PubMed Health⌕ Search

Biomedical subjects

L J DeGroot

Publications and source records attributed to L J DeGroot.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and characterization of the human thyroid hormone receptor beta 1 gene promoter.

The promoter region of the human (h) thyroid hormone receptor (TR) beta 1 gene was isolated from a human placenta genomic library. Primer extension and S1 nuclease mapping confirmed a single transcriptional start site. DNA sequence analysis of the 5' upstream region revealed the existence of a putative thyroid response element (TRE) which is highly homologous to TREs found in several thyroid hormone responsive genes. Binding of hTR protein to the promoter region of the hTR beta 1 gene was confirmed by gel mobility shift assay. A transient transfection study demonstrated that hTR activated the expression of a reporter gene containing the promoter sequence of the hTR beta 1 gene in a hormone dependent manner. The TRE in the hTR beta 1 gene promoter may be involved in the autoregulation of hTR beta 1 gene expression.

Base Sequence↗
PubMed1992

Admission screening by thyroid function tests in an acute general care teaching hospital.

To determine the incidence of unrecognized thyroid disease among admissions to a large acute care university teaching hospital, 364 samples taken on consecutive admissions were assayed for thyroid-stimulating hormone (TSH) and free thyroxine index (FTI). Patients with abnormal test results were further evaluated by determination of antimicrosomal and antithyroglobulin antibodies, and charts were reviewed for evidence of prior diagnosis of thyroid disease, especially severe illness, drug treatment that might affect thyroid function tests, and prior diagnosis of thyroid disease. Results of subsequent thyroid function tests performed during the patient's hospitalization were correlated with the admission serum assays, and data on subsequent testing during the following 6 months were also obtained. A total of 3.9% of patients had significantly depressed TSH, and 11.1% of values were significantly elevated. A total of 11.3% of patients had significantly low FTI values, and 1% had significantly elevated values. A total of 7.4% appeared to have the euthyroid sick syndrome, 5.8% appeared to have unrecognized or undertreated primary thyroid failure, 6% had apparent subclinical hypothyroidism, 2% were thyrotoxic, and 2.8% (all women) had suppressed TSH levels for inapparent reasons. Limiting testing to patients over 49 years of age, or to women, would have missed many individuals with abnormal test results. Considering widespread availability of tests, relative costs, and value of the information obtained, it is suggested that the FTI determination would provide an appropriate screening test for patients in a population such as this entering a large, acute care general hospital.

Diagnostic Tests, Routine↗
PubMed1992

Pemphigus vulgaris in siblings: HLA-DR4 and HLA-DQw3 and susceptibility to pemphigus.

BACKGROUND: The association of pemphigus vulgaris with the HLA serotypes, DR4 and DRw6, and with the DQ-beta chain alleles, DQw1 and DQw3, suggests that there is a genetic predisposition to this disease. However, familial cases of pemphigus vulgaris are exceedingly rare. OBJECTIVE: We studied two siblings with pemphigus vulgaris and three unaffected family members to determine whether an HLA allele was associated with the development of pemphigus vulgaris in this family. METHODS: We utilized restriction fragment length polymorphism methods using the HLA-DR beta 1, HLA-DQ alpha, and HLA-DW beta 1 genes as cDNA probes. RESULTS: We found that the affected siblings share the haplotype HLA-DR4 and the DQw.3.2 allele. CONCLUSION: Our findings of gene sharing among siblings with pemphigus vulgaris lend support to previous studies of unrelated Caucasian patients, which implicated DQw3 allele polymorphisms in conferring susceptibility to pemphigus.

Adult↗
PubMed1992

Expression and function of a human thyroid hormone receptor-derived DNA-binding domain protein.

DNA binding domain proteins (DBDP) were prepared using a pET construct containing an insert coding for amino acids 49-122 of human thyroid hormone receptor (hTR) alpha and 103-179 of hTR beta. These proteins were expressed in Escherichia coli strain BL21 (DE3)-plysS after induction by isopropyl-D-thiogalactopyranoside (IPTG). The hTR alpha and hTR beta DBDP contain respectively 79 and 82 amino acids, including an amino terminal 4 amino acid extension derived from pET-3a or the synthesized initiation codon. Using a gel shift assay, both DBDPs were found to bind to a DNA oligonucleotide containing a thyroid hormone response element (TRE). The DBDPs competed with full length hTR alpha 1 for binding to the oligonucleotide. Apo-DBDPs (Zn2+ released by low pH) failed to bind to the palindromic TRE. DNA binding is restored however if apo-DBDP is preincubated in 500 microM Zn2+. When the DBDPs were expressed in COS-7 cells using a pCB6+ expression vector, they did not induce expression of a TRE-CAT fusion gene. hTR DBDPs thus can bind to DNA, presumably as monomers, since they do not contain the leucine zipper-like motif for dimerization. In COS-7 cells, they fail to cause transactivation of a TRE-CAT fusion gene. It is inferred that this may be because the DBDPs are not translocated to the nucleus or lack a transactivation domain.

Amino Acid Sequence↗
PubMed1992

Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform.

We characterized four antipeptide polyclonal antibodies (abs) able to specifically recognize each thyroid hormone receptor (TR) isoform. The abs immunoprecipitated both the in vitro synthesized receptor and the receptor expressed in E. coli and their specificity was confirmed by competition studies and immunohistochemistry. Ab activity measured by enzyme-linked immunosorbent assay decreased after preabsorption of each ab with the immunizing peptide or the specific receptor protein expressed in E. coli. No specific activity was detectable in enzyme-linked immunosorbent assay, no nuclear staining was observed after affinity column immunoabsorption, and the specific bands obtained in Western blot analysis disappeared after preabsorption with the specific TR isoform expressed in E. coli. By immunohistochemical studies we detected coordinate expression of each receptor isoform in most tissues examined. However, in heart and muscle, the beta-isoform is expressed at a very low level compared to the alpha-isoform in spite of the significant TR beta mRNA levels previously demonstrated by Northern blot analysis. We also demonstrated a different pattern of distribution of alpha- and beta-isoforms in rat testis. In this tissue the TR alpha is significantly expressed in spermatogonia nuclei, but in spermatids the beta-isoform is predominant, and only the TR beta is detectable in mature spermatozoa.

Animals↗
PubMed1992

Recessive inheritance of thyroid hormone resistance caused by complete deletion of the protein-coding region of the thyroid hormone receptor-beta gene.

Generalized resistance to thyroid hormone is a syndrome of reduced responsiveness of target tissues to thyroid hormone. The determination of amino acid sequences of the human thyroid receptor-beta (hTR beta), deduced from cDNA sequencing, has enabled evaluation of the genetic basis for this syndrome. Distinct point mutations in the ligand-binding domain of hTR beta have been identified in affected members of unrelated families, producing single amino acid substitutions that result in products with decreased or no hormone-binding activity. Inheritance in these families was autosomal dominant. We now report the molecular basis of generalized resistance to thyroid hormone in a consanguineous family unique for its autosomal recessive mode of inheritance. Deletion of the entire coding region of both hTR beta alleles in homozygous affected members of the family was demonstrated by the failure to amplify the coding exons 3-8 by the polymerase chain reaction using primers specific for flanking intronic sequences and by the demonstration of the presence of only two noncoding exons in Southern blots hybridized with exon-specific probes. As expected, obligate heterozygotes were phenotypically normal, since, in contrast to alleles with point mutations, the deleted allele could not act in a dominant negative fashion. Survival and maintenance of a euthyroid state are presumably mediated through expression of the hTR alpha gene, present in affected subjects, and the maintenance of high thyroid hormone levels. Furthermore, the clinical manifestations were relatively more mild that those observed in a homozygous patient with a single amino acid deletion in the hTR beta gene.

Amino Acid Sequence↗
PubMed1992

Proliferative responses of peripheral blood mononuclear cells from patients with autoimmune thyroid diseases to synthetic peptide epitopes of human thyroid peroxidase.

In order to analyze T cell epitopes of human thyroid peroxidase (TPO), 60 peptides based on the sequence of TPO were synthesized and used as antigens in a peripheral blood mononuclear cell (PBMC)-proliferation assay. PBMCs were obtained from 19 patients with Graves' disease, 19 patients with Hashimoto's thyroiditis, and 24 normal subjects. Significant proliferation of PBMC to these peptides occurred only among the autoimmune thyroid disease (AITD) patients, whereas normal subjects did not respond to any of the peptides with a stimulation index over 2. Many peptides induced isolated positive responses and eight produced stimulation of PBMCs from multiple patients on comparison to control PBMC responses. To confirm the significance of reactivity to the peptides PBMCs from four patients were studied on two occasions, and the proliferative responses found to be reproducible. Four peptides, designated according to the amino acid sequence as p110-129, p211-230, p842-861, and p882-901, stimulated patients' PBMCs in a dose-dependent manner. The optimal concentration was 10 micrograms/ml. An anti-HLA-DR monoclonal antibody directed against a monomorphic determinant of the DR molecule was able to block the responses. A significant correlation was found between the PBMC responses to these peptides and responses to microsomal antigen (McAg)/TPO. These data suggest that four peptides corresponding to the amino acid sequences 110-129, 211-230, 842-861 and 882-901 are T cell epitopes of TPO.

Adult↗
PubMed1992

HLA class II genes in Graves' disease.

Inheritance of Graves' disease has been linked to the HA-DR3 gene product which may function in some specific way in antigen presentation. To determine whether the first extracellular domain of this protein, which is specifically involved in antigen presentation, has the same sequence in patients with Graves' disease and in normal individuals, we have amplified the second exon using the polymerase chain reaction, and then cloned and sequenced the DNA segment. In eight subjects with Graves' disease, sequences identical to prototypic reported sequence for DRB1*0301 were recovered, and in two individuals sequences varied by a few nucleotides, leading to 1-3 amino acid substitutions which did not occur in a pattern. Sequences identical to the prototypic sequence known as DRB3*0101, also previously known as DRw52, were also recovered. Thus the HLA-DRB1 and B3 genes present in patients with autoimmune disease appear to be the same as those present in the general population. These observations indicate that a unique allele is not present in patients with autoimmune disease, but rather that the normal DR3 allele itself, in a manner yet to be described, increases the probability of developing autoimmune thyroid disease.

Amino Acid Sequence↗
PubMed1992

Site-specific anti-c-erb A antibodies recognizing native thyroid hormone receptors: their use to detect the expression and localization of alpha and beta c-erb A proteins in rat liver.

The cell-specific expression and tissue distribution of c-erbA proteins alpha and beta is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the alpha or beta form of thyroid hormone receptors. The cDNAs coding for h c-erbA beta 1, alpha 1 and alpha 2 were transcribed and the mRNAs were translated in vitro in the presence of 35S-methionine, and then their reactivity with the antisera was evaluated. The antiserum anti-beta 62-81 immunoprecipitated only the beta 1 receptor. The antiserum anti-alpha 144-162 determined precipitation of both alpha 1 and alpha 2 proteins but not of the beta 1 receptor. Anti-alpha 2 431-451 produced a selective precipitation of alpha 2, and had no effect on alpha 1 or beta 1 receptor. In order to study the interaction of the antibodies with native T3 receptor we evaluated the binding of antibodies to rat liver T3 receptors by Sephacryl S300 chromatography: both antisera anti-beta 62-81 and anti-alpha 144-162 caused a partial shift of the labeled T3-receptor complex to a higher molecular form, while the antibody directed against c-erbA alpha 2 did not produce any significant shift. The anti-peptide antibodies were then immunopurified by affinity chromatography and used to immunolocalize the different forms of c-erb A proteins in adult and fetal rat liver, by a sensitive immunohistochemical technique. All 3 antibodies stained mainly the nuclei of the majority of adult liver cells. No staining was detectable when the original antiserum was deprived of anti-peptide antibodies by running through the affinity columns or when the antibodies were pre-absorbed with the homologous peptide. No significant staining was present in the liver from rat fetus.

Animals↗
PubMed1991

Screening of nineteen unrelated families with generalized resistance to thyroid hormone for known point mutations in the thyroid hormone receptor beta gene and the detection of a new mutation.

Generalized resistance to thyroid hormone (GRTH) is a syndrome characterized by impaired tissue responsiveness to thyroid hormone. Two distinct point mutations in the hormone binding domain of the thyroid hormone receptor (TR) beta have recently been identified in two unrelated families with GRTH. One, Mf, involves a replacement of the normal glycine-345 for arginine in exon 7 and another, Mh, replaces the normal proline-453 for histidine in exon 8. To probe for the presence of the Mf and Mh defect in 19 unrelated families with GRTH, we applied separate polymerase chain reactions using allele-specific oligonucleotide primers containing the normal and each of the two mutant nucleotides at the 3'-position. A total of 24 affected subjects and 13 normal family members were studied. The mode of inheritance was dominant in 13 families, was unknown in 5 families, and was clearly recessive in 1 family in which only the consanguineous subjects were affected. Primers containing the substitutions specific for Mf and Mh amplified exons 7 and 8, respectively, only in affected members of each of the two index families. Primers containing the normal sequences amplified exons 7 and 8 of the TR beta gene in all subjects except affected members of one family. In this family with recessively inherited GRTH, neither exon could be amplified using any combinations of primers and DNA blot revealed absence of all coding exons. These results indicate a major deletion of the TR beta gene, including both DNA and hormone binding domains. Since heterozygous members of this family are not affected, the presence of a single normal allele is sufficient for normal function of the TR beta. These data also support the hypothesis that in the dominant mode of GRTH inheritance the presence of an abnormal TR beta interferes with the function of the normal TR beta. Distinct mutations are probably responsible for GRTH in unrelated families.

Alleles↗
PubMed1991

The effect of hepatic enzyme-inducing drugs on thyroid hormones and the thyroid gland.

With the understanding that various drugs, industrial chemicals, and chemicals of environmental importance can increase thyroid hormone hepatic metabolism and excretion, it is important to consider whether compensation by the thyroid gland for this increased excretion can lead to stimulation of the hypothalamic-pituitary-thyroid axis and possibly secondary hyperplastic or neoplastic changes in the thyroid. The compounds discussed in this review all affect thyroid function by increasing biliary excretion of thyroid hormone metabolites. Numerous studies have been performed to elucidate the effect of these drugs on thyroid hormone equilibrium. Animals given PB compensate for the increased biliary excretion with an elevated TSH allowing maintenance of a euthyroid state. Human studies demonstrate increased thyroid hormone plasma clearance, but without an increased TSH. The effects of an experimental leukotriene antagonist are similar. In humans, diphenylhydantoin has been conclusively shown to cause a decrease in peripheral thyroid hormone levels, although without evident hypothyroidism or increase in TSH. Limited studies of rifampin and carbamazepine reveal similar results. Nicardipine in rats causes reproducible decreases in free T4 levels, although it does not clearly stimulate a rise in TSH levels. An experimental imidazole caused reversible lowering of peripheral thyroid hormone levels in rats; in this study TSH was not measured. Studies with aromatic hydrocarbons administered to rats reveal a general decrease in T4 levels, with a compensatory increase of TSH. The effects of chronic administration of a compound that can cause increased thyroid hormone metabolism with compensation via increased TSH production are of more than theoretical interest, as it has been well documented that constant stimulation of the thyroid gland in rats with supraphysiological levels of TSH causes goiter, thyroid hyperplasia, adenomas, and carcinomas. In humans with congenital metabolic thyroid deficiencies there is an increased incidence of thyroid neoplasia, suggesting an association with chronic increased TSH levels. In contrast, however, large epidemiological studies of areas of endemic goiter do not show an association of human thyroid cancer with iodine deficiency and presumed chronic thyroid gland stimulation. Histological evidence of thyroid follicular hyperplasia has been noted in rats after administration of phenobarbital, nicardipine, polycyclic hydrocarbons, and possibly an experimental imidazole. Increased thyroid gland size has been demonstrated in rats given phenobarbital, nicardipine, a leukotriene antagonist, and various polycyclic hydrocarbons. Thyroid carcinoma in rats has been associated with treatment with nicardipine and after exposure to several aromatic hydrocarbons.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Effects of zinc and other divalent metals on deoxyribonucleic acid binding and hormone-binding activity of human alpha 1 thyroid hormone receptor expressed in Escherichia coli.

Full-length human thyroid hormone receptor alpha 1 (hTR alpha 1) was expressed in Escherichia coli using a T7 expression system. While present in large amounts, the receptor was highly enriched in the insoluble fraction after cell lysis. We describe here the successful solubilization and refolding of the expressed receptor in a functional form in the presence of Zn2+. Using a DNA-cellulose binding assay and gel shift assay, we found that treatment of expressed receptor with 1 mM EDTA in the denaturing agent (5 M guanidine-HCl) results in the formation of aporeceptor that does not specifically recognize target DNA, while it does retain T3-binding activity. This aporeceptor recovered DNA-binding activity by adding Zn2+ during refolding. Zinc-induced restoration of DNA-binding activity occurred in a dose- and time-dependent manner. Moreover, once recovered, this DNA-binding activity persisted without Zn2+, even in the presence of 1 mM EDTA. These data indicate that the hTR alpha 1 molecule has a high affinity for Zn2+, and this metal coordination is essential for proper folding of TR protein into its native active structure.

Base Sequence↗
PubMed1991

Genetic linkage studies of thyroid peroxidase (TPO) gene in families with TPO deficiency.

We have conducted biochemical and genetic studies in five unrelated families (denoted A, C, R, P, and G), which included nine goitrous subjects (five borderline euthyroid and four hypothyroid) with complete (n = 6) or partial (n = 3) thyroid peroxidase (TPO) deficiency. Thyroid tissue was obtained from four subjects, respectively, in families A, C, and R. No iodide organification or iodide incorporation into protein was present in families A and C. The two affected siblings in family R had a low normal tissue thyroperoxidase activity. Using a 0.8-kilobase (kb) cDNA clone (pM5) encoding 30% of the cDNA of human TPO gene down-stream from basepair 730 and four restriction enzymes (TaqI, PstI, BglI, and BglII), we were unable to find any polymorphisms in family A. In another family (C) blood samples were obtained from only two family members, and consequently, it was not possible to determine linkage. DNA from families G, P, and C showed biallelic polymorphisms when digested with BglII, at 8.7 and 8.5 kb. In family R we detected two biallelic polymorphisms at 9.0 and 8.5 kb, and the 9.0-kb bands were clearly larger than 8.7-kb bands found in the other subjects. Also, there was an absence of a 4.0- to 3.9-kb band that may represent a partial gene deletion. With PstI-restricted DNA a possible deletion of 5.5-kb band was also present in affected siblings of family R. The logarithm of odds (Lod) score analyzed from the family with inbreeding (R) was compatible with linkage of disease and the TPO gene (Lod = 2.08). When this method was used with families G and P, the Lod score was inconsistent with linkage between disease and the TPO gene. These data suggest that the cause of TPO deficiency in these families is heterogeneous. However, the restriction fragment length polymorphism pattern of BglII-restricted DNA in the R family strongly suggests that a partial TPO gene deletion has occurred in this family.

Bacterial Proteins↗
PubMed1991

Genetic factors in autoimmune thyroid disease analyzed by restriction fragment length polymorphisms of candidate genes.

Possible genetic effects on the development of autoimmune thyroid disease were studied by analysis of restriction fragment length polymorphisms (RFLPs) of candidate genes. Peripheral blood leukocyte DNA was obtained from 65 caucasian patients with Graves' disease, 63 caucasian patients with Hashimoto's thyroiditis, and 65 caucasian controls. RFLP analysis was carried out on genomic DNA, using probes for DR beta, DQ alpha, DQ beta, DP alpha, DP beta, T-cell receptor TCR alpha, and thyroid peroxidase. The methodology allowed HLA-DR and DQ typing and provided information on specific RFLP patterns related to T cell receptor (TCR)alpha, DP alpha and -beta, and -beta, and thyroid peroxidase. HLA-DR3 frequency was significantly increased in patients with Graves' disease, as reported previously by others, but neither DNA-derived subtype of DR3 was differentially increased. HLA-DQw2 was also present in increased frequency because of its linkage disequilibrium with HLA-DR3. It is uncertain whether the primary susceptibility is with DQ, DR, or another nearby locus. Susceptibility was not related in these studies to genetic loci recognized in these studies involving DQ alpha, DP, TCR, or thyroid peroxidase. A significant linkage disequilibrium between DR3 and a specific DX alpha RFLP was observed in Graves' disease, but is believed to be representative of a generalized linkage disequilibrium between DR3 and DX alpha, rather than a specific abnormality in Graves' disease. Previous studies indicating association with specific TCR RFLPs could not be reproduced. The relative risk for carriers of HLA-DR3 subtype A in this study was 7.37-fold. RFLP analysis offers the possibility of investigating linkage in a variety of candidate genes as well as established genetic relationships for potentially important subtypes. While the significant relationship with HLA-DR3/DQw2 was reconfirmed, the involvement of other genes or haplotypes could not be established.

DNA↗
PubMed1991

Second operations for "completion" of thyroidectomy in treatment of differentiated thyroid cancer.

The role of elective completion thyroidectomy after lobectomy for differentiated thyroid cancers remains controversial. The potential benefit of tumor removal by the second procedure is considered by some to be overbalanced by a prohibitive operative morbidity rate. During a 20-year period at the University of Chicago Medical Center, 26 patients underwent completion thyroidectomy within a 6-month period of the original thyroid operation. This group represents 8% of the 326 patients who underwent surgery during that time for differentiated thyroid cancer (269 papillary and 57 follicular). Of the 26 patients, 18 had papillary and eight had follicular cancers. The average size was 2.5 cm, with 24 of 26 being greater than 1 cm in diameter. At the first operation, 81% of tumors were intrathyroidal. Eight percent had lymph node metastases and 12% manifested local invasion. Tumor was found in eight (31%) of 26 of the reoperative specimens. The incidence of tumor did not vary by histologic type but did differ according to the extent of the original operation. Cancer was found in 50% (three of six) of those who had undergone previous partial lobectomy, in 33% (five of 15) of those after a total lobectomy, and in none of five who had undergone a prior bilateral (although incomplete) thyroid resection. One permanent recurrent nerve injury occurred at the first operation. No additional recurrent nerve injuries or hypoparathyroidism occurred as a result of the second operation. Finally, no disease characteristic of the initial tumor (e.g., size, clinical class, tumor capsular invasion, multifocality, thyroiditis, or extrathyroidal tumor invasiveness) predicted the presence or absence of tumor on the second side. We conclude that completion thyroidectomy is appropriate for patients with lesions 1 cm or greater who have undergone lobectomy or less at the original operation, because 40% of such patients would be expected to have residual cancer. With care, this operation can be performed with minimal morbidity.

Adenocarcinoma↗