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The International Journal of... 2010The iodotyrosine dehalogenase1 (DEHAL1) enzyme is a transmembrane protein that belongs to the nitroreductase family and shows a highly conserved N-terminal domain....
The iodotyrosine dehalogenase1 (DEHAL1) enzyme is a transmembrane protein that belongs to the nitroreductase family and shows a highly conserved N-terminal domain. DEHAL1 is present in the liver, kidney and thyroid of mammals. DEHAL1 is known to act on diiodotyrosine (DIT) and monoiodotyrosine (MIT), and is involved in iodine recycling in relation to thyroglobulin. Here, we show the distribution of DEHAL1 during gastrulation to neurulation in developing chick. Immunocytochemistry using an anti-serum directed against the N-terminal domain (met(27)-trp(180) fragment) of human DEHAL1 revealed labelled cells in the embryonic ectoderm, embryonic endoderm, neural plate and in the yolk platelets of the chick embryo at gastrulation stage. Distinct DEHAL1 positive cells were located in the presumptive head ectoderm, presumptive neural crest, head mesenchymal cells and in the dorsal, lateral and ventral parts of neural tube during neurulation. Some cells located at the margin of the developing notochord and somites were also DEHAL1-positive. While the functional significance of this observation is not known, it is likely that DEHAL1 might serve as an agent that regulates cell specific deiodination of MIT and DIT before the onset of thyroidal secretion. The presence of DEHAL1 in different components of the chick embryo suggests its involvement in iodine turnover prior to the formation of functional thyroid.
Topics: Animals; Body Patterning; Chick Embryo; Diiodotyrosine; Ectoderm; Endoderm; Gastrulation; Gene Expression Regulation, Developmental; Hydrolases; Immunoblotting; Iodine; Membrane Proteins; Mesoderm; Monoiodotyrosine; Neural Crest; Neural Plate; Neural Tube; Neurulation; Thyroid Gland; Yolk Sac
PubMed: 21302259
DOI: 10.1387/ijdb.092932am -
PloS One 2015Considerable evidence indicates that sleep is essential for learning and memory. Drosophila melanogaster has emerged as a novel model for studying sleep. We previously...
Considerable evidence indicates that sleep is essential for learning and memory. Drosophila melanogaster has emerged as a novel model for studying sleep. We previously found a short sleeper mutant, fumin (fmn), and identified its mutation in the dopamine transporter gene. We reported similarities in the molecular basis of sleep and arousal regulation between mammals and Drosophila. In aversive olfactory learning tasks, fmn mutants demonstrate defective memory retention, which suggests an association between sleep and memory. In an attempt to discover additional sleep related genes in Drosophila, we carried out a microarray analysis comparing mRNA expression in heads of fmn and control flies and found that 563 genes are differentially expressed. Next, using the pan-neuronal Gal4 driver elav-Gal4 and UAS-RNA interference (RNAi) to knockdown individual genes, we performed a functional screen. We found that knockdown of the NMDA type glutamate receptor channel gene (Nmdar1) (also known as dNR1) reduced sleep. The NMDA receptor (NMDAR) plays an important role in learning and memory both in Drosophila and mammals. The application of the NMDAR antagonist, MK-801, reduced sleep in control flies, but not in fmn. These results suggest that NMDAR promotes sleep regulation in Drosophila.
Topics: Animals; Animals, Genetically Modified; Brain; Dizocilpine Maleate; Drosophila Proteins; Drosophila melanogaster; Gene Expression Regulation; Gene Knockdown Techniques; Monoiodotyrosine; Mutation; Oligonucleotide Array Sequence Analysis; Receptors, N-Methyl-D-Aspartate; Sleep
PubMed: 26023770
DOI: 10.1371/journal.pone.0128101 -
International Journal of Molecular... Dec 2018The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity....
The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNA. First, we constructed a naïve hybrid between archaeal tRNA and tRNA, which consisted of the acceptor and T stems of tRNA and the other parts of tRNA. This hybrid tRNA efficiently translated the UAG codon to 3-iodotyrosine in cells, when paired with a variant of the archaeal tyrosyl-tRNA synthetase. The amber suppression efficiency was slightly lower than that of the "bench-mark" archaeal tRNA suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNA: ZtRNA had an augmented D (auD) helix in a noncanonical form and the D and T loops bound by the standard tertiary base pairs, and YtRNA had a canonical auD helix and non-standard interloop interactions. It was then suggested that the ZtRNA scaffold could also support the glycylation and glutaminylation of tRNA. The synthetic diversity of tRNA would help create new tRNA⁻aminoacyl-tRNA synthetase pairs for reprogramming the genetic code.
Topics: Base Sequence; Codon, Terminator; Escherichia coli; Methanosarcina; Monoiodotyrosine; Nucleic Acid Conformation; RNA, Transfer
PubMed: 30587834
DOI: 10.3390/ijms20010092 -
The Journal of Biological Chemistry Jan 1984The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free...
The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.
Topics: Animals; Glutathione; Hydrogen Peroxide; Iodide Peroxidase; Iodine; Kinetics; Oxidation-Reduction; Peroxidases; Spectrophotometry, Ultraviolet; Swine; Thyroglobulin; Tyrosine
PubMed: 6706940
DOI: No ID Found -
International Journal of Molecular... Jan 2019Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein...
Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various strains genomically engineered for specific requirements.
Topics: Codon, Terminator; Escherichia coli; Escherichia coli Proteins; Monoiodotyrosine; Peptide Termination Factors; Protein Biosynthesis; RNA, Transfer; Subcellular Fractions
PubMed: 30678326
DOI: 10.3390/ijms20030492 -
The Journal of Biological Chemistry Mar 2018Thyroglobulin (TG) is the most abundant thyroid gland protein, a dimeric iodoglycoprotein (660 kDa). TG serves as the protein precursor in the synthesis of thyroid...
Thyroglobulin (TG) is the most abundant thyroid gland protein, a dimeric iodoglycoprotein (660 kDa). TG serves as the protein precursor in the synthesis of thyroid hormones tetraiodothyronine (T) and triiodothyronine (T). The primary site for T synthesis in TG involves an iodotyrosine acceptor at the antepenultimate Tyr residue (at the extreme carboxyl terminus of the protein). The carboxyl-terminal region of TG comprises a olinsterase-ike (ChEL) domain followed by a short unique tail sequence. Despite many studies, the monoiodotyrosine donor residue needed for the coupling reaction to create T at this evolutionarily conserved site remains unidentified. In this report, we have utilized a novel, convenient immunoblotting assay to detect T formation after protein iodination , enabling the study of T formation in recombinant TG secreted from thyrocytes or heterologous cells. With this assay, we confirm the antepenultimate residue of TG as a major T-forming site, but also demonstrate that the side chain of this residue intimately interacts with the same residue in the apposed monomer of the TG dimer. T formation in TG, or the isolated carboxyl-terminal region, is inhibited by mutation of this antepenultimate residue, but we describe the first substitution mutation that actually increases T hormonogenesis by engineering a novel cysteine, 10 residues upstream of the antepenultimate residue, allowing for covalent association of the unique tail sequences, and that helps to bring residues Tyr from apposed monomers into closer proximity.
Topics: Animals; Cattle; Halogenation; Mice; Protein Domains; Protein Multimerization; Thyroglobulin; Triiodothyronine
PubMed: 29440273
DOI: 10.1074/jbc.RA118.001786 -
Journal of Neurochemistry Dec 2011The highly reactive nature of dopamine renders dopaminergic neurons vulnerable to oxidative damage. We recently demonstrated that loss-of-function mutations in the...
The highly reactive nature of dopamine renders dopaminergic neurons vulnerable to oxidative damage. We recently demonstrated that loss-of-function mutations in the Drosophila gene Catecholamines up (Catsup) elevate dopamine pools but, paradoxically, also confer resistance to paraquat, an herbicide that induces oxidative stress-mediated toxicity in dopaminergic neurons. We now report a novel association of the membrane protein, Catsup, with GTP cyclohydrolase rate-limiting enzyme for tetrahydrobiopterin (BH(4)) biosynthesis and tyrosine hydroxylase, rate-limiting enzyme for dopamine biosynthesis, which requires BH(4) as a cofactor. Loss-of-function Catsup mutations cause dominant hyperactivation of both enzymes. Elevated dopamine levels in Catsup mutants coincide with several distinct characteristics, including hypermobility, minimal basal levels of 3,4-dihydroxy-phenylacetic acid, an oxidative metabolite of dopamine, and resistance to the vesicular monoamine transporter inhibitor, reserpine, suggesting that excess dopamine is synaptically active and that Catsup functions in the regulation of synaptic vesicle loading and release of dopamine. We conclude that Catsup regulates and links the dopamine synthesis and transport networks.
Topics: Administration, Oral; Animals; Animals, Genetically Modified; Antipsychotic Agents; Dopamine; Dopamine Agents; Dopaminergic Neurons; Drosophila; Drosophila Proteins; Electrochemistry; Female; GTP Cyclohydrolase; Green Fluorescent Proteins; Herbicides; Immunoprecipitation; Levodopa; Locomotion; Male; Monoiodotyrosine; Paraquat; Protein Transport; Reserpine; Synapses; Tyrosine 3-Monooxygenase; Vesicular Monoamine Transport Proteins
PubMed: 21985068
DOI: 10.1111/j.1471-4159.2011.07517.x -
Analytical Chemistry Sep 2018The fruit fly, Drosophila melanogaster, is a popular model organism for studying neurological processes and diseases due to the availability of sophisticated genetic...
The fruit fly, Drosophila melanogaster, is a popular model organism for studying neurological processes and diseases due to the availability of sophisticated genetic tools. While endogenous neurotransmitter release has been characterized in Drosophila larvae, here, we measured endogenous dopamine release in isolated adult Drosophila brains for the first time. Dopamine was measured with fast-scan cyclic voltammetry (FSCV), and acetylcholine or nicotine were used as the stimulus, as both interact with nicotinic acetylcholine receptors (nAChRs) to evoke endogenous dopamine release. Stimulations with 10 pmol of acetylcholine elicited 0.26 ± 0.05 μM dopamine, while 70 fmol nicotine stimulations evoked 0.29 ± 0.03 μM in the central complex. Nicotine-stimulated dopamine release lasted much longer than acetylcholine-stimulated release. Dopamine release is reduced in the presence of nAChR antagonist α-bungarotoxin and the sodium channel blocker tetrodotoxin, indicating release is mediated by nAChRs and exocytosis. The identity of dopamine was confirmed by using 3-iodotyrosine, a dopamine synthesis inhibitor, and by confirming that release was not changed in octopamine synthesis mutant flies, Tdc2 . Additionally, the half-decay time ( t) in fumin (67 ± 15 s), dopamine transporter mutant flies, was larger than in wild-type flies (16 ± 3.7 s) further proving that acetylcholine stimulation evokes dopamine release. This study demonstrates that stimulation of nAChRs can be used to elicit endogenous dopamine release in adult fly brains, which will be a useful technique for future studies probing dopamine changes during aging or in neurodegenerative diseases.
Topics: Acetylcholine; Animals; Brain; Bungarotoxins; Dopamine; Dopamine Antagonists; Drosophila melanogaster; Electrochemical Techniques; Exocytosis; Monoiodotyrosine; Nicotine; Nicotinic Antagonists; Octopamine; Receptors, Nicotinic; Reproducibility of Results; Sodium Channel Blockers; Tetrodotoxin
PubMed: 30073836
DOI: 10.1021/acs.analchem.8b02114 -
Developmental Biology Jun 1996Manipulation of dopamine levels by inhibition of tyrosine hydroxylase activity was accomplished in Drosophila melanogaster larval instars by feeding enzyme inhibitors...
Manipulation of dopamine levels by inhibition of tyrosine hydroxylase activity was accomplished in Drosophila melanogaster larval instars by feeding enzyme inhibitors for a 24-hr period. Behavioral assays performed immediately after treatment demonstrated that larval phototaxis, salt aversion, and heptanol preference were unaffected by reduced levels of dopamine. Within a few hours of treatment, the larvae ceased exploratory behavior and were unresponsive to external stimuli; these larvae eventually died. This behavior is strikingly similar to that displayed by dopamine-deficient transgenic mice. Treated larvae placed immediately onto normal food (to replenish dopamine levels) showed significant developmental delays and decreased fertility as adults. The lethality, developmental retardation, and decrease in fertility were reversed by addition of L-DOPA to inhibitor-containing food, suggesting that these effects were due solely to inhibition of tyrosine hydroxylation. Depletion of dopamine in newly eclosed females resulted in abnormally developed ovaries. These results suggest that the enzymatic function of tyrosine hydroxylase is vital and that reduced levels of dopamine result in akinesia and lethality, developmental retardation, and decreased fertility.
Topics: Animals; Behavior, Animal; Dopamine; Drosophila melanogaster; Enzyme Inhibitors; Female; Fertility; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; In Situ Hybridization; Larva; Methyltyrosines; Mice; Monoiodotyrosine; Ovary; RNA, Messenger; Tyrosine 3-Monooxygenase
PubMed: 8660862
DOI: 10.1006/dbio.1996.0128 -
The Biochemical Journal Jan 1972Insulin dissolved in aqueous or methanolic buffer was iodinated to give preparations containing an average of between one and five iodine atoms per insulin monomer. The...
Insulin dissolved in aqueous or methanolic buffer was iodinated to give preparations containing an average of between one and five iodine atoms per insulin monomer. The resultant preparations were fragmented in various ways and the ratio of tyrosine to monoiodotyrosine and di-iodotyrosine was determined in each fragment. This has allowed the distribution of iodine between the combined A-chain tyrosine residues and the individual B-chain tyrosine residues to be determined. The hormonal activity of each of these iodinated insulin preparations was measured from their effect on the production of (14)CO(2) from [1-(14)C]glucose by isolated adipose cells. The results were interpreted as meaning that the iodination of tyrosine residue A19 or B16 leads to the inactivation of insulin. Speculations are made about the nature of an interaction between insulin and a receptor site on the target tissue.
Topics: Animals; Biological Assay; Carbon Dioxide; Carbon Isotopes; Glucose; In Vitro Techniques; Insulin; Iodine; Methanol; Monoiodotyrosine; Receptors, Drug; Tyrosine
PubMed: 5075226
DOI: 10.1042/bj1260123