Iodination of proteins in TPO transfected thyroid cancer cells is independent of NIS.

This study shows that organification of radioiodide into proteins of thyroid cancer cells exogenously co-expressing the thyroid peroxidase (TPO) and the sodium/iodide symporter (NIS) is independent of NIS function. When administering (125) I to cells constitutively expressing either NIS, or TPO or NIS/TPO, next to iodide accumulation due to NIS activity, organification was exclusively observed in TPO expressing/co-expressing cells. The use of specific inhibitors for TPO and NIS showed that organification is strictly dependent of TPO and not of NIS. An identical pattern of iodoproteins migrating between approximately 75 and 200 kDa in all cell lines tested was observed. Among the five major iodoproteins, two polypeptides appear to be related and three are most probably unrelated, according to their peptide pattern. Our results significantly indicate that co-expression of TPO in NIS transfected cells mediates iodination on the one hand but on the other hand does not contribute to augmentation of a putative NIS-based radioiodide concentrator gene therapy.

Formation of potential antigens from radiographic contrast media.

The use of radiographic contrast media is occasionally accompanied by more or less serious adverse effects, evidently of complex etiology, following intravascular administration. Some of these reactions are suspected of having an allergic basis. The in vitro and in vivo formation of iodinated serum proteins following gamma irradiation in the presence of two commonly used radiographic contrast media is demonstrated. Non-toxic concentrations of ascorbate present during the irradiation is shown to prevent the formation of such iodo-proteins in vitro as well as in vivo. The amounts of potentially antigenic iodoprotein formed during radiographic procedures will certainly be very small, but this quantity may be sufficient to elicit a hypersensitivity reaction in cases when an individual has been previously sensitized to immunologically similar iodo-proteins, a mechanism that could account for certain rare and unpredictable reactions. The radiation induced formation of iodo-proteins may also serve as a model for the formation of iodine containing antigens mediated by a free radical mechanism, i.e. in the metabolism of iodinated compounds like erythrosine, a widely used colouring agent for certain foods.

Iodoprotein formation by rat mammary glands during pregnancy and early postpartum period.

Iodide organification by rat mammary glands was studied during the trimesters of pregnancy and early postpartum period. Organification was followed by measuring trichloroacetic acid (TCA) precipitation of delipidated tissue homogenates. The radiolabeled material was sensitive to proteolytic cleavage by a bacterial protease indicating that the 125I was protein-bound. Gel filtration column chromatography in the presence of sodium dodecyl sulfate (SDS) of delipidated mammary tissue homogenates of pregnant and postpartum rats reproducibly resolved several iodoproteins from free iodide. The Kav value for each iodoprotein peak was calculated and was used to estimate each subunit molecular weight which averaged 37,500, 25,100, and 8500. Another iodoprotein with a very large subunit molecular weight of greater than 300,000 was also detected in mammary tissue. Incorporation of 125I-iodide into the three smaller iodoproteins increased logarithmically from the start of the second trimester of pregnancy through the early postpartum period when approximately 20% of the total 125I uptake by mammary tissue was incorporated into protein. Hyperplasia, acinar development, and intracytoplasmic vacuolization of mammary tissue correlated with the increased incorporation of 125I-iodide into these iodoproteins. The characterization and quantitation of specific iodoproteins in mammary tissue may be important as organification of iodide is believed to be a marker for normal hormone-responsive cells.

Effects of TSH on iodination in rat thyroid follicles studied by autoradiography.

The possible functional relation between TSH-stimulated exocytosis and TSH-stimulated iodination in the thyroid gland was studied using quantitative EM autoradiography and cytochemistry. The study was performed in rats pretreated with thyroxine for 2 days. TSH, giving i.v. 10 min before sacrifice to thyroxine-treated rats, increased organification of 125I by about 50%. TSH decreased the number of peroxidase-positive vesicles in the apical cytoplasm and increased the width of the peroxidase reaction at the apical plasma membrane, suggesting a redistribution of peroxidase. EM autoradiography after labelling with [3H]leucine showed that TSH caused a rapid redistribution by exocytosis of newly synthesized protein to the follicle lumen. The protein deposited in the lumen remained to a large extent in the microvillus region. 10 min after injection of 125I, newly iodinated protein was distributed in a gradient in the lumen periphery. TSH, given 5 min before 125I, caused a significant increase in the labelling of the colloid in the microvillus region, indicating a selective incorporation of iodine into newly synthesized molecules deposited in this region by stimulated exocytosis. Our results confirm and extend earlier observations on a functional link between exocytosis and iodination. Redistribution of peroxidase as well as newly synthesized protein to the site of iodination might be of importance.

Resting human female breast tissue produces iodinated proteins.

Normal resting human breast tissue was obtained from immediate autopsies performed on six women who had died from head injuries sustained in accidents. Tissue samples containing epithelium were dissected asceptically and either fixed immediately or placed into culture. Samples in culture for 2 or 3 days were exposed to radioiodide for 4 hr in order to establish whether or not the isotope became incorporated into proteins. Light and electron microscope autoradiographs were prepared and evaluated. Radiolabeled secretory material was observed in both the terminal ductules and intralobular terminal ducts, but not within the larger ducts. Therefore the products in these separate compartments of the mammary epithelial tree differ in composition. Extensive gap junctions were discovered between adjacent myoepithelial cells in the terminal ductules and intralobular terminal ducts. These junctions probably serve to coordinate contractions which facilitate the movement of material from the most distal parts of the gland into larger ducts.

In vitro effects of arachidonic acid and of inhibitors of its metabolism on the dog thyroid gland.

Incubation of dog thyroid tissue with arachidonic acid (10 to 200 microM) led to the following events: --low conversion to prostaglandins E2 and F2 alpha: 0.07% and 0.02% per hour and 100 mg tissue, respectively --inhibition of the stimulatory effect of low concentrations of TSH on thyroid secretion: the secretory effect of supra-maximal concentrations of TSH and of dB-cAMP was unaffected --inhibition of the cyclic AMP accumulation induced by TSH: this effect was inhibited neither by indomethacin nor by ETYA; cyclic AMP accumulation in response to cholera toxin or PGE1 was unaffected --no effect on cyclic GMP level --stimulation of thyroid proteins iodination. ETYA, but not indomethacin, depressed the iodination of thyroid proteins in resting and stimulated tissue. These data show that arachidonic acid-or a metabolite-can modulate thyroid responsiveness to TSH and suggest that lipoxygenase-products of arachidonic acid metabolism could be involved in thyroid proteins iodination.

Some disturbances related to iodination and utilisation of thyroglobulin and 27 S iodoprotein in non-toxic multinodular goitre.

The relationships among the thyroid iodoproteins, their biosynthesis in vitro in thyroid slices and the histological structure of a multinodular, sporadic goitre was examined after strumectomy from a euthyroid 51-year-old woman. Using sedimentation methods 27S, thyroglobulin (TG), 12S and 3--8S proteins were found. Besides these, 4 to 5 other proteins with molecular weights between 165,000 and 36,000 daltons were detected by polyacrylamide gel electrophoresis. The concentration of soluble proteins was very low (3 mg/100 mg wet tissue), particularly TG (0.69 MG/100 MG). The sedimentation constant (18.3S) of goitrous TG was lower than mature 19S-TG and it was poorly iodinated (0.06% w/w). 27S iodoprotein was present in the goitre extract. Thyroid slices of goitre tissue incorporated 14C-leucine into proteins and synthesized TG and its subunits. Newly synthesized proteins were rapidly released from the microsomes. Pathohistologically, struma colloido-microfolliculare with dystrophic-proliferative changes was noted. In the large follicles which were filled with colloid, dystrophic alterations in thyrocytes and desquamation of the follicular epithelium were observed. In conclusion, it is suggested that the presence of poorly iodinated, immature TG in nontoxic multinodular, sporadic goitre, in the absence of iodine deficiency, probably results in disturbances in the utilization of TG from the follicular lumen. In the follicular lumen under these conditions it is possible that 27S protein is formed from pre-existing poorly iodinated TG. These findings suggest that inability to resorb colloid may be a cause of nodule formation.

Iodinated phospholipids and the in vitro iodination of proteins of dog thyroid gland.

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.

Preparation of monoiodotyrosine-13-glucagon.

Glucagon was iodinated with the lactoperoxidase method at pH 10.0 in the presence of propylene glycol using a substitution of 0.3 g-atom I/mol glucagon. Under these conditions the reactivity of the iodine to tyrosine at position 13 is found to be 4-fold that of the tyrosine at position 10. The amount of diiodotyrosine was less than one-twentieth that of the monoiodotyrosine at either tyrosine residue. Relatively pure monoiodo[125I]tyrosine-13-glucagon can be separated from other iodoglucagons by means of DEAE-chromatography. Such a homogeneous preparation with a known position of the iodine makes it possible to study a specific interaction between the monoiodoglucagon and the glucagon antisera or the glucagon receptor.

Localization of thyroid peroxidase and the site of iodination in rat thyroid gland.

The iodinated protein was localized in thyroid tissue slices by using radioautography. In unfixed tissue, the labelled protein was localized in the colloid, whereas, in tissue that was fixed before the 125I addition, the label was within the follicular cell. This localizes thyroid peroxidase largely on the endoplasmic reticulum of the cell.

Improved radioiodination of glucagon with the lactoperoxidase method. Influence of pH on iodine substitution.

To produce a 125I-labelled glucagon suitable for radioligand assays, we studied the influence of variations in the lactoperoxidase iodination method. Both the degree of iodine substitution and the formation of monoiodo- or diiodo-tyrosines were pH dependent. The substitution increased and the diiodo-/monoiodotyrosine ratio decreased when pH increased. These two factors affected the immunoreactivity of the iodoglucagon relatively independently of each other. It was found that iodination at pH 10.0 with an average of 0.3 gatom I/mol glucagon resulted in 125I-labelled glucagon with higher immunoreactivity and stability than that produced at the conventional pH 7.5 and 8.5.