Search PubMed⌕ Search

Biomedical subjects

S Lissitzky

Publications and source records attributed to S Lissitzky.

At least 37 records · Page 2Linked to original sources
PubMed1984

The relationships between ribosomal genes and fibrillar centers in thyroid cells cultivated in vitro.

Despite the fact that the fibrillar centers of the nucleolus and the chromosomal nucleolar organisers (NORs) are similarly stained with the NOR-silver technique, there remain some questions about the identification of fibrillar centers as NORs. The distinct delineation of the fibrillar centers in porcine thyroid cells allowed us to determine whether there was a numerical equivalence or correlation between fibrillar centers and NORs. Hybridization in situ and silver staining performed on pig chromosomes showed that pairs 8 and 10 contained rDNA sites. Silver staining of thyroid cells in electron microscopy showed that the fibrillar centers and their surrounding layer of dense fibrils were the sites of silver deposit. Chromatin fibers were demonstrated within the fibrillar centers through the aid of the osmiumammine reaction and with the oxidized diaminobenzidine technique. It was observed that in cultured thyroid cells the fibrillar centers could be identified in the light microscope as argyrophilic spherules, and easily counted. The number of fibrillar centers was variable according to culture conditions. In cells cultured for 5 hr, the mean number of fibrillar centers was 1.7. After 5 days of culture, the number of fibrillar centers increased, reaching a mean value of 5.93. When thyroid cells were stimulated with thyrotropin, the number of fibrillar centers again increased to a mean value of 7.54. These results demonstrate that the relationship between fibrillar centers and NORs is not a simple proportionality: the number of fibrillar centers increases with increased cellular activity. These data imply that in active cells each NOR may pass through several fibrillar centers.

Animals↗
PubMed1984

[Relation between the iodination of human thyroglobulin and the cleavage of the hormone peptide 26K N-terminal].

At moderate iodination levels (about 20 iodine atoms/mol) human thyroglobulin yields after reduction and alkylation a hormone (T4)-containing N-terminal peptide of 26K. Further iodination of the thyroglobulin in vitro results in the cleavage of this part of the molecule into smaller peptides of 22K and 18K. A precursor-product relationship between the 26K peptide segment and the latter was established by showing an identical N-terminal T4-containing sequence in the 3 peptides. Cleavage of peptide bonds in the 26K segment to give the smaller fragments could possibly be related to the formation of another hormone residue.

Amino Acid Sequence↗
PubMed1984

[Comparison of quantity of thyroxine in thyroglobulin and the peptide hormones derived from chromatography and immunoenzyme assay].

An evaluation of the thyroxine content in thyroglobulin and different derived thyroxine-containing peptides by enzyme immunoassay is presented. Results correlate very well with those obtained by ion-exchange chromatography which served as a reference method: r = 0.997, p less than 10(-6). Enzyme immunoassay is more rapid and sensitive than ion-exchange chromatography and then suitable for routine use. Furthermore, this paper shows, for the first time, that enzyme immunoassay can be used to perform direct thyroxine estimation in small peptide (Mr less than 2,500) without previous total enzymatic hydrolysis.

Animals↗
PubMed1983

Amino acid sequence around a hormonogenic tyrosine residue in the N-terminal region of human thyroglobulin after in vivo and in vitro iodination.

Reduced and S-alkylated human thyroglobulin (hTgb), normally iodinated, was previously shown by SDS/PAGE to contain a small peptide (Mr 26 000) rich in thyroxine. This peptide was not found when very poorly iodinated hTgb was treated under the same conditions but was present after iodination in vitro. Peptide 26 K was purified from in vivo and in vitro iodinated hTgb. The study of these peptides shows that: 1/ at the iodination levels studied (0.18 and 0.25% iodine) one of the preferential hormonogenic sites is the same in hTgb iodinated both in vivo and in vitro; 2/ the amino acid sequence around the thyroxine residue: Asn-Ile-Phe-Glu-T4-Gln-Val is identical with the previously described hormonogenic site of bovine thyroglobulin. Most probably the hormone-containing peptide 26 K is the N-terminal peptide of the hTgb chain.

Amino Acid Sequence↗
PubMed1983

In vitro and in vivo iodination of human thyroglobulin in relation to hormone release.

Reduced and S-alkylated thyroglobulin (Tgb) from different species were shown by SDS-PAGE to contain small peptides (from 45-9 kDa) rich in thyroxine. Several hypotheses were proposed to explain their origin. The polypeptide composition of iodine-poor (Tgb A) and normally iodinated (Tgb B) human Tgb prepared by two different procedures (one minimizing and the other favoring post-mortem proteolysis) was compared in the native state and after in vitro iodination. Results show that one of the hormonogenic sites of human Tgb is part of a domain of the molecule most susceptible to proteolysis, especially when it is very iodinated.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Amino acid sequence of the unique 3,5,3'-triiodothyronine-containing sequence from porcine thyroglobulin.

A unique T3-containing peptide was isolated by chemical and enzyme treatments from pTgb of different iodine contents and microsequenced: Leu-Ala-Ser-Lys-Ser-T3. This site can also form T4 to a small extent. It contains all the T3 of the protein and presents a special susceptibility to proteolysis thus suggesting a likely mechanism to explain that the thyroid secretes T3 preferentially to T4 in vivo.

Amino Acid Sequence↗
PubMed1983

The effect of triiodothyronine on fatty acid synthetase activity and content in differentiating ob17 preadipocytes.

The effect of triiodothyronine on the activity and amount of the key lipogenic enzyme fatty acid synthetase was studied in differentiating preadipocyte cells (ob17) isolated from ob/ob mouse epididymal fat pad. In the presence of physiological concentrations of insulin, the acquisition of adipose morphology was accompanied by a parallel increase (10--15-fold) in synthetase specific activity and radioimmunoassayable amount relative to soluble cellular proteins. Inclusion of T3 at confluence significantly enhanced synthetase activity and content, with a maximum of 1.5--2-fold above controls at the physiological 1.5 nM concentration, whether insulin is present or not. During adipose conversion, T3 increased the development of enzyme activity and after a longer lag period, the accumulation of the synthetase. Our results suggest that the stimulating effect of T3 upon synthetase activity could involve as a first step the activation of preexisting inactive synthetase molecules and as a second one an increased accumulation of activable synthetase. After longer culture periods, inactive radioimmunoassayable synthetase accumulated.

Adipose Tissue↗
PubMed1983

Specificity of monoclonal antibodies against human thyroglobulin; comparison with autoimmune antibodies.

Ten monoclonal antibodies (mAb) directed against human thyroglobulin (hTgb) were produced, purified and characterized. The mAb avidity for hTgb ranged from 10(-10) to 10(-6) M. The species specificity of the mAb was as follows: eight mAb reacted with monkey Tgb, three with dog Tgb and one with pig Tgb; none with bovine and ovine Tgb. The binding of mAb to hTgb was not significantly inhibited in the presence of Tgb carbohydrate moieties, tyrosine, iodotyrosines and iodothyronines. The topology of the antigenic determinants recognized by the 10 mAb on hTgb was explored by inhibition of Tgb binding of radiolabeled mAb by the other antibodies. Six distinct clusters of reactivity were described. Localization of the antigenic determinants recognized by mAb on hTgb was attempted using tryptic fragments of hTgb to inhibit the binding of mAb to hTgb. The inhibitory effect of hydrolysis products was different for each mAb but exhibited partial analogies between mAb of the same cluster of reactivity. Anti-hTgb autoimmune antibodies (aAb) purified from sera of Graves patients cross-reacted essentially with mAb of one out of the six clusters. These results demonstrate that the large number of antigenic determinants presented by the hTgb are not disseminated on the molecule but are clustered in antigenic regions. Furthermore, from the six antigenic regions evidenced in this paper, only one is involved in autoimmune antibody production in Grave's disease.

Animals↗
PubMed1983

Heterogeneity of the Graves' immunoglobulins directed toward the thyrotropin receptor-adenylate cyclase system.

The TSH-displacing and the thyroid-stimulating activities of Graves' immunoglobulins G (GIgG) have been accounted for by either homogeneous TSH receptor antibodies or heterogeneous antibodies directed toward the TSH receptor-adenylate cyclase system. To clarify this matter, the study of the interactions of GIgG preparations from 14 untreated Graves' patients with human thyroid membranes was undertaken. Dose-response curves of GIgG, diluted in IgG from normal subjects, were carried out in the [125I]TSH radioreceptor assay and the adenylate cyclase assay in the presence or absence of TSH. In the radioreceptor assay, GIgG were constantly negative in 3 cases (21%) and positive, depending of the dose, in 11 cases. In the adenylate cyclase assay, the dose-activity profiles in the absence of TSH were bell-shaped curves in 3 cases and sigmoid curves in 9 cases; in 2 cases (14%), GIgG preparations were devoid of any effect. Binding-isotherms and dose-activity profiles did not appear to share simple relationships. In the presence of TSH, GIgG preparations elicited a decrease in 6 cases, an increase in 3 cases, and no effect in 5 cases (36%) in the adenylate cyclase activity. The data obtained by radioreceptor assay and adenylate cyclase assay in the presence or absence of TSH were found statistically correlated (P less than 0.05 to P less than 0.001) but not linearly related, the points being scattered on specific parts of the diagrams. These observations could not be accounted for by TSH receptor antibodies in GIgG being an entity of constant properties, albeit varying in titer among patients. Rather, GIgG effects fit well the patterns of action of a heterogeneous ligand, as shown by computing a theoretical model for ligand heterogeneity with respect to binding equilibrium constant and intrinsic biological activity. Accordingly, GIgG activity in the TSH receptor-adenylate cyclase system could be attributed to heterogeneous antibodies varying with regard to binding constant and acting as stimulating or blocking antibodies of the adenylate cyclase.

Adenylyl Cyclases↗
PubMed1983

Quantitative in situ hybridization of 3H-labeled complementary deoxyribonucleic acid (cDNA) to the messenger ribonucleic acid of thyroglobulin in human thyroid tissues.

Thyroglobulin (Tgb) mRNA content was studied in human thyroid tissues using liquid hybridization and in situ hybridization. Liquid hybridization revealed no differences in mRNA content, except in the case of colloid adenoma in which a lower amount of Tgb mRNA was found. Conditions for quantitative in situ hybridization of [3H]DNA complementary to the mRNA of Tgb are described. In situ hybridization allowed correlation of the morpho-functional state of the follicles and their content of Tgb mRNA.

Adenoma↗
PubMed1983

A sensitive method for assaying thyroid stimulating immunoglobulins of Graves' disease: use of the guanyl nucleotide-amplified thyroid adenylate cyclase assay.

The purpose of this study was to develop and validate a sensitive method for evaluating adenylate cyclase stimulation by thyroid-stimulating antibodies (TSAb), based on the measurement of thyroid membrane adenylate cyclase activity in the presence of a non-hydrolyzable GTP analogue, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). The addition of Gpp(NH)p (10(-5)M) produced a 10-fold increase of the sensitivity of the system for both TSH and TSAb. Immunoglobulin G preparations from sera of 30 patients with Graves' disease were tested for the adenylate cyclase stimulation either in the presence or in the absence of Gpp(NH)p: a significant stimulation was observed in 27/30 patients when the GTP analogue was added to the system, while only 20/30 patients were positive in the absence of the nucleotide. The advantage of Gpp(NH)p addition was also evident in a large series which included 57 patients with Graves' disease, 15 with Hashimoto's thyroiditis or primary myxoedema and 22 normal subjects. In fact, 88% of patients with Graves' disease resulted positive, while no significant stimulation was elicited by Hashimoto's thyroiditis, primary myxoedema and by normal immunoglobulins. The sensitivity achieved in our system which employs thyroid plasma membranes was similar to that obtained by other investigators with the use of thyroid slices or thyroid cells in primary culture. Furthermore, methods based on thyroid plasma membranes are supposed to have a better reproducibility, since the same tissue preparation, if appropriately stored, may be used in several different tests.

Adenylyl Cyclases↗
PubMed1982

Structure-function relationship in thyroglobulin: amino acid sequence of two different thyroxine-containing peptides from porcine thyroglobulin.

From the cyanogen bromide (CNBr) treatment of porcine thyroglobulin a peptide of mol. wt. 15 000, CNBr-b1, was purified by gel filtration and ion-exchange chromatography. CNBr-b1 contained 50% of the thyroxine (T4) content of the protein. After digestion with trypsin and protease from Staphylococcus aureus V-8, thyroxine-containing peptides were purified and analyzed by microsequence analysis using the colored Edman's reagent dimethylaminoazobenzeneisothiocyanate . Two different sequences harboring T4 were identified: sequence 1, His-Asp-Asp-Asp-T4-Ala-Thr-(Glx,Gly)-Leu-Tyr-Phe-Ser-Ser-Arg, which contains 1 mol T4/mol peptide and sequence 2, Asp-(Tyr/MIT/DIT/T4)-Phe-Ile-Leu-X-Pro-Val-, which is a mixture of the same peptide at different levels of iodination and coupling. These sequences are likely to be representative of distinct hormonogenic sites, the former giving evidence of early iodinated tyrosine residues where preferential coupling into hormonal residues occurs especially at low iodine levels and the latter representing less reactive site(s) operative at higher iodine levels.

Amino Acid Sequence↗
PubMed1982

Reorganization of porcine thyroid cells into functional follicles in a chemically defined, serum- and thyrotropin-free medium.

In the serum-free, chemically defined medium NCTC 109, freshly isolated porcine thyroid cells aggregate and form functional follicles in culture even in the absence of thyrotropin. The follicular pattern observed under light and electron microscopy express the main morphological characteristics of in vivo thyroid cells. Follicles are large, replete with dense colloid, and the apical pole of cells is characterized by well-developed microvilli and the presence of aminopeptidase N. The index of iodide transport activity (125I-C/M ratio) decreases vs. days of culture to a resting value of about 1 or 2 at day 2. Addition of thyrotropin (200 microU/ml final concentration) at day 4 is followed by a 10-fold increase in iodide transport activity within 24 h and a 40-fold increase 4 d later. Incorporation and organification of iodide are dose dependent between 0 and 250 microU/ml thyrotropin; highest concentrations (4,000--16,000 muU/ml) are significantly inhibitory. In the absence of thyrotropin each cell synthesizes 8.2 pg thyroglobulin/d. Acute stimulation by thyrotropin at day 4 resulted in a slight decrease in the quantity of thyroglobulin present in the cell layer but in an increase in the total amount of thyroglobulin recovered in both cells and medium, reaching 34.3 pg/cell/d. The protein exported into the medium is thyroglobulin, as shown by SDS PAGE and immunological properties. Here we demonstrate that porcine thyroid cells can be maintained in culture as resting, highly differentiated, follicular-associated cells, sensitive to acute stimulation by thyrotropin.

Animals↗
PubMed1981

Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA.

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.

Biotransformation↗