Proteomic Analysis of Charcoal-Stripped Fetal Bovine Serum Reveals Changes in the Insulin-like Growth Factor Signaling Pathway.

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.

Late Onset of Non-islet Cell Tumor Hypoglycemia Managed via Multidisciplinary Treatment in a Patient with a Solitary Fibrous Tumor.

Solitary fibrous tumor (SFT) is a rare subtype of soft tissue sarcoma (STS). We herein describe a case of late onset of non-islet cell tumor hypoglycemia (NICTH) that was managed via multidisciplinary treatment in a patient with SFT. A 67-year-old man previously diagnosed with SFT 4 years prior to this presentation and treated with several rounds of surgery, presented with massive tumors. Eighteen months following his prescribed chemotherapy, the patient developed hypoglycemia. He was diagnosed with NICTH, after confirming the presence of high molecular weight insulin-like growth factor-2. This case suggests that paraneoplastic syndrome can occur even in cases of rare cancers, such as STS.

Magnetic Surfactants and Polymers with Gadolinium Counterions for Protein Separations.

New magnetic surfactants, (cationic hexadecyltrimethlyammonium bromotrichlorogadolinate (CTAG), decyltrimethylammonium bromotrichlorogadolinate (DTAG), and a magnetic polymer (poly(3-acrylamidopropyl)trimethylammonium tetrachlorogadolinate (APTAG)) have been synthesized by the simple mixing of the corresponding surfactants and polymer with gadolinium metal ions. A magnetic anionic surfactant, gadolinium tri(1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate) (Gd(AOT)3), was synthesized via metathesis. Both routes enable facile preparation of magnetically responsive magnetic polymers and surfactants without the need to rely on nanocomposites or organic frameworks with polyradicals. Electrical conductivity, surface tensiometry, SQUID magnetometry, and small-angle neutron scattering (SANS) demonstrate surface activity and self-aggregation behavior of the magnetic surfactants similar to their magnetically inert parent analogues but with added magnetic properties. The binding of the magnetic surfactants to proteins enables efficient separations under low-strength (0.33 T) magnetic fields in a new, nanoparticle-free approach to magnetophoretic protein separations and extractions. Importantly, the toxicity of the magnetic surfactants and polymers is, in some cases, lower than that of their halide analogues.

A novel two-chain IGF-II-derived peptide from purified ß-cell granules.

OBJECTIVE: Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic ß-cells. Here we isolated secretory granules from a ß-cell line, ßTC6-F7, and characterized the nature of the IGF-II-LI located therein. DESIGN: Secretory granules were isolated from cultured mouse ßTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH(2)-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. RESULTS: ßTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 106 cells compared to 340 ± 44 pmol insulin-LI per 106 cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37-40)IGF-II, which we termed 'vesiculin'. This molecule was also detectable in ßTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC(50) value of 131 nM ± 1.35. CONCLUSIONS: Vesiculin, des(37-40)IGF-II, is a novel two-chain insulin-like hormone and the major "IGF-II-like" peptide found in purified mouse ßTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species.

A genome-inspired DNA ligand for the affinity capture of insulin and insulin-like growth factor-2.

The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a. Here, we report on interactions of insulin and the highly homologous insulin-like growth factor-2 (IGF-2) with ILPR variants a, h, and i. Circular dichroism indicated intramolecular G-quadruplex formation for variants a and h. Affinity MALDI MS and surface plasmon resonance were used to compare protein capture and binding strengths. Insulin and IGF-2 exhibited high binding affinity for variants a and h but not i, indicating the involvement of intramolecular G-quadruplexes. Interaction between insulin and variant a was unique in the appearance of two binding interactions with K(D) approximately 10(-13) M and K(D) approximately 10(-7) M, which was not observed for insulin with variant h (K(D) approximately 10(-8) M) or IGF-2 with either variant (K(D)s approximately 10(-9) M). The results provide a basis for the design of DNA binding ligands for insulin and IGF-2 and support a new approach to discovery of DNA affinity binding ligands based on genome-inspired sequences rather than the traditional combinatorial selection route to aptamer discovery.

Co-expression and purification of recombinant human insulin-like growth factor II and insulin-like growth factor binding protein-6 in Pichia pastoris yeast.

For the preparation of the complex of IGF-II and IGFBP-6, a co-expression vector containing two copies of human IGF-II and IGFBP-6 expression cassette was constructed with alcohol oxidase (AOX1) promoter and secretion signal sequence of alpha-factor, and transformed to Pichia pastoris yeast. Through a purification procedure involving anion-exchange chromatography and gel filtration, a complex of IGF-II with IGFBP-6 was obtained. An additional C-terminal sequence of IGFBP-6 (CS-BP6) was found to be bound to this complex. Dynamic light scattering showed that this complex was very stable and homogenous in solution. Western blotting based on non-reducing Tricine-SDS-PAGE indicated that IGF-II expression coupled with IGFBP-6 might significantly avoid the mispairing of disulfide bonds compared with the IGF-II expressed alone.

Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2.

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca(2+) flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.

Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II.

The insulin-like growth factors, IGF-I and IGF-II, are single chain polypeptides, which are structurally related to proinsulin and promote proliferation and differentiation of cells in many vertebrate species. Previous attempts to produce recombinant salmon IGF-II (rsIGF-II) were compromised by low expression levels and co-purification of incorrectly cleaved protein with the authentic recombinant product. In this study, a gene containing the coding region for Atlantic salmon (Salmo salar) IGF-II was cloned into a modified pET32a expression vector and transformed into Escherichia coli BL21 trxB (DE3) cells. Upon growth and induction (with IPTG) of the transformant, recombinant salmon IGF-II (rsIGF-II) was expressed as an insoluble, 28kDa thioredoxin.sIGF-II fusion protein linked by a protease cleavage motif (trx.FAHY.sIGF-II) in inclusion bodies. The inclusion bodies were subsequently solubilized and the fusion protein was purified by Ni-affinity chromatography. Recombinant IGF-II (7.8kDa) was then released from the fusion partner using H64A subtilisin BPN' protease and purified by reversed-phase HPLC. Homogeneity of the final recombinant product was confirmed by N-terminal amino acid sequencing, ion-spray mass spectrometry, SDS-polyacrylamide gel electrophoresis, and analytical reversed-phase HPLC. The biological activity of rsIGF-II was demonstrated in cultured rat L6 myoblasts and was found to be approximately 9- and 5-fold less potent than recombinant human IGF-I and recombinant salmon IGF-I, respectively, a result similar to that demonstrated previously with other recombinant fish IGF-II's in non-homologous cell lines.

Production of tilapia insulin-like growth factor-2 in high cell density cultures of recombinant Escherichia coli.

An improved expression plasmid pET-insulin-like growth factor-2 (IGF2) was constructed and transferred into Escherichia coli BL21(DE3) for the expression of tilapia insulin-like growth factor-2. The recombinant insulin-like growth factor-2 was produced as inclusion bodies, and the recombinant insulin-like growth factor-2 content was as high as 10.3% of the total protein content. For production of recombinant insulin-like growth factor-2 in E. coli, pH-stat fed-batch cultures were used to achieve a high cell density culture. A cell concentration 183gl(-1) dry cell weight (DCW) was obtained after 30h cultivation and plasmid stability was maintained at high levels. Expression of insulin-like growth factor-2 was induced at three different cell concentrations, 50, 78.5, and 114.5gl(-1) dry cell weight. When cells were induced at a cell concentration of 114.5gl(-1) dry cell weight, the amount of insulin-like growth factor-2 produced was 9.69gl(-1) (11.3% of the total protein). Using a simple purification process including inclusion body isolation, denaturation, refolding and Ni-NTA affinity chromatography, 19.51mg of insulin-like growth factor-2 was obtained from a 22.5ml of culture, and the recovery yield was 20.5%. The biological activity of the purified IGF-2 was demonstrated as promoting the growth of four different cell lines by the colorimetric bioassay and the best growth stimulation ratio was obtained for the Balb/3T3 clone 31A cell line.

In vivo processed fragments of IGF binding protein-2 copurified with bioactive IGF-II.

Proteolysis of insulin-like growth factor binding proteins (IGFBPs), the major carrier of insulin-like growth factors (IGFs) in the circulation, is an essential mechanism to regulate the bioavailability and half-live of IGFs. Screening for peptides in human hemofiltrate, stimulating the survival of PC-12 cells, resulted in the isolation of C-terminal IGFBP-2 fragments and intact IGF-II co-eluting during the chromatographic purification procedure. The IGFBP-2 fragments exhibited molecular masses of 12.7 and 12.9kDa and started with Gly169 and Gly167, respectively. The fragments were able to bind both IGFs. The stimulatory effect of the purified fraction on the survival of the PC-12 cells could be assigned exclusively to IGF-II, since it was abolished by the addition of neutralizing IGF-II antibodies. We suggest that in the circulation IGF-II is not only complexed with intact IGFBP but also with processed IGFBP-2 fragments not impairing the biological activity of IGF-II.

Preptin derived from proinsulin-like growth factor II (proIGF-II) is secreted from pancreatic islet beta-cells and enhances insulin secretion.

Pancreatic islet beta-cells secrete the hormones insulin, amylin and pancreastatin. To search for further beta-cell hormones, we purified peptides from secretory granules isolated from cultured murine beta TC6-F7 beta-cells. We identified a 34-amino-acid peptide (3948 Da), corresponding to Asp(69)-Leu(102) of the proinsulin-like growth factor II E-peptide, which we have termed 'preptin'. Preptin, is present in islet beta-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated beta TC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while anti-preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.

Improved recovery of insulin-like growth factors (IGFs) from bovine colostrum using alkaline diafiltration.

Studies to develop a rapid, bioprocess-compatible method to recover low-molecular-mass growth factors from bovine colostrum are reported. Defatted bovine colostrum was subjected to tangential-flow filtration (TFF) using two different filters [polyether sulphone (PES) and regenerated cellulose (RC)] at pH 5.8, pH 8.0 and pH 8.0+0. 01 M NaCl. Single-pass TFF at pH 5.8 using a 100 kDa RC filter resulted in the loss of approx. 90% of insulin-like growth factor I (IGF-I) to non-specific filter adsorption. Comparison of 30 kDa RC and PES filters under single-pass conditions showed that yields of IGF-1 and IGF-II were highest with RC filters. Yields of IGF-I and protein from both filter types were increased at pH 8.0 and were greatest for the 30 kDa RC filter. Effects of adding large diluent volumes continuously during TFF (diafiltration) were tested at pH 5. 0 and 8.0. The use of 10 diafiltrate vols. at pH 8.0 resulted in the recovery of 15-28% of colostral IGF-1 from the RC 30 kDa permeates, 2-4-fold greater than under acidic conditions. Yields of IGF-II (39.6%) were unaffected by pH and at least 97% of total protein was retained by the 30 kDa filter at pH 8.0. Denaturing SDS/PAGE analysis of the alkaline RC 30 kDa permeates demonstrated two major regions of stained proteins at 10-13 kDa and 17-19 kDa. Acidic TFF permeates contained additional stained proteins at approximately 90, 48 and 37 kDa. Isoelectric focusing of these samples demonstrated the presence of proteins with isoelectric points of 8.2 and 8.6. The current study demonstrates a one-step bioprocess-compatible technique for the recovery of low-molecular-mass polypeptides from bovine colostrum. By using alkaline diafiltration with RC filters TFF provided optimal recovery of IGF-1 from colostrum.

Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant.

Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.

Identification of a potent neurotrophic substance for ciliary ganglionic neurons in fetal calf serum as insulin-like growth factor II.

When fetal calf serum (FCS) alone is used as a trophic support for cultured chicken parasympathetic ciliary ganglionic (cCG) neurons, it does not show any survival-promoting effects on these neurons. When FCS is applied to heparin-affinity chromatography, however, potent survival-promoting activity is obtained in the fraction eluted with 0.5 M NaCl. Using cCG neurons as a bioassay system, this neurotrophic activity was purified by a combination of heparin-affinity chromatography, gel filtration chromatography, and Sep-Pak C18 cartridge. The 40-50-kDa fractions from the gel filtration column with strong survival-promoting activity were shown to contain insulin-like growth factor II (IGF-II) by immunoblot analysis. By acidification, the survival-promoting activity and IGF-II were translocated together from the 40-50-kDa to the 7-10-kDa fractions, and the survival-promoting activity in the 7-10-kDa fractions was blocked by an anti-IGF-II neutralizing monoclonal antibody. These results indicate that the neurotrophic substance in 0.5 M NaCl-eluate from heparin-affinity chromatography is IGF-II and that mechanisms may exist in vivo for the activation of latent IGF-II, whose biological effects may be blocked by its specific binding proteins.

Capillary electrophoresis of insulin-like growth factors: enhanced ultraviolet detection using dynamically coated capillaries and on-line solid-phase extraction.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) are difficult to separate and measure as a result of their homology, both structurally and immunologically. A number of binding proteins (BPs) which interact with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly. Current methodology for measuring IGF is immuno-based and involves dissociation from the IGFs and removal of the binding proteins through sample acidification and removal by solid-phase adsorption. However, the net result is an assay that is time-consuming and, at best, semiquantitative. In an attempt to improve the reproducibility and accuracy of IGF-I and -II measurement, electrophoretic systems employing dynamically coated and bare silica capillaries were evaluated. Separations in bare silica capillaries in the presence or absence of the cationic additive, decamethonium bromide were ineffective for resolving IGF-I and IGF-II. However, when the capillary was coated dynamically with polybrene, IGF-I and -II could be resolved in a BSA sample matrix using a low pH buffer. Despite the fact that the IGFs could be resolved in the presence of an IGF-I analog used as an internal standard, polybrene recoating was required after as few as 12 runs and poor coating-to-coating reproducibility was observed. Use of polydiallyldimethylammonium chloride (PDMAC) as a dynamic cationic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -II. It was found that PDMAC need not be included in the separation buffer to obtain reproducible analyses regarding IGF separation. Subsequently, functionality remained intact for as many as 35-40 consecutive analyses before recoating was required. Without the need for PDMAC in the buffer, on-line solid-phase extraction-capillary electrophoresis could be accomplished for detection of IGF-I and -II at concentrations as low 195 ng/ml.

Purification, amino acid sequence and characterisation of kangaroo IGF-I.

Insulin-like growth factor-I (IGF-I) and IGF-II have been purified to homogeneity from kangaroo (Macropus fuliginosus) serum, thus this represents the first report of the purification, sequencing and characterisation of marsupial IGFs. N-Terminal protein sequencing reveals that there are six amino acid differences between kangaroo and human IGF-I. Kangaroo IGF-II has been partially sequenced and no differences were found between human and kangaroo IGF-II in the 53 residues identified. Thus the IGFs appear to be remarkably structurally conserved during mammalian radiation. In addition, in vitro characterisation of kangaroo IGF-I demonstrated that the functional properties of human, kangaroo and chicken IGF-I are very similar. In an assay measuring the ability of the proteins to stimulate protein synthesis in rat L6 myoblasts, all IGF-I proteins were found to be equally potent. The ability of all three proteins to compete for binding with radiolabelled human IGF-I to type-1 IGF receptors in L6 myoblasts and in Sminthopsis crassicaudata transformed lung fibroblasts, a marsupial cell line, was comparable. Furthermore, kangaroo and human IGF-I react equally in a human IGF-I RIA using a human reference standard, radiolabelled human IGF-I and a polyclonal antibody raised against recombinant human IGF-I. This study indicates that not only is the primary structure of eutherian and metatherian IGF-I conserved, but also the proteins appear to be functionally similar.

Insulin-like growth factors in poultry.

A large amount of research, primarily in mammals, has defined to a great extent the pleiotropic effects of the IGF system on growth, development, and intermediary metabolism. Similar elucidations in poultry were hindered to some extent by the absence of native peptides (IGF-I and IGF-II) until their purification, followed by the production of recombinant chicken IGFs. In many ways IGF physiology in birds is similar to that in other species, including but not limited to the fact that IGF-I synthesis is both GH- and GH-independent, and that autocrine-paracrine IGF action is evident. However, it is clear that several unique differences in IGF physiology exist between birds and mammals. For example, more IGF is present in the free form in chickens, and the biological responses to the IGFs is different in several metabolic pathways in birds compared to mammals. To date, no unique IGF-II receptor has been identified in birds. Despite an increasing understanding of the IGFs in aves, several important questions remain to be answered. What is the role of IGF-II in embryo development and posthatch growth? Does an IGF-II receptor entity exist in nonmammalian species? How does nutrition affect IGF-I and IGF-II gene expression, and can this information be used to enhance poultry production? What is the biochemical composition of the IGFBPs, and what are their roles in birds? Can the genetic variation present in poultry be used to positively modify IGF gene expression and physiology? How do the IGFs regulate intermediary metabolism? What is the role of the IGFs in the etiology of several disease states associated with rapid growth in poultry, including tibial dyschondroplasia, obesity, ascites, and spiking mortality syndrome? Answers to these questions are relevant to our understanding of the basic mechanisms of IGF physiology as well as possibly assisting in the amelioration of problems found in modern poultry production.