Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectro-photometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either the ‘apparent’ V or the ‘apparent’ K_m values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in ‘apparent’ K_m for choline and a 53% increase in ‘apparent’ V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanoltreated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondrial Mg²⁺. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and β-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.     

Effects of ethanol and other alkanols on passive proton influx in the yeast Saccharomyces cerevisiae

Ethanol, isopropanol, propanol and butanol enhanced the passive influx of protons into deenergized cells of Saccharomyces cerevisiae. The influx followed first-order kinetics with a rate constant that increased exponentially with the alkanol concentration. The exponential enhancement constants increased with the lipid solubility of the alkanols, which indicated hydrophobic membrane regions as the target sites. While the enhancement constants were independent of pH over the range tested (3.3–5.0), the rate constants decreased linearly with increasing extracellular proton concentration, indicating the presence of an additional surface barrier against proton penetration, the effectiveness of which increased with protonation. The alkanols affected the acidification curves of energized yeast suspensions in such a way that the final pH values were linear functions of the alkanol concentrations. These results were consistent with a balance between active and passive proton movements at the final pH, the exponential enhancement constants calculated from the slopes being nearly identical with those obtained with deenergized cells. It was concluded that passive proton influx contributes to the kinetics of acidification in S. cerevisiae and that uncoupling contributes to the overall kinetics of alkanol-inhibited secondary active transport across the yeast plasma membrane.     

Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo

Incorporation of 8 mol percent lactosylceramide into small unilamellar vesicles consisting of cholesterol and sphingomyelin in an equimolar ratio and containing [³H]inulin as a marker resulted in an increase in total liver uptake and a drastic change in intrahepatic distribution of the liposomes after intravenous injection into rats. The control vesicles without glycolipid accumulated predominantly in the hepatocytes, but incorporation of the glycolipid resulted in a larger stimulation of Kupffer-cell uptake (3.2-fold) than of hepatocyte uptake (1.2-fold). Liposome preparations both with and without lactosylceramide in which part of the sphingomyelin was replaced by phosphatidylserine, resulting in a net negative charge of the vesicles, were cleared much more rapidly from the blood and taken up by the liver to higher extents. The negative charge had, however, no influence on the intrahepatic distributions. The fast hepatic uptake of the negatively charged liposomes allowed competition experiments with substrates for the galactose receptors on liver cells. Inhibition of blood clearance and liver uptake of lactosylceramide-containing liposomes by $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ indicated the involvement of specific recognition sites for the liposomal galactose residues. This inhibitory effect of $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ was shown to be mainly the result of a decreased liposome uptake by the Kupffer cells, compatible with the reported presence of a galactose specific receptor on this cell type (Kolb-Bachofen et al. (1982) Cell 29, 859–866). The difference between the results on sphingomyelin-based liposomes as described in this paper and those on phosphatidylcholine-based liposomes as published previously (Spanjer and Scherphof (1983) Biochim. Biophys. Acta 734, 40–47) are discussed.     

Corporate sustainability for architecture engineering and construction (AEC) organizations: Framework,transition and implication strategies

This research aims to propose a regenerative sustainability framework for AEC organizations, not only to focus on delivering green certified projects, but to encourage a revitalized approach to systematically drive their sustainability initiative. To achieve this aim, the study conducts an extensive global review of sustainable assessment systems in various industries and organizations, such as corporate sustainable reports, green company competitions, and green-renowned stocks. After learning the best practices from other industries, a sustainable development framework for the AEC organizations is developed. The proposed framework constitutes 110 key indicators covering three-dimensions of sustainability—social, environmental, and economic, respectively—and four-elements of corporate development essentials—projects, operations, governance, and stakeholders. By comparing the proposed framework to existing AEC practices, the study identifies the skewed development of corporate sustainability in the AEC industry where much awareness have been given to project-level environmental matters, but the social dimension, such as social stakeholders and social governance, are seriously lacking and need to be prioritized. A case study of a multi-millions construction company in China was investigated to validate this framework in practice. The case analysis justifies the practical value of the proposed framework, and elaborates the future need of regenerative sustainable initiatives for AEC organizations. The study contributes to the development of corporate sustainability theory in the AEC industry, and also provides industrial practitioners and policy makers with a better understanding of the next generation of corporate sustainable performance and implementation strategies.     

Biosynthesis of (−)-ent-kaurenoic acid in Smallanthus sonchifolius and its effect against microbial biofilms

The biosynthetic pathway of (–)-ent-kaurenoic acid (1) was investigated by incorporation of 1-d-¹³C-glucose in Smallanthus sonchifolius (Asteraceae) plantlets. The ¹³C-enrichment pattern indicated that methylerythritol-4-phosphate (MEP) pathway is the biosynthetic pathway involved in diterpenoid biosynthesis. Our studies in S. sonchifolius reinforce that the biosynthesis of different classes of terpenes should not be compartmentalized into cytosol and plastids. Additionally, (–)-ent-kaurenoic acid showed antimicrobial activity against Staphylococcus aureus biofilm.     

Selection of Small Peptides, Inhibitors of Translation

Identification of small molecular weight compounds targeting specific sites in the ribosome can accelerate development of new antibiotics and provide new tools for ribosomal research. We demonstrate here that antibiotic-size short peptides capable of inhibiting protein synthesis can be selected by using specific elements of ribosomal RNA as a target. The ‘h18’ pseudoknot encompassing residues 500-545 of the small ribosomal subunit RNA was used as a target in screening a heptapeptide phage-display library. Two of the selected peptides could efficiently interfere with both bacterial and eukaryotic translation. One of these inhibitory peptides exhibited a high-affinity binding to the isolated small ribosomal subunit (K_d of 1.1 μM). Identification of inhibitory peptides that likely target a specific rRNA structure may pave new ways for validating new antibiotic sites in the ribosome. The selected peptides can be used as a tool in search of novel site-specific inhibitors of translation.     

Depolarization-induced release of l-glutamic acid from isolated-resealed synaptic membrane vesicles

l-Glutamic acid actively loaded into resealed brain synaptic membrane vesicles was rapidly released into the incubation medium following the introduction of KCl and CaCl₂, or nigericin, or veratridine into the external medium. The KCl-induced release was enhanced by the presence of low (0.1 mM), extravesicular [Ca²⁺]. Neither the KCl-induced nor the veratridine-stimulated l-glutamate efflux were carrier-mediated processes. Finally, the KCl-stimulated l-glutamate efflux was dependent on the ratio of intra- to extravesicular [K⁺]. The observations described in this study were indicative of depolarization-induced l-glutamate release from isolated synaptic plasma membrane vesicles.     

Recovery of methanotrophs from disturbance: population dynamics,evenness and functioning

Biodiversity is claimed to be essential for ecosystem functioning, but is threatened by anthropogenic disturbances. Prokaryotes have been assumed to be functionally redundant and virtually inextinguishable. However, recent work indicates that microbes may well be sensitive to environmental disturbance. Focusing on methane-oxidizing bacteria as model organisms, we simulated disturbance-induced mortality by mixing native with sterilized paddy soil in two ratios, 1:4 and 1:40, representing moderate and severe die-offs. Disturbed microcosms were compared with an untreated control. Recovery of activity and populations was followed over 4 months by methane uptake measurements, pmoA-qPCR, pmoA-based terminal restriction fragment length polymorphism and a pmoA-based diagnostic microarray. Diversity and evenness of methanotrophs decreased in disturbed microcosms, but functioning was not compromised. We consistently observed distinctive temporal shifts between type I and type II methanotrophs, and a rapid population growth leading to even higher cell numbers comparing disturbed microcosms with the control. Overcompensating mortality suggested that population size in the control was limited by competition with other bacteria. Overall, methanotrophs showed a remarkable ability to compensate for die-offs.     

Ca2+-induced isotropic motion and phosphatidylcholine flip-flop in phosphatidylcholine-cardiolipin bilayers

Ca²⁺ induces a structural change in phosphatidylcholine-cardiolipin bilayers, which is visualised by freeze-fracturing as lipidic particles associated with the bilayer and is detected by ³¹P-NMR as isotropic motion of the phospholipids. In this structure a rapid transbilayer movement of phosphatidylcholine and a highly increased permeability towards Mn²⁺ are observed.