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F. Monaco and M. Andreoli

ABSTRACT

Thyroglobulins (TG) from a "hot" human thyroid nodule and from Fisher rats have been purified and the effects of progressive removal of sialic acid and galactose on the immunoreactive properties of the proteins were studied. Terminal sialic acid and galactose were released by stepwise hydrolysis with neuraminidase and beta-galactosidase.

Agalacto-TG shows a slower electrophoretic mobility than native TG, but in polyacrylamide gel electrophoresis and immunoelectrophoresis it migrates in the same position as asialo-TG.

In immunodiffusion agalacto-TG forms a spur with native TG and asialo-TG when tested against anti 19S native TG or anti-asialo-TG sera.

It is thus shown that galactose in the terminal environment of the oligosaccharide chains of thyroglobulin is essential for the structural groups involved in the antigenic properties of thyroglobulin.

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F. Monaco, S. Grimaldi, G. Scuncio and M. Andreoli

ABSTRACT

In an experimental rat thyroid tumour (Wollman line 1-1C2) thyroglobulin (TG) was isolated from soluble iodoproteins by ammonium sulphate fractionation and density gradient ultracentrifugation. The isolated TG was characterized by polyacrylamide gel electrophoresis, microimmunodiffusion, immunoelectrophoresis, analytical ultracentrifugation and by chemical determination of sialic acid and iodine content.

Tumour thyroglobulin has a sedimentation coefficient of 17.7S, which is similar to that of enzymatically desialylated thyroglobulin (18.4S) in the normal rat; tumour TG shows a slower electrophoretic mobility than native TG and similar to rat desialylated TG.

In double immunodiffusion tumour TG is shown to precipitate with an identity reaction with both native and desialylated rat TG; in immunoelectrophoresis it has a slower migration than native TG and is very close to that of enzymatically desialylated TG. Tumour TG has an iodine content of 0.02 % and a sialic content of 0.15 %. Chromatography of the 125I-labelled tumour TG revealed the presence of mono- and diiodothyrosines, a very small amount of triiodothyronine but no thyroxine.

These results suggest that tumour TG, isolated in the experimental rat thyroid tumour, previously shown not to incorporate sialic acid into thyroglobulin, has a different electrophoretic mobility from native TG but is very similar to desialylated rat TG and has a very low sialic acid and iodine content as well as a striking decrease in thyroxine.

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M Centanni, R Cesareo, O Verallo, M Brinelli, G Canettieri, N Viceconti and M Andreoli

Abstract

Objective: The aim of the study was to analyse the relationship between the ocular parameters, namely intraocular pressure (IOP), and the early forms of subclinical hypothyroidism.

Design: Fifty-three subjects (9 male and 44 female) aged from 18 to 45 years (mean 32±7 years) were selected for this study. Twenty-nine met the criteria of subclinical hypothyroidism and 24 euthyroid subjects, age- and sex-matched, were used as controls.

Methods: All individuals underwent a complete ocular examination, including visual field examination and serial measurement of IOP by means of a Goldmann tonometer. A tonographic examination was also performed.

Results: The hypothyroid patients showed a substantially higher pressure in both eyes compared with control subjects (right eye=17·52±4·74 vs 13·42±1·95 mmHg, p<0.0001; left eye=17·55±3·99 vs 13·71±1·55 mmHg, p<0.0001). Indeed, the tonometric pressure exceeded 18 mmHg in 11 out of the 29 (38%) patients in the right eye and in 8 out of 29 (27%) patients in the left eye. The outflow index was normal in all subjects except in two hypothyroid patients. After two months of L-thyroxine (L-T4) replacement therapy, only one patient continued to show tonometric values above 18 mmHg and the hypothyroid patients showed a significant reduction in mean IOP in both eyes compared with pretreatment values (right eye=14·96± 1·32 mmHg. p<0.0097; left eye=15·03± 1·38 mmHg, p<0.0018). Treatment did not lead to any change in the outflow indices; however, the C value (outflow coefficient at the sclerocorneal corner) returned to normal in the two patients with increased pre-treatment tonographic values.

Conclusions: These findings indicate that the intraocular pressure is increased even in subclinical hypothyroid patients and that, at this early stage, the impairment is fully reversible with L-T4 therapy.

European Journal of Endocrinology 136 595–598

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M Centanni, G Canettieri, N Viceconti, R Sibilla, A Bei and M Andreoli

OBJECTIVE: We have studied the effect of tryptophan on cellular [(125)I]tri-iodothyronine (T3) uptake by mouse thymocytes. MATERIALS AND METHODS: Mouse thymocytes (20 x 10(6 )cells/ml) were suspended in Krebs-Ringer solution buffered by Tris-HCl and incubation (23 degrees C at pH7.45+/-0.6), in the presence or absence of 1mM tryptophan, was started by adding 25 pM [(125)I]T3. At the end of incubation, samples were cooled in ice, centrifuged over a 30% sucrose cushion and the cell-associated radioactivity was measured in the pellet. RESULTS: Tryptophan reduced both the total and the saturable fraction of [(125)I]T3 uptake by 44% (P=0.0009) and 60% (P=0.0006) respectively, following 1 min of incubation. This effect was specific and dose-dependent, being maximal at 5mM concentration (-82%). In contrast, the pre-exposure of cells to tryptophan for up to 2h had no effect on the subsequent uptake of [(125)I]T3, in the absence of tryptophan. The effect of D-tryptophan on saturable T3 uptake was not different from that obtained using the L-stereoisomer. Tryptophan reduced the V(max) of the initial rate of saturable [(125)I]T3 uptake by two-thirds without affecting the apparent K(m) (2.2 nM) of the process, thus indicating the non-competitive nature of the inhibition. In sodium-free medium the saturable [(125)I]T3 uptake was reduced by 43%. The inhibitory effect of tryptophan on [(125)I]T3 uptake was exerted in both the presence and the absence of sodium. In fact, the inhibitory effect of tryptophan on T3 transport was greater and significantly different (P=0.0046) from that obtained by sodium depletion alone. CONCLUSIONS: Tryptophan interferes with both the sodium-dependent and -independent components of [(125)I]T3 uptake by a dose-dependent, non-competitive mechanism which operates in cis-modality at the plasma membrane level of mouse thymocytes.

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FS Celi, G Canettieri, D Mentuccia, L Proietti-Pannunzi, A Fumarola, R Sibilla, V Predazzi, M Ferraro, M Andreoli and M Centanni

OBJECTIVE: The selenoenzyme type 2 iodothyronine 5' deiodinase (DII) catalyzes the conversion of thyroxine into its active form tri-iodothyronine (T3), modulating thyroid hormone homeostasis in a local, tissue-specific manner. The amphibian, rodent and human cDNAs encoding this enzyme have been recently cloned and expressed. At present, little information regarding the genomic structure of mammalian DII is available. DESIGN AND METHODS: The complete structure, including intron-exon junctions, of the human DII (hDII) gene was obtained by long PCR and rapid amplification of cDNA ends (RACE). Chromosomal assignment of the hDII gene was performed by fluorescence in situ hybridization using a highly specific probe. RESULTS AND CONCLUSIONS: Our data demonstrated that hDII is a single copy gene located on chromosome 14, position 14q24.3. The gene spans over 15 kb, and the 7 kb transcript is encoded by three exons of 149 bp, 273 bp and 6.6 kb separated respectively by two 274 bp and 7.4 kb introns. A restriction map of the hDII gene is also reported. These data will help in further studies of the role of DII in the maintenance of peripheral thyroid hormone homeostasis.