Cytochrome bd Protects Bacteria against Oxidative and Nitrosative Stress: A Potential Target for Next-Generation Antimicrobial Agents.

Cytochrome bd is a terminal quinol oxidase of the bacterial respiratory chain. This tri-heme integral membrane protein generates a proton motive force at lower efficiency than heme-copper oxidases. This notwithstanding, under unfavorable growth conditions bacteria often use cytochrome bd in place of heme-copper enzymes as the main terminal oxidase. This is the case for several pathogenic and opportunistic bacteria during host colonization. This review summarizes recent data on the contribution of cytochrome bd to bacterial resistance to hydrogen peroxide, nitric oxide, and peroxynitrite, harmful species produced by the host as part of the immune response to microbial infections. Growing evidence supports the hypothesis that bd-type oxidases contribute to bacterial virulence by promoting microbial survival under oxidative and nitrosative stress conditions. For these reasons, cytochrome bd represents a protein target for the development of next-generation antimicrobials.

The oxidation of cytochrome-c oxidase vesicles by hemoglobin.

Human hemoglobin has been used as a pro-oxidant for artificial unilamellar phospholipid vesicles, containing cytochrome-c oxidase inserted into the bilayer. This experimental system was suitable to follow directly the kinetics of lipid oxidation and the effects on both the vesicle membrane permeability and the functional state of cytochrome-c oxidase. Following mixing of vesicles with hemoglobin, an oxygen dependent, peroxyl radical mediated, rapid oxidation (taking a few minutes) of the lipid was found to occur. On a similar time scale the membrane became ion-leaky and cytochrome-c oxidase damaged. The pro-oxidant effects of hemoglobin in various oxidation and ligation states were studied and a mechanism, based on a ferric/ferryl redox cycle of the heme-iron is proposed to account for these observations.

Selective oxidation of Met-192 in bovine alpha-chymotrypsin. Effect on catalytic and inhibitor binding properties.

Catalytic and inhibitor binding properties of bovine alpha-chymotrypsin, in which the Met-192 residue has been converted by treatment with chloramine T to the sulfoxide derivative (Met(O)192 alpha-chymotrypsin), have been examined relative to the native enzyme (alpha-chymotrypsin), between pH 4.5 and 8.0 (mu = 0.1), and/or 5.0 degrees C and 40.0 degrees C. Values of kcat, k+2 and/or k+3 for the hydrolysis of all the substrates examined (i.e., tMetAcONp, ZAlaONp, ZLeuONp, ZLysONp and ZTyrONp) catalyzed by native and Met(O)192 alpha-chymotrypsin are similar, as well as values of Km for the hydrolysis of ZLeuONp, ZLysONp and ZTyrONp. On the other hand, Ks and Km values for the hydrolysis of ZAlaONp and tMetAcONp are decreased by about 5-fold. Met-192 oxidation does not affect the kinetic and thermodynamic parameters for the (de)stabilization of the complex formed between the proteinase and the bovine basic pancreatic trypsin inhibitor. On the other hand, the recognition process between between alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis is influenced by the oxidation event. Considering known molecular models, the observed catalytic and inhibitor binding properties of native and Met(O)192 alpha-chymotrypsin were related to the inferred stereochemistry of the proteinase-substrate and proteinase-inhibitor contact region(s).

The oxygen reactive species of cytochrome-c-oxidase: an alternative view.

In a recent review article Babcok and Wikström (Nature, 1992, 356, 301-309) proposed that the species of cytochrome-c-oxidase which binds molecular oxygen during turnover is the so-called mixed valence enzyme, in which the binuclear center cytochrome a3-CuB is reduced, while the cytochrome a/CuA sites are oxidized. This proposal is based on earlier work (Morgan and Wikström, Biochemistry 1991, 30, 948-958) in which it was found that the steady-state reduction levels of cytochrome c and cytochrome a in respiring rat liver mitochondria (sustained by ascorbate and TMPD) are quite different, the latter being much more oxidized than the former; evaluation of the steady-state reduction levels demanded a large correction due to the optical contribution of oxidized TMPD+ which overlaps with the cytochromes. We report below that application of transient spectroscopy and SVD analysis to respiring rat heart myocytes, under conditions in which the contribution of TMPD+ is very small or absent, allows to show that the steady-state reduction levels of cytochrome c and cytochrome a are comparable at all times accessible to measurement in the rapid-scanning stopped-flow spectrophotometer. Our conclusion, in agreement with previous results, is that mixed valence cytochrome-c-oxidase as defined above is not the prevailing oxygen binding species of cytochrome-c-oxidase, unless electron donation to cytochrome c becomes rate limiting.

Electron transfer and ligand binding in terminal oxidases. Impact of recent structural information.

A consensus structure for the active site of terminal oxidases has been recently proposed by Hosler et al. [(1993) J. Bioenerg. Biomem. 25, 121-135]. We exploit the novel structural information to propose a hypothesis for the large difference in the rate of internal electron transfer found when experiments are started either with the reduced or with the oxidized enzyme. This rationale also allows us to discuss the oxidation state of the prevailing oxygen reacting species with reference to the concentration of the two substrates (oxygen and cytochrome c) and to the structural state of the oxidase.

Reaction of the Hansenula anomala flavocytochrome b2 and cytochrome b2 core with inorganic outer sphere redox compounds.

The oxidation of reduced cytochrome b2 core and flavocytochrome b2 by three inorganic outer sphere compounds, Fe(CN)6(3-), Co(phen)3(3+) and Mn(CyDTA) (H2O)-, has been studied by stopped-flow. The reaction with Fe(CN)6(3-) is very rapid; the second order rate constants at 10 degrees C (pH 7) and I = 0.02 M are k = 1 x 10(8) M-1 s-1 and 1 x 10(7) M-1 s-1 for cytochrome b2 core and flavocytochrome b2, respectively. The reaction between cytochrome b2 core and Co(phen)3(3+), too fast at pH 7.0, has been characterized at 10 degrees C and pH 4.0; the second order rate constant is k = 2 x 10(7) M-1 s-1 and becomes 4 x 10(8) M-1 s-1 at pH 6.5. The reaction between flavocytochrome b2 and Co(phen)3(3+) has a second order rate constant k = 2 x 10(7) M-1 s-1 at pH 7.0, 10 degrees C. The oxidation of both proteins by Mn(CyDTA)(H2O)- is characterized by a second order rate constant k = 2.8 x 10(6) M-1 s-1 and 2.3 x 10(5) M-1 s-1 for cytochrome b2 core and flavocytochrome b2, respectively, at pH 7.0 and 10 degrees C. The reactivity of the b2 heme towards the outer sphere oxidants is higher than that reported for heme c in bacterial and eukaryotic cytochrome c. The larger delta E and the larger accessibility of the b2 heme can account for this result. The flavodehydrogenase domain seems to modulate the electron transfer also to these inorganic compounds, as found previously in the case of macromolecular electron acceptors.

Cell proliferation in the atherosclerotic plaques of cholesterol-fed rabbits. Part 3. Histological and radioautographic observations on glucocorticoids-treated rabbits.

Tritiated thymidine radioautography was employed to study the effect of cortisol and other glucocorticoids on cellular proliferation in the aorta and pulmonary artery of rabbits with cholesterol atherosclerosis. Labelled cell counts showed that glucocorticoids, even after one day and at a relatively low dose, decrease sharply the deoxyribonucleic acid synthesis in the intimal plaques. The hormonal influence on [3H]thymidine uptake seems to be a dose-dependent process. The relative potency of these steroids in inhibiting DNA synthesis in the plaques parallels closely their anti-inflammatory effectiveness. Conversely mineralocorticoids, including aldosterone and deoxycorticosterone, increase the rate of DNA synthesis in the plaques. It is concluded that the antiatherogenic effect of glucocorticoids on cholesterol-fed rabbits may be due, at least partly, to the inhibitory effect of these steroids on the DNA synthesis of the cellular components of the intimal plaques.

Effect of bepridil on the activity of cytochrome c oxidase in solution and in proteoliposomes.

The interaction between bepridil and mitochondrial cytochrome c oxidase has been studied using the purified enzyme either in aqueous suspensions in the presence of detergents, or embedded into phospholipid vesicles. The investigation, systematically extended to nonactin and valinomycin for comparison, showed that: (a) valinomycin and nonactin induce similar changes in the visible absorption spectrum of cytochrome oxidase; these changes are quite different from those induced by bepridil. (b) The three compounds have an effect on the functional properties of purified, solubilized oxidase which may be related to binding. In particular, bepridil displays a complex pH-dependent effect which at concentrations below 50 microM results in a stimulation of the activity of approximately 30% starting with the oxidized resting enzyme. At variance with valinomycin and nonactin, the stimulatory effect is the same, within the errors, for the detergent-suspended, the vesicle-embedded and even the Keilin-Hartree particles. (c) In the case of detergent-suspended oxidase, the stimulatory effect of bepridil is also similar whether the enzyme is in the resting or in the pulsed state. If the oxidase is embedded into vesicles, however, the pulsed state is significantly more sensitive to bepridil than the resting one. These results are discussed in the light of the possible role assigned to pulsed oxidase in the regulation of the electron flux through the respiratory chain.

Lonidamine-mediated respiratory changes in rat heart myocytes: a re-examination of the functional response of mitochondrial cytochrome c oxidase.

Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND.

Effects of oral clarithromycin and amoxycillin on interdigestive gastrointestinal motility of patients with functional dyspepsia and Helicobacter pylori gastritis.

BACKGROUND: Clarithromycin and amoxycillin are antibiotics commonly used in association for Helicobacter pylori eradication. Because this treatment, which lasts 1-2 weeks, is frequently associated with gastrointestinal symptoms, we investigated the effects of these antibiotics on gastrointestinal motility. PATIENTS AND METHODS: Gastroduodenal motility was recorded in 14 patients with functional dyspepsia and H. pylori gastritis by means of a low-compliance manometric system with four recording ports in the stomach and four in the duodenum. Two tablets of clarithromycin 250 mg (seven patients, clarithromycin group) or one of amoxycillin 1 g (seven patients, amoxycillin group), ground and dissolved in 20 mL of water, were given randomly and in double-blind manner 30 min after the end of the first activity front (AF) of the migrating motor complex (MMC) or, in the absence of AFs, after at least 200 min of recording. Recording continued until an AF was observed during the subsequent 200 min. RESULTS: Clarithromycin administration was followed by a typical gastroduodenal AF in a significantly higher number of patients than for amoxycillin administration. In addition, the time lag between clarithromycin administration and the appearance of AFs was 48 min +/- 8.5 (mean +/- s.d.), significantly shorter than after amoxycillin (121 min +/- 29). The clarithromycin-related duodenal AFs showed a duration of 6.6 min +/- 1.5, significantly longer than that of the spontaneous AFs (3.6 min +/- 1.2, P < 0.01), while the amoxycillin-related AFs were not significantly different from the spontaneous ones. CONCLUSION: Clarithromycin stimulated cyclic gastroduodenal motility, while amoxycillin was ineffective. It is likely that symptoms during the eradication treatment are due to this effect of clarithromycin.

Medication and psychotherapy in the treatment of chronic schizophrenia.

Current theory in the treatment of schizophrenia emphasizes medication and expresses considerable doubt about the value of psychotherapy. Yet the data that lead to this conclusion have been gathered in studies of carefully selected groups of cooperative patients who consent to treatment and participate well in scientific studies. In public psychiatry, where many patients are uncooperative and even coerced into treatment, we have found that attention to the patient's individual psychology is essential to success. We examine the role of the psychiatrist who provides psychotherapy and medication in the context of a comprehensive program for patients with chronic schizophrenia.

Structure and function of a molecular machine: cytochrome c oxidase.

Cytochrome c is responsible for over 90% of the dioxygen consumption in the living cell and contributes to the build-up of a proton electrochemical gradient derived by the vectorial transfer of electrons between cytochrome c and molecular oxygen. The metal ions found in cytochrome oxidases play a crucial role in these processes and have been extensively studied. In this review we present and discuss some of the relevant spectroscopic and kinetic properties of the prosthetic groups of cytochrome c oxidase.

Pulsed cytochrome c oxidase.

The identification of two functionally distinct states, called pulsed and resting, has led to a number of investigations on the conformational variants of the enzyme. However, the catalytic properties of cytochrome oxidase may depend on a number of experimental conditions related to the solvent as well as to the protocol followed to determine the turnover number of the enzyme. This paper reports results which illustrate that the steady-state differences between pulsed and resting oxidase may, or may not, be detected depending on experimental conditions.

Stopped-flow studies of cytochrome oxidase reconstituted into liposomes: proton pumping and control of activity.

The transient kinetics of proton pumping and the electron transfer properties of cytochrome oxidase inserted into small unilamellar vesicles have been investigated by stopped-flow spectrophotometry. In the presence of valinomycin, proton pumping and cytochrome c oxidation by cytochrome oxidase are synchronous up to rate constants of approximately 9 sec-1. Moreover, the enzyme depleted of subunit III ("three-less oxidase") was also shown to pump protons, although with a significantly smaller stoichiometry. Thus, subunit III is not the only (or even the main) proton channel, although it may be involved in the regulation of activity. The kinetics of cytochrome c oxidation by COV in the absence and in the presence of ionophores have been investigated. Analysis of the time course of the process in the transient and steady state phases indicates that the onset of control by the electrochemical gradient follows the transfer of four electrons, i.e., one complete turnover of the oxidase. Two possible alternative interpretations for the control of the turnover phase are presented and discussed.

Kinetic control of internal electron transfer in cytochrome c oxidase.

Two alternative hypotheses have been proposed to account for the relatively slow (ms) internal eT observed in the oxidized cyt c oxidase. The thermodynamic control hypothesis states that eT between cyt a and a3 is very fast (microsecond), but the apparent reduction of cyt a3 is slow because thermodynamics favors reduced cyt a. Whereas the kinetic control hypothesis states that inter-heme eT is intrinsically slow (ms), for the oxidized binuclear center. Monitoring by stopped flow the anaerobic reduction of the oxidized enzyme by ruthenium hexamine in the absence and presence of CO or NO, used as "trapping" ligands for cyt a3(2+), we found that the rate of formation of the cyt a3(2+)-NO adduct (k' approximately 20-25 s-1) is independent of the concentration of ruthenium hexamine and NO. We conclude that in the oxidized enzyme the two hemes are not in very rapid redox equilibrium and internal eT is kinetically controlled.

Effect of omeprazole on interdigestive gastroduodenal motility of patients with ulcer-like dyspepsia.

BACKGROUND/AIMS: As omeprazole, an antisecretory drug, is largely used in the treatment of acid-related diseases, we investigated its effects on the interdigestive gastroduodenal motility of ulcer-like dyspepsia. METHODOLOGY: Gastroduodenal motility was recorded manometrically in 9 patients complaining of ulcer-like dyspepsia with normal gastric emptying at scintigraphy, without ulcerative lesions and H. pylori infection at endoscopy, and without diseases known to affect gut motility (group ULD), and in a group of 9 healthy subjects as control (group C). After a period sufficient to record two consecutive phases III of the migrating motor complex (MMC) in patients of group ULD, omeprazole 20 mg was infused intravenously 30 min after the end of the last duodenal phase III and the recording was continued to the next one. RESULTS: With respect to the control group, the group ULD showed a significantly longer MMC cycle duration, a frequent absence of gastric phases III and a shorter duration of duodenal phases III. Omeprazole administration in group ULD was followed after 18.9 +/- 8.5 min by a typical gastroduodenal phase III and, consequently, the duration of the omeprazole-related cycle was significantly shortened. These omeprazole-related phases III started from the stomach in nearly all cases and showed a significantly longer duration at the duodenal level with respect to the spontaneous ones. CONCLUSIONS: Patients with ulcer-like dyspepsia showed a decrease in frequency and duration of gastroduodenal phases III. The omeprazole intravenous administration induced the anticipated appearance of an apparently normal gastroduodenal phase III, probably by suppressing the inhibitory action of acid secretion.

Interdigestive gastroduodenal motility in patients with ulcer-like dyspepsia: effect of ranitidine.

Interdigestive gastroduodenal motility was studied manometrically in 16 patients with ulcer-like dyspepsia due to hypersecretory gastroduodenitis (group A) and in a control group of 6 healthy subjects (group B). After a basal recording period sufficient to record at least two activity fronts (AF) of the migrating motor complex (MMC) of the gastroduodenal tract, we administered 100 mg of ranitidine intravenously to 8 patients of group A (group A1), and the same dose of ranitidine to the remaining 8 patients of group A (group A2) after pretreatment with cimetidine 200 mg i.v. to block the acid secretion. The interdigestive motility of patients with hypersecretory gastroduodenitis is characterized by a decrease in frequency and duration of the activity fronts of MMC, which may play a role in the pathogenesis of mucosal lesions. Ranitidine induced premature and prolonged activity fronts in all patients without antisecretory pretreatment, and in the majority of patients in whom the acid secretion was previously blocked.

The esophagus as a possible cause of chest pain in patients with and without angina pectoris.

In a series of 18 patients with angina pectoris, in whom treatment over at least 3 years with nitroderivatives and Ca-antagonists had become partially ineffective on chest pain, and in 18 patients with angina-like non-cardiac chest pain, the following examinations were carried out: upper gut x-ray and endoscopy, acid perfusion test, esophageal manometry, 24-hour esophageal pH monitoring associated with Holter recording. The presence or absence of coronary insufficiency was established by means of scintigraphic and ECG tests, Holter monitoring and coronary arteriography. In both groups the majority of patients had abnormal esophageal function, but in patients with angina pectoris treated for a long period of time the motility changes were prevalently reflux-related. With respect to the origin of chest pain, the esophagus was found to be the likely cause in 4 patients with angina pectoris, and the probable cause in another 10 of the same group; it was the likely cause in 7 patients without angina pectoris, and the probable cause in another 7 of the same group. As nitroderivatives and Ca-antagonists decrease the LES tone and the amplitude of esophageal pressure waves, long-term treatment with these drugs may be taken into account in the genesis of gastro-esophageal reflux and related changes, including esophageal pain.

["Esophageal" angina and angina pectoris]. / "Angina esofagea" ed angina pectoris.

In the last few years the non cardiac angina-like chest pain has encompassed more and more agitation not only in many patients but also in cardiologists, gastroenterologists and psychologists, as it involves socio-economic, pathophysiologic and therapeutic problems. The socio-economic aspect is well explained by the fact that in the USA at least 200,000 patients a year suffering from non cardiac angina-like chest pain, even when coronary arteriography has demonstrated normal coronary vessels, nevertheless continue to require cardiologic examinations and, if no one has clearly demonstrated the origin of their pain, they continue to live as invalids in constant fear of myocardial infarction. The discovery that the esophagus may be one of the causes of chest pain in these patients presenting with a previous diagnosis of "atypical" angina pectoris, unfortunately cannot resolve definitively the problem. An association of esophageal angina in patients with angina pectoris treated for long periods of time with Ca-antagonists and nitroderivatives has been described. In addition, the provocative or spontaneous tests to demonstrate the esophageal origin of chest pain give only a "likely" and not a "definite" diagnosis of esophageal angina. This also means to no "gold standard" text exist. Lastly, the "likely" diagnosis of esophageal angina is made in only about 50% of patients leaving the problem of the remaining 50% unanswered. These uncertainties induce some psychologists to assert that the cause of non cardiac angina-like chest pain is in the head ("panic disorder") and not in the esophagus, where the observed motor disorders should be an epiphenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)