Protective Effect of 5-hydroxytryptophan and α-Methyl DOPA against the Inhibitive Effect of Lithium on the Cicatrisation and the Regeneration of the Planarian Worm Dugesia tigrina and the Hydroid Clava squamata

The lithium ion has an inhibitive effect on cicatrisation and regeneration of Dugesia tigrina and Clava squamata while 5-hydroxytryptophan and α-methyl DOPA have a protective one. It seems that these substances as well as the biogenic amines 5-hydroxytryptamine and N-adrenaline play a role during the cell migrations which underly morphogenesis.     

Cholera toxin and thyrotropine can replace natural inducers required for the metamorphosis of larvae and buds of the scyphozoanCassiopea andromeda

Summary The scyphozoan medusaCassiopea andromeda forms free swimming planulae and buds that metamorphose into tentacle bearing sedentary polyps. About 30% of the planulae and 7% of the buds undergo such metamorphosis within 30 days in sterile natural seawater from the Red Sea. In sterile artificial sea water devoid of any organic substances, normal metamorphosis does not take place. This indicates that both the planulae and the buds require organic morphogenetic inducers present in the sea to settle and metamorphose. The addition of cholera toxin or thyrotropin to preparations of sterile artificial sea water, induced normal metamorphosis. These inducers enhanced the rate of metamorphosis and up to 100% of the planulae and buds formed polyps within 2–18 days. We conclude that our preparations of cholera toxin and thyrotropin mimic the action of natural inducers.     

A Simple Radioenzymatic Method to Measure Picogram Amounts of DOPA in Brain and Biological Fluids

A simple radioenzymatic method for the determination of DOPA is described. The method is based on the conversion of DOPA to 3-O-[methyl-³H]DOPA by catechol-O-methyltransferase in the presence of S-adenosyl-[methyl-³H]methionine and purification of the labelled product by Sephadex G10 and Dowex 50 W × 4 ion exchange resin. The method has been applied to the assay of endogenous DOPA in different brain areas and to measuring DOPA accumulation after inhibition of aromatic amino acid DOPA decarboxylase.     

Amine neuromediators,their precursors,and oxidation products in the culture of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The growth dynamics of the synthesis of monoamine neuromediators serotonin, norepinephrine, and dopamine in Escherichia coli K-12 was investigated for the first time using high performance liquid chromatography with electrodetection. Maximum (micromolar) concentrations of these compounds were detected in E. coli cells during the early growth phases; their intracellular content decreases after the transition to late growth phases. E. coli biomass contains (i) the substances DOPA and 5-hydroxytryptamine that serve in animal cells as neuromediator precursors and (ii) the products of their oxidative deamination. Presumably, the biosynthesis and degradation of monoamine neuromediators in bacterial cells involves enzyme systems analogous to those typical of animals. The culture fluid of E. coli contains micromolar concentrations of DOPA and nanomolar of serotonin, dopamine, and norepinephrine during the late growth phase. These concentrations are sufficient for animal/human receptors to bind them. This article deals with the potential biotechnological applications of the data obtained.     

Adsorption of chemically synthesized mussel adhesive peptide sequences containing DOPA on stainless steel

The adsorption of proteins at solid–liquid interfaces is important in biosensor and biomaterial applications. Marine mussels affix themselves to surfaces using a highly cross‐linked, protein‐based adhesive containing a high proportion of L‐3,4‐dihydroxyphenylalanine (DOPA) residues. In this work, the effect of DOPA residues on protein adhesion on stainless steel surfaces was studied using a quartz crystal microbalance with dissipation system. The adsorption of two repetitive peptide motifs, KGYKYYGGSS and KGYKYY, from the mussel Mytilus edulis foot protein 5 on stainless steel was studied before and after chemo‐enzymatic modification of tyrosine residues to DOPA using mushroom tyrosinase. Conversion from tyrosine to DOPA, evaluated by HPLC, was in the range 70–99%. DOPA‐modified sequences showed fourfold greater adhesion than unmodified M. edulis foot protein 5 motifs. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma

The addition of a commercially available sulfatase to the incubation mixture for the single isotope radioenzymatic assay of the catecholamines permits the simultaneous assay of the catecholamine sulfates in plasma. The sulfatase is compatible with the catechol-0-methyl transferase of the radioenzymatic assay and cleaves the sulfate to form the free catechol. With the multiple enzyme action it is possible to assay total (sulfated plus free) catecholamines and DOPA. A second series of assays performed in the absence of the sulfatase provides results for each of the free catecholamines and DOPA. In a study with ten normotensive male subjects supine free NE was 19% (range of 10–24%) of the total, E was 12% (range 6–16%), DA was 1% (range 0.5–2%) and DOPA was 45% (range 14–71%) of the total. Upon standing, free NE increased an average of 71% from 196±53 pg/ml (supine) to 336±69 pg/ml. Free NE (30%) and free E (18%) comprised a larger proportion of the total plasma NE and E in standing subjects than in the plasma of the supine subjects. Changes in the free levels and in the ratio of free to total levels of DA and DOPA were less than those noted for NE and E. This assay methodology may be useful in demonstrating individual variations in the ability to sulfate endogenous catechols.     

Purification of DOPA decarboxylase from bovine striatum

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30–60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit M_r = 56000 by SDS-PAGE) with a native M_r of 106000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.Abbreviations AADC Aromatic L-amino Acid Decarboxylase - CNS Central Nervous System - DOPA 3,4-dihydroxyphenylalanine - DTT Dithiothreitol, 5-HTP - 5-hydroxytryptophan - Mr relative molecular weight - PLP pyridoxal 5-phosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Part of this paper was presented at the 1987 Annual Pharmacology and Toxicology Conferences held at University of North Dakota School of Medicine, North Dakota, USA Res Commun Psychol Psychiat Behav 12: 227–228, 1987 (Abstr).     

Localization of dopamine in the freshwater hydrozoanHydra attenuata

Summary High performance liquid chromatography (HPLC), with electrochemical detection, is an analytical method sensitive enough to permit quantification of dopamine, dihydroxyphenylalanine (DOPA) and 5-S-cysteinyl DOPA in single or hemisected specimens ofHydra attenuata. Dopamine and 5-S-cysteinylDOPA appear to be the quantitatively predominant catechol compounds inH. attenuata, whereas DOPA is present in minor amounts. The presence of DOPA and 5-S-cysteinylDOPA, and the quantitative correlation between dopamine and these compounds in many specimens, suggests that dopamine inH. attenuata, as in higher animals, is formed through decarboxylation of DOPA. Contrary to the dopaminergic nerves in higher animals, DOPA inHydra seems to be oxidized and 5-S-cysteinyl DOPA is formed as a by-product. The oxidation of DOPA indicates that the hydroxylation of tyrosine into DOPA in the tissues ofH. attenuata is mediated by a tyrosinase rather than a tyrosine hydroxylase. Immunocytochemical methods demonstrate a highly variable distribution of dopamine in the tissues of different specimens ofH. attenuata. Dopamine immunoreactivity is confined to ectodermal tissue and can be found in several different cell types including nerve cells, battery cells, nematocytes, epithelial cells and interstitial undifferentiated cells. The large amounts of dopamine found in some specimens ofH. attenuata indicate some biological function, although its sporadic occurrence in neurites makes it less plausible as a generally utilized neurotransmitter in this animal.     

Morphogenetic effects of 9-cis-retinoic acid on the regenerating limbs of the axolotl

9-cis-retinoic acid has recently been found to be a high affinity ligand for the retinoic X receptor (RXR). RXRs are believed to be involved in metabolic activities rather than in morphogenetic ones. Interestingly, RXR has been found to form heterodimers involving other receptors from the steroid family, such as the thyroid hormone receptor, vitamin D receptor or retinoic acid receptors (RARs). In this paper we examined whether or not 9-cis-retinoic acid had any morphogenetic properties on the regenerating axolotl limb. It is shown that 9-cis-retinoic acid proximalized regenerating limbs and was somewhat more potent in this action than all-trans-retinoic acid. Based on these observations, the possible roles of other receptors during pattern formation is discussed. Correspondence to: P.A. Tsonis     

A sensitive fluorometric assay for protein-bound DOPA and related products of radical-mediated protein oxidation

Oxidative attack on proteins results in the hydroxylation of tyrosyl residues to protein-bound DOPA (3,4-dihydroxyphenylalanine). Existing methods for assaying protein-bound DOPA have poor sensitivity and numerous possible interferences, such that accurate determination (especially of very low DOPA concentrations) has required time-consuming acid hydrolysis and HPLC analysis with fluorometric detection. This work presents a sensitive and selective assay for peptide or protein-bound o-benzoquinones derived from DOPA based on fluorometric detection of ethylenediamine derivatives. Detection limits for protein-bound DOPA are in tbe range 0.53–4.70 ng/mL for the assay mixture, corresponding to sample DOPA concentrations of 0.59–5.30 ng/mL (representing a minimum of 6–54 pmole detected), depending on the particular protein/peptide under study. The assay response increases linearly with DOPA concentration, and also with the extent of radical exposure of the protein. The assay is a simple and fast way to assess DOPA formation and thus oxidative damage in a protein.     

Effect of 3,4-dihydroxyphenylalanine on Cu2+-induced Inactivation of HDL-associated Paraoxonase1 and Oxidation of HDL; Inactivation of Paraoxonase1 Activity Independent of HDL Lipid Oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Effects of Neurochemicals upon a Dinoflagellate Photoresponse*

SYNOPSIS Photoresponsiveness by Gymnodinium splendens Lebour was monitored quantitatively by a microscope-television system. Exposure to the catecholamines DOPA and Dopamine caused a decrease in light sensitivity, while 0.01 mM norepinephrine, epinephrine, or isoproterenol did not affect photoresponsiveness. Classical catecholamine blocking agents, dichloroisoproterenol, propranolol, and dibenzyline, and an inhibitor of DOPA synthesis, α-methyl-ρ-tyrosine, caused an increase in sensitivity. In addition, acetylcholine and an inhibitor of acetylcholinesterase activity, eserine, caused an increase in sensitivity, while an inhibitor of acetylcholine action atropine, had the opposite effect. These experiments suggest that G. splendens may have an antagonistic catecholamine-cholinergic system which participates in regulating photosensitivity.     

α-DIFLUOROMETHYL DOPA, A NEW ENZYME-ACTIVATED IRREVERSIBLE INHIBITOR OF AROMATIC L-AMINO ACID DECARBOXYLASE

DL-x-Difluoromethyl DOPA (DFMD, RMI 71801), an enzyme-activated irreversible inhibitor of aromatic L-amino acid decarboxylase in vitro, produces a rapid, long-lasting and dose-dependent inhibition of aromatic L-amino acid decarboxylase in peripheral tissues of mice when administered i.p. or orally. Doses of 500 mg/kg i.p. produce only very slight inhibition of the enzyme activity in mouse brain whilst inhibiting the enzyme activity of peripheral tissues by more than 90%. With L-[³H]-DOPA co-administration brain concentrations of L-[³H]DOPA and ³H-catecholamines are increased 3- to 8-fold concomitant with a decrease in the peripheral decarboxylation of L-[³H]DOPA. Under these conditions it is clear that the slight inhibition of enzyme activity in the brain is totally inadequate to inhibit the decarboxylation of L-DOPA in this organ. Similarly, the decarboxylation of exogenously supplied 5-hydroxytryptophan is inhibited peripherally with a consequent increase in brain serotonin concentrations. DFMD is another example of an enzyme-activated irreversible inhibitor which due to its novel and specific mechanism of action, may offer advantages over existing decarboxylase inhibitors.     

Anreicherung von Morphogenetischen Substanzen in Lichtpflanzen von Acetabularia Mediterranea Unter dem Einfluss von Puromycin

Zusammenfassung Unter dem Einfluß von Puromycin, das spezifisch aber reversibel die Proteinsynthese blockiert, erfolgt in belichteten kernhaltigen Pflanzenteilen von Acetabularia mediterranea eine Anreicherung morphogenetischer Substanzen. Wie Transplantationsversuche zwischen A. mediterranea und A. crenulata zeigen, betrifft diese Anreicherung sowohl die artspezifischen als auch die nichtartspezifischen morphogenetischen Substanzen. Die Anreicherung wird durch Actinomycin stark gehemmt.Es wird diskutiert, welche Schlußfolgerungen daraus für die Natur der artspezifischen morphogenetischen Substanzen gezogen werden können.

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Accumulation of morphogenetic substances in plants of Acetabularia mediterranea in light under the influence of puromycin

Summary Morphogenetic substances are accumulated in nucleated parts of the alga Acetabularia mediterranea in light, if they are treated with puromycin, which inhibits specifically but reversibel protein synthesis. Transplantation experiments between A. mediterranea and A. crenulata show, that there was an accumulation of both species specific morphogenetic substances and also of nonspecies specific ones. This accumulation is strongly inhibited if the parts are treated with actinomycin together with puromycin.The conclusions, which can be drawn from these results for the nature of the species specific substances, are discussed.

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An ultrastructural and cytochemical analysis of the cellular basis for tyrosine-derived collagen crosslinks in Leptogorgia virgulata (Cnidaria: Gorgonacea)

Summary Electron-microscopical autoradiography and cytochemical techniques have been used to identify the distinct and separate subcellular structures involved in the secretion of 1) procollagen, 2) dihydroxyphenylalanine (DOPA), which is a precursor of a collagen-crosslinking compound, and 3) DOPA oxidase, which converts DOPA to a putative crosslinking compound of collagen in the axial skeleton of the gorgonian coral Leptogorgia virgulata. Some skeletal-protein hydrolysates contain material that co-elutes with DOPA. The data indicate that these skeletogenic cells, corticocytes, are capable of modifying the number of non-reducible, tyrosine-derived crosslinkages of collagen by the secretion of a crosslinking compound that acts extracellularly on collagen. A mechanism for a cell-mediated control of the mechanical properties of collagen is thereby presented.     

A physico-chemical theory of sweet and bitter taste excitation based on the properties of the plasma membrane

Summary This paper is concerned with physical and chemical changes occurring in the taste buds due to the presence of sapid substances, and not with the nervous mechanism transmitting impulses to the brain. From this standpoint taste phenomena are closely allied with general cell physiology or pharmacology. It has been shown that the great majority of anesthetic or toxic substances have a bitter taste and it is considered that the same structural changes occur in the cell membranes in taste buds as in other cells of the body when such substances are present. From consideration of a large amount of experimental data on the taste of organic compounds it is shown that thesweet and bitter substances are chemically very closely related and there is no line of demarcation between them. In many homologous series there is a continuous (rather than discontinuous) transition from sweet to bitter. Sweet substances having a bitter after-taste are also common.It has been pointed out that it is a general property of anesthetics to be stimulants (i. e. to accelerate cell activities)when in low concentrations. The continuous transition from stimulant to narcotic action is correlated with the sweet-bitter transition. FollowingClowes, Lillie and others the cell membrane is looked upon as a balanced emulsion partly of the water in oil type and partly of the reverse type.The phenomena cited above have been explained in terms of adsorption and surface tension action of substances on this emulsion system and the consequent changes in cell membrane permeability due to changes in the phase ratio of the emulsion types.     

Mechanical stresses as factors in the autoregulation of morphogenesis

Since morphogenetic processes are nonlinear, feedback must be essential for their regulation. Two concepts of feedback in morphogenesis are developed: 1) inhibition by diffusing morphogenetic substances and 2) action of mechanical strain resulting from morphogenetic movements. The data on the role of mechanical strain in formation of integral structure of embryonic tissues, primary demarcation of embryonic epithelia and mesenchymal rudiments and bending of epithelial layers are presented. Evolutionary aspects of morphogenetic role of mechanical strain and its possible use in applied biotechnology are discussed.     

Free and peptide-bound DOPA can inhibit initiation of low density lipoprotein oxidation

Hydroxyl radicals have been shown to convert free tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA) which has reducing properties. During protein or peptide oxidation such reducing species are also formed from tyrosine residues. Free DOPA or peptide-bound DOPA (PB-DOPA) is able to promote radical-generating events, facilitating the damage of biomolecules such as nucleic acids. Radical induced lipid oxidation in low density lipoprotein (LDL) transforms the lipoprotein into an atherogenic particle. As PB-DOPA has been found in atherosclerotic plaques, we tested the ability of free and PB-DOPA to influence LDL oxidation. Free DOPA, in contrast to tyrosine had strong inhibitory action on both, the copper-ion initiated and metal ion independent (AAPH-induced) lipid oxidation. Free DOPA also inhibited LDL oxidation induced by the copper transport protein ceruloplasmin. To test if PB-DOPA was also able to inhibit LDL oxidation, DOPA residues were generated enzymatically in the model peptides insulin and tyr-tyr-tyr, respectively. PB-DOPA formation substantially increased the ability of both molecules to inhibit LDL oxidation by copper or AAPH. We hypothesize that DOPA-peptides and -proteins may have the potential to act as efficacious antioxidants in the atherosclerotic plaque.     

Insect Melanogenesis. II. Inability of Manduca Phenoloxidase to Act on 5, 6-Dihydroxyindole-2-Carboxylic Acid1

Eumelanins in animals are biosynthesized by the combined action of tyrosinase, 3, 4-dihydroxyphenylalanine (DOPA)chrome isomerase, and other factors. Two kinds of eumelanins were characterized from mammalian systems; these are 5,6-dihydroxyindole (DHI)-melanin and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin. In insects, melanin biosynthesis is initiated by phenoloxidase and supported by DOPAchrome isomerase (decarboxylating). Based on the facts that DOPA is a poor substrate for insect phenoloxidases and DHI is the sole product of insect DOPAchrome isomerase reaction, it is proposed that insects lack DHICA-melanin. Accordingly, the phenoloxidase isolated from the hemolymph of Manduca sexta failed to oxidize DHICA. Control experiments reveal that mushroom tyrosinase, as well as laccase, which is a contaminant in the commercial preparations of mushroom tyrosinase, are capable of oxidizing DHICA. Neither the whole hemolymph nor the cuticular extracts of M. sexta possessed any detectable oxidase activity towards this substrate. Thus, insects do not seem to produce DHICA-eumelanin. A useful staining procedure to localize DHICA oxidase activity on gels is also presented.     

Wirkungen von Colchicin auf die Morphogenese vonAcetabularia

Zusammenfassung Es wird über die Wirkungen von Colchicin auf verschiedene morphogenetische Prozesse bei kernhaltigen und kernlosen Zellen der GrünalgeAcetabularia berichtet.Das polare Stielwachstum dieser Zellen wird durch das Pseudo-Alkaloid nicht oder nur sehr wenig beeinflußt. Dagegen wird bei kernhaltigen und bei kernlosen Zellen die Hutbildung gehemmt. Die Hemmung ist bei den kernhaltigen Zellen reversibel, bei den kernlosen Zellen aber irreversibel.Aus Transplantationsexperimenten ergab sich, daß Colchicin die Bildung und die Funktionen der die Artspezifität eines Merkmales determinierenden morphogenetischen Substanzen nicht beeinflußt. Die Hemmung der Hutbildung durch Colchicin ist demnach auf eine Hemmung der Funktionen der artunspezifischen Substanzen zurückzuführen, die im Plasma gespeichert werden und für dieAuslösung von Formbildungsprozessen verantwortlich sind. Es wird angenommen, daß sie in einer Form von Kern in das Cytoplasma gelangen, die Colchicin nicht zu binden vermag. Möglicherweise stellen sie spezielle Proteine dar, die durch spezifische Änderungen ihrer Struktur morphogenetisch aktiv und damit erst durch Colchicin beeinflußbar werden. Zu entsprechenden Folgerungen führten Befunde aus Versuchen über die Beeinflußbarkeit der Cystenbildung aus dem Hutkammerplasma.

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Actions of colchicine on morphogenesis ofAcetabularia

Summary The paper reports on interactions of colchicine with certain morphogenetic processes in nucleate and enucleated cells of the green algaeAcetabularia.The strictly polarized stalk growth was found to be only slightly affected by the pseudoalkaloid. Cap formation, however, was strongly inhibited. The inhibition effect was reversible in nucleate cells, but irreversible in the enucleated cells.According to results of grafting experiments it is concluded that colchicine exerts no effect on the production and on the function of the morphogenetic substances which determine the special properties of the species. It appears that the inhibition is caused by the stable inactivation of rather unspecific substances, which are necessary for theinitiation of certain morphogenetic events during cell ontogeny. The production of these substances by the nucleus, however, is not affected by colchicine. Experimental evidence was received that they are released from the nucleus into the cytoplasm in a precursor stage, which is not affected by the pseudo-alkaloid. It is proposed that these substances represent protein which is able to become functionally active in morphogenesis by specific structural changes.Similar conclusions were drawn from results of experiments on the influence of colchicine on the differentiation of cysts from cap ray plasm.The behaviour of the substances which are influenced by colchicine and which are involved in the formation of a new polarity axis of a cell, shows similarities to that of the nonspecies specific morphogenetic substances.