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B Rapoport

Publications and source records attributed to B Rapoport.

At least 163 records · Page 9Linked to original sources
PubMed1990

The functional expression of recombinant human thyrotropin receptors in nonthyroidal eukaryotic cells provides evidence that homologous desensitization to thyrotropin stimulation requires a cell-specific factor.

TSH desensitization involves decreased coupling of the TSH receptor to the adenylate cyclase regulatory protein, Gs. There is evidence that a desensitization protein in thyroid cells plays a role in this process. The molecular cloning of the human TSH receptor and its stable expression in Chinese hamster ovary (CHO-TSHR) cells allowed us to test whether or not TSH desensitization can occur in a nonthyroidal cell. Similar to human thyroid cells, maximal stimulation of cAMP levels in CHO-TSHR cells was attained after 30-60 min of exposure to bovine TSH. Unlike in human thyroid cells, however, preincubation of CHO-TSHR cells with TSH for 12-16 h did not decrease the subsequent cAMP response to a 1-h pulse of TSH stimulation. That is, the human TSH receptor in CHO-TSHR cells does not undergo functional desensitization. Scatchard plot analysis of specific TSH binding to the CHO-TSHR cells revealed high and low affinity sites (Ka of 1.8 +/- 0.4 x 10(9) M-1 and 1.4 +/- 0.3 x 10(7) M-1, respectively), with approximately 10(5) TSH receptors per cell. This is 10- to 100-fold greater than the number of TSH receptors estimated to be present on human thyroid cells. Untransfected CHO cells exhibited only the low affinity binding site. Prior exposure of CHO-TSHR cells to bovine TSH or to (Bu)2cAMP for periods up to 24 h did not reduce [125I]TSH binding to these cells. In summary, desensitization of the adenylate cyclase response to TSH stimulation does not occur in nonthyroidal cells expressing a human TSH receptor with normal functional and TSH binding characteristics. These data support the concept that a cell-specific protein may be involved in homologous TSH desensitization.

Animals↗
PubMed1990

Recombinant human thyroid peroxidase generated in eukaryotic cells: a source of specific antigen for the immunological assay of antimicrosomal antibodies in the sera of patients with autoimmune thyroid disease.

Recombinant, enzymatically active human thyroid peroxidase (hTPO) generated in nonthyroidal eukaryotic cells was compared with Graves' thyroid microsomes as a source of antigen for the immunological detection of antimicrosomal/anti-hTPO antibodies. Enzyme-linked immunosorbent assay of 51 sera, selected to produce a balanced distribution of antimicrosomal antibody (anti-MSA) levels, revealed (at 1:100 serum dilution) a moderately good correlation between anti-MSA and anti-hTPO antibody levels (r = 0.668; P less than 0.001). However, a number of sera with high anti-MSA levels yielded markedly discordant values between the two assays. A much lower correlation was observed between antithyroglobulin and anti-hTPO antibody levels (r = 0.315; P less than 0.05). At higher serum dilutions (1:1,000 and 1:10,000), at which low affinity, high capacity binding reactions will be reduced, the correlation between anti-MSA and anti-hTPO antibody values was greatly improved (r = 0.906 and 0.902, respectively; P less than 0.001), and there were no longer widely discrepant values between the two assays. In summary, the present study indicates that recombinant hTPO expressed in nonthyroidal cells provides an unlimited source of human TPO of unvarying quality for anti-hTPO antibody assays. This material offers increased specificity over standard anti-MSA assays that use thyroid cell microsomes as antigen.

Antigen-Antibody Reactions↗
PubMed1990

Evidence for the highly conformational nature of the epitope(s) on human thyroid peroxidase that are recognized by sera from patients with Hashimoto's thyroiditis.

To define the epitope(s) on human thyroid peroxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease, we constructed and screened a human TPO cDNA sublibrary containing 3.8 million random fragments of human TPO cDNA, each 200-500 basepairs in length. These fragments would code for TPO polypeptides of 66-166 amino acid residues. The validity of this approach was first tested with a murine monoclonal antibody against the denatured human thyroid microsomal antigen (TPO). Analysis of the nucleotide sequence of 14 clones selected from this library enabled molecular identification of the epitope recognized by this monoclonal antibody. In contrast to the data obtained with the monoclonal antibody, sera from patients with Hashimoto's thyroiditis containing polyclonal antimicrosomal/TPO antibodies did not recognize the TPO protein fragments generated by this library. These results differ from previous data obtained with recombinant human TPO fragments generated as bacterial fusion proteins. Our data suggest that, contrary to previous concepts, the natural B-cell epitope(s) on human TPO may be highly conformational (requiring a complex 3-dimensional structure) or may be discontinuous (formed by distant regions of the linear polypeptide chain being brought into apposition by protein folding).

Amino Acid Sequence↗
PubMed1990

Characterization, by molecular cloning, of smaller forms of thyroid peroxidase messenger ribonucleic acid in human thyroid cells as alternatively spliced transcripts.

The mRNA for human thyroid peroxidase (hTPO) is 3.1 kilobases (kb) in size, coding for a protein of 933 amino acids. However, there is controversy as to whether other hTPO mRNA transcripts exist in thyroid cells. There is one report of the existence of 2.1- and 1.7-kb transcripts (hTPO mRNA species I and II), representing up to half of the hTPO mRNA in TSH-stimulated human thyroid cells. On the other hand, numerous other studies have only observed 3.1-kb hTPO mRNA transcripts. The nature of these putative 2.1- and 1.7-kb mRNA transcripts, if present, is unknown. We now report the isolation and characterization of two smaller hTPO cDNA species, designated hTPO cDNA I and II. cDNA I and II are 1753 and 1044 basepairs (bp) in size, with open reading frames of only 225 and 174 amino acids, respectively. Comparison of the nucleotide sequences of cDNA I and II with available hTPO genomic sequence reveals that cDNA I consists of exons 1-6 (654 bp) and the 5'-end of intron 6 (1099 bp); cDNA II contains exons 1-5 (486 bp) and an unidentified DNA tract of 558 bp further down-stream, presumably an intron. Confirmation that cDNA I and II correspond to mRNA transcripts I and II, respectively, was provided by Northern blot analysis with DNA probes specific for cDNA I and II. hTPO mRNA transcripts I and II are present in TSH-stimulated and TSH-deprived human thyroid cells in culture as well as in intact thyroid tissue. In summary, the present data 1) demonstrate directly that hTPO mRNA transcripts I and II exist in human thyroid cells, 2) explain the discrepant data in the literature regarding their existence, 3) elucidate their molecular structure, 4) indicate that they are generated by alternative splicing, and 5) demonstrate that hTPO mRNA I and II exist in the thyroid gland in vivo as well as in thyroid cells in culture.

Base Sequence↗
PubMed1990

Generation of a biologically active, secreted form of human thyroid peroxidase by site-directed mutagenesis.

Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.

Animals↗
PubMed1990

An abnormal splice donor site in one allele of the thyroid peroxidase gene in FRTL5 rat thyroid cells introduces a premature stop codon: association with the absence of functional enzymatic activity.

Low stringency screening of an FRTL5 cDNA library with a human thyroid peroxidase (TPO) cDNA probe yielded two different types of TPO cDNA clones. One type contained the full-length structural gene, but there was an A at the first nucleotide of an intronic splice donor site that leads to alternate splicing, with the retention of part of an intron. This 54-basepair retained intron fragment contains a premature inframe stop codon that would truncate the protein at its carboxyl-terminus by 71% with the loss of enzymatic activity. The second type of TPO cDNA does not contain the retained intron fragment and premature stop codon, but it is not full-length and lacks 580-680 basepairs at its 5' end. However, Northern blot analysis reveals only full-length copies (3.2 kilobases) of TPO mRNA in FRTL5 cells, and this 5' truncation is, therefore, an artifact of library construction. The relative proportions of the two types of TPO mRNA in FRTL5 cells was determined by the polymerase chain reaction, using as template single stranded cDNA generated by reverse transcription of FRTL5 mRNA. Slightly more than half of the TPO mRNA in the FRTL5 cells represented the form with the abnormal splice donor site. The relative proportions of the two TPO mRNA forms was not influenced by TSH stimulation of the FRTL5 cells, and the proportion remained unaltered even after the FRTL5 cells were subcloned by limiting dilution. At the genomic level, we used allele-specific oligonucleotides for the mutant and normal forms of TPO, and found FRTL5 cells to have both normal and abnormal TPO alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1990

Thyrotropin and adenosine 3',5'-monophosphate stimulate the activity of the ferritin-H promoter.

Fragments of the rat ferritin-H 5'-flanking region up to 1 kilobase in length were generated by the polymerase chain reaction using FRTL5 rat thyroid cell genomic DNA as template. Ferritin-H 5'-flanking region fragments of 219, 351, 666, and 1046 basepairs (bp), ligated up-stream to the reporter gene luciferase, were transiently transfected into FRTL5 thyroid cells and NIH-3T3 mouse fibroblasts. In both cell types, constitutive (nonstimulated) ferritin-H promoter activity increased progressively with constructs containing increasing lengths of 5'-flanking region. TSH or (Bu)2cAMP (dBcAMP) stimulation of FRTL5 cells transfected with the shorter (219 and 351 bp) ferritin-H 5'-flanking region fragments increased promoter activity 2- to 3-fold. However, with the longer DNA segments (666 and 1046 bp), the extent of TSH stimulation was less. Exposure of transfected NIH-3T3 cells to dBcAMP mimicked in all respects the effects of TSH and dBcAMP on ferritin-H promoter activity in FRTL5 cells. Transcription initiation sites in the luciferase reporter gene were unaffected by the length of the ferritin-H 5'-flanking region included in the construct or by dBcAMP stimulation. Plasmid constructs with 45 bp of the ferritin-H 5'-flanking region containing a potential cAMP response element did not reveal any promoter activity or dBcAMP responsiveness in this region. Gel shift mobility assays with the -219 bp ferritin-H 5'-flanking region fragment and NIH-3T3 nuclear proteins revealed specific protein-DNA interaction. Reduced DNA mobility was inhibited by excess unlabeled probe DNA, but not by DNA fragments corresponding to the recognition sites for a variety of known trans-activating factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Studies on the role of c-fos in TSH-stimulated thyroid cell proliferation.

TSH stimulates proliferation of FRTL5 rat thyroid cells, and also increases c-fos mRNA levels in these cells. We therefore investigated the role of c-fos in TSH-mediated FRTL5 cell proliferation. FRTL5 cells were stably transfected with plasmid constructs that transcribe, under the control of the dexamethasone-inducible MMTV promoter, a 5'-fragment (84 nucleotides) of c-fos mRNA, either in the sense or in the antisense orientation. Four c-fos antisense clones (A1, A2, A3, A4), one sense clone (S2), and wild-type (untransfected) FRTL5 cells were studied. Southern blot analysis indicated similar levels of transgenome in all transfected cell lines. Antisense clone A3 exhibited the greatest dexamethasone-induced growth inhibition, two times (59% vs. 30%) greater than that in control, sense c-fos-transfected or wild-type FRTL5, cells. Consistent with the degree of growth inhibition, northern blot analysis revealed that c-fos antisense clone A3 expressed the highest levels of c-fos antisense transcript. However, we did not observe dexamethasone induction of the antisense c-fos-beta globin hybrid mRNA in any clone, including clone A3. Taken together, our results provide circumstantial evidence that c-fos, at least in part, may play a role in TSH-mediated thyroid cell growth.

Animals↗
PubMed1989

Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor.

We screened a human thyroid cDNA library with two synthetic oligonucleotides based on the reported amino acid sequence of the 3rd and 4th transmembrane domains of a putative human TSH receptor and related receptors. The nucleotide sequence of a 4 kb clone revealed an open reading frame of 764 amino acids (86,816 Daltons) with a putative signal peptide, seven transmembrane domains, five potential glycosylation sites, and a very short intracytoplasmic region. Homology with the extracellular domain of the pig LH/CG receptor was only 33%. Chinese hamster ovary cells stably transfected with this cDNA in an expression vector generated a functional receptor, able to activate adenylate cyclase, specifically in response to TSH stimulation.

Amino Acid Sequence↗
PubMed1989

Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen.

Animals↗
PubMed1989

Thyrotropin regulation of thyroid peroxidase messenger ribonucleic acid levels in cultured rat thyroid cells: evidence for the involvement of a nontranscriptional mechanism.

The influence of TSH on thyroid peroxidase (TPO) gene expression was investigated in FRTL5 rat thyroid cells. Cultured in the presence of TSH, these cells express a TPO mRNA species of 3.1 kilobases. TSH withdrawal from the culture medium led to a decline in TPO mRNA levels over 24 h. In contrast, no decline in beta-actin mRNA levels occurred after 24 h of incubation in TSH-free medium. TSH (1 mU/ml) added to FRTL5 cells cultured in the absence of TSH increased TPO mRNA levels 7- to 9-fold compared to levels in control cells. This effect of TSH on TPO mRNA accumulation in FRTL5 cells was time related (it was already present after 4 h and was maximal after 24 h of cell exposure to TSH), dose related (0.01 and 1 mU/ml were, respectively, the lowest and the maximally effective doses), and independent of new protein synthesis, in that it was not prevented by cycloheximide (100 microM). cAMP analogues [8-bromo-cAMP and (Bu)2cAMP] also increased TPO mRNA levels, although to a lesser degree than TSH. Run-on transcription analysis in nuclei prepared from FRTL5 cells previously cultured in the presence or absence of TSH did not reveal any difference in TPO mRNA transcripts. These results suggest that TSH regulates the level of TPO mRNA in FRTL5 cells, in part via the second messenger cAMP and by a nontranscriptional mechanism. This TSH effect may represent a primary site of TSH action in regulating TPO bioactivity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Evidence for a potential common T-cell epitope between human thyroid peroxidase and human thyroglobulin with implications for the pathogenesis of autoimmune thyroid disease.

In order to explore the possibility that, in autoimmune thyroid disease, anti-thyroglobulin (Tg) and anti-thyroid peroxidase (TPO) antibodies arise concurrently because they share a common T-cell epitope, we performed a detailed comparative analysis of the cDNA nucleotide sequences corresponding to these two genes. We discovered an 8 amino acid region (Leu-Ser-Glu-Asp-Leu-Leu-Ser- Ile in human TPO) in which there were 6 identical and 2 conserved amino acid residues when compared with human Tg. This remarkably similar region is near the amino-terminus of human TPO (residues 119-126) and the carboxyl-terminus of human Tg (residues 2763-2770). A second feature of interest was that this region of homology conforms to the Rothbard algorithm for a T-cell epitope. Third, probing of the Swiss-protein data bank (10,008 proteins containing 2,952,765 amino acids) with the human TPO region revealed greater homology for human Tg than for any other eukaryotic protein. Two bacterial proteins (E. coli aminopeptidase N and stringent starvation protein) had higher homology scores from human TPO than did human Tg. Our findings provide circumstantial evidence that human TPO and human Tg, and possibly certain bacterial proteins, do indeed share common T-cell epitopes that may play a role in the pathogenesis of autoimmune thyroid disease.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H.

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.

Animals↗
PubMed1988

IGF-I increases c-fos expression in FRTL5 rat thyroid cells by activating the c-fos promoter.

We studied the effect of IGF-1 on c-fos mRNA expression in FRTL5 rat thyroid cells. IFG-1, or calf serum, transiently increased c-fos mRNA levels in quiescent FRTL5 cells, with the latter being more potent. This increase was prevented by actinomycin D. FRTL5 cells were stably transfected with the c-fos promoter linked upstream to the reporter gene chloramphenicol acetyl transferase (CAT). Calf serum and IGF-1 increased c-fos promoter activity in a prolonged manner, and to an equal degree. Our data indicate that IGF-1, as well as additional factor(s) in serum, increase c-fos mRNA levels, at least in part, by activation of the c-fos promoter.

Animals↗
PubMed1988

A suppressor of transcriptional activity is present upstream from the rat c-Ha-ras promoter.

We report the presence of an element in the rat c-Ha-ras 5'-flanking region that has a suppressive effect on the promoter of this gene. Promoter activity was determined using constructs of different regions of the c-Ha-ras 5'-flanking region linked to the reporter gene chloramphenicol acetyl transferase (CAT) transfected into NIH-3T3 cells. The presence of the tract 400 base-pairs upstream from the promoter region reduced promoter activity tenfold. This suppressor element was effective in either orientation and was not produced by other DNA fragments of similar length. Transfection with different amounts of plasmid suggested that a trans-acting factor in limiting amounts was acting on the suppressor region. Gel-retardation assays demonstrated that a nuclear protein was present in 3T3 cells that specifically interacted with the 400-base fragment containing the suppressor element.

Animals↗
PubMed1988

TSH stimulates the activity of the c-fos promoter in FRTL5 rat thyroid cells.

C-fos promoter activity was examined in FRTL5 rat thyroid cells transiently transfected with a chimeric gene containing the c-fos promoter region linked upstream of the receptor gene chloroamphenicol acetyl transferase (CAT). Thyroid-stimulating hormone (TSH) stimulation of transfected cells increased CAT activity 2- to 3-fold. TSH did not stimulate CAT activity driven by the Rous sarcoma virus (RSV) promoter. Both forskolin and 8-Br-cyclic AMP also stimulated CAT activity, and were not additive with TSH stimulation. The kinetics of c-fos-driven CAT activity in response to TSH and cyclic AMP inducers were similar, and stimulation was reversible. These data therefore provide the first evidence that TSH and cyclic AMP stimulate the activity of the c-fos promoter in FRTL5 cells.

Animals↗
PubMed1987

The functional activity of the rat c-Ha-ras promoter requires the coordinate involvement of multiple elements.

Promoter activity in the rat c-Ha-ras promoter region was assessed in NIH 3T3 cells using the chloramphenicol acetyltransferase (CAT) reporter gene. Promoter activity was orientation dependent. Deletion of the GC box closet to the transcriptional start site, or the upstream GC box and the two CAAT boxes, greatly diminished promoter activity. The GC boxes in the SV40 early promoter could not functionally replace the downstream c-Ha-ras GC box, but specifically competed for expression of rat c-Ha-ras promoter activity. Serum-stimulated growth of 3T3 cells stably transfected with p035-ras-CAT was not associated with increased promoter activity. These data indicate that the GC and CAAT boxes in the rat c-Ha-ras gene are related to constitutive, and not regulatory, promoter activity, and that these elements are involved in a coordinate manner.

Acetyltransferases↗