNAP). Simultaneous generation of these two radicals by either xanthine oxidase/SNAP or the sydnonimine SIN‐1 in the presence of low‐density lipoprotein (LDL) results in the depletion of α‐tocopherol and formation of its oxidised product α‐tocopheroquinone. The mechanism of oxidation required both the formation of nitric oxide and superoxide. In contrast to the promotion of LDL oxidation by transition metals the oxidation of LDL by SIN‐1 was not sensitive to the addition of exogenous lipid hydroperoxide.

Prevalence of Tetracycline Resistance Genes in Oral Bacteria

ABSTRACT Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common tet gene identified was tet(M), which was found in 79% of all the isolates. The second most common gene identified was tet(W), which was found in 21% of all the isolates, followed by tet(O) and tet(Q) (10.5 and 9.5% of the isolates, respectively) and then tet(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had tet(L) and 1% carried tet(A) and tet(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of tet(W) in oral bacteria and the first report to show that tet(O), tet(Q), tet(A), and tet(S) can be found in some oral species.

Cytochrome c oxidase rapidly metabolises nitric oxide to nitrite

Previous studies have shown that the addition of nitric oxide to cytochrome c oxidase rapidly generates spectral changes compatible with the formation of nitrite at the binuclear haem:copper centre. Here we directly demonstrate nitrite release following nitric oxide addition to the enzyme. The nitrite complex is kinetically inactive and the off rate for nitrite was found to be slow (0.024 min−1). However, the presence of reductants enhances the off rate and enables cytochrome oxidase to catalyse the rapid oxidation of nitric oxide to nitrite free in solution. This may play a major role in the mitochondrial metabolism of nitric oxide.

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 a 3/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 a 3. 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.

Novel Tetracycline Resistance Determinant from the Oral Metagenome

ABSTRACT A major drawback of most studies on how bacteria become resistant to antibiotics is that they concentrate mainly on bacteria that can be cultivated in the laboratory. In the present study, we cloned part of the oral metagenome and isolated a novel tetracycline resistance gene, tet(37), which inactivates tetracycline.

A common mechanism for the interaction of nitric oxide with the oxidized binuclear centre and oxygen intermediates of cytochrome c oxidase.

The reactions of nitric oxide (NO) with fully oxidized cytochrome c oxidase (O) and the intermediates P and F have been investigated by optical spectroscopy, using both static and kinetic methods. The reaction of NO with O leads to a rapid (∼100 s−1) electron ejection from the binuclear center to cytochrome a and CuA. The reaction with the intermediates P and F leads to the depletion of these species in slower reactions, yielding the fully oxidized enzyme. The fastest optical change, however, takes place within the dead time of the stopped-flow apparatus (∼1 ms), and corresponds to the formation of the F intermediate (580 nm) upon reaction of NO with a species that we postulate is at the peroxide oxidation level. This species can be formulated as either Fe5+ = O CuB 2+or Fe4+ = O CuB 3+, and it is spectrally distinct from the P intermediate (607 nm). All of these reactions have been rationalized through a mechanism in which NO reacts with CuB 2+, generating the nitrosonium species CuB 1+ NO+, which upon hydration yields nitrous acid and CuB 1+. This is followed by redox equilibration of CuB with Fea/CuA or Fea3 (in which Fea and Fea3 are the iron centers of cytochromes a and a 3, respectively). In agreement with this hypothesis, our results indicate that nitrite is rapidly formed within the binuclear center following the addition of NO to the three species tested (O, P, and F). This work suggests that nitrosylation at CuB 2+ instead of at Fea3 2+ is a key event in the fast inhibition of cytochrome c oxidase by NO.

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°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 O2 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.

Tyrosine Residues as Redox Cofactors in Human Hemoglobin IMPLICATIONS FOR ENGINEERING NONTOXIC BLOOD SUBSTITUTES

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves “through-protein” electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the α and β chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the α chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the β chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.