The effect of serum TSH on rat thyroid peroxidase mRNA levels was studied in order to investigate the regulation of thyroid peroxidase gene expression in vivo. A nearly full-length rat thyroid peroxidase cDNA clone was isolated from a bacteriophage cDNA library synthesized using poly A+ RNA isolated from the thyroids of propylthiouracil-treated rats. cDNA probes derived from this clone were used to study rat thyroid peroxidase mRNA levels in response to the level of serum TSH. Two major rat thyroid peroxidase mRNA bands were detected on Northern blots of total cellular RNA (at 3.2 kb and at 3.7kb). Injection of thyroxine, which lowered the levels of serum TSH, also lowered the steady-state levels of both rat thyroid peroxidase mRNAs, whereas treatment with methimazole, which increased serum TSH, increased both rat thyroid peroxidase mRNA levels. In hypophysectomized rats 10 days postoperative, very low levels of thyroid peroxidase mRNA were observed. Injection of bovine TSH (1 IU/day) increased rat thyroid peroxidase mRNA expression, preferentially in the 3.2 kb band. These results clearly demonstrate that TSH regulates rat thyroid peroxidase mRNA levels in vivo.
Ronald P Magnusson, Bo Yu and Veronica Brennan
C. Magnusson, H. Billig, P. Eneroth, P. Roos and T. Hillensjö
Several studies have shown differences in gonadotrophin receptor content between different granulosa cell (gc) populations within the ovarian follicle, but little is known about the gonadotrophin sensitivity in gc from different parts of the follicle. This study is an investigation of progestin synthesis and responsiveness to highly purified human and partly purified rat gonadotrophins. It involves short-term culture of rat cumulus and mural gc obtained from preovulatory follicles of PMSG-treated immature rats. The responsiveness to FSH in terms of progestin accumulation was similar in the two gc populations, whereas the responsiveness to LH was greater in the mural gc than in the cumulus. The morphological response in the cumulus cells (mucification) correlated with the steroidogenic response. The pattern of progestin synthesis differed between the two gc populations. In the cumulus gc progesterone was dominant, whereas 20α-dihydroprogesterone was the main progestin secreted in the mural gc. The difference in responsiveness to gonadotrophins correlates well with the reported differences in receptor content in the two gc populations.
Basil Rapoport, Hideshi Hirayu, Pui Seto and Ronald P. Magnusson
Abstract. The molecular cloning of certain antigens to thyroid autoantibodies present in patients with autoimmune thyroid disease would be of great value in further understanding the pathogenesis of these diseases, and in devising new approaches for their treatment. The ideal method for molecular cloning is to first obtain a partial amino acid sequence of a purified protein and to construct an oligonucleotide probe for screening an appropriate cDNA library. Unfortunately in the case if thyroid autoimmunity, important antigens such as the TSH receptor and the microsomal antigen have not been purified. An alternate approach devised by Young & Davis (1983) is to construct a cDNA library in a vector that expresses the encoded proteins. The library can then be screened with an antibody as a probe. We have constructed cDNA libraries in gt11 using mRNA purified from human, pig and rat thyroid cells. Our experiences in constructing and screening these libraries will be described. The advantages of this system are 1) the protein does not have to be purified, 2) previously unknown antigens may be identified. The disadvantages are 1) lack of specificity with antibody selection, 2) because the cDNA is inserted in the β-galactosidase gene in the vector the antigen is expressed as a fusion protein. This may disturb the tertiary structure of the antigen and alter its antigenicity, 3) cDNA inserts frequently only contain part of the antigen molecule, and may therefore lack important epitopes; polyclonal antibody may therefore be preferable to monoclonal, 4) only 1 in 6 cDNA inserts will be in the correct reading frame for antigen expression, 5) the expressed protein is not glycosylated, 6) antibody absorption or purification is necessary to reduce the high background of antibody binding to bacterial host protein products. Despite these disadvantages the system has been used to clone numerous proteins. Thus far we have been successful in cloning 3 newly recognized autoimmune thyroid disease-related antigens that are of potential importance in further understanding the nature of these diseases.
J. Weeke, L. Dybkjær, K. Granlie, S. Eskjær Jensen, E. Kjærulff, P. Laurberg and B. Magnusson
Serum T4, T3, rT3, free T4, free T3 and TSH were measured during and after normal pregnancy in 20 women. Special methodological precautions were taken to avoid interference of other hormones and protein alterations in the assays. Serum T4 and T3 were steadily increasing during the last part of the 1st trimester, and remained high and nearly stable during the 2nd and 3rd trimester of the gestation period. The high levels were approximately 1.5 times the values measured 10 weeks post-partum. Serum rT3 was elevated already during the last part of the 1st trimester and remained high throughout pregnancy, compared to the post-partum value. Serum free T4 and free T3 were slightly elevated in early pregnancy. The values decreased gradually during pregnancy and were slightly depressed during the 3rd trimester. A gradual increase in serum TSH was observed during pregnancy and the 2nd and 3rd trimester values were significantly higher than the post-partum value. The mean values for serum TSH, free T4 and free T3 remained always well within the normal range. Thus small variations in serum free iodothyronines and TSH occur during normal pregnancy, the alterations observed in the last trimester of the gestation period resembling those of a slight thyroid insufficiency. These trends in variation of the reference values are worth to remember in the diagnosis of borderline hypo- or hyperthyroidism and in the balanced treatment of pregnant women with thyroid dysfunction.
UH Jansson, B Kristiansson, P Magnusson, L Larsson, K Albertsson-Wikland and R Bjarnason
OBJECTIVE: In children with coeliac disease, the ingestion of gluten causes small intestinal inflammation and a clinical picture of malabsorption, weight reduction and short stature. Decreased alkaline phosphatase (ALP) during gluten challenge was found in a previous study. ALP is a marker of bone formation and ALP activities are correlated with growth velocity. The aim of this study was to characterise the previously observed decrease of total ALP by investigating three specific bone ALP isoforms (bone/intestinal, B1 and B2) and three specific liver ALP isoforms (L1, L2 and L3) and, moreover, to correlate these ALP isoforms with other growth factors and growth markers. In addition, we also studied the association with possible weight changes, small intestinal mucosa inflammation, sex, age and gluten dose during gluten challenge. MATERIALS AND METHODS: Bone and liver ALP isoforms, IGF-I, IGF-binding protein (IGFBP)-3 and serum cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) were measured together with change in weight and small intestinal mucosa histopathology in 54 children with earlier enteropathy who participated in a 4-week gluten challenge. RESULTS: We observed a decreased total ALP activity after 4 weeks of gluten challenge, 7.8+/-1.8 to 6.5+/-1.7 microkat/l (means +/- s.d.), which was mainly due to a reduction of the bone ALP isoforms. The sum of all three bone ALP isoforms decreased from 6.3+/-1.7 to 5.1+/-1.6 microkat/l. The decreased activities of the bone ALP isoforms correlated with the observed reductions of IGF-I (r=0.74, P<0.001), IGFBP-3 (r=0.51, P<0.001) and ICTP (r=0.57, P<0.001). The decrease of the growth factors and growth markers correlated with weight reduction, but when influences from the change in weight were adjusted for, the partial correlation of the small intestinal mucosa inflammation was significant for IGF-I (r=-0.56, P<0.001) and IGFBP-3 (r=-0.55, P<0.001). CONCLUSION: The decrease of total ALP was due to a reduction of bone ALP. The decrease of IGF-I and IGFBP-3 was independently correlated with weight change and small intestinal inflammation.