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Background and Objectives TGF-β1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-β1 from virally inactivated human platelets. Study Design and Methods Apheresis platelet concentrates (N = 12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X-45; 31 °C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7·5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1 M NaCl–PBS buffer pH 7·5 (DEAE-eluate). The content in TGF-β1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions. Results Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-β1 at a mean concentration of about 102 ng/ml, whereas EGF, b-FGF were at about 0·72 and 0·18 ng/ml, respectively. The content in TnBP and Triton X-45 was

Background Human platelet growth factors (HPGF) are essential for tissue regeneration and may replace fetal bovine serum (FBS) in cell therapy. No method for the manufacture of standardized virally inactivated HPGF has been developed yet. Study design and methods Platelet concentrates (PC) were subjected to solvent/detergent (S/D) treatment (1% TnBP/1% Triton X-45), oil extraction, hydrophobic interaction chromatography and sterile filtration. Platelet-derived growth factor (PDGF)-AB, -BB and -AA, transforming growth factor-s1 (TGF-s1), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and vascular endothelium growth factor (VEGF) were measured by ELISA. Composition in proteins and lipids was determined, protein profiles were obtained by SDS-PAGE, and TnBP and Triton X-45 were assessed by gas chromatography and high-performance liquid chromatography, respectively. Cell growth promoting activity of HPGF was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using human embryonic kidney (HEK293A) fibroblast and Statens Seruminstitute rabbit corneal (SIRC) epithelial cell lines. Results The GF preparation contained a mean of 16·66, 2·04, 1·53, 72·19, 0·33, 48·59 and 0·44 ng/ml of PDGF-AB, -BB, -AA, TGF-s1, EGF, IGF-1 and VEGF, respectively. The protein profile was typical of platelet releasates and had less than 2 p.p.m. of residual S/D agents. MTS assay of HEK293A and SIRC cultures showed that the GF preparation at 10% and 0·1% (v/v), respectively, could successfully replace 10% FBS for cell proliferation. Cell-stimulating activity of HPGF on HEK293A was over twice that of PC releasates. Conclusion Standardized and functional virally inactivated HPGF can be prepared from human PC for possible applications in cell therapy and regenerative medicine.

Polymerase chain reaction (PCR) amplification was used to detect cytomegalovirus (CMV) in 1000 urine specimens from Chinese newborns for defining the incidence of congenital CMV infection in the Chinese population. The major immediate-early and the late antigen genes of CMV were amplified and detected by gel electrophoresis. There were 18 congenitally infected infants found when tests were performed with one or both primer pairs. Comparing with tissue culture, PCR of both primer sets provided a sensitivity of 94%, a specificity of 100% and a predictive value of positive result of 100%.

The growth-promoting activity of GH, the principal hormonal determinant of body size, is mediated by insulin-like growth factor I (IGF-I). Most of the IGF-I in plasma circulates in a 150-kDa complex that contains IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). The 150-kDa complex serves as a reservoir of IGF-I and determines its bioavailability to the tissues. Formation of the 150-kDa complex depends upon the synthesis of ALS, which is synthesized primarily in liver and is regulated by GH. The present study demonstrates that GH stimulates ALS gene transcription in rat liver and ALS promoter activity in a rat hepatoma cell line. ALS messenger RNA (mRNA) and ALS nuclear transcripts were decreased to similar extents in the livers of GH-deficient hypophysectomized rats. GH increased hepatic ALS mRNA within 3–4 h to about 65% of the levels seen in sham-operated control rats. To confirm that GH stimulated ALS gene transcription, we transiently transfected an ALS promoter-luciferase reporter gene construct into H4-II-E rat hepatoma cells and primary rat hepatocytes. Recombinant human GH (hGH) stimulated promoter activity about 3-fold. In contrast, basal promoter activity was lower, and GH stimulation was absent when the ALS reporter construct was transfected into GH-responsive 3T3-F442A mouse preadipocyte fibroblasts. GH stimulation of ALS promoter activity in H4-II-E cells was mediated by functional GH receptors; nonprimate (rat and bovine) GH gave identical stimulation to hGH, and stimulation by hGH occurred at physiological concentrations. Reverse transcriptase-PCR analysis indicated that GH receptor mRNA was present in H4-II-E cells at approximately 40% of the level seen in rat liver. GH also induced the expression of the endogenous c-fos gene, indicating that the signaling pathway necessary for the activation of gene expression by GH was intact in H4-II-E cells. Thus, H4-II-E cells are a GH-responsive liver cell line that should provide a useful system in which to study the molecular mechanism of transcriptional regulation by GH of ALS and other hepatic genes.

The PC12 cell line established from a rat pheochromocytoma has been extensively studied as a model of neuronal differentiation. Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic for PC12 cells under serum-starved conditions. IGF activity is modulated by a family of six IGF-binding proteins (IGFBPs). It recently was reported that PC12 cells produced an IGFBP that had a marked preferential binding affinity for IGF-II over IGF-I. We now show that the main IGFBP produced by PC12 cells is rat IGFBP-6 and compare its properties with those of human IGFBP-6. The predominant IGFBP in medium conditioned by undifferentiated and differentiated PC12 cells migrated on sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis with an apparent molecular mass of 22.5-25 kilodaltons and was recognized by polyclonal antiserum to rat IGFBP-6 by immunoblotting. Rat IGFBP-6 mRNA (1.4 kilobases) was detected by Northern hybridization of total RNA extracted from PC12 cells using a rat IGFBP-6 cDNA probe. Rat IGFBP-6, like human IGFBP-6, is O-glycosylated; incubation with neuraminidase, fucosidase, and O-glycanase reduced its apparent molecular mass to 21 kilodaltons. Competitive binding studies of rat and human IGFBP-6 with [125I]IGF-II and unlabeled IGF-II or IGF-I demonstrated that both IGFBPs bound IGF-II with similar affinities (Ka, 1.5-1.8 x 10(11) M-1) and bound IGF-I with approximately 25- to 35-fold lower affinity than IGF-II. Thus, differences in amino acid sequence, such as deletion of nine amino-terminal residues (including two conserved cysteine residues) in rat IGFBP-6 compared with human IGFBP-6, do not alter its binding characteristics. PC12 cells should provide a useful system to define the regulation of IGFBP-6 expression and the role of IGFBP-6 in modulating IGF action.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of six proteins that modulate the biological activity of IGF-I and IGF-II and determine their bioavailability to tissues. One of the IGFBPs, IGFBP-1, is distinctive in the dynamic response of its levels in human plasma to metabolic changes. Parallel changes occur in IGFBP-1 mRNA and IGFBP-1 transcription in rat liver. Using the well differentiated H4-II-E rat hepatoma cell line as a model system, we demonstrated previously that IGFBP-1 transcription is positively regulated by dexamethasone and negatively regulated by insulin. We now examine the effect of the protein synthesis inhibitor, cycloheximide, on the hormonal regulation of IGFBP-1 gene expression. Preincubation of H4-II-E cells with 10.7 microM cycloheximide for 1.5 h did not prevent the induction of IGFBP-1 mRNA and IGFBP-1 transcription (determined in nuclear run-on assays) by dexamethasone. By contrast, cycloheximide treatment abolished the decrease in IGFBP-1 mRNA induced by insulin. Insulin rapidly decreased IGFBP-1 transcription in the absence of cycloheximide (> 50% inhibition in 20 min) and caused a similar decrease in cells pretreated with cycloheximide. Cycloheximide alone also decreased IGFBP-1 transcription. Similar results were observed with a second protein synthesis inhibitor, anisomycin, which also prevented the insulin-induced decrease in IGFBP-1 mRNA without abolishing the insulin-induced inhibition of IGFBP-1 transcription. These results suggest that although insulin decreases IGFBP-1 gene transcription in the presence of protein synthesis inhibitors, IGFBP-1 mRNA levels are maintained because of stabilization of the mRNA. Stabilization was demonstrated directly in actinomycin D-treated cells, where the t1/2 of IGFBP-1 mRNA increased from approximately 2 to approximately 20 h in the presence of cycloheximide; insulin did not affect IGFBP-1 mRNA turnover. Thus, cycloheximide-sensitive labile proteins contribute to the maintenance of basal IGFBP-1 promoter activity and the rapid turnover of IGFBP-1 mRNA, which determine the dynamic regulation of IGFBP-1 gene expression.

Insulin-like growth factor-binding protein-1 (IGFBP-1) can inhibit or potentiate IGF action. The biological activity of IGFBP-1 is determined by many factors, including its abundance in tissues and plasma, posttranslational modifications, and localization. IGFBP-1 levels in human plasma are highly regulated. They are increased after acute fasting and in diabetes, and are rapidly reversed by refeeding and insulin treatment, respectively. Similarly, IGFBP-1 mRNA is increased in the liver of severely diabetic and ketotic rats and decreased after 4 days of insulin treatment. Insulin rapidly decreases IGFBP-1 mRNA and IGFBP-1 transcription in rat hepatoma cells. The present study asks whether the increase in IGFBP-1 mRNA in diabetic rat liver reflects increased gene transcription, whether insulin decreases IGFBP-1 mRNA through a transcriptional or posttranscriptional mechanism, and whether this decrease is sufficiently rapid to account for the dynamic fluctuations in plasma IGFBP-1. Rats were injected ip with 100 mg/kg streptozotocin and used 7 days later when they were hyperglycemic and failed to gain weight, but were not ketotic. Hepatic IGFBP-1 mRNA levels were 13.6 +/- 5.3-fold greater in diabetic than control liver and decreased to the low levels in nondiabetic controls within 1 h after insulin treatment. In run-on transcription assays, IGFBP-1 transcription was 12.6 +/- 1.5-fold greater in nuclei from diabetic than control liver and decreased to low control levels by 1 h after insulin injection. Normalization of hepatic IGFBP-1 mRNA in insulin-treated diabetic animals did not require restoration of euglycemia. IGFBP-1 mRNA and IGFBP-1 gene transcription also were increased in the kidney of diabetic ketotic rats. We propose that the dynamic regulation of IGFBP-1 gene transcription in diabetes and after insulin treatment, by determining the availability of IGFBP-1 in tissues and plasma, may be a critical factor in the modulation of IGF action.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 μM. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is ∼13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl β- d -maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an αβγ protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.

Insulin-like growth factor binding protein-1 (IGFBP-1) is distinctive among the IGFBPs in human plasma in its dynamic regulation by metabolic changes [reviewed in 1]. IGFBP-1 levels are increased by acute fasting 2,3 and in diabetes 4,5, and normalized by refeeding and insulin treatment, respectively. IGFBP-1 also is increased by prolonged exercise, growth hormone deficiency and pregnancy 1, and following insulin-induced hypoglycemia 6.

IGFBP-1 and IGFBP-2 mRNAs are increased in the livers of streptozotocin-diabetic rats. A corresponding increase is observed in transcription of the IGFBP-1 but not the IGFBP-2 gene, indicating that the increase in steady-state levels of IGFBP-2 mRNA is a post-transcriptional effect. IGFBP-1 and IGFBP-2 mRNAs also differ in the rapidity of their response to insulin treatment: hepatic IGFBP-1 mRNA is normalized within 1 h, IGFBP-2 mRNA decreases more slowly. These differences suggest that IGFBP-2 may provide more chronic adaptation to metabolic change than IGFBP-1.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of proteins that specifically bind IGF-I and IGF-II, determine their bioavailability to tissues, and modulate their actions in target tissues. Levels of IGFBPs in plasma and IGFBP mRNAs in liver are highly regulated with developmental age and metabolic status. We now demonstrate that the increase in IGFBP-2 mRNA in fasted adult rat liver and in the liver of normal neonatal rats reflects an increased rate of transcription. When adult rats were fasted for 2-3 days, IGFBP-2 mRNA was increased in liver, but not in brain or kidney. The increase in hepatic IGFBP-2 mRNA was observed after only 1 day of fasting. Levels decreased by half after 6 h of refeeding and returned to their low starting values after 2 days of refeeding. Transcription-elongation experiments indicated that transcription of the IGFBP-2 gene was increased in fasted liver. The rate of transcription increased 9.2- +/- 3.5-fold for transcripts labeled in exon 1 and 6.6- +/- 2.4-fold for transcripts labeled in exons 2, 3, and 4, suggesting that fasting causes a uniform increase in the number of RNA polymerase II molecules along the length of the IGFBP-2 gene. We infer from these results that the regulation of IGFBP-2 gene transcription in fasting occurs at the level of initiation rather than elongation. IGFBP-2 gene transcription also was increased 3.8- +/- 1.2-fold (exon 1) and 2.9- +/- 0.9-fold (exons 2, 3, and 4) in nuclei from 2-day postnatal rat liver compared with adult rat liver, consistent with the greater abundance of IGFBP-2 mRNA in neonatal rat liver.

Virtually all of the insulin-like growth factors (IGFs) in extracellular fluids and cell culture medium occur complexed to specific IGF-binding proteins (IGFBPs).1,2 The IGFBPs are a family of proteins that bind IGF-I and IGF-II but are unrelated to IGF receptors. Four IGFBPs have been cloned from human and rat sources,1–8 and partial protein sequence information is available forafifthlGFBP.9–11 OthermembersoftheIGFBPfamilyundoubtedlyexist,12butspecific assignment must await amino acid or nucleotide sequencing.

Insulin-like growth factor-II (IGF-II) is the predominant insulin-like growth factor in fetal and neonatal rat serum and tissues. In serum, it occurs complexed to a 30-kDa nonglycosylated IGF-binding protein (IGFBP) that is immunologically related to the IGFBP in BRL-3A rat liver cells (rIGFBP-2). Levels of rIGFBP-2 and IGF-II decrease in rat serum after birth. Using a recently isolated cDNA clone for rIGFBP-2 as hybridization probe, we now compare the expression of rIGFBP-2 and IGF-II in fetal tissues and the effects of hypophysectomy and fasting on the abundance of these mRNAs in adult rat liver. rIGFBP-2 mRNA is expressed at high levels in term gestation liver and at lower levels in other tissues. The ratio of rIGFBP-2 to IGF-II mRNAs in stomach, kidney, and lung is similar to that seen in liver, whereas IGF-II mRNA is more abundant than rIGFBP-2 mRNA in muscle, intestine, heart, and skin. Both mRNAs are more abundant in fetal tissues than in the corresponding tissues from adult rats. Dexamethasone treatment of 4-day-old rats for 4 days caused a greater (90%) decrease in hepatic IGF-II mRNA than in rIGFBP-2 mRNA (50%), suggesting subtle differences in the developmental regulation of the two mRNAs. Even more striking differences were observed in the regulation of the two mRNAs in adult rats after hypophysectomy or fasting. Hepatic rIGFBP-2 mRNA was increased 10- to 20-fold compared to age-matched control rats, whereas IGF-II mRNA was not increased. A parallel increase in serum rIGFBP-2 was observed, suggesting that this regulation may result at least in part from the increased abundance of rIGFBP-2 mRNA. Thus, in addition to modulating the stimulation of growth and differentiation by IGF-II in fetal tissues, rIGFBP-2 may play a homeostatic role during catabolic states in the adult rat.

A synthetic insulin-like compound consisting of the A-chain of insulin extended at its carboxyl terminus with the hexapeptide “D-domain” of insulin-like Growth Factor II, linked via disulfide bonds to a B-chain corresponding to the “B-domain” of insulin-like Growth Factor I, has been examined for insulin-like metabolic activity and for mitogenic activity. The synthetic material ( A 27 insulin B IGF-I ) is less potent than insulin in metabolic assays, and less potent than both insulin and IGF-I in mitogenic assays. It is proposed that neither the “D-domain” nor the “B-domain” of the IGFs is a major contributor to mitogenic activity. Their presence in the same molecule does not result in significant growth-promoting activity.

The biological effects of the insulin-like growth factors, IGF-I and IGF-II, on their receptors are modulated by IGF-binding proteins. Recently, we isolated a cDNA clone for one member of the family of IGF-binding proteins, BP-3A, a 30 kilodalton (kDa) protein synthesized by the BRL-3A rat liver cell line. BP-3A is related to but distinct from two other cloned IGF-binding proteins, the human amniotic fluid binding protein and the glycosylated binding subunit of the 150 kDa IGF-binding protein complex in serum. It is expressed in multiple nonneural tissues and in serum in the fetal rat and decreases after birth, similar to the developmental pattern of IGF-II expression. IGF-I, IGF-II, and their receptors are expressed in brain. The present study examines the expression of BP-3A in the rat central nervous system. By Northern blot analysis, BP-3A mRNA is present at high levels in brain stem, cerebral cortex, and hypothalamus from 21-day gestation rats and, like IGF-II mRNA, persists in adult rat brain. The site of BP-3A mRNA synthesis was localized by in situ hybridization to coronal sections of adult rat brain using 35S-labeled oligonucleotides, 48 bases in length, complementary and anticomplementary to the coding region of BP-3A. Specific hybridization of the BP-3A probe was observed exclusively to the choroid plexus extending from the level of the medial preoptic nucleus to the arcuate nucleus of the hypothalamus, similar to the previously reported preferential localization of IGF-II mRNA to the choroid plexus. Synthesis of BP-3A mRNA by choroid plexus suggested that BP-3A might be secreted into the cerebrospinal fluid. A 30 kDa IGF-binding protein was demonstrated in rat cerebrospinal fluid that is recognized by antibodies to BP-3A and, like purified BP-3A, has equal affinity for IGF-I and IGF-II. By analogy with other transport proteins synthesized by the choroid plexus, BP-3A may facilitate the secretion of IGF-II to the cerebrospinal fluid and modulate its biological actions at distant sites within the brain.

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.

The single insulin-like growth factor II (IGF-II) gene is transcribed into multiple RNA species in most fetal and neonatal rat tissues. For IGF-II to serve as a local growth factor in fetal tissues, IGF-II RNA must be translated into pre-pro-rat (r) IGF-II, and the biosynthetic precursor processed to smaller biologically active forms. IGF-II RNA extracted from fetal rat liver, muscle, intestine, lung, and stomach, from rat placenta, and from fetal or neonatal mouse liver and lung directed the synthesis of 22,000 mol wt pre-pro-IGF-II in a reticulocyte lysate cell-free translation system. A biosynthetic precursor of this size had been observed previously in translation of RNA from BRL-3A rat liver cells and is predicted by the nucleotide sequence of cDNA clones encoding rIGF-II. Consistent with the developmental pattern of expression of IGF-II RNA observed in hybridization studies, RNA from adult rat liver, muscle, and intestine did not direct the synthesis of pre-pro-rIGF-II. To determine whether the IGF-II biosynthetic precursor was processed to smaller biologically active IGF-II, term fetal rat tissues were extracted with acid-ethanol, the extracts were fractionated by acid gel filtration, and the IGF pools were examined in a RIA specific for IGF-II. Levels of 1-2 micrograms/g were observed in liver, limb, lung, intestine, and brain; lower levels were observed in heart and kidney. In general, the levels of immunoreactive IGF-II corresponded to the levels of IGF-II mRNA. These results suggest that IGF-II mRNA is translated, and pre-pro-IGF-II processed to mature IGF-II in different fetal rat tissues. In contrast to IGF-I, in which alternative RNA splicing generates possible precursor molecules containing different COOH-terminal propeptide segments, we find no evidence for an IGF-II precursor in rat tissues other than 22,000 mol wt pre-pro-rIGF-II.