A personal view on the early history of the insulin-like growth factors.

Salmon and Daughaday, when trying to set up an in vitro assay for Growth Hormone (GH), failed to obtain a direct effect on sulphate uptake in cartilage of hypophysectomized (hypox) rats. They recognised that this was not the consequence of poor methodology or materials, but an encrypted message from the examined system. They decided to turn around and to try and decipher it. Treatment with GH appeared to render hypox rat serum active in stimulating sulphation in hypox rat cartilage. They proposed that GH induced an intermediary substance, responsible for this biological effect: sulphation factor (SF), later renamed to Somatomedin(s) (SM). This hypothesis met with great criticism and very few took on to study this hypothetical substance. Besides disbelief, slow progress was also due to initial lack of a practical assay and to the failure to find a tissue with enriched concentration from which to extract the activity. From experimental evidence, the concept gradually evolved that SF/SM was insulin-like and might be identical to NSILA (non-suppressible insulin-like activity). This again generated controversy. This characteristic was too far away from the known effects of GH to be readily acceptable as a physiological phenomenon. The subsequent recognition of the distinct characteristics of the receptors for SM/NSILA and insulin, the discovery of the SM/NSILA binding proteins and, much later, a beginning understanding of their interactions, modifications and breakdown, have gradually resolved this apparent contradiction. When the sequence of two NSILA molecules became known, they were named IGF-I and -II. Structural similarity with proinsulin and identity of IGF-I with SM-C and -A were established and it was found that Multiplication Stimulating Activity (MSA), a growth factor isolated from fetal calf serum and subsequently from conditioned media of a rat liver cell line, was the rat equivalent of IGF-II. Structure-function relations could be studied, a quest which is not yet brought to an end. Meanwhile, the endocrine profile of SF/SM had gradually emerged by measuring plasma levels with bioassays. The main determinants were found to be age, body size, GH and the nutritional state. Later, radioimmunoassays were developed, enabling consolidation and detailing of these early observations, and allowing explorations at the tissue level. As another aspect of the endocrine paradigm, in vivo effects of IGFs were studied. The initial demonstration of an effect of crude preparations on longitudinal growth in experimental animals raised heavy scepticism, since the effect might have been an artefact caused by contaminants. It took confirmation with highly purified preparations and biosynthetic IGF-I to ease this concern. Still, not until recent years it was demonstrated, by knocking out the genes, that a true physiological and not a pharmacological effect had been induced previously. When it was found that most tissues produce SMs and are sensitive to their actions, the concept emerged that IGFs may have para- and autocrine functions. Early experiments with combinations of growth factors in cell cultures had begun to define their specific roles in the cell cycle as competence or progression factors. SM-C fell in the latter category. Still, the awareness grew that, for obtaining physiologically meaningful results on the role of IGFs in complex, dynamic and tissue-specific environments, involving interactions of many hormones and growth factors, the intactness of tissue was a prerequisite. One result of this approach was the discovery of a direct interaction of GH with cartilage, leading, in concert with IGFs, to a clonal expansion of the cartilage cells of the growth plate. The isolation and sequencing of the IGF-I and -II genes, and later, of six IFG-BPs initiated the gradual elucidation of structure and function at the DNA and RNA level and the study of natural and synthetic IGF-variants. The generation of transgenic animals became feasible

Plasma levels of insulin-like growth factor (IGF)-I, IGF-II and IGF-binding protein-3 in the evaluation of childhood growth hormone deficiency.

BACKGROUND: Traditionally, measurement of plasma IGF-I and more recently of IGFBP-3 are used to distinguish GHD from idiopathic short stature in slowly growing children, using a single blood sample. In earlier studies it was claimed that IGFBP-3 was superior to IGF-I, but more recently doubts around this claim have arisen. The role of serum IGF-II has never been studied extensively. On theoretical grounds, it can also be hypothesized that molar ratios of these peptides might be of additional value. DESIGN: Retrospective, multicentre, cohort study. PATIENTS: 96 children evaluated for short stature. METHODS: Serum IGF-I, IGF-II, IGFBP-3 and various molar ratios were, after correction for age and sex using SD scores, compared to the maximum serum GH peak after two standard provocation tests using four different methods (t-test, chi2, likelihood ratios and ROC curves). In addition, the correlations between these parameters and the short-term (1 year) and long-term (3 years) response to GH therapy were calculated. RESULTS: IGF-I performed better than IGFBP-3, but the best results were achieved by the molar ratio IGF-I:IGF-II. However, IGFBP-3 correlated better with the short-term response to GH therapy than IGF-I or the ratios, and none of the parameters investigated was found to be related to the response of long-term GH therapy.

Solid-phase assay for insulin-like growth factor (IGF) binding to IGF-binding protein-3: application to the study of the effects of antibodies and heparin.

The availability and activity of insulin-like growth factors (IGF-I and IGF-II) are largely determined by a group of IGF-binding proteins (IGFBPs). We have developed a new assay to characterize the interaction between the IGFs and IGFBP-3. In this assay, recombinant IGFBP-3 (5 ng) was immobilized on plastic microtitre wells, after which radiolabelled IGF-I or -II was allowed to bind. The assay is highly specific, since neither IGF bound to control wells blocked with albumin. By constructing both saturation and competition binding curves, equivalence of binding between the radiolabelled and native IGF ligands could be demonstrated. From these curves, reliable specific activities of the tracers were calculated. Scatchard plots of both types of data produced identical results for dissociation constants and number of binding sites. The affinity of IGF-II was twice as high as the affinity of IGF-I (dissociation constants of 44 and 102 pM respectively). The assay was used to show that polyclonal anti-IGFBP-3 antibodies could block binding of IGF. Alkylating agents and NaCl were without effect, but chaotropic salts such as CaCl2 and NaSCN decreased IGF binding to IGFBP-3. IGFBP-1 and IGFBP-3, but not an N-terminal fragment of IGFBP-3, could effectively block binding of both IGF-I and IGF-II to the solid-phase IGFBP-3. Increasing concentrations of heparin had little or no effect on IGF-I binding, but strongly inhibited IGF-II binding. This was shown to be a consequence of a decrease in both the affinity and the number of binding sites. Possibly, the interaction of IGFBP-3 with heparin or heparin-like structures in vivo can lead to the selective release of IGF-II from this binding protein. Our results with heparin also suggest that the binding sites on IGFBP-3 for IGF-I and IGF-II are not completely identical. This assay can be applied to the study of various aspects of the interaction between the IGFs and IGFBP-3, such as the effects of interfering substances and structure-function relationships of both moieties of the complex.

Characterization of O-glycosylated precursors of insulin-like growth factor II by matrix-assisted laser desorption/ionization mass spectrometry.

High molecular weight precursors of insulin-like growth factor II (IGF-II) were isolated from Cohn fraction IV of human plasma by ultrafiltration, affinity chromatography and reversed-phase high-performance liquid chromatography. Molecular weight determination by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of two high molecular weight IGF-II preparations revealed heterogeneous glycosylation. A combination of enzymatic degradation and MALDI-MS were applied for further structural characterization of the glycosylated precursors of IGF-II. The first step was molecular weight determination of intact high molecular weight IGF-IIs prior to and after treatment with neuraminidase and O-glycosidase. This, together with a comparison of molecular weight information available from the cDNA, revealed that both high molecular weight IGF-II species contain an identical C-terminal extension of 20 residues but different degrees of glycosylation. Second, comparative Endo Glu-C digestion of the preparations prior to and after enzymatic release of carbohydrates and subsequent remeasurement of the molecular weight by MALDI-MS confirmed the primary structure of precursor IGF-II1-87. The O-linked carbohydrates were found to be associated with the C-terminal extension and the heterogeneity was identified as varied sialylated forms of one and two HexNAc-Hex groups.

Translational control of gene expression.

Translational regulation of mRNA is an important step in the control of gene expression. In a general way, the efficiency of the translational apparatus can be influenced either positively or negatively by changing the level or the activity of rate-limiting protein factors taking part in the process of translation. But translational control can also be very specific, affecting only a single mRNA or class of mRNA molecules. In most of these cases regulation takes place at the level of initiation of translation, which is often attributable to structural peculiarities of the mRNA in question, especially of the 5'-untranslated region or leader. This review summarizes the mechanisms which lie at the root of translational control. A better understanding of these mechanisms will eventually provide us with new drugs and antisense oligonucleotide technology, aimed at influencing the level of expression of single proteins. These developments are of interest to basic researchers and clinicians alike, because they may profoundly change the ways in which we treat, e.g. viral infections and malignancies in the future.

Insulinlike growth factor-II/mannose 6-phosphate receptor is expressed on CCl4-exposed rat fat-storing cells and facilitates activation of latent transforming growth factor-beta in cocultures with sinusoidal endothelial cells.

Transforming growth factor beta (TGF-beta), a potent fibrogenic cytokine, is secreted in latent form. We examined which cell type in both normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver bears surface type II IGF/mannose 6-phosphate (IGF-II/M6P) receptor, known to facilitate activation of TGF-beta. In addition, the role of the IGF-II/M6P receptor in activation of latent TGF-beta was investigated in a coculture system with sinusoidal endothelial cells. Northern hybridization analysis for IGF-II/M6P receptor messenger RNA (mRNA) was performed on total RNA of different isolated and purified liver cell types. In normal liver, cells expressed little IGF-II/M6P receptor mRNA. In fibrotic liver, we found significant expression only in fat-storing cells. The presence of IGF-II/M6P receptors was established by [125I]IGF-II binding assays on freshly isolated fat-storing cells from normal and CCl4-exposed rat livers. We found specific binding of [125I]IGF-II only on CCl4 exposed fat-storing cells. As determined by polyacrylamide gel electrophoresis after affinity labeling, the specific binding involved 220 kD type II IGF receptors. Scatchard analysis revealed the presence of 3,043 +/- 1,378 IGF-II/M6P high-affinity receptors/fat-storing cell, with a Kd of 387 = 165 pmol/L. With a mink lung epithelial cell (Mv1Lu) proliferation inhibition assay, inhibition of proliferation (a measure of active TGF-beta function) was determined using conditioned media of activated fat-storing cells, cocultures of fat-storing cells, and endothelial cells and pure endothelial cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteins binding to the leader of the 6.0 kb mRNA of human insulin-like growth factor 2 influence translation.

The leader of the 6.0 kb human insulin-like growth factor 2 (IGF-2) mRNA, leader 3, has been reported to partially repress translation. In the regulation of this phenomenon, RNA-binding proteins may play a role. Using UV-irradiation crosslinking, we found specific binding of four proteins (57, 43, 37 and 36 kDa) to this leader. Binding of these proteins to RNA proved to be highly sensitive to the potassium chloride concentration in the buffer solution, each protein having its own optimum. The 57 kDa protein was indistinguishable by size, binding properties and immunoprecipitation from the polypyrimidine tract binding protein (PTB), first described as a nuclear protein binding to the polypyrimidine tracts (PPTs) in introns. Cross-competition experiments showed that leader 3 has a much higher affinity for this 57 kDa protein than the PPT on which PTB was originally characterized. By competition with different fragments of leader 3, we were able to localize the binding of the 57 kDa protein to a 162 nt RNA fragment (AsnI-PvuII) in the 3'-part of the leader. When placed before a chloramphenicol acetyltransferase (CAT) open reading frame, this RNA fragment stimulated translation in reticulocyte lysate 3-fold, while other fragments of leader 3 repressed translation. The efficient translation directed by the 162 nt AsnI-PvuII fragment fused to CAT could be repressed by adding free AsnI-PvuII RNA fragment, indicating that the high translation efficiency of the AsnI-PvuII-CAT synthetic mRNA was due to the binding of protein and not to the structure of the RNA itself.

Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus.

In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5'). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one II5' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5' without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs II5 and II6 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5'. Inbreeding of 2 lines containing construct II5' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting. In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action.

Co-administration of IGF-binding protein-3 differentially inhibits the IGF-I-induced total body and organ growth of Snell dwarf mice.

In mammals IGF-I is part of a 150-kDa binding protein complex, which also contains a glycosylated acid-labile protein (ALS) and a glycosylated acid-stable IGF binding subunit IGFBP-3. Administration of free IGF-I in vivo induces not only acute insulin-like effects but also growth stimulation. Since co-injection with IGFBP-3 only partially blocked the hypoglycemic response of free IGF-I in hypophysectomized rats, we were interested in the growth stimulating activity of the IGFI-IGFBP-3 complex in pituitary-deficient mice compared to that obtained by IGF-I alone. Therefore, the effects of subcutaneously administered IGF-I, IGFBP-3 and the IGF-I-IGFBP-3 complex on somatic growth and organ growth of pituitary-deficient Snell dwarf mice were studied after 4 weeks of treatment. Treatment with IGF-I alone induced a significant increase in body length and weight, as well as in weights of the submandibular salivary glands, kidneys and quadriceps femoris muscles as compared to buffer treated controls. No significant changes were found in liver, brain, heart and thymus. IGFBP-3 alone had no effect. However, the stimulating effects of IGF-I alone on body length and weight, as well as on the weight of the kidneys, were fully neutralized by co-injection with IGFBP-3. In contrast, the weights of submandibular salivary glands and m. quadriceps femoris were increased by treatment with the complex compared to controls and not significantly different from animals treated with IGF-I alone. Our data show that in GH-deficient mice administration of IGFBP-3 differentially inhibits the IGF-I induced body and organ growth. This calls for extra vigilance when exploring presumed advantages of administering an IGF-I-IGFBP-3 complex to GH-deficient individuals in order to obtain stimulation of growth.

Influence of the four leader sequences of the human insulin-like-growth-factor-2 mRNAs on the expression of reporter genes.

The human insulin-like-growth-factor-2 (IGF-2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader-2-containing and leader-4-containing mRNAs are completely polysomal, whereas mRNAs possessing leader-3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF-2 leader sequences on expression of the gene, IGF-2 leader-luciferase and leader-chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader-1, leader-2 and leader-4 were very similar, both at the level of mRNA and protein. Leader-3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328-906 of the leader sequence. The remaining 5' part of the leader sequence was efficient both in RNA expression and in translation, but the 3' part of the leader (nucleotides 906-1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA. To evaluate the effect of the IGF-2 leaders on in vitro translation, leader-chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader-1-chloramphenicol acetyltransferase mRNA translated over 20-fold less efficiently, whereas leader-2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3-5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader-3-containing mRNAs in reticulocyte lysates was barely detectable. The whole 5' region of leader-3, up to nucleotide 614, could be shown to be repressive. Only leader-4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader-1 and leader-2, this L4-chloramphenicol acetyltransferase mRNA translated in a cap-dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue. We conclude that all four IGF-2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF-2 mRNAs differentially.

Insulin-like growth factors-I and -II and their binding proteins during postnatal development of dwarf Snell mice before and during growth hormone and thyroxine therapy.

The ontogeny of serum insulin-like growth factors (IGFs)-I and -II and their binding proteins (IGFBPs) was studied in normal and dwarf Snell mice. IGF-I concentrations in serum of normal mice increased between 4 and 8 weeks of age; dwarf mice had very low serum IGF-I levels. In both normals and dwarfs, serum IGF-II levels were highest soon after birth and dropped steadily thereafter. Western ligand blots of serum IGFBPs with 125I-IGF-II as tracer revealed the expected bands of 41.5, 38.5, 30-32 and 24 kDa. In normal mice the IGFBP-3 doublet was already detectable at 2 weeks of age, and its intensity increased with age. In dwarf mice the IGFBP-3 doublet was hardly detectable. The changes of IGFs and their IGFBPs were studied in sera of dwarf mice after treatment with growth hormone (GH) and/or thyroxine (T4) for 4 weeks. In spite of a comparable growth response obtained using these hormones, serum IGF-I was increased only by GH treatment; a small but significant decrease of serum IGF-II was obtained following GH or T4 treatment. An increase of the IGFBP-3 doublet was only obtained with GH; T4 and GH + T4 had no effect. The rise of IGFBP-3 after GH treatment was accompanied by the formation of the IGFBP 150 kDa complex, as measured by neutral gel chromatography. The size distribution of 125I-IGF-II was restored to normal, while with 125I-IGF-I only a small peak at 150 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant insulin-like growth factor-II inhibits the growth-stimulating effect of growth hormone on the liver of Snell dwarf mice.

The actions and interactions of recombinant insulin-like growth factor-I and -II (IGF-I and IGF-II), alone or in combination with human GH on body growth and the growth of several organs were studied in the Snell dwarf mouse. IGF-I and -II stimulate to a similar extent sulfate incorporation into cartilage, and both IGFs increase body length and weight. IGF-II as well as IGF-I have clear effects on the size of the submandibular salivary glands, kidneys, and spleen. IGF-II, however, did not influence the weight of the lung, in contrast with IGF-I. GH treatment alone resulted in growth of the liver, whereas both IGFs were inactive. Surprisingly, IGF-II and, to a lesser extent, IGF-I inhibited GH-induced growth of the liver. Glycogen storage in the liver was decreased by treatment with IGF-II alone or in combination with GH, as shown by histological examination. It was not affected by GH, IGF-I, or GH plus IGF-I. Also, the size of the centrilobular hepatocytes was decreased by treatment with IGF-II and IGF-II plus GH; GH alone had a hypertrophic effect, whereas IGF-I or GH plus IGF-I had none. In contrast to GH, IGFs did not increase polyploidy. Treatment with IGF-II increased the level of IGFBP-3, as did IGF-I or GH treatment, as shown by Western ligand blotting. The IGFs appeared to have a greater effect on the induction of 38.5-kilodalton IGFBP-3 than GH, suggesting a different role in the regulation of glycosylation. In conclusion, IGF-I and IGF-II as well as GH have a stimulatory effect on general body growth and are effective in the stimulation of serum IGFBP-3, sulfate incorporation into cartilage, as well as the growth of specific organs in Snell dwarf mice. Both IGFs, alone or in combination with GH, show distinct effects on the growth of the liver with respect to several histological parameters, which require further exploration.

Differential polysomal localization of human insulin-like-growth-factor-2 mRNAs in cell lines and foetal liver.

Examination of the association of insulin-like-growth-factor-2 mRNAs with polyribosomes in five cell lines revealed that greater than 50% of the total mRNA population was present in the untranslated free mRNP fraction for each cell line. Of the different subtypes of insulin-like-growth-factor-2 messengers, the least abundant mRNAs, starting with exon 4 (leader 2, 5.0 kb) and exon 6 (leader 4, 4.8 kb), were found in the polysomes only, while the most abundant transcript, starting with exon 5 (leader 3, 6.0 kb and 2.1 kb) was found predominantly in the untranslated fractions. 20-30% of leader 3 mRNAs, however, were in the larger polysomes (four or more ribosomes), indicating that a subpopulation of this mRNA can be translated efficiently. The peak fraction for the leader 4 insulin-like-growth-factor-2 mRNA (4.8 kb) in the polysomes was migrating faster in the sucrose gradients than the peak fractions of leader 2 and 3 mRNAs (5.0 kb and 6.0 kb), implying that more ribosomes were associated with this type of mRNA. In foetal liver, the situation was similar, though in this case the leader 2 mRNA was most heavily loaded with polysomes. Treatment of cells with low concentrations of cycloheximide caused the polysomal RNAs to shift to even larger polysomes while the untranslated fraction of the leader 3 mRNAs stayed in the untranslated fractions. These results indicate that, both in established cell lines and in foetal liver, insulin-like-growth-factor-2 translation is influenced both by mRNP sequestration and differential translation initiation efficiency of the insulin-like-growth-factor-2 mRNAs.

Insulin-like growth factors (IGFs) and IGF binding protein-3 display disulfide isomerase activity.

Insulin-like growth factors (IGF-I and -II) bind with high affinity to IGF-binding proteins (IGFBPs). IGFBP-3 contains vicinal cysteines in sequence which is similar to the active sites in thioredoxin and protein disulfide isomerase. We tested if, in analogy with these redox enzymes, IGFBP-3 could catalyze the isomerization of intramolecular disulfide bridges in protein substrates. IGFBP-3 (30 microM) was able to reactivate reduced ribonuclease at a rate of 38% of that of thioredoxin. Also recombinant IGF-I induced the regeneration of ribonuclease activity. Thiol redox reactions are known to play a role in regulating conformational changes in the insulin receptor and possibly also in the IGF-I receptor. Therefore, the intrinsic isomerase activities of IGF-I may be important in the activation of its receptor. The observed effects of IGFBP-3 may help to elucidate the mechanism by which this binding protein can modulate the actions of IGF-I.

Body proportions in individuals with Turner syndrome. The Dutch Growth Hormone Working Group.

The body proportions in 191 individuals with Turner syndrome (TS) were investigated. At 3 years of age the mean sitting height in TS was normal, thereafter trunk growth was impaired, resulting in a standard deviation score (SDS) of -2.4 in the adult. From 3 to 12 years of age the mean SDS of leg length increased from -2.7 to -3.6; and then fell to -2.5. At 3 years of age the ratio of sitting height to leg length was 3.2 standard deviations (SD) above the normal mean. Thereafter the ratio slowly approached the normal percentiles. It was +0.6 SD in 15- to 18-year-old women. Thereafter it increased to 1.7 for adults with TS. Knemometric measurements in 32 individuals with TS and 32 controls revealed that in TS the upper legs were relatively shorter than the lower legs. We conclude that children with TS, and to a lesser extent adults, have a disproportionately short stature with relatively short legs whereas body proportions are almost normal in adolescents.

Influence of treatment on the maturation of the somesthetic pathway in infants with primary congenital hypothyroidism during the first year of life.

To assess the influence of treatment on the development of the somesthetic pathway in infants with congenital hypothyroidism receiving early treatment, median nerve somatosensory evoked potentials were measured during the 1st y of life. Twenty-nine infants were studied with six to seven somatosensory evoked potential tests per infant. The cervical latency (N13) divided by arm length and the first (N19) and second (N32) cephalic latencies as well as N13-N32 latency were measured. At diagnosis, all components showed a small but significant delay, which was not related to thyroxine (T4) levels before treatment. During treatment, T4 ranged from 50 to 290 nmol/L. At 12 mo, the cervical latency divided by arm length had normalized, whereas N19 and N13-N32 were more abnormal than at diagnosis. For N19, these abnormalities were related to a slow initial rise of T4 (< or = 100 nmol/L after 1 wk of treatment) and the initial N19 values. Abnormal N13-N32 values were associated with high T4 values during treatment (> 200 nmol/L) and the type of congenital hypothyroidism (partial or total deficiency in T4 production). Induction of therapy with l-triiodothyronine rather than l-thyroxine and the occurrence of low T4 values (< 100 nmol/L) after the 4th wk of therapy had no such effect. Our data suggest that, for normal CNS development, euthyroidism should be reached as soon as possible by adequate induction therapy. Thereafter, T4 supplementation should be strictly dosed, keeping the serum T4 values within narrow limits around the mean normal for age, because overtreatment, like initial undertreatment, may lead to CNS abnormalities at the end of the first year.

Site-specific cleavage of IGF-II mRNAs requires sequence elements from two distinct regions of the IGF-II gene.

The human insulin-like growth factor II (IGF-II) gene constitutes a complex transcriptional unit that contains nine exons and four promoters. Expression of the IGF-II gene yields a family of mRNAs that all encode prepro-IGF-II. In addition, a stable 1.8 kb RNA is formed that is derived from the 3' untranslated region of exon 9. Recently, we have shown that this RNA species arises by site-specific endonucleolytic cleavage of IGF-II mRNAs and not by transcription from a separate promoter. In the present study we establish that two widely separated sequence elements of approximately 300 nucleotides, both located within exon 9, are required for this cleavage reaction. The first element encompasses about 200 nucleotides upstream and 100 nucleotides downstream of the cleavage site, while the second element is located within a region of 330 nucleotides about 2 kb upstream of the cleavage site. Interestingly, site-specific cleavage also occurred when a fragment from exon 9 of the IGF-II gene containing these two elements was inserted into the 3' untranslated part of the beta-globin gene. Apparently, the expressed hybrid beta-globin-IGF-II mRNA contains all the regulatory elements to confer site-specific endonucleolytic cleavage.