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The Journal of Clinical Investigation Oct 1973Benign and malignant nodules in human thyroid glands, which did not concentrate iodide in vivo, were also unable to accumulate iodide in vitro. The mean...
Benign and malignant nodules in human thyroid glands, which did not concentrate iodide in vivo, were also unable to accumulate iodide in vitro. The mean thyroid-to-medium ratio (T/M) in seven benign nodules was 0.8+/-0.2 compared with 7+/-2 in adjacent normal thyroid tissue. In four malignant thyroid nodules, the mean T/M was 0.5+/-0.1 compared with 11+/-4 in adjacent normal thyroid. Despite the inability of such nodules to concentrate iodide, iodide organification was present but was only one-half to one-third as active as in surrounding normal thyroid. Thyroid-stimulating hormone (TSH) increased iodide organification equally in both benign nodules and normal thyroid although it had no effect in three of the four malignant lesions. The reduction in organification is probably related to the absence of iodide transport, since incubation of normal thyroid slices with perchlorate caused similar diminution in iodide incorporation but no change in the response to TSH. Monoiodotyrosine (MIT) and di-iodotyrosine (DIT) accounted for most of the organic iodide in both the nodules and normal tissue. The MIT/DIT ratio was similar in normal and nodule tissue. The normal tissue contained much more inorganic iodide than the nodules, consistent with the absence of the iodide trap in the latter tissue. The thyroxine content of normal thyroid was 149+/-17 mug/g wet wt and 18+/-4 mug/g wet wt in the nodules. The transport defect in the nodules was not associated with any reduction in total, Na(+)-K(+)- or Mg(++)-activated ATPase activities or the concentration of ATP. Basal adenylate cyclase was higher in nodules than normal tissue. Although there was no difference between benign and malignant nodules, the response of adenylate cyclase to TSH was greater in the benign lesions. These studies demonstrate that nonfunctioning thyroid nodules, both benign and malignant, have a specific defect in iodide transport that accounts for their failure to accumulate radioactive iodide in vivo. In benign nodules, iodide organification was increased by TSH while no such effect was found in three of four malignant lesions, suggesting additional biochemical defects in thyroid carcinomas.
Topics: Adenoma; Adenosine Triphosphatases; Adenosine Triphosphate; Adenylyl Cyclases; Animals; Biological Transport; Carcinoma, Papillary; Cattle; Diiodotyrosine; Humans; Iodides; Iodine Radioisotopes; Magnesium; Monoiodotyrosine; Ouabain; Perchlorates; Potassium; Sodium; Thyroid Gland; Thyroid Neoplasms; Thyroxine
PubMed: 4353998
DOI: 10.1172/JCI107430 -
The Journal of Biological Chemistry Feb 1996In this study, we identify a transport system for tyrosine, the initial precursor of melanin synthesis, in the melanosomes of murine melanocytes. Melanosomes preloaded...
In this study, we identify a transport system for tyrosine, the initial precursor of melanin synthesis, in the melanosomes of murine melanocytes. Melanosomes preloaded with tyrosine demonstrated countertransport of 10 microM [3H]tyrosine, indicating carrier-mediated transport. Melanosomal tyrosine transport was saturable, with an apparent Km for tyrosine transport of 54 microM and a maximal velocity of 15 pmol of tyrosine/unit of hexosaminidase/min. Transport was temperature-dependent (Ea = 7.5 kcal/mol) and showed stereospecificity for the l-isomer of tyrosine. Aromatic, neutral hydrophobic compounds (such as tryptophan and phenylalanine), as well as the small, bulky neutral amino acids (such as leucine, isoleucine, and methionine) competed for tyrosine transport. Tyrosine transport was inhibited by the classical system L analogue, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and by monoiodotyrosine, but not by cystine, lysine, glutamic acid, or 2-(methylamino)-isobutyric acid. Tyrosine transport showed no dependence on Na+ or K+, and did not require an acidic environment or the availability of free thiols. These results demonstrate the existence of a neutral amino acid carrier in murine melanocyte melanosomes which resembles the rat thyroid FRTL-5 lysosomal system h. This transport system is critical to the function of the melanosome since tyrosine is the essential substrate required for the synthesis of the pigment melanin.
Topics: Amino Acids; Animals; Binding, Competitive; Biological Transport; Calorimetry; Cell Line; Cytoplasmic Granules; Kinetics; Melanocytes; Mice; Mice, Inbred C57BL; Microscopy, Electron; Tritium; Tyrosine; beta-N-Acetylhexosaminidases
PubMed: 8626732
DOI: 10.1074/jbc.271.8.4002 -
The Journal of Cell Biology Oct 1972The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and...
The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 10(6) cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7-9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-(125)I, cell-associated isotope disappears with a t (1/2) of 20-30 hr and they release monoiodotyrosine-(125)I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.
Topics: Animals; Binding Sites; Cell Membrane; Cells, Cultured; Half-Life; Histocytochemistry; Inclusion Bodies; Iodine Isotopes; Lysosomes; Macrophages; Mice; Monoiodotyrosine; Peritoneum; Peroxidases; Pinocytosis; Plants; Protein Binding; Solubility; Time Factors
PubMed: 4347251
DOI: 10.1083/jcb.55.1.186 -
Learning & Memory (Cold Spring Harbor,... 1998Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely...
Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely impaired the ability of these flies to modify their courtship responses to immature males. Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminishes with time. Dopamine is also required for normal female sexual receptivity; dopamine-depleted females show increased latency to copulation. One kilobase of 5' upstream information from the Drosophila tyrosine hydroxylase (DTH) gene, when fused to the Escherichia coli beta-galactosidase reporter and transduced into the genome of Drosophila melanogaster, is capable of directing expression of the reporter gene in the mushroom bodies, which are believed to mediate learning acquisition and memory retention in flies. Ablation of mushroom bodies by treatment of newly hatched larva with hydroxyurea resulted in the inability of treated mature adult males to cease courtship when placed with untreated immature males. However, functional mushroom bodies were not required for the dopaminergic modulation of an innate behavior, female sexual receptivity. These data suggest that dopamine acts as a signaling molecule within the mushroom bodies to mediate a simple form of learning.
Topics: Animals; Animals, Genetically Modified; Conditioning, Psychological; Dopamine; Drosophila melanogaster; Enzyme Inhibitors; Female; Genes, Reporter; Male; Monoiodotyrosine; Neurons; Neuropil; Olfactory Pathways; Promoter Regions, Genetic; Sexual Behavior, Animal; Tyrosine 3-Monooxygenase
PubMed: 10454380
DOI: No ID Found -
Endocrinology Oct 2012Thyroid hormone (TH) plays critical roles during vertebrate postembryonic development. TH production in the thyroid involves incorporating inorganic iodide into...
Thyroid hormone (TH) plays critical roles during vertebrate postembryonic development. TH production in the thyroid involves incorporating inorganic iodide into thyroglobulin. The expression of iodotyrosine deiodinase (IYD; also known as iodotyrosine dehalogenase 1) in the thyroid gland ensures efficient recycling of iodine from the byproducts of TH biosynthesis: 3'-monoiodotyrosine and 3', 5'-diiodotyrosine. Interestingly, IYD is known to be expressed in other organs in adult mammals, suggesting iodine recycling outside the thyroid. On the other hand, the developmental role of iodine recycling has yet to be investigated. Here, using intestinal metamorphosis as a model, we discovered that the Xenopus tropicalis IYD gene is strongly up-regulated by TH during metamorphosis in the intestine but not the tail. We further demonstrated that this induction was one of the earliest events during intestinal metamorphosis, with IYD being activated directly through the binding of liganded TH receptors to a TH response element in the IYD promoter region. Because iodide is mainly taken up from the diet in the intestine and the tadpole stops feeding during metamorphosis when the intestine is being remodeled, our findings suggest that IYD transcription is activated by liganded TH receptors early during intestinal remodeling to ensure efficient iodine recycling at the climax of metamorphosis when highest levels of TH are needed for the proper transformations of different organs.
Topics: Animals; Gene Expression Regulation, Developmental; Intestinal Mucosa; Iodide Peroxidase; Larva; Metamorphosis, Biological; Receptors, Thyroid Hormone; Thyroid Gland; Thyroid Hormones; Xenopus laevis
PubMed: 22865369
DOI: 10.1210/en.2012-1308 -
The Journal of Biological Chemistry Jan 1984In order to study whether hormone-sensitive tyrosine aminotransferase exists in tissues other than liver, we have devised means to separate the liver-specific enzyme...
In order to study whether hormone-sensitive tyrosine aminotransferase exists in tissues other than liver, we have devised means to separate the liver-specific enzyme from other enzymes that transaminate tyrosine and to distinguish between the authentic enzyme and the principal "pseudotyrosine aminotransferases," which are the isoenzymes of aspartate aminotransferase. We accomplish this by suppressing proteolysis of the authentic enzyme using a buffer of pH 8.0 containing 0.1 M potassium chloride; enzyme extracted from liver in this buffer migrates as a single peak during chromatography on hydroxylapatite and represents the undegraded native form. A much smaller peak of tyrosine aminotransferase activity elutes at higher ionic strength and corresponds to a mixture of mitochondrial aspartate aminotransferase and partially degraded tyrosine aminotransferase. Cytosolic aspartate aminotransferase, in contrast, adsorbs weakly to the hydroxylapatite column and transaminates tyrosine very poorly although it readily utilizes monoiodotyrosine. The aspartate aminotransferase isoenzymes separate completely from tyrosine aminotransferase during chromatography on DEAE-Sepharose CL-6B. By combining these techniques with the use of specific antibodies, we show that brain, heart, and kidney do not contain tyrosine aminotransferase. Furthermore, we locate both isoenzymes of aspartate aminotransferase on polyacrylamide gels and show that both react histochemically as tyrosine aminotransferases when monoiodotyrosine is used as substrate. Use of these techniques, therefore, permits unambiguous identification of tyrosine aminotransferase and its separation from the background of nonspecific transamination.
Topics: Animals; Aspartate Aminotransferases; Chromatography, Ion Exchange; Female; Glucocorticoids; Isoenzymes; Kidney; Liver; Male; Myocardium; Rats; Rats, Inbred Strains; Tissue Distribution; Tyrosine Transaminase
PubMed: 6142885
DOI: No ID Found -
The Journal of Neuroscience : the... Sep 2004Coordinated flight in winged insects requires rhythmic activity of the underlying neural circuit. Here, we show that Drosophila mutants for the inositol...
Coordinated flight in winged insects requires rhythmic activity of the underlying neural circuit. Here, we show that Drosophila mutants for the inositol 1,4,5-trisphosphate (InsP(3)) receptor gene (itpr) are flightless. Electrophysiological recordings from thoracic indirect flight muscles show increased spontaneous firing accompanied by a loss of rhythmic flight activity patterns normally generated in response to a gentle puff of air. In contrast, climbing speed, the jump response, and electrical properties of the giant fiber pathway are normal, indicating that general motor coordination and neuronal excitability are much less sensitive to itpr mutations. All mutant phenotypes are rescued by expression of an itpr(+) transgene in serotonin and dopamine neurons. Pharmacological and immunohistochemical experiments support the idea that the InsP(3) receptor functions to modulate flight specifically through serotonergic interneurons. InsP(3) receptor action appears to be important for normal development of the flight circuit and its central pattern generator.
Topics: Animals; Animals, Genetically Modified; Calcium Channels; Drosophila Proteins; Drosophila melanogaster; Electromyography; Fenclonine; Flight, Animal; Hot Temperature; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Interneurons; Monoiodotyrosine; Motor Neurons; Muscles; Phenotype; Pupa; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Serotonin; Transgenes; Wings, Animal
PubMed: 15356199
DOI: 10.1523/JNEUROSCI.0656-04.2004 -
The Journal of Biological Chemistry Mar 1989Monoiodotyrosine (MIT) crosses the lysosomal membrane of rat FRTL-5 thyroid cells by a carrier-mediated process. In egress studies, MIT lost from inside lysosomes was...
Monoiodotyrosine (MIT) crosses the lysosomal membrane of rat FRTL-5 thyroid cells by a carrier-mediated process. In egress studies, MIT lost from inside lysosomes was quantitatively recovered outside lysosomes as MIT, indicating that the compound was transported intact across the lysosomal membrane. In uptake studies, [125I]MIT entry required intact lysosomes and exhibited saturation kinetics. The apparent Km for MIT was approximately 1.5 microM and the Vmax was approximately 0.24 pmol/unit hexosaminidase/min. Countertransport of MIT was demonstrated, with an initial velocity of [125I]MIT uptake which reached a maximum at high intralysosomal MIT loading. Nonradioactive MIT and diiodotyrosine competed to approximately equivalent extents with [125I]MIT for uptake in countertransport experiments. The existence of a lysosomal MIT carrier in thyroid cells may explain how this product of thyroglobulin catabolism is transported to the cytosol for iodine salvage and reutilization.
Topics: Animals; Biological Transport; Carrier Proteins; Cell Line; Cell-Free System; Diiodotyrosine; Kinetics; Lysosomes; Monoiodotyrosine; Rats; Thyroid Gland
PubMed: 2925666
DOI: No ID Found -
The Journal of Biological Chemistry Apr 1986Vasoactive intestinal polypeptide (VIP) was labeled with sodium [125I]iodide using the chloramine-T method and subsequently purified by reverse-phase high performance...
Vasoactive intestinal polypeptide (VIP) was labeled with sodium [125I]iodide using the chloramine-T method and subsequently purified by reverse-phase high performance liquid chromatography. Three main 125I-labeled peaks designated A, B, and C resulted from the radioiodination and purification procedures. They were characterized by electrophoresis of tryptic fragments; Edman degradation (for Peaks A and C); enzymatic digestion to amino acids by leucine aminopeptidase, carboxypeptidase Y and Pronase; and treatment with cyanogen bromide. Peak A corresponds to VIP monoiodinated on Tyr10 and with the Met17 residue oxidized to methionine sulfoxide. This [mono[125I]iodo-Tyr10,MetO17]VIP displays the following characteristics. 1) It constitutes quantitatively the major product of the iodination procedure (62.5%); 2) it is well resolved from other labeled and unlabeled products; 3) it is stable (2 months at -20 degrees C); 4) it possesses a high specific activity (2050 Ci/mmol); 5) it maintains the biological activity of native VIP; and 6) it binds to antibody and membrane recognition sites in a specific, saturable, and reversible manner. Reduction of [mono[125I]iodo-Tyr10, Met-O17]VIP to [mono[125I]iodo-Tyr10]VIP does not improve the performance of the tracer in a radioimmunoassay. The method described in this article is simple and rapid and yields a molecular form of 125I-labeled VIP that has been fully characterized and is suitable for use in biological studies.
Topics: Animals; Carboxypeptidases; Chloramines; Chromatography, High Pressure Liquid; Cross Reactions; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; Glycogen; Hydrolysis; Iodine Radioisotopes; Kinetics; Leucyl Aminopeptidase; Methionine; Mice; Monoiodotyrosine; Peptide Fragments; Pronase; Radioimmunoassay; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Synaptic Membranes; Tosyl Compounds; Trypsin; Vasoactive Intestinal Peptide
PubMed: 3007497
DOI: No ID Found -
The Journal of Clinical Investigation Jan 1973Butanol-insoluble iodinated compounds in the urine of patients with congenital goiters have been generally regarded as iodopeptides. Monoiodohistidine (MIH) and...
Butanol-insoluble iodinated compounds in the urine of patients with congenital goiters have been generally regarded as iodopeptides. Monoiodohistidine (MIH) and diiodohistidine (DIH) were identified from the urine of four patients with congenital goitrous hypothyroidism. From radioiodine studies, 40-70% of the urinary radioactivity was in the iodide-free fraction from which about 40% was identified as MIH and DIH by crystallizations to a constant specific activity. Iodotyrosines were simultaneously identified in the urine. However the presence of an iodotyrosine-deiodinase activity was demonstrated in the two removed goiters with a normal K(m) for MIT. In vivo iodotyrosine deiodination was normal for hypothyroid subjects. No thyroglobulin was identified in the thyroids from these patients. The major iodoprotein was iodoalbumin which, after in vivo labeling, contained 84-89% of the total soluble protein radioactivity. The thyroxine content of the goiter iodoalbumins and other iodoproteins was extremely low. Iodohistidines were identified in comparable proportions in the iodoalbumin and in the other iodoproteins isolated from each goiter. The average iodohistidine content of these proteins as crystallizable MIH and DIH was in the individual cases 15 and 4% of the in vivo incorporated radioiodine. DIH was identified in all iodoprotein fractions. The mean DIH/MIH ratios from the individual cases were 1.16 and 0.35. The corresponding DIT/MIT ratios were 3.19 and 1.45, respectively. The major consequence of this thyroglobulin defect is the iodination of inappropriate proteins (mainly albumin) resulting in low yields of thyroxine and high yields of iodohistidines. Iodohistidines from the goiter iodoproteins were not deiodinated and, at least for MIH, were quantitatively excreted in the urine of these patients. From the MIH iodoalbumin content and the MIH urinary excretion, goiter iodoalbumin turnover estimates were made and, although elevated, could not maintain a normal thyroxine secretion. The urinary excretion of iodohistidines easily demonstrated by column chromatography is offered as a test for detecting this variety of congenital goiter.
Topics: Adult; Albumins; Albuminuria; Child; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Female; Goiter; Histidine; Humans; Hypothyroidism; Immunodiffusion; Iodides; Iodoproteins; Male; Monoiodotyrosine; Peroxidases; Thyroglobulin; Thyroid Gland
PubMed: 4629905
DOI: 10.1172/JCI107154