The role of ubiquinones in regulating lipid metabolism in rat thymocytes

The effects of ubiquinones Q-1, Q-2, Q-8 and Q-9 on lipid biosynthesis in thymocytes in vitro were studied during incubation of cells with ubiquinones within the concentration range of 1-100 mM. A 2-fold inhibition of cholesterol synthesis in thymocytes by ubiquinone Q-9 occurred, when the exogenous ubiquinone concentration in the medium was no less than 40 mM. Incubation of thymocytes with unrelated ubiquinone (Q-1 and Q-2-40 and 100 mM, respectively) resulted in the inhibition of cholesterol synthesis. The inhibiting effect was of the same order of magnitude as that during incubation with ubiquinone Q-9. Ubiquinone Q-8 showed a tendency to inhibit cholesterol synthesis in rat thymocytes. The inhibiting effect of ubiquinone on cholesterol metabolism in thymocytes is specific and is not coupled with fatty acid metabolism.     

Possible involvement of 3-hydroxymethylglutaryl-CoA reductase in determining the side-chain length of ubiquinone in rat heart.

The biosynthetic mechanism for determining the side-chain length of ubiquinone in rat heart mitochondria was investigated. The biosynthesis of nonaprenyl ubiquinone (UQ-9) and decaprenyl ubiquinone (UQ-10) in the mitochondria from rat hearts previously perfused with mevalonolactone was accelerated depending on the concentration of mevalonolactone. Furthermore the synthesis ratio between UQ-10 and UQ-9 (UQ-10/UQ-9) increased in accordance with the increasing concentration of mevalonolactone used. In addition, an enhancement of the synthesis ratio (UQ-10/UQ-9) was observed when the rats were treated with isoproterenol to increase the activity of 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme which forms mevalonate. Moreover, the addition of isopentenyl pyrophosphate, which is a metabolite of mevalonate, elevated the synthetic ratios UQ-10/UQ-9 in intact mitochondria and decaprenyl pyrophosphate/solanesyl pyrophosphate in the partially purified polyprenyl pyrophosphate synthetase from rat heart. These results suggest that the HMG-CoA reductase could be involved as a determining factor of the side-chain length of ubiquinone in rat heart.     

A role of ubiquinone in energy conservation in mitochondria

Short chain ubiquinones (Q-3) uncouple oxidative phosphorylation in rat heart mitochondria, as shown by polarimetric experiments, and abolish P:O ratios in succinate driven oxidative phosphorylaton. The uncoupling is reversed by long chain ubiquinones (Q-7). Furthermore, short chain ubiquinones abolish oligomycin sensitivity of ATPase; the inhibition is restored by Q-7. The extraction of endogenous ubiquinone from mitochondria reversibly lowers oligomycin sensitivity of ATPase.     

On the sidedness of the ubiquinone redox cycle. Kinetic studies in mitochondrial membranes

The inhibition of NADH oxidation but not of succinate oxidation by the low ubiquinone homologs UQ-2 and UQ-3 is not due to a lower rate of reduction of ubiquinone by NADH dehydrogenase: experiments in submitochondrial particles and in pentane-extracted mitochondria show that UQ-3 is reduced at similar rates using either NADH or succinate as substrates. The fact that reduced UQ-3 cannot be reoxidized when reduced by NADH but can be reoxidized when reduced by succinate may be explained by a compartmentation of ubiquinone.Using reduced ubiquinones as substrates of ubiquinol oxidase activity in intact mitochondria and in submitochondrial particles we found that ubiquinol-3 is oxidized at higher rates in submitochondrial particles than in mitochondria. The initial rates of ubiquinol oxidation increased with increasing lengths of isoprenoid side chains in mitochondria, but decreased in submitochondrial particles. These findings suggest that the site of oxidation of reduced ubiquinone is on the matrix side of the membrane; reduced ubiquinones may reach their oxidation site in mitochondria only crossing the lipid bilayer: the rate of diffusion of ubiquinol-3 is presumably lower than that of ubiquinol-7 due to the differences in hydrophobicity of the two quinones.     

Effect of ubiquinone-homologs on the sensitivity of mitochondrial ATPase to energy transfer inhibitors.

Short-chain ubiquinone (UQ-3) abolishes oligomycin sensitivity of ATPase in submitochondrial particles and the effect is reversed by long-chain ubiquinone (UQ-7). Ubiquinone-3 also abolishes DCCD sensitivity of ATPase in submitochondrial particles but the effect is not reversed by long-chain ubiquinones. These data suggest that ubiquinone interferes with energy transfer process by interaction with mitochondrial ATPase.     

Characterization of cytochrome bo3 activity in a native-like surface-tethered membrane

We have developed a simple native-like surface-tethered membrane system to investigate the activity of cbo(3) (cytochrome bo(3)), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner-membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo(3) substrate UQ-10 (ubiquinol-10). Tethered membranes are formed by self-assembly from vesicles on to gold electrodes functionalized with cholesterol derivatives. cbo(3) activity was monitored using CV (cyclic voltammetry) with electron transfer to cbo(3) mediated by UQ-10. The apparent K(m) for oxygen with this system is 1.1+/-0.4 microM, in good agreement with values reported in the literature for whole-cell experiments and for purified cbo(3). Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo(3) activity. The activity of cbo(3) with long-chain ubiquinones appears to be different from previous reports using short-chain substrate analogues such as UQ-1 in that typical Michaelis-Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.     

Biogenesis of ubiquinone in rats--an alternate origin of the benzoquinone moiety

The addition of cyclohexane carboxylic acid (CCA) to the incubation medium results in a dilution of the radioactivity incorporated into ubiquinone-9 (UQ-9) from 1-¹⁴C-benzoate by rat liver slices. This effect is more pronounced when the slices are preincubated prior to addition of the labeled precursor. A similar dilution by CCA of label incorporation, is observed using U-¹⁴C-tyrosine, but not either CH₃-¹⁴C-methionine or 2-¹⁴C-mevalonate, as precursors. UQ-9, but not cholesterol, isolated from liver slices incubated with ring-U-¹⁴C-CCA is found to be labeled. The extent of labeling of UQ-9 by this precursor is enhanced by the presence of an excess of mevalonate in the incubation medium and decreased by the addition of p-hydroxybenzoate. These results suggest that aromatisation of cyclohexane derivatives may serve as a possible source of the benzoquinone nucleus of UQ-9 in the rat.     

A phylogenetic study of ubiquinone Q-8 species of the genera Candida,Pichia, and Citeromyces based on 18S ribosomal DNA sequence divergence

To clarify phylogenetic relationships among species of the anamorphic ascomycetous genus Candida with ubiquinone Q-8, we determined complete sequences of 18S ribosomal RNA genes (18S rDNAs) from the type strains of 20 species of the genus Candida and 7 of the teleomorphic ascomycetous genera Pichia and Citeromyces, which have Q-8 as the major ubiquinone. Q-8-forming Candida species were divided into six clusters and were phylogenetically distant from a group of Candida species that included the type species of the genus. One Q-8-forming species from each of the genera Pichia, Citeromyces, or Clavispora was included in five of six clusters. Cluster 1 comprised C. ishiwadae, C. ernobii, C. karawaiewii, C. anatomiae, C. populi, and Pichia holstii. Cluster 2 comprised C. globosa and its teleomorph, Citeromyces matritensis. Cluster 3 comprised C. molischiana and Pichia capsulata. Cluster 4 comprised C. silvanorum, C. sequanensis, C. fennica, C. entomophila, C. homilentoma, C. rhagii, C. gotoi, and Pichia burtonii. Cluster 5 comprised C. fructus, C. musae, and C. lusitaniae (anamorph of Clavispora lusitaniae). Cluster 6 comprised C. stellata, C. lactiscondensi, C. galacta, and C. incommunis and was a heterogeneous group with large interspecific divergence. Pichia pastoris was quite divergent and phylogenetically distant from other Pichia species examined. Pichia methanolica and its synonym, P. cellobiosa, which have both Q-7 and Q-8 as major ubiquinones, were closely associated with Q-7-forming Williopsis salicorniae. Based on this comparative analysis of 18S rDNA sequences, it is evident that Q-8 Candida species and Q-8 Pichia species are polyphyletic.     

Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles.

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.     

Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.     

Production of isoprenoid compounds in the facultative methylotroph Protomonas extorquens

Screening of a strain which contained a large amount of ubiquinone Q-10 and a variety of isoprenoid compounds using different culture conditions and mutations was carried out.Protomonas extorquens TK 0045, which was found to contain carotenoid pigments, Hop-22(29)-ene, and Hopan-22-ol, was selected on the basis of cell yield and the content of ubiquinone Q-10. The contents of ubiquinone and sterols increased as the age of the culture increased, and reached a maximum level during the stationary phase.The contents of ubiquinone, sterols and carotenoid pigments, and ubiquinone homologs produced by P. extorquens TK 0045 were varied using mutagenesis. Mutants that had increased or decreased contents of carotenoid pigments were obtained with a high frequency. Most mutants had varying contents of other isoprenoid compounds. The ubiquinone homologs obtained by mutagenesis varied with a high frequency, and mutants which possessed increased levels of ubiquinone Q-9, Q-11 or Q-12 were isolated. However, the major ubiquinone component in these mutants was Q-10 the same as that in the wild strain. The production of ubiquinone was increased considerablyby repeated mutagenesis, with the content of ubiquinone produced by the third generation mutant (strains HB-5) being approximately 3.3 mg·g dry cell⁻¹ (2.5 times that of the wild strain). The acquisition of mutants exhibiting altered synthesis of carotenoid pigments would be useful for increasing the content of ubiquinone Q-10 in bacterial cells.     

Differentiation of neuroblastoma cells induced by an inhibitor of mevalonate synthesis: relation of neurite outgrowth and acetylcholinesterase activity to changes in cell proliferation and blocked isoprenoid synthesis

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, stimulates neurite outgrowth and acetylcholinesterase (ACE) activity in C1300 (Neuro-2A) murine neuroblastoma cells. Sprouting of neurites began within 4-8 h, before changes in cell proliferation could be detected by [3H]thymidine incorporation or flow cytometry. In contrast, the increase in ACE activity was temporally correlated with suppression of DNA synthesis, which occurred after 8 h. The activity of the membrane marker enzyme phosphodiesterase I was not stimulated by mevinolin. Suppression of protein synthesis with cycloheximide blocked the induction of ACE activity but only partially inhibited neurite outgrowth in the mevinolin-treated cultures. When mevinolin was removed from the culture medium, most of the cells retracted their neurites within 2 h, but ACE activity did not decline until DNA synthesis began to return to control levels after 10 h. Similarly, retraction of neurites in differentiated cells exposed to colchicine was not accompanied by a decrease in ACE activity. DNA histograms suggested that mevinolin arrests neuroblastoma cells in both the G1 and G2/M compartments of the cell cycle. Other cytostatic drugs that arrest cells at different stages of the cell cycle did not cause Neuro-2A cells to form neurites such as those seen in the mevinolin-treated cultures. When incorporation of [3H]acetate into isoprenoid compounds was studied in cultures containing mevinolin in concentrations ranging from 0.25 microM to 25 microM, the labeling of cholesterol, dolichol, and ubiquinone was suppressed by 90% or more at all concentrations. However, significant growth arrest and cell differentiation were observed only at the highest concentrations of mevinolin. Supplementing the medium with 100 microM mevalonate prevented the cellular response to mevinolin, but additions of cholesterol, dolichol, ubiquinone, or isopentenyl adenine were generally ineffective. The cholesterol content of neuroblastoma cells incubated with 25 microM mevinolin for 24 h was not diminished, and protein glycosylation, measured by [3H]mannose incorporation, was decreased only after 24 h at high mevinolin concentration. These studies suggest that the stimulation of neurite outgrowth and the increase in ACE activity induced by mevinolin are independent phenomena. Whereas neurite outgrowth is not related directly to the effects of mevinolin on cell cycling, the induction of ACE is correlated with the inhibition of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana

Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.     

Effect of dicumarol,a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 μM dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro , was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 μM dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Comparison between a submerged membrane bioreactor and a conventional activated sludge system on treating ammonia-bearing inorganic wastewater

A submerged membrane bioreactor (SMBR) and a conventional activated sludge system (CAS) were compared in parallel over a period of 210 days on treating synthetic ammonia-bearing inorganic wastewater under similar conditions. Except for a short period of pH control failure, almost complete conversion of NH(4)(+)?N to NO(3)(-)?N was constantly achieved over an NH(4)(+)?N concentration range from 180 to 1300mgl(-1) at a hydraulic retention time (HRT) of 24h in the SMBR, compared to an average conversion ratio of 95.0% in the CAS. Scanning electron micrographs (SEMs) demonstrated the accumulation of extracellular polymeric substances (EPSs) in the SMBR. Ubiquinone-8 (UQ-8), followed by UQ-10, UQ-7 and UQ-9, was the dominant ubiquinone in both the systems. The dominant menaquinone in the SMBR was menaquinone-6 (MK-6), while that in the CAS was MK-7, indicating that some differences existed between the two systems in terms of microbial community structure. Soluble microbial products (SMPs) tended to accumulate, and then biodegrade in SMBR.     

Ubiquinone system of the form-genusChrysosporium

The ubiquinone (coenzyme Q: Q) system of 17 strains of the form-genusChrysosporium was analyzed by high performance liquid chromatography (HPLC) and found to show a heterogeneous distribution of the major ubiquinone. Q-9, Q-10 or Q-10(H₂) was found to be the major ubiquinone in 3, 9 and 5 strains, respectively. It was further demonstrated that the teleomorphs of the species characterized by Q-9 and Q-10 could be classified into two separate families, Arthrodermataceae (Q-9) and Onygenaceae (Q-10), which were defined within the revised order Onygenales by Currah. Teleomorphs ofChrysosporium species having Q-10(H₂) have not been found. This paper also includes the ubiquinone system of dermatophytes which relate to the form-genusChrysosporium morphologically.     

Effect of Dicumarol,a NAD(P)H: Quinone Acceptor Oxidoreductase 1 (DT-diaphorase) Inhibitor on Ubiquinone Redox Cycling in Cultured Rat Hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 &#119 M dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro, was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 &#119 M dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Inhibition of ubiquinone synthesis in isolated rat heart under an ischemic condition

1. The biosynthesis of ubiquinone (UQ) in isolated rat heart under ischemic and hypoxic conditions was investigated. 2. Under ischemic perfusion, a greater amount of biosynthetic intermediates, 3-nonaprenyl and 3-decaprenyl-4-hydroxybenzoate (PPHBs) was accumulated and a smaller amount of UQ-9 and -10 was synthesized when compared with normal conditions. 3. The accumulation of PPHBs was observed without forming UQs during anaerobic perfusion. 4. Hydroxylation which is the following reaction of PPHBs for the biosynthesis of UQ in rat heart, was proceeded by the monooxygenase(s) depending upon the oxygen concentrations.     

Inactivation of adenosine 5''-triphosphate synthesis and reduced-form nicotinamide adenine dinucleotide dehydrogenase activity in Escherichia coli by near-ultraviolet and violet radiations.

Near-ultraviolet (near-UV) light (300 to 380 nm) is a significant component of sunlight and has a variety of effects on biological systems. The present work is an attempt to identify chromophores (molecular absorbers of light) and targets (critical damaged molecules) for inhibition of adenosine triphosphate (ATP) synthesis in Escherichia coli by near UV. The fluence of 334 nm required for 37% survival of net ATP synthesis (F37) in E. coli AB2463 in succinate medium is 140 kJ/m2. The action spectrum for this inactivation is almost structureless, exhibiting a smooth transition from high efficiency at 313 nm to low efficiency at 405 nm. The action spectrum for inhibition of net ATP synthesis is consistent with the chromophore being either ubiquinone Q-8 or vitamin K2. The fluence required is consistent with ubiquinone Q-8 also being a target molecule. The activity of reduced nicotinamide adenine dinucleotide dehydrogenase in extracts of E. coli B is also inactivated by near UV and shows an F37 of about 40 kJ/m2. The action spectrum for this effect is quite structureless; it shows high efficiency at 313 nm and low efficiency at 435 nm. The data do not suggest a target molecule for this action, although it is possible that ubiquinone Q-8 absorbs the near-UV energy and then passes it on to some other target molecule. The data further indicate that inactivation of the oxidative phosphorylation system is not a primary factor in near-UV-induced growth delay in E. coli.     

Analysis of microbial community structure in a biofilm on membrane surface in the submerged membrane bioreactor treating domestic wastewater on the basis of respiratory quinone profiles

The objective of this study was to investigate the microbial community structure of the biofouling film formed on hollow-fiber membrane surfaces in the submerged membrane bioreactor (SMBR) with a nitrification-denitrification process. In this experiment, aeration was conducted intermittently (60 min off, 90 min on) cyclic anoxic and oxic conditions in the SMBR. The dominant quinone types of biofilm on the membrane surface in an intermittently aerated SMBR were ubiquinone (UQs)-8, -10, followed by menaquinones (MKs)-8(H4), -8(H2) and -7, but those of suspended microorganisms were UQ-8, UQ-10 followed by MKs-8, -9(H4) and -6. The change in quinone profiles of biofilm on the membrane surface suggested that UQ-9, MK-7, MK-8(H2) and MK-8(H4) contributed to microbiological fouling in the intermittently aerated SMBR treating domestic wastewater. The microbial diversities of suspended microorganisms and biofilm, calculated based on the composition of all quinones, were 9.5 and 10.9, respectively.