Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

NdhO,a Subunit of NADPH Dehydrogenase,Destabilizes Medium Size Complex of the Enzyme in Synechocystis sp. Strain PCC 6803

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.     

Modified structure and kinetics of cytochrome-c oxidase in fibroblasts from patients with Leigh syndrome

In this study we compared the properties of cytochrome-c oxidase (COX) in cultured fibroblasts from two patients with Leigh Syndrome with COX from control fibroblasts. The fibroblasts from patients showed decreased growth reates and elevated lactate production. COX activity of patients fibroblasts was about 25% of control. Kinetic studies with isolated mitochondria showed a higher K_m for cytochrome c and a markedly reduced molecular turnover of COX from patients, indicating a different structure of the enzyme. A biphasic change of COX activity was obtained by titration of dodecylmaltoside solubilized mitochondria from control fibroblasts with increasing concentrations of anions. With patient mitochondria we found only the inhibiting phase of COX activity and, in contrast to control mitochondria, irreversible inhibition of COX activity by guanidinium chloride. ELISA titrations with monoclonal antibodies to subunit II, IV, Vab, VIac and VIIab indicated a normal amount of mitochondrial coded subunit II, but a reduced amound of nuclear coded subunits. The data indicate incompletely assembled nuclear coded subunits of COX from patient fibroblasts.     

Effect of micellar lipids on rabbit intestinal brush-border membrane phospholipid bilayer integrity studied by31P NMR

Summary The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe³¹P NMR. The broad. asymmetric lineshape of the³¹P NMR spectrum of isolated brush-border vesicles demostrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18mm), demonstrating that the vesicles are highly stable, corresponding to their biological function. However, the emergence of a narrow peak superimposed on the broad lineshape indicates that a small proportion of the membrane phospholipids has reached isotropic motion, which may correspond to external or internal micellar structures. Incubation with mixed micelles of fatty acids and taurochlorate show that long-chain fatty acids enhance the membrane-perturbing effect of taurocholate while short-chain, watersoluble fatty acids do not, suggesting a difference in the absorption mechanisms.     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Immuno-isolation and culture of biliary epithelial cells from normal human liver

Summary Biliary epithelial cells (BEC) lining the intra-hepatic biliary ducts are the site of damage in several immunologically mediated liver diseases. BEC are difficult to isolate since they represent only 5% of the total cell number in normal liver. In this communication, a novel method for their isolation from normal liver is presented using a monoclonal antibody (HEA125) with specificity for an epithelial cell surface glyco-protein reported to be expressed in liver only by biliary epithelium. By combining differential density centrifugation and immuno-magnetic separation using HEA125 pure BEC (10⁵ cells/g fresh tissue) were prepared routinely. These cells were maintained in culture for up to 4 weeks with significant increases in cell numbers. The ability to prepare BEC from human liver offers an opportunity to develop In Vitro models to investigate the aetiology of diseases in intra-hepatic biliary epithelium. EDITOR’S STATEMENT This is a novel application to purification of specific liver cell types directly from tissue. It is well-suited for rapid communication because of its novelty and potential utility to investigators.     

Chronic In Vivo Sodium Azide Infusion Induces Selective and Stable Inhibition of Cytochrome c Oxidase

Abstract: The effect of chronic subcutaneous infusion of sodium azide on the activity of mitochondrial respiratory chain enzymes was investigated in Sprague-Dawley rats. Treatment with ∼1 mg/kg/h sodium azide induced chronic, partial inhibition of cytochrome c oxidase, whereas the activities of respiratory complexes I and III were not significantly affected. The inhibition of cytochrome c oxidase was evident by 7 days after infusion began, and the effect was stable for at least 3 weeks. The selectivity of azide for cytochrome c oxidase is discussed in the context of other findings of azide effects on enzymes. The results of the present study indicate that the sodium azide infusion paradigm described here provides a useful tool for the evaluation of selective and stable cytochrome oxidase inhibition in vivo.     

Biological diversity and cultural heritage

In this paper some examples of the development of communities of microorganisms and plants on historic buildings and montiments are shown. When the building stones differ from the surrounding natural substrata, an increase in the biological diversity of the area is produced. In some cases, monuments can come to constitute a true refuge for a few species when the natural habitat is threatened. It is suggested that biological diversity, when it does not represent a threat for the cultural heritage, should be considered worthy of preservation.     

Sedimentology of the Upper Triassic reef complex at the Hochkönig Massif (Northern Calcareous Alps,Austria)

Summary The Upper Triassic Dachsteinkalk of the Hochk?nig Massif, situated 50 km south of Salzburg in the Northern Calcareous Alps, corresponds to a platform margin reef complex of exceptional thickness. The platform interior limestones form equally thick sequences of the well known cyclic Lofer facies. Sedimentation in the reef complex was not so strongly controlled by low-amplitude sea-level oscillations as was the Lofer facies. The westernmost of the 8 facies of the reef complex is an oncolite-dominated lagoon, in which wave-resistant stromatolite mounds with a relief of a few metres were periodically developed. The transition to the central reef area is accomplished across the back-reef facies. In the back-reef facies patch reefs and calcisponges appear. The proportion of coarse bioclastic sediment increases rapidly over a few hundred metres before the central reef area is encountered. The central reef area consists of relatively widely spaced small patch reefs that did not develop wave-resistant reef framework structures. The bulk of the sediment in the central reef area is coarse bioclastic material, provided by the dense growth of reef organisms and the wave-induced disintegration of patch reefs. Collapse of the reef margin is recorded by the supply of large blocks of patch reef material to the upper reef slope. Additionally, coarse, loose bioclastic debris was supplied to the upper reef slope and this was incorporated into debris flows on the reef slope and turbidites found at the base of the slope and in the off-reef facies. Partially lithified packstones and wackestones of the lower to middle reef slope were modified by mass movement to form breccia and rudstone sheets. The latter reach out hundreds of metres into the off-reef facies environment. A reef profile is presented which was derived by the restoration of strike and dip information. In conjunction with constraints imposed by sedimentary facies related to slope processes, the angle of slope in the reef margin area ranged from 11° to 5°, forming a concave (dished downwards) slope. Water depth estimations require that the central reef area did not develop in water of less than 10 metres depth. At the reef margin water depths were about 30 metres, at the base of the reef slope 200 metres and deepening in the off-reef facies to 250 metres. While previous work on reef complexes from this type of setting suggests growth in a heavily storm-dominated environment, the present author finds little evidence for the storm generation of the fore reef breccias, although there is good evidence for storm-influenced sedimentation and reworking in the central reef area. Post-depositional processes were characterised by continued slope processes causing brecciation and hydraulic injection of red internal sediments downwards into the reef slope and off-reef limestones. Hydrothermal circulation caused a number of phases of post-depositional (diagenetic) brecciation. There appears not to have been an important period of emergence at the Triassic/Jurassic boundary.