Search PubMed⌕ Search

Biomedical subjects

Y Nagayama

Publications and source records attributed to Y Nagayama.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor transcript.

In the present study, we report the molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor (hTSHR) mRNA transcript, which has previously been detected on Northern blot analysis of human thyroid cells. The smaller hTSHR cDNA, designated hTSHR cDNA-I, is approximately 1 kb in size and encodes a protein of 253 amino acids. Comparison of the nucleotide sequence of hTSHR cDNA-I with available hTSHR genomic sequence data reveals that the cDNA-I contains exons 1-8 and unidentified DNA tract, presumably an intron. Thus, the hTSHR cDNA-I encodes for the N-terminal half of the extracellular domain of the hTSHR (approximately 60%). The truncated TSHR-I may be secreted and function as a TSH binding protein.

Alternative Splicing↗
PubMed1992

[The TSH receptor gene and the pathogenesis of Graves' disease].

The molecular cloning of the thyrotropin (TSH) receptor cDNA has led to advances in understanding the structure and function of the molecule and the pathogenesis of autoimmune thyroid disease. The recombinant TSH receptors expressed on mammalian cells are now available for TSAb and TBIAb assays. TBI assay in this system is much more sensitive than the conventional method. The binding sites for TSH and TSAb/TBIAb have been studied with epitope library, synthetic peptides, anti-peptide sera and mutagenesis. Some of these data, however, are confusing and undefined. The binding sites for TSH and TSAb/TBIAb are very likely to span the entire region of the extracellular domain of the TSH receptor with discontinuous contact points, and seem to be different from each other on N-terminal half, but similar on C-terminal half, of the extracellular domain of the receptor. However, the determination of the precise amino acids involved may be very difficult without monoclonal anti-TSH receptor antibodies.

Amino Acid Sequence↗
PubMed1992

Obstructive jaundice caused by polypoid inflammatory granuloma in a consanguineous case.

Granuloma in the biliary system is a rare condition. The majority of the reported cases concern postsurgical granuloma. We note a patient with obstructive jaundice due to inflammatory granuloma in whom a filling defect was found at the bifurcation of the common hepatic duct by preoperative cholangiography. There have been no reported cases with similar findings. In addition to these features, this case was further characterized by consanguinity, repeated spontaneous abortions, and severe vascular lesions found in many organs that suggested the presence of a hereditary defect.

Bile Duct Diseases↗
PubMed1992

Role of amino acids 261-418 in proteolytic cleavage of the extracellular region of the human thyrotropin receptor.

Previous in vivo cross-linking studies of TSH to the recombinant TSH receptor revealed that the receptor exists at least in part as a single chain glycoprotein of approximately about 100 kilodaltons (kDa), with intramolecular disulfide bonds. TSH also binds to a 54-kDa amino-terminal fragment of the TSH receptor (cleaved up-stream of amino acid residue 317). In the present study in order to better understand the structure of the TSH receptor, we covalently cross-linked radiolabeled TSH to six TSH-LH receptor extracellular region chimeras and the wild-type TSH receptor expressed on Chinese hamster ovary cells in vivo. In these chimeras, different regions of the TSH receptor were substituted with the homologous regions of the LH receptor. When analyzed under nonreducing conditions by polyacrylamide gel electrophoresis, the TSH-cross-linked products were similar to all TSH-LH receptor chimeras and the wild-type receptor. In contrast, differences among the receptors were noted when the TSH-cross-linked products were examined under reducing conditions. With the exception of two chimeras, as noted previously with the wild-type receptor, two TSH-cross-linked products were observed, representing TSH cross-linked to a holoreceptor of about approximately 100 kDa and a fragment of the receptor of about approximately 54 kDa. However, in the two chimeras in which both domains D and E (amino acids 261-418) of the TSH receptor were substituted, only the holoreceptor and not the smaller fragment was detected. Substitution of domains ABC (amino acids 1-260) did not prevent proteolytic cleavage of the TSH receptor. In conclusion, amino acids 261-418 are necessary for proteolytic cleavage of the extracellular region of the human TSH receptor.

Endopeptidases↗
PubMed1992

Role of the carboxyl-terminal half of the extracellular domain of the human thyrotropin receptor in signal transduction.

We studied the role of the carboxyl-terminus of the extracellular region of the human TSH receptor in signal transduction (cAMP generation). For this purpose, we introduced homologous substitutions of smaller segments within amino acids 261-418 (domains D and E) of the TSH receptor with the corresponding amino acids of the rat LH/CG receptor. Amino acids 317-366 were not investigated in view of previous data indicating their noninvolvement. Mutant TSH receptor cDNAs, in a eukaryotic expression vector, were stably transfected into Chinese hamster ovary cells. Eight of nine plasmid constructs expressed TSH receptors that could be detected by radiolabeled TSH binding; six of these were of high affinity similar to the wild-type receptor and, therefore, provided informative data on signal transduction. Despite high affinity TSH binding, five of six TSH receptor mutants displayed a diminished cAMP response to TSH stimulation, suggesting the involvement of broad segments of domains DE in signal transduction. Amino acids 270-278 and 287-297 were particularly important in this respect. The conformation conferred by these segments of the TSH receptor, therefore, appears to be involved in transducing a signal from the extracellular to the intracellular region of the receptor.

Amino Acid Sequence↗
PubMed1992

Thyroid stimulatory autoantibodies in different patients with autoimmune thyroid disease do not all recognize the same components of the human thyrotropin receptor: selective role of receptor amino acids Ser25-Glu30.

To study the interaction between TSH, autoantibodies and the amino-terminal half of the TSH receptor extracellular region (amino acids 1-260; domains ABC), we constructed 20 LH/CG chimeric receptor cDNAs. The prototypic receptor for modification contained domains ABC of the TSH receptor and domains DE of the LH/CG receptor. Segments (6-13 amino acids) within the ABC domains were replaced with the corresponding amino acids of the rat LH/CG receptor. Fifteen of the 20 chimeric receptors could be expressed functionally in Chinese hamster ovary cells. Twelve retained both high affinity TSH binding and normal signal transduction. These 12 receptors were tested with a panel of 10 patients' immunoglobulin G (IgG) samples containing potent TSH receptor stimulatory activity. With 11 of the receptor variants, the cAMP responses were similar to those with the prototype receptor (TSH-LHR-6). However, the -A1 variant of TSH-LHR-6 (Ser25-Glu30) responded poorly to 6 of 10 IgGs. The same pattern was observed when the IgGs were tested for their ability to inhibit [125I] TSH binding to the receptor variants, suggesting qualitative differences between the different stimulatory TSH receptor autoantibodies. Therefore, we examined the dose-response relationship of 2 IgGs that were approximately equipotent when tested with TSH-LHR-6 and its -A1 variant and another 2 IgGs that displayed greatly diminished potency with respect to the -A1 variant. Despite dilution to nearly undetectable levels, the relative potencies of the 4 IgG samples for both types of receptors remained similar. These data demonstrate directly that stimulatory TSH receptor autoantibodies do not all recognize the same components of the TSH receptor. The segment of the TSH receptor discriminated by these autoantibodies is between amino acids Ser25-Glu30.

Amino Acid Sequence↗
PubMed1992

The thyrotropin receptor 25 years after its discovery: new insight after its molecular cloning.

The molecular cloning and functional expression of the TSH receptor has led to rapid advances in understanding the structure and function of the molecule. Knowledge of its genomic structure provides information on the evolutionary origin of the TSH receptor as well as on the functional organization of its extracellular domain, which is responsible for ligand binding. A beginning has been made in defining the discontinuous contact points for TSH in this extracellular region, but determination of all of the amino acids involved will be difficult. The binding sites of TSH receptor autoantibodies do not appear to be identical to the TSH binding site. Two of the six potential glycosylation sites in the extracellular domain are important in the expression of a functional receptor. Disulfide bonding contributes toward maintenance of the three-dimensional structure of the receptor. Recent evidence suggests that the TSH receptor exists as a single polypeptide chain without subunits. Significant progress has been made in understanding the intracellular regions of the TSH receptor that are involved in signal transduction. Although still in the distant future, we are closer to the goal of understanding precisely how TSH interacts with and activates its receptor. More importantly from the clinical perspective, we are closer to defining the B cell, and ultimately T cell, epitopes on the TSH receptor that are recognized by the immune system. This information may ultimately facilitate the development of immunological approaches to treating Graves' disease, which will be an improvement over thyroid gland destruction and consequent hypothyroidism, the most common form of therapy at the present time.

Amino Acid Sequence↗
PubMed1992

Studies on the role of amino acids 38-45 in the expression of a functional thyrotropin receptor.

We previously reported that deletion or substitution of a unique eight-amino acid tract (residues 38-45) in the extracellular domain of the human TSH receptor led to the loss of specific ligand binding to the surface of transfected cells. In the present study we analyzed this region in more detail. Using site-directed mutagenesis of the TSH receptor cDNA, we substituted amino acid residues 38-45, either in three overlapping groups of four amino acids each or individually. The resultant TSH receptor mutant cDNAs were stably transfected into Chinese hamster ovary cells, and the cells were tested for their TSH-binding ability. Our data demonstrate that amino acid residues 38-40 and 42-45 in this region of the human TSH receptor can be substituted without alteration in receptor function and are, therefore, not critical in forming or maintaining the TSH-binding site. However, substitution of Cys41, either alone or together with adjacent amino acids, leads to the loss of TSH binding to its receptor. These data suggest a central role for the amino acid in position 41 in preserving the biological function of the TSH receptor.

Amino Acid Sequence↗
PubMed1992

Decrease in renal function following decreased fibrinogen and raised fibrin degradation products in lupus nephritis with nephrotic syndrome.

To clarify the relationship between renal function and laboratory indicators of abnormal blood coagulation in lupus nephritis with nephrotic syndrome, we studied 28 patients with this disease over the 11 years. Follow-up tests included serial determination of plasma fibrinogen, serum fibrin degradation products (FDP), serum creatinine and endogenous creatinine clearance (Ccr) as well as other parameters. Either decreased fibrinogen, elevated FDP, or both were found in 13 cases. Taking as a criterion of decreased renal function an increase in serum creatinine above 0.2 mg/100 ml combined with a decrease in Ccr by more than 30% below the immediately preceding values, 11 of the above 13 patients showed a marked deterioration in renal function concurrently or within two weeks after the episodes of clotting disorder. Two other patients showed a less marked decrease in renal function. The 15 patients whose circulating fibrinogen and FDP remained normal did not show deterioration in renal function. The difference in renal function decrease between the groups with and without abnormal hemocoagulation indicators is significant. Our preliminary results indicate that when fibrinogen and FDP indicate intravascular hypercoagulation deterioration in renal function in patients with lupus nephritis is probable.

Adolescent↗
PubMed1992

[Infection and asthma. V. Study of childhood asthma and viral infection by cytological analysis of nasal smears].

Cytological analysis of nasal smears was carried out for 295 asthmatic children aged 2 to 15 years old at the time of asthma attack and non-attack, in combination with blood tests and chest roentgenograms. Damaged ciliated cells (ciliocytophhthoria, CCP) were looked for as a marker of viral infections. The appearance rate of greater than 2+ of CCP was higher among children during attack (38/70, 54.3%) than that among children not suffering an attack (6/29, 20.7%); this was pronounced among young children under five. However, the results of blood examinations and chest roentgenograms did not correlate with the rate of positive signs of infections in CCP positive- or negative-attack. These results suggest that the cytological examination of nasal smears in useful in evaluating the presence of viral infections during asthma aggravation among young children, whereas evaluation of these infections may be difficult by blood tests or chest roentgenograms.

Adolescent↗
PubMed1991

A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

Amino Acid Sequence↗
PubMed1991

Eleven amino acids (Lys-201 to Lys-211) and 9 amino acids (Gly-222 to Leu-230) in the human thyrotropin receptor are involved in ligand binding.

Our previous studies involving chimeric thyrotropin-lutropin/choriogonadotropin (TSH-LH/CG) receptors suggest that multiple segments spanning the entire extracellular domain of the human TSH receptor contribute to the TSH binding site. Nevertheless, the mid-region (segment C, amino acid residues 171-260) of the receptor extracellular domain is particularly important in TSH binding. In the present studies, we constructed seven new chimeric receptors in order to analyze segment C in further detail. Seven small segments spanning segment C of the TSH receptor were replaced with the counterpart of the rat LH/CG receptor. These mutant receptors were stably introduced into Chinese hamster ovary cells and were tested for hormone binding and cAMP responsiveness to hormone stimulation. The results indicate that 11 amino acids of the TSH receptor (Lys-201 to Lys-211) and the corresponding region of the LH/CG receptor (Thr-202 to Ile-212) are important for specific TSH and human CG binding, respectively. In addition, nine amino acids of the TSH receptor (Gly-222 to Leu-230) are also involved in TSH binding. A further conclusion from these data is that TSH and human CG bind to partially overlapping sites on their respective receptor molecules.

Amino Acid Sequence↗
PubMed1991

Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for thyrotropin receptor function.

To define the sites in the extracellular domain of the human thyrotropin (TSH) receptor that are involved in TSH binding and signal transduction we constructed chimeric thyrotropin-luteinizing hormone/chorionic gonadotropin (TSH-LH/CG) receptors. The extracellular domain of the human TSH receptor was divided into five regions that were replaced, either singly or in various combinations, with homologous regions of the rat LH/CG receptor. The chimeric receptors were stably expressed in Chinese hamster ovary cells. The data obtained suggest that the carboxyl region of the extracellular domain (amino acid residues 261-418) and particularly the middle region (residues 171-260) play a role in signal transduction. The possibility is also raised of an interaction between the amino and carboxyl regions of the extracellular domain in the process of signal transduction. With respect to hormone binding, substitution of the entire extracellular domain of the LH/CG receptor for the corresponding region of the TSH receptor resulted in high-affinity human CG binding with complete loss of TSH binding. Surprisingly, however, there was at least one chimera with a substitution at each of the five domains that still retained high-affinity TSH binding. Substitution of residues 1-170 of the TSH receptor with the corresponding region of the LH/CG receptor was associated with the retention of high-affinity TSH binding but ligand specificity was lost in that TSH and human CG could interact functionally with the receptor. In summary, these studies suggest that the middle region and carboxyl half of the extracellular domain of the TSH receptor are involved in signal transduction and that the TSH-binding region is likely to span the entire extracellular domain, with multiple discontinuous contact sites.

Animals↗
PubMed1991

Clinical observations on lower respiratory tract infections with special reference to serum IgE levels.

To examine if the occurrence of wheezing among children with lower respiratory tract infections is associated with atopic predisposition, serum levels of IgE were determined for groups of children with or without wheezing, during infection with Mycoplasma pneumoniae or respiratory syncytial virus (RSV). In M. pneumoniae infection, more than 60% of wheezy children over 2 years of age showed a high IgE level (200 IU/mL or more), while IgE levels were less than 200 IU/mL in 73% of non-wheezy children. In RSV infection, the incidence of high IgE levels among wheezy children was nearly double that of non-wheezy children, although the difference was not significant. These results suggest that in children an allergic disposition is involved in the occurrence of wheezing during the course of lower respiratory tract infections.

Age Factors↗
PubMed1991

Differences and similarities in the diagnosis and treatment of Graves' disease in Europe, Japan, and the United States.

In three separate studies, members of the American Thyroid Association (ATA), the European Thyroid Association (ETA), and the Japan Thyroid Association (JTA) were surveyed by questionnaire on their management of Graves' disease. The aim was to determine how expert clinical thyroidologists employ diagnostic procedures and the three different therapies that are available for this disorder. In this report, we identify, summarize, compare, and contrast similarities and differences in the results of these surveys in these three different regions of the world. In general, ATA members used fewer diagnostic tests than did their European or Japanese colleagues. For the index patient, radioiodine was the therapy of choice for 69% of ATA respondents but only 22% and 11% of ETA and JTA respondents, respectively. In contrast, only 30.5% of ATA respondents chose antithyroid drugs as first-line therapy compared to 77% of ETA and 88% of JTA respondents. There was consensus on the relative lack of a role for thyroidectomy except for narrow indications. The implications of these differing approaches for the diagnosis and treatment of hyperthyroidism due to Graves' disease are discussed.

Adolescent↗
PubMed1991

Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors.

We examined the relative effects of thyrotropin (TSH) and TSH receptor autoantibodies in the sera of patients with autoimmune thyroid disease on three TSH-lutropin/chorionic gonadotropin (LH/CG) receptor extracellular domain chimeras. Each chimera binds TSH with high affinity. Only the chimera with TSH receptor extracellular domains ABC (amino acids 1-260) had a functional (cAMP) response to thyroid stimulatory IgG. The chimeras with TSH receptor domains CD (amino acids 171-360) and DE (amino acids 261-418) were unresponsive. The lack of response of the chimera with TSH receptor domains DE was anticipated because it fails to transduce a signal with TSH stimulation, unlike the other two chimeras. A different spectrum of responses occurred when the TSH-LH/CG chimeras were examined in terms of autoantibody competition for TSH binding. IgG with TSH binding-inhibitory activity when tested with the wild-type TSH receptor also inhibited TSH binding to the chimera with TSH receptor domains DE. Dramatically, however, these IgG did not inhibit TSH binding to the chimera with TSH receptor domains CD, and had weak or absent activity with the chimera with TSH receptor domains ABC. Chimeras with TSH receptor domains ABC and DE were equally effective in affinity-purifying IgG with thyroid-stimulatory and TSH binding-inhibitory activities. Nonstimulatory IgG with TSH binding-inhibitory activity inhibited the action of stimulatory IgG on the wild-type TSH receptor, but not with the chimera containing TSH receptor domains ABC. In summary, TSH receptor autoantibodies and TSH bind to regions in both domains ABC and DE of the TSH receptor extracellular region. Stimulatory and inhibitory TSH receptor autoantibodies, as well as TSH, appear to bind to different sites in domains ABC, but similar sites in domains DE, of the receptor. Alternatively, TSH and the different TSH receptor antibodies bind with differing affinities to the same site in the ABC region.

Autoantibodies↗