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ABSTRACT

Insulin-like growth factor-II (IGF-II) is a polypeptide mitogen which is believed to play an important role in fetal development. The human and rat IGF-II genes are complex transcription units, which contain multiple promoters and polyadenylation sites and which exhibit alternate splicing of their primary transcripts. In order to study IGF-II gene expression during chick embryonic development, we screened a 10-day chick embryo cDNA library with a human IGF-II cDNA probe. We isolated a clone, designated cigf, that was comprised, in part, of sequences homologous to the second coding exon of the human, mouse and rat IGF-II genes. Comparison of the nucleotide sequence of cigf with that of the corresponding genomic clone indicated that cigf was derived from a spliced antisense transcript of the chicken IGF-II gene, which overlapped the second coding exon. Northern blotting experiments with single-stranded RNA probes synthesized using cigfDNA as a template showed that stage 22 and stage 36 chick embryos contained sense strand IGF-II transcripts of 1.4, 2.2, 4.7 and 7.0kb and antisense strand transcripts of 0.7, 1.3, 1.8, 2.5, 4.9, 6.0 and 8.0kb. The pattern of sense strand IGF-II transcripts was similar to that previously found in rat fetal tissues. Whilst there are precedents for the transcription of both strands of a single gene, this is the first evidence for antisense transcription of an IGF gene. The functions of the antisense transcripts remain to be determined. These findings demonstrate a further level of complexity in the IGF-II transcription unit and indicate that studies of IGF-II transcript distribution performed with double-stranded probes should be interpreted with caution. They also suggest explanations for the recent finding that IGF-II peptides are present at much lower levels in embryos than expected from the high levels of IGF-II transcripts present.

ABSTRACT

The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)₁₆.d(AG)₂₁) in the 5′ untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3′ UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA.

The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3′ UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3′ UTR, allowing transcription to continue into the 3′ UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA. This would then result in an alternative 412 amino acid version of the mouse TRH-R protein, with 95% homology to the rat TRH-R. This study focused on the structural differences in the intracellular COOH-terminal tail of the receptor, which is known to be a functionally important domain in other members of the G protein-coupled receptor family. We have also recently characterized the human TRH-R cDNA, which revealed a third variant at the COOH terminus. Comparisons between mouse, rat and human TRH-Rs show that the amino acid sequences are virtually identical. However, significant differences between these species exist at the COOH terminus, with each TRH-R having a unique form of the COOH-terminal tail, beginning at exactly the same site and encoding 1, 20 and 6 amino acids in the mouse, rat and human respectively.

ABSTRACT

A monoclonal antibody (BPL-M23) to insulin-like growth factor-I (IGF-I) was obtained following immunization of BALB/c mice with human IGF-I conjugated to ovalbumin. The affinity constant of BPL-M23 for IGF-I was 10·5 litres/nmol and the cross-reactivities of IGF-II, multiplication-stimulating activity III-2 and insulin were 08, 003 and less than 0·0001 % respectively. Porcine, bovine, ovine and rabbit sera, but not rat or mouse sera, showed substantial reactivity with the antibody.

Comparison of radioimmunoassay analyses of 54 human serum samples from normal subjects and acromegalic and GH-deficient patients using BPL-M23 and a polyclonal rabbit antiserum (R557A) to human IGF-I showed a high correlation, indicating the usefulness of the monoclonal antibody in radioimmunoassay.

Monoclonal antibody BPL-M23 was capable of abolishing the sulphation, mitogenic and insulin-like activities of IGF-I in in-vitro bioassays, suggesting that these activities may rely upon the same receptor-binding site which is near to the antibody-binding site.

ABSTRACT

Thyroid hormones are essential for the normal growth and development of many tissues. In the rat, hypothyroidism is associated with growth impairment, and hyperthyroidism with the development of a hypercatabolic state and skeletal muscle wasting but, paradoxically, cardiac hypertrophy. The mechanism by which thyroid hormone produces cardiac hypertrophy and myosin isoenzyme changes remains unclear. The role of IGF-I, an anabolic hormone with both paracrine and endocrine actions, in producing cardiac hypertrophy was investigated during this study in hyperthyroid, hypothyroid and control rats. A treated hypothyroid group was also included in order to assess the effect of acute normalization of thyroid function.

Body weight was significantly lower in the hyperthyroid (mean±s.e.m.; 535·5±24·9 g, P<0·05), hypothyroid (245·3±9·8 g, P<0·001) and treated hypothyroid (265·3±9·8 g, P<0·001) animals when compared with controls (618·5±28·6 g). Heart weight/body weight ratios were, however, significantly increased in the hyperthyroid (2·74 ± 0·11×10⁻³, P<0·01) and treated hypothyroid (2·87±0·07 ×10⁻³, P<0·001) animals when compared with controls (2·26±0·03 × 10⁻³). Serum IGF-I concentrations were similar in the control and hyperthyroid rats (0·91±0·07 vs 0·78±0·04 U/ml, P=0·26), but bioactivity was reduced by 70% in hyperthyroid serum, suggesting a circulating inhibitor of IGF. Serum IGF-I levels (0·12±0·03 U/ml, P<0·001) and bioactivity (0·12±0·04 U/ml, P<0·001) were significantly lower in the hypothyroid group. Liver IGF-I mRNA levels were not statistically different in the control and hyperthyroid animals, but were significantly reduced in the hypothyroid animals (P<0·05 vs control). Heart IGF-I mRNA levels were similar in the control and hypothyroid rats, but were significantly increased in the hyperthyroid and treated hypothyroid animals (increased by 32% in hyperthyroidism, P<0·05; increased by 57% in treated hypothyroidism, P<0·01). Cardiac IGF-I was significantly elevated in hyperthyroidism (0·16±0·01 U/mg heart tissue, P<0·01), was low in hypothyroidism (0·08±0·01 U/mg, P<0·01) and was normalized in the treated hypothyroid group (0·11 ± 0·01 U/mg vs control, 0·13±0·01 U/mg).

Low body mass during both hypothyroidism and hyperthyroidism is therefore associated with reduced systemic IGF bioactivity. In hypothyroidism there is a primary defect in the endocrine function of IGF-I, while in hyperthyroidism serum IGF bioactivity is reduced in the presence of normal endocrine production of this anabolic hormone. In contrast, the paracrine actions of IGF-I are increased in the heart during hyperthyroidism, and this hormone appears to play a part in the development of hyperthyroid cardiac hypertrophy.

ABSTRACT

pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO₃− ions, FRTL-5 cells regulate intracellular pH (pH_i) by an Na⁺/H⁺ antiport but HCO₃−-dependent exchangers cannot operate. We have investigated pH_i regulation (by microfluorimetry and the pH sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6′)-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO₃− ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na⁺/H⁺ antiporter and the stilbene derivative disodium 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) for HCO₃ −-dependent mechanisms), ionic substitution and by NH₄ ⁺/NH₃ (10mm) acid loading. Basal pH_i was lower in the presence (7·3±0·058, mean±s.d., n= 14) than in the absence (7·59±0·078, n=10) of HCO₃ ions. In HCO₃ −-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pH_i of 7·35±0·06. This recovery was Na⁺-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO₃ ⁻-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na⁺ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na⁺ from HCO₃ ⁻-containing media acidified the cells (ΔpH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl⁻ alkalinized the cells (ΔpH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na⁺ and presence of amiloride, alkalinization was still seen on the removal of Cl⁻, albeit at a diminished rate (i.e. ΔpH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pH_i by two Na⁺-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na⁺-independent, Cl⁻/HCO₃− mechanism.