Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Benign multicystic mesothelial proliferation of the peritoneum: immunohistochemical and electron microscopical study of a case and review of the literature.

We report a case of benign multicystic mesothelial proliferation (the so-called multicystic peritoneal mesothelioma) arising multifocally in the abdomen of a 46-year-old white man. His anamnesis showed an 8-year history of intermittent pain in the right lower abdominal quadrant. Mucin stains, immunohistochemistry, and electron microscopy confirmed the mesothelial origin of the lesion. Review of the available literature allowed us to find another 85 reported cases of benign multicystic mesothelial proliferations of the peritoneum. Out of these cases, eighteen only occurred in men, the majority being reported in middle-aged women mostly with complaints of abdominal pain. Electron microscopy or immunohistochemistry are needed to make a differential diagnosis towards other multicystic lesions, such as peritoneal cystic lymphangioma. Although multicystic mesothelial proliferations of the peritoneum have often been regarded as benign neoplasms, the true nature--neoplastic or hyperplastic--of these lesions still remains greatly elusive. Therefore, we believe that the unbinding term benign multicystic mesothelial proliferation (first used with regard to the unique hitherto reported case arisen in the pleural cavity) should be considered at present more appropriate to indicate even these peritoneal lesions.     

The electron transport system of Alcaligenes eutrophus H16

In a previous work (Kömen et al. 1991) it has been concluded that membrane fragments isolated from autotrophically grown Alcaligenes eutrophus H16 contain several iron-sulphur centres along with haems of a-, b-, c-, and d-type. These redox components have been proposed to be part of a branched respiratory chain leading to multiple membrane bound oxidases. Here, some of the respiratory activities catalyzed by membrane fragments from wild type cells of A. eutrophus (H16) and, for comparison, Paracoccus denitrificans, have been investigated through the use of electron transport inhibitors. Cyanide (CN^-) titration curves indicated that in A. eutrophus H16 oxidation of succinate and H₂ preferentially proceeds via the cytochrome c oxidase(s) branch (I ₅₀=2 · 10^-5 M) whereas the NADH dependent respiration started being inhibited at higher CN^- concentrations (I ₅₀=5 · 10^-4 M). In membranes isolated from both, cells harvested at late growth-phase (OD 12) and from a mutant deficient in cytochrome c oxidase activity (A. eutrophus RK1), respiration was insensitive to low CN^- concentrations (< 10^-4 M), and it was sustained by the high catalytic activities of two quinol oxidases. These alternative oxidases of b- (formally o-) and d-type showed different sensitivities to KCN (I ₅₀=10^-3 M and 10^-2 M, respectively). Interestingly, the cytochrome c oxidase(s) dependent respiration of H16 membranes was insensitive to antimycin A but largely inhibited by myxothiazol (10^-6 M). This, and previous work (Kömen et al. 1991), suggest that although the respiratory chain of A. eutrophus is endowed with a putative bc ₁ complex, its biochemical nature and role in respiration of this organism are apparently different from those of P. denitrificans. The peculiarity of the respiratory chain of A. eutrophus is confirmed by the rotenone insensitivity of the NADH oxidation in both protoplasts and membrane fragments from wild type and soluble hydrogenase deficient cells (HF14 and HF160). A tentative model of the respiratory chain of autotrophically grown A. eutrophus is presented.     

Oxidation-reduction thermodynamics of the acceptor quinone complex in whole-membrane fragments from Chloroflexus aurantiacus

Oxidation-reduction thermodynamic equilibria involving the quinone-acceptor complex have been examined in whole-membrane fragments from Chloroflexus aurantiacus. The primary quinone acceptor was titrated by monitoring the amount of cytochrome c554 photooxidized by a flash of light as a function of the redox potential. In contrast to previous data obtained in purified plasma membranes, in which the primary quinone acceptor exhibited a midpoint potential equal to -50 mV at pH 8.2, in whole-membrane fragments it titrated at -210 mV (pH 8.0), with a pH dependence of -60 mV/pH up to a pK value of 9.3. o-Phenanthroline, an inhibitor of electron transfer from the primary to the secondary quinone acceptor, shifted the Em/pH curve of the primary acceptor to higher redox potentials. The midpoint potential of the secondary quinone acceptor and its dependence on pH has been determined by comparing the kinetics of the charge recombination processes within the reaction center complex in the presence and in the absence of o-phenanthroline. It is concluded that both the primary and the secondary quinone acceptors interact with a proton, with pK values of 9.3 and of approximately 10.2 respectively. At physiological pH the electron appears to be stabilized on the secondary with respect to the primary quinone acceptor by approximately 60 meV.     

Functional Characterization and Partial Purification of the Ubiquinol-Cytochrome c Oxidoreductase from Higher Plant Mitochondria (Helianthus tuberosus)

The functional and thermodynamic characteristics of the ubiquinolcytochrome (Cyt) c oxidoreductase in a Cyt b/c₁-enriched fraction (defined S-1) isolated from Jerusalem artichoke mitochondria (JAM) (Helianthus tuberosus), have been analyzed. Fraction S-1, obtained through deoxycholate-KCl fractionation procedure, contained one Cyt of c type (formally c₁ with Em_7.0 of +240 millivolts), two b type Cyt with Em_7.0 values of +100 and −25 millivolts, ferredoxin-like centers presumably linked to succinic- and NADH-dehydrogenases, and a Rieske-type iron sulfur center (g_y = 1.89). The ubiquinol-dependent Cyt c reduction by fraction S-1 showed sensitivity to antimycin A, myxothiazol, and n-2-hepthyl-1-hydroxyquinoline N-oxide with I₅₀ of 12 nanomolar, 30 nanomolar, and 0.1 micromolar, respectively. Oxidation-induced extra b type reduction, a widespread phenomenon of bacterial and mitochondrial respiratory systems, has also been observed in both intact mitochondria and S-1 fraction. The data seem to blur previous experiments in which both spectral and functional differences between higher plant and mammalian mitochondria have been underlined.     

Effects of myxothiazol and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole on the respiratory pathways of the phytopathogenic fluorescent bacteria Pseudomonas cichorii and Pseudomonas aptata

Myxothiazol inhibited the electron transport in the cytochrome b/c segment of membrane particles from Pseudomonas cichorii. A residual NADH-oxidation due to the presence of an alternative pathway via cytochrome o (Em,7=+250 mV) was sensitive to the quinone analog 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). This latter inhibitor was equally effective in blocking the linear respiratory chain of Pseudomonas aptata, a strain deficient in cytochromes of c type and Rieske iron-sulphur centre. The analysis of the oxido-reduction kinetic patterns of cytochromes indicated that, among the b type haems present in P. aptata, only cyt. o could be reduced by ubiquinol-1 in a reaction insensitive to both antimycin A and myxothiazol but inhibited by UHDBT. This latter finding has been correlated to the fact that P. aptata exhibits a defective b/c complex. In membranes from P. cichorii, in which the absorption maximum of dithionite reduced cytochrome(s) b shifted by 2–3 nm in the presence of antimycin A and/or myxothiazol, the electron flow through the b/c oxidoreductase complex has tentatively been arranged in a proton motive Q-cycle like mechanism.Non standard abbreviations UHDBT 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole - cyt. cytochrom - Em, 7 mid-point potential at pH 7.0 - b/c complex ubiquinol-cyt. c oxidoreductase     

Effect of oxygen limitation on the formation of the electron transport system of the phytopathogenic fluorescent bacteriumPseudomonas cichorii

The composition of the membrane-bound electron transport system of the phytopathogenic bacteriumPseudomonas cichorii underwent modification in response to oxygen supply. Growth adaptation to low oxygen concentrations was characterized by repression of cytochromes involved in ubiquinol-cyt.c oxidoreductase and cyt.c oxidase activities. By contrast, cyto.o, i.e., the alternative cyanide-insensitive oxidase ofP. cichorii, was unaffected by low oxygen tension. Noa-type cytochromes could be detected at any stage of growth.     

Effects of 3,5-Dibromo-4-Hydroxybenzonitrile (Bromoxynil) on Bioenergetics of Higher Plant Mitochondria (Pisum sativum)

The herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was tested on mitochondria from etiolated pea (Pisum sativum L. cv Alaska) stems. This compound when used at micromolar concentrations ([almost equal to]20 [mu]M) inhibited malate- and succinate-dependent respiration by intact mitochondria but not oxidation of exogenously added NADH. Bromoxynil did not affect the activities of the succinic and the internal NADH dehydrogenases. Analyses of the effects induced by this herbicide on the membrane potential, [delta]pH, matrix Ca2+ movements, and dicarboxylate transport demonstrated that bromoxynil is likely to act as an inhibitor of the dicarboxylate carrier. In addition, bromoxynil caused a mild membrane uncoupling at concentrations [greater than or equal to]20 [mu]M. No effect on the ATPase activity was observed.     

Cytochrome b type oxidases in the respiratory chains of the phytopathogenic fluorescent bacteria Pseudomonas cichorii and Pseudomonas aptata

Membrane fragments from the phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata have been examined. A branched respiratory chain is operative in P. cichorii whereas a linear electron transport system characterizes the related bacterium P. aptata. Both species contain several b type cytochromes resolved by redox titration analysis, but no a type components may be detected. In contrast, only P. cichorii is endowed with c type cytochromes and hence with cytochrome c oxidase activity. Among the b type cytochromes, two high-potential components, with Em_7.0 at +250 mV and +380 mV, have been kinetically characterized and tentatively associated with cyanideresistant and cytochrome c oxidase activities, respectively. Cytochrome b-250 should correspond to the spectrally detectable cytochrome o whereas cytochrome b-380 is functionally similar to cytochrome b-410 described in Rhodopseudomonas capsulata. This conclusion seems to blur previous reported data on other obligate aerobes in which cytochrome o has been generally associated with cytochrome c oxidase and also suggests that a more accurate reconsideration of the actual physiological role of cyt. o in bacterial respiration is necessary. Furthermore the question arises whether cyt. b-410 like oxidases, i. e. high-potential b's similar to cyt. b-410 of R. capsulata, may be widely distributed among aerobes rather than restricted to facultative photosynthetic prokaryotes.     

Relations of single semicircular canals to the pontine reticular formation.

The effects of afferent vestibular impulses on single pontine reticular formation units and on a small filament of the IIIrd cranial nerve were recorded with tungsten microelectrodes in 40 curarized guinea pigs. Single-shock and repetitive electrical stimulations were applied by means of stimulating electrodes inserted bilaterally into the perilymphatic space of single ampullae of the anterior and lateral semicircular canals. The reticular unitary response consisted mainly in excitation of the resting discharge rate: most units showed vestibular convergence being affected by separate stimulation of the single four ampullae. the reticular evoked field and unitary potentials accounted for latency values ranging from 0.3 to 2.5 msec. As for the early latencies they can be interpreted as responses mediated by direct vestibulo-reticular fibres. A delimited vestibular projection field in the parameidan pontine reticular formation was not identified.