A Mutagenic and Biological Study of Rat Insulin-Like Growth Factor-Binding Protein-2

We have undertaken a mutagenic and cell biological study of the properties of the rat insulin like growth factor binding protein-2 (IGFBP-2). This 32kDa protein belongs to a family of six high affinity IGFBPs (IGFBP 1-6) which are well characterised in both rodents and humans. In initial studies we mutated two highly conserved proline residues in the N-terminal region of IGFBP-2 to create the mutant proteins P20A-IGFBP-2 and P22A-IGFBP-2 and expressed these proteins along with wild type rat IGFBP-2 in the baculovirus/insect cell system. We found that this expression system was able to produce correctly folded, functional rat IGFBP-2 and using solution phase equilibrium binding techniques we demonstrated that the affinity of these mutated proteins for both IGF-I and IGF-II was similar to that of the wild type protein. We conclude that, although highly conserved in all IGFBP species, these proline residues are not involved in the IGF ligand binding site of rat IGFBP-2. In order to investigate the biological functions of IGFBP-2 in the regulation of IGF action we undertook experiments to establish a cell culture model in which the appropriate components of the IGF axis were present to allow such an investigation to take place. In initial experiments we screened several cell lines for the presence of cell or extra-cellular matrix associated IGFBP-2 (a phenomenon which limited reports in the literature had previously described). For one of these cell lines Clone 9 - a cell line derived from rat liver- we demonstrated clearly by Western blotting, ligand blotting and immunoprecipitation the association of IGFBP-2 with the surface of the cell line although experiments to determine the chemical nature of this interaction proved inconclusive. In addition to the demonstration of cell membrane associated IGFBP-2 we also identified the presence of IGF-I receptor on these cells and we were also able to show that these cells secrete IGF-I (but not IGF-II) into the surrounding medium. The identification of these components of the IGF axis in Clone 9 cells encouraged us in biological experiments to examine the possible modulatory role of cell-membrane associated and secreted IGFBP-2 on the modulation of IGF-I activity at the IGF-I receptor. We initially established that Clone 9 cells do indeed respond to exogenous IGF-I both in short term signalling events (acute tyrosine phosphorylation of a 180kDa protein) and in longer term metabolic/mitogenic assays as determined by MTT-formazan dye conversion. Further, we demonstrate, by the use of the IGF-I analogue des(l -3) IGF-I (which does not interact with IGFBPs but retains wild type potency at the IGF-I receptor) that IGF-I activity is inhibited both acutely and in longer term assays by the presence of IGFBP-2. We postulate that the former inhibition may be due to the association of IGFBP-2 with the plasma membrane of Clone 9 cells while the latter may be more related to the accumulation of IGFBP-2 in the conditioned medium. In order to further test and clarify this hypothesis we finally report on some preliminary experiments aimed at stably transfecting Clone 9 cells with sense or antisense IGFBP-2 constructs. Initial characterisation of mixed populations of such cells show no difference in basal growth rates between control, antisense and sense transfected cells although we observed reduced secretion of IGFBP-2 into conditioned medium by antisense transfected cells. We believe that the use of these cells will aid in the clarification of the role of IGFBP-2 in the regulation of the autocrine and perhaps paracrine role of IGF-I. In the final chapter of this thesis we expand on those areas of the current work which we feel would benefit from further research

Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201–218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (KD) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigatio

3.2 I gruppi di ricerca

Incontrarsi per caso e scoprirsi colleghe Il progetto sulla ricerca/azione ha seguito una strutturazione tale da permettere al gruppo di insegnanti partecipanti una formazione e una riflessione preliminari sul percorso che si stava per presentare, e successivamente dichiararsi rispetto all’adesione al percorso di ricerca. La formazione proposta ha previsto due seminari in cui sono state presentate le tematiche e il progetto di ricerca; abbiamo poi svolto un incontro di animazione teatrale che..

2.5 Esperienze di laboratorio per i bambini

Un percorso di ricerca/azione in una relazione educativa L’esperienza di ricerca/azione è frutto di un percorso che si è sviluppato a partire dall’intenzione di mettere al centro il laboratorio come metodologia che colora tutte le attività, e la narrazione come elemento che incontra i bambini ogni giorno in una pratica educativa fondamentale nella loro crescita. L’idea, inoltre, è che ciascun bambino possieda punti di forza diversi e che il suo sviluppo avvenga in modo differenziato. Proprio ..

The automation control systems for the efficiency of metro transit lines

The cities are becoming increasingly larger and more complex. This also determines increased requirements on mass transit systems. In this case, the operators have to cope with rapidly growing traffic flows and passengers' rising expectations. Their success is measured against factors such as safety, punctuality, convenience and energy efficiency. In order to successfully meeting these challenges, it is necessary to consider the Intelligent and future-oriented mass transit solutions. The aim of this work is to compare the performances between two types of subway, traditional ones and the automatic ones considering in one case the Line 1 and Line 5 to Milan

Boys are like Girls: Insights in the Gender Digital Divide in Higher Education in Switzerland and Europe

This paper explores the differences between boys and girls in their approach to ICT. Data were gathered from students in the final grades of high school in the Swiss Canton Ticino as part of a wider European project, with a particular focus on the potential uptake of a study or professional career in ICT. Preliminary focus groups with teachers and interviews with women with a job in ICT were followed by a quali-quantitative study which involved 539 students. Results indicate that there are no big gender differences when it comes to ICT use and perception. Small differences are found in the perception of the gender digital divide, while relevant differences were found in the perception and values attached to future professional or academic careers in ICT

Abstract

The aim of DICE is the improvement of communication between culture-providers (institutions, museums, etc.) and different categories of users (scholars, publishers, culturaltourism promoters, etc.), trying to “translate ” into a common vocabulary the patrimony of content held by each source of information. The result is a shared virtual space, allowing access to different sources of information, integrated in a peer-to-peer philosophy, in order to create an effective working environment for professional users. DICE provides a technical infrastructure, a software platform, an organizational and cultural model, with a flexible and scalable approach that allows the collection of information from all the actors involved