Studies on 9-amino-6-chloro-2-methoxy acridine fluorescence quenching and enhancement in relation to energy transduction in spheroplasts and intact cells of the cyanobacterium Plectonema boryanum

The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O₂ proceeds via the thylakoid membrane and terminates at a CN^- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA 9-amino-6-chloro-2-methoxy acridine - chl a chlorophyll a - DBMIB 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DNP dinitrophenol - DNP-INT dinitrophenyl ether of 2-iodo-4-nitrothymol - FCCP carbonylcyanide-p-trifluoro-methoxy phenylhydrazone - S-13 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide - tricine N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine - Tris Tris (hydroxymethyl) amino methane     

In vivo dopamine receptor binding studies with a non-radioactively labeled agonist,dipropyl-5, 6-ADTN

Evidence is presented that a low dose of peripherally administered N, N-dipropylamino-5, 6-dihydroxytetralin (DiPr-5, 6-ADTN) specifically labels dopamine (DA) receptors in rat brain.Concentrations of this potent DA receptor agonist were determined by a highly selective method using reversed phase liquid chromatography and amperometric detection. The binding characteristics satisfy all criteria regarding saturability, stereospecificity, regional distribution and relation with pharmacological effects that are associated with DA receptor interactions. A rough estimation of the density of binding sites in the striatum resulted in values of 60–70 pmol/g. Lesioning the nigrostriatal pathway does not significantly alter the amount of ligand bound, nor do pretreatments with serotonergic, α-adrenergic or β-adrenergic antagonists. DiPr-5, 6-ADTN has thus been shown to be a useful ligand for labeling central DA receptors and a powerful tool in the study of DA-ergic mechanisms.     

Comparative study of methodologies for obtaining β-glucosidase immobilized on dextran-modified silica

In this study, two procedures for the immobilization of β-glucosidase on silica are compared. The first approach comprises a preliminary stabilization of β-glucosidase by coupling with dextran dialdehyde and subsequent immobilization of the obtained β-glucosidase dextran dialdehyde with aminopropylsilica. In the second approach, β-glucosidase is immobilized on silica modified with a dextran-dialdehyde coating. Enzyme immobilized via coupling with dextran dialdehyde and subsequent attachment with aminopropylsilica show a remarkably enhanced thermostability. Enzyme immobilized by the alternative approach demonstrated an inferior thermoresistance. The difference in behavior of the immobilized enzyme obtained via these two methods can be explained considering the number of links between the enzyme and carrier. Enzyme immobilized on dextran dialdehydecoated silica is fixed via a limited number of links. On the other hand, with soluble β-glucosidase-dextran conjugates, the enzyme configuration is already stabilized via a high number of links with the dextran backbone. It is clear from this study that the sequence of reactions in immobilizing enzymes on silica support via a dextran-dialdehyde linker has a significant effect on the final properties.     

Biotechniques for air pollution control

This paper gives an overview of present biological techniques for the treatment of off-gases and the techniques that are being developed at the moment. The characteristics, advantages, disadvantages, costs and application area are discussed and compared. Biological off-gas treatment is based on the absorption of volatile contaminants in an aqueous phase or biofilm followed by oxidation by the action of microorganisms. Biofilters, bioscrubbers and biotrickling filters are used for elimination of odour and bioconvertable volatile organic and inorganic compounds and are enjoying increasing popularity. This popularity is a result of the low investment and operational costs involved compared to physico-chemical techniques and the elimination efficiencies that can be obtained. The operational envelop is still extending to higher concentrations and gas flow rates (exceeding 200,000 m³ h^–1) and a broader spectrum of degradable compounds. Research and development on the use of membranes and the addition of activated carbon or a second liquid phase to the biological systems may lead to a more efficient elimination of hydrophobic compounds and buffering of fluctuating loads. Shorter adaptation periods can be obtained by inoculation with specialized microorganisms. Improved design and operation are made possible by the growing insights in the kinetics and microbiology and supported by the development of models describing biological off-gas treatment. In conclusion, biotechniques are efficient and cost effective in treating off-gases with concentrations of biodegradable contaminants up to 1–5 g/m³. They could play a justified and important role in air pollution control in the coming years.Abbreviations VOC volatile organic compound - NO _x gaseous oxides of nitrogen     

Impact of grazing and atmospheric nitrogen deposition on the vegetation of dry coastal dune grasslands

Abstract. A five-year experimental study was carried out to examine the combined effects of grazing and atmospheric nitrogen deposition on the vegetation of three dry dune grasslands: one short species-rich, one short species-poor, and one predominated by tall graminoids. Additional fertilization with nitrogen had no significant effect, neither in grazed nor in non-grazed plots. Exclusion of grazing by rabbits resulted in an increase in the frequency of perennial graminoids and a decrease in the frequency of annual graminoids and herbs. Nevertheless, species diversity remained the same in the species-rich grassland. During the experiment, the above-ground biomass increased in all nongrazed plots and the amount of bare soil and mosses decreased. The vegetation changes occurred mainly within one year after the exclusion of grazing. An exception is the grass-dominated site where the amount of Calamagrostis epigejos increased gradually from ca. 20 % in the first two years to about 50 % in the fourth and fifth year. Grazing by rabbits seems essential to prevent graminoids to become predominant in the dry dunes. If graminoids are dominant, grazing by horses can be an appropriate method to restore the original grassland vegetation. After six months of grazing by horses the grass-dominated site showed a decrease of the frequency of perennial graminoids, from 95 % to 80 %, and an increase of the frequency of perennial herbs, from 2.5 % to between 13 and 20 %.     

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome $\text{b}\text{c}$ redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O₂ reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.