Tg Synthesis Research Articles

Hydrogen peroxide-induced production of a 40 kDa immunoreactive thyroglobulin fragment in human thyroid cells: the onset of thyroid autoimmunity?

We recently reported that, during in vitro thyroid-hormone synthesis, H(2)O(2) stress cleaved thyroglobulin (Tg) into C-terminal peptides. These peptides were found to contain the immunodominant region of Tg recognized by Tg autoantibodies from patients with an autoimmune thyroid disease. To test the hypothesis that Tg fragmentation is an early upstream initiating event involved in Tg autoimmune response and the consequence of oxidative injuries, we studied the effect of H(2)O(2) stress on human thyroid cells. In culture conditions allowing Tg synthesis and iodine organification by the cells, we found that bolus addition of increasing millimolar doses of H(2)O(2) induced a dose-response appearance of floating cells in the culture medium. These cells apparently resulted from a necrotic process, and they bore iodinated Tg fragments. These fragments were found to be similar to those previously obtained in vitro from purified Tg. In both cases, Tg peptides were recognized by a well-defined monoclonal antibody directed to the immunodominant region of Tg. The smallest immunoreactive Tg peptide had a molecular mass of 40 kDa and entered human thyrocytes more efficiently than the entire Tg. These data suggest that thyrocytes exposed to locally increased H(2)O(2) doses accumulate fragmented Tg for further delivery into surrounding living thyrocytes in the course of an autoimmune response.

Hydrogen peroxide-induced production of a 40 kDa immunoreactive thyroglobulin fragment in human thyroid cells: the onset of thyroid autoimmunity?

We recently reported that, during in vitro thyroid-hormone synthesis, H2O2 stress cleaved thyroglobulin (Tg) into C-terminal peptides. These peptides were found to contain the immunodominant region of Tg recognized by Tg autoantibodies from patients with an autoimmune thyroid disease. To test the hypothesis that Tg fragmentation is an early upstream initiating event involved in Tg autoimmune response and the consequence of oxidative injuries, we studied the effect of H2O2 stress on human thyroid cells. In culture conditions allowing Tg synthesis and iodine organification by the cells, we found that bolus addition of increasing millimolar doses of H2O2 induced a dose–response appearance of floating cells in the culture medium. These cells apparently resulted from a necrotic process, and they bore iodinated Tg fragments. These fragments were found to be similar to those previously obtained in vitro from purified Tg. In both cases, Tg peptides were recognized by a well-defined monoclonal antibody directed to the immunodominant region of Tg. The smallest immunoreactive Tg peptide had a molecular mass of 40kDa and entered human thyrocytes more efficiently than the entire Tg. These data suggest that thyrocytes exposed to locally increased H2O2 doses accumulate fragmented Tg for further delivery into surrounding living thyrocytes in the course of an autoimmune response.

Genética molecular do hipotireoidismo congênito

O hipotireoidismo congênito (HC) ocorre, mundialmente, em 1/3000-4000 neonatos e pode ser classificado em permanente ou transitório. O HC primário é responsável pela maioria dos afetados, enquanto o secundário e terciário são raros. Nos países iodo-suficientes, a disgenesia tireóidea (DT) é a causa mais freqüente de HC. Os defeitos hereditários da síntese hormonal ocorrem em minoria de crianças portadoras de HC. Fatores ambientais, genéticos e auto-imunes concorrem na etiologia do HC, mas na maioria dos casos de DT a causa é obscura. Atribui-se aos genes envolvidos na ontogenia da glândula tireóidea, como os fatores de transcrição TITF1, TITF2, PAX-8 e receptor de TSH (TSHR), função patogenética na DT. Até o momento não foi descrita anormalidade no gene TITF1 como causa de HC, enquanto foram identificadas mutações no PAX-8 em cinco recém-nascidos com DT. Embora não envolvidas na DT, mutações inativadoras do TSHR podem produzir espectro de defeitos congênitos oscilando entre hipertirotropinemia com eutireoidismo e hipotireoidismo com hipoplasia glandular. A clonagem dos genes envolvidos na biossíntese dos hormônios tireóideos, como o da tireoperoxidase (TPO) e tireoglobulina (Tg), permitiu a identificação de mutações responsáveis por alguns casos de bócio e hipotireoidismo decorrente de defeito de incorporação de iodeto ou anormalidades na síntese de Tg. Recentemente, foi demonstrada a base molecular do defeito de transporte ativo de iodeto e da síndrome de Pendred, respectivamente, devidas a mutações no gene NIS (simportador de sódio e iodeto) e no gene PDS (pendrina). Em conclusão, grande parte dos pacientes com HC e DT não tem esclarecida, ainda, a causa molecular desta síndrome.

Endoplasmic Reticulum (ER)-associated Degradation of Misfolded N-Linked Glycoproteins Is Suppressed upon Inhibition of ER Mannosidase I

To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyroglobulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhibitors of ER mannosidase I (but not other glycosidases) acutely suppressed Cog Tg degradation and also perturbed the ERAD process for Tg reduced with dithiothreitol as well as for gamma-carboxylation-deficient protein C expressed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaperones calnexin and calreticulin to monoglucosylated oligosaccharides. Notably, the undegraded protein fraction remained completely microsome-associated. In pulse-chase studies, kifunensine-sensitive degradation was still inhibitable even 1 h after Tg synthesis. Intriguingly, chronic treatment with kifunensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary cells and did not lead to significant induction of the ER unfolded protein response. We hypothesize that, in a manner not requiring lectin-like activity of calnexin/calreticulin, the recognition or processing of a specific branched N-linked mannose structure enhances the efficiency of glycoprotein retrotranslocation from the ER lumen.

Treating the patient with differentiated thyroid cancer with thyroglobulin-positive iodine-131 diagnostic scan-negative metastases: Including comments on the role of serum thyroglobulin monitoring in tumor surveillance

Differentiated thyroid cancer (DTC) patients, especially the 10% to 15% at high risk of cancer-related death, should have long-term monitoring for detection of recurrence or metastasis. Conventional radiologic and ultrasonographic imaging is useful for localization of recurrent or persistent disease. For patients who have had ablation of residual thyroid tissue, measurement of serum thyroglobulin (Tg) levels and radioactive iodine (RAI) imaging provide highly sensitive tools for early detection. Serum Tg is reliable only in the absence of Tg autoantibodies. Sensitivity increases with TSH stimulation, either by withdrawal of thyroxine (T4) therapy, or administration of recombinant TSH (rTSH). In some patients, serum Tg levels are positive but the RAI whole body scan (WBS) is negative. In these patients, either the recurrent tumor is too small and below the sensitivity of the diagnostic scan, or there is a dissociation between Tg synthesis and the iodine-trapping mechanism. Recent literature suggests that empiric high-dose RAI therapy of Tg-positive diagnostic scan-negative patients may result in a high rate of visualization of uptake in posttherapy scans (PTS). Evidence for subsequent improvement of parameters of disease activity has also been presented. Almost all such reported cases had micrometastases that were not visualized by conventional imaging. In our experience, aggressive macrometastases with negative diagnostic WBS do not show significant uptake after therapeutic doses of RAI. The small size of micrometastases in the first group of patients and a possible defect of the iodine-trapping mechanism in the second group may explain this apparent discrepancy. Based on presently available information, a generalized recommendation for RAI therapy of Tg-positive, diagnostic scan-negative patients should await further studies. Meanwhile, in some high-risk patients, in the absence of alternative therapies, empiric RAI therapy is justified.

Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells

PtdCho accumulation is a periodic, S phase-specific event that is modulated in part by cell cycle-dependent fluctuations in CTP:phosphocholine cytidylyltransferase (CCT) activity. A supply of fatty acids is essential to generate the diacylglycerol (DG) precursors for phosphatidylcholine (PtdCho) biosynthesis but it is not known whether the DG supply is also coupled to the cell cycle. Although the rate of fatty acid synthesis in a macrophage cell line was dramatically stimulated in response to the growth factor, CSF-1, it was not regulated by the cell cycle. Increased fatty acid synthesis correlated with elevated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) steady-state mRNA levels. Cellular fatty acid synthesis was essential for membrane PL synthesis. Cerulenin inhibition of endogenous fatty acid synthesis also inhibited PtdCho synthesis, which was not relieved by exogenous fatty acids. Inhibition of CCT activity by the addition of lysophosphatidylcholine (lysoPtdCho) or temperature-shift of a conditionally defective CCT diverted newly synthesized DG to the TG pool where it accumulated. Enforced expression of CCT stimulated PtdCho biosynthesis and reduced TG synthesis. Thus, the cellular DG supply did not regulate PtdCho biosynthesis and CCT activity governs the partitioning of DG into either the PL or TG pools, thereby controlling both PtdCho and TG biosynthesis.

Genomic organization of the 3' region of the human thyroglobulin gene.

The genomic organization of the 3' end of the human Thyroglobulin (Tg) gene has not previously been characterized. We isolated and characterized seventeen lambda phage clones from a human genomic library that included nucleotides 6263 to 8410 of the Tg mRNA, encompassing the last thirteen 3' exons of the Tg gene. The region contained exons ranging in size from 94 to 222 nucleotides, split by introns of 1 to 64 kb. We estimate a total of 48 exons in the Tg gene. All the intron-exon boundaries were sequenced. We found that the splicing sequences diverged considerably from the 3' and 5' consensus. However, the GT-AG rule was perfectly respected in all the exons. A total of 5788 intronic bases and most of the sequences contained in the 13 exons were analyzed (1846 bases). One sequence variation, TT to CC at positions 8377-8378, was found in the 3' untranslated segment. The three tyrosine residues involved in thyroid hormones synthesis (amino acids 2554, 2568, and 2747) at the carbosyl termini of Tg, are encoded by exons 44, 45, and 48. The knowledge of the precise organization of the Tg gene should help to direct studies of Tg gene mutations in families in which a defect in the synthesis of Tg occurs.

Effects of Insulin on Lipid Metabolism of Larvae and Metamorphosing Landlocked Sea Lamprey,Petromyzon marinus

This study was designed to examine the role of insulin (INS) in regulating changes in lipid metabolism of larval and metamorphosing landlocked lamprey,Petromyzon marinus.Larvae and stage 6 metamorphosing individuals were injected intraperitoneally once per day for 2 days with either saline (0.6%), bovine INS (100 ng/g body weight), or alloxan (0.2 mg/g body weight). Insulin administration resulted in depressed plasma fatty acid (FA) levels, whereas alloxan injection elevated plasma FA levels at both life cycle intervals. In larvae, INS-induced hypolipidemia was attended by increased lipid concentration in kidney and muscle, reduced rates of lipolysis in kidney, liver, and muscle (as indicated by decreased triacylglycerol lipase activity), and, to a lesser extent, by higher rates of lipogenesis in kidney and muscle (as evidenced by higher acetyl-CoA carboxylase and/or diacylglycerol acyltransferase activities). In general, the effects of alloxan were opposite of those of INS. The alloxan-induced increase in plasma FA was supported by an enhanced rate of lipolysis in the kidney, a relatively lower rate of fatty acid synthesis in kidney, liver, and muscle, and a relatively lower renal rate of TG synthesis. In stage 6 metamorphosing lamprey, the INS-induced decline in plasma FA was attended by reduced renal and hepatic rates of lipolysis and by enhanced lipogenesis, as indicated by increased renal and hepatic rates ofde novofatty acid synthesis and hepatic and muscular rates of TG synthesis. In contrast, the increase in plasma FA induced by alloxan in stage 6 animals was supported by reduced TG synthsis in liver. Immunocytochemistry revealed that alloxan was not cytotoxic to pancreatic β cells, suggesting that the effects of alloxan were extrapancreatic in the time frame of our study. Because insulin-induced lipogenesis and antilipolysis is similar to the pattern of lipid metabolism (phase I) displayed by lamprey during their spontaneous metamorphosis, INS may play a role, possibly in concert with other factors, in coordinating metamorphosis-associated changes in lipid metabolism.

In vivo expression of thyroid transcription factor-1 RNA and its relation to thyroid function and follicular heterogeneity: identification of follicular thyroglobulin as a feedback suppressor of thyroid transcription factor-1 RNA levels and thyroglobulin synthesis.

We used in situ hybridization to evaluate thyroid transcription factor-1 (TTF-1) RNA expression in individual follicles and related this to thyroglobulin (Tg) synthesis in vivo, as estimated by immunohistochemical analysis. We studied the thyroids of Wistar rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels, but affects follicular function and Tg accumulation in the follicular lumen very differently. We show that TTF-1 RNA levels in vivo correlate directly with an increase in the cytoplasmic accumulation of Tg within the cells of individual follicles. Because TTF-1 increases Tg gene expression, RNA levels, and protein synthesis in thyroid cell cultures and because there is no correlation with TSH-increased Tg degradation within the follicular lumen, the increased cytoplasmic accumulation of Tg in vivo is interpreted to reflect TTF-1-increased Tg synthesis. Increases in serum TSH levels in the PTU or T4 treated animals did not always correlate with increases in this measure of increased Tg synthesis; and TSH levels did not always correlate with changes in TTF-1 RNA levels that would be expected to accompany increased Tg synthesis. As one possibility, this suggested there might be a hitherto unrecognized suppressor of TTF-1 RNA levels and TSH-induced Tg synthesis in individual follicles. The immunohistochemical data suggested that this suppressor might be follicular Tg itself. Supporting this possibility, we show that physiological concentrations of highly purified 19S follicular Tg decrease TTF-1 RNA levels in rat FRTL-5 thyroid cells and inhibit the action of TSH to increase Tg synthesis. We therefore suggest that follicular Tg is a feedback autoregulator of thyroid function that can counterregulate TSH actions on thyroid function in vivo and in thyroid cells in culture. We suggest this phenomenon contributes to follicular heterogeneity in vivo.

Effects of Somatostatin on Lipid Metabolism of Larvae and Metamorphosing Landlocked Sea Lamprey,Petromyzon marinus

This study was designed to examine the role of somatostatin in regulating changes in lipid metabolism of larvae and metamorphosing landlocked sea lamprey,Petromyzonmarinus. Larvae and animals in late metamorphosis (stage 6 on a 7-stage scale) were injected intraperitoneally once per day for 2 days with either saline (0.6%) or somatostatin-14 (SS-14; 500 ng/g body wt). Injection of SS-14 into larval and stage 6 metamorphosing animals resulted in elevated plasma fatty acids levels. In larvae, SS-14-induced hyperlipidemia was supported by enhanced lipolysis, as indicated by increased triacylglycerol lipase (TGL) activity in the liver and kidney. Mobilization of larval renal lipid was accompanied by reduced TG synthesis, as indicated by decreased diacylglycerol acyltransferase (DGAT) activity. In stage 6 metamorphosing lamprey, SS-14 did not significantly affect TGL activity; however, SS-14 significantly reduced fatty acid synthesis, as measured by acetyl-CoA carboxylase activity, in kidney, liver, and muscle, as well as muscular TG synthesis. SS-14-stimulated lipid depletion is reminiscent of the pattern of lipid metabolism displayed byP. marinusduring their spontaneous metamorphosis—an observation which suggests that somatostatin may play a role in metamorphosis-associated changes in lipid metabolism in this species.

Effects of Propyithiouracil (PTU) Administration on the Synthesis and Secretion of Thyroglobulin in the Rat Thyroid Gland: A Quantitative Immuno-electron Microscopic Study Using Immunogold Technique.

To clarify the effects of an antithyroid drug on the kinetics of thyroglobulin synthesis, secretion, and reabsorption in the thyroid follicles, propylthiouracil (PTU) was administered to rats and the thyroid glands were examined by a refined post-embedding immunogold technique during and after withdrawal of PTU. Seven-wk-old male Wistar rats were administered with S mg of PTU/d through a gastric tube, and sacrificed at 1 and 2 wk of administration and at 1, 2, and 3 d, and 1, 2, 3, and 4 wk, after discontinuation. The administration of PTU caused a remarkable dilatation of the rER and Golgi apparatus, but these areas gradually recovered after withdrawal of PTU. During the experiment, no significant change in the density of thyroglobulin (Tg) was observed except for a transient increase immediately after withdrawal of PTU. The expression of Tg on subapical vesicles (SV) and follicular colloid took a relatively parallel course; increasing during administration of PTU and decreasing with a transient peak immediately after treatment was discontinued. In contrast to the remarkable changes in the morphology of compartments involved in Tg synthesis, the development of colloid droplets and formation of secondary lysosomes were suppressed during and after discontinuing administration of PTU. However, the basic pattern of the gradient of Tg density among the cellular compartments was essentially retained in the experimental group. Thus the present immunoelectron-microscopic study provided evidence that administration of PTU stimulates the synthesis and secretion of Tg in the follicular epithelium in vivo, and, also, suppresses reabsorption and degradation of Tg. Further, it was speculated that the density gradient of Tg among the compartments involved in Tg synthesis, secretion and storage is regulated by an unknown constitutive mechanism and not by the thyroid-stimulating hormone (TSH)-TSH receptor-mediated system.

脂質代謝に及ぼす多価不飽和脂肪酸の影響に関する研究

We investigated the metabolism of n-6 and n-3 fatty acids and their effect on lipid metabolism. Rat hepatocytes were a good model for the study of Δ5-desaturation, while HepG2 cells did not have this activity. We found that sesamin, a lignan of sesame seed, inhibited the activity of Δ5-desaturation in n-6 fatty acids but not in n-3 fatty acids. This result was confirmed in vivo using rats fed diets rich in n-6 and n-3 fatty acids. Sesamin also inhibited the increase of n-3 fatty acids in the liver caused by a n-3 fatty acid-rich diet. Sesamin had a unique function in changing the balance of intermediate metabolites of n-6 and n-3 fatty acids. We also examined the effect of fatty acids on lipid metabolism in HepG2 cells. Linolenic acid (LLA) decreased the synthesis and secretion of TG from HepG2 cells while linoleic acid (LA) and γ-linolenic acid (GLA) reduced only the secretion of TG. LA increased the binding and uptake of LDL. LLA had a strong effect in promoting the catabolism of cholesterol. These results suggest that a change in n-6/n-3 balance caused by a dietary component such as sesamin can affect lipid metabolism.

HighTg polyimide nanofoams derived from pyromellitic dianhydride and 1,1-bis(4-aminophenyl)-1-phenyl-2,2,2-trifluoroethane

New routes for the synthesis of high Tg thermally stable polymer foams with pore sizes in the nanometer regime have been developed. Foams were prepared by casting well-defined microphase-separated block copolymers comprised of a thermally stable block and a thermally labile material. At properly designed volume fractions the morphology provides a matrix of the thermally stable material with the thermally labile material as the dispersed phase. Upon thermal treatment, the thermally unstable block undergoes thermolysis generating pores, the size and shape of which are dictated by the initial copolymer morphology. Triblock copolymers comprised of a high Tg, amorphous polyimide matrix with poly(propylene oxide) as the thermally decomposable coblock, were prepared. The copolymer synthesis was conducted through the poly(amic acid) precursor and subsequent cyclodehydration to the polyimide by either thermal or chemical means. Dynamic mechanical analysis confirmed microphase separated morphologies for all copolymers, irrespective of the propylene oxide block lengths investigated. Upon decomposition of the thermally labile coblock, a 9–18% reduction in density was observed, consistent with the generation of a foam which was stable to 400°C. © 1996 John Wiley & Sons, Inc.

Thyrotropin controls dolichol-linked sugar pools and oligosaccharyltransferase activity in thyroid cells

We previously showed that thyroglobulin (Tg) glycosylation is enhanced 1.5-fold under thyrotropin (TSH) stimulation, corresponding to an increased number of oligosaccharide chains per molecule of Tg. Now the steps involving dolichol components and oligosaccharyltransferase activity have been studied. Porcine thyroid cells were cultured on porous bottom filters with or without TSH and incubated with [ 14C]mevalonate. Under TSH regulation, the level of the whole of dolichol components was increased 1.25-fold without modifying their distribution. Dolichol, and free and monosaccharide-linked dolichyl-phosphate, represented respectively 40% and 45% of total dolichol components while dolichyl-pyrophosphate-oligosaccharide represented 3% only. A marked enhancement (4.2-fold) of oligosaccharyltransferase activity occurred in stimulated cells, which could correspond to the addition of the two TSH effects: stimulation of Tg synthesis (3-fold) and of Tg glycosylation (1.5-fold). The amount of lipid carriers appeared to be insufficiently increased but no component is a limiting step, suggesting that the turnover of dolichol derivatives may be increased under TSH control through their use by higher amounts of Tg.

Effects of iodide on thyroglobulin biosynthesis in FRTL-5 cells.

Iodide inhibits several thyroid parameters through an organic intermediate, and this process has been related to thyroid autoregulation. The aim of this study was to determine the effect of iodine on thyroglobulin (Tg) synthesis in the rat thyroid cell line FRTL-5. TSH stimulated amino acid incorporation into the cells by 400% and iodine had no effect on this parameter. No effect of TSH or iodide on [35S] methionine incorporation into protein was found under our experimental conditions (approximately 80% of total [35S]methionine incorporated was found in TCA-precipitable material). TSH caused an increase in Tg synthesis, after 1 h, while iodide partially blocked the effect of TSH (control 6.4% of TCA precipitable radioactivity; TSH 10.7%; iodide 8.4%). After 24 h, the protein released into the medium was measured. TSH stimulated total protein liberation and iodide inhibited this parameter. TSH stimulated total RNA content, and iodide caused an inhibition. Northern analysis did not show inhibition by iodide of TSH-stimulated Tg mRNA levels. The present results show an inhibitory effect of excess iodide on TSH-stimulated thyroglobulin biosynthesis in FRTL-5 cells.

Hyperinsulinemia and triglyceride-rich lipoproteins.

Hypertriglyceridemia is the most frequent form of hyperlipidemia seen in diabetes. Because hypertriglyceridemia and hyperinsulinemia often coexist in the general population and because patients with NIDDM generally are hyperinsulinemic, we have undertaken a series of in vivo studies to examine the effects of hyperinsulinemia on VLDL production. These studies showed that chronic hyperinsulinemia is accompanied by increased VLDL production and that this occurs even when plasma free fatty acid (FFA) levels have fallen. By contrast, acute hyperinsulinemia is accompanied by a reduction in VLDL production, and this reduction is, at least in part, mediated by an associated reduction in the availability of plasma FFAs as a substrate for VLDL-triglyceride (TG). The studies also raise the possibility that the difference in the dependence of VLDL production on plasma FFAs in acute versus chronic hyperinsulinemia results from an increase in hepatic lipogenic enzymes and from the availability of an alternate substrate such as fructose. The overall effect of hyperinsulinemia on VLDL production is postulated to reflect both the effect of insulin on apolipoprotein B production and the hepatic synthesis of TG from either plasma FFAs or newly made fatty acids.

Long-term observation of serum thyroglobulin after resection of nontoxic goiter and relation to ultrasonographically demonstrated relapse.

The object was to carry out a prospective study of the changes in serum thyroid hormones and thyroglobulin (Tg) following resection of nontoxic goiter and to investigate if there was a correlation to the pattern of relapse. A group of 39 consecutive patients, mainly with nodular, nontoxic goiter, were studied for 13 years following thyroidectomy. No thyroid hormone replacement therapy was given after surgery. The preoperative serum Tg level was elevated. After operation the mean serum Tg declined to a nadir of 43 micrograms/L at 1 year and subsequently increased to 90 micrograms/L at 10 years, with large individual differences. In 19 patients the serum Tg increased, in 1 it decreased, and in 19 no significant alteration was observed. Serum free thyroxine and triiodothyronine indices decreased following resection but achieved normal levels within 6 to 12 months. Serum thyroid-stimulating hormone increased after resection, with a peak level 1 month after surgery, but it returned to normal levels at 1 year and remained stable for the rest of the period. At 13 years after resection the thyroid volume was determined by ultrasonography in 30 of the patients. In 10 patients the thyroid volume was enlarged (>/= 28 ml). In this group a rise of average serum Tg after resection [DeltaTg(10-1 year)] of 133 micrograms/L was observed, compared to 26 micrograms/L in the 20 patients without sonographic relapse (volume < 28 ml). A positive correlation was demonstrated between serum DeltaTg(10-1 year) postsurgically and thyroid volume 13 years after surgery. However, an overlap was observed between the groups with and without ultrasonographic relapse, probably in part due to large differences in the Tg synthesis activity of different follicle cell clones. It is concluded that repeated serum Tg determinations may provide biochemical evidence of increased growth activity of thyroid remnants monitored after goiter resection.

Tyrosine β-Glucosyltransferase in the tobacco hornworm, Manduca sexta (L.): properties, tissue localization, and developmental profile

Tyrosine β-glucosyltransferase activity in larval tissues of Manduca sexta was determined by reversed-phase HPLC to quantify the product tyrosine glucoside [β- d-glucopyranosyl- O- l-tyrosine, (TG)] formed in incubation mixtures. Synthesis of TG occurred only in fat body preparations, with most of the enzyme activity (95%) in the 15,000 g pellet of homogenates. Other tissues were devoid of activity but did support glucosylation of phenolic substrates other than tyrosine. Activity was greatest with uridine 5′-diphosphoglucose (UDPG) as the glucose donor. However, thymidine 5′-diphosphoglucose (dTDPG) afforded approximately one fourth the amount of tyrosine glucosylation provided by UDPG. Magnesium ions at concentrations up to 15 mM stimulated activity, whereas Ca 2+ and Mn 2+ were only slightly stimulatory at 5 mM but progressively inhibited TG synthesis at higher concentrations as did Co 2+. Maximal rates of glucosylation occurred in the pH range 7.5–9.0. The enzyme was highly specific for tyrosine, because no glucosylation occurred for a number of tyrosine derivatives. Several natural and synthetic mono- and diphenols were found to be weak to moderate inhibitors of the enzyme. In a study of tyrosine β-glucosyltransferase activity during development, no TG synthesis occurred in enzyme preparations from first, second, third, or fourth larval instars, although glucosylation of p-nitrophenol and 4-hydroxycoumarin did occur. Activity was not detected in the fat body of newly ecdysed fifth instars, but low levels were observed 36 h later. The rate of tyrosine glucosylation continued to increase to a peak at 4 days, but then decreased to very low levels after cessation of larval feeding. Low activity was also observed in the pharate adult fat body about 1 day before eclosion. Therefore, tyrosine β-glucosyltransferase activity occurs primarily in the fat body of fifth stadium larvae for synthesis of the pupal cuticle tanning precursor tyrosine glucoside.

Lipid esterification and synthesis by the IEC-6 intestinal epithelial crypt cell line: effect of transforming growth factor β

The capacity of immature intestinal epithelial crypt cells to synthesize lipids and the factors that promote their differentiation remain largely unknown. We examined the profile of lipids synthesized by a normal rat intestinal epithelial crypt cell line (IEC-6) and determined the effects of transforming growth factor beta (TGF beta), a putative crypt cell differentiating factor, on their lipid handling. Incubation of IEC-6 cells with [14C]oleic acid (20 h) resulted in lipid esterification and synthesis, mainly as triglycerides (TGs, 57 +/- 0.6%) and phospholipids (PLs, 30 +/- 0.6%), with a PL/TG ratio of 0.53. When cells were pulsed (2.5 h) with [14C]oleic acid and then maintained 20 h in medium alone, a significant elevation of the PL/TG ratio (10.2 +/- 1.3, p < 0.01) was observed, primarily accounted for by a significant decrease of the TG fraction (p < 0.01). IEC-6 cells secreted only trace amounts of lipids under the latter experimental condition. Incubation with TGF beta (20 h) significantly inhibited IEC-6 cell proliferation but did not promote the expression of cell sucrase activity. TGF beta induced a significant increase in the cellular composition of PL (p < 0.05) and a decrease in the TG fraction (p < 0.02), after a 2.5-h pulse of [14C]oleic acid. Lipid production was unaffected by TGF beta during the 20-h incubation with [14C]oleic acid. Lipid secretion into the medium remained negligible in the presence of TGF beta, after 2.5 h of incubation with substrate as above. Our findings suggest that immature crypt IEC-6 cells are capable of lipid esterification and synthesis but secrete minute amounts of lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Tiglylglycine excreted in urine in disorders of isoleucine metabolism and the respiratory chain measured by stable isotope dilution GC-MS

Tiglyglycine (TG), an intermediate product of the catabolism of isoleucine, is increased in the urine of patients with beta-ketothiolase deficiency or with disorders of propionate metabolism. It is also implicated as a useful diagnostic marker in disorders of the respiratory chain. We present a method for the synthesis of TG and tiglyl[13C, 15N]glycine and the development of a stable isotope dilution mass spectrometric assay for TG. We compare data from controls with that from subjects with beta-ketothiolase deficiency and propionyl-CoA carboxylase deficiency, and with six patients with enzyme-confirmed disorders of the respiratory chain. TG was increased in the urine from all of the patient groups. The increased TG excretion did not persist in one patient with a respiratory chain defect, which suggests that, in some patients, multiple sample analysis may be necessary to identify a respiratory chain defect. This is the first urinary compound to be implicated as a potential marker of disorders of the respiratory chain.