Photobiochemistry without light

Summary Efficient excited state formation — much higher than that hitherto expected — may occur in organelles and in intact cells. Excited triplet states can be enzymatically generated in high yields by different routes. An example is the oxidation of isobutanal to acetone and formic acid, catalyzed by horseradish peroxidase. Other enzymatic systems that generate triplet carbonyls are linear aliphatic aldehydes when oxidized by peroxidase/O₂, or the indole-3-acetic acid/peroxidase/O₂-reaction. The latter is widespread in plants.This new field — photobiochemistry without light — has led to a growing awareness of the idea that cells may utilize excited states to trigger photochemical processes even in the dark. Such phenomena are of considerable importance, also for the understanding of weak photon emission from biological systems.     

Respiratory burst in peritoneal exudate cells in response to a modified tuftsin.

Intraperitoneal administration of tuftsin-M [Thr-Lys-Pro-Arg-NH-(CH2)2-NH-CO-C15H31] to Balb/C mice has been shown to induce a respiratory burst in the peritoneal exudate cells. The macrophages exhibited enhanced levels of O2-, H2O2, NADPH oxidase and myeloperoxidase, but the activities of superoxide dismutase, catalase and glutathione peroxidase remained virtually unchanged. The magnitude of the oxidative burst depended directly on the dose of tuftsin-M; higher activity was observed at higher doses of the peptide. Tuftsin-M enhanced the generation of both O2- and H2O2 under in vitro conditions, as did phorbol myristate acetate. These results suggest that tuftsin-M could enhance non-specific defence against infections by activating the macrophages.     

Hydrogen peroxide generation in Trypanosoma cruzi.

Homogenates from T. cruzi epimastigotes produced 3.4 pmoles H2O2/min 10(6) cells, as detected by the cytochrome c peroxidase assay. Addition of NADH or NADPH increased H2O2 production by a factor of 3 and 5, respectively. When supplemented with NADH and NADPH, the mitochondrial, microsomal and supernatant fractions produced H2O2, the soluble fraction and the mitochondrial membranes being apparently the main generators of H2O2. The epimastigote homogenates showed cyanide-sensitive superoxide dismutase activity, equivalent to 0.28 microgram bovine superoxide dismutase per mg homogenate protein.     

Differential influences of various arsenic compounds on glutathione redox status and antioxidative enzymes in porcine endothelial cells

The cellular response and detoxification mechanisms in porcine endothelial cells (PAECs) to arsenic trioxide (As2O3), sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4) were investigated. NaAsO2 at 20 microM for 72 h increased Cu/Zn superoxide dismutase activity resulting in elevated intracellular hydrogen peroxide levels, but As2O3 and Na2HAsO4 did not. Trivalent arsenic compounds increased intracellular oxidized glutathione (GSSG) and total glutathione (GSH) and cellular glutathione peroxidase (cGPX) and glutathione S-transferase (GST) activity, but not glutathione reductase activity. The increased cGPX activity resulted in an elevated cellular GSSG content. Na2HAsO4 increased the cellular GSSG level at 72 h compared to controls. These results imply that the increased GSH content responding to the oxidative stress by trivalent arsenic compounds may be mainly related to the regulation of GSH turnover. The increased GST activity implies that the elevated intracellular GSH level responding to the oxidative stress may be used to conjugate arsenic in PAECs and facilitate arsenic efflux.     

Alpha 1-adrenergic stimulation of ketogenesis and fatty acid oxidation is associated with inhibition of lipogenesis in rat hepatocytes

The effect of norepinephrine on fatty acid synthesis (3H2O incorporation into fatty acids), on fatty acid oxidation to CO2 and on ketogenesis was studied in isolated hepatocytes of fed rats. After incubation with norepinephrine (50 microM), lipogenesis was lower (5.7 +/- 1.1 nmoles 3H2O incorporated into fatty acids/mg dry weight/30 min) than in controls (7.5 +/- 1.7; n = 6, p less than 0.02). In contrast, (1-14C) palmitate conversion into total ketone bodies was increased to 10.9 +/- 1.8 nmoles/mg/30 min with norepinephrine, vs 8.5 +/- 1.6 in controls (p less than 0.05), and more (1-14C) palmitate was converted to 14CO2 with norepinephrine than in controls (1.48 +/- 0.10 nmoles/mg/30 min vs 1.06 +/- 0.11, p less than 0.05). The inhibitory effect of norepinephrine on lipogenesis was abolished by addition of the alpha 1-receptor blocker prazosin, but not by alpha 2 or beta-blockers. The results demonstrate that the ketogenic effect of norepinephrine is coupled with an inhibitory effect on lipogenesis which may be explained by diminished activity of acetyl-CoA carboxylase, diminished formation of malonyl-CoA and decreased activity of carnitine palmitoyl transferase I.     

Fine structural relation between pancreatic excretory ductules and intercellular spaces

Summary Horse-radish peroxidase injected into the femoral vein of intact rats, or infused at 30 cm H₂O pressure into the main pancreatic duct of intact dogs, entered easily the interstitial spaces surrounding acini and acinar cells. The latter are interconnected at their luminal segments by zonulae occludentes. These junctions form a barrier to tracer penetrating from the interstitium towards the lumen of terminal ductules. However, the intraductally infused peroxidase entered the interstitial spaces, probably through the pressure injured acinar cells, as did colloidal carbon particles when infused intraductally.     

The glutathione peroxidases

There are several proteins in mammalian cells that can metabolize hydrogen peroxide and lipid hydroperoxides. These proteins include four selenium-containing glutathione peroxidases that are found in different cell fractions and tissues of the body. This review considers the structure and distribution of the selenoperoxidases and how this relates to their biological function. The functions of the selenoperoxidases were originally studied in systems where their activity was manipulated by changing dietary selenium levels. More recently, molecular techniques have allowed overexpression of selenoperoxidases in cell lines and animals. Additionally, cellular glutathione peroxidase knockout mice have been used to investigate the functions of this protein. From this work it is clear that the selenoperoxidases are involved in cell antioxidant systems. However, they also have more subtle functions in ensuring the regulation and formation of arachadonic acid metabolites that are derived from hydroperoxide intermediates. The range of biological processes, which are potentially dependent on optimal selenoperoxidase activity in mammals, emphasises the importance of achieving adequate selenium intake in the diet.     

Proton-induced dismutation of superoxide in aprotic media by bile pigments

Cyclic voltammetry of molecular oxygen in aprotic media (dimethylformamide) and in the presence of bilirubin and other bile pigments shows that superoxide anion (O2-.) undergoes proton-induced dismutation. Lactam hydrogens of bile pigments are sufficiently acid to induce (O2-.) disproportionation to O2 and H2O2. Because of its characteristic lipophilic behavior, a biological role for natural bilirubin similar to that of other non-enzymatic lipophilic scavengers of (O2-.) is suggested.     

Evaluation of models for the mechanism of action of 4-hydroxyphenylpyruvate dioxygenase

Summary 4-Hydroxyphenylpyruvic acid was oxygenated with various complexes of oxygen with Fe²⁺, superoxide ion, hydroperoxide anion, triplet and singlet oxygen. Oxidation occurred giving varying amounts of the 4-hydroxy derivatives of benzaldehyde, benzoic acid, phenol, phenylacetic acid and 4-carboxymethylquinone, but no homogentisic acid. 4-Hydroxyphenylperacetic acid was prepared and shown not to undergo self-oxidation. Its ferrous salt decomposed to 4-hydroxybenzyl alcohol. It is concluded that thea-keto carboxylic function is the site of oxygenation in the enzyme process and that a trioxalanone rather than a peracid intermediate may be implicated.Acknowledgments. We thank a referee for drawing our attention to the work of Hawkinsin and we are indebted to the Swiss National Science Foundation for the support of this work (grant No 2.418.0.79).     

Effects of scavengers of superoxide radicals, hydrogen peroxide, singlet oxygen and hydroxyl radicals on malondialdehyde generation from arachidonic acid by bovine seminal vesicle microsomes

Superoxide dismutase, catalase and sodium formate did not inhibit the formation of malondialdehyde (MDA) from arachidonic acid, suggesting that O2-., H2O2 and OH. are not involved in the enzymatical oxidation of arachidonic acid. Sodium azide was found to be an inhibitor of MDA production.     

Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate acetate stimulated human leukocytes

A Ginkgo biloba extract (Gbe) containing flavonoids, among other compounds, was tested for the release of activated oxygen species (O-2, H2O2, OH.) during the stimulation of human neutrophils (PMNs) by a soluble agonist. The extract slows down O2 consumption (respiratory burst) of stimulated cells by its inhibitory action on NADPH-oxidase, the enzyme responsible for the reduction of O2 to O-2. Consequently, superoxide anion (O-.2) and hydrogen peroxide (H2O2) production is significantly decreased when the PMNs stimulation is done in the presence of the extract at concentrations of 500, 250 and 125 micrograms/ml. Moreover, the hydroxyl radical generation (OH.) is very much decreased at concentrations as low as 15.6 micrograms Gbe/ml, which indicates that the extract also has free radical scavenging activity. Gbe is able at least to reduce very severely the activity of myeloperoxidase contained in neutrophils. This enzyme, secreted into the intra and extracellular medium, catalyzes the oxidation of chloride (Cl-) by H2O2 to yield strong oxidants (HOCl, chloramines) which are implicated in inflammatory processes.     

Optical probes and techniques for O<Subscript>2</Subscript> measurement in live cells and tissue

In recent years, significant progress has been achieved in the sensing and imaging of molecular oxygen (O(2)) in biological samples containing live cells and tissue. We review recent developments in the measurement of O(2) in such samples by optical means, particularly using the phosphorescence quenching technique. The main types of soluble O(2) sensors are assessed, including small molecule, supramolecular and particle-based structures used as extracellular or intracellular probes in conjunction with different detection modalities and measurement formats. For the different O(2) sensing systems, particular attention is paid to their merits and limitations, analytical performance, general convenience and applicability in specific biological applications. The latter include measurement of O(2) consumption rate, sample oxygenation, sensing of intracellular O(2), metabolic assessment of cells, and O(2) imaging of tissue, vasculature and individual cells. Altogether, this gives the potential user a comprehensive guide for the proper selection of the appropriate optical probe(s) and detection platform to suit their particular biological applications and measurement requirements.     

Zum Nachweis von Aminosäuren im 10−10-Mol-Maßstab. Trennung von 1-Dimethylamino-naphthalin-5-sulfonyl-aminosäuren auf Dünnschichtchromatogrammen

Summary Some solvent systems are described for the separation of 1-dimethyl-amino-naphthalene-5-sulphonylamino acids on thin layer chromatograms. 10^–10 mole per amino acid can be detected on a two-dimensional chromatogram, when the yellow fluorescence of these compounds is excited by a UV-lamp.     

The action of substance P on mesencephalic reticular and substantia nigral neurones of the rat.

Extracellular recordings were made from neurones in the mesencephalic reticular formation and substantianigra of the rat which was anaesthetized with urethane 1.5-2 g/kg i.p. Out of 44 cells tested 42 were excited by Substance P applied iontophoretically and in some cases this excitation was rapid. Evidence is presented for Substance P as a putative excitatory transmitter onto reticular and nigral neurones possibly released from primary sensory afferents.     

Control of respiration in skeletal muscle at rest

The suggestion is made that, under resting conditions in situ, muscle cell respiration is dependent on the way O2 and substrates are distributed to the cells by the microcirculation. (Delivery is measured as arterial-blood concentration multiplied by flow to the organ.) Microscale heterogeneity of this distribution, which is more marked but less stable than the more easily demonstrated larger-scale heterogeneity (0.1 to 0.5-g sampling grain), might indeed ration O2 and substrates in a large population of the cells of a resting organ at any given moment, and microscale heterogeneity of distribution may thus take part in the normal control of cell respiration.     

Involvement of reactive oxygen species in the microsomal S-oxidation of thiobenzamide

Superoxide dismutase, catalase and methional proved capable of inhibiting the microsomal oxidation of thiobenzamide, which is most probably catalyzed by the flavin-containing monooxygenase. This indicates that excited oxygen species (e.g. X O-2,H2O2, X OH) are involved in the catalytic cycle of this enzymatic reaction. CO, which inhibits the cytochrome P-450-dependent oxygen radical formation, had no effect on the oxidation reaction, suggesting that the source of the reactive oxygen species is not the microsomal mixed-function oxidase.     

Metabolism of amphibian spermatozoa in relation to their motility.

Bufo arenarum spermatozoa were able to sustain motility both under aerobic and anaerobic conditions. In aerobiosis, the oxygen consumption varies between 2.6 and 4.2 microliter O2/10(8) cells/h at 30 degree C. The synthesis of lactic acid by anaerobic spermatozoa demonstrated the existence of an active glycolytic pathway.     

The interaction of superoxide with nitric oxide destabilizes hypoxia-inducible factor-1α

In renal carcinoma cells (RCC4) hypoxia inducible factor-1 (HIF-1) is constitutively expressed due to a von Hippel Lindau protein deficiency, but can be degraded by calpain, independently of the 26S proteasome, when exposed to hypoxia/nitric oxide (NO). In this study we examined molecular mechanisms to explain calpain activation. The inability of hypoxia/NO to degrade HIF-1α in respiratory-deficient RCC4-ρ0 cells pointed to the requirement for mitochondria-derived reactive oxygen species. A prerequisite for O ₂ ⁻ in combination with NO to destabilize HIF-1α was corroborated in RCC4-p0 cells, when the redox cycler 2,3-dimethoxy-1,4-naphthoquinone was used as a source of superoxide. Degradation of HIF-1α required intracellular calcium transients and calpain activation. Using uric acid to interfere with signal transmission elicited by NO/O ₂ ⁻ blocked HIF-1α degradation and attenuated a calcium increase. We conclude that an oxidative signal as a result of NO/O ₂ ⁻ coformation triggers a calcium increase that activates calpain to degrade HIF-1α, independently of the proteasome. Received 14 August 2007; received after revision 4 October 2007; accepted 22 October 2007     

Infusion at constant rate in vivo

The alpha-amylase of mycelial cells of Aspergillus oryzae exists in a particular form in 8000 g pellet. The lysosomal localization of acid phosphatase is confirmed by electron microscopy. The purification of lysosomes by discontinuous gradient of sucrose in D2O shows that alpha-amylase activity is bound to these particles.     

Antioxidant survey to assess antagonism to redox stress using a prokaryotic and an eukaryotic system

Using a prokaryote (Escherichia coli) and a metazoa-resembling eukaryote (Ochromonas danica), we surveyed antioxidants which might overcome redox stress imposed by menadione sodium bisulphite (MD) and buthionine sulphoximine (BSO). BSO oxidant stress was evident only inO. danica; MD oxidant stress was evident in both organisms. Glutathione, its precursors, e.g. cysteine, homocysteine, and 2-oxo-4-thiazolidine carboxylic acid, and red blood cells, emerged as prime antioxidants for relieving BSO and MD oxidant stress. BSO and MD oxidant activity and antioxidant-annulling effect inO. danica were judged comparable to those found in animal cells whereas the resultsE. coli were not entirely equivalent. TheO. danica system emerged as a practical, rapid, and useful system for pinpointing oxidant stressors and antioxidants, and shows promise for studies with mammalian systems.