Attenuated kallikrein-related peptidase activity disrupts desquamation and leads to stratum corneum thickening in human skin equivalent models.

BACKGROUND: Epidermal homeostasis is maintained through the balance between keratinocyte proliferation, differentiation and desquamation; however, human skin equivalent (HSE) models are known to differentiate excessively. In native tissue, proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 cleave the extracellular components of corneodesmosomes; proteins corneodesmosin, desmocollin 1 and desmoglein 1, loosening the cellular connections and enabling desquamation. The actions of KLK7 are tightly controlled by protease inhibitors, skin-derived antileucoproteinase (SKALP) and lymphoepithelial Kazal-type-related inhibitor (LEKTI), which also inhibits KLK5, localizing protease activity to the stratum corneum. OBJECTIVES: To investigate the mechanisms that inhibit the desquamation cascade in HSE models. METHODS: Human skin tissue and HSE models were investigated using gene microarray, real-time polymerase chain reaction (PCR), immunohistochemistry and Western blot analysis to examine key components of the desquamation pathway. To elucidate proteolytic activity in HSEs and native skin, in situ and gel zymography was performed. RESULTS: Histological analysis indicated that HSE models form a well-organized epidermis, yet develop an excessively thick and compact stratum corneum. Gene microarray analysis revealed that the desquamation cascade was dysregulated in HSE models and this was confirmed using real-time PCR and immunohistochemistry. Immunohistochemistry and Western blot indicated overexpression of LEKTI and SKALP in HSEs. Although KLK7 was also highly expressed in HSEs, zymography indicated that protease activation and activity was lower than in native skin. CONCLUSIONS: These findings demonstrate that stratum corneum thickening is due to inhibited KLK5 and KLK7 activation and a subsequent lack of corneodesmosome degradation in the HSE model epidermis.

Choose wisely: Network, ontology and annotation resources for the analysis of Staphylococcus aureus omics data.

Staphylococcus aureus (S. aureus) is a prominent human and livestock pathogen investigated widely using omic technologies. Critically, due to availability, low visibility or scattered resources, robust network and statistical contextualisation of the resulting data is generally under-represented. Here, we present novel meta-analyses of freely-accessible molecular network and gene ontology annotation information resources for S. aureus omics data interpretation. Furthermore, through the application of the gene ontology annotation resources we demonstrate their value and ability (or lack-there-of) to summarise and statistically interpret the emergent properties of gene expression and protein abundance changes using publically available data. This analysis provides simple metrics for network selection and demonstrates the availability and impact that gene ontology annotation selection can have on the contextualisation of bacterial omics data.

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

Stratum basale keratinocyte expression of the cell-surface glycoprotein CDCP1 during epidermogenesis and its role in keratinocyte migration.

BACKGROUND: Epidermogenesis and epidermal wound healing are tightly regulated processes during which keratinocytes must migrate, proliferate and differentiate. Cell-to-cell adhesion is crucial to the initiation and regulation of these processes. CUB-domain-containing protein (CDCP)1 is a transmembrane glycoprotein that is differentially tyrosine phosphorylated during changes in cell adhesion and survival signalling, and is expressed by keratinocytes in native human skin, as well as in primary cultures. OBJECTIVES: To investigate the expression of CDCP1 during epidermogenesis and its role in keratinocyte migration. METHODS: We examined both human skin tissue and an in vitro three-dimensional human skin equivalent model to examine the expression of CDCP1 during epidermogenesis. To examine the role of CDCP1 in keratinocyte migration we used a function-blocking anti-CDCP1 antibody and a real-time Transwell™ cell migration assay. RESULTS: Immunohistochemical analysis indicated that in native human skin CDCP1 is expressed in the stratum basale and stratum spinosum. In contrast, during epidermogenesis in a three-dimensional human skin equivalent model, CDCP1 was expressed only in the stratum basale, with localization restricted to the cell-cell membrane. No expression was detected in basal keratinocytes that were in contact with the basement membrane. Furthermore, an anti-CDCP1 function-blocking antibody was shown to disrupt keratinocyte chemotactic migration in vitro. CONCLUSIONS: These findings delineate the expression of CDCP1 in human epidermal keratinocytes during epidermogenesis and demonstrate that CDCP1 is involved in keratinocyte migration.

A mathematical model of wound healing and subsequent scarring.

Wound healing is a complex process involving the delicate interaction between elements that vary widely in nature and size scales, from the nanometre level, such as molecules, to cells measured in micrometres, and fibres with width and length measured on both scales. Hybrid approaches, where each species is represented by a model on an appropriate size scale, have received attention recently. In this study, we provide a review of earlier work on such hybrid models of wound healing. General models for each of the element types involved in dermal wound healing used in this research are described: cells, modelled as discrete individuals; chemicals, modelled as continua; and fibres, modelled with a novel tensorial representation. Techniques for integrating such disparate models are outlined. A six-species model (fibrin, collagen, macrophages, fibroblasts, transforming growth factor-beta (TGF-beta) and tissue plasminogen activator) of dermal wound healing is presented. The role of the cytokine TGF-beta in the healing cascade is investigated using the model, along with its role in the degree of scarring in the healed tissue.

Increased matrix metalloproteinase-9 (MMP-9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer.

BACKGROUND: The pathology of chronic wounds is often characterized by elevated levels of proinflammatory cytokines [e.g. tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta], proteases [e.g. matrix metalloproteinases (MMPs)] and neutrophil elastase. MMPs specifically have been implicated by a number of studies as the major protease family responsible for the degradation of key factors critical to the ulcer's ability to heal. OBJECTIVES: To assess individual MMPs in chronic wound fluid (CWF) in order to develop improved treatments for chronic ulcers. METHODS: Collagen type I and IV zymography, immunoprecipitation followed by a substrate activity assay, and an indirect enzyme-linked immunosorbent assay were all used to analyse MMP levels in CWF. RESULTS: Our studies demonstrate that there is excessive protease activity in CWF compared with both human serum and acute wound fluid (AWF), which can be specifically attributed to MMPs as determined through a MMP-inhibitor study. Multiple MMPs were immunoprecipitated from the CWF samples and MMP-9 was identified as the predominant protease in CWF, with significantly elevated activity levels in CWF compared with AWF. In addition, the clinical status of the ulcer is directly associated with the amounts of MMP-9 present in the wound fluid. Therefore, this study suggests that higher levels of MMP-9 in chronic wound fluid correlate with a clinically worse wound. CONCLUSIONS: In view of these results, it is hypothesized that a specific inhibitor of MMP-9 could potentially be more therapeutically effective than general MMP inhibitors in modulating chronic ulcers towards a healing state.

Development of an enhanced proteomic method to detect prognostic and diagnostic markers of healing in chronic wound fluid.

BACKGROUND: Chronic venous leg ulcers are a significant cause of pain, immobility and decreased quality of life for patients with these wounds. In view of this, research efforts are focused on multiple factors in the wound environment to obtain information regarding the healing of ulcers. OBJECTIVES: Chronic wound fluid (CWF), containing a complex mixture of proteins, is an important modulator of the wound environment, and therefore we hypothesized that these proteins may be indicators of the status of wounds and their potential to heal or otherwise. To explore this we developed and validated a proteomic approach to analyse CWF. METHODS: In this study, pooled CWF was depleted of high abundant proteins using immunoaffinity chromatography. The flow-through and bound fractions were collected, concentrated, desalted and analysed using a range of techniques. Each fraction was further separated using two-dimensional (2D) gel electrophoresis and 2D liquid chromatography and analysed using mass spectrometry (MS). RESULTS: Western blot analysis against three high abundant proteins confirmed the selective removal of these proteins from CWF. Critically, one-dimensional and 2D gel electrophoresis indicated that subsequent removal of these proteins enhanced the ability to detect proteins in low abundance in CWF. Further, MS demonstrated that depletion of these abundant proteins increased the detection of other proteins in these samples. CONCLUSIONS: Results obtained indicate that this approach significantly improves separation of proteins present in low concentrations in CWF. This will facilitate the identification of biomarkers in samples collected from patients with ulcers and lead to improved patient therapies and wound care approaches.

Production, in vitro characterisation, in vivo clearance, and tissue localisation of recombinant barramundi (Lates calcarifer) insulin-like growth factor II.

Recombinant barramundi insulin-like growth-factor-II (bIGF-II) has been produced in Escherichia coli after modification of an expression plasmid that coded for a chicken IGF-II fusion protein. The bIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, and refolded. The protein was then released from the fusion protein by cleavage with subtilisin BPN'. Finally the protein was purified to homogeneity with a number of HPLC steps. In vitro analysis of recombinant bIGF-II demonstrated decreased potency in stimulating protein synthesis when compared to human and barramundi IGF-I (bIGF-I). The in vivo distribution of radiolabeled bIGF-II and bIGF-I in the circulation and tissue uptake of radiolabeled bIGF-II was also compared in juvenile barramundi (Lates calcarifer). Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that bIGF-II was degraded faster than bIGF-I. Moreover, neutral gel chromatography of these samples suggested this difference may be due to reduced affinity of bIGF-II, compared to blGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi circulation. Based on these results, it would appear that elements important in the function of IGFs have been well conserved during vertebrate evolution. However, to clearly define the IGF system in fish it will be necessary to characterise the IGFBPs present and to determine how they influence the biological actions of native IGFs.

IGFs stimulate zebrafish cell proliferation by activating MAP kinase and PI3-kinase-signaling pathways.

Insulin-like growth factor (IGF)-I and -II have been cloned from a number of teleost species, but their cellular actions in fish are poorly defined. In this study, we show that both IGF-I and -II stimulated zebrafish embryonic cell proliferation and DNA synthesis in a concentration-dependent manner, whereas insulin had little mitogenic activity. Affinity cross-linking and immunoblotting studies revealed the presence of IGF receptors with the characteristics of the mammalian type I IGF receptor. Competitive binding assay results indicated that the binding affinities of the zebrafish IGF-I receptors to IGF-I, IGF-II, and insulin are 1.9, 2.6, and >190 nM, indicating that IGF-I and -II bind to the IGF-I receptor(s) with approximately equal high affinity. To further investigate the cellular mechanism of IGF actions, we have studied the effects of IGFs on two major signal transduction pathways: mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase). IGFs activated MAPK in zebrafish embryonic cells in a dose-dependent manner. This activation occurred within 5 min of IGF-I stimulation and disappeared after 1 h. IGF-I also caused a concentration-dependent activation of protein kinase B, a downstream target of PI3 kinase, this activation being sustained for several hours. Inhibition of MAPK activation by the MAPK kinase inhibitor PD-98059 inhibited the IGF-I-stimulated DNA synthesis. Similarly, use of the PI3 kinase inhibitor LY-294002 also inhibited IGF-I-stimulated DNA synthesis. When both the MAPK and PI3 kinase pathways were inhibited using a combination of these compounds, the IGF-I-stimulated DNA synthesis was completely negated. These results indicate that both IGF-I and -II are potent mitogens for zebrafish embryonic cells and that activation of both the MAPK and PI3 kinase-signaling pathways is required for the mitogenic action of IGFs in zebrafish embryonic cells.

Comparison of recombinant barramundi and human insulin-like growth factor (IGF)-I in juvenile barramundi (Lates calcarifer): in vivo metabolic effects, association with circulating IGF-binding proteins, and tissue localisation.

The in vivo actions of human and fish insulin-like growth factor (IGF)-I have been compared to extend the understanding of the metabolism of IGFs in fish and to identify potential differences in their actions. The effects of acute administration of these proteins on the incorporation of glucose into muscle glycogen and leucine into liver protein in juvenile barramundi were investigated. In these in vivo metabolic assays, both baramundi IGF-I (bIGF-I) and human IGF-I (hIGF-I) increase the incorporation of D-[14C]glucose into muscle glycogen and [14C]leucine into liver protein. The distribution of radio-labeled human and barramundi IGF-I in the circulation and their uptake by tissue was also compared in juvenile barramundi (Lates calcarifer). Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that hIGF-I was degraded faster than bIGF-I. Neutral gel chromatography of these samples suggested that this difference is due to reduced affinity of hIGF-I, compared to bIGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi. Tissue uptake of [125I]-labeled hIGF-I and bIGF-I was similar except that [ 125I]bIGF-I uptake by the kidney exceeded that of hIGF-I. It is suggested that while some of the in vivo actions of IGFs in fish are conserved, functional differences between mammalian and teleostean IGFs exist, particularly with respect to their interactions with fish IGFBPs.

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein.

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(1-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/IGF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

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.

Assessment of developmental changes in chicken and turkey insulin-like growth factor-II by homologous radioimmunoassay.

The development of a homologous RIA for chicken insulin-like growth factor-II (cIGF-II) and its application to investigate the developmental changes in IGF-II in the chicken and turkey are described. A double-antibody RIA has been developed using recombinantly derived cIGF-II as antigen, radiolabelled tracer and standard. Serial dilutions of chicken and turkey plasma were parallel to serial dilutions of cIGF-II standard. We have also established that acid/ethanol extraction of chicken and turkey plasma reduced possible interference of insulin-like growth factor-binding proteins in the RIA. Consumption of a low-protein diet by male chickens lowered plasma IGF-I twofold, whereas IGF-II levels were unchanged. Food withdrawal evoked an increase in circulating IGF-II, while IGF-I levels were reduced. Refeeding returned both growth factors to normal circulating concentrations. During chick embryo incubation, plasma IGF-II levels were tenfold higher than those of IGF-I. In the turkey embryo, plasma IGF-II concentrations were higher than those of IGF-I. During the post-hatch period. IGF-II levels declined with age in chickens. In the growing turkey, IGF-II levels were consistently higher than IGF-I levels. The application of the homologous RIA to monitor plasma levels during embryonic development and post-hatch growth in avian species will provide more accurate comparisons of results from studies on the role of IGF-II in growth and metabolism of domestic birds.

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.

Evolution of insulin-like growth factor-I (IGF-I) action: in vitro characterization of vertebrate IGF-I proteins.

While there is considerable structural evidence that IGFs share a long evolutionary history, less is known about the conservation of IGF action. These studies have primarily been hampered by the small amounts of purified IGFs that have been available for testing. More recently, however, we have adopted recombinant strategies to produce milligram quantities of IGFs for biological studies. Thus we have been able to compare the properties of rat, kangaroo, chicken, salmon and barramundi IGF-I, proteins that differ from human IGF-I by 3, 6, 8, 14 and 16 amino acids respectively. While we have found that the IGF-I proteins exhibit similar biological activities and type-I IGF receptor binding affinities, regardless of whether mammalian, avian or piscine cell lines are used, there was a trend suggesting that the fish proteins at least, were most effective in studies using homologous systems. Thus, salmon IGF-I was not as potent as human IGF-I in bioassays in mammalian cells, but was as effective as human IGF-I in piscine cells. As expected, the IGF-I proteins competed poorly for binding to type-2 receptors present on ovine placental membranes. Interestingly however, the two fish IGF-I proteins exhibited greater affinity for this receptor than the other IGF-I proteins, hence reminiscent of the results previously found with recombinant hagfish IGF. Despite these small differences, these results taken together indicate that the IGF-I proteins appear to have been remarkably conserved in both structure and in vitro action during vertebrate radiation.

Insulin-like growth factor-I effect on chicken hepatoma cells (LMH) is inhibited by endogenous IGF-binding proteins.

LMH chicken hepatoma cells show type 1 IGF receptors and a 28 kDa IGF-binding protein (IGFBP) on their membranes. They also secrete large amounts of the 28 kDa IGFBP. Following overnight incubation in serum-free medium, human IGF-I was markedly less effective than insulin in stimulating amino acid (AIB) uptake. Chicken and human IGF-I were equipotent, consistent with their equipotency in inhibiting [125I]IGF-I binding to wheat germ agglutinin-purified IGF receptors or membrane solubilized IGFBP. When cells were supplied with fresh medium, cell-associated IGFBP were unaffected, but the level of soluble IGFBP was largely reduced. This potentiated the effect of IGF-I on AIB uptake. The effect of chicken Long-[Arg3]-IGF-I, which exhibited low affinity for the IGFBP, was unchanged. In fresh or conditioned medium, this analog was more potent than IGF-I, suggesting that both soluble and membrane-bound 28 kDa IGFBP inhibited the effect of IGF-I.

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.

Evolution of insulin-like growth factor (IGF) function: production and characterization of recombinant hagfish IGF.

While there is considerable structural evidence that insulin-like growth factors (IGFs) share a long evolutionary history, little is known about the conservation of IGF function. In order to address this, we have made recombinant hagfish IGF, hence allowing characterization of an IGF from a representative of the primitive vertebrate class, Agnatha. The production of recombinant hagfish IGF has been complicated by a number of factors including the requirement of a longer leader peptide for fusion protein expression, reduced solubility of the protein, as well as problems in the refolding procedure. However, we were able to produce a small quantity of hagfish IGF with an N-terminal glycine addition which is biologically active. Furthermore, N-terminal amino acid sequencing and mass spectrometry confirm that we have produced hagfish IGF. In vitro assessment of recombinant hagfish IGF in cultured cells indicates that hagfish IGF indeed shares functional properties with mammalian IGFs. Thus, hagfish IGF stimulates protein synthesis in rat myoblasts, but 20- and 5-fold more peptide, respectively, is required to achieve the same half-maximal responses as with human IGF-I (hIGF-I) or IGF-II (hIGF-II). Hagfish IGF also competes for binding to the type-1 IGF receptor present both on rat myoblasts and on salmon embryo fibroblasts, though with somewhat lower affinity than either hIGF-I or hIGF-II. However, studies investigating binding to the IGF-II-specific type-2 receptor suggest that hagfish IGF may in fact be more closely related to IGF-I than to IGF-II. These results indicate that motifs important for functions associated with mammalian IGFs appear to have evolved prior to the Agnathans diverging from the main line of vertebrate evolution 550 million years ago. Accordingly, we now have functional as well as structural evidence that the IGFs have a long evolutionary history.

Plasma clearance and tissue distribution of labelled chicken and human IGF-I and IGF-II in the chicken.

The metabolic clearance of chicken IGF-I (cIGF-I), cIGF-II, human IGF-I (hIGF-I), and hIGF-II was examined in the chicken using 125I-labelled growth factors. Superose-12 chromatography of plasma collected at 7.5 min post-infusion revealed peaks of radioactivity corresponding to 150 and 43 kDa and unbound tracer. Statistical analysis of trichloroacetic acid (TCA)-precipitable radioactivity in sequential plasma samples as well as following chromatography of the same samples revealed that clearance of the radiolabelled peptides followed an apparent triphasic pattern. The close similarity of the individual chromatographically defined pools in their clearance rate compared with the three components described by TCA precipitation strongly suggested their identity. Both free 125I-labelled cIGF-II (3.11 min) and hIGF-II (3.01 min) were cleared at a greater rate than their IGF-I counterparts. Unbound hIGF-I was cleared at a greater rate than cIGF-I (4.45 vs 5.66 min respectively). A similar pattern for clearance was evident in the radio-labelled growth factors associated with the 43 kDa component, although at a longer half-life. There was no difference in the apparent clearance of the radiolabelled growth factors associated with the 150 kDa component between IGF-I or -II or between species. Analysis of the chromatographic profiles of radioactive IGF-I peptides complexed to serum proteins versus those bound to labelled IGF-II peptides revealed the presence of a large molecular mass binding protein in vivo. Ligand blotting of chicken serum determined that a binding protein with a mass of 70 kDa was detectable with 125I-IGF-II probes only, and was not present in pig serum. In addition, tissue uptake of 125I-cIGF-I and -II was evaluated. Similar patterns of tissue distribution and uptake were observed for 125I-cIGF-I and -II, except that cIGF-II uptake by the liver exceeded that of 125I-cIGF-I at 15 min post-infusion. The rank order of tissue distribution was as follows: kidney > testis > heart > liver > pancreas > small intestine > cartilage > bursa > gizzard > leg muscle > breast muscle > brain. We conclude from these studies that the clearance of IGFs from the compartments identified in blood and the potential target tissues is dependent on their interactions with IGF-binding proteins and receptors.