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Sample records for 3,4-dihydroxyphenylalanine

  1. Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

    Han, Q.; Ding, H; Robinson, H; Christensen, B; Li, J

    2010-01-01

    3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  2. Encapsulation of phenylalanine and 3,4-dihydroxyphenylalanine into β-cyclodextrin: Spectral and molecular modeling studies

    Antony Muthu Prabhu, A.; Suresh Kumar, G. S.; Fatiha, Madi; Sorimuthu, S.; Sundar Raj, M.

    2015-01-01

    Encapsulation behavior of phenylalanine (PA) and 3,4-dihydroxyphenylalanine (DPA) with β-cyclodextrin (β-CD) were analyzed by UV-visible, Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), and molecular modeling methods. The stoichiometric ratio of the inclusion complexes was found to be 1:1 and the binding constant was evaluated using the Benesi-Hildebrand equation. FT-IR, PXRD, and SEM results confirmed the formation of inclusion complexes. PM3 calculations suggest that orientation B is more favored for PA and orientation A is more favored for DPA. The hydrophobic and H-bond interaction between PA/DPA and β-CD plays an important role in the inclusion complexes. NBO analysis confirmed that mutual interactions formed between occupied and vacant orbitals of both host (β-CD) and guest (PA/DPA) molecules which is the driving force for the formation of inclusion complexes.

  3. Effects of 3-O-methyldopa, L-3,4-dihydroxyphenylalanine metabolite, on locomotor activity and dopamine turnover in rats.

    Onzawa, Yoritaka; Kimura, Yasuhiro; Uzuhashi, Kengo; Shirasuna, Megumi; Hirosawa, Tasuku; Taogoshi, Takanori; Kihira, Kenji

    2012-01-01

    It has been well known that 3-O-methyldopa (3-OMD) is a metabolite of L-3,4-dihydroxyphenylalanine (L-DOPA) formed by catechol O-methyltransferase (COMT), and 3-OMD blood level often reaches higher than physiological level in Parkinson's disease (PD) patients receiving long term L-DOPA therapy. However, the physiological role of 3-OMD has not been well understood. Therefore, in order to clarify the effects of 3-OMD on physiological function, we examined the behavioral alteration in rats based on locomotor activity, and measured dopamine (DA) and its metabolites levels in rats at the same time after 3-OMD subchronic administration. The study results showed that repeated administrations of 3-OMD increased its blood and the striatum tissue levels in those rats, and decreased locomotor activity in a dose dependent manner. Although 3-OMD subchronic administration showed no significant change in DA level in the striatum, DA metabolite levels, such as 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA) were significantly decreased. After 3-OMD washout period (7 d), locomotor activity and DA turnover in those rats returned to normal levels. Furthermore, locomotor activity and DA turnover decreased by 3-OMD administration were recovered to normal level by acute L-DOPA administration. These results suggested that 3-OMD affect to locomotor activity via DA neuron system. In conclusion, 3-OMD itself may have a disadvantage in PD patients receiving L-DOPA therapy. PMID:22863920

  4. Endogenous 3, 4- Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical

    Zhang, Xu; Monroe, Matthew E.; Chen, Baowei; Chin, Mark H.; Heibeck, Tyler H.; Schepmoes, Athena A.; Yang, Feng; Petritis, Brianne O.; Camp, David G.; Pounds, Joel G.; Jacobs, Jon M.; Smith, Desmond J.; Bigelow, Diana J.; Smith, Richard D.; Qian, Weijun

    2010-06-02

    Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3, 4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first global proteome survey of endogenous site-specific modifications, i.e, DOPA and its further oxidation product dopaquinone (DQ) in mouse brain and heart tissues. Results from LC-MS/MS analyses included 203 and 71 DOPA-modified tyrosine sites identified from brain and heart, respectively, with a false discovery rate of ~1%; while only a few nitrotyrosine containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 57 and 29 DQ modified peptides were observed from brain and heart, respectively; nearly half of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal-binding properties, consistent with metal catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondria-associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation suggesting potential disruption of signaling pathways. Structural aspects of DOPA-modified tyrosine sequences are distinct from those of nitrotyrosines suggesting that each type of modifications provides a marker for different in vivo reactive chemistries and can be used to predict sensitive protein targets. Collectively, the results suggest that these modifications are linked with mitochondrially-derived oxidative stress, and may serve as sensitive markers for disease pathologies.

  5. Amino acid-type interactions of L-3,4-dihydroxyphenylalanine with transition metal ions: An experimental and theoretical investigation

    Mandal, Shilpi; Das, Gunajyoti; Askari, Hassan

    2015-11-01

    This paper reports the synthesis of the Ni+2, Cu+2 and Zn+2 complexes of L-3,4-dihydroxyphenylalanine (L-dopa) using a solvent-free solid-state grinding procedure. The synthesized complexes are characterized by elemental, molar conductance, EDAX-SEM, TG/DTA, infrared, electronic absorption, fluorescence and XRD analyses; confirming a 1:2 metal-ligand stoichiometry of the complexes and involvement of the carboxyl and amino groups in complex formation. Phase-diagram and the kinetic parameters of the interactions between L-dopa and the metal ions are also explored. Molecular structures of the metal complexes are modeled within the framework of density functional theory in a vacuum and implicit aqueous environment using the most stable L-dopa conformers determined at the MP2/6-311++G(d,p) level. The gas and aqueous phase metal-binding affinities; theoretical IR and UV-vis spectral aspects; partial atomic charges; Wiberg bond indices; HOMO-LUMO energy gaps and dipole moments of the L-dopa conformers as well as their complexes are calculated and analyzed at B3LYP/6-311++G(d,p) level. The singlet state of the Ni(L-dopa)2 complex is found to be more favorable from thermodynamic perspectives as compared to the triplet state. Use of BHandHLYP and dispersion-corrected B3LYP (at DFT-D2 level) methods in conjugation with the 6-311++G(d,p) basis set affords us to accurately predict the binding affinity order of the three Lewis acids investigated, assess the influence of metal-aromatic π interactions on the thermodynamic stability of metalated L-dopa, and explore the effectiveness of the aforesaid methodologies in predicting a certain set of spectral and electronic properties of bioactive molecules. UV-vis titration and docking studies reveal that the metal complexes of L-dopa are able to bind to the surface of DNA.

  6. Studies on Preparation of Poly(3,4-Dihydroxyphenylalanine-Polylactide Copolymers and the Effect of the Structure of the Copolymers on Their Properties

    Dongjian Shi

    2016-03-01

    Full Text Available Properties of copolymers are generally influenced by the structure of the monomers and polymers. For the purpose of understanding the effect of polymer structure on the properties, two kinds of copolymers, poly(3,4-dihydroxyphenylalanine-g-polylactide and poly(3,4-dihydroxyphenylalanine-b-polylactide (PDOPA-g-PLA and PDOPA-b-PLA were designed and prepared by ring-opening polymerization of lactide with pre-prepared PDOPA as the initiator and the amidation of the functional PLA and PDOPA oligomer, respectively. The molecular weight and composition of the copolymers could be adjusted by changing the molar ratio of LA and DOPA and were confirmed by gel permeation chromatography (GPC and proton nuclear magnetic resonance (1H NMR spectra. The obtained copolymers with graft and block structures showed high solubility even in common organic solvents. The effects of the graft and block structures on the thermal and degradation properties were also detected. The PDOPA-g-PLA copolymers showed higher thermal stability than the PDOPA-b-PLA copolymers, due to the PDOPA-g-PLA copolymers with regular structure and strong π-π stacking interactions among the intermolecular and intramolecular chains. In addition, the degradation results showed that the PDOPA-g-PLA copolymers and the copolymers with higher DOPA composition had quicker degradation speeds. Interestingly, both two kinds of copolymers, after degradation, became undissolved in the organic solvents because of the oxidation and crosslinking formation of the catechol groups in the DOPA units during degradation in alkaline solution. Moreover, fluorescent microscopy results showed good biocompatibility of the PDOPA-g-PLA and PDOPA-b-PLA copolymers. The PDOPA and PLA copolymers have the potential applications to the biomedical and industrial fields.

  7. Determination of free and conjugated catecholamines and L-3,4-dihydroxyphenylalanine in plasma and urine: evidence for a catechol-O-methyltransferase inhibitor in uraemia

    A sensitive, accurate and reproducible method has been developed for the determination of free and conjugated catecholamines and L-3,4-dihydroxyphenylalanine in plasma and urine. The assay involves the enzymatic conversion of these compounds to their radio-labelled O-methylated derivatives using catechol-O-methyltransferase and S-adenosyl-L-[methyl-3H]methionine. Recoveries of 75 +- 5% for dopamine, 70 +- 5% for adrenaline and 65 +- 5% for noradrenaline were obtained. The sensitivities were 0.5 pg for adrenaline and noradrenaline and 5-7 pg for dopamine and dihydroxyphenylalanine. Measurements of conjugated catecholamines were performed after mild acid hydrolysis for 20 min at 950 C. During this procedure no degradation of the catecholamines was observed. This assay led to the discovery of a dialyzable factor in the plasma of chronic uraemic patients which inhibits catechol-O-methyltransferase activity in vitro. The mean 22% inhibition observed for unhydrolyzed plasma increased to 42% after hydrolysis. The identity of this inhibitor which exists as an inactive conjugated form, probably a sulphate ester, and its implication in physiopathological disorders remain to be established. (Auth.)

  8. The protein oxidation product 3,4-dihydroxyphenylalanine (DOPA) mediates oxidative DNA damage

    Morin, B; Davies, Michael Jonathan; Dean, R T

    1998-01-01

    of the present work was to investigate whether DOPA, and especially PB-DOPA, can mediate oxidative damage to DNA. We chose to generate PB-DOPA using mushroom tyrosinase, which catalyses the hydroxylation of tyrosine residues in protein. This permitted us to study the reactions of PB-DOPA in the virtual absence...

  9. Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima

    Belli, Sabina I; Wallach, Michael G; Luxford, Catherine; Davies, Michael Jonathan; Smith, Nicholas C

    2003-01-01

    The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control...

  10. Dityrosine, 3,4-dihydroxyphenylalanine (DOPA), and radical formation from tyrosine residues on milk proteins with globular and flexible structures as a result of riboflavin-mediated photo-oxidation

    Dalsgaard, Trine K; Nielsen, Jacob H; Brown, Bronwyn E;

    2011-01-01

    Riboflavin-mediated photo-oxidative damage to protein Tyr residues has been examined to determine whether protein structure influences competing protein oxidation pathways in single proteins and protein mixtures. EPR studies resulted in the detection of Tyr-derived o-semiquione radicals, with this...

  11. Application of Organometallic Catalysis to the Commercial Production of L-DOPA.

    Knowles, W. S.

    1986-01-01

    Shows how asymmetric organometallic catalysts can be used to make complex organic molecules with extremely high enantioselectivity. The molecule considered is l-3, 4-dihydroxyphenylalanine (L-DOPA), an amino acid which was found to be effective in the treatment of Parkinson's disease. (JN)

  12. Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C(U)-L-tyrosine incorporation into melanin

    Husain, I.; Vijayan, E.; Ramaiah, A.; Pasricha, J.S.; Madan, N.C.

    1982-03-01

    Tyrosinase activity (Monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) in vitiligo and normal epidermal homogenates of skin from human beings was measured by estimating beta 3,4-dihydroxyphenylalanine (dopa) by a highly sensitive fluorometric method described in this paper. The tyrosine activity in the vitiligo skin was about 4 to 37% of corresponding normal skin. The activity of tyrosinase in normal human skin from different individuals and from different regions of the body was in the range of 4 to 140 picomoles of beta 3,4-dihydroxyphenylalanine formed per min/mg protein of epidermal homogenate. The enzyme from vitiligo and normal skin was severely inhibited by substance(s) of low molecular weight. The enzyme exhibits a lag of about 4 hr in the absence of added beta 3,4-dihydroxyphenylalanine and 1 hr in presence of 5 microM dopa. Tyrosinase from the normal and vitiligo skin was inhibited by excess concentration of tyrosine. The homogenates from vitiligo skin could synthesize melanin from C14(U)-L-Tyrosine. The rate of tyrosine incorporation into melanin by the epidermal homogenates is increased by 3,4-dihydroxyphenylalanine (dopa) disproportionate to its effect on tyrosinase activity. Based on the data presented in this paper it is concluded that melanocytes are present in the vitiligo skin. A tentative hypothesis is put forward to explain the lack of melanin synthesis by the vitiligo skin under in vivo conditions, although melanocytes are present.

  13. Oxidation of DNA, proteins and lipids by DOPA, protein-bound DOPA, and related catechol(amine)s

    Pattison, David I; Dean, Roger T; Davies, Michael Jonathan

    2002-01-01

    Incubation of free 3,4-dihydroxyphenylalanine (DOPA), protein-bound DOPA (PB-DOPA) and related catechols with DNA, proteins and lipids has been shown to result in oxidative damage to the target molecule. This article reviews these reactions with particular emphasis on those that occur in the...

  14. Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C(U)-L-tyrosine incorporation into melanin

    Tyrosinase activity (Monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) in vitiligo and normal epidermal homogenates of skin from human beings was measured by estimating beta 3,4-dihydroxyphenylalanine (dopa) by a highly sensitive fluorometric method described in this paper. The tyrosine activity in the vitiligo skin was about 4 to 37% of corresponding normal skin. The activity of tyrosinase in normal human skin from different individuals and from different regions of the body was in the range of 4 to 140 picomoles of beta 3,4-dihydroxyphenylalanine formed per min/mg protein of epidermal homogenate. The enzyme from vitiligo and normal skin was severely inhibited by substance(s) of low molecular weight. The enzyme exhibits a lag of about 4 hr in the absence of added beta 3,4-dihydroxyphenylalanine and 1 hr in presence of 5 microM dopa. Tyrosinase from the normal and vitiligo skin was inhibited by excess concentration of tyrosine. The homogenates from vitiligo skin could synthesize melanin from C14(U)-L-Tyrosine. The rate of tyrosine incorporation into melanin by the epidermal homogenates is increased by 3,4-dihydroxyphenylalanine (dopa) disproportionate to its effect on tyrosinase activity. Based on the data presented in this paper it is concluded that melanocytes are present in the vitiligo skin. A tentative hypothesis is put forward to explain the lack of melanin synthesis by the vitiligo skin under in vivo conditions, although melanocytes are present

  15. Serotonergic modulation of receptor occupancy in rats treated with L-DOPA after unilateral 6-OHDA lesioning

    Nahimi, Adjmal; Høltzermann, Mette; Landau, Anne M.;

    2012-01-01

    Recent studies suggest that l-3,4 dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID), a severe complication of conventional L-DOPA therapy of Parkinson's disease, may be caused by dopamine (DA) release originating in serotonergic neurons. To evaluate the in vivo effect of a 5-HT(1A) agonist...

  16. Cross-linking Chemistry of Squid Beak*

    Miserez, Ali; Rubin, Daniel; Waite, J. Herbert

    2010-01-01

    In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of th...

  17. Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.

    Horellou, P; Guibert, B; Leviel, V; Mallet, J

    1989-01-01

    Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resist...

  18. Asymmetric Pictet-Spengler Reactions:Synthesis of Tetrahydroisoquinoline Derivatives from L-DOPA

    Ye WANG; Zhan Zhu LIU; Shi Zhi CHEN; Xiao Tian LIANG

    2004-01-01

    The cis-1-substituted-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid esters 3 can be obtained in a highly diastereoselective fashion through 1,3-induction Pictet- Spengler (P-S) cyclization of the L-DOPA (3,4-dihydroxyphenylalanine) methyl ester with aromatic or aliphatic aldehydes under acidic conditions. Their epimers 4 are also obtained as minor products.

  19. 1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in Torula corallina, and DHN-Melanin Inhibits Erythrose Reductase

    Lee, Jung-Kul; Jung, Hyung-Moo; Kim, Sang-Yong

    2003-01-01

    The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which sug...

  20. Detection of Melanin-Like Pigments in the Dimorphic Fungal Pathogen Paracoccidioides brasiliensis In Vitro and during Infection

    Gómez, Beatriz L.; Nosanchuk, Joshua D.; Díez, Soraya; Youngchim, Sirida; Aisen, Philip; Cano, Luz E.; Restrepo, Angela; Casadevall, Arturo; Hamilton, Andrew J.

    2001-01-01

    Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with l-3,4-dihydroxyphenylalanine (l-DOPA) produced pigmente...

  1. Protection of Melanized Cryptococcus neoformans from Lethal Dose Gamma Irradiation Involves Changes in Melanin's Chemical Structure and Paramagnetism

    Abdelahad Khajo; Ruth A Bryan; Matthew Friedman; Burger, Richard M.; Yan Levitsky; Arturo Casadevall; Magliozzo, Richard S.; Ekaterina Dadachova

    2011-01-01

    Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans), which uses L-3,4-dihydroxyphenylalanine (L-DOPA) to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50...

  2. Selective and validated spectrophotometric methods for the determination of nicorandil in pharmaceutical formulations

    Rahman, Nafisur; Ahmad, Yasmin; Azmi, Syed Najmul Hejaz

    2004-01-01

    Two simple and sensitive validated spectrophotometric methods have been described for the assay of nicorandil in drug formulations. Method A is based on the reaction of the drug with phloroglucinol-sulfanilic acid reagent in sulfuric acid medium to give yellow-colored product, which absorbs maximally at 425 nm. Method B uses the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with DL-3,4-dihydroxyphenylalanine (DL-dopa) in the presence of nicorandil as oxidan...

  3. α,β-Dehydro-Dopa: A Hidden Participant in Mussel Adhesion

    Mirshafian, Razieh; Wei, Wei; Israelachvili, Jacob N.; Waite, J. Herbert

    2016-01-01

    Dopa (L-3,4-dihydroxyphenylalanine) is a key chemical signature of mussel adhesive proteins, but its susceptibility to oxidation has limited mechanistic investigations as well as practical translation to wet adhesion technology. To investigate peptidyl-Dopa oxidation, the highly diverse chemical environment of Dopa in mussel adhesive proteins was simplified to a peptidyl-Dopa analogue, N-acetyl-Dopa ethyl ester. On the basis of cyclic voltammetry and UV–vis spectroscopy, the Dopa oxidation pr...

  4. D-amino acid oxidase activator gene (DAOA) variation affects cerebrospinal fluid homovanillic acid concentrations in healthy Caucasians

    Andreou, Dimitrios; Saetre, Peter; Werge, Thomas;

    2012-01-01

    The D-amino acid oxidase activator (DAOA) protein regulates the function of D-amino oxidase (DAO), an enzyme that catalyzes the oxidative deamination of D-3,4-dihydroxyphenylalanine (D-DOPA) and D-serine. D-DOPA is converted to L-3,4-DOPA, a precursor of dopamine, whereas D-serine participates in...... dopamine turnover in healthy individuals, suggesting that disturbed dopamine turnover is a possible mechanism behind the observed associations between genetic variation in DAOA and behavioral phenotypes in humans....

  5. Adhesion of Mussel Foot Protein Mefp-5 to Mica: An Underwater Superglue†

    Danner, Eric W.; Kan, Yajing; Hammer, Malte U.; Israelachvili, Jacob N.; Waite, J. Herbert

    2012-01-01

    Mussels have a remarkable ability to attach their holdfast, or byssus, opportunistically to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms. Mytilus edulis foot protein-5 (Mefp-5) is one of several proteins in the byssal adhesive plaque of the mussel M. edulis. The high content of 3,4 dihydroxyphenylalanine (Dopa) (~30 mol%) and its localization near the plaque-substrate interface have often prompted speculation that Mefp-5 plays a key role in adhesion. Using ...

  6. Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures▿

    Walker, Claire A; Gómez, Beatriz L.; Mora-Montes, Héctor M.; Mackenzie, Kevin S; Munro, Carol A.; Brown, Alistair J. P.; Gow, Neil A. R.; Kibbler, Christopher C.; Odds, Frank C.

    2010-01-01

    The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes tha...

  7. Identification and Molecular Characterization of the Homogentisate Pathway Responsible for Pyomelanin Production, the Major Melanin Constituents in Aeromonas media WS

    He Wang; Yunqian Qiao; Baozhong Chai; Chenxi Qiu; Xiangdong Chen

    2015-01-01

    The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine) based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate). Gene products of phhA (encodes phenylalanine hydroxylase), ty...

  8. Cloning and Random Mutagenesis of the Erwinia herbicola tyrR Gene for High-Level Expression of Tyrosine Phenol-Lyase

    Katayama, Takane; Suzuki, Hideyuki; Koyanagi, Takashi; Kumagai, Hidehiko

    2000-01-01

    Tyrosine phenol-lyase (Tpl), which can synthesize 3,4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that the tpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrR gene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tpl were screened for by use of the lac ...

  9. Zur Synthese radiofluorierter aromatischer Aminosäuren mittels Isotopenaustausch am Beispiel von 6-[18F]Fluor-L-DOPA

    Wagner, Franziska Martina

    2007-01-01

    Zur in vivo Erfassung des präsynaptischen Dopamin-Metabolismus sowie von bestimmten Tumortypen mittels Positronen-Emission-Tomographie wird in der nuklearmedizinischen Diagnostik das L-DOPA Analogon 6-[18F]Fluor-L-3,4-dihydroxyphenylalanin (6-[18F]Fluor-L-DOPA), eines der wenigen etablierten Radiopharmaka, eingesetzt. Die z.Z. übliche Herstellung des Radiotracers durch elektrophile Markierung erlaubt es nur, geringe Aktivitätsmengen bei hohem Kostenaufwand zu produzieren. Bisherige nukleophil...

  10. Plasma dihydroxyphenylalanine (DOPA) is independent of sympathetic activity in humans

    Eldrup, E; Christensen, N J; Andreasen, J;

    1989-01-01

    To clarify the origin of plasma DOPA (3,4-Dihydroxyphenylalanine), the relationship between plasma DOPA and acute or chronic changes in sympathetic activity has been studied. Plasma DOPA and noradrenaline (NA) concentrations were measured by reverse-phase high-performance liquid chromatography with...... electrochemical detection. Administration of clonidine to healthy men decreased plasma NE markedly compared to no drug. Plasma DOPA decreased slightly but significantly with time, but values were identical after clonidine compared to no drug. Baseline plasma NE concentrations were significantly reduced in...... diabetic patients with autonomic neuropathy compared to diabetics without neuropathy, whereas baseline plasma DOPA concentrations were similar in the three groups investigated: 6.55 (5.03-7.26, median [interquartile range], n = 8) nmol l-1 in diabetics with neuropathy, 7.41 (5.79-7.97, n = 8) nmol l-1 in...

  11. Ultrastructural and Histochemical Characterization of the Zebra Mussel Adhesive Apparatus

    Farsad, Nikrooz

    Since their accidental introduction into the Great Lakes in mid- to late-1980s, the freshwater zebra mussels, Dreissena polymorpha, have colonized most lakes and waterways across eastern North America. Their rapid spread is partly attributed to their ability to tenaciously attach to hard substrates via an adhesive apparatus called the byssus, resulting in serious environmental and economic impacts. A detailed ultrastructural study of the byssus revealed a 10 nm adhesive layer at the attachment interface. Distributions of the main adhesive amino acid, 3,4-dihydroxyphenylalanine (DOPA), and its oxidizing (cross-linking) enzyme, catechol oxidase, were determined histochemically. It was found that, upon aging, DOPA levels remained high in the portion of the byssus closest to the interface, consistent with an adhesive role. In contrast, reduced levels of DOPA corresponded well with high levels of catechol oxidase in the load-bearing component of the byssus, presumably forming cross-links and increasing the cohesive strength.

  12. Adaptive synergy between catechol and lysine promotes wet adhesion by surface salt displacement

    Maier, Greg P.; Rapp, Michael V.; Waite, J. Herbert; Israelachvili, Jacob N.; Butler, Alison

    2015-08-01

    In physiological fluids and seawater, adhesion of synthetic polymers to solid surfaces is severely limited by high salt, pH, and hydration, yet these conditions have not deterred the evolution of effective adhesion by mussels. Mussel foot proteins provide insights about adhesive adaptations: Notably, the abundance and proximity of catecholic Dopa (3,4-dihydroxyphenylalanine) and lysine residues hint at a synergistic interplay in adhesion. Certain siderophores—bacterial iron chelators—consist of paired catechol and lysine functionalities, thereby providing a convenient experimental platform to explore molecular synergies in bioadhesion. These siderophores and synthetic analogs exhibit robust adhesion energies (Ead ≥-15 millijoules per square meter) to mica in saline pH 3.5 to 7.5 and resist oxidation. The adjacent catechol-lysine placement provides a “one-two punch,” whereby lysine evicts hydrated cations from the mineral surface, allowing catechol binding to underlying oxides.

  13. Iron-Clad Fibers: A Metal-Based Biological Strategy for Hard Flexible Coatings

    Harrington, Matthew J.; Masic, Admir; Holten-Andersen, Niels; Waite, J. Herbert; Fratzl, Peter

    2010-04-01

    The extensible byssal threads of marine mussels are shielded from abrasion in wave-swept habitats by an outer cuticle that is largely proteinaceous and approximately fivefold harder than the thread core. Threads from several species exhibit granular cuticles containing a protein that is rich in the catecholic amino acid 3,4-dihydroxyphenylalanine (dopa) as well as inorganic ions, notably Fe3+. Granular cuticles exhibit a remarkable combination of high hardness and high extensibility. We explored byssus cuticle chemistry by means of in situ resonance Raman spectroscopy and demonstrated that the cuticle is a polymeric scaffold stabilized by catecholato-iron chelate complexes having an unusual clustered distribution. Consistent with byssal cuticle chemistry and mechanics, we present a model in which dense cross-linking in the granules provides hardness, whereas the less cross-linked matrix provides extensibility.

  14. Artificial pheomelanin nanoparticles and their photo-sensitization properties.

    Pyo, Jung; Ju, Kuk-Youn; Lee, Jin-Kyu

    2016-07-01

    Pheomelanin-type nanoparticles (PMNPs) were synthesized through a simple oxidative polymerization of 3,4-dihydroxyphenylalanine (DOPA) in the presence of cysteine by KMnO4. The synthesized PMNPs had a diameter of approximately 100nm, exhibited high dispersion stability in neutral water and various culture media and possessed similar morphology to naturally occurring pheomelanins. The efficiency of photoinduced generation of hydroxyl radicals from PMNPs was determined and related in vitro cell experiments that were carried out, with data being compared to those from eumelanin-type nanoparticles (EMNPs) and natural sepia melanin nanoparticles. Endocytosed PMNPs showed the highest phototoxicity (~50% viability) to UV-irradiated HeLa cells, confirming the direct relationship between phototoxic efficiency and the generation of hydroxyl radicals through the complex processes of the O2 sensitization. PMID:27173400

  15. Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

    Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

    2003-01-01

    83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions. The......Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has...... material obtained from advanced human atherosclerotic lesions are shown to contain elevated levels of oxidized amino acids [3,4-dihydroxyphenylalanine (DOPA), di-tyrosine, 2-hydroxyphenylalanine ( o-Tyr)] when compared with healthy (human and pig) arterial tissue. These matrix-derived materials account for...

  16. Diagnosing dopamine-responsive dystonias.

    Malek, N; Fletcher, N; Newman, E

    2015-10-01

    The clinical spectrum of dopamine-responsive dystonias (DRDs) has expanded over the last decade to comprise several distinct disorders. At the milder end of the clinical spectrum is the autosomal-dominant guanosine triphosphate cyclohydrolase deficiency syndrome (GTPCH-DRD), and at the more severe end is the much less common autosomal recessive tyrosine hydroxylase deficiency syndrome (TH-DRD), with intermediate forms in between. Understanding the pathophysiology of DRDs can help in their optimal diagnosis and management. These are conditions with the potential to be either underdiagnosed when not considered or overdiagnosed if there is an equivocal L-dopa (levo-3,4-dihydroxyphenylalanine) response. In this article, we discuss the clinical phenotypes of these disorders, and we outline how investigations can help in confirming the diagnosis. PMID:26045581

  17. Synthesis of L-[β-11C]amino acids using immobilized enzymes

    L-[β-11C]-3,4-dihydroxyphenylalanine(L-[β-11C]DOPA) and L-[β-11C]-5-hydroxytryptophan(L-[β-11C]-5-HTP) were synthesized in one step with immobilized thermostable enzymes (alanine racemase, D-amino acid oxidase, and β-tyrosinase or tryptophanase) on an aminopropyl-CPG carrier in a single column and by passing D,L-[3-11C]alanine through the column with coenzymes and other substrates. L-[β-11C]DOPA and L-[β-11C]-5-HTP could be obtained at yields of 53% and 60%, respectively, by optimizing the amounts and the ratios of the enzymes used, the reaction temperature, the pH, and the flow rate. Moreover, the same immobilized enzyme column could be used repeatedly

  18. Effects of Quercetin on Mushroom Tyrosinase and B16-F10 Melanoma Cells

    Ken-ichi Nihei

    2007-05-01

    Full Text Available In searching for tyrosinase inhibitors from plants using L-3,4-dihydroxyphenylalanine (L-DOPA as a substrate, quercetin was found to be partially oxidized to the corresponding o-quinone under catalysis by mushroom tyrosinase (EC 1.14.18.1. Simultaneously, L-DOPA was also oxidized to dopaquinone and both o-quinones were further oxidized, respectively. The remaining quercetin partially formed adducts with dopaquinone through a Michael type addition. In general, flavonols form adducts with dopaquinone as long as their 3-hydroxyl group is free. Quercetin enhanced melanin production per cell in cultured murine B16-F10 melanoma cells, but this effect may be due in part to melanocytotoxicity. The concentration leading to 50% viable cells lost was established as 20 μM and almost complete lethality was observed at 80 μM.

  19. Norepinephrine and its metabolites are involved in the synthesis of neuromelanin derived from the locus coeruleus.

    Wakamatsu, Kazumasa; Tabuchi, Keisuke; Ojika, Makoto; Zucca, Fabio A; Zecca, Luigi; Ito, Shosuke

    2015-11-01

    In order to elucidate the chemical structure of black to brown pigments, neuromelanins (NMs), in the substantia nigra (SN) and the locus coeruleus (LC) in the central nervous system of humans and other mammalian species during aging, chemical degradative methods are powerful tools. HPLC analysis after hydroiodic acid hydrolysis detected aminohydroxyphenylethylamines, aminohydroxyphenylacetic acids, and aminohydroxyethylbenzenes, which confirmed that SN-NM and LC-NM contain melanin derived not only from dopamine and norepinephrine (NE) but also from several other catecholic metabolites, such as 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylethanol, and 3,4-dihydroxyphenylethylene glycol, in addition to the corresponding Cys-derivatives in varying degrees. However, hydroiodic acid hydrolysis showed that LC-NM produced the same degradation products as were detected in SN-NM. Thus, we needed to develop a new chemical detection method to validate the existence of NE in LC-NM. In the present study, we report that HCl hydrolysis of LC-NM in the presence of thioglycolic acid yields new products arising from substitution of the hydroxyl group by thioglycolic acid at the benzyl position of NE and cysteinyl-NE. This is the first chemical evidence showing that NE and cysteinyl-NE are incorporated into LC-NM. Using the chemical degradation methods for the determination of catechols in neuromelanin (NM), we have shown that dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylethanol (DOPE), and 3,4-dihydroxyphenylalanine (DOPA) are mainly responsible for the structure of NM from substantia nigra (SN), while norepinephrine (NE), 3,4-dihydroxymandelic acid (DOMA), and 3,4-dihydroxyphenylethylene glycol (DOPEG) are additionally responsible for the structure of NM from locus coeruleus (LC). PMID:26156066

  20. Confined Flocculation of Ionic Pollutants by Poly(L-dopa)-Based Polyelectrolyte Complexes in Hydrogel Beads for Three-Dimensional, Quantitative, Efficient Water Decontamination.

    Yu, Li; Liu, Xiaokong; Yuan, Weichang; Brown, Lauren Joan; Wang, Dayang

    2015-06-16

    The development of simple and recyclable adsorbents with high adsorption capacity is a technical imperative for water treatment. In this work, we have successfully developed new adsorbents for the removal of ionic pollutants from water via encapsulation of polyelectrolyte complexes (PECs) made from positively charged poly(allylamine hydrochloride) (PAH) and negatively charged poly(l-3,4-dihydroxyphenylalanine) (PDopa), obtained via the self-polymerization of l-3,4-dihydroxyphenylalanine (l-Dopa). Given the outstanding mass transport through the hydrogel host matrixes, the PDopa-PAH PEC guests loaded inside can effectively and efficiently remove various ionic pollutants, including heavy metal ions and ionic organic dyes, from water. The adsorption efficiency of the PDopa-PAH PECs can be quantitatively correlated to and tailored by the PDopa-to-PAH molar ratio. Because PDopa embodies one catechol group, one carboxyl group, and one amino group in each repeating unit, the resulting PDopa-PAH PECs exhibit the largest capacity of adsorption of heavy metal ions compared to available adsorbents. Because both PDopa and PAH are pH-sensitive, the PDopa-PAH PEC-loaded agarose hydrogel beads can be easily and completely recovered after the adsorption of ionic pollutants by adjusting the pH of the surrounding media. The present strategy is similar to the conventional process of using PECs to flocculate ionic pollutants from water, while in our system flocculation is confined to the agarose hydrogel beads, thus allowing easy separation of the resulting adsorbents from water. PMID:25981870

  1. The preosteoblast response of electrospinning PLGA/PCL nanofibers: effects of biomimetic architecture and collagen I.

    Qian, Yunzhu; Chen, Hanbang; Xu, Yang; Yang, Jianxin; Zhou, Xuefeng; Zhang, Feimin; Gu, Ning

    2016-01-01

    Constructing biomimetic structure and incorporating bioactive molecules is an effective strategy to achieve a more favorable cell response. To explore the effect of electrospinning (ES) nanofibrous architecture and collagen I (COL I)-incorporated modification on tuning osteoblast response, a resorbable membrane composed of poly(lactic-co-glycolic acid)/poly(caprolactone) (PLGA/PCL; 7:3 w/w) was developed via ES. COL I was blended into PLGA/PCL solution to prepare composite ES membrane. Notably, relatively better cell response was delivered by the bioactive ES-based membrane which was fabricated by modification of 3,4-dihydroxyphenylalanine and COL I. After investigation by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, contact angle measurement, and mechanical test, polyporous three-dimensional nanofibrous structure with low tensile force and the successful integration of COL I was obtained by the ES method. Compared with traditional PLGA/PCL membrane, the surface hydrophilicity of collagen-incorporated membranes was largely enhanced. The behavior of mouse preosteoblast MC3T3-E1 cell infiltration and proliferation on membranes was studied at 24 and 48 hours. The negative control was fabricated by solvent casting. Evaluation of cell adhesion and morphology demonstrated that all the ES membranes were more favorable for promoting the cell adhesion and spreading than the casting membrane. Cell Counting Kit-8 assays revealed that biomimetic architecture, surface topography, and bioactive properties of membranes were favorable for cell growth. Analysis of β1 integrin expression level by immunofluorescence indicated that such biomimetic architecture, especially COL I-grafted surface, plays a key role in cell adhesion and proliferation. The real-time polymerase chain reaction suggested that both surface topography and bioactive properties could facilitate the cell adhesion. The combined effect of biomimetic architecture with enhanced

  2. The preosteoblast response of electrospinning PLGA/PCL nanofibers: effects of biomimetic architecture and collagen I

    Qian YZ

    2016-08-01

    Full Text Available Yunzhu Qian,1,2 Hanbang Chen,1 Yang Xu,1 Jianxin Yang,2 Xuefeng Zhou,3 Feimin Zhang,1 Ning Gu3 1Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, 2Center of Stomatology, The Second Affiliated Hospital of Soochow University, Suzhou, 3School of Biological Science and Medical Engineering, Southeast University, Nanjing, People’s Republic of China Abstract: Constructing biomimetic structure and incorporating bioactive molecules is an effective strategy to achieve a more favorable cell response. To explore the effect of electrospinning (ES nanofibrous architecture and collagen I (COL I-incorporated modification on tuning osteoblast response, a resorbable membrane composed of poly(lactic-co-glycolic acid/poly(caprolactone (PLGA/PCL; 7:3 w/w was developed via ES. COL I was blended into PLGA/PCL solution to prepare composite ES membrane. Notably, relatively better cell response was delivered by the bioactive ES-based membrane which was fabricated by modification of 3,4-dihydroxyphenylalanine and COL I. After investigation by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, contact angle measurement, and mechanical test, polyporous three-dimensional nanofibrous structure with low tensile force and the successful integration of COL I was obtained by the ES method. Compared with traditional PLGA/PCL membrane, the surface hydrophilicity of collagen-incorporated membranes was largely enhanced. The behavior of mouse preosteoblast MC3T3-E1 cell infiltration and proliferation on membranes was studied at 24 and 48 hours. The negative control was fabricated by solvent casting. Evaluation of cell adhesion and morphology demonstrated that all the ES membranes were more favorable for promoting the cell adhesion and spreading than the casting membrane. Cell Counting Kit-8 assays revealed that biomimetic architecture, surface topography, and bioactive properties of membranes were favorable for cell

  3. Management of L-dopa overdose in the competitive inhibition state

    Hinz M

    2014-07-01

    Full Text Available Marty Hinz,1 Alvin Stein,2 Ted Cole3 1Clinical Research, NeuroResearch Clinics, Inc., Cape Coral, FL, USA; 2Stein Orthopedic Associates, Plantation, FL, USA; 3Cole Center for Healing, Cincinnati, OH, USA Abstract: The amino acid L-3,4-dihydroxyphenylalanine (L-dopa is prescribed for conditions where increased central and/or peripheral dopamine synthesis is desired. Its administration can establish dopamine concentrations higher than can be achieved from an optimal diet. Specific indications include Parkinson's disease and restless leg syndrome. The interaction between serotonin and dopamine exists in one of two distinctly different physiologic states: the endogenous state or the competitive inhibition state. Management with L-dopa in the competitive inhibition state is the focus of this paper. In the past, control of the competitive inhibition state was thought to be so difficult and complex that it was described in the literature as functionally “meaningless”. When administering L-dopa without simultaneous administration of serotonin precursors, the patient is in the endogenous state. Experience gained with patient outcomes during endogenous L-dopa administration does not allow predictability of L-dopa outcomes in the competitive inhibition state. The endogenous approach typically increases the daily L-dopa dosing value in a linear fashion until symptoms of Parkinson's disease are under control. It is the novel observations made during treatment with the competitive inhibition state approach that L-dopa dosing values above or below the optimal therapeutic range are generally associated with the presence of the exact same Parkinson's disease symptoms with identical intensity. This recognition requires a novel approach to optimization of daily L-dopa dosing values from that used in the endogenous state. This paper outlines that novel approach through utilization of a pill stop. This approach enhances patient safety through its ability to

  4. Inhibitory Kinetics of p-Substituted Benzaldehydes on Polyphenol Oxidase from the Fifth Instar of Pieris Rapae L.

    2007-01-01

    Polyphenol oxidase (PPO) is the enzyme responsible for enzymatic browning during the growth of insects. It is also involved in defense reactions and is related with immunities in insects. PPO,a metalloenzyme oxidase, catalyzes the oxidation of o-diphenol to o-quinone. The present paper describes the effects of benzaldehyde and its p-substituted derivatives on the activity of PPO from the fifth instar of Pieris rapae L. PPO from the fifth instar of Pieris rapae L. was purified using ammonium sulfate fractionation and chromatography on Sephadex G-100. The enzyme kinetics was characterized using L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate. The results show that benzaldehyde, p-hydroxybenzaldehyde, p-chlorobenzaldehyde, and p-cyanobenzaldehyde can inhibit the PPO activity for the oxidation of L-DOPA. The inhibitor concentration leading to 50% activity lost, IC50, was estimated to be 5.90, 5.62, 2.83, and 2.91 mmol/L for the four tested inhibitors, respectively. Kinetic analyses show that the inhibitory effects of these compounds are reversible. Benzaldehyde, p-hydroxybenzaldehyde, and p-chlorobenzaldehyde are noncompetitive inhibitors while p-cyanobenzaldehyde is a mixed-type inhibitor. The inhibition constants were determined for all four inhibitors.p-chlorobenzaldehyde and p-cyanobenzaldehyde were more potent inhibitors than the other compounds. These results provide a basis for developing PPO inhibition-based pesticides.

  5. New Whitening Constituents from Taiwan-Native Pyracantha koidzumii: Structures and Tyrosinase Inhibitory Analysis in Human Epidermal Melanocytes

    Rong-Dih Lin

    2015-12-01

    Full Text Available Nontoxic natural products useful in skin care cosmetics are of considerable interest. Tyrosinase is a rate-limiting enzyme for which its inhibitor is useful in developing whitening cosmetics. Pyracantha koidzumii (Hayata Rehder is an endemic species in Taiwan that exhibits tyrosinase-inhibitory activity. To find new active natural compounds from P. koidzumii, we performed bioguided isolation and studied the related activity in human epidermal melanocytes. In total, 13 compounds were identified from P. koidzumii in the present study, including two new compounds, 3,6-dihydroxy-2,4-dimethoxy-dibenzofuran (9 and 3,4-dihydroxy-5-methoxybiphenyl-2ʹ-O-β-d-glucopyranoside (13, as well as 11 known compounds. The new compound 13 exhibited maximum potency in inhibiting cellular tyrosinase activity, the protein expression of cellular tyrosinase and tyrosinase-related protein-2, as well as the mRNA expression of Paired box 3 and microphthalmia-associated transcription factor in a concentration-dependent manner. In the enzyme kinetic assay, the new compound 13 acted as an uncompetitive mixed-type inhibitor against the substrate l-3,4-dihydroxyphenylalanine and had a Km value against this substrate of 0.262 mM, as calculated using the Lineweaver–Burk plots. Taken together, our findings show compound 13 exhibits tyrosinase inhibition in human melanocytes and compound 13 may be a potential candidate for use in cosmetics.

  6. The Use of the Calcitonin Minimal Recognition Module for the Design of DOPA-Containing Fibrillar Assemblies

    Galit Fichman

    2014-08-01

    Full Text Available Amyloid deposits are insoluble fibrous protein aggregates, identified in numerous diseases, which self-assemble through molecular recognition. This process is facilitated by short amino acid sequences, identified as minimal modules. Peptides corresponding to these motifs can be used for the formation of amyloid-like fibrillar assemblies in vitro. Such assemblies hold broad appeal in nanobiotechnology due to their ordered structure and to their ability to be functionalized. The catechol functional group, present in the non-coded L-3,4-dihydroxyphenylalanine (DOPA amino acid, can take part in diverse chemical interactions. Moreover, DOPA-incorporated polymers have demonstrated adhesive properties and redox activity. In this work, amyloid-like fibrillar assemblies were formed through the self-assembly of a pentapeptide containing DOPA residues, Asp-DOPA-Asn-Lys-DOPA. The design of this peptide was based on the minimal amyloidogenic recognition motif of the human calcitonin hormone, Asp-Phe-Asn-Lys-Phe, the first amyloidogenic pentapeptide identified. By substituting phenylalanine with DOPA, we obtained DOPA-functionalized amyloid-like assemblies in water. Electron microscopy revealed elongated, linear fibril-like nanometric assemblies. Secondary structure analysis indicated the presence of amyloid-characteristic β-sheet structures as well as random coil structures. Deposition of silver on the DOPA-incorporated assemblies suggested redox activity and demonstrated the applicative potential of this novel nanobiomaterial.

  7. Chromatographic assay to study the activity of multiple enzymes involved in the synthesis and metabolism of dopamine and serotonin.

    Morgan, Lindsay D; Baker, Hannah; Yeoman, Mark S; Patel, Bhavik Anil

    2012-03-21

    Serotonin and dopamine are crucial regulators of signalling in the peripheral and central nervous systems. We present an ex-vivo, isocratic chromatographic method that allows for the measurement of tyrosine, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), tryptophan, 5-hydroxytryptophan (5-HTP), serotonin and 5-hydroxy-3-indoleacetic acid (5-HIAA) in a model central nervous (CNS) system, to study the role of key enzymes involved in the synthesis and metabolism of serotonin and dopamine. By utilising a sample splitting technique, we could test a single CNS sample at multiple time points under various pharmacological treatments. In, addition, we were able to conduct this assay by utilising the endogenous biochemical components of the CNS to study the synthesis and metabolism of serotonin and dopamine, negating the requirement of additional enzyme activators or stabilisers in the biological matrix. Finally we utilised NSD-1015, an aromatic amino acid decarboxylase enzyme inhibitor used to study the synthesis of dopamine and serotonin to monitor alterations in levels of key neurochemicals. 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015) was able to reduce levels of serotonin and dopamine, whilst elevating precursors L-DOPA and 5-HTP. PMID:22290325

  8. Neuropharmacology of beclamide and related compounds

    Darmani, N.A.

    1988-01-01

    In order to determine the acute and prolonged effects of beclamide on brain monoamine levels and turnover, assay procedures were developed to separate noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT) and their major precursors (tryptophan,3,4-dihydroxyphenylalanine (DOPA)) and metabolites (normetanephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylacetic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxyindoleacetic acid (5-HIAA)) using an HPLC-ECD technique. Beclamide acutely increased striatal DA turnover and levels of the major DA metabolites by 3-fold and it also decreased the steady state concentration of DA by a similar factor. On continued beclamide administration (5 days), steady state concentrations of striatal NA, DOPAC, NVA and 5-HIAA were reduced in contrast to DA and 5-HT. The present investigation also showed that in vitro, beclamide, its metabolites (dihydroxybeclamide, m-hydroxybeclamide, p-hydroxytbeclamide) and its analogue aminobeclamide, did not displace the following tritiated radioligands: /sup 3/(H)-clonidine, /sup 3/(H)-dihydroalprenolol, /sup 3/(H)-spiperone, /sup 3/(H)-5-HT and therefore lacked affinity for ..cap alpha../sub 2/, ..beta.., D/sub 2/, 5-HT/sub 1/ and 5-HT/sub 2/ binding sites in the rat temporal cortex, striatum, hippocampus and frontal cortex respectively.

  9. The effect of L-dopa on the potentiation of radiation damage to human melanoma cells

    Since L-dopa (L-3,4-dihydroxyphenylalanine) has been shown to possess a selective toxicity for melanoma cells both in vitro and in vivo, we have examined the combined effect of L-dopa and radiation on human melanoma cells. It was found that the combined use of L-dopa potentiated the radiation cytotoxicity to HMV-I human melanoma cells, compared with the response seen in non-melanoma HeLa S3 cells. In HMV-I cells during their exponential phase, L-dopa decreased the shoulder width of the radiation survival curve significantly. In addition, L-dopa significantly inhibited the repair of potentially lethal damage (PLD) in HMV-I cells during their plateau phase. When the distributions of the G1, S, and G2-M cells were measured 24 h after combined L-dopa and radiation treatment, there was significant increase in the accumulation of cells in the G2-M phase of the cell cycle, compared to cells that received either L-dopa or radiation treatment only. (author)

  10. Resolving Non-Specific and Specific Adhesive Interactions of Catechols at Solid/Liquid Interfaces at the Molecular Scale.

    Utzig, Thomas; Stock, Philipp; Valtiner, Markus

    2016-08-01

    The adhesive system of mussels evolved into a powerful and adaptive system with affinity to a wide range of surfaces. It is widely known that thereby 3,4-dihydroxyphenylalanine (Dopa) plays a central role. However underlying binding energies remain unknown at the single molecular scale. Here, we use single-molecule force spectroscopy to estimate binding energies of single catechols with a large range of opposing chemical functionalities. Our data demonstrate significant interactions of Dopa with all functionalities, yet most interactions fall within the medium-strong range of 10-20 kB T. Only bidentate binding to TiO2 surfaces exhibits a higher binding energy of 29 kB T. Our data also demonstrate at the single-molecule level that oxidized Dopa and amines exhibit interaction energies in the range of covalent bonds, confirming the important role of Dopa for cross-linking in the bulk mussel adhesive. We anticipate that our approach and data will further advance the understanding of biologic and technologic adhesives. PMID:27374053

  11. Influence of gamma-radiation on the nutritional and functional qualities of lotus seed flour.

    Bhat, Rajeev; Sridhar, Kandikere Ramaiah; Karim, Alias A; Young, Chiu C; Arun, Ananthapadmanabha B

    2009-10-28

    In the present study, we investigated the physicochemical and functional properties of lotus seed flour exposed to low and high doses of gamma-radiation (0-30 kGy; the dose recommended for quarantine and hygienic purposes). The results indicated raw seed flour to be rich in nutrients with minimal quantities of antinutritional factors. Irradiation resulted in a dose-dependent increase in some of the proximal constituents. The raw and gamma-irradiated seeds meet the Food and Agricultural Organization-World Health Organization recommended pattern of essential amino acids. Some of the antinutritional factors (phytic acid, total phenolics, and tannins) were lowered with gamma-irradiation, while the seed flours were devoid of lectins, L-3,4-dihydroxyphenylalanine, and polonium-210. The functional properties of the seed flour were significantly improved with gamma-radiation. gamma-radiation selectively preserved or improved the desired nutritional and functional traits of lotus seeds, thus ensuring a safe production of appropriate nutraceutically valued products. PMID:19778060

  12. Properties of diphenolase from Vanilla planifolia (Andr.) shoot primordia cultured in vitro.

    Debowska, R; Podstolski, A

    2001-07-01

    Properties of diphenolase (PPO, EC1.10.3.1) from vanilla (Vanilla planifolia Andr.) shoot primordia culture were investigated. Two pH optima of the enzyme extraction at pH 6 and 8 were found. Nevertheless, the enzymes shared the same optimum pH of activity-between pH 3 and 4. Sodium dodecyl sulfate slightly improved diphenolase extraction but caused a 3-fold increase in its specific activity. The extracts of pH 6 and 8.0 revealed three isozyme bands after polyacrylamide gel electrophoresis-two of them were similar in both extracts and two distinct. The enzyme showed high thermal stability-no loss was observed after 120 min at 50 degrees C. Diethyldithiocarbamic acid, ethylenediaminetetracetic acid disodium salt, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, L-ascorbic acid, dithiothreitol, glutathione (reduced), and beta-mercaptoethanol were found to be potent inhibitors of the diphenolase studied. The enzyme showed also monophenolase activity. Km and Vmax were calculated with monophenols [p-coumaric acid, 3-(p-hydroxyphenyl)propionic acid, 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid] and with diphenols (caffeic acid, hydrocaffeic acid, chlorogenic acid, 4-methylcatechol, protocatechuic aldehyde and acid, and 3,4-dihydroxyphenylalanine). The highest Vmax was found with 4-hydroxybenzyl alcohol and the greatest affinity to protocatechuic acid, respectively-the most abundant monophenol and one of the least abundant o-diphenols in the studied Vanilla tissue. PMID:11453787

  13. The adhesive protein of Choromytilus chorus (Molina, 1782) and Aulacomya ater (Molina, 1782): a proline-rich and a glycine-rich polyphenolic protein.

    Burzio, L A; Saéz, C; Pardo, J; Waite, J H; Burzio, L O

    2000-06-15

    The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure. PMID:11004549

  14. Cross-linking chemistry of squid beak.

    Miserez, Ali; Rubin, Daniel; Waite, J Herbert

    2010-12-01

    In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance ((1)H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed. PMID:20870720

  15. Cross-linking Chemistry of Squid Beak*

    Miserez, Ali; Rubin, Daniel; Waite, J. Herbert

    2010-01-01

    In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (1H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed. PMID:20870720

  16. Targeted predation of extrafloral nectaries by insects despite localized chemical defences.

    Gish, Moshe; Mescher, Mark C; De Moraes, Consuelo M

    2015-10-01

    Extrafloral (EF) nectaries recruit carnivorous arthropods that protect plants from herbivory, but they can also be exploited by nectar thieves. We studied the opportunistic, targeted predation (and destruction) of EF nectaries by insects, and the localized chemical defences that plants presumably use to minimize this effect. In field and laboratory experiments, we identified insects that were possibly responsible for EF nectary predation in Vicia faba (fava bean) and determined the extent and accuracy of the feeding damage done to the EF nectaries by these insects. We also performed biochemical analyses of plant tissue samples in order to detect microscale distribution patterns of chemical defences in the area of the EF nectary. We observed selective, targeted feeding on EF nectaries by several insect species, including some that are otherwise not primarily herbivorous. Biochemical analyses revealed high concentrations of l-3,4-dihydroxyphenylalanine, a non-protein amino acid that is toxic to insects, near and within the EF nectaries. These results suggest that plants allocate defences to the protection of EF nectaries from predation, consistent with expectations of optimal defence theory, and that this may not be entirely effective, as insects limit their exposure to these defences by consuming only the secreting tissue of the nectary. PMID:26446809

  17. Pharmacological treatment with L-DOPA may reduce striatal dopamine transporter binding in in vivo imaging studies.

    Nikolaus, S; Antke, C; Hautzel, H; Mueller, H-W

    2016-01-01

    Numerous neurologic and psychiatric conditions are treated with pharmacological compounds, which lead to an increase of synaptic dopamine (DA) levels. One example is the DA precursor L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted to DA in the presynaptic terminal. If the increase of DA concentrations in the synaptic cleft leads to competition with exogenous radioligands for presynaptic binding sites, this may have implications for DA transporter (DAT) imaging studies in patients under DAergic medication. This paper gives an overview on those findings, which, so far, have been obtained on DAT binding in human Parkinson's disease after treatment with L-DOPA. Findings, moreover, are related to results obtained on rats, mice or non-human primates. Results indicate that DAT imaging may be reduced in the striata of healthy animals, in the unlesioned striata of animal models of unilateral Parkinson's disease and in less severly impaired striata of Parkinsonian patients, if animal or human subjects are under acute or subchronic treatment with L-DOPA. If also striatal DAT binding is susceptible to alterations of synaptic DA levels, this may allow to quantify DA reuptake in analogy to DA release by assessing the competition between endogenous DA and the administered exogenous DAT radioligand. PMID:26642370

  18. α,β-Dehydro-Dopa: A Hidden Participant in Mussel Adhesion.

    Mirshafian, Razieh; Wei, Wei; Israelachvili, Jacob N; Waite, J Herbert

    2016-02-01

    Dopa (l-3,4-dihydroxyphenylalanine) is a key chemical signature of mussel adhesive proteins, but its susceptibility to oxidation has limited mechanistic investigations as well as practical translation to wet adhesion technology. To investigate peptidyl-Dopa oxidation, the highly diverse chemical environment of Dopa in mussel adhesive proteins was simplified to a peptidyl-Dopa analogue, N-acetyl-Dopa ethyl ester. On the basis of cyclic voltammetry and UV-vis spectroscopy, the Dopa oxidation product at neutral to alkaline pH was shown to be α,β-dehydro-Dopa (ΔD), a vinylcatecholic tautomer of Dopa-quinone. ΔD exhibited an adsorption capacity on TiO2 20-fold higher than that of the Dopa homologue in the quartz crystal microbalance. Cyclic voltammetry confirmed the spontaneity of ΔD formation in mussel foot protein 3F at neutral pH that is coupled to a change in protein secondary structure from random coil to β-sheet. A more complete characterization of ΔD reactivity adds a significant new perspective to mussel adhesive chemistry and the design of synthetic bioinspired adhesives. PMID:26745013

  19. Enzymatic properties of phenoloxidase from Pieris rapae (Lepidoptera) larvae

    CHAO-BIN XUE; WAN-CHUN LUO; QING-XI CHEN; QIN WANG; LI-NA KE

    2006-01-01

    The kinetic parameters of partially purified phenoloxidase (PO, EC. 1.14.18.1) from the 5th instar larvae of Pieris rapae (Lepidoptera) were determined, using L-3, 4-dihydroxyphenylalanine (L-DOPA) as substrate. The optimal pH and temperature of the enzyme for the oxidation of L-DOPA were determined to be at pH 7.0 and at 42℃,respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37℃. At pH 6.8 and 37℃, the Michaelis constant (Km) and maximal velocity (Vm) of the enzyme for the oxidation of L-DOPA were determined to be 0.80 mmol/L and 1.84 μmol/ L/min, respectively. Tetra-hexylresorcinol and 4-dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC50 of 1.50 and 1.12μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47μmol/L, respectively.

  20. Dopamine Reuptake Inhibitors in Parkinson's Disease: A Review of Nonhuman Primate Studies and Clinical Trials.

    Huot, Philippe; Fox, Susan H; Brotchie, Jonathan M

    2016-06-01

    Striatal dopamine deficiency is the core feature of the pathology of Parkinson's disease (PD), and dopamine replacement with l-3,4-dihydroxyphenylalanine (l-DOPA) is the mainstay of PD treatment. Unfortunately, chronic l-DOPA administration is marred by the emergence of dyskinesia and wearing-off. Alternatives to l-DOPA for alleviation of parkinsonism are of interest, although none can match the efficacy of l-DOPA to date. Catechol-O-methyltransferase and monoamine oxidase inhibitors are currently used to alleviate wearing-off, but they do not increase "on-time" without exacerbating dyskinesia. Alternate approaches to dopamine replacement in parkinsonism generally (and to wearing-off and dyskinesia, specifically) are therefore urgently needed. Inasmuch as they increase synaptic dopamine levels, dopamine transporter (DAT) inhibitors, whether they are selective or have actions on noradrenaline or serotonin transporters, theoretically represent an attractive way to alleviate parkinsonism per se and potentially enhance l-DOPA antiparkinsonian action (provided that sufficient dopamine terminals remain within the striatum). Several nonhuman primate studies and clinical trials have been performed to evaluate the potential of DAT inhibitors for PD. In this article, we review nonhuman primate studies and clinical trials, we summarize the current knowledge of DAT inhibitors in PD, and we propose a hypothesis as to how tailoring the selectivity of DAT inhibitors might maximize the benefits of DAT inhibition in PD. PMID:27190169

  1. Mussel-mimetic protein-based adhesive hydrogel.

    Kim, Bum Jin; Oh, Dongyeop X; Kim, Sangsik; Seo, Jeong Hyun; Hwang, Dong Soo; Masic, Admir; Han, Dong Keun; Cha, Hyung Joon

    2014-05-12

    Hydrogel systems based on cross-linked polymeric materials which could provide both adhesion and cohesion in wet environment have been considered as a promising formulation of tissue adhesives. Inspired by marine mussel adhesion, many researchers have tried to exploit the 3,4-dihydroxyphenylalanine (DOPA) molecule as a cross-linking mediator of synthetic polymer-based hydrogels which is known to be able to achieve cohesive hardening as well as adhesive bonding with diverse surfaces. Beside DOPA residue, composition of other amino acid residues and structure of mussel adhesive proteins (MAPs) have also been considered important elements for mussel adhesion. Herein, we represent a novel protein-based hydrogel system using DOPA-containing recombinant MAP. Gelation can be achieved using both oxdiation-induced DOPA quinone-mediated covalent and Fe(3+)-mediated coordinative noncovalent cross-linking. Fe(3+)-mediated hydrogels show deformable and self-healing viscoelastic behavior in rheological analysis, which is also well-reflected in bulk adhesion strength measurement. Quinone-mediated hydrogel has higher cohesive strength and can provide sufficient gelation time for easier handling. Collectively, our newly developed MAP hydrogel can potentially be used as tissue adhesive and sealant for future applications. PMID:24650082

  2. Chromium(VI) reduction by catechol(amine)s results in DNA cleavage in vitro

    Pattison, D I; Davies, Michael Jonathan; Levina, A;

    2001-01-01

    (VI)/catechol(amine) mixtures with plasmid DNA have been investigated to model a potential route to Cr(VI)-induced genotoxicity. Reduction of Cr(VI) by 3,4-dihydroxyphenylalanine (DOPA) (1), dopamine (2), or adrenaline (3) produces species that cause extensive DNA damage, but the products of similar reactions with catechol (4......) or 4-tert-butylcatechol (5) do not damage DNA. The Cr(VI)/catechol(amine) reactions have been studied at low added H(2)O(2) concentrations, which lead to enhanced DNA cleavage with 1 and induce DNA cleavage with 4. The Cr(V) and organic intermediates generated by the reactions of Cr(VI) with 1 or 4......-acetyl-Tyr with tyrosinase, respectively) were correlated with the DNA cleaving abilities of the products of these reactions. The reaction of Cr(VI) with enzymatically generated 1 produced significant amounts of H(2)O(2) and caused significant DNA damage, but the N-acetyl-DOPA did not. The extent of in vitro DNA...

  3. Pharmacological treatments inhibiting levodopa-induced dyskinesias in MPTP-lesioned monkeys: brain glutamate biochemical correlates

    NicolasMorin

    2014-08-01

    Full Text Available Antiglutamatergic drugs can relieve Parkinson’s disease (PD symptoms and decrease L-3,4-dihydroxyphenylalanine (L-DOPA-induced dyskinesias (LID. This review reports relevant studies investigating glutamate receptor subtypes in relation to motor complications in PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP-lesioned monkeys. Antagonists of the ionotropic glutamate receptors, such as NMDA and AMPA receptors, display antidyskinetic activity in PD patients and animal models such as the MPTP monkey. Metabotropic glutamate 5 (mGlu5 receptor antagonists were shown to reduce the severity of LID in PD patients as well as in already dyskinetic non-human primates and to prevent the development of LID in de novo treatments in non-human primates. An increase in striatal post-synaptic NMDA, AMPA and mGlu5 receptors is documented in PD patients and MPTP monkeys with LID. This increase can be prevented in MPTP monkeys with the addition of a specific glutamate receptor antagonist to the L-DOPA treatment and also with drugs of various pharmacological specificities suggesting multiple receptor interactions. This is yet to be well documented for presynaptic mGlu4 and mGlu2/3 and offers additional new promising avenues.

  4. FDOPA-PET as a paradigm of molecular imaging in oncology; FDOPA-PET als Paradigma molekularer Bildgebung in der Onkologie

    Brink, I.; Hentschel, M.; Neumann, H.P.H.; Schaefer, O.; Moser, E. [Radiologische Universitaetsklinik, Abt. Nuklearmedizin, Universitaetsklinikum Freiburg (Germany); Medizinische Klinik, Abt. Nephrologie und Allgemeinmedizin, Universitaetsklinikum Freiburg (Germany); Abt. Diagnostische Radiologie, Universitaetsklinikum Freiburg (Germany)

    2007-03-15

    In recent years, positron emission tomography with 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) has become a wide-spread method in the diagnostics of Parkinson's disease. The amino acid is an important component in protein metabolism. As a precursor in the synthesis of catecholamines it is also of use in metabolic imaging of a variety, mostly neuroendocrine, tumors. The specific uptake mechanisms make FDOPA a paradigm of metabolic imaging. The current review assesses the value of the tracer in the diagnostics of different oncological diseases. It summarizes own experiences and the published results of oncological FDOPA PET-studies. Above all, there is a very high impact of FDOPA in the staging of pheochromocytomas and paragangliomas as well as serotonin-positive neuroendocrine tumors of the gastroentero-pancreatic system (NET-GEPs). Additionally, FDOPA extends the diagnostic possibilities in recurrent medullary thyroid cancer. In the imaging of tumors of the central nervous system, FDOPA represents an alternative to {sup 11}C-labelled PET-tracers. First reports show a high accuracy in the differentiation of radiation induced necrosis and recurrent disease in both high and low grade brain tumors. Furthermore, there is a correlation between the uptake of FDOPA and the expression of proliferation markers. Today, the noninvasive differentiation of focal and diffuse congenital hyperinsulinism has therapeutic consequences. In cases of focal disease, the extent of pancreas resection can be limited resulting in better prognosis without diabetes mellitus. (orig.)

  5. In vivo protective effect of Uridine, a pyrimidine nucleoside, on genotoxicity induced by Levodopa/Carbidopa in mice.

    Orenlili Yaylagul, Esra; Cansev, Mehmet; Celikler Kasimogullari, Serap

    2015-08-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that affects millions of people all over the world. Motor symptoms of PD are most commonly controlled by L-3,4-dihydroxyphenylalanine (Levodopa, L-DOPA), a precursor of dopamine, plus a peripherally-acting aromatic-L-amino-acid decarboxylase (dopa decarboxylase) inhibitor, such as carbidopa. However, chronic treatment with a combination of Levodopa plus carbidopa has been demonstrated to cause a major complication, namely abnormal involuntary movements. On the other hand, the effect of this treatment on bone marrow cells is unknown. Therefore, in this study, we aimed to investigate possible genotoxic effects of Levodopa and Carbidopa using male Balb/C mice. Our results showed that Levodopa alone or in combination with carbidopa caused genotoxicity in in vivo micronucleus test (mouse bone marrow) and Comet assay (blood cells). Furthermore, we showed that simultaneous administration of uridine, a pyrimidine nucleoside, reversed the genotoxic effect of Levodopa and Carbidopa in both assays. Our data show for the first time that Levodopa plus carbidopa combination causes genotoxicity which is reversed by uridine treatment. These findings might enhance our understanding for the complications of a common Parkinson's treatment and confer benefit in terms of reducing a possible genotoxic effect of this treatment. PMID:25976300

  6. Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

    Jiang, Jingwei; Zhou, Zunchun; Dong, Ying; Sun, Hongjuan; Chen, Zhong; Yang, Aifu; Gao, Shan; Wang, Bai; Jiang, Bei; Guan, Xiaoyan

    2014-02-01

    Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses. PMID:24355405

  7. Studies on the sulphation of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds by rat tissues

    Jenner, William N.; Rose, Frederick A.

    1973-01-01

    The formation of sulpho-conjugates of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds was examined in preparations of rat tissues. Liver high-speed-supernatant preparations readily transferred sulphate from adenosine 3′-phosphate 5′-sulphato-phosphate to dopamine under standard conditions. The main product was identified as the 3-O-sulphate. The preparation also sulphated the 3- and 4-methoxy derivatives but to a lesser extent (44% and 95% respectively) relative to dopamine. Brain preparations possessed only half the activity of liver but formed both the 3- and 4-O-sulphates in the molar ratio of 1.7:1. l-3,4-Dihydroxyphenylalanine (l-dopa) in both tissue preparations did not yield any significant amount of sulpho-conjugate when the dopa decarboxylase present was inhibited. The sulphotransferase activity of preparations was doubled in the presence of dithiothreitol and it was concluded that l-tyrosine methyl ester sulphotransferase was the enzyme involved. A method for the preparation of authentic dopamine 3-O-sulphate and 4-O-sulphate was developed. PMID:4798178

  8. Anti-melanogenic effects of resveratryl triglycolate, a novel hybrid compound derived by esterification of resveratrol with glycolic acid.

    Park, Soojin; Seok, Jin Kyung; Kwak, Jun Yup; Choi, Yun-Hyeok; Hong, Seong Su; Suh, Hwa-Jin; Park, Woncheol; Boo, Yong Chool

    2016-07-01

    Resveratrol is known to inhibit cellular melanin synthesis by multiple mechanisms. Glycolic acid (GA) is used in skin care products for its excellent skin penetration. The purpose of this study was to examine the anti-melanogenic effects of resveratryl triglycolate (RTG), a novel hybrid compound of resveratrol and GA, in comparison with resveratrol, GA, resveratryl triacetate (RTA) and arbutin. Resveratrol, RTG, and RTA inhibited the catalytic activity human tyrosinase (TYR) more potently than arbutin or GA did. Their cytotoxic and anti-melanogenic effects were examined using murine melanoma B16/F10 cells and human epidermal melanocytes (HEMs). The cytotoxicity of RTG was similar to that of resveratrol and RTA. RTG at 3-10 μM decreased melanin levels and cellular TYR activities in α-melanocyte-stimulating hormone-stimulated B16/F10 cells, and L-tyrosine-stimulated HEMs. RTG also suppressed mRNA and protein expression of TYR, tyrosinase-related protein 1, L-3,4-dihydroxyphenylalanine chrome tautomerase, and microphthalmia-associated transcription factor (MITF) in HEMs stimulated with L-tyrosine. This study suggests that, like resveratrol and RTA, RTG can attenuate cellular melanin synthesis effectively through the suppression of MITF-dependent expression of melanogenic enzymes and the inhibition of catalytic activity of TYR enzyme. RTG therefore has potential for use as a cosmeceutical ingredient for skin whitening. PMID:27059716

  9. Identification and molecular characterization of the homogentisate pathway responsible for pyomelanin production, the major melanin constituents in Aeromonas media WS.

    He Wang

    Full Text Available The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate. Gene products of phhA (encodes phenylalanine hydroxylase, tyrB and aspC (both encode aromatic amino acid aminotransferase, and hppD (encodes 4-hydroxyphenylpyruvate dioxygenase constitute a linear pathway of converting phenylalanine to HGA and disruption of any one of these genes impairs or blocks pigmentation of A. media strain WS. This HGA biosynthesis pathway is widely distributed in Aeromonas, but HGA is only detectable in the cultures of pigmented Aeromonas species. Heterologous expression of HppD from both pigmented and non-pigmented Aeromonas species in E. coli leads to the production of pyomelanin and thus pigmentation, suggesting that most Aeromonas species have the critical enzymes to produce pyomelanin through HGA. Taken together, we have identified a widely conserved biosynthesis pathway of HGA based pyomelanin in Aeromonas that may be responsible for pigmentation of many Aeromonas species.

  10. The role of vitamin D in melanogenesis with an emphasis on vitiligo

    Khalid AlGhamdi

    2013-01-01

    Full Text Available Vitiligo is a common pigmentary disorder caused by the destruction of functional melanocytes. Vitamin D is an essential hormone synthesized in the skin and is responsible for skin pigmentation. Low levels of vitamin D have been observed in vitiligo patients and in patients with other autoimmune diseases. Therefore, the relationship between vitamin D and vitiligo needs to be investigated more thoroughly. We reviewed the literature to date regarding the role of vitamin D in skin pigmentation. Our review revealed that vitamin D deficiency has been identified in many conditions, including premature and dysmature birth, pigmented skin, obesity, advanced age, and malabsorption. Vitamin D increases melanogenesis and the tyrosinase content of cultured human melanocytes by its antiapoptotic effect. However, a few growth-inhibitory effects on melanocytes were also reported. Vitamin D regulates calcium and bone metabolism, controls cell proliferation and differentiation, and exerts immunoregulatory activities. Vitamin D exerts its effect via a nuclear hormone receptor for vitamin D. The topical application of vitamin D increased the number of L-3,4-dihydroxyphenylalanine-positive melanocytes. The topical application of vitamin D yields significant results when used in combination with phototherapy and ultraviolet exposure to treat vitiligo in humans. Vitamin D decreases the expression of various cytokines that cause vitiligo. In conclusion, application of vitamin D might help in preventing destruction of melanocytes thus causing vitiligo and other autoimmune disorders. The association between low vitamin D levels and the occurrence of vitiligo and other forms of autoimmunity is to be further evaluated.

  11. Augmentation by L-Dopa of growth inhibition and melanin formation of X-irradiated Harding-Passey melanoma cells in culture

    Treatment of exponentially proliferating melanogenic Harding-Passey melanoma cells in monolayer culture (HPM-73 line) with a single dose of X-irradiation (up to 8 Gy) or continuously (for several weeks) with L-3,4-dihydroxyphenylalanine (L-Dopa) up to 5x10-4 M resulted in a dose-dependent inhibition of cell proliferation, but not in death of all cells. Actually, 8 Gy-irradiated or L-Dopa (2x10-4 M)-treated cultures finally reached the cell number and cell density of controls. However, a combination of a single dose of radiation (8 Gy) followed by L-Dopa (2x10-4 M)-treatment resulted in destruction of all cells. Melanin formation was stimulated by L-dopa-treatment or X-irradiation, and was further elevated by the combined application of radiation and L-Dopa-exposure. Whether the effects of exogenously applied L-Dopa, an intermediary metabolite of melanin synthesis, are due to the conversion to growth-inhibitory metabolites (quinones, radicals, etc.) inside or outside the cell, was discussed. The latter might result from release (due to membrane damage or cell desintegration) of tyrosinase or/and melanosomes into the culture medium with the consequence of extracellular synthesis of potentially cytotoxic metabolites from medium substrates. Further, endocytosis of exogenous melanosomes and tyrosinase with potentially harmful effects is feasible. An application of such a combination therapy of melanoma to clinical medicine should be considered. (orig.)

  12. Searching for indole derivatives as potential mushroom tyrosinase inhibitors.

    Ferro, Stefania; Certo, Giovanna; De Luca, Laura; Germanò, Maria Paola; Rapisarda, Antonio; Gitto, Rosaria

    2016-06-01

    Tyrosinase is a copper-containing enzyme widely distributed in nature, involved in the biosynthesis of melanin whose role is to protect the skin from ultraviolet damage. A great interest has been shown on the melanin involvement in malignant melanoma and other carcinogenetic processes. These phenomena have encouraged the research of tyrosinase inhibitors useful in therapeutic field as well as in foods and cosmetics to prevent browning. The idea was to screen our "in house" database to select suitable lead compounds for the discovery of potential drug-inhibiting enzyme. The obtained biological results demonstrated that compounds containing 4-fluorobenzyl moiety at N - 1 position of indole system showed the best activity. In addition, the role of the portion linked to the carbonyl group at C - 3 was discussed. A Lineweaver-Burk kinetic analysis of the most active indoles, CHI 1043 and derivative 4, showed a mixed-type inhibition in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) as substrate. PMID:25826148

  13. An evaluation of extracts of five traditional medicinal plants from Iran on the inhibition of mushroom tyrosinase activity and scavenging of free radicals.

    Khazaeli, P; Goldoozian, R; Sharififar, F

    2009-10-01

    This study aimed to evaluate the free radical scavenging and inhibition properties of five medicinal plants, including Quercus infectoria Olive., Terminalia chebula Retz., Lavendula stoechas L., Mentha longifolia L., Rheum palmatum L., toward the activity of mushroom tyrosinase using L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate.The methanol extracts of Q. infectoria and T. chebula showed strong radical scavenging effect in 2,2'-dipheny L-1-picrylhydrazyl (DPPH) assay(IC50 = 15.3 and 82.2 microg mL)1 respectively).These plants also showed inhibitory effects against the activity of mushroom tyrosinase in hydroxylation of L-tyrosine (85.9% and 82.2% inhibition,respectively). These two plants also inhibited the oxidation of l-DOPA similar to kojic acid as positive control (IC50 = 102.8 and 192.6 microg mL)1 respectively). In general Q. infectoria and T. chebula significantly inhibited tyrosinase activity and DPPH radical. Both activities were concentration dependant but not in linear manner. It is needed to study the cytotoxicity of these plant extracts in pigment cell culture before further evaluation and moving to in vivo conditions. PMID:19467035

  14. FDOPA-PET as a paradigm of molecular imaging in oncology

    In recent years, positron emission tomography with 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) has become a wide-spread method in the diagnostics of Parkinson's disease. The amino acid is an important component in protein metabolism. As a precursor in the synthesis of catecholamines it is also of use in metabolic imaging of a variety, mostly neuroendocrine, tumors. The specific uptake mechanisms make FDOPA a paradigm of metabolic imaging. The current review assesses the value of the tracer in the diagnostics of different oncological diseases. It summarizes own experiences and the published results of oncological FDOPA PET-studies. Above all, there is a very high impact of FDOPA in the staging of pheochromocytomas and paragangliomas as well as serotonin-positive neuroendocrine tumors of the gastroentero-pancreatic system (NET-GEPs). Additionally, FDOPA extends the diagnostic possibilities in recurrent medullary thyroid cancer. In the imaging of tumors of the central nervous system, FDOPA represents an alternative to 11C-labelled PET-tracers. First reports show a high accuracy in the differentiation of radiation induced necrosis and recurrent disease in both high and low grade brain tumors. Furthermore, there is a correlation between the uptake of FDOPA and the expression of proliferation markers. Today, the noninvasive differentiation of focal and diffuse congenital hyperinsulinism has therapeutic consequences. In cases of focal disease, the extent of pancreas resection can be limited resulting in better prognosis without diabetes mellitus. (orig.)

  15. Dopamine- and cAMP-regulated phosphoprotein of 32-kDa (DARPP-32)-dependent activation of extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) signaling in experimental parkinsonism.

    Santini, Emanuela; Feyder, Michael; Gangarossa, Giuseppe; Bateup, Helen S; Greengard, Paul; Fisone, Gilberto

    2012-08-10

    Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia. PMID:22753408

  16. Dopamine- and cAMP-regulated Phosphoprotein of 32-kDa (DARPP-32)-dependent Activation of Extracellular Signal-regulated Kinase (ERK) and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Experimental Parkinsonism*

    Santini, Emanuela; Feyder, Michael; Gangarossa, Giuseppe; Bateup, Helen S.; Greengard, Paul; Fisone, Gilberto

    2012-01-01

    Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia. PMID:22753408

  17. On the use of [18F]DOPA as an imaging biomarker for transplanted islet mass

    Islet transplantation is being developed as a potential cure for patients with type 1 diabetes. There is a need for non-invasive imaging techniques for the quantification of transplanted islets, as current transplantation sites are associated with a substantial loss of islet viability. The dopaminergic metabolic pathway is present in the islets; therefore, we propose Fluorine-18 labeled L-3,4-dihydroxyphenylalanine ([18F]DOPA) as a biomarker for transplanted islet mass. The expression of enzymes involved in the dopaminergic metabolic pathway was investigated in both native and transplanted human islets. The specific uptake of [18F]DOPA in islets and immortalized beta cells was studied in vitro by selective blocking of dopa decarboxylase (DDC). Initial in vivo positron emission tomography (PET) imaging of viable subcutaneous human islets was performed using [18F]DOPA. DDC and vesicular monoamine transporter 2 are co-localized with insulin in the native human pancreas, and the expression is retained after transplantation. Islet uptake of the [18F]DOPA could be modulated by inhibiting DDC, indicating that the uptake followed the normal dopaminergic metabolic pathway. In vivo imaging revealed [18F]DOPA uptake at the site of the functional islet graft. Based on the in vitro and in vivo results presented in this study, we propose to further validate [18F]DOPA-PET as a sensitive imaging modality for imaging extrahepatically transplanted islets. (author)

  18. Cardiovascular actions of DOPA mediated by the gene product of ocular albinism 1.

    Goshima, Yoshio; Nakamura, Fumio; Masukawa, Daiki; Chen, Sandy; Koga, Motokazu

    2014-01-01

    l-3,4-Dihydroxyphenylalanine (DOPA) is the metabolic precursor of dopamine, and the single most effective agent in the treatment of Parkinson's disease. One problem with DOPA therapy for Parkinson's disease is its cardiovascular side effects including hypotension and syncope, the underlying mechanisms of which are largely unknown. We proposed that DOPA is a neurotransmitter in the central nervous system, but specific receptors for DOPA had not been identified. Recently, the gene product of ocular albinism 1 (OA1) was shown to possess DOPA-binding activity. It was unknown, however, whether or not OA1 is responsible for the actions of DOPA itself. Immunohistochemical examination revealed that OA1 was expressed in the nucleus tractus solitarii (NTS). OA1-positive cells adjacent to tyrosine hydroxylase-positive cell bodies and nerve fibers were detected in the depressor sites of the NTS. OA1 knockdown using oa1-specific shRNA-adenovirus vectors in the NTS reduced the expression levels of OA1 in the NTS. The prior injection of the shRNA against OA1 suppressed the depressor and bradycardic responses to DOPA but not to glutamate in the NTS of anesthetized rats. Thus OA-1 is a functional receptor of DOPA in the NTS, which warrants reexamination of the mechanisms for the therapeutic and untoward actions of DOPA. PMID:25185585

  19. Levodopa-induced dyskinesias in Parkinson’s disease: emerging treatments

    Bargiotas P

    2013-10-01

    Full Text Available Panagiotis Bargiotas, Spyridon KonitsiotisDepartment of Neurology, University of Ioannina, Ioannina, GreeceAbstract: Parkinson’s disease therapy is still focused on the use of l-3,4-dihydroxyphenylalanine (levodopa or l-dopa for the symptomatic treatment of the main clinical features of the disease, despite intensive pharmacological research in the last few decades. However, regardless of its effectiveness, the long-term use of levodopa causes, in combination with disease progression, the development of motor complications termed levodopa-induced dyskinesias (LIDs. LIDs are the result of profound modifications in the functional organization of the basal ganglia circuitry, possibly related to the chronic and pulsatile stimulation of striatal dopaminergic receptors by levodopa. Hence, for decades the key feature of a potentially effective agent against LIDs has been its ability to ensure more continuous dopaminergic stimulation in the brain. The growing knowledge regarding the pathophysiology of LIDs and the increasing evidence on involvement of nondopaminergic systems raises the possibility of more promising therapeutic approaches in the future. In the current review, we focus on novel therapies for LIDs in Parkinson’s disease, based mainly on agents that interfere with glutamatergic, serotonergic, adenosine, adrenergic, and cholinergic neurotransmission that are currently in testing or clinical development.Keywords: motor fluctuations, dopaminergic/nondopaminergic systems, pharmacotherapy

  20. Mussel-inspired soft-tissue adhesive based on poly(diol citrate) with catechol functionality.

    Ji, Yali; Ji, Ting; Liang, Kai; Zhu, Lei

    2016-02-01

    Marine mussels tightly adhering to various underwater surfaces inspires human to design adhesives for wet tissue adhesion in surgeries. Characterization of mussel adhesive plaques describes a matrix of proteins containing 3,4-dihydroxyphenylalanine (DOPA), which provides strong adhesion in aquatic conditions. Several synthetic polymer systems have been developed based on this DOPA chemistry. Herein, a citrate-based tissue adhesives (POEC-d) was prepared by a facile one-pot melt polycondensation of two diols including 1,8-octanediol and poly(ethylene oxide) (PEO), citric acid (CA) and dopamine, and the effects of hydrophilic and soft PEO on the properties of adhesives were studied. It was found that the obtained adhesives exhibited water-soluble when the mole ratio of PEO to 1,8-octanediol was 70%, and the equilibrium swelling percentage of cured adhesive was about 144%, and degradation rate was in the range of 1-2 weeks. The cured adhesives demonstrated soft rubber-like behavior. The lap shear adhesion strength measured by bonding wet pig skin was in the range of 21.7-33.7 kPa, which was higher than that of commercial fibrin glue (9-15 kPa). The cytotoxicity tests showed the POEC-d adhesives had a low cytotoxicity. Our results supports that POEC-d adhesives, which combined strong wet adhesion with good biodegradability, acceptable swelling ratio, good elasticity and low cytotoxicity, have potentials in surgeries where surgical tissue adhesives, sealants, and hemostatic agents are used. PMID:26704547

  1. Antibacterial polyelectrolyte micelles for coating stainless steel.

    Falentin-Daudré, Céline; Faure, Emilie; Svaldo-Lanero, Tiziana; Farina, Fabrice; Jérôme, Christine; Van De Weerdt, Cécile; Martial, Joseph; Duwez, Anne-Sophie; Detrembleur, Christophe

    2012-05-01

    In this study, we report on the original synthesis and characterization of novel antimicrobial coatings for stainless steel by alternating the deposition of aqueous solutions of positively charged polyelectrolyte micelles doped with silver-based nanoparticles with a polyanion. The micelles are formed by electrostatic interaction between two oppositely charged polymers: a polycation bearing 3,4-dihydroxyphenylalanine units (DOPA, a major component of natural adhesives) and a polyanion (poly(styrene sulfonate), PSS) without using any block copolymer. DOPA units are exploited for their well-known ability to anchor to stainless steel and to form and stabilize biocidal silver nanoparticles (Ag(0)). The chlorine counteranion of the polycation forms and stabilizes biocidal silver chloride nanoparticles (AgCl). We demonstrate that two layers of micelles (alternated by PSS) doped with silver particles are enough to impart to the surface strong antibacterial activity against gram-negative E. coli. Moreover, micelles that are reservoirs of biocidal Ag(+) can be easily reactivated after depletion. This novel water-based approach is convenient, simple, and attractive for industrial applications. PMID:22506542

  2. Sugary interfaces mitigate contact damage where stiff meets soft

    Yoo, Hee Young; Iordachescu, Mihaela; Huang, Jun; Hennebert, Elise; Kim, Sangsik; Rho, Sangchul; Foo, Mathias; Flammang, Patrick; Zeng, Hongbo; Hwang, Daehee; Waite, J. Herbert; Hwang, Dong Soo

    2016-06-01

    The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina's foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+-DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.

  3. In vitro antibacterial analysis of phenoloxidase reaction products from the sea cucumber Apostichopus japonicus.

    Jiang, Jingwei; Zhou, Zunchun; Dong, Ying; Cong, Cong; Guan, Xiaoyan; Wang, Bai; Chen, Zhong; Jiang, Bei; Yang, Aifu; Gao, Shan; Sun, Hongjuan

    2014-08-01

    Three phenoloxidases (POs) of Apostichopus japonicus, AjPOs (AjPO1, AjPO2 and AjPO3), were partially purified from the coelomocytes with an electrophoretic method, and then employed for the in vitro antibacterial analysis. Using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, AjPO1 and AjPO2-derived compounds inhibited the growth of Vibrio splendidus and Staphylococcus aureus, while AjPO3-derived compounds only inhibited the growth of V. splendidus. When dopamine was used as a substrate, AjPO1 and AjPO3-derived compounds inhibited the growth of V. splendidus and Vibrio harveyi, while AjPO2-derived compounds only inhibited the growth of V. splendidus. Moreover, AjPO1-derived compounds showed stronger inhibition in V. harveyi than AjPO3-derived compounds did. However, all of the three AjPO reaction products showed no inhibitions on the growth of Pseudoalteromonas nigrifaciens, Shewanella baltica, Micrococcus lysodeikticus, Streptococcus dysgalactiae and Nocardiopsis sp. with L-DOPA or dopamine as a substrate. Scanning electron microscope (SEM) observation of V. harveyi treated by AjPOs and dopamine showed that AjPO1-derived compounds resulted in massive bacteriolysis, AjPO2-derived compounds caused no obvious alteration on bacterial morphology, and AjPO3-derived compounds increased the ratio of spheroidal bacteria. All these results suggested that AjPO reaction products derived by L-DOPA and dopamine had different but limited antibacterial spectrum, and the different antibacterial effects observed among three AjPOs resulted from the different reaction products generated by AjPOs with the same substrate. PMID:24931626

  4. From the cell to the clinic: a comparative review of the partial D₂/D₃receptor agonist and α2-adrenoceptor antagonist, piribedil, in the treatment of Parkinson's disease.

    Millan, Mark J

    2010-11-01

    Though L-3,4-dihydroxyphenylalanine (L-DOPA) is universally employed for alleviation of motor dysfunction in Parkinson's disease (PD), it is poorly-effective against co-morbid symptoms like cognitive impairment and depression. Further, it elicits dyskinesia, its pharmacokinetics are highly variable, and efficacy wanes upon long-term administration. Accordingly, "dopaminergic agonists" are increasingly employed both as adjuncts to L-DOPA and as monotherapy. While all recognize dopamine D(2) receptors, they display contrasting patterns of interaction with other classes of monoaminergic receptor. For example, pramipexole and ropinirole are high efficacy agonists at D(2) and D(3) receptors, while pergolide recognizes D(1), D(2) and D(3) receptors and a broad suite of serotonergic receptors. Interestingly, several antiparkinson drugs display modest efficacy at D(2) receptors. Of these, piribedil displays the unique cellular signature of: 1), signal-specific partial agonist actions at dopamine D(2)and D(3) receptors; 2), antagonist properties at α(2)-adrenoceptors and 3), minimal interaction with serotonergic receptors. Dopamine-deprived striatal D(2) receptors are supersensitive in PD, so partial agonism is sufficient for relief of motor dysfunction while limiting undesirable effects due to "over-dosage" of "normosensitive" D(2) receptors elsewhere. Further, α(2)-adrenoceptor antagonism reinforces adrenergic, dopaminergic and cholinergic transmission to favourably influence motor function, cognition, mood and the integrity of dopaminergic neurones. In reviewing the above issues, the present paper focuses on the distinctive cellular, preclinical and therapeutic profile of piribedil, comparisons to pramipexole, ropinirole and pergolide, and the core triad of symptoms that characterises PD-motor dysfunction, depressed mood and cognitive impairment. The article concludes by highlighting perspectives for clarifying the mechanisms of action of piribedil and other

  5. Acute L: -DOPA effect on hydroxyl radical- and DOPAC-levels in striatal microdialysates of parkinsonian rats.

    Nowak, Przemysław; Kostrzewa, Rose Anna; Skaba, Dariusz; Kostrzewa, Richard M

    2010-04-01

    The object of the current study was to determine the effect of L: -3,4-dihydroxyphenylalanine (L: -DOPA) on the in vivo striatal microdialysate levels of the respective dopamine and serotonin metabolites 3,4-dihydroxyphenlalanine (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) and hydroxyl radical level (HO(*); 2,3- and 2,5-dihydroxybenzoic acid, 2,3- and 2,5-DHBA) in adult rats made parkinsonian by treatment at 3 days after birth with the neurotoxin 6-hydroxydopamine (6-OHDA; 66.7 microg, base form, on each side; desipramine pretreatment, 1 h). Using HPLC/ED we found that in 6-OHDA-lesioned rats the basal striatal extraneuronal level of DOPAC was dramatically reduced and constituted only approximately 4.5% of referenced value (intact rats). Conversely, the striatal microdialysate level of 5-HIAA was elevated 2-fold in 6-OHDA-lesioned rats. Acute L: -DOPA (60 mg/kg i.p.; S-carbidopa pretreatment, 12.5 mg/kg i.p., 30 min) produced a rapid rise in the extraneuronal DOPAC in both tested groups but to a much greater extent in intact rats (P DOPA did not enhance HO(*) production; acute 6-OHDOPA treatment (60 mg/kg i.p.) also did not alter HO(*) production. In summary, L: -DOPA, an effective drug in ameliorating PD symptoms, did not acutely pose a risk for HO(*) generation in parkinsonian rats. We conclude that L: -DOPA is not likely to generate reactive oxygen species in humans nor is L: -DOPA likely to accelerate PD in humans. PMID:19760476

  6. Binding of dopamine and 3-methoxytyramine as l-DOPA metabolites to human alpha(2)-adrenergic and dopaminergic receptors.

    Alachkar, Amal; Brotchie, Jonathan M; Jones, Owen T

    2010-07-01

    The ability of l-3,4-dihydroxyphenylalanine (l-DOPA), l-DOPA-methyl ester and their major metabolites, dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic (HVA), 3-O-methyldopa and 3-methoxytyramine (3-MT) to bind to alpha(2) adrenergic and D1 and D2 dopamine receptors was assessed by radioligand binding to cloned human receptors expressed in cell lines. As anticipated, dopamine bound with high affinity to D1 (IC(50) 1.1 + or - 0.16 microM) and D2 (IC(50) 0.7 + or - 0.3 microM) dopamine receptors. However, dopamine also bound with high affinity to alpha(2A) (IC(50) was 2.6 + or - 0.5 microM), alpha(2C) (IC(50) 3.2 + or - 0.7 microM). 3-MT bound to alpha(2A) with high affinity (IC(50), 3.6 + or - 0.2 microM) though moderate affinity to alpha(2)c, D1 and D2 receptors (values of IC(50) were 55 + or - 14, 121 + or - 43, 36 + or - 14 microM, respectively). l-DOPA-methyl ester bound with high affinity to alpha(2) (IC(50) 17-36 microM) but not dopamine receptors (IC(50) 0.9-2.5 mM). l-DOPA, 3-O-methyldopa and DOPAC had no observable effect on binding to any of the receptors tested. These data suggest that the effects of l-DOPA in Parkinson's disease may result from actions of its metabolites dopamine and 3-MT on both dopaminergic and non-dopaminergic receptors. These findings may provide explanations for the differences between l-DOPA and dopamine receptor agonists in mediating anti-parkinsonian effects and propensity to be associated with dyskinesia and motor complications such as wearing-off and on-off. PMID:20302892

  7. Blood withdrawal affects iron store dynamics in primates with consequences on monoaminergic system function.

    Hyacinthe, C; De Deurwaerdere, P; Thiollier, T; Li, Q; Bezard, E; Ghorayeb, I

    2015-04-01

    Iron homeostasis is essential for the integrity of brain monoaminergic functions and its deregulation might be involved in neurological movement disorders such as the restless legs syndrome (RLS). Although iron metabolism breakdown concomitantly appears with monoaminergic system dysfunction in iron-deficient rodents and in RLS patients, the direct consequences of peripheral iron deficiency in the central nervous system (CNS) of non-human primates have received little attention. Here, we evaluated the peripheral iron-depletion impact on brain monoamine levels in macaque monkeys. After documenting circadian variations of iron and iron-related proteins (hemoglobin, ferritin and transferrin) in both serum and cerebrospinal fluid (CSF) of normal macaques, repeated blood withdrawals (RBW) were used to reduce peripheral iron-related parameter levels. Decreased serum iron levels were paradoxically associated with increased CSF iron concentrations. Despite limited consequences on tissue monoamine contents (dopamine - DA, 3, 4-dihydroxyphenylacetic acid - DOPAC, homovanillic acid, L-3, 4-dihydroxyphenylalanine - L-DOPA, 5-8 hydroxytryptamine - 5-HT, 5-hydroxyindoleacetic acid - 5-HIAA and noradrenaline) measured with post-mortem chromatography, we found distinct and region-dependent relationships of these tissue concentrations with CSF iron and/or serum iron and/or blood hemoglobin. Additionally, striatal extracellular DA, DOPAC and 5-HIAA levels evaluated by in vivo microdialysis showed a substantial increase, suggesting an overall increase in both DA and 5-HT tones. Finally, a trending increase in general locomotor activity, measured by actimetry, was observed in the most serum iron-depleted macaques. Taken together, our data are compatible with an increase in nigrostriatal DAergic function in the event of iron deficiency and point to a specific alteration of the 5-HT/DA interaction in the CNS that is possibly involved in the etiology of RLS. PMID:25662508

  8. Positron Emission Tomography Imaging Demonstrates Correlation between Behavioral Recovery and Correction of Dopamine Neurotransmission after Gene Therapy

    In vivo gene transfer using viral vectors is an emerging therapy for neuro-degenerative diseases with a clinical impact recently demonstrated in Parkinson's disease patients. Recombinant adeno-associated viral (rAAV) vectors, in particular, provide an excellent tool for long-term expression of therapeutic genes in the brain. Here we used the [11C]raclopride [(S)-(-)-3, 5-dichloro-N-((1-ethyl-2-pyrrolidinyl)methyl)-2-hydroxy- 6-methoxybenzamide] micro-positron emission tomography (PET) technique to demonstrate that delivery of the tyrosine hydroxylase (TH) and GTP-cyclohydrolase 1 (GCH1) enzymes using an rAAV5 vector normalizes the increased [11C]raclopride binding in hemi-parkinsonian rats. Importantly, we show in vivo by micro-PET imaging and postmortem by classical binding assays performed in the very same animals that the changes in [11C]raclopride after viral vector-based enzyme replacement therapy is attributable to a decrease in the affinity of the tracer binding to the D2 receptors, providing evidence for reconstitution of a functional pool of endogenous dopamine in the striatum. Moreover, the extent of the normalization in this non-invasive imaging measure was highly correlated with the functional recovery in motor behavior. The PET imaging protocol used in this study is fully adaptable to humans and thus can serve as an in vivo imaging technique to follow TH+GCH1 gene therapy in PD patients and provide an additional objective measure to a potential clinical trial using rAAV vectors to deliver L-3, 4-dihydroxyphenylalanine in the brain. (authors)

  9. Positron Emission Tomography Imaging Demonstrates Correlation between Behavioral Recovery and Correction of Dopamine Neurotransmission after Gene Therapy

    Leriche, L.; Besret, L.; Gregoire, M.C.; Deglon, N.; Hantraye, Ph. [1Centre National de la Recherche Scientifique, Unite de Recherche Associee 2210, 91401 Orsay (France); Leriche, L.; Besret, L.; Gregoire, M.C.; Deglon, N.; Hantraye, Ph. [2Commissariat a l' energie Atomique (CEA), Biomedical Imaging Institute, Molecular Imaging Research Center, 92265 Fontenay-aux-Roses (France); Bjorklund, T.; Breysse, N.; Carlsson, T.; Kirik, D. [Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University, SE-221 84, Lund (Sweden); Dolle, F. [CEA, Biomedical Imaging Institute, Service Hospitalier Frederic Joliot, 91406 Orsay (France); Mandel, R.J. [5Department of Neuroscience, McKnight Brain Institute and Gene Therapy Centre, College of Medicine, University of Florida, Gainesville, Florida 32610 (US); Kirik, D. [LundUniversity Bio-Imaging Center, Faculty of Medicine, SE-221 84 Lund (Sweden)

    2009-07-01

    In vivo gene transfer using viral vectors is an emerging therapy for neuro-degenerative diseases with a clinical impact recently demonstrated in Parkinson's disease patients. Recombinant adeno-associated viral (rAAV) vectors, in particular, provide an excellent tool for long-term expression of therapeutic genes in the brain. Here we used the [{sup 11}C]raclopride [(S)-(-)-3, 5-dichloro-N-((1-ethyl-2-pyrrolidinyl)methyl)-2-hydroxy- 6-methoxybenzamide] micro-positron emission tomography (PET) technique to demonstrate that delivery of the tyrosine hydroxylase (TH) and GTP-cyclohydrolase 1 (GCH1) enzymes using an rAAV5 vector normalizes the increased [{sup 11}C]raclopride binding in hemi-parkinsonian rats. Importantly, we show in vivo by micro-PET imaging and postmortem by classical binding assays performed in the very same animals that the changes in [{sup 11}C]raclopride after viral vector-based enzyme replacement therapy is attributable to a decrease in the affinity of the tracer binding to the D2 receptors, providing evidence for reconstitution of a functional pool of endogenous dopamine in the striatum. Moreover, the extent of the normalization in this non-invasive imaging measure was highly correlated with the functional recovery in motor behavior. The PET imaging protocol used in this study is fully adaptable to humans and thus can serve as an in vivo imaging technique to follow TH+GCH1 gene therapy in PD patients and provide an additional objective measure to a potential clinical trial using rAAV vectors to deliver L-3, 4-dihydroxyphenylalanine in the brain. (authors)

  10. Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

    Burke, Kelly A; Roberts, Dane C; Kaplan, David L

    2016-01-11

    Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants. PMID:26674175

  11. Cardiovascular changes in atherosclerotic ApoE-deficient mice exposed to Co60 (γ radiation.

    Prem Kumarathasan

    Full Text Available BACKGROUND: There is evidence for a role of ionizing radiation in cardiovascular diseases. The goal of this work was to identify changes in oxidative and nitrative stress pathways and the status of the endothelinergic system during progression of atherosclerosis in ApoE-deficient mice after single and repeated exposure to ionizing radiation. METHODS AND RESULTS: B6.129P2-ApoE tmlUnc mice on a low-fat diet were acutely exposed (whole body to Co60 (γ (single dose 0, 0.5, and 2 Gy at a dose rate of 36.32 cGy/min, or repeatedly (cumulative dose 0 and 2 Gy at a dose-rate of 0.1 cGy/min for 5 d/wk, over a period of 4 weeks. Biological endpoints were investigated after 3-6 months of recovery post-radiation. The nitrative stress marker 3-nitrotyrosine and the vasoregulator peptides endothelin-1 and endothelin-3 in plasma were increased (p<0.05 in a dose-dependent manner 3-6 months after acute or chronic exposure to radiation. The oxidative stress marker 8-isoprostane was not affected by radiation, while plasma 8-hydroxydeoxyguanosine and L-3,4-dihydroxyphenylalanine decreased (p<0.05 after treatment. At 2Gy radiation dose, serum cholesterol was increased (p = 0.008 relative to controls. Percent lesion area increased (p = 0.005 with age of animal, but not with radiation treatment. CONCLUSIONS: Our observations are consistent with persistent nitrative stress and activation of the endothelinergic system in ApoE-/- mice after low-level ionizing radiation exposures. These mechanisms are known factors in the progression of atherosclerosis and other cardiovascular diseases.

  12. Multifunctional Electrochemical Platforms Based on the Michael Addition/Schiff Base Reaction of Polydopamine Modified Reduced Graphene Oxide: Construction and Application.

    Huang, Na; Zhang, Si; Yang, Liuqing; Liu, Meiling; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

    2015-08-19

    In this paper, a new strategy for the construction of multifunctional electrochemical detection platforms based on the Michael addition/Schiff base reaction of polydopamine modified reduced graphene oxide was first proposed. Inspired by the mussel adhesion proteins, 3,4-dihydroxyphenylalanine (DA) was selected as a reducing agent to simultaneously reduce graphene oxide and self-polymerize to obtain the polydopamine-reduced graphene oxide (PDA-rGO). The PDA-rGO was then functionalized with thiols and amines by the reaction of thiol/amino groups with quinine groups of PDA-rGO via the Michael addition/Schiff base reaction. Several typical compounds containing thiol and/or amino groups such as 1-[(4-amino)phenylethynyl] ferrocene (Fc-NH2), cysteine (cys), and glucose oxidase (GOx) were selected as the model molecules to anchor on the surface of PDA-rGO using the strategy for construction of multifunctional electrochemical platforms. The experiments revealed that the composite grafted with ferrocene derivative shows excellent catalysis activity toward many electroactive molecules and could be used for individual or simultaneous detection of dopamine hydrochloride (DA) and uric acid (UA), or hydroquinone (HQ) and catechol (CC), while, after grafting of cysteine on PDA-rGO, simultaneous discrimination detection of Pb(2+) and Cd(2+) was realized on the composite modified electrode. In addition, direct electron transfer of GOx can be observed when GOx-PDA-rGO was immobilized on glassy carbon electrode (GCE). When glucose was added into the system, the modified electrode showed excellent electric current response toward glucose. These results inferred that the proposed multifunctional electrochemical platforms could be simply, conveniently, and effectively regulated through changing the anchored recognition or reaction groups. This study would provide a versatile method to design more detection or biosensing platforms through a chemical reaction strategy in the future. PMID

  13. Targeting the D1-N-methyl-D-aspartate receptor complex reduces L-dopa-induced dyskinesia in 6-hydroxydopamine-lesioned Parkinson’s rats

    Song L

    2016-02-01

    Full Text Available Lu Song,1,* Zhanzhao Zhang,2,* Rongguo Hu,1 Jie Cheng,1 Lin Li,1 Qinyi Fan,1 Na Wu,1 Jing Gan,1 Mingzhu Zhou,1 Zhenguo Liu11Department of Neurology, Xinhua Hospital, 2Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workAbstract: L-3,4-dihydroxyphenylalanine (L-dopa remains the most effective therapy for Parkinson’s disease (PD, but its long-term administration is associated with the development of debilitating motor complications known as L-dopa-induced dyskinesia (LID. Enhanced function of dopamine D1 receptor (D1R and N-methyl-d-aspartate receptor (NMDAR is believed to participate in the pathogenesis of LID. Given the existence of physical and functional interactions between D1R and NMDAR, we explored the effects of uncoupling D1R and NMDA GluN1 (GluN1 interaction on LID by using the Tat-conjugated interfering peptide (Tat-D1-t2. In this study, we demonstrated in 6-hydroxydopamine (6-OHDA-lesioned PD rat model that intrastriatal injection of Tat-D1-t2 alleviated dyskinetic behaviors and downregulated the phosphorylation of DARPP-32 at Thr34 induced by levodopa. Moreover, we also showed intrastriatal administration of Tat-D1-t2 elicited alterations in membranous GluN1 and D1R expression. These findings indicate that D1R/GluN1 complexes may be a molecular target with therapeutic potential for the treatment of dyskinesia in Parkinson’s patients.Keywords: 6-hydroxydopamine, Parkinson’s disease, dyskinesia, L-dopa, D1 receptor, NMDA, protein–protein interaction

  14. Inhibition of glycogen synthase kinase-3 reduces L-DOPA-induced neurotoxicity

    The neurotoxicity of L-3,4-dihydroxyphenylalanine (L-DOPA), used for the treatment of Parkinson's disease, remains controversial. Although there are many reports suggesting that long-term treatment of L-DOPA causes neuronal death, an increasing body of recent evidence has proposed that L-DOPA might be neuroprotective rather than neurotoxic. We investigated the effect of L-DOPA on neuronally differentiated PC12 (nPC12) cells by treating cells with various concentrations of L-DOPA for 24 h. We also studied whether glycogen synthase kinase (GSK)-3 activation is related to L-DOPA-induced neurotoxicity by simultaneously treating cells with several concentrations of L-DOPA and a GSK-3 inhibitor for 24 h. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, cell counting kit-8, and DAPI staining all showed that L-DOPA decreased nPC12 cell viability at high concentrations. In addition, 100 μM L-DOPA treatment significantly increased the activity of GSK-3 and death signals including cytochrome c, activated caspase-3 and cleaved PARP, and decreased survival signals including heat shock transcription factor-1 in a concentration-dependent manner. Treatment with GSK-3 inhibitor VIII or lithium chloride prevented L-DOPA-induced cell death. Together, these results suggest that L-DOPA induces neuronal cell death at high concentrations and that the neurotoxic effect of L-DOPA might be mediated in part by GSK-3 activation

  15. An Ultra-Violet Tolerant Wild-Type Strain of Melanin-Producing Bacillus thuringiensis

    Sansinenea

    2015-07-01

    Full Text Available Background Bacillus thuringiensis is the most successful biological control agent used in agriculture, forestry and mosquito control. However, the insecticidal activity of the B. thuringiensis formulation is not very stable and rapidly loses its biological activity under field conditions, due to the ultraviolet radiation in sunlight. Melanin is known to absorb radiation therefore photo protection of B. thuringiensis based on melanin has been extensively studied. Objectives The aim of this study was to find a wild type strain of naturally melanin-producing B. thuringiensis to avoid any mutation or manipulation that can affect the Cry protein content. Materials and Methods Bacillus thuringiensis strains were isolated from soils of different States of Mexico and pigment extraction was followed by lowering the pH to 2 using 1N HCl. Pigment was characterized by some chemical tests based on its solubility, bleaching by H2O2 and flocculation with FeCl3, and using an Infrared (IR spectrum. Ultraviolet (UV irradiation experiment was performed to probe the melanin efficacy. Results ELI52 strain of B. thuringiensis was confirmed to naturally produce melanin. The Cry protein analysis suggested that ELI52 is probably a B. thuringiensis subsp. israelensis strain with toxic activity against the Diptera order of insects. Ultra Violet protection efficacy of melanin was probed counting total viable colonies after UV radiation and comparing the results with the non-producing melanin strain L-DOPA (L-3, 4-dihydroxyphenylalanine was also detected in the culture. ELI52 strain showed an antagonistic effect over some common bacteria from the environment. Conclusions ELI52 wild-type strain of B. thuringiensis is a good bio-insecticide that produces melanin with UV-resistance that is probably toxic against the Diptera order of insects and can inhibit the growth of other environmental bacteria.

  16. Comparison of Monoamine Oxidase Inhibitors in Decreasing Production of the Autotoxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde in PC12 Cells.

    Goldstein, David S; Jinsmaa, Yunden; Sullivan, Patti; Holmes, Courtney; Kopin, Irwin J; Sharabi, Yehonatan

    2016-02-01

    According to the catecholaldehyde hypothesis, the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to the loss of nigrostriatal dopaminergic neurons in Parkinson's disease. Monoamine oxidase-A (MAO-A) catalyzes the conversion of intraneuronal dopamine to DOPAL and may serve as a therapeutic target. The "cheese effect"-paroxysmal hypertension evoked by tyramine-containing foodstuffs-limits clinical use of irreversible MAO-A inhibitors. Combined MAO-A/B inhibition decreases DOPAL production in rat pheochromocytoma PC12 cells, but whether reversible MAO-A inhibitors or MAO-B inhibitors decrease endogenous DOPAL production is unknown. We compared the potencies of MAO inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and auto-oxidation. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All three drugs also increased dopamine and norepinephrine, decreased 3,4-dihydroxyphenylalanine, and increased cysteinyl-dopamine concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine spontaneous oxidation. In conclusion, clorgyline, rasagiline, and selegiline decrease production of endogenous DOPAL. At relatively high concentrations, the latter drugs probably lose their selectivity for MAO-B. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in Parkinson

  17. Polymer Composition and Substrate Influences on the Adhesive Bonding of a Biomimetic, Cross-Linking Polymer

    Matos-Pérez, Cristina R.; White, James D.; Wilker, Jonathan J.

    2012-01-01

    Hierarchical biological materials such as bone, sea shells, and marine bioadhesives are providing inspiration for the assembly of synthetic molecules into complex structures. The adhesive system of marine mussels has been the focus of much attention in recent years. Several catechol-containing polymers are being developed to mimic the cross-linking of proteins containing 3,4-dihydroxyphenylalanine (DOPA) used by shellfish for sticking to rocks. Many of these biomimetic polymer systems have been shown to form surface coatings or hydrogels, however bulk adhesion is demonstrated less often. Developing adhesives requires addressing design issues including finding a good balance between cohesive and adhesive bonding interactions. Despite the growing number of mussel mimicking polymers, there has been little effort to generate structure-property relations and gain insights on what chemical traits give rise to the best glues. In this report, we examined the simplest of these biomimetic polymers, poly[(3,4-dihydroxystyrene)-co-styrene]. Pendant catechol groups (i.e., 3,4-dihydroxystyrene) were distributed throughout a polystyrene backbone. Several polymer derivatives were prepared, each with a different 3,4-dihyroxystyrene content. Bulk adhesion testing showed where the optimal middle ground of cohesive and adhesive bonding resides. Adhesive performance was benchmarked against commercial glues as well as the genuine material produced by live mussels. In the best case, bonding was similar to cyanoacrylate “Krazy” or “Super” glue. Performance was also examined using low (e.g., plastics) and high (e.g., metals, wood) energy surfaces. Adhesive bonding of poly[(3,4-dihydroxystyrene)-co-styrene] may be the strongest of reported mussel protein mimics. These insights should help us to design future biomimetic systems, thereby bringing us closer to development of bone cements, dental composites, and surgical glues. PMID:22582754

  18. l-DOPA-induced dyskinesia is associated with a deficient numerical downregulation of striatal tyrosine hydroxylase mRNA-expressing neurons.

    Klietz, Martin; Keber, Ursula; Carlsson, Thomas; Chiu, Wei-Hua; Höglinger, Günter U; Weihe, Eberhard; Schäfer, Martin K-H; Depboylu, Candan

    2016-09-01

    l-3,4-Dihydroxyphenylalanine (l-DOPA) is the therapeutic gold standard in Parkinson's disease. However, most patients develop debilitating abnormal involuntary movements termed l-DOPA-induced dyskinesia (LID) as therapy-complicating side effects. The underlying mechanisms of LID pathogenesis are still not fully understood. Recent evidence suggests an involvement of striatal tyrosine hydroxylase (TH) protein-expressing neurons, as they are capable of endogenously producing l-DOPA and possibly dopamine. The aim of this study was to elucidate changes of TH transcription in the striatum and nucleus accumbens that occur under experimental conditions of LID. Mice with a unilateral 6-hydroxydopamine-induced lesion of the medial forebrain bundle were treated daily with l-DOPA for 15days to provoke dyskinesia. In situ hybridization analysis revealed a significant numerical decrease of TH mRNA-positive neurons in the striatum and nucleus accumbens of mice not exhibiting LID, whereas dyskinetic animals failed to show this reduction of TH transcription. Interestingly, similar changes were observed in intact non-deafferentiated striata, demonstrating an l-DOPA-responsive transcriptional TH regulation independently from nigrostriatal lesion severity. Consolidation with our previous study on TH protein level (Keber et al., 2015) impressively highlights that LID is associated with both a deficient downregulation of TH transcription and an excessive translation of TH protein in intrastriatal neurons. As TH protein levels in comparison to mRNA levels showed a stronger correlation with development and severity of LID, antidyskinetic treatment strategies should focus on translational and posttranslational modulations of TH as a promising target. PMID:27320210

  19. Protection of melanized Cryptococcus neoformans from lethal dose gamma irradiation involves changes in melanin's chemical structure and paramagnetism.

    Abdelahad Khajo

    Full Text Available Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans, which uses L-3,4-dihydroxyphenylalanine (L-DOPA to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50 to 80% of the cells did not lead to a detectable loss of melanin integrity according to EPR spectra of melanin radicals. Moreover, upon UV-visible (Xe-lamp illumination of melanized cells, the increase in radical population was unchanged after γ-irradiation. Gamma-irradiation of frozen cell suspensions and storage of samples for several days at 77 K however, produced melanin modification noted by a reduced radical population and reduced photoresponse. More direct evidence for structural modification of melanin came from the detection of soluble products with absorbance maxima near 260 nm in supernatants collected after γ-irradiation of cells and cell-free melanin. These products, which include thiobarbituric acid (TBA-reactive aldehydes, were also generated by Fenton reagent treatment of cells and cell-free melanin. In an assay of melanin integrity based on the metal (Bi(+3 binding capacity of cells, no detectable loss in binding was detected after γ-irradiation. Our results show that melanin in C. neoformans cells is susceptible to some damage by hydroxyl radical formed in lethal radioactive aqueous environments and serves a protective role in melanized fungi that involves sacrificial breakdown.

  20. Antioxidant Activity of Phenolic Compounds from Fava Bean Sprouts.

    Okumura, Koharu; Hosoya, Takahiro; Kawarazaki, Kai; Izawa, Norihiko; Kumazawa, Shigenori

    2016-06-01

    Fava beans are eaten all over the world and recently, marketing for their sprouts began in Japan. Fava bean sprouts contain more polyphenols and l-3,4-dihydroxyphenylalanine (l-DOPA) than the bean itself. Our antioxidant screening program has shown that fava bean sprouts also possess a higher antioxidant activity than other commercially available sprouts and mature beans. However, the individual constituents of fava bean sprouts are not entirely known. In the present study, we investigated the phenolic compounds of fava bean sprouts and their antioxidant activity. Air-dried fava bean sprouts were treated with 80% methanol and the extract was partitioned in water with chloroform and ethyl acetate. HPLC analysis had shown that the ethyl acetate-soluble parts contained phenolic compounds, separated by preparative HPLC to yield 5 compounds (1-5). Structural analysis using NMR and MS revealed that the compounds isolated were kaempferol glycosides. All isolated compounds had an α-rhamnose at the C-7 position with different sugars attached at the C-3 position. Compounds 1-5 had β-galactose, β-glucose, α-rhamnose, 6-acetyl-β-galactose and 6-acetyl-β-glucose, respectively, at the C-3 position. The amount of l-DOPA in fava bean sprouts was determined by the quantitative (1) H NMR technique. The l-DOPA content was 550.45 mg ± 11.34 /100 g of the raw sprouts. The antioxidant activities of compounds 2-5 and l-DOPA were evaluated using the 2,2-diphenyl-1-picrylhydrazyl scavenging assay. l-DOPA showed high antioxidant activity, but the isolated kaempferol glycosides showed weak activity. Therefore, it can be suggested that l-DOPA contributed to the antioxidant activity of fava bean sprouts. PMID:27155370

  1. Imaging DA release in a rat model of L-DOPA-induced dyskinesias: a longitudinal in vivo PET investigation of the antidyskinetic effect of MDMA.

    Lettfuss, Nadine Y; Fischer, Kristina; Sossi, Vesna; Pichler, Bernd J; von Ameln-Mayerhofer, Andreas

    2012-10-15

    In the context of Parkinson's disease, motor symptoms result from the degeneration of nigrostriatal neurons. Dopamine (DA) replacement using l-3,4-dihydroxyphenylalanine (L-DOPA) has been the treatment of choice in the early stages of the disease. However, with disease progression, patients suffer from motor complications, which have been suggested to arise from DA released from serotonergic terminals according to the false neurotransmitter hypothesis. The synthetic amphetamine derivative (±) 3,4-methylenedioxymethamphetamine (MDMA) has been shown to significantly inhibit dyskinesia in humans and in animal models of PD. In this study, we examined the effect of MDMA on L-DOPA-induced DA release by using [(11)C]raclopride kinetic modeling to assess alterations in DA neurotransmission in a rat model of L-DOPA-induced dyskinesia (LID) in a longitudinal in vivo PET study. Rats were submitted to 6-OHDA lesions, and the lesions were confirmed to be sufficiently severe based on the performance during stepping tests and [(11)C]methylphenidate PET scans. The rats underwent two [(11)C]raclopride PET sessions before (baseline) and after two weeks of chronic L-DOPA treatment (priming). L-DOPA priming led to strong abnormal involuntary movements (AIMs). In group 1, L-DOPA priming reduced L-DOPA-induced DA release in the lesioned striatum with no effect on the healthy side, while the concomitant administration of L-DOPA and MDMA (group 2) increased the DA levels in the lesioned and healthy striatum. In addition, behavioral analysis, which was performed two weeks after the second PET session, confirmed the antidyskinetic effect of MDMA. Our data show that L-DOPA-induced DA release is attenuated in the Parkinsonian striatum after chronic L-DOPA pretreatment and that the antidyskinetic mechanism of MDMA does not depend primarily on dopaminergic neurotransmission. PMID:22766162

  2. Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp.: a comparative study of wild-type and genetically manipulated strains

    The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed

  3. Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson's disease.

    Bortolanza, Mariza; Cavalcanti-Kiwiatkoski, Roberta; Padovan-Neto, Fernando E; da-Silva, Célia Aparecida; Mitkovski, Miso; Raisman-Vozari, Rita; Del-Bel, Elaine

    2015-01-01

    l-3, 4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of L-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with L-DOPA (30 mg/kg plus benserazide 7.5 mg/kg, for 21 days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30 mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with L-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. L-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of L-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinson's disease. PMID:25447229

  4. Effect of siRNA-induced silencing of cellular prion protein on tyrosine hydroxylase expression in the substantia nigra of a rat model of Parkinson's disease.

    Wang, X; Yang, H A; Wang, X N; Du, Y F

    2016-01-01

    The most significant pathological feature of Parkinson's disease (PD) is the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra. Currently, available treatments for PD cannot prevent the loss of DA neurons. Tyrosine hydroxylase (TH) expressed in substantia nigra neurons catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the rate-limiting step of DA biosynthesis. Major reasons for PD occurrence include decreased TH activity in the substantia nigra and secondary DA suppression. Decreased TH activity and the resulting suppression of DA synthesis (or neurotransmission) in the substantia nigra are key factors underlying the development of PD. Cellular prion protein (PRP) is a membrane glycoprotein expressed in the central nervous system. Although the sequence of PRP is highly conserved, its physiological function is unclear. The purpose of this study was to investigate the effect of PRP-targeted small interfering RNA (siRNA) on TH expression in a rat model of PD. Thirty male Wistar rats were injected with 6-hydroxydopamine (6-OHDA) to generate a model of PD. The rats then received injections of PRP-siRNA or nonsense siRNA in the lateral ventricles. Substantia nigra samples were collected for quantification of PRP and TH expression using real-time polymerase chain reaction and western blotting. PRP-siRNA decreased PRP expression in the substantia nigra. TH expression was decreased in PD model rats but was increased after PRP silencing. We conclude that PRP-siRNA may increase TH expression in vivo and may therefore exert protective effects on neurons in a model of PD. PMID:27173342

  5. Behavioural sensitisation during dopamine replacement therapy in Parkinson's disease is reminiscent of the addicted brain.

    Biagioni, F; Pellegrini, A; Ruggieri, S; Murri, L; Paparelli, A; Fornai, F

    2009-01-01

    The intermittent oral intake of the dopamine (DA) precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is the classic therapy of Parkinson's disease (PD). In this way, the drug precursor can be metabolised into the active neurotransmitter DA. Although this occurs throughout the brain, the therapeutic relief is believed to be due to restoring extracellular DA levels within the dorsal striatum (more in the putamen than the caudate nucleus) which lacks endogenous DA as a consequence of the disease process. However, differing from physiological DA transmission, this therapeutic pattern leads to abnormal peaks of non-synaptic DA, which are supposed to trigger behavioural sensitisation expressed as abnormal involuntary movements. A similar pattern of abnormal DA stimulation occurs during methamphetamine (METH) intake. In the present review we will provide evidence showing the similarities between METH- and L-DOPA-induced DA stimulation with an intact and denervated striatum respectively. This comparison will encompass various features; the timing, the areas and the amount of extracellular DA levels which reveal surprising homologies. Such an overlapping between L-DOPA in PD and METH will be further analysed to critically assess the commonalities concerning the following points: abnormal receptor stimulation, recruitment of altered transduction pathways, abnormal gene expression, alterations in the phenotype of striatal neurons, and the establishment of behavioural sensitisation which appear as distinct phenomena (i.e. abnormal involuntary movements in PD and drug addiction in METH abuse); nonetheless, this may also lead to common behavioural alterations (METH-like addictive behaviours in PD patients during the course of DA replacement therapy in subsets of PD patients). PMID:19754404

  6. Yokukansan, a Traditional Japanese Medicine, Enhances the L-DOPA-Induced Rotational Response in 6-Hydroxydopamine-Lesioned Rats: Possible Inhibition of COMT.

    Ishida, Yasushi; Ebihara, Kosuke; Tabuchi, Masahiro; Imamura, Sachiko; Sekiguchi, Kyoji; Mizoguchi, Kazushige; Kase, Yoshio; Koganemaru, Go; Abe, Hiroshi; Ikarashi, Yasushi

    2016-01-01

    The aim of the present study was to investigate the effects of the traditional Japanese medicine yokukansan (YKS) on the function of dopamine (DA) in the rat nigrostriatal system. Unilateral 6-hydroxydopamine lesions were produced in the rat nigrostriatal system. Despite a marked loss in the striatal immunoreactivity of tyrosine hydroxylase on the lesion side, striatal serotonin (5-HT) immunoreactivity was not affected. Treatment using L-3,4-dihydroxyphenylalanine (L-DOPA) in conjunction with benserazide for 15 d induced abnormal involuntary movements (AIMs) such as locomotive (rotational response), axial, forelimb, and orolingual movements in the lesioned rats. The L-DOPA-induced locomotive and axial, but not forelimb and orolingual, AIMs were significantly increased and prolonged by the pre-administration of YKS. We next investigated the effects of YKS on the production of DA from L-DOPA in 5-HT synthetic RIN 14B cells. RIN 14B cells produced DA and its metabolite, 3-methoxytyramine (3-MT), following L-DOPA treatment. YKS significantly augmented DA production and inhibited its metabolism to 3-MT in a manner similar to the catechol-O-methyltransferase (COMT) inhibitor entacapone. YKS and some alkaloids (corynoxeine: CX, geissoschizine methyl ether: GM) in Uncaria hook, a constituent herb of YKS, also inhibited COMT activity, indicating that the augmenting effect of YKS on L-DOPA-induced DA production in 5-HT synthetic cells was due to the inhibition of COMT by CX and GM. Our results suggest that YKS facilitates the DA supplemental effect of L-DOPA, and that COMT inhibition by CX and GM contributes, at least in part, to the effects of YKS. PMID:26725433

  7. Antidyskinetic Effect of 7-Nitroindazole and Sodium Nitroprusside Associated with Amantadine in a Rat Model of Parkinson's Disease.

    Bortolanza, Mariza; Bariotto-Dos-Santos, Keila D; Dos-Santos-Pereira, Maurício; da-Silva, Célia Aparecida; Del-Bel, Elaine

    2016-07-01

    Amantadine is the noncompetitive antagonist of N-methyl-D-aspartate, receptor activated by the excitatory neurotransmitter glutamate. It is the only effective medication used to alleviate dyskinesia induced by L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson's disease patients. Unfortunately, adverse effects as abnormal involuntary movements (AIMs) known as L-DOPA-induced dyskinesia limit its clinical utility. Combined effective symptomatic treatment modalities may lessen the liability to undesirable events. Likewise drugs known to interfere with nitrergic system reduce AIMs in animal models of Parkinson's disease. We aimed to analyze an interaction between amantadine, neuronal nitric oxide synthase inhibitor (7-nitroindazole, 7NI), and nitric oxide donor (sodium nitroprusside, SNP) in 6-hydroxydopamine-(6-OHDA)-lesioned rats (microinjection in the medial forebrain bundle) presenting L-DOPA-induced dyskinesia (20 mg/kg, gavage, during 21 days). We confirm that 7NI-30 mg/kg, SNP-2/4 mg/kg and amantadine-40 mg/kg, individually reduced AIMs. Our results revealed that co-administration of sub-effective dose of amantadine (10 mg/kg) plus sub-effective dose of 7NI (20 mg/kg) potentiates the effect of reducing AIMs scores when compared to the effect of the drugs individually. No superior benefit on L-DOPA-induced AIMs was observed with the combination of amantadine and SNP. The results revealed that combination of ineffective doses of amantadine and 7NI represents a new strategy to increase antidyskinetic effect in L-DOPA-induced AIMs. It may provide additional therapeutic benefits to Parkinson's disease patients from these disabling complications at lower and thus safer and more tolerable doses than required when either drug is used alone. To close, we discuss the paradox of both nitric oxide synthase inhibitor and/or donor produced AIMs reduction by targeting nitric oxide synthase. PMID:27053252

  8. Overexpression of L-Type Amino Acid Transporter 1 (LAT1 and 2 (LAT2: Novel Markers of Neuroendocrine Tumors.

    Susi Barollo

    Full Text Available 6-18F-fluoro-L-3,4-dihydroxyphenylalanine (18F-FDOPA PET is a useful tool in the clinical management of pheochromocytoma (PHEO and medullary thyroid carcinoma (MTC. 18F-FDOPA is a large neutral amino acid biochemically resembling endogenous L-DOPA and taken up by the L-type amino acid transporters (LAT1 and LAT2. This study was conducted to examine the expression of the LAT system in PHEO and MTC.Real-time PCR and Western blot analyses were used to assess LAT1 and LAT2 gene and protein expression in 32 PHEO, 38 MTC, 16 normal adrenal medulla and 15 normal thyroid tissue samples. Immunohistochemistry method was applied to identify the proteins' subcellular localization.LAT1 and LAT2 were overexpressed in both PHEO and MTC by comparison with normal tissues. LAT1 presented a stronger induction than LAT2, and their greater expression was more evident in PHEO (15.1- and 4.1-fold increases, respectively than in MTC (9.9- and 4.1-fold increases, respectively. Furthermore we found a good correlation between LAT1/2 and GLUT1 expression levels. A positive correlation was also found between urinary noradrenaline and adrenaline levels and LAT1 gene expression in PHEO. The increased expression of LAT1 is also confirmed at the protein level, in both PHEO and MTC, with a strong cytoplasmic localization.The present study is the first to provide experimental evidence of the overexpression in some NET cancers (such as PHEO or MTC of L-type amino acid transporters, and the LAT1 isoform in particular, giving the molecular basis to explain the increase of the DOPA uptake seen in such tumor cells.

  9. Identification and functional characterization of a novel low affinity aromatic-preferring amino acid transporter (arpAT). One of the few proteins silenced during primate evolution.

    Fernández, Esperanza; Torrents, David; Zorzano, Antonio; Palacín, Manuel; Chillarón, Josep

    2005-05-13

    We have identified in silico arpAT, a gene encoding a new member of the LSHAT family, and cloned it from kidney. Co-expression of arpAT with the heavy subunits rBAT or 4F2hc elicited a sodium-independent alanine transport activity in HeLa cells. L-tyrosine, l-3,4-dihydroxyphenylalanine (L-DOPA), L-glutamine, L-serine, L-cystine, and L-arginine were also transported. Kinetic and cis-inhibition studies showed a K(m) = 1.59 +/- 0.24 mM for L-alanine or IC50 in the millimolar range for most amino acids, except L-proline, glycine, anionic and D-amino acids, which were not inhibitory. L-DOPA and L-tyrosine were the most effective competitive inhibitors of L-alanine transport, with IC50 values of 272.2 +/- 57.1 and 716.3 +/- 112.4 microM, respectively. In the small intestine, arpAT mRNA was located at the enterocytes, in a decreasing gradient from the crypts to the tip of the villi. It was also expressed in neurons from different brain areas. Finally, we show that while the arpAT gene is conserved in rat, dog, and chicken, it has become silenced in humans and chimpanzee. Actually, it has been recently reported that it is one of the 33 recently inactivated genes in the human lineage. The evolutionary implications of the silencing process and the roles of arpAT in transport of L-DOPA in the brain and in aromatic amino acid absorption are discussed. PMID:15757906

  10. Cloning and random mutagenesis of the Erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase.

    Katayama, T; Suzuki, H; Koyanagi, T; Kumagai, H

    2000-11-01

    Tyrosine phenol-lyase (Tpl), which can synthesize 3, 4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that the tpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrR gene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tpl were screened for by use of the lac reporter system in E. coli. The most increased transcription of tpl was observed for the strain with the mutant tyrR allele involving amino acid substitutions of alanine, cysteine, and glycine for valine-67, tyrosine-72, and glutamate-201, respectively. A tyrR-deficient derivative of E. herbicola was constructed and transformed with a plasmid carrying the mutant tyrR allele (V67A Y72C E201G substitutions). The resultant strain expressed Tpl without the addition of tyrosine to the medium and produced as much of it as was produced by the wild-type strain grown under tyrosine-induced conditions. The regulatory properties of the mutant TyrR(V67A), TyrR(Y72C), TyrR(E201G), and TyrR(V67A Y72C E201G) proteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrR(V67A) protein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector. PMID:11055921

  11. Blood-brain transfer and metabolism of 6-[18F]fluoro-L-dopa in rat

    In a study designed to reveal the rates of blood-brain transfer and decarboxylation of fluoro-L-3,4-dihydroxyphenylalanine (FDOPA), we discovered a major discrepancy between the DOPA decarboxylase activity reported in the literature and the rate of FDOPA decarboxylation measured in the study. Donor rats received intravenous injections of 6 mCi fluorine-18-labeled FDOPA. The donor rats synthesized methyl-FDOPA. Arterial plasma, containing both FDOPA and methyl-FDOPA, was sampled from the donor rats at different times and reinjected into recipient rats in which it circulated for 20 s. The blood-brain clearance of the mixture of labeled tracers in the plasma was determined by an integral method. The individual permeabilities were determined by linear regression analysis, according to which the average methyl-FDOPA permeability in the blood-brain barrier was twice that of FDOPA, which averaged 0.037 ml g-1 min-1. The permeability ratio was used to determine the fractional clearance from the brain of FDOPA (and hence of methyl-FDOPA), which averaged 0.081 min-1. In the striatum, the measured average FDOPA decarboxylation rate constant (kD3) was 0.010 min-1, or no more than 1% of the rate of striatal decarboxylation of DOPA measured in vitro and in vivo. We interpreted this finding as further evidence in favor of the hypothesis that striatum has two dopamine (DA) pools, of which only DA in the large pool is protected from metabolism. Hence, no more than 1% of the quantity of fluoro-DA theoretically synthesized was actually retained in striatum

  12. Ascorbyl coumarates as multifunctional cosmeceutical agents that inhibit melanogenesis and enhance collagen synthesis.

    Kwak, Jun Yup; Park, Soojin; Seok, Jin Kyung; Liu, Kwang-Hyeon; Boo, Yong Chool

    2015-09-01

    L-Ascorbic acid (AA) and p-coumaric acid (p-CA) are naturally occurring antioxidants that are known to enhance collagen synthesis and inhibit melanin synthesis, respectively. The purpose of this study was to examine hybrid compounds between AA and p-CA as multifunctional cosmeceutical agents. Ascorbyl 3-p-coumarate (A-3-p-C), ascorbyl 2-p-coumarate (A-2-p-C), and their parent compounds were tested for their effects on cellular melanin synthesis and collagen synthesis. At 100 μM, A-3-p-C and A-2-p-C decreased melanin content of human dermal melanocytes stimulated by L-tyrosine, by 65 and 59%, respectively, compared to 11% inhibition of AA and 70% inhibition of p-CA. A-3-p-C and A-2-p-C were less effective than p-CA but more effective than AA at inhibiting tyrosinase activity. A-3-p-C and A-2-p-C were more effective than p-CA at inhibiting the autoxidation of L-3,4-dihydroxyphenylalanine. At 100-300 μM, A-3-p-C and A-2-p-C augmented collagen release from human dermal fibroblasts by 120-144% and 125-191%, respectively, compared to 126-133% increase of AA and 120-146% increase of p-CA. They increased procollagen type I C-peptide release (A-3-p-C, and A-2-p-C) like AA, and decreased matrix metalloproteinase 1 level (A-2-p-C) like p-CA, implicating that they might regulate collagen metabolism by multiple mechanisms. This study suggests that A-3-p-C and A-2-p-C could be used as multifunctional cosmeceutical agents for the attenuation of certain aspects of skin aging. PMID:26078014

  13. Polymer composition and substrate influences on the adhesive bonding of a biomimetic, cross-linking polymer.

    Matos-Pérez, Cristina R; White, James D; Wilker, Jonathan J

    2012-06-01

    Hierarchical biological materials such as bone, sea shells, and marine bioadhesives are providing inspiration for the assembly of synthetic molecules into complex structures. The adhesive system of marine mussels has been the focus of much attention in recent years. Several catechol-containing polymers are being developed to mimic the cross-linking of proteins containing 3,4-dihydroxyphenylalanine (DOPA) used by shellfish for sticking to rocks. Many of these biomimetic polymer systems have been shown to form surface coatings or hydrogels; however, bulk adhesion is demonstrated less often. Developing adhesives requires addressing design issues including finding a good balance between cohesive and adhesive bonding interactions. Despite the growing number of mussel-mimicking polymers, there has been little effort to generate structure-property relations and gain insights on what chemical traits give rise to the best glues. In this report, we examine the simplest of these biomimetic polymers, poly[(3,4-dihydroxystyrene)-co-styrene]. Pendant catechol groups (i.e., 3,4-dihydroxystyrene) are distributed throughout a polystyrene backbone. Several polymer derivatives were prepared, each with a different 3,4-dihyroxystyrene content. Bulk adhesion testing showed where the optimal middle ground of cohesive and adhesive bonding resides. Adhesive performance was benchmarked against commercial glues as well as the genuine material produced by live mussels. In the best case, bonding was similar to that obtained with cyanoacrylate "Krazy Glue". Performance was also examined using low- (e.g., plastics) and high-energy (e.g., metals, wood) surfaces. The adhesive bonding of poly[(3,4-dihydroxystyrene)-co-styrene] may be the strongest of reported mussel protein mimics. These insights should help us to design future biomimetic systems, thereby bringing us closer to development of bone cements, dental composites, and surgical glues. PMID:22582754

  14. Molecular weight effects upon the adhesive bonding of a mussel mimetic polymer.

    Jenkins, Courtney L; Meredith, Heather J; Wilker, Jonathan J

    2013-06-12

    Characterization of marine biological adhesives are teaching us how nature makes materials and providing new ideas for synthetic systems. One of the most widely studied adhering animals is the marine mussel. This mollusk bonds to wet rocks by producing an adhesive from cross-linked proteins. Several laboratories are now making synthetic mimics of mussel adhesive proteins, with 3,4-dihydroxyphenylalanine (DOPA) or similar molecules pendant from polymer chains. In select cases, appreciable bulk bonding results, with strengths as high as commercial glues. Polymer molecular weight is amongst several parameters that need to be examined in order to both understand biomimetic adhesion as well as to maximize performance. Experiments presented here explore how the bulk adhesion of a mussel mimetic polymer varies as a function of molecular weight. Systematic structure-function studies were carried out both with and without the presence of an oxidative cross-linker. Without cross-linking, higher molecular weights generally afforded higher adhesion. When a [N(C4H9)4](IO4) cross-linker was added, adhesion peaked at molecular weights of ~50,000-65,000 g/mol. These data help to illustrate how changes to the balance of cohesion versus adhesion influence bulk bonding. Mussel adhesive plaques achieve this balance by incorporating several proteins with molecular weights ranging from 6000 to 110,000 g/mol. To mimic these varied proteins we made a blend of polymers containing a range of molecular weights. Interestingly, this blend adhered more strongly than any of the individual polymers when cross-linked with [N(C4H9)4](IO4). These results are helping us to both understand the origins of biological materials as well as design high performance polymers. PMID:23668520

  15. Dopamine signaling regulates the projection patterns in the mouse chiasm.

    Chen, Tingting; Hu, Yunlong; Lin, Xiaotan; Huang, Xinping; Liu, Bin; Leung, Peggy; Chan, Sun-On; Guo, Deyin; Jin, Guangyi

    2015-11-01

    Ocular albinism (OA) is characterized by inadequate L-3, 4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA) in the eyes. This study investigated DA-related signaling pathways in mouse chiasm projection patterns and the potential role of ocular albinism type 1 (OA1) and dopamine 1A (D1A) receptors in the optic pathway. In embryonic day (E) E13-E15 retina, most L-DOPA and OA1-positive cells were distributed among Müller glial cells on E13 and retinal ganglion cells (RGC) on E14. In the ventral diencephalon, OA1 and L-DOPA were strongly expressed on the optic chiasm (OC) and optic tract (OT), respectively, but weak on the optic stalk (OS). At E13-E15, DA and D1A staining was predominately expressed in radially arranged cells with a neuronal expression pattern. In the ventral diencephalon, DA and D1A were strongly expressed on the OC, OT and OS. Furthermore, L-DOPA significantly inhibited retinal axon outgrowth in both the dorsal nasal (DN) and ventral temporal (VT) groups. DA inhibited retinal axon outgrowth, which was abolished by the D1A antagonist SCH23390. Brain slice cultures indicated that L-DOPA inhibited axons that crossed at the OC of E13 embryos, which was not abolished by DA. L-DOPA also inhibited axons that crossed at the OC of albino mice. Albino mice exhibited reduced ipsilateral retinal projections compared with C57 pigmented mice. No significant difference was identified in the uncrossed projections of albino mice following L-DOPA and DA expression. Furthermore, transcription factor Zic family member 2 (Zic2) upregulated OA1 mRNA expression. Our findings provide critical insights into DA-related signaling in retinal development. PMID:26363092

  16. Chemoselective O-acylation of hydroxyamino acids and amino alcohols under acidic reaction conditions: History, scope and applications

    Tor E. Kristensen

    2015-04-01

    Full Text Available Amino acids, whether natural, semisynthetic or synthetic, are among the most important and useful chiral building blocks available for organic chemical synthesis. In principle, they can function as inexpensive, chiral and densely functionalized starting materials. On the other hand, the use of amino acid starting materials routinely necessitates protective group chemistry, and in reality, large-scale preparations of even the simplest side-chain derivatives of many amino acids often become annoyingly strenuous due to the necessity of employing protecting groups, on one or more of the amino acid functionalities, during the synthetic sequence. However, in the case of hydroxyamino acids such as hydroxyproline, serine, threonine, tyrosine and 3,4-dihydroxyphenylalanine (DOPA, many O-acyl side-chain derivatives are directly accessible via a particularly expedient and scalable method not commonly applied until recently. Direct acylation of unprotected hydroxyamino acids with acyl halides or carboxylic anhydrides under appropriately acidic reaction conditions renders possible chemoselective O-acylation, furnishing the corresponding side-chain esters directly, on multigram-scale, in a single step, and without chromatographic purification. Assuming a certain degree of stability under acidic reaction conditions, the method is also applicable for a number of related compounds, such as various amino alcohols and the thiol-functional amino acid cysteine. While the basic methodology underlying this approach has been known for decades, it has evolved through recent developments connected to amino acid-derived chiral organocatalysts to become a more widely recognized procedure for large-scale preparation of many useful side-chain derivatives of hydroxyamino acids and related compounds. Such derivatives are useful in peptide chemistry and drug development, as amino acid amphiphiles for asymmetric catalysis, and as amino acid acrylic precursors for preparation of

  17. Bioactivity of phytochemicals in some lesser-known plants and their effects and potential applications in livestock and aquaculture production systems.

    Makkar, H P S; Francis, G; Becker, K

    2007-10-01

    possible use as antibacterial, antioxidant, anticarcinogenic and antipest agents; (v) Jatropha curcas toxic variety with high levels of various phytochemicals such as trypsin inhibitor, lectin, phytate and phorbol esters in seeds limiting the use of seed meal in fish and livestock diets; and the use of phorbol esters as bio-pesticidal agent; and (vi) lesser-known legumes such as Entada phaseoloides seeds containing high levels of trypsin inhibitor and saponins, Sesbania aculeate seeds rich in non-starch polysaccharides and Mucuna pruriens var. utilis seeds rich in l-3,4-dihydroxyphenylalanine and their potential as fish feed; Cassia fistula seeds as a source of antioxidants; and the use of Canavalia ensiformis, C. gladiata and C. virosa seeds containing high levels of trypsin inhinitor, lectins and canavanine. The paper also presents some challenges and future areas of work in this field. PMID:22444893

  18. Preliminary Study on Purification and Identification of Aromatic Acid Amino L-Dopa From Malaysia Freshwater Green Mussel Byssus

    L-DOPA (L-3, 4-dihydroxyphenylalanine) is a type of aromatic amino acid which can be detected by using acidic extraction and purification method involving adhesive byssus green mussel protein. The main objective of this study is to identify and purify the aromatic amino acid L-DOPA via the utilization of gel Sephadex G-200 filtration chromatography based on two types of acidic and basic mobile phase solution. The crushing and homogenizing for adhesive byssus green mussel were conducted using a mortar and a pestle with the aid of liquid nitrogen. The samples that had been crushed were then mixed and dissolved in perchloric acid 0.7 %, 1.0 % and 1.5 % (v/ v) (pre-treatment) prior to the extraction process. The extraction was carried out by centrifuging the extracts at 11,000 rpm for about 10 mins and at a temperature of 10 degree Celsius to obtain supernatant S1. The supernatant was mixed with acetone and sulphuric acid and centrifuged for the second time to produce a pellet and then it was dissolved in the respective mobile phase solutions prior to purification process. Purification was later performed using two mobile phase solutions which were acetic acid 5 % (v/ v) and NaOH 1 M. The absorbance (abs) value of each purified protein extract fractions was collected and analysed at 214 nm to 400 nm with the help of UV-spectrophotometer. The highest abs value was selected for identification and verification of amino acid L-DOPA in the purified solution. Verification was carried out by utilizing high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). The results showed that the use of 0.7 % (v/ v) perchloric acid and 5 % (v/ v) acetic acid for pre-treatment process and mobile phase solution of purification process respectively, yielded the highest effluent abs profile at a wavelength of 260 nm. TLC analysis proved the existence of several important amino acids besides L-DOPA which were tyrosine and phenylalanine after 78 hrs of collection of

  19. Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders.

    dos Santos-Pinto, José Roberto Aparecido; Lamprecht, Günther; Chen, Wei-Qiang; Heo, Seok; Hardy, John George; Priewalder, Helga; Scheibel, Thomas Rainer; Palma, Mario Sergio; Lubec, Gert

    2014-06-13

    Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk

  20. {sup 18}F-Fluorodihydroxyphenylalanine vs other radiopharmaceuticals for imaging neuroendocrine tumours according to their type

    Balogova, Sona [Comenius University and St. Elisabeth Institute, Department of Nuclear Medicine, Bratislava (Slovakia); Hopital Tenon, AP-HP and Universite Pierre et Marie Curie, Department of Nuclear Medicine, Paris (France); Talbot, Jean-Noel; Michaud, Laure; Huchet, Virginie; Kerrou, Khaldoun; Montravers, Francoise [Hopital Tenon, AP-HP and Universite Pierre et Marie Curie, Department of Nuclear Medicine, Paris (France); Nataf, Valerie [Hopital Tenon, AP-HP, Department of Radiopharmacy, Paris (France)

    2013-06-15

    6-Fluoro-({sup 18}F)-L-3,4-dihydroxyphenylalanine (FDOPA) is an amino acid analogue for positron emission tomography (PET) imaging which has been registered since 2006 in several European Union (EU) countries and by several pharmaceutical firms. Neuroendocrine tumour (NET) imaging is part of its registered indications. NET functional imaging is a very competitive niche, competitors of FDOPA being two well-established radiopharmaceuticals for scintigraphy, {sup 123}I-metaiodobenzylguanidine (MIBG) and {sup 111}In-pentetreotide, and even more radiopharmaceuticals for PET, including fluorodeoxyglucose (FDG) and somatostatin analogues. Nevertheless, there is no universal single photon emission computed tomography (SPECT) or PET tracer for NET imaging, at least for the moment. FDOPA, as the other PET tracers, is superior in diagnostic performance in a limited number of precise NET types which are currently medullary thyroid cancer, catecholamine-producing tumours with a low aggressiveness and well-differentiated carcinoid tumours of the midgut, and in cases of congenital hyperinsulinism. This article reports on diagnostic performance and impact on management of FDOPA according to the NET type, emphasising the results of comparative studies with other radiopharmaceuticals. By pooling the results of the published studies with a defined standard of truth, patient-based sensitivity to detect recurrent medullary thyroid cancer was 70 % [95 % confidence interval (CI) 62.1-77.6] for FDOPA vs 44 % (95 % CI 35-53.4) for FDG; patient-based sensitivity to detect phaeochromocytoma/paraganglioma was 94 % (95 % CI 91.4-97.1) for FDOPA vs 69 % (95 % CI 60.2-77.1) for {sup 123}I-MIBG; and patient-based sensitivity to detect midgut NET was 89 % (95 % CI 80.3-95.3) for FDOPA vs 80 % (95 % CI 69.2-88.4) for somatostatin receptor scintigraphy with a larger gap in lesion-based sensitivity (97 vs 49 %). Previously unpublished FDOPA results from our team are reported in some rare NET, such as

  1. Chemoselective O-acylation of hydroxyamino acids and amino alcohols under acidic reaction conditions: History, scope and applications.

    Kristensen, Tor E

    2015-01-01

    Amino acids, whether natural, semisynthetic or synthetic, are among the most important and useful chiral building blocks available for organic chemical synthesis. In principle, they can function as inexpensive, chiral and densely functionalized starting materials. On the other hand, the use of amino acid starting materials routinely necessitates protective group chemistry, and in reality, large-scale preparations of even the simplest side-chain derivatives of many amino acids often become annoyingly strenuous due to the necessity of employing protecting groups, on one or more of the amino acid functionalities, during the synthetic sequence. However, in the case of hydroxyamino acids such as hydroxyproline, serine, threonine, tyrosine and 3,4-dihydroxyphenylalanine (DOPA), many O-acyl side-chain derivatives are directly accessible via a particularly expedient and scalable method not commonly applied until recently. Direct acylation of unprotected hydroxyamino acids with acyl halides or carboxylic anhydrides under appropriately acidic reaction conditions renders possible chemoselective O-acylation, furnishing the corresponding side-chain esters directly, on multigram-scale, in a single step, and without chromatographic purification. Assuming a certain degree of stability under acidic reaction conditions, the method is also applicable for a number of related compounds, such as various amino alcohols and the thiol-functional amino acid cysteine. While the basic methodology underlying this approach has been known for decades, it has evolved through recent developments connected to amino acid-derived chiral organocatalysts to become a more widely recognized procedure for large-scale preparation of many useful side-chain derivatives of hydroxyamino acids and related compounds. Such derivatives are useful in peptide chemistry and drug development, as amino acid amphiphiles for asymmetric catalysis, and as amino acid acrylic precursors for preparation of catalytically

  2. Processable Conducting Polyaniline, Carbon Nanotubes, Graphene and Their Composites

    Wang, Kan

    Good processability is often required for applications of conducting materials like polyaniline (PANI), carbon nanotubes (CNTs) and graphene. This can be achieved by either physical stabilization or chemical functionalization. Functionalization usually expands the possible applications for the conducting materials depending on the properties of the functional groups. Processable conducting materials can also be combined with other co-dissolving materials to prepare composites with desired chemical and physical properties. Polyanilines (PANI) doped with dodecylbenzenesulfonic acid (DBSA) are soluble in many organic solvents such as chloroform and toluene. Single wall carbon nanotubes (SWCNTs) can be dispersed into PANI/DBSA to form homogeneous solutions. PANI/DBSA functions as a conducting surfactant for SWCNTs. The mixture can be combined with two-parts polyurethanes that co-dissolve in the organic solvent to produce conducting polymer composites. The composite mixtures can be applied onto various substrates by simple spray-on methods to obtain transparent and conducting coatings. Graphene, a single layer of graphite, has drawn intense interest for its unique properties. Processable graphene has been produced in N-methyl-2-pyrrolidone (NMP) by a one-step solvothermal reduction of graphite oxide without the aid of any reducing reagent and/or surfactant. The as-synthesized graphene disperses well in a variety of organic solvents such as dimethylsulfoxide (DMSO), ethanol and tetrahydrogenfuran (THF). The conductivity of solvothermal reduced graphite oxide is comparable to hydrazine reduced graphite oxide. Attempts were made to create intrinsically conducting glue comparable to mussel adhesive protiens using polyaniline and graphene. Mussels can attach to a variety of substrates under water. Catechol residue in 3,4-dihydroxyphenylalanine (L-DOPA) is the key to the wet adhesion. Tyrosine and phosphoserine with primary alkyl amine groups also participate in adhesion. A

  3. A Multi-tracer Dopaminergic PET Study of Young-Onset Parkinsonian Patients With and Without Parkin Gene Mutations

    The impact of parkin gene mutations on nigrostriatal dopaminergic degeneration is not well established. The purpose of this study was to characterize by PET using 18F-fluoro-L-3, 4- dihydroxyphenylalanine (18F-fluoro-L-DOPA), 11C-PE2I, and 11C-raclopride the pattern of dopaminergic lesions in young-onset Parkinson disease (YOPD) patients with or without mutations of the parkin gene and to correlate the clinical and neuro-psychologic characteristics of these patients with PET results. Methods: A total of 35 YOPD patients were enrolled (16 with parkin mutation, 19 without). The uptake constant (Ki) of 18F-fluoro- L-DOPA and the binding potential (BP) of 11C-PE2I (BPDAT) and of 11C-raclopride (BPD2) were calculated in the striatum. Comparisons were made between the 2 groups of YOPD and between controls and patients. For each radiotracer, parametric images were obtained, and statistical parametric mapping (SPM) analysis using a voxel-by-voxel statistical t test was performed. Correlations between the cognitive and motor status and PET results were analyzed. Results: In YOPD patients, 18F-fluoro-L-DOPA Ki values were reduced to 68% (caudate) and 40% (putamen) of normal values (P ≤ 0.0001). This decrease was symmetric and comparable for non-parkin and parkin patients. No correlation was found between the Ki values and cognitive or motor status. 11C-PE2I BPDAT values in YOPD patients were decreased to 56% (caudate) and 41% (putamen) of normal values (P ≤ 0.0001) and did not differ between the 2 YOPD populations. The mean 11C-raclopride BPD2 values were reduced to 72% (caudate) and 84% (putamen) of the normal values (P ≤ 0.02) and did not differ between non-parkin and parkin patients. SPM analyses showed in patients an additional decrease of 11C-raclopride in the frontal cortex and a decrease of 18F-fluoro-L-DOPA and 11C-PE2I uptake in the substantia nigra bilaterally (P ≤ 0.05, false-discovery rate-corrected). Conclusion: Carriers of parkin mutations are

  4. Enzymic sulphation of dopa and tyrosine isomers by HepG2 human hepatoma cells: stereoselectivity and stimulation by Mn2+.

    Suiko, M; Sakakibara, Y; Nakajima, H; Sakaida, H; Liu, M C

    1996-02-15

    HepG2 human hepatoma cells, labelled with [35S]sulphate in media containing L-3,4-dihydroxyphenylalanine (L-dopa), (D-dopa), DL-m-tyrosine or D-p-tyrosine, were found to produce the [35S]sulphated forms of these compounds. Addition to the labelling media of m-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor, greatly enhanced the production of L-dopa O-[35S]sulphate and DL-m-tyrosine O-[35S]sulphate, with a concomitant decrease in the formation of dopamine O-[35S]sulphate and m-tyramine O-[35S]sulphate. With 3'-phosphoadenosine 5'-phospho[35S]sulphate as the sulphate donor., HepG2-cell cytosol was shown to contain enzymic activity catalysing the sulphation of L-dopa, D-dopa, L-m-tyrosine, D-m-tyrosine, L-p-tyrosine and D-p-tyrosine. The pH optimum of the enzyme, designated dopa/tyrosine sulphotransferase, was determined to be 8.75 with D-m-tyrosine as the substrate. The enzyme exhibited stereoselectivity for the D-form of dopa or tyrosine isomers. Addition of 10mM MnCl2 to the reaction mixture resulted in a remarkable stimulation of dopa/tyrosine sulphotransferase activity, being as high as 267.8 times with D-p-tyrosine as the substrate. Quantitative assays revealed L-dopa, D-dopa and D-m-tyrosine to be better substrates than L-p-tyrosine. When the HepG2-cell cytosol was subjected to DEAE Bio-Gel and hydroxyapatite column chromatography, dopa/tyrosine sulphotransferase was co-eluted with the thermolabile 'M-form' phenol sulphotransferase. Furthermore dopa/tyrosine sulphotransferase displayed properties similar to that of the M-form phenol sulphotransferase with respect to thermostability and sensitivity to 2,6-dichloro-4-nitrophenol. Whether the M-form phenol sulphotransferase is truly (solely) responsible for the dopa/tyrosine sulphotransferase activity present in HepG2 cells remains to be clarified. PMID:8660277

  5. Enhanced adhesion of dopamine methacrylamide elastomers via viscoelasticity tuning.

    Chung, Hoyong; Glass, Paul; Pothen, Jewel M; Sitti, Metin; Washburn, Newell R

    2011-02-14

    We present a study on the effects of cross-linking on the adhesive properties of bio-inspired 3,4-dihydroxyphenylalanine (DOPA). DOPA has a unique catechol moiety found in adhesive proteins in marine organisms, such as mussels and polychaete, which results in strong adhesion in aquatic conditions. Incorporation of this functional group in synthetic polymers provides the basis for pressure-sensitive adhesives for use in a broad range of environments. A series of cross-linked DOPA-containing polymers were prepared by adding divinyl cross-linking agent ethylene glycol dimethacrylate (EGDMA) to monomer mixtures of dopamine methacrylamide (DMA) and 2-methoxyethyl acrylate (MEA). Samples were prepared using a solvent-free microwave-assisted polymerization reaction and compared to a similar series of cross-linked MEA materials. Cross-linking with EGDMA tunes the viscoelastic properties of the adhesive material and has the advantage of not reacting with the catechol group that is responsible for the excellent adhesive performance of this material. Adhesion strength was measured by uniaxial indentation tests, which indicated that 0.001 mol % of EGDMA-cross-linked copolymer showed the highest work of adhesion in dry conditions, but non-cross-linked DMA was the highest in wet conditions. The results suggest that there is an optimal cross-linking degree that displays the highest adhesion by balancing viscous and elastic behaviors of the polymer but this appears to depend on the conditions. This concentration of cross-linker is well below the theoretical percolation threshold, and we propose that subtle changes in polymer viscoelastic properties can result in significant improvements in adhesion of DOPA-based materials. The properties of lightly cross-linked poly(DMA-co-MEA) were investigated by measurement of the frequency dependence of the storage modulus (G') and loss modulus (G''). The frequency-dependence of G' and magnitude of G'' showed gradual decreases with the

  6. Volumetric assessment of recurrent or progressive gliomas: comparison between F-DOPA PET and perfusion-weighted MRI

    Cicone, Francesco [Sant' Andrea Hospital, Rome (Italy). Unit of Nuclear Medicine; Rome Sapienza Univ. (Italy). Dept. of Surgical and Medical Sciences and tranlational Medicine; Research Centre Juelich (Germany). Inst. of Neureoscience and Medicine; Filss, Christian P.; Langen, Karl-Josef [Research Centre Juelich (Germany). Inst. of Neureoscience and Medicine; RWTH Aachen Univ. Hospital (Germany). Dept. of Nuclear Medicine; Minniti, Giuseppe; Scaringi, Claudia [Rome Sapienza Univ. (Italy). Dept. of Surgical and Medical Sciences and tranlational Medicine; Sant' Andrea Hospital, Rome (Italy). Unit of Radiotherapy; Rossi-Espagnet, Camilla; Bozzao, Alessandro [Sant' Andrea Hospital, Rome (Italy). Unit of Neuroradiology; Rome Sapienza Univ. (Italy). Dept. of Neurosciences, Mental Health and Sensory Organs (Ne.S.M.O.S.); Papa, Annalisa; Scopinaro, Francesco [Sant' Andrea Hospital, Rome (Italy). Unit of Nuclear Medicine; Rome Sapienza Univ. (Italy). Dept. of Surgical and Medical Sciences and tranlational Medicine; Galldiks, Norbert [Research Centre Juelich (Germany). Inst. of Neureoscience and Medicine; Cologne Univ. (Germany). Dept. of Neurology; Shah, N. Jon [Research Centre Juelich (Germany). Inst. of Neureoscience and Medicine

    2015-05-01

    To compare the diagnostic information obtained with 6-[{sup 18}F]-fluoro-l-3,4-dihydroxyphenylalanine (F-DOPA) PET and relative cerebral blood volume (rCBV) maps in recurrent or progressive glioma. All patients with recurrent or progressive glioma referred for F-DOPA imaging at our institution between May 2010 and May 2014 were retrospectively included, provided that macroscopic disease was visible on conventional MRI images and that rCBV maps were available for comparison. The final analysis included 50 paired studies (44 patients). After image registration, automatic tumour segmentation of both sets of images was performed using the average signal in a large reference VOI including grey and white matter multiplied by 1.6. Tumour volumes identified by both modalities were compared and their spatial congruence calculated. The distances between F-DOPA uptake and rCBV hot spots, tumour-to-brain ratios (TBRs) and normalized histograms were also computed. On visual inspection, 49 of the 50 F-DOPA and 45 of the 50 rCBV studies were classified as positive. The tumour volume delineated using F-DOPA (F-DOPA{sub vol} {sub 1.6}) greatly exceeded that of rCBV maps (rCBV{sub vol} {sub 1.6}). The median F-DOPA{sub vol} {sub 1.6} and rCBV{sub vol} {sub 1.6} were 11.44 ml (range 0 - 220.95 ml) and 1.04 ml (range 0 - 26.30 ml), respectively (p < 0.00001). Overall, the median overlapping volume was 0.27 ml, resulting in a spatial congruence of 1.38 % (range 0 - 39.22 %). The mean hot spot distance was 27.17 mm (±16.92 mm). F-DOPA uptake TBR was significantly higher than rCBV TBR (1.76 ± 0.60 vs. 1.15 ± 0.52, respectively; p < 0.0001). The histogram analysis showed that F-DOPA provided better separation of tumour from background. In 6 of the 50 studies (12 %), however, physiological uptake in the striatum interfered with tumour delineation. The information provided by F-DOPA PET and rCBV maps are substantially different. Image interpretation is easier and a larger tumour extent

  7. Accuracy of F-DOPA PET and perfusion-MRI for differentiating radionecrotic from progressive brain metastases after radiosurgery

    Cicone, Francesco; Papa, Annalisa; Scopinaro, Francesco [Sant' Andrea Hospital, Rome (Italy). Unit of Nuclear Medicine; ' ' Sapienza' ' Univ., Rome (Italy). Dept. of Surgical and Medicine Sciences and Translational Medicine; Minniti, Giuseppe; Scaringi, Claudia; Maurizi Enrici, Riccardo [' ' Sapienza' ' Univ., Rome (Italy). Dept. of Surgical and Medicine Sciences and Translational Medicine; Sant' Andrea Hospital, Rome (Italy). Unit of Radiotherapy; Romano, Andrea; Tavanti, Francesca; Bozzao, Alessandro [Sant' Andrea Hospital, Rome (Italy). Unit of Neuroradiology; Rome Univ. (Italy). Dept. of Neurosciences, Mental Health and Sensory Organs (Ne.S.M.O.S.)

    2015-01-15

    We assessed the performance of 6-[{sup 18}F]-fluoro-l-3,4-dihydroxyphenylalanine (F-DOPA) PET for differentiating radionecrosis (RN) from tumour progression (PD) in a population of patients with brain metastases, treated with stereotactic radiosurgery. The accuracy of F-DOPA PET was compared with that of perfusion-weighted magnetic resonance (perfusion-MR). In 42 patients with a total of 50 brain metastases from various primaries F-DOPA PET/CT was performed because of suspected radiological progression at the site of previously irradiated brain metastasis. Several semiquantitative PET parameters were recorded, and their diagnostic accuracy was compared by receiver operating characteristic curve analyses. The diagnosis was established by either surgery or follow-up. A comparison was made between F-DOPA PET and perfusion-MR sequences acquired no more than 3 weeks apart. Definitive outcome was available in 46 of the 50 lesions (20 PD, 26 RN). Of the 46 lesions, 11 were surgically excised while in the remaining 35 lesions the diagnosis was established by radiological and clinical criteria. The best diagnostic performance was obtained using the semiquantitative PET parameter maximum lesion to maximum background uptake ratio (SUVL{sub max}/Bkgr{sub max}). With a cut-off value of 1.59, a sensitivity of 90 % and a specificity of 92.3 % were achieved in differentiating RN from PD lesions (accuracy 91.3 %). Relative cerebral blood volume (rCBV) derived from perfusion-MR was available for comparison in 37 of the 46 metastases. Overall accuracy of rCBV was lower than that of all semiquantitative PET parameters under study. The best differentiating rCBV cut-off value was 2.14; this yielded a sensitivity of 86.7 % and a specificity of 68.2 % (accuracy 75.6 %). F-DOPA PET is a highly accurate tool for differentiating RN from PD brain metastases after stereotactic radiosurgery. In this specific setting, F-DOPA PET seems to perform better than perfusion-MR. (orig.)

  8. Extração e dosagem da atividade da polifenoloxidase do café Extraction and activity determination of polyphenoloxidase in coffee

    Paulo Mazzafera

    2002-12-01

    significant interference of phenols present in the extracts. Consequentely, the data reported in the literature are not reproducible. PFO activity differentiated Soft coffee from Hard and Rio, but not between the lost two. Soft coffee presented higher PFO activity. For an accurate activity determination, antioxidants and phenol complexation is essential during extraction, as well as their elimination by exclusion chromatography. However, using this procedure and O2 consumption, PFO activity could still not differentiate the three coffee qualities, except Soft from the other two. Instead of 3,4 - dihydroxyphenylalanine, it is suggested that chlorogenic acid (5-caffeoylquinic acid should be used as substrate.

  9. Subthalamic Nucleus Deep Brain Stimulation Modulate Catecholamine Levels with Significant Relations to Clinical Outcome after Surgery in Patients with Parkinson’s Disease

    Yamamoto, Tatsuya; Uchiyama, Tomoyuki; Higuchi, Yoshinori; Asahina, Masato; Hirano, Shigeki; Yamanaka, Yoshitaka; Kuwabara, Satoshi

    2015-01-01

    Aims Although subthalamic nucleus deep brain stimulation (STN-DBS) is effective in patients with advanced Parkinson’s disease (PD), its physiological mechanisms remain unclear. Because STN-DBS is effective in patients with PD whose motor symptoms are dramatically alleviated by L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, the higher preoperative catecholamine levels might be related to the better clinical outcome after surgery. We aimed to examine the correlation between the preoperative catecholamine levels and postoperative clinical outcome after subthalamic nucleus deep brain stimulation. The effectiveness of STN-DBS in the patient who responded well to dopaminergic medication suggest the causal link between the dopaminergic system and STN-DBS. We also examined how catecholamine levels were modulated after subthalamic stimulation. Methods In total 25 patients with PD were enrolled (Mean age 66.2 ± 6.7 years, mean disease duration 11.6 ± 3.7 years). Mean levodopa equivalent doses were 1032 ± 34.6 mg before surgery. Cerebrospinal fluid and plasma catecholamine levels were measured an hour after oral administration of antiparkinsonian drugs before surgery. The mean Unified Parkinson’s Disease Rating Scale scores (UPDRS) and the Parkinson’s disease Questionnaire-39 (PDQ-39) were obtained before and after surgery. Of the 25 patients, postoperative cerebrospinal fluid and plasma were collected an hour after oral administration of antiparkinsonian drugs during on stimulation at follow up in 11 patients. Results Mean levodopa equivalent doses significantly decreased after surgery with improvement in motor functions and quality of life. The preoperative catecholamine levels had basically negative correlations with postoperative motor scores and quality of life, suggesting that higher preoperative catecholamine levels were related to better outcome after STN-DBS. The preoperative plasma levels of L-DOPA had significantly negative correlations with

  10. Depressive-like behavior observed with a minimal loss of locus coeruleus (LC) neurons following administration of 6-hydroxydopamine is associated with electrophysiological changes and reversed with precursors of norepinephrine.

    Szot, Patricia; Franklin, Allyn; Miguelez, Cristina; Wang, Yangqing; Vidaurrazaga, Igor; Ugedo, Luisa; Sikkema, Carl; Wilkinson, Charles W; Raskind, Murray A

    2016-02-01

    Depression is a common co-morbid condition most often observed in subjects with mild cognitive impairment (MCI) and during the early stages of Alzheimer's disease (AD). Dysfunction of the central noradrenergic nervous system is an important component in depression. In AD, locus coeruleus (LC) noradrenergic neurons are significantly reduced pathologically and the reduction of LC neurons is hypothesized to begin very early in the progression of the disorder; however, it is not known if dysfunction of the noradrenergic system due to early LC neuronal loss is involved in mediating depression in early AD. Therefore, the purpose of this study was to determine in an animal model if a loss of noradrenergic LC neurons results in depressive-like behavior. The LC noradrenergic neuronal population was reduced by the bilateral administration of the neurotoxin 6-hydroxydopamine (6-OHDA) directly into the LC. Forced swim test (FST) was performed three weeks after the administration of 6-OHDA (5, 10 and 14 μg/μl), animals administered the 5 μg/μl of 6-OHDA demonstrated a significant increase in immobility, indicating depressive-like behavior. This increase in immobility at the 5 μg/μl dose was observed with a minimal loss of LC noradrenergic neurons as compared to LC neuronal loss observed at 10 and 14 μg/μl dose. A significant positive correlation between the number of surviving LC neurons after 6-OHDA and FST immobile time was observed, suggesting that in animals with a minimal loss of LC neurons (or a greater number of surviving LC neurons) following 6-OHDA demonstrated depressive-like behavior. As the 6-OHDA-induced loss of LC neurons is increased, the time spent immobile is reduced. Depressive-like behavior was also observed with the 5 μg/μl dose of 6-OHDA with a second behavior test, sucrose consumption. FST increased immobility following 6-OHDA (5 μg/μl) was reversed by the administration of a single dose of L-1-3-4-dihydroxyphenylalanine (DOPA) or l-threo-3

  11. On the synthesis of radiofluorinated amino acids by isotope exchange based on the example of 6-[{sup 18}F]Fluor-L-DOPA; Zur Synthese radiofluorierter aromatischer Aminosaeuren mittels Isotopenaustausch am Beispiel von 6-[{sup 18}F]Fluor-L-DOPA

    Wagner, F.M.

    2008-06-15

    In nuclear medical diagnosis, 6-[{sup 18}F]fluoro-L-3,4-dihydroxyphenylalanine (6-[{sup 18}F]fluoro-LDOPA), an analogue of L-DOPA, is one of the few established radiopharmaceuticals used for the in vivo investigation of the presynaptic dopaminergic metabolism and of some kind of tumours via Positron Emission Tomography (PET). The presently used method of preparation of the radiotracer by electrophilic labelling is limited to low amounts of activity at high costs. Known nucleophilic syntheses, however, result either in insufficient enantiomeric purity or the known multi-step syntheses are hard to automate, due to their complexity. During this work a novel, easy to automate alternative for the preparation of 6-[{sup 18}F]fluoro-L-DOPA, was developed and evaluated, using a direct nucleophilic {sup 18}F-fluorination of a protected amino acid derivative. The resulting product has a very high enantiomeric purity. At first, the general suitability of the (S)-BOC-BMI-derivatives for the synthesis of {sup 18}F-labelled amino acids, used in this work, was investigated using a less complex precursor, which resulted in the amino acid 6-[{sup 18}F]fluoro-L-m-tyrosin via acidic hydrolysis. The preparation of a useful precursor for the nucleophilic {sup 18}F-isotope substitution, namely the (2S,5S)-tert.-butyl- 5-(2-fluoro-5-formylbenzyl)-2-tert.-butyl-3-methyl-4-oxoimidazolidine-1-carboxylate, was investigated in three general different ways. At first it was tried to obtain this product via formylation after coupling with the BOC-BMI, secondly via {alpha},{beta}-dehydro amino acid derivatives and finally via a systematic multi-step synthesis. Only the last mentioned way resulted in a precursor with sufficient purity that could be labelled. The radiochemical yield of the isotopic exchange was about 60 %. In the next step, the presented concept was modified to synthesize a precursor for the preparation of 6-[{sup 18}F]fluoro-L-DOPA. Only a combination of the protecting groups

  12. Beta-scission of alkoxyl radicals on peptides and proteins can give rise to backbone cleavage and loss of side-chains

    Full text: Exposure of proteins to radicals in the presence of O2 brings about multiple changes including side-chain oxidation, backbone fragmentation, cross-linking, unfolding, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes and formation of new reactive groups (e.g. hydroperoxides and 3,4-dihydroxyphenylalanine). All of these processes can result in loss of structural or enzymatic activity. The mechanisms that give rise to backbone cleavage are only partly understood. Whilst it is known that direct hydrogen atom abstraction at a-carbon sites gives backbone cleavages it has also been proposed that initial attack at side-chain sites might also give rise to backbone cleavage. In this study we have examined whether initial attack at the β- (C-3) position can give rise to α-carbon radicals (and hence backbone cleavage) via the formation, and subsequent β- scission, of C-3 alkoxyl radicals. This process has been observed previously with protected amino acids in organic solvents (J. Chem. Soc. Perkin Trans. 2, 1997, 503-507) but the occurrence of such reactions with proteins in aqueous solution has not been explored. Alkoxyl radicals were generated at the C-3 position of a variety of protected amino acids and small peptides by two methods: metal-ion catalysed decomposition of hydroperoxides formed as a result of γ-radiolysis in the presence of O2, and UV photolysis of nitrate esters. In most cases radicals have been detected by EPR spectroscopy using nitroso and nitrone spin traps, which can be assigned by comparison with literature data to α-carbon radicals; in some case assignments were confirmed by the generation of the putative species by other routes. With Ala peptide hydroperoxides and nitrate esters, and MNP as the spin trap, the major radical detected in each case has been assigned to the adduct of an α-carbon radical with partial structure - NH-.CH-C(O) - consistent with the rapid occurrence of the above reaction

  13. On the synthesis of radiofluorinated amino acids by isotope exchange based on the example of 6-[18F]Fluor-L-DOPA

    In nuclear medical diagnosis, 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-[18F]fluoro-LDOPA), an analogue of L-DOPA, is one of the few established radiopharmaceuticals used for the in vivo investigation of the presynaptic dopaminergic metabolism and of some kind of tumours via Positron Emission Tomography (PET). The presently used method of preparation of the radiotracer by electrophilic labelling is limited to low amounts of activity at high costs. Known nucleophilic syntheses, however, result either in insufficient enantiomeric purity or the known multi-step syntheses are hard to automate, due to their complexity. During this work a novel, easy to automate alternative for the preparation of 6-[18F]fluoro-L-DOPA, was developed and evaluated, using a direct nucleophilic 18F-fluorination of a protected amino acid derivative. The resulting product has a very high enantiomeric purity. At first, the general suitability of the (S)-BOC-BMI-derivatives for the synthesis of 18F-labelled amino acids, used in this work, was investigated using a less complex precursor, which resulted in the amino acid 6-[18F]fluoro-L-m-tyrosin via acidic hydrolysis. The preparation of a useful precursor for the nucleophilic 18F-isotope substitution, namely the (2S,5S)-tert.-butyl- 5-(2-fluoro-5-formylbenzyl)-2-tert. -butyl-3-methyl-4-oxoimidazolidine-1-carbox= yl ate, was investigated in three general different ways. At first it was tried to obtain this product via formylation after coupling with the BOC-BMI, secondly via α,β-dehydro amino acid derivatives and finally via a systematic multi-step synthesis. Only the last mentioned way resulted in a precursor with sufficient purity that could be labelled. The radiochemical yield of the isotopic exchange was about 60 %. In the next step, the presented concept was modified to synthesize a precursor for the preparation of 6-[18F]fluoro-L-DOPA. Only a combination of the protecting groups benzyl and THP resulted in the useful precursor (2S,5S