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Superoxide as an obligatory, catalytic intermediate in photosynthetic reduction of oxygen by adrenaline and dopamine.

The superoxide anion radical is known to be the first product of photosynthetic reduction of oxygen mediated by a variety of electron carriers. The effectiveness of many of these electron carriers as herbicides, and the toxicity of the superoxide they produce, have been suggested to rule out oxygen reduction as a physiological component of normal photosynthesis. Here results with isolated spinach chloroplasts are presented that demonstrate that the related catecholamines adrenaline and dopamine mediate photosynthetic reduction of oxygen. Complete inhibition by added superoxide dismutase of light-dependent oxygen uptake by isolated chloroplasts and of the electron transport it supports indicates that superoxide is an obligatory catalytic intermediate, not a product, in adrenaline- and dopamine-mediated oxygen reduction. These compounds might function as chemical analogues of a proposed natural mediator, or oxygen-reducing factor, that allows oxygen reduction to participate in energy transduction in photosynthesis. The identity of the putative natural mediator and the role of oxygen reduction in photosynthesis are discussed. The fully oxidized form of adrenaline, adrenochrome, also acts as a mediator of photosynthetic oxygen uptake, but only by reducing oxygen to superoxide.

Animals↗

On the functional of neuromelanin.

The hypothesis is presented, based on the chemical structure of neuromelanin, that one of its functions in the catecholamine neurons in the brain is to protect the cell against toxic quinones (such as dopaminchrome and noradrenochrome, or their dihydroxy isomers) produced from the catecholamines dopamine and noradrenaline (and possibly adrenaline) during the course of prostaglandin synthesis by the enzyme prostaglandin H synthetase, or possibly by spontaneous oxidation. One aminochrome-adrenochrome-has been shown to be neurotoxic and to have psychotomimetic properties in humans. Depending on the site of production these compounds may be involved in the pathogenesis of Parkinson's disease or schizophrenia.

Brain↗

Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination.

Hydrogen peroxide steady state levels of 5 micromolar were determined in soybean (Glycine max) embryonic axes incubated for 2 hours and in axes pretreated with aminotriazole or cyanide, where these levels were 50 and 1 micromolar, respectively. The activities of catalase (105 picomoles H(2)O(2) per minute per axis), peroxidase (10-44 picomoles H(2)O(2) per minute per axis), glutathione peroxidase (3 picomoles H(2)O(2) per minute per axis) and superoxide dismutase (3.5 units per axis), were also determined. Catalase seems to be the most important H(2)O(2) consuming enzyme at the physiological concentration of H(2)O(2). A short treatment with aminotriazole, while substantially increasing H(2)O(2) level, did not affect the growth of the axes. The production of superoxide anion by the mitochondria isolated from soybean axes was measured from the superoxide dismutase-sensitive rate of adrenochrome formation in the presence of NADH or succinate as substrate and amounted to 1.3 and 0.8 nanomole O(2) (-) per minute per milligram protein, respectively. According to the stoichiometry of O(2) (-) and H(2)O(2) dismutation reactions, it is apparent that about 0.9 to 1.5% of the total oxygen uptake proceeds through the formation of the free intermediates of the partial reduction of oxygen.

Journal Article↗

Initiation of the polypeptide chain by reticulocyte cell-free systems. Survey of different inhibitors of translation.

In order to elucidate the mechanism of action of inhibitors that block the initiation of protein synthesis in mammalian systems, we have studied the following steps: (a) formation of the ternary complex Met-tRNAr-IF-E2-GTP, (b) binding of the initiator Met-tRNAf to the 40-S ribosomal subunit in the presence of initiation factors and dependent or not on the addition of mRNA, (c) formation of the initiation complex with 80-S ribosomes and (d) formation of the first peptide bond. Adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and showdomycin block the formation of the ternary complex Met-tRNAf-IF-E2-GTP. Edeine A1, aurintricarboxylic acid and polydextran sulphate block the binding of the mRNA to the 40-S ribosomal subunit. Pactamycin induces the formation of stable smaller initiation complexes which are unable to go through the subsequent steps of initiation. Stimulation of the binding of the initiator Met-tRNAf to the 80-S ribosome in the presence of initiation factors is observed with sparsomycin and antibiotics of the sesquiterpene family (verrucarin A, trichodermin and trichothecin). However, these antibiotics block the reaction of the bound Met-tRNAf with puromycin. Narciclasine has no effect on the binding of the initiator to the ribosome but strongly blocks its reaction with puromycin. We have developed a simple technique to detect the Met-tRNAf-40-S-subunit-poly(A, G, U) initiation complexes by chromatography on Sepharose 6B columns. The requirements for the formation of such complexes measured by this technique and its comparison with the sucrose gradient centrifugation method are described.

Animals↗

SOME ACTIONS OF CENTRALLY ACTIVE AND OTHER DRUGS ON THE TRANSMISSION OF SINGLE NERVE IMPULSES THROUGH THE ISOLATED SUPERIOR CERVICAL GANGLION PREPARATION OF THE RABBIT.

The effect of some centrally-active and other drugs on the transmission of single nerve impulses through the isolated superior cervical ganglion preparation of the rabbit has been studied by recording both preganglionic and postganglionic action potentials. Block of conduction in the axon could be distinguished from block of the synaptic mechanism. The drugs did not appear to exert any one characteristic form of blocking action. A continuous spectrum of drug action linked an agent such as meprobamate which acted predominantly on the synapse to benactyzine which acted mainly by blocking axonal conduction. The drugs have been divided into three groups. Group I: hexamethonium, meprobamate, paraldehyde, amylobarbitone, methylpentynol and azacyclonal; these acted relatively selectively at the ganglion. Group II: N714C (the cis-isomer of chlorprothixene), prochlorperazine, methylpentynol carbamate, pipradrol, promethazine, perphenazine and procaine; the action of these drugs on the ganglion could be accounted for entirely in terms of their axonal depressant action. Group III: chlorprothixene, promazine, N720 (dihydrochlorprothixene), chlorpromazine, hydroxyzine and benactyzine; these drugs also blocked axonal conduction but in addition they appeared to exert a "facilitating" action at the ganglionic synapse. The actions of adrenaline, adrenochrome, iproniazid, ergotoxine, mescaline and lysergic acid diethylamide on transmission were also studied. The implications of the modifications of ganglionic transmission produced by these drugs is discussed.

Action Potentials↗

A spectropolarimetric assay of (-)-adrenaline in compendial formulations.

A method is described for the determination of (-)-adrenaline in certain formulations containing adrenaline hydrogen tartrate at concentrations down to 0.18 mg ml-1 (1:10 000 adrenaline). The assay is based upon a spectropolarimetric measurement at 249 nm of sample solutions, suitably treated to remove interfering substances. The rotation of the sample solutions is corrected for the rotation of the tartaric acid species which is determined by a difference rotation measurement on equimolar sample solutions at pH 1.1 and pH 5.6. The concentration of (-)-adrenaline in the sample is calculated from the net rotation at 249 nm due to the (-)-adrenaline and the total concentration of adrenaline (+)-and (-)-isomers) determined by a published spectrofluorimetric method. The assay is specific for (-)-adrenaline in the presence of (+)-adrenaline, (+)-tartaric acid, adrenaline sulphonic acid and low levels of adrenochrome.

Aerosols↗

Effect of catecholamines on the bactericidal activity of polymorphonuclear leukocytes.

Epinephrine, norepinephrine, and dihydroxyphenylalanine at 8 mm concentrations prevented iodination of zymosan by intact neutrophils and decarboxylation of l-alanine by leukocyte sonic extracts. The same concentration of epinephrine also reduced bactericidal activity of the leukocyte against Staphylococcus aureus, Enterobacter cloacae, and Proteus rettgeri without decreasing phagocytosis of bacteria. Spectral studies indicated that epinephrine interferes with the myeloperoxidase-mediated reactions by competing for available H(2)O(2) via its enzymatic oxidation to adrenochrome. These findings support a mechanism in which H(2)O(2) plays an important role in the bactericidal activity of the leukocyte.

Aldehydes↗

Laccase and melanization in clinically important Cryptococcus species other than Cryptococcus neoformans.

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.

Cryptococcosis↗

The endogenous biosynthesis of prostaglandins by brain tissue in vitro.

The capacity for biosynthesis of prostaglandin F2alpha (PGF2alpha) and prostaglandin E2 (PGE2) from endogenous precursors by brain tissue slices and homogenates was measured by a gas chromatography - mass fragmentography method using deuterated prostaglandins as internal standards. Mean biosynthesis in rat cerebral cortex slices incubated for 60 min was 60.2 ng PGF2alpha and 17.4 ng PGE2 per 100 mg of tissue. The corresponding values for homogenates were 78.1 ng and 28.9 ng. Synthetic capacity of cat cerebral cortex was considerably greater but that of human tissue was smaller than that found in rat brain. Cat cerebellum in contrast to other regions synthesized more PGE2 than PGF2alpha. The time-course of prostaglandin formation in slices was linear for the initial 60 min. Catabolism in cerebral tissues was found to be very small. Prostaglandins formed or added to the incubation media distributed between tissue and medium in a manner indicating some specific binding as well as nonspecific solubilization in tissue lipids. Norepinephrine, 3,4-L-dihydroxyphenylalanine, dopamine, adrenochrome and apomorphine greatly stimulated PGF2alpha formation probably through a nonenzymatic reduction of endoperoxides. Norepinephrine added to homogenates appeared to stabilize the fatty acid cyclo-oxygenase preventing it from inactivation. Indomethacin and Ketoprofen were potent inhibitors of biosynthesis. Paracetamol was found to be a less potent synthetase inhibitor than aspirin. The biosynthetic capacity of brain tissue in vitro appears to be orders of magnitude more than that of normal brain in situ.

Animals↗

In vitro cytotoxicity of melanized poly(2-hydroxyethyl methacrylate) hydrogels, a novel class of ocular biomaterials.

Due to their ability to absorb ultraviolet and visible radiation, we have proposed the melanized poly(2-hydroxyethyl methacrylate) hydrogels as biomaterials suitable for the manufacture of soft artificial intraocular lenses. Their biocompatibility has not been evaluated so far. In this study, poly(2-hydroxyethyl methacrylate) containing various amounts of adrenochrome-melanin were synthesized and the cytotoxicity of their aqueous extracts was assessed by using four in vitro testing techniques (trypan blue dye exclusion, inhibition of DNA synthesis, lactate dehydrogenase release, and inhibition of cell growth). Assays were based on incubation with human choroidal fibroblasts. By the first three techniques, no cytotoxicity was found in the extracts. The inhibition of cell growth test showed a slight cellular protein loss, however only in the extracts of polymers with high melanin content. The results suggest that the release of potentially toxic agents from melanized hydrogels into an aqueous medium is not significant. However, when an assay in collagen gel was carried out in the presence of specimens of melanized hydrogels, a toxic reaction was clearly revealed. This can be caused by a delayed release of toxic molecules from melanin, or by some other mechanism. The use of melanin-containing polymers as implant materials becomes questionable and further research is necessary.

Biocompatible Materials↗

The salivary catechol oxidase/peroxidase activities of the mosquito Anopheles albimanus.

Salivary gland homogenates from adult female Anopheles albimanus mosquitoes relaxed aortic rings preconstricted with noradrenaline (NA). This relaxation is slow and is due to destruction of NA. Incubation of NA with the homogenate yielded a product with a spectrum consistent with the corresponding adrenochrome. Oxidation of NA was enhanced by a superoxide generation system and inhibited by the combined action of superoxide dismutase and catalase. Additionally, peroxidase activity on both synthetic (o-dianisidine) and biologically active (serotonin) substrates was also present in the salivary gland homogenates, this latter activity requiring hydrogen peroxide. Noradrenaline oxidation, serotonin and o-dianisidine peroxidation and vasodilation all co-elute with a heme protein of relative molecular mass 50,000, as determined by molecular sieving chromatography. Peroxidase activity was localized in the posterior (female-specific) lobes of salivary glands and was also detected in nitrocellulose membranes probed by hungry mosquitoes. Protein and peroxidase activities were significantly lower in salivary glands of mosquitoes after probing and feeding on blood. It is suggested that adult female Anopheles albimanus mosquitoes contain a salivary heme peroxidase that functions during blood finding and blood feeding by destroying hemostatically active biogenic amines released by the vertebrate host during tissue destruction.

Animals↗

[Examination of non-transmitter effects of catecholamines on clonal cells derived from Drosophila CNS].

The effects of catecholamines (CAs) other than their transmitter action were investigated using clonal neuronal cells, ML-DmBG2-c2, derived from Drosophila in the larval central nervous system (CNS). All catecholamines tested, adrenaline (AD), dopamine (DA), noradrenaline (NA) and isoproterenol (ISO), prevented any increase in the number of cells during 2- to 7-day culture. alpha-, beta-adrenergic and dopaminergic antagonists did not block the effects of CAs on the number of cells. Adrenochrome, a product of the oxidative degradation of AD, also prevented any increase in the number of cells, as AD did. The effect of AD was partially blocked by an antioxidant, dithiothreitol (DTT). These results suggest that the inhibition of the increase in cell numbers by CA might be mediated by CAs themselves and/or oxidative products in the CA metabolic process. It is concluded that CAs inhibit cell proliferation but do not induce cell death in the Drosophila clonal cells.

Animals↗

HSV-induced reactivation: contribution of epinephrine after corneal iontophoresis.

A modified, bilateral iontophoresis technique was used to evaluate herpes simplex virus (HSV) reactivation and epinephrine distribution and concentration in full-thickness corneas and in trigeminal ganglia of rabbits. Infectious virus was recovered from 58% of epinephrine-induced eyes 24-96 hours after iontophoresis. A histochemical adrenochrome oxidation method localized epinephrine exclusively in the corneal epithelium and anterior stromal lamellae after iontophoresis. Epinephrine introduced iontophoretically into the corneal epithelium and stroma did not reach the ganglion level as detected by the assay. Radiometric-enzymatic assay of corneal and ganglionic tissues demonstrated a transient increase in total corneal catecholamine levels from an average of 417 micrograms to an average of 778 micrograms ; concentrations returned to approximate control values 24 hours after iontophoresis. Ganglion total catecholamine levels increased from an average of 606 micrograms to an average of 1211 micrograms for a 12-hour period and remained elevated at 24 hours after iontophoresis. Epinephrine acting directly on the corneal epithelium and epinephrine metabolites at the ganglion level could act synergistically to induce ocular and ganglionic HSV reactivation.

Animals↗

An unusual NAD(P)H-dependent O2-.-enerating redox system in hepatoma 22a nuclei.

Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.

Animals↗

Inhibition of O2-.- and HO.- mediated processes by a new class of free radical scavengers: the N-acyl dehydroalanines.

N-phenylacetyl dehydroalanines are captodative olefins. They inhibit two processes mediated by superoxide anion (O2-.) in a concentration dependent manner: reduction of NBT to blue formazan and oxidation of epinephrine to adrenochrome. They also inhibit in a dose related way the degradation of deoxyribose produced during either the Fenton reaction or the radiolysis of water, which are the two experimental sources of hydroxyl radical (HO.) production. Based on the results obtained with superoxide dismutase, mannitol, thiourea, and uric acid, we postulate that these competitive inhibitory effects suggest a reaction between the dehydroalanine derivatives and the two oxygen derived radicals. Hydroxyl free radical is scavenged more efficiently than superoxide anion. Substitution of the phenyl ring by methoxy groups does not modify significantly the activity. These molecules possess three target active sites which can react with free radicals.

Alanine↗

Hydrogen peroxide dependent oxidative degradation of DNA by copper epinephrine.

The hydrogen peroxide dependent oxidation of the epinephrine-copper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers. The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H2O2 and it preserves the capacity over a wide range of complex:DNA ratios. The rate of DNA clevage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when "free" copper ions are available, catecholamines may induce oxidative degradation of DNA and other biological macromolecules.

Copper↗

[Novel approach to the study od adrenaline auto-oxidation and its use for the measurements of superoxide dismutase activity].

The intensive rise of absorption maximum at 347 nm (wave number 28.8) was found during autoxidation of low concentrations of adrenaline (230 microM) in alkaline pH (10.65) under room temperature and in the absence of additional sources of superoxide. It was stated that the appearance of this product of adrenaline oxidation, which was not reported earlier, is considerably more rapid than formation of adrenochrome (absorbtion at 480 nm) and is inhibited by superoxide dismutase (SOD). It was shown that erythrocyte hemolysate and some studied antioxidants (ascorbate and cysteine) inhibit formation of this substance like SOD. The determination of the described substance can be used for measurement of both: activity of SOD or antioxidant activity of different biological materials. The possibility to use available not expensive chemicals (0.1% adrenaline solution from drug store, 0.2 M bicarbonate buffer) serves as an additional advantage of the proposed method.

Epinephrine↗

[The influence of natural inhibitors of radical reactions on autooxidation of adrenaline].

In the presence of EDTA soluble antioxidants (1 muM reduced glutathion, 3 muM cystein, 1 muM ascorbic acid) inhibited the autoxidation of epinephrine at pH 10.2; as to alpha-tocopherol (40 muM) and the oxidative forms of these antioxidants - they were ineffective. The inhibitory effect of superoxide dismutase was much greater than that of the antioxidants testsd. The appearance of adrenochrome during the free radical autoxidation of epinephrine proceeded without any participation of the hydroxyl radical.

Antioxidants↗