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.

Morfofisiologia do tecido intertubular e das células de Leydig de jaguatirica (Leopardus pardalis) adulta / Morphophisiology of the intertubular tissue and Leydig cells in adult ocelots (Leopardus pardalis)

Estudou-se o espaço intertubular e descreveu-se seu arranjo em jaguatirica adulta (Leopardus pardalis). Para tal, colheram-se fragmentos dos testículos, de cinco jaguatiricas adultas, provenientes de cativeiro, obtidos por biópsia incisional. O compartimento intertubular correspondeu a 19,4 por cento do parênquima, sendo 3,9 por cento ocupado por células de Leydig. Estes se apresentaram uninucleados, com o núcleo arredondado e nucléolo único, e com grande quantidade de grânulos pigmentares no citoplasma. O compartimento intertubular apresentou padrão do tipo II, e o tecido conjuntivo foi o componente mais abundante do tecido intertubular. Observou-se pequeno percentual de células de Leydig na espécie estudada, e o número médio por grama de testículo, 33,39 x10(6), apresentou-se dentro da amplitude descrita para a maioria dos mamíferos.

The aim of this study was to do a quantitative investigation of the intertubular space and describe its arrangement in adult ocelots (Leopardus pardalis). In this experiment we used testicular fragments, obtained through biopsy from five adult ocelots maintained in captivity. The intertubular compartment corresponded to 19.4 percent of the testicular parenchyma, and 3.9 percent of this compartment was occupied by Leydig cells. The Leydig cells showed to be uninucleate, with rounded nuclei and single nucleoli, with a large amount of pigment granules in their cytoplasm. The intertubular compartment showed a clear pattern of type II and the conjunctive tissue was the most abundant component of the intertubular tissue. Despite the small unit volume of Leydig cells in adult ocelots, the average number per gram of testis (33.39 x106) was within the range described for most mammals.

Morphine but not fentanyl and methadone affects mitochondrial membrane potential by inducing nitric oxide release in glioma cells.

We have observed that treatment of human glioma cells with morphine in the nanomolar range of concentration affects the mitochondrial membrane potential. The effect is specific to morphine and is mediated by naloxone-sensitive receptors, and is thus better observed on glioma cells treated with desipramine; moreover, the mitochondrial impairment is not inducible by fentanyl or methadone treatment and is prevented by the nitric oxide (NO) synthase inhibitor L-NAME. We conclude that in cultured glioma cells, the morphine-induced NO release decreases the mitochondrial membrane potential, as one might expect based on the rapid inhibition of the respiratory chain by NO. The identification of new intra-cellular pathways involved in the mechanism of action of morphine opens additional hypotheses, providing a novel rationale relevant to the therapy and toxicology of opioids.

Trichomonas vaginalis degrades nitric oxide and expresses a flavorubredoxin-like protein: a new pathogenic mechanism?

Besides possessing many physiological roles, nitric oxide (NO) produced by the immune system in infectious diseases has antimicrobial effects. Trichomoniasis, the most widespread non-viral sexually transmitted disease caused by the microaerophilic protist Trichomonas vaginalis, often evolves into a chronic infection, with the parasite able to survive in the microaerobic, NO-enriched vaginal environment. We relate this property to the finding that T. vaginalis degrades NO under anaerobic conditions, as assessed amperometrically. This activity, which is maximal (133 +/- 41 nmol NO/10(8) cells per minute at 20 degrees C) at low NO concentrations (< or = 1.2 microM), was found to be: (i) NADH dependent, (ii) cyanide insensitive and (iii) inhibited by O(2). These features are consistent with those of the Escherichia coli A-type flavoprotein (ATF), recently discovered to be endowed with NO reductase activity. Using antibodies against the ATF from E. coli, a protein band was immunodetected in the parasite grown in a standard medium. If confirmed, the expression of an ATF in eukaryotes suggests that the genes coding for ATFs were transferred during evolution from anaerobic Prokarya to pathogenic protists, to increase their fitness for the microaerobic, parasitic life style. Thus the demonstration of an ATF in T. vaginalis would appear relevant to both pathology and evolutionary biology. Interestingly, genomic analysis has recently demonstrated that Giardia intestinalis and other pathogenic protists have genes coding for ATFs.

Aplicação de formulação do fungo predador de nematóides Monacrosporium thaumasium (Drechsler, 1937) no controle de nematóides de bovinos / Control of bovine gastrointestinal nematodes using formulation of the nematode-trapping fungus Monacrosporium thaumasium (Drechsler, 1937)

A viabilidade de uma formulação do fungo Monacrosporium thaumasium (Drechsler, 1937) foi avaliada no controle biológico de nematóides parasitos gastrintestinais de bovinos. Dois grupos de sete bezerras cada, mestiças Holandês x Zebu, de quatro a seis meses de idade, foram colocados em pastagens de Cynodon dactilon. No grupo A, cada animal recebeu 20g de pellets (formulação granulada) de M. thaumasium via oral, duas vezes por semana, durante quatro meses, com início na estação chuvosa (outubro, 2001). No grupo B (controle), as bezerras não receberam nenhum tratamento. As contagens de ovos por grama de fezes (OPG) e das larvas infectantes encontradas na pastagem do grupo B foram significativamente maiores (P<0,05) do que as do grupo A e a diferença entre o OPG dos animais dos grupos A e B, no final do experimento, foi de 88,8 por cento. O gênero Cooperia foi o mais prevalente em ambas as pastagens. Conclui-se que a aplicação de pellets de M. thaumasium na dosagem e periodicidade de aplicação usadas foi eficiente no controle de nematóides parasitos gastrintestinais de bovinos.

Control of respiration by nitric oxide in Keilin-Hartree particles, mitochondria and SH-SY5Y neuroblastoma cells.

The pattern of cytochrome c oxidase inhibition by nitric oxide (NO) was investigated polarographically using Keilin-Hartree particles, mitochondria and human neuroblastoma cells. NO reacts with purified cytochrome c oxidase forming either a nitrosyl- or a nitrite-inhibited derivative, displaying distinct kinetics and light sensitivity of respiration recovery in the absence of free NO. Keilin-Hartree particles or cells, respiring either on endogenous substrates alone or in the presence of ascorbate, as well as state 3 and state 4 mitochondria respiring on glutamate and malate, displayed the rapid recovery characteristic of the nitrite derivative. All systems, when respiring in the presence of tetramethyl-p-phenylenediamine, were characterised by the slower, light-sensitive recovery typical of the nitrosyl derivative. Together the results suggest that the reaction of NO with cytochrome c oxidase in situ follows two alternative inhibition pathways, depending on the electron flux through the respiratory chain.

The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity.

The cytochrome cbb3 is an isoenzyme in the family of cytochrome c oxidases. This protein purified from Pseudomonas stutzeri displays a cyanide-sensitive nitric oxide reductase activity (Vmax=100+/-9 mol NO x mol cbb3(-1) x min(-1) and Km=12+/-2.5 microm), which is lost upon denaturation. This enzyme is only partially reduced by ascorbate, and readily re-oxidized by NO under anaerobic conditions at a rate consistent with the turnover number for NO consumption. As shown by transient spectroscopy experiments and singular value decomposition (SVD) analysis, these results suggest that the cbb3-type cytochromes, sharing structural features with bacterial nitric oxide reductases, are the enzymes retaining the highest NO reductase activity within the heme-copper oxidase superfamily.

Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects.

The reactions of nitric oxide (NO) with the turnover intermediates of cytochrome c oxidase were investigated by combining amperometric and spectroscopic techniques. We show that the complex of nitrite with the oxidized enzyme (O) is obtained by reaction of both the "peroxy" (P) and "ferryl" (F) intermediates with stoichiometric NO, following a common reaction pathway consistent with P being an oxo-ferryl adduct. Similarly to chloride-free O, NO reacted with P and F more slowly [k approximately (2-8) x 10(4) M(-1) s(-1)] than with the reduced enzyme (k approximately 1 x 10(8) M(-1) s(-1)). Recovery of activity of the nitrite-inhibited oxidase, either during turnover or after a reduction-oxygenation cycle, was much more rapid than nitrite dissociation from the fully oxidized enzyme (t(1/2) approximately 80 min). The anaerobic reduction of nitrite-inhibited oxidase produced the fully reduced but uncomplexed enzyme, suggesting that reversal of inhibition occurs in turnover via nitrite dissociation from the cytochrome a(3)-Cu(B) site: this finding supports the hypothesis that oxidase may have a physiological role in the degradation of NO into nitrite. Kinetic simulations suggest that the probability for NO to be transformed into nitrite is greater at low electron flux through oxidase, while at high flux the fully reduced (photosensitive) NO-bound oxidase is formed; this is fully consistent with our recent finding that light releases the inhibition of oxidase by NO only at higher reductant pressure [Sarti, P., et al. (2000) Biochem. Biophys. Res. Commun. 274, 183].

The ratio between the fast and slow forms of bovine cytochrome c oxidase is changed by cholate or nucleotides bound to the cholate-binding site close to the cytochrome a3/CuB binuclear centre.

We determined the fraction of 'slow' and 'fast' conformations of bovine cytochrome c oxidase, following the kinetics of cyanide binding to the oxidized enzyme. We investigated whether treatment of heart mitochondrial particles with different commercially available types of cholate (standard and ultrapure) can affect the fraction of cytochrome c oxidase in the two states. Compared to standard cholate, the use of ultra-pure cholate for solubilization of heart mitochondrial particles significantly increased the fraction of the fast enzyme. Complete homogeneity (approximately 100% fast) was observed when cytochrome c oxidase was solubilized with ultra-pure cholate from heart mitochondrial particles pre-equilibrated with AMP; equilibration with ADP yielded a much smaller fraction of fast enzyme (approximately 35%). These observations are discussed on the basis of the structural relationships between the known cholate-binding site and the binuclear cytochrome a3-CuB site: variation in the occupancy of this binding site with cholate or nucleotides may modify reactivity of the oxidized binuclear centre towards cyanide.

Nitric oxide and cytochrome c oxidase: mechanisms of inhibition and NO degradation.

NO inhibits mitochondrial respiration by reacting with either the reduced or the oxidized binuclear site of cytochrome c oxidase, leading respectively to accumulation of cytochrome a(2+)(3)-NO or cytochrome a(3+)(3)-NO(-)(2) species. Exploiting the unique light sensitivity of the cytochrome a(2+)(3)-NO, we show that under turnover conditions, depending on the cytochrome c(2+) concentration, either the cytochrome a(2+)(3)-NO or the nitrite-bound enzyme is formed. The predominance of one of the two inhibitory pathways depends on the occupancy of the turnover intermediates. In the dark, the respiration recovers at the rate of NO dissociation (k' = 0.01 s(-1) at 37 degrees C). Illumination of the sample speeds up recovery rate only at higher reductant concentrations, indicating that the inhibited species is cytochrome a(2+)(3)-NO. When the reaction occurs with the oxidized binuclear site, light has no effect and NO is oxidized to harmless nitrite eventually released in the bulk, accounting for catalytic NO degradation.

The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications.

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions. The rate of NO consumption and N(2)O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 +/- 0.7 mol NO/mol ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at [NO] approximately 50 microM and 20 degrees C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant. We also show that for ba(3) oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar Cu(B)(+) coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb(3) terminal oxidases. Our findings represent functional evidence in support of this hypothesis.

Mechanism of S-nitrosothiol formation and degradation mediated by copper ions.

Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu(2+), but not Fe(3+), induces in the presence of NO a fast S-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu(+) is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu(2+), the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu(2+) in S-nitrosothiol formation with the known destabilizing effect of Cu(+), providing a unique simple picture where the redox state of copper determines either the NO release from S-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation.

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.

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy.

With the electro-driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer-aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady-state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic-to-measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20-fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.

Effect of intravenous clarithromycin on interdigestive gastroduodenal motility of patients with functional dyspepsia and Helicobacter pylori gastritis.

Gastroduodenal motility of 16 patients complaining of functional dyspepsia and Helicobacter pylori gastritis was recorded by means of a low-compliance manometric system with four recording ports in the stomach and four in the duodenum. Clarithromycin (CLA) 250 mg (group A: 8 patients) or normal saline solution (group B: 8 patients) was infused intravenously randomly and in double-blind manner 30 min after the end of the first recorded activity front (AF) of the migrating motor complex or, in the absence of AFs, after 200 min of recording, continuing the recording until an AF was observed during the subsequent 200 min. CLA administration was followed by a typical gastroduodenal AF in a significantly higher number of patients than saline administration. In addition, the time-lag between the drug administration and the appearance of AFs was 22 min +/-7.4 (mean +/- SD), significantly shorter than after saline (109+/-56 min) and the CLA-related duodenal AFs showed a duration of 7.4 min +/-1.6 in group A, significantly longer than that of the spontaneous AFs (3.5 min +/-1), while in group B AF duration after saline was not significantly different from that of the spontaneous ones. In conclusion, clarithromycin is able to stimulate cyclic interdigestive gastroduodenal motility. This prokinetic property of clarithromycin is not unexpected because it is a macrolide like erythromycin, the prokinetic activity of which is well known, and could be utilized for therapeutic uses.

Nitric oxide and cellular respiration.

The role of nitric oxide (NO) as a signalling molecule involved in many pathophysiological processes (e.g., smooth muscle relaxation, inflammation, neurotransmission, apoptosis) has been elaborated during the last decade. Since NO has also been found to inhibit cellular respiration, we review here the available information on the interactions of NO with cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. The effect of NO on cellular respiration is first summarized to present essential evidence for the fact that NO is a potent reversible inhibitor of in vivo O2 consumption. This information is then correlated with available experimental evidence on the reactions of NO with purified COX. Finally, since COX has been proposed to catalyze the degradation of NO into either nitrous oxide (N2O) or nitrite, we consider the putative role of this enzyme in the catabolism of NO in vivo.

Permeability of rat heart myocytes to cytochrome c.

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 microM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c.

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.