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G Vassart

Publications and source records attributed to G Vassart.

At least 145 records · Page 8Linked to original sources
PubMed1994

Molecular genetics of thyroid diseases.

Over the past 20 years, recombinant deoxyribonucleic acid technology has led to the cloning of three major thyroid specific protein genes (thyroglobulin, thyroperoxidase and thyrotrophin receptor), which happen to be also the main thyroid autoantigens implicated in thyroid diseases. In this context, the impact that molecular genetics has had on the understanding of aetiopathogeny and diagnosis of thyroid diseases is summarized, with special emphasis on a recently discovered genetic mechanism responsible for toxic nodules. One fruitful outcome of the basic research on thyroid-specific gene expression has been the possibility of targeting the expression of a series of genes to the thyroid in transgenic mice. The result is the availability of mouse models mimicking human thyroid diseases such as destructive hypothyroidism, hyperactive thyroid adenomas and thyroid cancers exhibiting varying levels of differentiation.

Animals↗
PubMed1994

Overexpression of the extracellular domain of the thyrotrophin receptor in bacteria; production of thyrotrophin-binding inhibiting immunoglobulins.

The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21-414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting. To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1994

Importance of the extracellular domain of the human thyrotrophin receptor for activation of cyclic AMP production.

The mechanism by which the TSH receptor is activated is unknown. Current knowledge leads us to consider that G protein-coupled receptors are activated by positioning of their ligand in the pocket formed by the hydrophobic transmembrane segments. Furthermore, activation of an N-terminally truncated LH receptor lacking most of the extracellular domain has been described, suggesting the existence of a mechanism involving a direct interaction between LH and the transmembrane segments. The high conservation of the transmembrane segments among G protein-coupled receptors is a strong indication for a common mechanism of receptor activation. To test this hypothesis for the TSH receptor we have constructed four N-terminally truncated TSH receptor mutants with 5 or 69 amino acids of the extracellular domain joined to signal peptide regions consisting of the first 23 or 33 amino acids. The four fragments were amplified by PCR and subcloned into pBSK+. Sequences were confirmed after subcloning in M13. After joining the four fragments in pBSK+, the four TSH receptor constructs were subcloned in pSVL and transiently or stably expressed in COS and Chinese hamster ovary (CHO) cells respectively. In contrast to results obtained for the LH receptor, stimulation of the transfectants with 10 microM human chorionic gonadotrophin or 350 mU TSH/ml did not increase cyclic AMP (cAMP) concentrations in cultures of transiently transfected COS cells or stably transfected CHO cells. However, mRNA for the TSH receptor could be detected by RNase protection assay in all stable transfectants used for stimulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The human and dog 5-HT1D receptors can both activate and inhibit adenylate cyclase in transfected cells.

The cloned human serotonin 1D (5-HT1D) receptor has been shown to inhibit adenylate cyclase while the corresponding cloned dog receptor has been characterized by its enhancement of cAMP accumulation. To resolve this apparent discrepancy, the human 5-HT1D receptor has been cloned and expressed in Chinese hamster ovary (CHO) cells and the corresponding dog receptor expressed in mutant Y1 adrenal (Y1 Kin-8) cells. It is shown that both receptors when activated by sumatriptan depress forskolin induced adenosine 3'5'-cyclic monophosphate (cAMP) accumulation by a pertussis toxin sensitive mechanism, presumably involving Gi (the adenylate cyclase inhibitory GTP transducing protein). In the absence of forskolin, the dog receptor enhances cAMP accumulation, thus activating Gs (the adenylate cyclase stimulatory GTP transducing protein). When its overriding action on Gi is blocked by pertussis toxin pretreatment, the human receptor also enhances cAMP accumulation. Thus both 5-HT1D receptors activate markedly Gi and to a lesser extent Gs and can exert opposite effects on the same effector system, adenylate cyclase.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1993

Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas.

The pituitary hormone thyrotropin stimulates the function, expression of differentiation and growth of thyrocytes by cyclic AMP-dependent mechanisms. Tissue hyperplasia and hyperthyroidism are therefore expected to result when activation of the adenylyl cyclase-cAMP cascade is unregulated. This is observed in several situations, including when somatic mutations impair the GTPase activity of the G protein Gsa (ref 6, 7). Such a mechanism is probably responsible for the development of a minority of monoclonal hyperfunctioning thyroid adenomas. Here we identify somatic mutations in the carboxy-terminal portion of the third cytoplasmic loop of the thyrotropin receptor in three out of eleven hyperfunctioning thyroid adenomas. These mutations are restricted to tumour tissue and involve two different residues (aspartic acid at position 619 to glycine in two cases, and alanine at position 623 to isoleucine in one case). The mutant receptors confer constitutive activation of adenylyl cyclase when tested by transfection in COS cells. This shows that G-protein-coupled receptors are susceptible to constitutive activation by spontaneous somatic mutations and may thus behave as proto-oncogenes.

Adenoma↗
PubMed1993

The high-affinity interleukin 8 receptor gene (IL8RA) maps to the 2q33-q36 region of the human genome: cloning of a pseudogene (IL8RBP) for the low-affinity receptor.

The selective amplification by polymerase chain reaction (PCR) of gene fragments corresponding to new G-protein-coupled receptors resulted in the cloning of 18 orphan members of this gene family. Of these, three human clones amplified from genomic DNA (HGMP03, HGMP04, and HGMP05) were shown to be structurally related. Genomic clones corresponding to HGMP03 and HGMP05 were isolated and their putative coding region sequenced. Following the characterization of two interleukin 8 (IL-8) receptors, HGMP03 appeared to encode the high-affinity IL-8 receptor, whereas the partial clone HGMP04 encodes the low-affinity IL-8 receptor. Comparison with the cDNA sequence suggests that the high-affinity receptor gene is split by an intron in the 5' untranslated region. The high-affinity receptor gene was mapped by in situ hybridization to the 2q33-q36 region of the human genome. The HGMP05 locus turned out to be a pseudogene for the low-affinity IL-8 receptor (87% identity), with multiple frameshifts and point mutations introducing stop codons. Southern blotting on genomic DNA did not allow the further detection of related loci in the human genome.

Amino Acid Sequence↗
PubMed1993

Lack of evidence supporting the presence of mRNA for the thyrotropin receptor in extra-ocular muscle.

The search for autoantigenic targets in thyroid associated ophthalmopathy (TAO) and a possible link with the thyroid continues. Several authors have presented data suggesting that extrathyroidal tissues, e.g. extraocular muscle (EOM), may contain a functional thyrotropin receptor (TSH-R). We have applied Northern blotting and PCR amplification in an attempt to demonstrate the mRNA for the TSH-R in EOM, which although predominantly composed of muscle, may contain a significant proportion of fibroblasts. In Northern blots of poly(A) mRNA of normal human thyroid, EOM and skeletal muscle (SM) probed with a p32 labeled hTSH-R cDNA, transcripts of 4.6 and 4.4 kb were observed only in the thyroid. PCR amplification was performed on thyroid and EOM cDNAs using pairs of primers designed to amplify exons 1-->9, the extracellular domain (ECD), and exon 10, the intracellular domain (ICD). Both the ECD and ICD were amplified from a thyroidal cDNA library but only the ICD in the EOM cDNA library, this probably being due to the inevitable contamination with genomic DNA. These results do not support the hypothesis that the TSH receptor, as such, would be an autoantigen of EOM involved in TAO.

Adipose Tissue↗
PubMed1993

Human thyroid tissue do not express thyroalbumin.

Thyroid tissue total RNAs from multinodular goiter (G2) and from hereditary goiter with defective Tg synthesis (JNA) were hybridized with a 5'albumin cDNA probe (F-47), a 3' albumin cDNA probe (B-44) and a thyroglobulin cDNA probe (phTgM3). JNA refers to tissue obtained from a patient with virtual absence of Tg in thyroid tissue and the presence of increased concentration of an albumin-like labeled protein in the thyroid. No hybridization signal was detected in both G2 and JNA with albumin probes at Northern Blot studies. Those results were confirmed by dot-blot analysis of total RNA where no hybridization signal was detected in G2 and JNA. To confirm that thyroid tissues do not express thyroalbumin total RNA from JNA and normal control thyroid tissue (C) were amplified by PCR using albumin and Tg primers. An expected fragment of 592 bp was observed in a human liver sample with the albumin primers. However JNA and C samples showed absence of an amplification product of the same size. We concluded that thyroid cells do not contain the albumin transcript. Albumin is probably taken up from circulation and iodinated by the thyroid follicular cell with subsequent release of iodoalbumin into the circulation.

Albumins↗
PubMed1993

Human, bovine, canine and rat thyroglobulin promoter sequences display species-specific differences in an in vitro study.

The proximal promoter regions of the thyroglobulin gene from man, beef, dog and rat were compared by transient expression in primary cultured dog thyrocytes. All four promoter regions were able to control properly the expression of a reporter gene in response to cyclic AMP stimulation. Surprisingly, despite extensive sequence conservation, the transcriptional activities of these four mammalian thyroglobulin promoters were differently affected by equivalent mutations. Homologous sequence elements from these promoter regions also exhibited distinct binding characteristics in mobility-shift experiments conducted in the presence of nuclear proteins from bovine thyroids. Our observations show that the highly conserved thyroglobulin promoters may exhibit unexpected functional differences in a specific assay and indicate that some of the molecular mechanisms involved in the control of thyroglobulin gene expression have evolved differently within mammals.

Animals↗
PubMed1993

Olfactory receptors are displayed on dog mature sperm cells.

Olfactory receptors constitute a huge family of structurally related G protein-coupled receptors, with up to a thousand members expected. We have shown previously that genes belonging to this family were expressed in the male germ line from both dog and human. The functional significance of this unexpected site of expression was further investigated in the present study. We demonstrate that a few dog genes representative of various subfamilies of olfactory receptors are expressed essentially in testis, with little or no expression in olfactory mucosa. Other randomly selected members of the family show the expected site of expression, restricted to the olfactory system. Antibodies were generated against the deduced amino acid sequence of the most abundantly expressed olfactory receptor gene in dog testis. The purified serum was able to detect the gene product (DTMT receptor) in late round and elongated spermatids, as well as in the cytoplasmic droplet that characterizes the maturation of dog sperm cells, and on the tail midpiece of mature spermatozoa. Western blotting further confirmed the presence of a 40-kD immunoreactive protein in the membrane of mature sperm cells. Altogether , these results demonstrate that the main expression site of a subset of the large olfactory receptor gene family is not olfactory mucosa but testis. This expression correlates with the presence of the corresponding protein during sperm cell maturation, and on mature sperm cells. The pattern of expression is consistent with a role as sensor for unidentified chemicals possibly involved in the control of mammalian sperm maturation, migration, and/or fertilization.

Amino Acid Sequence↗
PubMed1993

The OLFR1 gene encoding the HGMP07E putative olfactory receptor maps to the 17p13-->p12 region of the human genome and reveals an MspI restriction fragment length polymorphism.

Olfactory receptors are believed to be encoded by an extremely large subfamily of G protein-coupled receptors. A human gene (OLFR1) encoding a member of this family was cloned from a genomic library by cross-hybridization with a gene fragment obtained by the polymerase chain reaction. The nucleotide sequence of a 3.4-kb EcoRI fragment was determined, and the protein sequence was deduced from the single open reading frame. The gene was assigned by in situ hybridization of metaphase chromosomes to the 17p13-->p12 region of the human genome, in proximity to the tumor-suppressor gene encoding p53. When used as a probe on Southern blots under moderately stringent conditions, it hybridizes to at least three closely related genes. A restriction fragment length polymorphism was detected after MspI digestion. Mendelian segregation of the gene was assessed in three CEPH families, and linkage analysis confirmed the localization of the locus.

Amino Acid Sequence↗
PubMed1993

Defective thyroglobulin synthesis and secretion causing goiter and hypothyroidism.

The integrity of the Tg structure as a protein is essential for adequate synthesis of thyroid hormone. Also a large supply of iodine and of thyroid hormone is stored into the Tg molecule and available for secretion on demand. Mutations in Tg gene or hyposialylated Tg due to a defective sialyltransferase activity would cause a structurally defective protein and severely impair the functional ability of Tg. In this review we attempt to cover the abnormalities in the synthesis of Tg described in both animals and man. Hereditary congenital goiter with or without hypothyroidism is the phenotypic major clinical finding in these species. Affected animals include sheep, bovine cattle, bongo antelope, goats, and mice. As in man the inheritance mode is autosomal recessive. In most animal studies structurally abnormal Tg is present. The molecular basis for the defective Tg synthesis was attributable to nonsense mutation in exon 9 (Afrikander cattle) and in exon 8 (Dutch goats). In man the Tg defective synthesis has been reported in 89 subjects and frequently more than one sibling is affected in a given generation. Characteristically these patients exhibit hereditary congenital goiter with relatively low Tg levels that do not increase after stimulation with bovine TSH. High PBI concentrations with low serum T4 values indicate the serum presence of iodinated proteins (mainly iodoalbumin). Also iodinated peptides are frequently excreted into the urine. Tissue studies confirm that there is an absent Tg peak at gel filtration, and virtually no immunoassayable Tg is present in the tissue extracts. The molecular basis of these defects have been recently reported in a patient and includes low tissue Tg mRNA probably due to premature degradation of a defective Tg mRNA. The responsible mutation is a cytosine to thymine transition creating a stop codon at position 1510. The point mutation is removed by the preferential accumulation of a 171 nt deleted Tg mRNA. In another subject molecular studies revealed that exon 4 was missing from the major Tg transcript due to a cytosine to guanine transversion at position minus 3 in the acceptor splice site of intron 3. It is anticipated that other mutations responsible for these defects will be identifiable in the near future.

Amino Acid Sequence↗
PubMed1993

Detection of thyroid-stimulating antibody using Chinese hamster ovary cells transfected with cloned human thyrotropin receptor.

A clone of Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) was used to optimize an assay for thyroid-stimulating antibody (TSAb), measuring adenylate cyclase stimulation by purified immunoglobulin G from patients with Graves' disease. Optimal sensitivity to bovine TSH (1 mU/L) and TSAb was obtained using hypotonic buffer and measuring extracellular cAMP. In time-response experiments, TSAb stimulation was maximal after 2 h of incubation in hypotonic buffer. Under these conditions, a significant stimulation by Graves' immunoglobulin G was obtained with 33 of 35 (94%) samples from patients with untreated Graves' disease and with 21 of 23 (91%) from patients who relapsed after a course of antithyroid drugs. On the other hand, TSAb was detected in only 12 of 20 (60%) patients who were euthyroid during methimazole treatment and in 4 of 11 (36%) who were euthyroid after a course of antithyroid drugs. All samples from Graves' patients were also tested for TSAb activity on FRTL-5 cells. The results of cAMP stimulation in FRTL-5 and CHO-R showed a fairly good correlation (r = 0.60; P < 0.0001). In particular, of the 58 patients with active Graves' disease (35 with untreated hyperthyroidism and 23 relapsed after methimazole), 43 (74%) were positive in both assays, 3 (5%) were negative in both, 11 (19%) were negative in FRTL-5 and positive in CHO-R, and 1 (1.7%) was negative in CHO-R and positive in FRTL-5. In conclusion, CHO cells transfected with the cloned human TSH receptor are suitable for the clinical assay of TSAb. The sensitivity of this assay is higher than that obtained using FRTL-5 cells, having the additional advantages of expressing the human TSH receptor and requiring less cumbersome procedures for cell culture.

Adenylyl Cyclases↗
PubMed1993

A nonsense mutation causes human hereditary congenital goiter with preferential production of a 171-nucleotide-deleted thyroglobulin ribonucleic acid messenger.

Defective or impaired thyroglobulin (Tg) synthesis usually results in congenital goitrous hypothyroidism, virtual absence of Tg in thyroid tissue, and the presence of an elevated concentration of iodoalbumin. The final result of these abnormalities is a decreased rate of T3 and T4 synthesis. We have previously reported two siblings with this syndrome that was attributable to decreased levels of thyroid tissue Tg mRNA, resulting in decreased translation of a fully mature Tg. Further molecular studies in this family are the subject of this report. The Tg mRNA from normal and goitrous thyroid tissue was first reverse transcribed and divided into five overlapping portions from positions 57-8448, and the resulting cDNAs were amplified by polymerase chain reaction and analyzed by agarose gel electrophoresis. The amplification of nucleotides (nt) 4502-5184 from control thyroid tissue Tg mRNA showed a predominant fragment of 683 basepairs (bp) and a minor fragment of 512 bp. This latter fragment contained a 171-nt deletion that mapped between positions 4567 and 4737 of the Tg mRNA. In contrast, the fragment predominantly present in the congenital goiter was 512 bp. The sequencing of the 683-bp fragment revealed that the responsible mutation is a cytosine to thymine transition, creating a stop codon at position 1510. This results in loss of a TaqI restriction site. The point mutation is, thus, removed from a portion of the transcripts by the preferential accumulation in the goiter of a 171-nt-deleted Tg mRNA. The reading frame is maintained and is potentially fully translatable into a Tg polypeptide chain shorter by 57 residues. The presence of the deleted Tg mRNA in normal thyroid tissue, albeit at a low level, strongly suggests that the deleted mRNA sequence corresponds to a complete exon. Our studies suggest that the shorter, alternatively spliced Tg mRNA predominates in the goitrous tissue and probably has a shorter half-life. This would explain the tissue's low Tg mRNA levels, previously reported. Moreover, translation of the mutated transcript would generate a severely truncated Tg polypeptide with limited ability to generate thyroid hormone, resulting in congenital goitrous hypothyroidism.

Amino Acid Sequence↗
PubMed1993

[Molecular genetics of thyroid diseases].

Molecular cloning of the genes implicated in the synthesis of thyroid hormones and in the control of thyroid growth and function has led to the identification of mutations responsible for hereditary thyroid diseases (congenital hypo- and hyperthyroidism) and for hyperfunctional thyroid adenomas.

Congenital Hypothyroidism↗
PubMed1993

Thyroperoxidase mRNA in quiescent and proliferating thyroid epithelial cells: expression and subcellular localization studied by in situ hybridization.

Using in situ hybridization procedure, we have investigated the regulation and the cellular localization of thyroperoxidase (TPO) messenger RNA accumulation as a marker of differentiation in dog thyroid epithelial cells in primary culture. The response to different mitogens (TSH acting through cAMP, EGF and TPA) has been compared. TPO mRNA accumulation was exquisitely dependent on a continuous TSH/cAMP stimulation. It was induced within 1 h in the whole cell population from a very low basal level. This effect was inhibited by the cAMP-independent mitogens EGF and TPA. By contrast, the TSH-induction of TPO mRNA accumulation was observed irrespectively of the proliferative activity of the cells, i.e. in the presence or the absence of insulin, which is required for mitogenesis. The short half-life of TPO mRNA (+/- 2 h) implies that it was continuously transcribed during TSH/cAMP-dependent cell cycling. As compared to another thyroid differentiation marker, thyroglobulin mRNA (Pohl et al., J. Cell Biol. 111, 663-672 (1990)), TPO mRNA accumulation differed by the rapidity of its control by cAMP, the pattern of its intercellular heterogeneity, and the unexpected segregation to a perinuclear region, probably the nuclear envelope that constitutes a specialized part of the endoplasmic reticulum. Despite these differences, both TPO and thyroglobulin gene transcriptions are unequivocally compatible with the cell cycle when induced by cAMP, at variance with the generally observed antagonism between growth and differentiation expression.

Animals↗