Analysis of a uranium solution for evaluating the total number of fissions in the JCO criticality accident in Tokai-mura.

The uranium solution in the precipitation tank in the JCO's uranium conversion facility was analyzed in order to evaluate the total number of fissions in the criticality accident. Two analytical groups at JAERI performed chemical analyses independently in order to check the validity of the results: the concentration of the fission products (95Zr, 99Mo, 103Ru, 131I, 140Ba, etc), uranium, boron and impurity elements in the solution. The analytical results obtained by the two groups were almost in agreement within the analytical error. The number of fissions per one gram of uranium in the accident was determined to be (1.5 +/- 0.1 ) x 10(14). Also, the total number of events was evaluated to be (2.5 +/- 0.1) x 10(18) fissions using the total amount of uranium (16.6 kg) fed into the precipitation tank at the accident.

Cloning and characterization of the novel thyroid and eye muscle shared protein G2s: autoantibodies against G2s are closely associated with ophthalmopathy in patients with Graves' hyperthyroidism.

Serum autoantibodies against eye muscle antigens are closely linked with thyroid-associated ophthalmopathy (TAO), although their significance is unclear. The two antigens that are most often recognized are eye muscle membrane proteins with molecular masses of 55 and 64 kDa, as determined from immunoblotting with crude human or porcine eye muscle membranes. We cloned a fragment of the 55-kDa protein by screening an eye muscle expression library with affinity-purified anti-55 kDa protein antibody prepared from a TAO patient's serum. A complementary DNA (cDNA) encoding a novel protein, which we have called G2s, was sequenced on both strands, and its size was 411 bp. The open reading frame of G2s corresponded to a 121-amino acid peptide with a size of 1.4 kb. Using the rapid amplification of 5'-cDNA ends technique we were able to clone an additional 0.3 kb of the protein. G2s did not share significant homologies with any other entered protein in computer databases and had one putative transmembrane domain. Using the 1.4 kb cDNA as probe in Northern blotting of a panel of messenger ribonucleic acids prepared from human tissues, the parent protein was shown to correspond to a large molecule of about 5.8 kb with a calculated molecular mass of approximately 220 kDa, consistent with earlier immunoblot studies performed in the absence of reducing agents. G2s was strongly expressed in eye muscle, thyroid, and other skeletal muscle and to a lesser extent in pancreas, liver, lung, and heart muscle, but not in kidney or orbital fibroblasts. We tested sera from patients with Graves' hyperthyroidism with and without ophthalmopathy and from control patients and subjects for antibodies against a G2s fusion protein by immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting, antibodies reactive with G2s were identified in 70% of patients with TAO of less than 3 yr duration, 53% with TAO of more than 3 yr duration, 36% with Graves' hyperthyroidism without evident ophthalmopathy, 17% with Hashimoto's thyroiditis, 3% with type 1 diabetes, 23% with nonimmunological thyroid disorders, and 16% of normal subjects. The prevalences, compared to normal values, were significant for the two groups of patients with TAO, but not for the other groups. Tests were positive in 54% of patients with active TAO, 33% with chronic ophthalmopathy, 36% with Graves' hyperthyroidism, 54% with Hashimoto's thyroiditis, 23% with type 1 diabetes, and in 11% of normal subjects using enzyme-linked immunosorbent assay. The antibodies predicted the development of the ocular myopathy subtype of TAO in six of seven patients and the congestive ophthalmopathy subtype in seven of eight patients, respectively, with Graves' hyperthyroidism studied prospectively during and after antithyroid drug therapy. Antibodies reactive with G2s may be early markers of ophthalmopathy in patients with Graves' hyperthyroidism. Because G2s is expressed in both thyroid and eye muscle, immunoreactivity against a shared epitope in the two tissues may explain the well known link between thyroid autoimmunity and ophthalmopathy.

Significance of serum antibodies reactive with flavoprotein subunit of succinate dehydrogenase in thyroid associated orbitopathy.

AIMS: Thyroid associated orbitopathy (TAO) is an autoimmune disorder of extraocular muscles and orbital connective tissue. Identification of the principal target antigens would help the understanding of the pathogenesis of the disease and possibly lead to the development of specific therapies in the future. The purpose of this study was to measure serum antibodies against the flavoprotein subunit of succinate dehydrogenase in patients with TAO and correlate their presence with factors of TAO. METHODS: Sera of patients with active TAO of 6 months' duration or less were tested for antibodies against the flavoprotein subunit of succinate dehydrogenase. Clinical data were obtained by retrospective review of patients' charts. Enzyme linked immunosorbent assay was used to test sera for serum antibodies against purified succinate dehydrogenase. RESULTS: 38 patients with TAO and 32 healthy age and sex matched controls were included in the study. Anti-flavoprotein antibodies were detected in 24 out of 38 patients with TAO (63.16%) and in five out of 32 healthy controls (15.63%) (p<0.01). Neither age, sex, duration of thyroid disease, thyroid status, treatment of thyroid disease, smoking history, duration of orbitopathy, activity of orbitopathy, nor the presence of lid retraction were significantly associated with the presence of serum anti-flavoprotein antibodies (p>0.05). However, the total number of rectus muscles affected in both eyes of the patients was significantly correlated with the finding of a positive antibody test (p<0.05). CONCLUSIONS: Serum antibodies reactive with the flavoprotein subunit of succinate dehydrogenase are associated with extraocular muscle involvement in active TAO of recent onset.

Serum antibodies against the flavoprotein subunit of succinate dehydrogenase are sensitive markers of eye muscle autoimmunity in patients with Graves' hyperthyroidism.

Thyroid-associated ophthalmopathy is an autoimmune disorder of the extraocular muscles and orbital connective tissue, which is usually associated with Graves' hyperthyroidism. Well-studied markers of ophthalmopathy are eye muscle membrane antigens, reportedly of approximately 64-kDa molecular mass. One, originally identified only as the 64-kDa protein, has recently been shown to be the flavoprotein (Fp) subunit of mitochondrial succinate dehydrogenase, which has a correct molecular mass of 67 kDa. We have used purified beef heart Fp as antigen in an enzyme-linked immunosorbent assay for cross-reactive human autoantibodies. Sera have been screened from patients with thyroid-associated ophthalmopathy classified according to activity and presence or not of eye muscle disease, and from those with Graves' hyperthyroidism without eye involvement. Also examined were serum samples taken periodically from 20 patients with Graves' hyperthyroidism during 24 months of treatment of their hyperthyroidism with antithyroid drugs. Four of these patients had ophthalmopathy at the onset, 12 developed ophthalmopathy, and 4 did not develop any eye signs during treatment. Anti-Fp subunit antibodies were detected in 73% of patients with active ophthalmopathy and evidence of eye muscle involvement but only in 25% if there was only congestive ophthalmopathy. These values were 0% and 11% for patients with chronic ophthalmopathy, with or without eye muscle dysfunction, respectively. The antibodies were also detected in 14% of patients with Graves' hyperthyroidism without evident ophthalmopathy, 11% of patients with nonimmunologic thyroid disorders, 12% of type I diabetics, and 12% of age- and sex-matched normal subjects. Significantly, appearance of anti-Fp antibodies predicted the development of ophthalmopathy in 5 of the 6 patients with Graves' hyperthyroidism, who developed eye muscle dysfunction after treatment of the hyperthyroidism, and coincided with the onset of eye muscle signs in the other patient. Antibodies were not detected in any of 6 patients who developed congestive ophthalmopathy without evidence of eye muscle damage or in 4 patients who did not develop any eye signs. In conclusion, we have shown a close relationship between eye muscle disease and serum antibodies against the Fp subunit of succinate dehydrogenase in patients with Graves' hyperthyroidism.

A 63 kDa skeletal muscle protein associated with eye muscle inflammation in Graves' disease is identified as the calcium binding protein calsequestrin.

It is generally accepted that thyroid-associated ophthalmopathy (TAO) is an autoimmune disease of the eye muscle (EM) and the surrounding orbital connective tissue in which circulating antibodies play an important role. Antibodies against EM membrane proteins of 63-67kDa mol. wt. seem to be the best markers of ophthalmopathy in patients with autoimmune thyroid disease. We purified a 63 kDa EM protein using SDS-polyacrylamide gel electrophoresis technology and TAO patients' sera as probes, digested the protein with cyanogen bromide and sequenced immunoreactive peptides. We also screened a human EM library with a rabbit antiserum against 63-65 kDa proteins and affinity purified antibodies from a TAO patient's serum that reacted with a 55 kDa EM membrane protein. From partial sequence information and from DNA sequencing of positive cDNA clones, the protein was identified as calsequestrin, a 63 kDa calcium binding protein localized in the sarcoplasmic reticulum of the muscle fiber. As determined by Northern blotting, calsequestrin was expressed in EM and other skeletal muscle but not thyroid or fibroblasts. Calsequestrin is different from the "64 kDa protein", which has been identified as succinate dehydrogenase flavoprotein subunit, which has a corrected mol. wt. of 67 kDa. Serum antibodies against calsequestrin were found in 40% of patients with clinically active TAO, but in only 4% of those with stable eye disease, and in 5% of normal subjects, by immunoblotting. Although it is possible that autoimmunity against calsequestrin plays a role in the progressive EM damage that characterizes ophthalmopathy it is more likely that the antibodies are secondary to a reaction against some other cell membrane protein, such as the novel thyroid and eye muscle shared protein G2s or the TSH receptor.

Thyroid-associated ophthalmopathy in black South African patients with Graves' disease: relationship to antiflavoprotein antibodies.

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins of 64 kd are good markers of ophthalmopathy in patients with thyroid autoimmunity. The 64-kd protein is now shown from a partial sequence to be the flavoprotein subunit (Fp) of mitochondrial succinate dehydrogenase. Hyperthyroidism due to Graves' disease is increasing in incidence among urban black female Africans, possibly because of exposure to environmental risk factors such as increased dietary iodine ingestion and stress. Ophthalmopathy is frequently observed in this clinical context, but its association with serum autoantibodies reactive with Fp has not been examined. We studied 19 black South African patients with Graves' disease during the course of prolonged antithyroid drug administration, of whom 10 had congestive ophthalmopathy, but no clinical evidence for eye muscle damage at the onset. Anti-Fp antibodies were detected in 2 of these patients, as well as in 2 of the 9 patients who did not have overt eye disease. Additionally, the antibodies became positive in 3 patients with ophthalmopathy in whom tests were negative initially, remained positive in 1 patient throughout the study period and became negative in 1 patient with positive tests initially. Ophthalmopathy did not develop in any of the 9 patients who lacked this complication on presentation. The reasons why we failed to demonstrate a close relationship between anti-Fp antibodies and the eye muscle component of ophthalmopathy are unclear although one possibility is that ocular myopathy is an uncommon manifestation in African thyrotoxic patients compared with those of Caucasian origin. The relationship between anti-Fp antibodies and eye muscle inflammation in patients with thyroid autoimmunity of different ethnic origins and environmental settings, needs to be addressed in a large prospective study.

Eye muscle antibodies in patients with ocular myasthenia gravis: possible mechanism for eye muscle inflammation in acetylcholine-receptor antibody-negative patients.

Myasthenia gravis is an organ-specific autoimmune disorder generally thought to be caused by an antibody-mediated attack against the skeletal muscle nicotinic acetylcholine (Ach) receptor (AchR) at the neuromuscular junction. Extraocular muscle weakness and double vision are present in about 90% of patients with myasthenia gravis and are the predominant complaints in about 20% of patients, when the condition is called ocular myasthenia gravis (OMG). While serum antibodies against the AchR are detected in most patients with generalized myasthenia gravis (GMG), they are not found in about one-third of patients with the ocular variety, and epidemiological, clinical, and serological studies suggest that OMG and GMG are two separate diseases. Both forms of myasthenia gravis are sometimes associated with thyroid autoimmunity or thyroid-associated ophthalmopathy (TAO). We have therefore tested the sera of patients with GMG and OMG by Western blotting for antibodies against porcine eye muscle membrane proteins in general, and by enzyme-linked immunosorbent assays (ELISA) specifically for reaction with two skeletal muscle antigens which are prominent marker antigens for TAO, namely, the calcium-binding protein calsequestrin and the so-called "64-kDa protein." The 64-kDa protein has recently been identified as the flavoprotein subunit of mitochondrial succinate dehydrogenase. Patients with ophthalmopathy and myasthenia were excluded. Nine of the patients had associated Graves' hyperthyroidism without evident ophthalmopathy and one had Hashimoto's thyroiditis. Antibodies against porcine eye muscle membrane antigens of M(r) 15-110 kDa were detected in patients with GMG or OMG, one or more antibodies being detected in 100% of patients with GMG and in 88% of those with OMG. The most frequently found antibodies were those targeting eye muscle membrane proteins of 15, 67, and 110 kDa. Antibodies reactive with purified calsequestrin (63 kDa) were detected in 21% of patients with OMG but in no patient with GMG. Antibodies recognizing purified succinate dehydrogenase (67 kDa) were found in 42% of patients with OMG, in 100% (5 of 5) of patients with GMG, and in 48% of all patients with myasthenia gravis not associated with Graves' hyperthyroidism. There was no close correlation between any eye muscle-reactive antibody and antibodies against the AchR in either group of myasthenic patients. The findings support the notion that immunoreactivity against skeletal muscle proteins other than the AchR may play a role in the development of the muscle weakness in AchR antibody-negative patients with OMG and GMG, although it is unlikely that any of the antibodies demonstrated in this study are directly implicated. Similarly, while the demonstration of antibodies reactive with eye muscle antigens associated with TAO in patients with OMG raises the possibility that the link between the ocular lesions of myasthenia gravis and Graves' disease may be autoimmunity against a common antigen(s), it is more likely that both disorders are mediated by cytotoxic T cells recognizing another cell membrane antigen, such as the novel thyroid and eye muscle shared protein G2s, and that serum antibodies reactive with succinate dehydrogenase Fp subunit and calsequestrin are markers of an immune-mediated eye muscle reaction.

Role of the eye muscles in thyroid eye disease: identification of the principal autoantigens.

Thyroid-associated ophthalmopathy (TAO) is a progressive orbital disorder associated with Graves' hyperthyroidism and, less often, Hashimoto's thyroiditis in which autoantibodies react with orbital antigens and lead to exophthalmos and eye muscle inflammation. Eye muscle (EM) membrane proteins initially reported as 55 and 64 kd are the best markers of ophthalmopathy. The "64-kd protein" is now shown to be the flavoprotein subunit of mitochondrial succinate dehydrogenase and to have a correct molecular weight of 67 kd. We have cloned a fragment of a novel eye muscle protein, which we call G2s, and sequenced 1.4 kb of the full length cDNA. G2s does not share any significant homologies with other reported proteins. The 5.9 kb G2s mRNA, that corresponds to a protein of approximately 220 kd, is expressed in EM, other skeletal muscle and thyroid, but not in other tissues tested. We have also cloned and sequenced a 63-kd eye muscle protein identified as the calcium binding protein calsequestrin. Antibodies against calsequestrin were found in 40% of patients with active ophthalmopathy, but in 0% of normal subjects. Finally, we have sequenced a 19 amino acid fragment of a 55-kd porcine eye muscle membrane protein that exactly matched porcine and human sarcalumenin, a 160-kd glycoprotein localized in the lumen of the longitudinal sarcoplasmic reticulum of the skeletal muscle fiber where it binds calcium. A 53-kd glycoprotein fragment of the molecule corresponds to the 55-kd protein. In a preliminary study, serum antibodies against purified sarcalumenin were detected in 40% of patients with active TAO of less than 1 year duration, but in no controls tested. We porpose that the primary autoantigen in TAO is G2s, which would also explain the association of ophthalmopathy with thyroid autoimmunity, and that antibodies against the intracellular proteins flavoprotein, calsequestrin, and sarcalumenin are secondary markers of an immune-mediated reaction in eye muscle in patients with thyroid autoimmunity.

[Echocardiographic observation of appearance and progression of mitral annular calcification in a patient with familial obstructive hypertrophic cardiomyopathy].

A 37-year-old man with familial hypertrophic obstructive cardiomyopathy (HOCM) developed mitral annular calcification (MAC) during the follow-up period. At the age of 23 years, a systolic murmur and electrocardiographic abnormality including giant T wave inversion were detected incidentally. His elder brother also had HOCM. Catheterization disclosed a left ventricular outflow pressure gradient of 25 mmHg and thickened interventricular septum. Echocardiography showed asymmetric septal hypertrophy and systolic anterior motion of the mitral valve. The patient was followed up by repeated echocardiography from age 37 years and the onset of MAC (2 mm in thickness) was found at age 48 years. One year later, the MAC had progressed markedly (5 mm) without other remarkable changes in the M-mode echocardiogram, except mitral regurgitation (at age 41 years), left ventricular apical as well as posterior wall hypertrophy (age 43 years) and left atrial enlargement (age 46 years). The left ventricular inflow velocity at the atrial contraction period decreased significantly concomitantly with MAC. MAC is a rare complication in young and middle aged patients. The onset and progression of MAC is still obscure in HOCM. This patient showed that sudden onset and rapid progression of MAC can occur in young patients with HOCM.

Reevaluation of the prevalences of serum autoantibodies reactive with "64-kd eye muscle proteins" in patients with thyroid-associated ophthalmopathy.

Serum autoantibodies reactive with eye muscle proteins of "64 kilodaltons (kd)" are frequently found in patients with Graves' hyperthyroidism and thyroid-associated ophthalmopathy (TAO). Earlier, we cloned a 64-kd protein that was identified as calsequestrin, a calcium-binding protein localized in the sarcoplasmic reticulum of striated muscle and extensively studied another cloned 64-kd protein, called 1D, which is expressed in thyroid and eye muscle, and some other tissues. Using a monoclonal antibody against calsequestrin, a polyclonal antibody against 1D and a TAO patient serum reactive with the "64-kd protein," as probes, we performed Western blots of porcine eye muscle membrane. We identified three different proteins in the 63 to 67 kd molecular weight range that were targeted by antibodies in sera from patients with TAO. It was not possible to differentiate antibodies reactive with calsequestrin and 1D because these two proteins have very similar molecular weights--63 to 64 kd--and band appearance in Western blotting. A 67-kd protein was most frequently recognized by TAO patients' sera. Serum antibodies reactive with the 67-kd protein were detected in 73% of patients with active TAO of 1 year duration or less, in 37% of patients with TAO of more than 3 years' duration, in 35% with Graves' hyperthyroidism without evident ophthalmopathy, in 30% of patients with Hashimoto's thyroiditis, and in 16% of normal subjects. Serum antibodies reactive with calsequestrin/1D were detected in 47% of patients with active TAO of less than 1 year, in 22% of patients with TAO longer than 3 years, 17% with Graves' hyperthyroidism without evident ophthalmopathy, in 10% of patients with Hashimoto's thyroiditis, and in 21% of normal subjects. The prevalence of anti-67-kd protein antibodies in TAO patients corresponded to those reactive with the so called "64-kd protein" that we have reported previously. In conclusion, we were able to improve the accuracy of the Western blots by comparing the molecular weight of positive bands using specific antibodies reactive with eye muscle antigens as probes. The previously recognized, and extensively studied, "64-kd protein" is now shown to have a molecular weight of 67 kd. The role of the various eye muscle antibodies in the diagnosis and management of the ophthalmopathy associated with Graves' hyperthyroidism needs to be addressed in prospective studies using purified or recombinant full-length proteins.

The 64-kilodalton eye muscle protein is the flavoprotein subunit of mitochondrial succinate dehydrogenase: the corresponding serum antibodies are good markers of an immune-mediated damage to the eye muscle in patients with Graves' hyperthyroidism.

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine eye muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine eye muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that eye muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and eye muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the eye muscle fiber and may support the idea that the extraocular muscles are targets of the autoimmune reactions of TAO.

Role of eye muscle antibody measurement in diagnosis of thyroid-associated ophthalmopathy: a laboratory update.

OBJECTIVE: To review the current role of measurement of serum eye muscle antibodies in thyroid-associated ophthalmopathy (TAO). METHODS: We conducted laboratory studies to determine the prevalences of serum autoantibodies reactive with eye muscle antigens in patients with active and inactive TAO, Graves' hyperthyroidism, and Hashimoto's thyroiditis as well as in normal subjects. RESULTS: The two antigens most often recognized in immunoblotting with crude human or porcine eye muscle membranes by serum autoantibodies in patients with TAO are eye muscle membrane proteins of 55 and 64 kd. One 64-kd eye muscle protein has recently been cloned by screening a human eye muscle expression library with two different antibody probes and identified from a computer gene bank search as the calcium-binding protein calsequestrin. A fragment of a 220-kd eye muscle protein, called G2s, has also been cloned by screening the eye muscle library with affinity-purified antibodies reactive with a 55-kd eye muscle membrane protein. The prevalences of autoantibodies reactive with these two antigens in our study groups were as follows. Antibodies against calsequestrin were detected in 38% of patients with TAO for <1 year, in 17% of those with TAO for >3 years, in 17% of patients with Graves' hyperthyroidism without ophthalmopathy, in 12% of patients with Hashimoto's thyroiditis without ophthalmopathy, and in 21% of normal subjects. Antibodies reactive with the 64-kd protein were demonstrated in 62% of patients with recent-onset active TAO, in 33% with eye disease for >3 years, in 39% of patients with Graves' hyperthyroidism without ophthalmopathy, in 25% of patients with Hashimoto's thyroiditis, and in 16% of normal control subjects. Antibodies reactive with G2s fusion protein were detected in 67% of patients with recent-onset active TAO, in 46% of patients with Graves' hyperthyroidism, and in 20% of normal subjects. Antibodies reactive with the parent protein, of which G2s is a fragment, may be markers of early eye muscle swelling and inflammation, whereas those reactive with the 64-kd protein and, less often, calsequestrin are associated with established eye disease. CONCLUSION: Measurement of serum eye muscle antibodies is recommended as an aid to the early diagnosis of ophthalmopathy in predisposed patients and first-degree relatives of patients with TAO as well as to monitor active or progressive eye disease.

Point mutations in the thyrotropin receptor in human thyroid tumors.

The mechanism of the impaired response to thyrotropin (TSH) in thyroid tumor cells was investigated by searching for structural changes in the TSH receptor (TSH-R) in neoplastic thyroid tissues in humans. Total RNA was prepared from 34 thyroid tissue samples (four normal, six adenoma, six follicular cancer, and 18 papillary cancer) and reverse- transcribed into single-stranded cDNA, which was then used as a template for the polymerase chain reaction and subjected to single-strand conformation polymorphism (SSCP) analysis. Two fragments, FRAG (468-692) (nucleotides 468 to 692, corresponding to the mid-portion of the receptor extracellular domain) and FRAG (2044-2295) (nucleotides 2044 to 2295, corresponding to the COOH-terminal, cytoplasmic domain of the TSH-R cDNA) showed differences in electrophoretic mobility among the various thyroid tissue samples. Direct sequencing revealed Phe197 (TTC) --> Ile(ATC), and Asp219 (GAT) --> Glu(GAG) substitutions in FRAG (468-692) from two papillary cancers. Three types of substitution were identified in FRAG(2044-2295): Asn715 (AAC) --> Asp(GAC) from one papillary cancer, Lys723 (AAG) --> Met(ATG) from one papillary cancer, and Asp 727 (GAC) --> Glu(GAG) from one normal tissue sample, one follicular cancer, and four papillary cancers. These results suggest that there exist structural changes in TSH-R in some cases of thyroid neoplastic tissue.

Isolation of a cDNA whose expression is markedly increased in malignantly transformed FRTL cells and neoplastic human thyroid tissues.

We have cloned a cDNA whose mRNA levels are increased in malignantly transformed rat thyroid FRTL cells (FRTL-Tc cells). We constructed a cDNA library from FRTL-Tc cells in lambda gt10 and screened the cDNAs by differential plaque filter hybridization. Twenty-five thousand clones were screened and one cDNA (C140) was selected which corresponded to a mRNA whose expression was 5.8 times higher in FRTL-Tc cells than in FRTL cells. A 0.8 kb specific C140 mRNA was detected by Northern blot analysis of FRTL-Tc and FRTL mRNAs. The C140 cDNA was sequenced and found to encode a protein of 227 amino acids. We have found that C140 mRNA is conserved in human thyroid cells, but it is encoded by a smaller 0.7 kb transcript. C140 mRNA was highly expressed in neoplastic thyroid tissues and weakly in normal thyroid tissues in the same patients. Additionally, we found that C140 mRNA was also increased in the thyroid tissue of a patient with Graves' disease. These results suggest that C140 expression might be higher in rapidly growing thyroid cells than in normal cells, and might provide a new aspect for the study of thyroid tumours.

Recombinant thyrotropin stimulates cAMP formation in CHO-K1 cells expressing recombinant chorionic gonadotropin receptor.

Using CHO-K1 cells expressing rat thyrotropin-receptor (CHO-rTSH-R cells) or rat chorionic gonadotropin-receptor (CHO-rCG-R cells), we have examined their reactivity or cross-reactivity to recombinant human thyrotropin (TSH) or human chorionic gonadotropin (hCG). TSH stimulated cAMP formation in CHO-rTSH-R cells dose-dependently, and the maximum response was obtained at 1 mIU/ml. hCG also increased cAMP in the cells from 10(4) mIU/ml, and the maximum stimulation was observed at 10(6) mIU/ml. 10(7) mIU of hCG had the same potency with 1 mIU of TSH to rTSH-R. On the other hand, 10(-1) mIU/ml of hCG increased cAMP content in CHO-rCG-R cells and reached the maximum level at 10(2) mIU/ml. TSH also showed the stimulatory activity to CHO-rCG-R cells. It increased cAMP formation in CHO-rCG-R cells dose-dependently from 10 mIU/ml to 10(3) mIU/ml. Thirty mIU of TSH was calculated to be equivalent to 1 mIU of hCG to CG-R. These results indicate that TSH and hCG cross-react each other with rTSH-R or rCG-R.

Thyrotropin receptor in non-thyroid tissues.

Thyrotropin receptor (TSH-R), the main target for the autoantibody in Graves' disease, has been thought to be a thyroid-specific protein. However, we successfully obtained the cDNA fragments of TSH-R from rat retro-orbital tissues and adipose tissues by using polymerase chain reaction methods. Sequencing has revealed that the nucleotide sequence of the cDNAs from these non-thyroid tissues was identical to that from the thyroid. TSH-R peptide antibody, which recognizes rat TSH-R, stained a 104 kDa protein from the retro-orbital tissues and the adipose tissues. The band was not detected with the antibody preabsorbed with the peptide. These results suggest that the message is translated to make a TSH-R protein even in these non-thyroid tissues.

[Study of the distribution of chrome in 17 Cr stainless steel by autoradiography using 51Cr (author's transl)].

The segregation of alloying elements in steels even as small as undetectable in X-ray microanalysers seems to affect physical properties for instance, the ridging phenomenon in a 17 Cr stainless steel sheet due to fine wrinkles of the surface by drawing, the mechanism of which is not yet clear. Study was made of a method for detecting small segregations by autoradiography using 51Cr for the 17 Cr stainless steel. In results. (1) in the steel containing a 0.025% carbon or no carbon content, a small variation in photographic density relating to the concentration of chromium was found. (2) In the case of the steel with a 0.05% carbon content, similar one to commercial steel, a clear photographic density variation was observed which was considered to be a cause of the ridging. (3) The autoradiography is suitable to investigation of the small segregation undetected by an X-ray microanalyser.