PubMed Health⌕ Search

Biomedical subjects

O E Janssen

Publications and source records attributed to O E Janssen.

At least 37 records · Page 2Linked to original sources
PubMed2003

[Drug treatment of immune hyperthyroidism (Basedow disease). Patient selection, long-term follow-up and prevention of recurrence].

Since theraphy of Graves' disease is not directed towards the cause of the disease, medical theraphy is still the first choice and symptomatically effective in treating hyperthyroidism. Antithyroid drugs are effective in restoring euthyroidism in >90% of the patients durning 4-6 weeks, but 30-50% of the patients experience relapse after withdrawal. Previous prospective randomized studies evaluated prognostic parameters and the use of levothyroxine for prevention of relapse of hyperthyroidism. Recent studies have addressed the period after withdrawal amd focused on the natural course of disease. The results of a recent prospective randomized and controlled multicenter study were as follows: supplementation of levothroxine does not prevent relapse of hyperthyroidism. Basal TSH (4 weeks after withdrawal of antithyroid drugs) is the best prognostic marker. Smoking and positive TSH-receptor-antibodies at the end of antithyroid theraphy are other risk factors.

Antithyroid Agents↗
PubMed2003

Specificity of the neuroendocrine response to orgasm during sexual arousal in men.

We have demonstrated that sexual activity produces transient sympathoadrenal activation and a pronounced, long-lasting increase in prolactin in men and women. However, by analyzing endocrine alterations at 10-min intervals, a precise assignment of these changes to the pre-, peri- and postorgasmic periods was not possible. Thus, the current study aimed to accurately differentiate the endocrine response to sexual arousal and orgasm in men using an automatic blood collection technique with 2-min sampling intervals. Blood was drawn continuously before, during and after orgasm over a total period of 40 min in 10 healthy subjects and were compared with samples obtained under a control condition. Sexual activity induced transient increases of plasma epinephrine and norepinephrine levels during orgasm with a rapid decline thereafter. In contrast, prolactin levels increased immediately after orgasm and remained elevated throughout the experiment. Although oxytocin was acutely increased after orgasm, these changes were not consistent and did not reach statistical significance. Vasopressin, LH, FSH and testosterone plasma concentrations remained unaltered during sexual arousal and orgasm. These data confirm that prolactin is secreted after orgasm and, compared with oxytocin, seems to represent a more reliable and sustained marker for orgasm in man. The results further reinforce a role for prolactin either as a neuroendocrine reproductive reflex or as a feedback mechanism modulating dopaminergic systems in the central nervous system that are responsible for appetitive behavior.

Adolescent↗
PubMed2002

The tumour necrosis factor-alpha induced vascular permeability is associated with a reduction of VE-cadherin expression.

BACKGROUND: Vascular permeability is controlled by endothelial cell-to-cell junctions. Vascular endothelial (VE)-cadherin, the major component of adherence junctions (AJ) in the endothelium, is the target of the permeability-increasing agent tumour necrosis factor-a (TNF-alpha). We investigated whether TNF-alpha regulates the synthesis of VE-cadherin on the transcriptional level. MATERIALS AND METHODS: Human endothelial cells, isolated from aorta (aEC) and umbilical cord (HUVEC), were exposed to TNF-alpha (200 U/ml) for 6 h or 12 h, with and without subsequent incubation with TNF-alpha for 24 h. VE-cadherin mRNA was evaluated by semi-quantitative RT-PCR. The VE-Cadherin protein expression was analyzed by flow cytometry (FACS). RESULTS: The VE-cadherin amplification curve of TNF-alpha treated cells was shifted to the right compared to controls, indicating a lower mRNA amount. The number of cycles at which half-maximal amplification (N50) was achieved, was lower for control aEC (30.7) than for TNF-alpha treated aEC (33.0). The N50 of HUVEC treated with TNF-alpha for 12 h and 12 h + 24 h medium (N50 = 32.1), was higher compared to controls (N50 = 29.7) and cells treated with TNF-alpha for 6 h (N50 = 30.8). As determined by FACS analysis, incubation with TNF-alpha caused a small decrease of VE-cadherin protein expression from 7.44 to 6.05 mean channel intensity. CONCLUSION: Our results indicate that TNF-alpha affects VE-cadherin gene expression on the transcriptional level, inducing a downregulation of the VE-cadherin expression.

Antigens, CD↗
PubMed2002

Long-term follow-up of thyroid nodule growth.

Benign thyroid nodules are common in iodine deficient countries. Although many recent studies have addressed the molecular basis and short-term outcome of treatment in nodular thyroid disease, data on the long-term follow-up of thyroid nodule growth are widely lacking. The aim of the present study was to evaluate the long-term behaviour of benign thyroid nodules growth. We followed 109 consecutive patients seen at yearly intervals in our Outpatient Clinic for at least 3 years (range 3-12 years, mean 4.9 +/- 2.6 years) presenting with 139 benign nodules in uni- or multinodular goiters. The size of the nodules and thyroid glands was analysed retrospectively. The study included a spectrum of benign thyroid nodules, 86 functioning and 53 non-functioning. 27 patients were treated with levothyroxine, 8 with iodide and 16 with a combination of both. 58 patients were not treated mainly because of thyroid functional autonomy. Patients with overt hyperthyroidism or suspected malignancy by fine-needle aspiration were excluded from the study. The nodules and glands were assessed by ultrasonography at yearly intervals and documented by photoprints. Relevant growth was defined as an increase in nodule volume of at least 30%. For statistical analyses, Cox Proportional Hazard Model and life-table analyses according to Kaplan-Meier were performed. Most thyroid nodules grew slowly but continuously during follow-up. After about 3 years, half of the nodules had increased their volume by at least 30%. Growth of the nodules was significantly faster than of the corresponding thyroid glands (p < 0.0001). Age and sex of the patients and size or function of the nodules at initial presentation were not significantly related to their growth. Suppression of TSH did not affect growth of the nodules irrespective of the source of thyroid hormones, endogenous or by administration of levothyroxine. In conclusion, benign thyroid nodules have a slow intrinsic growth potential, which is apparently higher than that of the non-nodular tissue. In this study, not only nodular but even non-nodular goiter growth continues in the majority of patients. Exogeneous factors, including therapy with levothyroxine and/or iodide, appear to have little effect on the growth behaviour.

Adolescent↗
PubMed2002

Role of conventional ultrasound and color Doppler sonography in the diagnosis of medullary thyroid carcinoma.

Early diagnosis of medullary thyroid carcinoma (MTC) can improve long-term prognosis. MTC can be detected at an early stage by calcitonin screening in all patients with thyroid nodules. This approach, however, is controversial due to high costs and a limited specificity of calcitonin. It was the aim of the present study to investigate whether ultrasonography may contribute to the diagnosis of MTC in patients with thyroid nodules. The study included 19 patients with newly diagnosed MTC. Ten patients had sporadic MTC, 7 had multiple endocrine neoplasia (MEN) type 2A, and 2 patients had MEN 2B. In all subjects conventional ultrasound, and in 14 patients color Doppler sonography were performed before primary surgery. For comparison, ultrasound appearance of 139 benign thyroid nodules was evaluated. In conventional ultrasound, MTC in 17/19 (89%) patients were hypoechoic, contained intranodular calcifications, and had no "halo sign". The combination of these criteria were found in only 8/139 (6%) of benign thyroid nodules. Intranodular blood flow was found in 11/14 patients with MTC (79%), perinodular blood flow in 7/14 MTC (50%). In conclusion, conventional ultrasound reveals a combination of hypoechogenicity, intranodular calcifications, and absence of "halo sign" in the vast majority of MTC. Since this sonographic pattern only rarely occurs in benign thyroid nodules, the results indicate that thyroid ultrasound can contribute to the diagnosis of MTC. In addition, the findings may have implications for calcitonin screening in nodular thyroid disease.

Adult↗
PubMed2002

RT-PCR-based detection of circulating calcitonin-producing cells in patients with advanced medullary thyroid cancer.

In patients with medullary thyroid carcinoma (MTC) the clinical course of disease ranges from rapid tumor progression to long-lasting stable disease. The purpose of the present study was to investigate whether circulating tumor cells can be detected in the peripheral blood of patients with MTC by RT-PCR targeted to calcitonin (CT) mRNA and whether the results of this method are correlated with disease manifestation and metastatic potential. Blood samples from 19 consecutive patients with MTC and elevated CT levels were analyzed. Four had newly diagnosed MTC, and 15 patients had undergone total thyroidectomy. Six of 19 patients had detectable CT mRNA by RT-PCR. CT levels in the CT mRNA-positive patients were significantly higher than those in CT mRNA-negative patients [2,239-265,313 pg/ml; median 80,921 pg/ml (n = 6) vs. 70-46,787 pg/ml; median, 932 pg/ml (n = 13); P = 0.006]. CT mRNA was detectable in 5 of 8 patients with distant metastases, in 1 of 6 patients with local/regional lymph node metastases, but in none of the patients with newly diagnosed, organ-confined MTC (n = 2) or surgically treated MTC without tumor manifestation by various imaging studies (n = 3). In peripheral blood from 10 healthy volunteers and 21 patients with benign thyroid nodules, no CT RNA could be detected. In conclusion, an RT-PCR-based procedure was established to detect circulating CT-producing cells in the peripheral blood of patients with MTC. RT-PCR results seem to reflect tumor spread and aggressiveness and thus may help with early identification of patients with disseminated and rapidly progressive disease.

Adult↗
PubMed2001

Three novel mutations causing complete T(4)-binding globulin deficiency.

Inherited T(4)-binding globulin deficiency is caused by mutations in the T(4)-binding globulin gene located on the X chromosome. We describe herein three novel mutations in three different families producing complete T(4)-binding globulin deficiency. The proposita of a family from Harwichport is a female with XO Turner's syndrome who expressed only the mutant T(4)-binding globulin allele. Her T(4)-binding globulin sequence has a 19-nucleotide deletion in the distal portion of exon 4. This causes a frameshift and a premature stop at codon 384 of the mature protein. Structure analysis with the Swiss PDB-Viewer revealed that this mutation removes beta-strand s5B from the core of the T(4)-binding globulin molecule, leading to a severe folding defect that is likely to prevent synthesis and secretion. The propositi of complete T(4)-binding globulin deficiency 7 and 8 were 7-month-old and 3-wk-old male infants who were identified because of low serum T(4) levels detected during neonatal screening. Sequencing of complete T(4)-binding globulin deficiency 7 revealed a single nucleotide deletion, a G at position 2690 in exon 3. This leads to an alteration of the amino acid sequence starting at codon 283 and a premature stop at codon 301. Complete T(4)-binding globulin deficiency 8 also has a deletion of the first nucleotide of exon 4, a G at position 3358. This leads to a frameshift and a premature stop at codon 374. As in the case of complete T(4)-binding globulin deficiency J, which has also a nucleotide deletion but downstream (position 3421) and a stop at codon 374, these two T(4)-binding globulin mutants undoubtedly have a defect in intracellular transport and therefore fail to be secreted. This explains the lack of T(4)-binding globulin in the hemizygous affected subjects.

Adolescent↗
PubMed2000

Identification of thyroxine-binding globulin-San Diego in a family from Houston and its characterization by in vitro expression using Xenopus oocytes.

T4-binding globulin (TBG) is a liver glycoprotein that transports iodothyronines in serum. Several TBG variants with reduced T4 binding affinity have been described, all of which are also characterized by reduced serum TBG concentrations and reduced heat stability. Their loss of binding thus appears to be due to a general defect of the molecule. We now report the occurrence of a variant TBG, detected in a family from Houston, TX, with half the normal T4 binding affinity and heat stability but normal serum concentration and isoelectric focussing pattern. The propositus was identified by reduced total T4 and T3 serum levels. All family members were euthyroid, and inheritance followed an X-linked pattern. Sequence analysis of the TBG gene of the propositus and his heterozygous mother revealed two amino acid substitutions: serine 23 with threonine (S23T), and the known polymorphism leucine 283 with phenylalanine (L283F). These substitutions are identical to those of TBG-San Diego (TBG-SD), a variant with similar properties except for a reduced serum concentration. Expression of recombinant TBG-SD/H with the S23T substitution in Xenopus oocytes reproduced the binding defect and heat lability. The amount of TBG-SD/H synthesized and secreted by the oocytes was not different from that of normal TBG. The difference in serum TBG concentrations in affected members of the San Diego and Houston families thus does not appear to be due to an error in the measurement of TBG, but may be related to differences in the rates of degradation.

Adult↗
PubMed2000

Effects of growth hormone replacement therapy on levels of cortisol and cortisol-binding globulin in hypopituitary adults.

OBJECTIVE: To determine if human growth hormone (hGH) replacement therapy alters pharmacokinetics of hydrocortisone (CS) substitution in hypopituitary adults. DESIGN: To this aim, we analysed serum and salivary CS profiles 270 min after oral CS administration at baseline and 6 and 12 months after initiation of hGH replacement therapy. METHODS: Serum IGF-I, cortisol-binding globulin (CBG), thyroxine-binding globulin (TBG) and sex hormone-binding hormone (SHBG) were measured using commercially available radioimmunoassays. In-house immunofluorometric assays were employed for measurements of CS and hGH. RESULTS: hGH replacement did not change total serum CS bioavailability (area under the serum cortisol profile curve). Interference of orally administered CS with salivary measurement of free CS (fCS) caused significant bias. Therefore, fCS levels were calculated from their total CS and cortisol-binding globulin (CBG) levels. CBG decreased by approximately 30% after both 6 and 12 months of hGH replacement therapy (n=20, P<0.01). A significant negative correlation between deltaCBG (CBG6months-CBGbaseline) and deltaIGF-I (IGF-I6months-IGF-Ibaseline) was observed (P=0.04). The calculated values of free CS tended to increase with physiological hGH replacement, but this effect was marginal and did not reach statistical significance. In contrast to the CBG concentrations, plasma levels of sex hormone-binding globulin and thyroxine-binding globulin were essentially stable. CONCLUSION: Given that no clinically relevant alterations in pharmacokinetics of CS were evoked by initiation of hGH replacement in hypopituitary adults, we conclude that CS substitution does not require dose adjustment after initiation of hGH replacement.

Adult↗
PubMed1999

Modularity of serpins. A bifunctional chimera possessing alpha1-proteinase inhibitor and thyroxine-binding globulin properties.

An exciting application of protein engineering is the creation of proteins with novel functions by the retrofitting of native proteins. Such attempts might be facilitated by the idea of a mosaic architecture of proteins out of structural units. Even though numerous theoretical concepts deal with the delineation of structural "modules," their potential in the design of proteins has not yet been sufficiently exploited. To address this question we used a gain of function approach by designing modular chimeric molecules out of two structurally homologous but functionally diverse members of the superfamily of serine-proteinase inhibitors, alpha1-proteinase inhibitor and thyroxine-binding globulin. Substitution of two of four alpha1-proteinase inhibitor modules (Lys222 to Leu288 and Pro362 to Lys394, respectively), identified by alpha-backbone distance analysis, with their thyroxine-binding globulin homologues resulted in a bifunctional chimera with inhibition of human leukocyte elastase and high affinity thyroxine binding. To our knowledge, this is the first report on a bifunctional chimera engineered from modules of homologous globular proteins. Our results demonstrate how a modular concept can facilitate the design of new functional proteins by swapping structural units chosen from members of a protein superfamily.

Humans↗
PubMed1999

Characterization of the thyroxine-binding site of thyroxine-binding globulin by site-directed mutagenesis.

The principal transport protein for T4 in human blood, thyroxine-binding globulin (TBG), binds T4 with an exceptionally high affinity (Ka = 10(10) M(-1)). Its homology to the superfamily of the serpins has recently been used in the design of chimeric proteins, providing experimental evidence that an eight-stranded beta-barrel domain encompasses the ligand-binding site. We have now characterized the T4 binding site by site-directed mutagenesis. Sequence alignment of TBG from several species revealed a phylogenetically highly conserved stretch of amino acids comprising strands 2B and 3B of the beta-barrel motif. Mutations within this region (Val228Glu, Cys234Trp, Thr235Trp, Thr235Gln, Lys253Ala, and Lys253Asp), designed to impose steric hindrance or restriction of its mobility, had no significant influence on T4 binding. However, binding affinity was 20-fold reduced by introduction of an N-linked glycosylation site at the turn between strands 2B and 3B (Leu246Thr) without compromising the proper folding of this mutant as assessed by immunological methods. In most other serpins, this glycosylation site is highly conserved and has been shown to be crucial for cortisol binding of corticosteroid-binding globulin, the only other member of the serpins with a transport function. The ligand-binding site could thus be located to a highly aromatic environment deep within the beta-barrel. The importance of the binding site's aromatic character was investigated by exchanging phenylalanines with alanines. Indeed, these experiments revealed that substitution of Phe249 in the middle of strand 3B completely abolished T4 binding, while the substitution of several other phenylalanines had no effect.

Amino Acid Sequence↗
PubMed1996

Thyroxine-binding globulin: organization of the gene and variants.

Thyroxine-binding globulin (TBG), the principal thyroid hormone transport protein in human serum, is synthesized by the liver and secreted into the bloodstream as a 54-kD acidic glycoprotein made up of a single polypeptide chain of 395 amino acids and four heterosaccharide units. The carbohydrate chains are important for the correct posttranslational folding, secretion and degradation of the molecule but are not required for hormone binding. TBG, encoded by a single gene copy located on Xq22, consists of five exons spanning 5.5 kbp. An upstream sequence of 218 nucleotides containing a hepatocyte nuclear factor 1 binding motif imparts to the gene a strong liver-specific transcriptional activity. Inherited TBG defects produce three phenotypes based on the level of TBG in serum of affected hemizygotes: complete TBG deficiency (TBG-CD), partial TBG deficiency (TBG-PD) and TBG excess (TBG-E). The molecular basis of the TBG defect has been identified in 12 of 16 known TBG variants. TBG-CD is caused by either premature termination of translation or an amino acid substitution resulting in failure of secretion. Point mutations resulting in single amino acid substitutions are responsible for the alteration of the properties and/or concentration of TBG-PD variants. Gene duplication and triplication has been recently identified in subjects with TBG-E.

Base Sequence↗
PubMed1995

Molecular and structural characterization of the heat-resistant thyroxine-binding globulin-Chicago.

Thyroxine-binding globulin (TBG) is the main transport protein for thyroxine (T4) in blood. It shares considerable sequence homology with alpha 1-antitrypsin (AT) and other members of the serine proteinase inhibitor (serpin) superfamily of proteins. The crystallographic structure of AT has been determined and was found to represent the archetype of the serpins. This model has been used for structure-function correlations of TBG. Sequence analysis of the heat-resistant variant TBG-Chicago (TBG-CH) revealed a substitution of the normal tyrosine 309 with phenylalanine. For further analysis, vectors containing the coding regions of normal TBG (TBG-N) and TBG-CH were constructed, transcribed in vitro, and expressed in Xenopus oocytes. Both TBGs were secreted into the culture medium and could not be distinguished by gel electrophoresis. Scatchard analysis of T4 binding to TBG-N and -CH revealed no significant differences in binding affinity. The rate of heat denaturation of TBGs was determined by measurement of residual T4 binding capacity after incubation at 60 degrees C for various periods of time. The half-life values of denaturation of TBG-N and -CH were 7 and 132 min, respectively. The tyrosine 309 to phenylalanine substitution of TBG-CH involves a highly conserved phenylalanine residue of the serpins. The respective phenylalanine 312 of AT ties the alpha-helix hI1 to the molecule, thus stabilizing the tertiary structure. A substitution with tyrosine would disrupt this interaction. Accordingly, stabilization of the TBG molecule by replacement of tyrosine with phenylalanine in position 309 causes the increased heat stability of TBG-CH.

Amino Acid Sequence↗
PubMed1995

Insulin-like growth factor I messenger ribonucleic acid expression in porcine thyroid follicles is regulated by thyrotropin and iodine.

Insulin-like growth factor I (IGF-I) has been shown to be released from thyrocytes in vitro. We investigated IGF-I mRNA expression during treatment with thyrotropin (TSH), forskolin and potassium iodide (KI) in intact porcine thyroid follicles ex vivo. Porcine thyroid follicles were prepared by collagenase digestion and cultured in the presence of TSH, forskolin or KI. After different incubation times, mRNA was isolated and examined by Northern hybridization with a porcine IGF-I cDNA probe of 405 bp in length. In untreated follicles no IGF-I mRNA was found, whereas in follicles stimulated with TSH an IGF-I mRNA of 7.0 kb was detected after 24 h, which persisted for another 24 h. Forskolin treatment mimicked the TSH effect, indicating that IGF-I mRNA expression may be stimulated by the adenylate cyclase pathway. Preincubation of the porcine follicles with KI decreased dose dependently the TSH-induced IGF-I mRNA expression, with complete inhibition at 10 mumol/l KI. These results suggest that TSH acts via the cAMP pathway to enhance IGF-I mRNA expression, which then may lead to an autocrine IGF-I stimulation. The IGF-I mRNA expression is under negative control of iodide.

Adenylyl Cyclases↗
PubMed1993

The relative expression of mutant and normal thyroid hormone receptor genes in patients with generalized resistance to thyroid hormone determined by estimation of their specific messenger ribonucleic acid products.

Generalized resistance to thyroid hormone (GRTH), is a syndrome of reduced tissue responsiveness to thyroid hormone. So far, mutations linked to GRTH have been only detected in the hormone-binding domain of the human thyroid hormone receptor (hTR)-beta gene. Although there is no doubt that these mutations result in abnormal hTRs, there is a conspicuous lack of correlation between the severity of clinical manifestations and the degree of functional impairment of the mutant hTRs. In this work we examined whether variable expression of mutant genes relative to the normal genes could explain the observed discrepancies. The relative amounts of mutant and normal hTR beta and normal hTR alpha messenger RNAs in fibroblasts from normal subjects and those from individuals with GRTH were estimated by coamplification of their complementary DNA products. Heterozygous subjects with GRTH from two families manifesting differences in the severity of clinical manifestations expressed equally both normal and mutant hTR beta alleles. Furthermore, there was no compensatory increase in the expression of the normal hTR alpha gene in these individuals nor in fibroblasts from members of a third family with homozygous deletion of the hTR beta gene. In vitro treatment with thyroid hormone did not affect the results. It is concluded that the apparent discrepancies between the functional impairment of the mutant hTRs and the clinical manifestations of GRTH are not due to quantitative differences in the expression of the normal or mutant hTR genes but more likely to variations in the interactions of the mutant hTRs with the normal hTR beta, hTR alpha and nuclear stabilization factors.

Alleles↗
PubMed1993

Human thyroxine-binding globulin gene: complete sequence and transcriptional regulation.

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. To characterize the human TBG (hTBG) gene, we studied its genomic organization, promoter activity, and regulation. To this purpose, we isolated from liver a complete hTBG cDNA clone containing the 5'-untranslated region and localized the transcription start site (TSS). The analysis of genomic clones revealed that the hTBG gene consists of five exons and that its exon-intron organization is similar to that of other members of the serine protease inhibitor family. The first exon (exon 0) is a short noncoding sequence located 1.62 kilobase pairs (kbp) upstream from exon 1. Potential cis-acting transcriptional regulatory elements including a TATA box, a CAAT box, and a hepatocyte nuclear factor-1 binding motif were identified in the upstream region. A reporter gene in which 3.2 kbp of the 5'-flanking region, including exon 0, was inserted upstream of the bacterial chloramphenicol acetyltransferase gene showed significant activity when transfected into a hepatblastoma-derived (HepG2) cell line. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, down-regulated the promoter activity by more than 80% and completely inhibited hTBG synthesis, whereas thyroid hormone, glucocorticoid, estrogen, and nicotinic acid had little, if any, effect. A series of 5'-deletions revealed that the fragment -218 to +4 from the TSS had the highest promoter activity, nearly 1000-fold greater than the promoterless chloramphenicol acetyltransferase construct. When nonhepatocyte-derived cell lines (CV-1 and CHO) were tested, promoter activity was reduced by a factor of 100, showing that the promoter works in liver-specific manner. The region -218 to -102 contains liver-specific enhancer elements, since deletion to nucleotide -101 resulted in a profound reduction of the promoter activity in HepG2 cells but not in CV-1 or CHO cells. On the other hand, mutational disruption of the putative hepatocyte nuclear factor-1 site (located 65 bp upstream of the TSS) completely abolished the promoter activity in all cell lines, indicating that this site is absolutely required for the transcription of the hTBG gene.

Amino Acid Sequence↗
PubMed1992

In vitro expression of thyroxine-binding globulin (TBG) variants. Impaired secretion of TBGPRO-227 but not TBGPRO-113.

Thyroxine-binding globulin (TBG) is a glycoprotein that transports thyroid hormones in blood. Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency. In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes. Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed. TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in thyroxine (T4) binding. TBG-CD5 had altered electrophoretic mobility and did not bind T4. TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly. Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M. The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion.

Animals↗