WO2005074966A1

WO2005074966A1 - A method of modulating cellular uptake and molecules useful for same

Info

Publication number: WO2005074966A1
Application number: PCT/AU2005/000150
Authority: WO
Inventors: Otto Schmidt
Original assignee: Adelaide Research & Innovation Pty. Ltd.
Priority date: 2004-02-06
Filing date: 2005-02-07
Publication date: 2005-08-18

Abstract

The present invention relates generally to a method of regulating the cellular endocytosis process and, more particularly, to a method for regulating cellular endocytosis by regulating the leverage mediated endocytosis mechanism and to molecules for use therein. Still more particularly, the method of the present invention is directed to regulation of the extracellular driving force of endocytosis which is dependent on the interaction of soluble adhesion molecules, endocytosis molecules and membrane anchored molecules. The method of the present invention is useful,inter alia, in the treatment and/or prophylaxis of conditions characterised by the aberrant, unwanted or otherwise inappropriate cellular endocytosis of a molecule. Further, this method provides for the rational design of means of intracellularly delivering a molecule such as, but not limited to, a drug. This method also provides for, inter alia, the rational design of means of manipulating cellular signalling processing and means of disease reduction, disease protection and toxin resistance management strategies in animals and plants.

Description

A METHOD FOR MODULATING CE?LLULAR UPTAKE AND MOLECULES USEFUL FOR SAME

FIELD OF THE INVENTION

The present invention relates generally to a method of regulating the cellular uptake process and, more particularly, to a method for regulating cellular uptake by regulating a leverage mediated mechanism and to molecules for use therein. Still more particularly, the method of the present invention is directed to regulation of the extracellular driving force of cellular uptake which is dependent on the interaction of soluble adhesion molecules, hinge molecules and membrane anchored molecules. The method of the present invention is useful, inter alia, in the treatment and/or prophylaxis of conditions characterised by the aberrant, unwanted or otherwise inappropriate cellular uptake of a molecule. Further, this method provides for the rational design of means of intracellularly delivering a molecule such as, but not limited to, a drug. This method also provides for, inter alia, the rational design of means of manipulating cellular signalling processing, cell-shape changes and means of disease reduction, disease protection and toxin resistance management strategies in animals and plants.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Endocytotic reactions are derived from ancient cellular functions that enable the cell surface to interact with external environments, ranging from nutrient uptake to defense reactions. Since endocytotic processes of single cell organisms resemble those in plants and animals (Rupper et al., Biochimica et Biophysica Ada General Subjects 3:205-216, 2001 ; Janssen et ah, Biochimica et Biophysica Acta General Subjects 3:228-233, 2001 ; D'Hondt et al, Annual Review of Genetics 34:255-295, 2000), some of the fundamental steps in endocytosis may predate multicellular organisms. It seems possible, therefore, that some cell-cell interactions in multicellular organisms may be derived from primeval uptake mechanisms.

Many fundamental problems in cell biology that appear to be fundamentally interconnected at the mechanics of cellular membrane changes remain unsolved. For example, the question of self-recognition in single cell or multicellular organisms remains a question particularly in the light of recent genome sequences revealing a relative limited number of innate recognition molecules which are not sufficient to explain the observed power of discrimination evident in histo-incompatibility in mammals, self-incompatibility in plants and mating type separation in single cell organisms.

These recognition processes are part of basic cellular mechanisms of membrane sculpturing, associated with cellular uptake reactions, cell-cell interactions, cell mobility and membrane protrusions, such as filopodia, nerve axon and pollen tube formation. Membrane shape-changes are not restricted to the cell surface but are an important aspect of protein secretion and retrograde protein transport. For example, vesicle formation in the ER and Golgi requires mechanistic forces that can identify and pinch off ER membrane areas that contain functionally active proteins for further processing. Likewise, incoming endosomes merge with cytoplasmic vesicles and separate sub-vesicles for recycling involving decision processes, which fractions of proteins are recycled and which are left behind for proteolytic degradation.

Finally, the osmofragility of membranes are intimately associated with membrane-shape changes. For example endosome formation is associated with pH- and ion changes, but the mechanical reasons for the apparent osmofragility during uptake reactions is not known. Likewise, the osmofragility in cholesterol-containing membranes caused by amphipathic peptides, such as the bee toxin melittin, and pore-forming toxins, such as hemolysin from E. coli or endotoxins from B. thuringiensis, is not clear. Current models of cellular processes, such as endocytosis, are based on a two-step process that involves ligand interactions with cell surface molecules, which activate cytoplasmic responses leading to engulfment of liquid and solid substrates. Uptake of soluble components (pinocytosis and macropinocytosis) are receptor-mediated processes involving clathrin, which usually occur independently of actin polymerization. In contrast, phagocytosis of microbes and large particles occurs by actin-mediated mechanisms and is usually independent of clathrin (Aderem et ah, Annual Review of Immunology 17:593-623, 1999). Both, "trigger" and receptor-mediated endocytosis (RME) mechanisms assume that the driving forces for intemalization are located in the cytoplasm with the phagocytotic cup induced by specific signalling pathways and carried out by cytoplasmic "motor" proteins. Accordingly, present models of receptor-ligand interactions leading to cell-shape changes are all based on the activation of cytoplasmic 'motor' proteins to mediate the observed changes in membrane curvature. In other words, the ligand-receptor interaction in current model has only signalling function that in turn activates a cytoplasmic response. However, some uptake reactions involving membrane invaginations are difficult to explain within the context of intracellular devices. For example, clathrin is clearly involved in shaping endosomal vesicles, but requires an initial curvature of the membrane to function. These and other processes appear to require extracellular driving forces (Altschuler et al, Molecular Biology of the Cell 11:819-831, 2000). Conversely, although the role of fibres, such as actin and myosin, is apparent in vesicle transport, a direct role in uptake reactions is not clear (Durrbach et al, Journal of Cell Science 109:457-465,1996; Hasson, J Cell Sci 116:3453-3461, 2003).

Present models of receptor-mediated uptake reactions, where receptors and ligands are only involved in signalling and where changes in membrane curvature are caused by intracellular motor proteins are faced with a conundrum; Some oligomeric adhesion molecules cause cells to spread if immobilised on artificial substrates but to detach when added to spread cells in a soluble form. For example, counter-adhesive proteins in vertebrates, such as thrombospondin (Adams, J. Cell Sci. 108(5):1977-1990, 1995 ; Adams et al Journal of Molecular Biology 328, 479-494, 2003.; Chen et al, Matrix Biology 19, 597-614., 2000) cause mammalian cells to spread on substrates coated with thromospondin but detach already spread cells when applied in soluble form (Adams, Annual Review of Cell and Developmental Biology 17:25-51, 2001). In the concept of the current model where receptors receive a signal and respond by mobilising cytoplasmic motor protein, the observation is a puzzle, since the same ligand triggers two opposite responses. In vertebrates, the observation that thrombospondin interacts with a multitude of receptors has been used to explain the conundrum. The assumption is that these molecules interact with different receptors invoking distinct signalling pathways (Chandrasekaran et al, Mol Biol Cell 11:2885-2900, 2000; Greenwood and Murphy, Microscopy Research & Technique 43:420-432, 1998) depending on the exposure of cells to immobilized and soluble thrombospondin (Adams, 1995 supra; Goicoechea et al, JBiol Chem 277:37219- 37228, 2002).

However, in Drosophila cell lines, where hemomucin is the only lectin-binding glycoprotein (Theopold et al, J Biol 271:12708-12715, 1996; Theopold et al, Insect Biochem Mol Bio 31:189-197, 2001) immobilized lectin causes extensive spreading (Figure 33A), whereas soluble lectin added to spread cell causes detachment and actin cytoskeleton breakdown (Fig. 33B).

Another difficulty with cytoplasmic driving forces as a sole source of energy is that different and sometime opposite cellular operations may be difficult to separate at a regulatory and functional level (Etienne-Manneville and Hall, Nature 420:629-635, 2002). For example, membrane attachment and invagination leading to uptake are performed by the same receptors in close proximity (Geffen et al, Journal of Biological Chemistry 268:20772-20777, 1993).

Finally, many processes involving cell-shape changes are mediated by receptors, which lack signalling capacity, either because they are GPI-anchored or are recombinant proteins with deleted cytoplasmic domains. Some specific applications of this current thinking are further described as follows. 1. Pore forming toxins

A general assumption is that many pore-forming toxins are inserted into the membrane as a monomer and assembled inside the membrane bilayer into a pore-forming complex (Olson et al, Nature Structural Biology 6:134-140, 1999). The main issue for understanding bacterial toxicity is how soluble toxins convert into membrane-spanning protein complexes (Lacy and Stevens, Current Opinion in Structural Biology 8:778-784, 1998). Most current models assume that toxin monomers interact with membrane receptors, and in the process insert the toxin into the membrane via structural changes of receptor or toxin molecules (Bhakdi et al, Archives of Microbiology 165:73-79, 1996). However, progress towards understanding toxicity mechanisms may have been hampered by the use of artificial membrane preparations or purified membrane vesicles in most bioassays for toxicity. For example, high concentrations of toxin monomers are required to obtain insertion into artificial membranes, which may not reflect events in vivo (Cortaj arena et al, Journal of Biological Chemistry 276:12513-12519, 2001). Results obtained with the isolated α 4- loop-α 5 hairpin from the endotoxin from Bacillus thuringiensis showed that this peptide is extremely active as an antibacterial peptide (Gerber and Shai, Journal of Biological Chemistry 275:23602-23607, 2000). The question is then, why are most of these peptides specific to bacterial but not to eukaryotic membranes? One argument is that the positive net charge of antibacterial peptides enables binding and permeation of negatively charged phospholipid membranes of bacteria but not to zwitterionic membranes, which are the major constituents of the outer leaflet of erythrocytes (Shai, Biopolymers 66:236-248, 2002). But there are important exceptions, which suggest that charge alone is not important but that the secondary and tertiary structure of the peptide is crucial for the insertion into the membrane. This is apparent in some antimicrobial peptides that are active in bacteria and in mammalian cells, such as melittin, its hybrid cecropin A peptides and its diastereomeric analogs (Merrifield et al, PNAS 92:3449-3453, 1995). Apart from the observation that changes in peptide sequence are more likely to destroy its activity to eukaryotic than to prokaryotic cells, there are no identifiable amino acid sequences that are responsible for the specificity (Hancock and Rozek, FEMS Microbiology Letters 206:143- 149, 2002). Another argument put forward to explain peptide-specificities against bacterial membranes is that cholesterol found in eukaryotic membranes protects against the action of antibacterial peptides (Boman, J Intern Med 254:197-215, 2003). Although this has been confirmed in artificial membrane systems, it raises the question as to why a large portion of antibacterial peptides, such as defensins (Hoffmann and Reichart, Nature Immunology 3:121-126, 2002), dermaseptins (Shai, 2002 supra ), cathelicidines (Zanetti et al, Ann NY Acad Sci 832:147-162, 1997), pardaxin analogs (Shai, 2002 supra), and tachystatin (Osaki et al, Journal of Biological Chemistry 274:26172-26178, 1999) are also active against fungal membranes, which contain cholesterol-related ergosterol. Another observation which is difficult to understand in the context of current models is the non- lytic mode of action, such as the transfer of some peptides through the membrane into the underlying cytoplasm, where active peptides interfere with a diverse range of metabolic processes (Hancock and Rozek, 2002 supra). In fact, some scientists have argued that the lytic action may not be the primary cause of death for bacteria (Boman, 2003 supra), since antibacterial peptides may have already affected the viability of bacteria before the apparent disruption of the membrane .

2. Tolerance to poreforming toxins

In work leading up to the present invention it was observed that Bt-tolerance is indeed associated with an immune induction in lepidopteran larvae, which can be transmitted to the next generation by a maternal effect. In the context of current models of toxicity and resistance mechanisms, this observation is puzzling. It suggests that in addition to receptor-inactivation, other resistance or tolerance mechanisms exist that involve immune- related proteins, such as pro-coagulants or post-translational modification enzymes.

3. Immune suppression and the role of counter-adhesion molecules

Soluble counter-adhesion molecules, such as thrombospondins (Chen et al, 2000, supra), SPARC (Yan and Sage, J Histochem Cytochem 47:1495-1506, 1999) and tenascin (Midwood and Schwarzbauer, Molecule Biol Cell 13:3601-3613, 2002), destabilize cell- matrix contacts by inhibiting focal contact formation and assembly and prevent cell adhesion to glass or fibronectin substrates. Conversely, immobilized counter-adhesion molecules promote adhesion (Bornstein, J Clin Invest 107:929-934, 2001) in ways that are different from focal contacts (Adams, 1995 supra). Although the mode of action of vertebrate counter-adhesion molecules and similar molecules in insects (Adams et al, 2003 supra) is not known, the assumption is that immobilized and soluble anti-adhesion molecules interact with different receptors invoking distinct signalling pathways (Chandrasekaran et al, 2000 supra), depending on the exposure of cells to the immobilized or soluble form (Adams, 1995 supra; Goicoechea et al, 2002 supra).

Some oligomeric lectins act as adhesion molecules by promoting spreading on an artificial surface, but on different substrate conditions act as counter-adhesion molecules by detaching already spread cells. For example, the pioneering work of the Rizki's demonstrated that lectins cause spreading of Drosophila cells on a glass surface and cause cell fusion of neighbouring cells (Rizki et al, Journal of Cell Science 18:113-142, 1975). Similarly Drosophila cells (and other cells as well) will spread more extensively when plated on immobilised lectins (Rogers et al, J Cell Biol 162:1079-1088, 2003). Since hemocyte-like cells secrete extracellular matrix-like substances (Gullberg et al, Developmental Dynamics 199: 116-128, 1994) that allow them to attach to artificial surfaces, such as glass or plastic, it is possible that lectins spread cells on surfaces coated with conditioned cell-culture medium, because there are many external binding sites readily available to connect to cell-bound glycoproteins. However, soluble lectins detach and round up cells that are spread directly on a glass surface. Again this was first detected in fat body cells, where detachment and associated rearrangements of actin-cytoskeleton was observed after lectin applications (Rizki and Rizki, Nature 303:340-342, 1983).

A conundrum also exists in polarised cells, where impairment of actin-containing microfilaments by cytochalasin D, a fungal actin-capping protein (Cooper, J Cell Biol 105:1473-1478, 1987), selectively inhibits the capacity of cells to take up membrane- bound and fluid-phase markers applied to the apical surface, without affecting uptake from the basolateral surface (Gottlieb et al, J Cell Biol 120:695-710, 1993). The authors concluded that 'the ankyrin-mediated linkage of some basolateral membrane proteins to the underlying cytoskeleton, which is triggered by the establishment of cell-cell contacts, appears to prevent the uptake of those proteins and thus contribute to their metabolic stabilisation'. Since then numerous examples have been found in polarised epithelial cells, in motile cells and during cell-spreading, where the role of actin-cytoskeleton appears to be counter-intuitive (Woodring et al., J Cell Sci 116:2613-2626, 2003). For example, c-Abl seems to have a negative role in cell migration but positively contributes to filopodia formation, membrane ruffling and neurite extension (Woodring et al, 2003 supra). Likewise, the Ena/NASP proteins decrease cell motility yet positively regulate actin polymerisation (Krause et al, J Cell Sci 115:4721-4726, 2002). This suggests that intracellular signals and the perceived function of motor proteins may not always be reliable indicators of cell shape changes, particularly in cells where receptors perform different tasks simultaneously.

4. Self-incompatibility and directed tip-growth

One of the first cytological signs of S-protein inhibition in incompatible pollen is the inactivation of actin cables at the periphery and growth arrest (Staiger and Franklin-Tong, J Exp Bot 54:103-113, 2003). Paradoxically, an immediate subsequent effect is an intense actin-staining at the tip due to the branched actin mesh in the current centre of the tube moving into a distal direction (Geitmann et al, Plant Cell 12:1239-1252, 2000). This is difficult to understand in the context of current theories of signal-mediated actin- depolymerisation.

Another problem is how cellular protrusions extending by tip growth, such as filopodia, nerve axons and plant rootlets, are directed by external clues. This problem has been difficult to unravel using current models of signalling and response mechanisms.

5. Immune recognition and histocompatibility

Current models of receptor-mediated cellular reactions are based on two-step processes, where individual ligand-receptor interactions constitute specific signals, which precede cellular responses, such as cell-shape changes carried out by cytoplasmic motor proteins. One of the problems with this model is that signal-specificities are based on the assumption that distinct protein-protein interactions, usually a ligand interacting with a corresponding receptor, provide the specificity of the observed responses. This paradigm, which is based on antibodies and pattern recognition molecules interacting with specific receptors (Janeway, Approaching the asymptode? Evolution and revolution in immunology (Plainview), 1989; Medzhitov and Janeway, Science 296:298-300, 2002), cellular 'danger' signals (Matzinger, Science 296:301-304, 2002) or diagnostic proteolytic 'surveillance' peptides (Johnson et al, Trends in Immunology 24:19-24, 2003), are limited in their capacity to discriminate, because genome projects have revealed that not enough protein variation is available to achieve the observed power of discrimination (Klein, Scandinavian Journal of Immunology 29:499-505, 1989), (Klein, Immunogenetics 50:116- 123, 1999). For example, the recognition of abiotic substances, which have not previously been encountered by an organism, is difficult to explain with a limited number of receptors. Moreover, long-standing attempts to integrate developmental, sensory, and innate immune recognition processes in single and multi-cellular organisms, are difficult to implement with individual ligand-receptor recognition systems associated with highly specific tasks (Klein, 1999 supra). Finally and most importantly, the basic mechanism of recognition of 'self with a limited set of innate recognition molecules is a major challenge. For example, some angiosperm plants are able to recognise and mount self-incompatibility (SI) reactions when pollen from the same plant interact with flower tissues, but form pollen tubes when pollen from different individuals of the same species are present. The genes involved in the Sl-reaction are clustered at the SI-locus and transmitted to the next generation as a single Mendelian unit. Likewise, the transplantation of tissues among mammals is only possible between two genetically related individuals. Again the group of genes responsible for recognition of 'self are clustered at the MHC-locus and transmitted as a genetic unit. Like in Si-plants the genetic diversity among MHC-alleles is relatively high within a population, but the actual receptor proteins available for two interacting cells are based to two sets of parental MHC-genes each. It is obvious that the degree of discrimination achieved in these systems is difficult to reconcile with receptor-specific recognition models on a one-to-one basis. Accordingly, there is a need to elucidate the functional nature of the cellular uptake mechanism in order to provide a platform for the rational design of means of modulating this system. In work leading up to the present invention, it has been determined that the cellular uptake of a molecule is effected by an extracellular protein assembly, termed the "leverage mediated uptake mechanism" which is comprised of multiple proteins with the potential to achieve highly specific outcomes based on combinatorial diversity.

In this model, soluble adhesion molecules (SAMs), such as tetrameric lectins, cross-link membrane-anchored molecules (MARMs) aroimd lipoproteins or bulky hinge molecules (HMs) leveraging MARMs to cause a local inversion of the membrane curvature and formation of an internal endosome or phagosome (Figure 4). This "leverage-mediated" (LM) uptake mechanism involves lateral clustering of MARMs by SAMs, generating the configurational energy which can drive the reaction towards intemalization of the complex. Thus the model describes a novel receptor-assembly that can generate mechanical energy causing an inverse curvature of the membrane and receptor- intemalisation upstream of signalling. The complex acts like a cellular engine that drives extracellular processes using configurational energy, instead of chemical energy (GTP or ATP) used by cytoplasmic motor proteins). The elucidation of this mechanism now facilitates the development of therapeutic, prophylactic and diagnostic methods directed to any number of objectives including, but not limited to, the rational design of means of manipulating cellular signalling processes and cell-shape changes, means of modulating intracellular delivery of a molecule such as a drug, means of disease reduction, disease protection and toxin resistance management strategies in both animals and plants. SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

One aspect of the present invention is directed to a method of regulating the uptake of an extracellular molecule by a cell, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

Another aspect of the present invention more particularly provides a method of regulating the uptake of a soluble adhesion molecule by a cell said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

In yet another aspect there is provided a method of regulating the uptake of an extracellular molecule by a cell said method comprising modulating one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(ii) the interaction of said soluble adhesion molecule with one or more hinge molecules;

(iii) the localisation of the hinge molecule or the complex of (ii) proximally to both the surface membrane of said cell and one or more membrane anchored molecules;

(iv) the interaction of said membrane-anchored molecule with said soluble adhesion molecule; and (v) the lateral clustering of said membrane anchored molecules relative to said hinge molecules.

wherein the subject cell's leverage-mediated uptake mechanism is modulated.

Still another aspect of the present invention provides a method of regulating the uptake of an exfracellular molecule by a cell in a subject, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

In still another aspect there is provided a method of regulating the uptake of an extracellular molecule by a cell in the subject said method comprising modulating one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(iii) the localisation of the hinge molecule or the complex of (ii) proximally to both a surface membrane of said cell and one or more membrane anchored molecules;

(iv) the interaction of said membrane anchored molecule with said soluble adhesion molecule; and

(v) the lateral clustering of said membrane anchored molecules relative to said hinge molecules.

wherein the subject cell's leverage mediated uptake mechanism is modulated.

Yet another aspect of the present invention is directed to a method for regulating cellular signalling, which cellular signalling is induced and/or otherwise regulated by the assembly of an extracellular complex, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

In yet another aspect there is provided a method for regulating cellular signalling, which cellular signalling is induced and/or otherwise regulated by the assembly of an extracellular complex, said method comprising modulating the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(iii) the localisation of the hinge molecule or the complex of (i) proximally to both the surface membrane of said cell and one or more membrane anchored molecules;

(v) the lateral clustering of said membrane anchored molecules relatively to said hinge molecules;

wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

Still yet another aspect of the present invention is directed to a method for regulating cellular signalling in a subject, which cellular signalling is induced and/or otherwise regulated by the assembly of an extracellular complex, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

Still another aspect of the present invention is directed to a method for the intracellular delivery of a molecule to a cell, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

In a further aspect there is provided a method for the intracellular delivery of a molecule to a cell, said method comprising modulating the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule; (ii) the interaction of said soluble adhesion molecule with one or more hinge molecules;

In another further aspect there is provided a method for the intracellular delivery of a molecule to a cell in a subject, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(iv) the interaction of said membrane anchored molecule with said soluble adhesion molecule; and (v) the lateral clustering of said membrane anchored molecules relatively to said hinge molecules;

Still another further aspect of the present invention is directed to a method for downregulating the microbial infection of a cell, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements downregulates the functioning of the leverage mediated uptake mechanism.

In yet another further aspect there is provided a method for downregulating the microbial infection of a cell, said method, comprising modulating the function of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism. In another aspect there is provided a method for downregulating the microbial infection of a cell in a subject said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

Another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a conditioning subject, which condition is characterised by the aberrant, unwanted or otherwise inappropriate cellular uptake of an extracellular molecule, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

In yet another aspect there is provided a method for the treatment and/or prophylaxis of a condition in the subject, which condition is characterised by the aberrant, unwanted or otherwise inappropriate cellular uptake of an extracellular molecule, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

wherein the subject cell's leverage mediated uptake mechanism is modulated.

Another aspect of the present invention contemplates the use of an agent, as hereinbefore defined, in the manufacture of a medicament for the treatment of a condition in a subject, which condition is characterised by aberrant, unwanted or otherwise inappropriate uptake of a molecule, wherein said agent modulates the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable earners and/or diluents. Said agents are referred to as the active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the frequency of macropinocytosis in Drosophila mbn-2 cells induced by oligomeric lectins. After addition of lectins at various concentrations, the relative number of cells with macropinocytosis was monitored over different time points as indicated. Tetrameric lectins, such as Vicia vilosa lectin (WL, GalNA-specific) and peanut agglutinin (PNA, Gal, GalNAc-specific) showed significant higher rates of macropinocytosis, whereas monomeric winged bean lectin (WB, GalNAc- specific) and hexameric Helix pomatia lectin (HPL, GalNAc-specific) were less effective.

Figure 2 is a graphical representation of macropinocytosis induction in mbn2-cells over time. Data from figure 2 shown in relative percentages over four time points.

Figure 3 is a graphical representation of macropinocytosis induction in mbn2-cel\s by oligomeric lectins is reduced in the presence of monomeric WBL. WB was able to compete for cell surface binding sites when applied before or together with tetrameric lectins. In this diagram the induction of macropinocytosis was monitored in the presence of both monomeric and tetrameric lectins.

Figure 4 is an illustration of an extracellular driving force based on a leverage-mediated pinocytosis mechanisms and its comparison to zipper-mediated phagocytosis of large objects. A) Lectin-mediated phagocytosis of an object containing lectin-specific surface glycodeterminants, which are connected by lectins (orange symbols) to lectin-binding glycoprotein receptors on the cell surface. B) The same principle can be applied at the molecular level, where bending of receptors around a glycoprotein resembles zipper- mechanisms, causing an inverse curvature of the membrane. The basis for the molecular driving force is a leverage of receptors around bulky molecules (LM-mechanism). The LM-mechanism can be applied to any dimeric or oligomeric adhesion molecules that cross-link receptors across molecules that act like a 'hinge'. Figure 5 is an image of low-density gradient centrifugation of activated CrylAc in the presence of cell-free hemolymph. Gut juice-activated CrylAc was mixed with a lipophorin fraction from Galleria mellonella hemolymph and separated by low-density gradient- centrifugation. Densities were measured in a control gradient by weighting 1ml volumes from each fraction. Note the enrichment of high molecular weight CrylAc complex in fraction 11-15 in contrast to the monomeric toxin, which is homogeneously spread over the gradient.

Figure 6 is a schematic representation of hemomucin with stmctural and functional features of MARMs. The putative lipophorin-binding domain of hemomucin (Theopold and Schmidt, J Insect Physiol, 43:661-61 A, 1997), which has some similarity to the plant enzyme strictosidine synthase (Fabbri et al., Biochemical & Biophysical Research Communications 271:191-196, 2000) shows a high proportion of hydrophobic amino acids. Figure 7 is an image of a Western blot of tissue-specific proteins extracts incubated with antibodies against hemomucin and PNA. The 100 kDa protein is visible in all tissues except in salivary glands where the size is different, and ovaries where an additional band is visible. PNA-staining only shows the modified band in the ovary extracts.

Figure 8 is an image demonstrating that Bt-toxin CrylA binds to hemomucin. Western blots containing proteins extracts from Schneider cells were incubated with HPL (H) and activated CrylA in two concentrations (Tl) and T2) and bound toxin identified with antibodies against the toxin. As a control blots were incubated with anti-toxin antibodies only (C). Note the specific staining of the hemomucin band in the two blots incubated with toxin.

Figure 9 is an image of HPL-staining of gut cells. Whole gut tissues were incubated with FITC-conjugated HPL and inspected under indirect UN-light using confocal microscopy. Transverse optical section showing epithelial cells (left) with basement membrane (heavily stained layer in the upper part of the picture) and gut lumen (lower part of the picture). Gracing optical section of gut tissue (right) with columnar cells showing intensive vesicular staining.

Figure 10 is an image of protoxin activation with gut juice extracts and trypsin. Protoxin purified from B. thuringiensis subsp. Kurstaki HD73 was solubilized in a solution containing 30 mM Na₂Co₃ and 1% mercaptoethanol at pH 9.5 ( ronson et al. Appl Env. Microbiol 65:2503-2507, 1999) and digested with trypsin or gut juice extracts.

Figure 11 Melanisation reactions in H. armigera strains that are resistant and susceptible to low levels of Bt-toxin. A) Cell-free hemolymph from 3 instar caterpillars was diluted in PBS -solution and relative absorbance measured over 30 min. Note a slight reduction of relative absorbance in hemolymph from the resistant strain due to coagulation reactions. B) Gut extracts without gut contents were measured in the presence of lOOmM 3,4- dihydroxyphenylalanine (DOPA). Although the shape of absorbance curves changed slightly between hemolymph from individual caterpillars, the differences between Bt- resistant and susceptible caterpillars, the difference in absorbance between the two strains always exceeded 200 units, with hemolymph from susceptible caterpillars showing variability due to possible immune-induction by wounding or infection.

Figure 12 Dynamics of adhesion and LM-uptake reactions. Receptors able to interact with oligomeric adhesion molecules can either attach to external binding sites or engage in lateral cross-linking leading to internalisation. Given that receptors are already aligned on two-dimensional membranes, whereas external binding sites are usually distributed over three dimensions, receptor-internalisation is favoured over adhesion. Receptors can be stabilised on the cell surface by anchoring to the actin-cytoskeleton. Inactivation of actin- cytoskeleton by cytochalasin D prevents phagocytosis (Nilcinskas et al, Journal of Insect Physiology 43 : 1149-1159, 1997) and causes cells to detach (Glatz et al, Journal of Insect Physiology, 50:955-963, 2004), which may be due to the removal of actin-anchorage of receptors on the cell surface. We suggest that lectin mediates receptor-intemalisation, whereas actin-cytoskeleton stabilise receptors on the cell surface allowing contacts with external glycodeterminants. Figure 13 is a schematic illustration of putative leverage-mediated (LM) mechanisms.

A) Protein assemblies, consisting of lipoproteins and multimeric lectins, interacting with membrane-anchored molecules, such as the Drosophila immune receptor hemomucin

(Theopold et al, Journal of Biological Chemistry 271(22):12708-15, 1996). A putative complex consisting of hemomucin binding to lipophorin at the strychdosidine synthase domain (Theopold et al, Journal of Insect Physiology 43:667-674, 1997; Li et al, Insect Biochemistry and Molecular Biology 32(8):919-928, 2002) and hemomucin binding to lectin at the mucin-domain (Theopold et al, 1996, supra) can rearrange the receptor and create configurational energy. The oligomeric lectin may interact with lipophorin before forming a complex with the receptor. Alternatively, lipophorin may interact with the receptor before lectin binding.

B) The complex can act as a template around which hemomucin molecules are bent, creating a concentric ring of twisted receptors, which cause an inverse curvature of the membrane. Twisting of cell surface receptors may also destabilise cytoplasmic linkages between intracellular receptor-domains and the actin cytoskeleton. Protein interactions are depicted schematically to emphasize possible leverage-mediated processes.

C) Putative globular stmctures as part of lectin-mediated coagulation reactions in insects. Oligomeric lectins interacting with hemolymph components to form globular stmctures, resemble coagulation products with pattern recognition molecules, which may include lipophorin, phenoloxidase (Li et al, 2002. supra), active and inactive serine proteases (Yu et al, Insect Biochemistry and Molecular Biology 33(2): 197 '-208, 2003).

Figure 14 Relationship between LM-uptake, coagulation reactions and phagocytosis.

A) The formation of coagulation globules in invertebrates is dependent on soluble pro- coagulant components interacting with soluble adhesion molecules. A putative anangement of larger pro-coagulant molecules, which may include lipophorin and phenoloxidase (Li et al, 2002, supra), with oligomeric adhesion molecules, forming spherical shapes (see also Fig. 13C). The interaction of soluble adhesion molecules with glycoprotein receptors on the cell surface leading to uptake reactions is conceptually related to the sphere-shaped configuration causing an inverse curvature of the membrane.

B) Possible LM-based cell-cell interactions and phagocytosis. Cell adhesion and membrane sculpturing at the site of contact between two cells. LM-uptake and cell adhesive attachments providing a balance of forces, where two cells form a straight line at the contact site. The balance is affected by membrane properties, such as phosphatidylserine distribution on the membrane bilayers, or receptor recycling and receptor anchorage to actin-cytoskeleton. If LM-uptake reactions between the two cells are out of balance, the interaction leads to phagocytosis of foreign or apoptotic cells (Zingg et al, LUBLMB Life 49(5):397-403, 2000). Local imbalances at the site of cell-cell interactions also play a role in development (Greco et al, Cell 2001; 106:633-645; Gibson et al, Cell 2000; 103(2):343-350) and wound healing (Jacinto et al, Nature Cell Biology 2001; 3:117-123; Theopold et al, Trends in Immunology 25:289-294, 2004).

Figure 15 Constitutive macropinocytosis and lectin-mediated clustering in insect hemocytes from Helicoverpa armigera. A) Live hemocytes are spread on a glass surface and incubated with a buffer solution containing FITC-conjugated GalNAc-specific lectin from Helix pomatia (HPL) and inspected under indirect UN-light. Clustering of fluorescence stain is visible after a few minutes (anows), before detachment is observed. After few hours the fluorescence is visible inside large endosomal vesicles (not shown). Next to the clusters are non-stained macropinocytosis vesicles (anowheads), formed by actin-mediated membrane 'ruffling' as part of a constitutive macropinocytosis. (Johannes et al, Traffic 2002; 3:443-451) B) Conesponding phase contrast picture. C) Live hemocytes stained with GalNac-specific FITC-conjugated HPL. Under these conditions hemocytes show surface staining under indirect UN-light. D) Live hemocytes after staining with FITC-conjugated Gal/GalΝ Ac-specific peanut agglutinin (PΝA) and inspected under indirect UN-light. Under these conditions the hemocyte surface is less stained but shows small intensely stained globules from discharged hemocyte granules. E) Fat body extract from H. armigera caterpillars mixed with FITC-conjugated GalNAc-specific lectin (HPL). Although fibres are stained with GalNac-specific lectins, they also form in the absence of the lectin. F) Fat body extract from H. armigera caterpillars mixed with FITC-conjugated Gal-specific lectin (PNA). Coagulation reactions in arthropods appear to produce fibrous structures in the absence of and round spherical stmctures (globules) in the presence of external or hemolymph-specific (Castro et al, Insect Biochemistry 17:513-523, 1987) oligomeric Gal-specific lectins. Gal-containing glycoproteins proteins are absent in hemolymph but are secreted into extra-hemolymph stmctures, such as egg-shells (Figure 7) or perittophic membranes.

Figure 16 Pore-forming toxins are oligomeric adhesion molecules with anti-bacterial peptides attached. Examples are endotoxins from the soil bacterium B. thuringiensis, where lectin domains are attached to amphipathic peptide domains with anti-bacterial activity (Szabo et al, International Journal of Peptide & Protein Research 1993; 42(6):527-532; Gerber and Shai, 2000, supra). LM-uptake mechanisms provide the configurational energy for insertion of oligomeric channels into cholesterol-containing membranes, by pushing the pore-forming peptide complex into the membrane bilayer. In the process, lipophilic domains may be involved in opening a membrane gap to the cytoplasm allowing ions and water to pass from the endosome into the cytoplasm, causing osmofragility and pΗ-changes.

Figure 17 Mortality rate in baculovirus treated caterpillars from Bt-resistant and susceptible strains. H. armigera 3^rd instar larvae were fed on artificial food mixed with a suspension of 10⁷/ml of Autographa californica multiple nuclear polyhedrosis vims (_^4cMNPN). H. armigera is semipermissive to _^4cMΝPN. The vims titer, resulting in low mortality rates, was chosen to avoid saturation of coagulation molecules, reflecting resistance against relatively low Bt-toxin levels. Each treatment was repeated three times with at least 20 caterpillars each. The difference in mortality rates was highly significant for each time point. No mortality was observed in non-treated insects. Figure 18 Schematic illustration of putative leverage-mediated mechanisms. A) LM- uptake driven by extracellular uptake complexes are initiated by soluble adhesion molecules. Assemblies, consisting of lipoproteins and multimeric lectins, interact with membrane-anchored molecules, such as hemomucin molecules. B) A putative complex consisting of hemomucin binding to lipophorin at the strychdosidine synthase domain (Li et al, 2002, supra; Theopold and Schmidt, Journal of Insect Physiology, 43:667 -674, 1997) and hemomucin binding to lectin at the mucin- domain (Theopold et al., Journal of Biological Chemistry 271:12708-15, 1996) can create configurational energy. Note that the three-dimensional stmcture of the lectin-lipophorin complex is not known. The schematic drawing is used as an example for the recmitment of receptors to the uptake complex. The complex can act as a template around which hemomucin molecules are bent, creating a concentric ring of twisted receptors, which cause an inverse curvature of the membrane. Twisting of cell surface receptors destabilises cytoplasmic linkages between intracellular receptor-domains and the actin cytoskeleton. LM-reanangements of cytoplasmic molecules are the molecular basis for intracellular signalling processes. Note that signalling is a possible outcome of LM-uptake reactions, whereas it is a precondition for RME. C) Co-localisation of receptors around membrane-spanning ABC-transporters (Wu and Horvitz, Cell 93:951-960, 1998; Zhou et al, Cell 104:43-56, 2001). D) LM-process induced by oligomeric adhesion molecules interacting with co-localised receptors around membrane-spanning molecules. E) Attachment of receptors to cytoplasmic proteins, such as G-proteins (Matsuo et al, Biochemical Journal 315:505-512, 1996) or scaffold proteins (Gil et al, J. Cell Biol. 2003; 162(4):719-730). F) LM-process induced by oligomeric adhesion molecules interacting with receptors anchored to cytoplasmic proteins.

Figure 19 LM-assemblies with adhesive and uptake properties. Receptors with adhesive properties, such as hemomucin, are associated with LM-complexes, which drive the inverse curvature of the membrane (red arrows). To maintain a presence of adhesive receptors on the cell surface the cytoplasmic domain is engaged in nucleation of actin cables, which provide a stabilising counter-force against the LM-uptake reactions. Once the receptor has made contact with external binding sites, the contact can only be broken with the help of LM-assemblies with anti-adhesion properties. Figure 20 Quality control in protein secretion and retrograde transport. The functional properties of putative LM-assemblies in the ER and Golgi essential for vesicle formation and quality control, Proteins that are not engaged in LM-vesicle formation are marked and digested or returned into the cytoplasm. The process of endosome formation, recycling and retrograde transport, involves disassembly of LM-complexes following the destabilization of oligomeric adhesion molecules at low pH- and Calcium concentrations. This allows new LM-complexes to form particularly with the merger of endosomes with other cytoplasmic vesicles. Again proteins that are able to quickly assemble into new LM-complexes are transported and recycled, whereas proteins that are left behind are marked, degraded or transferred across the membrane into the cytoplasm for further processing by the proteasome.

Figure 21 Schematic depiction of Trypanosoma cruzi invasion into mammalian cells, a) T. cruzi approaching a mammalian cell initiating contacts, which cause the parasite to release trans-sialase. b) Trans-sialase activity moves sialic acid residues from mammalian to parasite surface molecules, c) In the presence of lectins, the mucin-like glycoproteins on the mammalian cell are cross-linked, whereas the parasite is protected from lectin binding by sialic acid modifications. Lateral cross-linking and LM-complex formation causes the host cell membrane to bent around the parasite or membrane vesicles that are in the process to exocytose to form a membrane invagination in which the parasite is engulfed.

Figure 22 Morphology and cytoskeleton changes of hemocytes from Pieris rapae spread on a glass surface and subsequently treated with cytochalasin D (cyt D), Helix pomatia lectin (ΗPL) and cyt D and ΗPL combined. After treatment cells were fixed in the presence of non-ionic detergent and stained with FITC-conjugated phalloidin and inspected under confocal microscopy. Note the formation of stress fibres in spread cells, which are absent in treated cells. Both cytochalasin D and ΗPL-freated cells retreated from their attachment sites and formed round or spindle-formed shapes. Whereas cytochalasin D-treated cells accumulated actin at the periphery, ΗPL-treated cells showed irregular staining which was absent from cell extensions. Note that the combined treatment resembled cytochalasin D-treatment, which is in agreement with observations in separate treatments where HPL-effects were delayed compared to cytochalsin D-treatment.

Figure 23 HPL-internalisation in the presence of cytochalasin D. Cytochalasin D was applied to spread P. rapae cells together with TRITc-conjugated HPL and after cells were fixed in the absence of detergents, the cells were incubated with FITc-conjugated HPL to stain the surface and inspected under a confocal microscope. For comparison a group of cells from Fig. 22 is shown with TRITC-labelled uptake in the presence of cytochalasin D (left panel). The left HPL panel shows a single hemocyte where the optical section was through the centre of the cell, which had vesicles predominantly at the cortex but some towards the nucleus. The surface FITC-staining was relatively weak with clusters that co- located with TRITC-staining. The right HPL panel shows a small aggregate of cells, where one of the internal cells was heavily stained and spreading after incubation with HPL. FITC staining was weak but relatively high over the spreading cell, which was also stained on the surface with TRITC.

Figure 24 Lectin-staining on the hemocyte surface of HPL-treated cells. Cytochalasin D and TRITC-conjugated HPL were applied to spread P. rapae hemocytes until cells were spindle-shaped (see Fig. 22). Hemocytes were then treated with paraformaldehyde and stained with FITC-conjugated HPL to visualise lectin-binding receptors on the cell surface. The picture shows a small hemocyte aggregate at the time of cytochalasin D and HPL- treatments, where one or two cells were surrounded by other hemocytes and only exposed to cytochalasin D and HPL after sunounding cells detached. HPL-uptake (TRITC) was visible in the sunounding hemocytes, which had spindle-formed cell-shapes, whereas hemocytes inside the aggregate were labelled on the surface (arrow) and showed some spreading. ?HPL-surface staining (FITC) was reduced due to receptor-intemalisation, except in cells that were sureounded by other cells at the time of treatment.

Figure 25 F-actin and hemocyte spreading after lectin treatment. G. mellonella hemolymph containing hemocytes was isolated in PBS or treated with lectin (HPL) and hemocytes were separated from plasma by repeated washes and allowed to spread on a glass surface. Hemocytes were fixed and actin-cytoskeleton visualized with FITC- conjugated phalloidin. A) Hemocytes from hemolymph isolated in PBS/PTU. Note the evenly spread actin-cytoskeleton with a gap of staining over the nucleus. B) Hemocytes from lectin-treated hemolymph. Note the reduced spreading and the dotted phalloidin- staining over the cytoplasm and around the nucleus.

Figure 26 is a depiction of dynamic interactions between lectin-binding receptors on the cell surface, which can either make contacts to external binding sites (adhesion or phagocytosis), or cluster on the cell surface to internalise (uptake). Since some lectins internalise receptors from the cell surface and in the process appear to depolymerize actin- cytoskeleton, continued lectin-mediated uptake will cause depletion of cell surface receptors (immune suppression), as recycling of membrane-vesicles to the periphery requires actin-fibers.

Figure 27 Lectin-mediated uptake in the hemocyte-like Drosophila mbn-2 cell line in the form of induced macropinocytosis. These cells have hemomucin as the only lectin-binding glycoprotein on the cell surface. Glycodeterminants on the hemomucin receptor can be recognised by specific lectins, such as GalNAc-specific Helix pomatia lectin (HPL) (Theopold et al., Journal of Biological Chemistry; 271(22): 12708-15, 1996), Gal-specific peanut agglutinin (PNA) (Theopold et al., Insect Biochem. Mol Biol. 31:189-197, 2001) and GlcNAc-specific Concanavalin A (ConA). After attachment to the glass surface, cells were treated with medium containing PNA or with medium without lectin. In lectin-treated cells some cells react with enhanced spreading, whereas others detach and round up. The diversity of responses may be due to differences in protein secretion activities within the cell population.

Figure 28 Insertion of pore-forming toxins into the membrane. LM-uptake mechanism involving insertion of oligomeric channels of pore-forming toxins, such as Bt-toxin.

Leverage-mediated uptake reactions may push the toxin complex into the membrane bilayer. In the process, lipophilic toxin domains may be involved in opening a membrane gap to the cytoplasm allowing ions and water to pass from the endosome into the cytoplasm, causing osmofragility.

Figure 29 Mature Bt-toxins form oligomeric complexes. Protoxin activation with gut juice extracts. Protoxin purified from B. thuringiensis subsp. kurstaki HD73 was solubilized in a solution containing 30 mM Na₂CO₃ and 1 % mercaptoethanol at pH 9.6 (Aronson et al., 1999) and digested with gut juice extracts. A) Protoxin and gut juice- extract from the lepidopteran species Pieris rapae were incubated for 30' (1), 1 h (2), 2h (3) and five hours (4) and extracted at 65°C in SDS -containing buffer and analysed by SDS-PAGE. The mature toxin (69 kDa) is predominant initially, but is replaced by a 60 kDa protein. Both proteins appear to form hetero-oligomeric complexes, which form a cluster of nanow bands above the 250 kDa marker band. The relative amounts and distribution of these nanow bands are correlated with the relative composition of the 60 and 69 kDa bands. B) is an image of mature Bt-toxins forming oligomeric complexes. More specifically, this figure depicts a western blot of gut juice-activated protoxin after incubation of one hour (1) and five hours (2). Non-toxic recombinant protein (3) Protoxin (4), Marker (M), Gut juice (GJ), gut juice activated protoxin (as in 1) extracted at 100°C. Extraction at 100°C in SDS-containing buffers eliminated the bands above 250 kDa.

Figure 30 Mechanism of uptake and transduction of toxin components into the cytoplasm. Oligomeric lectins (protective antigen in anthrax) interact with cell surface receptors causing LM-uptake reactions. In this or subsequent endosomal compartments an emerging membrane gap between lipoproteins may facilitate release the toxin into the cytoplasm.

Figure 31 Retrograde protein transport and protein recycling. Endosomal changes in pH or ion-content may inactivate LM-components, such a Ca-dependent lectins and dissociate LM-complexes. After merging with cytoplasmic vesicles, containing different sets of oligomeric adhesion molecules or receptors, new LM-assemblies may create sub-vesicular compartments, which form new vesicles that can recycle to the cell surface or process further by merging with other vesicles or ER. Figure 32 is a schematic representation of receptor dynamics based on LM-mechanisms. Receptor-distributions on the cell surface can be described in mathematical terms using a system of interconnected reactions that are in steady-state conditions when cells are in homeostasis. Changes in conditions that are known to affect receptor-stabilisation or LM- complex formation provide a basis for predictions of cell activities and the in silico simulation of cell behaviour.

Figure 33 (a) is an image of actin-cytoskeleton in Schneider cells stained with FITC- conjugated phalloidin using confocal microscopy. A) Two cells at different stages of spreading on a surface containing Concavalin A immobilised on a poly-Lysine coated glass surface. The left cell is not (yet) spread and may represent the degree of spreading on a poly-Lysine coated surface. The right cell shows extreme spreading, which is only found on lectin-coated surfaces, (b) Two cell at different stages of detachment after addition of soluble Concavalin A to cells spread on immobilised Concavalin A. The lower left cell is in the process of detachment with few actin granules visible. The upper right cell shows strong evidence of actin-depolymerisation with bright actin granules in the cytoplasm.

Figure 34 is a schematic representation of the detachment of single cells from adhesive tissue connections. The balance of forces between two neighbouring cells raises the question of how cells dissociate from each other during tissue remodelling and cellular division. A) Notch-Delta interactions. Proteolytic cleavage by secreted proteases cuts adhesive connections between the neural precursor and sunounding epithelial cells during determination of neuroblasts in Notch-expressing cells. B) Another possible avenue is the secretion of counter-adhesion molecules, which internalize adhesive receptors on the membrane surfaces of opposing cells, leading to local detachment. Note that in this model receptor turn-over at focal adhesion points is affected by counter-adhesion molecules, which promote receptor-internalization rather than attachment to external binding sites. Cells secreting counter-adhesion molecules are therefore able to round up without being taken up by adjacent cells. Figure 35 is a schematic representation of the detachment of single cells from adhesive contacts. A) Single cells undergoing cell division. B) Neuroblast cells after separation from adjacent cells are able to migrate out of the epithelial context using external cues or the polarized secretion of counter adhesive molecules.

Figure 36 is a schematic representation of directional cell mobility. A) Polar secretion of counter-adhesion molecules predicts detachment of neighbouring cells on one side of the cell, while spreading continues on the other side, where more adhesion molecules remain on the cell surface. B) Gradients of binding-site densities on the substrate predicts movement towards higher densities based on an increased likelihood of receptors attachments in the presence of higher binding-site numbers and a concomitant addition of receptors and membrane material to the cell surface.

Figure 37 is a schematic representation depicting membrane protrusion by tip growth is dependent on the dynamics of membrane vesicles. Exocytosis reactions of Golgi-derived membrane vesicles provide new membrane material to the tip, which in turn is internalised by LM-uptake reactions. If exocytosis prevails the tip will grow, but if the rates of LM- uptake and exocytosis are equal the pollen will not form a protmsion. The model predicts the LM-assemblies with the power to overcome actin-stabilisation of surface receptors will prevent tip growth in self-compatible reactions, whereas LM-assemblies with less power allow the maintenance of receptors on the cell surface long enough for contacts to external binding sites to be made and for protmsions to grow. Since receptors are stabilised by adhesion, tip growth is expected to extend towards high binding site densities.

Figure 38 is a schematic representation depicting the membrane-vesicle flow within the cytoplasm. A) The process of endosome formation, recycling and retrograde transport involves disassembly of LM-complexes following the destabilization of oligomeric adhesion molecules at low pH- and Calcium concentrations. This allows new LM- complexes to form, particularly with the merger of endosomes with other cytoplasmic vesicles. Proteins that are able to quickly assemble into new LM-complexes are transported and recycled, whereas proteins that are left behind are marked, degraded or transferred across the membrane into the cytoplasm for further processing by the proteasome. B) The functional properties of putative LM-assemblies in the ER and Golgi essential for vesicle formation are marked and digested or returned into the cytoplasm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the identification of a novel mechanism of cellular uptake termed the "leverage-mediated uptake mechanism". The uptake of exfracellular molecules by this method is driven by an extracellular complex, which is formed, in one example, by oligomeric soluble adhesion molecules aggregating membrane anchored molecules around a hinge molecule. This causes an inverse curvature of the cell membrane and ultimately intemalization of the complex. These determinations have now facilitated, inter alia, the development of improved methods of regulating the cellular intemalization of extracellular soluble molecules.

Accordingly, one aspect of the present invention is directed to a method of regulating the uptake of an extracellular molecule by a cell, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

Reference to "uptake" should be understood as a reference to the interaction with the cell membrane or any molecule anchored to or otherwise associated with the cell membrane (for example, a receptor molecule) of a molecule or molecular complex which is located extracellularly or across the membrane in intercellular vesicles. The subject molecule may be ultimately internalised, partially or fully inserted into the cell's bilipid membrane or it may transiently or permanently remain at the cell surface. The various functional outcomes which may occur or which require said cellular uptake to occur are hereinafter discussed in detail. Accordingly, reference to "molecule" in this regard should be understood as a reference to any proteinaceous or non-proteinaceous molecule or complex thereof.

The present invention is directed to "regulating" the uptake of a molecule. By "regulating" is meant inducing or otherwise agonising (upregulating) or inhibiting or otherwise antagonising (downregulating) the subject uptake. As detailed above, the present invention is predicated on the identification of a previously undescribed uptake mechanism. Accordingly, by "leverage mediated uptake mechanism" is meant a reference to the cellular uptake mechanism, which functions to take up extracellular molecules via the impact of an extracellular driving force. More particularly, it has been determined that molecules which are to be the subject of uptake cross-link membrane anchored molecules around bulky molecule complexes (hinge molecules) thereby leveraging the membrane anchored molecules such as to cause an inverse curvature of the cell membrane and, ultimately, the desired functional outcome, for example, cellular signalling or the formation of an internalised phagosome (this latter outcome being schematically represented in Figure 13C, 14A). The lateral clustering of the membrane-anchored molecules is thought to generate the configurational energy which drives the reaction towards intemalization of the subject molecule. Without limiting the present invention to any one theory or mode of action, although this mechanism was originally observed to function in invertebrate cells, it has now been determined that this mechanism is utilised to facilitate the cellular uptake of an extracellular molecule in both invertebrate, vertebrate species and in all single and multicellular organisms, which have the capacity to, for example, internalise extracellular material by a membrane-invagination process.

Leverage mediated uptake also provides the configurational energy to potentially dislodge MARMs from attachments to cytoplasmic proteins, which provides the mechanistic basis for intracellular signalling. In this model, a signal to the interior of the cell is achieved if the extracellular complex is able to tilt MA?RMs enough to overcome the intracellular anchorage (Figure 13B).

The model also predicts the formation of cages (or coagulation globules) formed by SAMs and HMs under conditions which form multiple complexes in the absence of MARMs, which can either inactivate toxins, preferably oligomeric toxins, (Figure 13C, 29) or be used for drag delivery since globules may be internalised by cells involved in clearance of coagulation products . Accordingly, reference to "subject" hereinafter should be understood to include human, primate, livestock animal (eg. sheep, pig, cow, horse, donkey), laboratory test animal (eg. mouse, rabbit, rat, guinea pig), companion animal (eg. dog, cat), captive wild animal (eg. fox, kangaroo, deer), avies (eg. chicken, geese, duck, emu, ostrich), reptile, fish, insects, any other invertebrate species, plants and all other single and multi-cellular organisms.

Reference to "stmctural or functional elements" of the subject leverage mediated uptake mechanism should be understood as a reference to any one or more stmctural or functional elements which directly or indirectly act to facilitate the functioning of this mechanism. In this regard, reference to "stmctural elements" should be understood as a reference to elements, the physical properties of which facilitate the functioning of this mechanism. Examples of stmctural elements include, but are not limited to:

(i) The extracellular proteinaceous or non-proteinaceous molecule or molecule complex, which is the subject of uptake.

Without limiting the present invention to any one theory or mode of action, the subject molecule is preferably a soluble multimeric adhesion molecule such as a dimer, trimer, or teframer. It is the multimeric nature of this molecule, which is thought to facilitate its interaction with multiple membrane anchored molecules and thereby the lateral clustering of these membrane anchored molecules around one or more proximally located hinge molecules, thereby facilitating the inverse curvature of the cell surface membrane. The subject molecule is preferably a multimeric soluble molecule.

As detailed hereinafter, to the extent that the extracellular molecule of interest is a monomeric molecule, it may be necessary to appropriately modify this molecule such that it exhibits multimeric characteristics which would enable it to initiate the extracellular driving force which induces the leverage mediated uptake mechanism. More detailed discussion in relation to this issue are provided hereinafter. It should be understood that reference to "extracellular molecule" is a reference to the molecule which it is desired to be the subject of cellular uptake, inespective of whether that molecule (in its native or unmodified form) is a monomer or a multimer. However, due to the properties of the multimeric soluble molecule, which properties are required to initiate the leverage mediated uptake mechanism, a multimeric soluble molecule which can initiate this mechanism is hereinafter refened to as a "soluble adhesion molecule". Accordingly, it should be understood that the soluble adhesion molecule may conespond to the native form of an extracellular molecule of interest. Alternatively, the soluble adhesion molecule may conespond to an extracellular molecule (such as a monomeric exfracellular molecule) which has undergone some form of modification in order to render it a soluble adhesion molecule.

The soluble adhesion molecule (which may conespond to a modified or unmodified exfracellular molecule of interest) may be any molecule which can interact with a membrane anchored molecule. Examples of soluble adhesion molecules are provided in Tables 1 and 2. However, it should be understood that the soluble adhesion molecules which may utilise the leverage mediated uptake mechanism are not limited to this list and, as detailed above, may be any soluble adhesion molecule capable of interacting with a membrane anchored molecule to mediate LM- mechanisms. For example, drags, hormones, growth factors, antigens, modulators of intracellular signalling, immune regulators and pore forming toxins. Both proteinaceous and non-proteinaceous molecules (such as nucleic acid molecules and chemical compounds) can function as soluble adhesion molecules. Accordingly, the nature of the soluble adhesion molecule is limited only by the existence of a membrane anchored molecule which will interact sufficiently to induce the onset of the leverage mediated uptake mechanism.

(ii) The membrane anchored molecule. Reference to "membrane anchored molecule" should be understood as a reference to a molecule which, irrespective of its primary function, can also function in a receptor-like capacity in that it can associate with a soluble adhesion molecule (in either a specific or non-specific manner) such that clustering around one or more proximally located hinge molecules is facilitated and thereby the inverse curvature of the membrane to which the membrane anchored molecule is attached occurs. Accordingly, it should be understood that the subject membrane anchored molecule is not necessarily an antigen specific receptor, such as a T-cell receptor or immunoglobulin molecule, for example, but can function to interact and associate with one or more types or classes of soluble adhesion molecules. In addition to exemplifying soluble adhesion molecules, Tables 1 and 2 also exemplify membrane anchored molecules which are thought to sufficiently interact with these soluble adhesion molecules to induce the onset of the leverage mediated uptake mechanism and thereby mediate cellular uptake of the soluble adhesion molecules. It should be understood, however, that the scope of potential soluble adhesion molecules and membrane anchored molecules is in no way limited to the molecules detailed in Tables 1 and 2, which are merely intended to provide exemplification in this regard.

Rather, given a soluble adhesion molecule of interest, it would be a matter of routine procedure for the person of skill in the art to determine the nature of an appropriate membrane anchored molecule based on either the cunently known physical and functional properties of soluble adhesion molecules and membrane anchored molecules, in general, or via the performance of routine assays, such as high throughput binding assays, to screen for same. For example, one criteria for specific interactions of LM-components is the functionality of the complex to perform a cellular function, such as cell-shape changes and signalling.

An important property of MARMs is its integration into the lipid bilayer of the cellular membrane. Without limiting the present invention to any one theory or mode of action, two types of attachments are known: One where the protein is covalently linked to a lipid moiety, which is inserted into the lipid bilayer (GPI- anchored protein). Another attachment is provided by the insertion of part of the protein into the bilayer, which can comprise an intracellular protein domain separated from the exfracellular domain by a fransmembrane domain. The intracellular domain can be attached to cytoplasmic proteins, such as actin cytoskeleton, other scaffolds or cytoplasmic proteins, such as GTPases. IVIARMs with intracellular protein domains will only be tilted from their position if extracellular forces are strong enough to overcome the intracellular anchorage by cytoplasmic proteins. It should also be understood that the function of MARMs can also be represented by glycolipids.

(iii) The hinge molecule. Reference to "hinge molecule" should be understood as a reference to any molecule which exhibits physical properties which enable it to associate with soluble adhesion molecules and membrane anchored molecules such that upon interaction of the soluble adhesion molecule with the membrane anchored molecule, an inverse curvature of the membrane is induced. Without limiting the present invention in any way, the interaction of the soluble adhesion molecule and the membrane anchored molecule results in clustering of the membrane anchored molecules around one or more molecules which are located membrane-proximally to the binding site of the oligomeric adhesion molecule and therefore leads to leverage of the membrane anchored molecule over the hinge molecule due to the relatively larger size of the hinge molecule around which the membrane anchored molecules are clustered, relative to the size of the soluble adhesion molecule to which they bind. Examples of molecules which can act as hinge molecules include, but are not limited to, insect lipophorin-like protein (such as apolipophorin), modified apolipophorin, hexamerin-like glycoproteins, lipocalins, pentraxins or related gene products. Conesponding human proteins include, for example, apolipoprotein³¹⁰⁰, apolipoprotein E, macroglobulin and other such molecules.

It should be understood that the exfracellular, soluble adhesion molecules, membrane anchored molecules and hinge molecules of the present invention may be either proteinaceous or non-proteinaceous molecules. A proteinaceous molecule may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening. Said proteinaceous molecule may be a peptide, polypeptide or protein or parts thereof. The protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. The protein may also comprise a subunit of a larger molecule. For example, the subject hinge molecule may be a lipoprotein such as a lipophorin, consisting of proteins and lipids. Said non-proteinaceous molecule may be also derived from natural sources, such as for example natural product screening, or may be chemically or otherwise synthesized. It should also be understood that the hinge molecule may be of any suitable shape. For example, it may be conical in shape, as shown for some coagulation proteins (Hall et al Proc. Natl. Acad. Sci. (USA) 96:1965-1970, 1999), which not only facilitates globule formation as part of coagulation reactions (Figure 13C), but also the leverage mediated process (Figure 13B).

Reference to "functional elements" of the leverage mediated uptake mechanism should be understood as a reference to any one or more of the elements, the mechanism of action of which facilitates the functioning or specific modulation of this mechanism. Examples of functional elements include, but are not limited to:

(i) interaction of the soluble adhesion molecule with one or more of the hinge molecules;

(ii) interaction of one or more hinge molecules or soluble adhesion molecule-hinge molecule complexes with the cell surface membrane and/or membrane anchored molecule;

(iii) interaction of the membrane anchored molecule with the soluble adhesion molecule;

(iv) clustering of the membrane anchored molecule around the hinge molecule, and possibly binding thereto, pursuant to membrane anchored molecule-soluble adhesion molecule interaction and leverage thereof over the hinge molecule such that inverse curvature of the cell membrane is facilitated.

(v) intercalation of biologically active peptides inside the LM-complex, creating new functional LM-properties either by mediating pore-formation (e.g. melittin) or altering the configurational energy of the complex, which affects cellular properties (e.g. vaso-active peptide).

(vi) interaction of any of the LM-complex molecules vith chaperones, which can modify the stmctural features of the proteins.

Reference to "interaction" and/or "association" in this regard should be understood as a reference to any form of interaction and/or association, whether or not it involves the formation of a formal molecular bonding mechanism. To the extent that a bonding mechanism is involved, such as would occur in terms of the interaction of a soluble adhesion molecule with one or more membrane anchored molecules, bonding may be covalent or non-covalent. In relation to non-covalent bonding mechanisms, there may occur ionic bonds, hydrogen bonds, electrostatic bonds or interaction by virtue of van der Waals forces. In terms of an interaction which does not involve the formation of an actual bond, the present invention encompasses means of association which result in the proximal location of molecules such that one or more of the functional objectives of the present invention are met. For example, the positioning of one or more hinge molecules proximally to the membrane anchored molecule and cell surface membrane is an example of an "association" or "interaction" which does not necessarily involve the formation of formal bonds. Without limiting the present invention in any way, such an association may be facilitated by the minimal occunence of repulsive forces and/or steric hindrance. However, in some circumstances the membrane anchored molecule will form an interactive bonding mechanism with a hinge molecule.

In accordance with this understanding, the present invention more particularly provides a method of regulating the uptake of a soluble adhesion molecule by a cell said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

The present invention is directed to regulation of the leverage mediated uptake mechanism based on modulating one or more stmctural or functional elements of this mechanism. In this regard, reference to "regulation" has been hereinbefore defined and should be understood as a reference to the structural and functional assembly and all forms of upregulating or downregulating the leverage mediated uptake mechanism in relation to a given exfracellular molecule of interest. "Modulation" of any one or more stmctural or functional elements should be understood as a reference to altering the physical features of a stmctural element or upregulating, downregulating or otherwise modulating the actions of a functional element. Examples, in this regard, include but are not limited to:

(i) Modifying a monomeric extracellular molecule of interest such that it effectively functions as a multimeric soluble adhesion molecule, thereby facilitating its uptake via the leverage mediated uptake mechanism. This is an important process in all known biological systems where SAMs and HMs are regulated from "within" by proteolytic cascades. A list of possible SAMs regulated from within is given in Table 3. Methods of achieving such an objective would be well known to the person of skill in the art. For example, it may be desirable to couple a monomeric extracellular molecule of interest to a multimeric lectin, thereby effectively utilising the lectin as a earner, such that cells expressing membrane anchored molecules which will interact with the lectin would thereby also effectively take up the monomeric molecules interests. Alternatively, it may be desirable to induce complexing multiple monomeric extracellular molecules of interest such that they effectively form a multimeric complex which can function as a soluble adhesion molecule.

Alternatively, large objects (such as bacteria or abiotic particles), which may not have sufficient adhesive sites to support a "zipper-mediated" uptake reaction (and therefore inverse membrane curvature in a tight fitting phagosome) over one or more hinge molecules, may be coupled to a soluble adhesion molecule (such as lectin) in order to facilitate the onset of the leverage mediated uptake mechanism, which will then engulf the soluble adhesion complex and with it the bacterium in a loosely attached phagosome.

(ii) Further to (i), in order to facilitate the uptake of an extracellular molecule or object of interest by a specific cell type, the person of skill in the art may elect to couple the subject molecule to a soluble adhesion molecule which is of a type known to interact with membrane anchored molecules present on the surface of a cell of interest. The person of skill in the art is thereby provided with an opportunity to design highly specific and directed cell delivery systems.

(iii) Modulation of the interaction of a soluble adhesion molecule with one or more hinge molecules. Specifically, either upregulating or downregulating such interaction can lead, respectively, to the up or down-regulation of the functioning of the leverage mediated uptake mechanism. For example, inhibiting the interaction of a soluble adhesion molecule with a hinge molecule may be of particular value where the subject soluble adhesion molecule is a toxin which would, upon cellular uptake, for example, lead to lysis of the cell. Conversely, facilitating the interaction of a soluble adhesion molecule, such as a dmg, with a hinge molecule "which will also interact with the membrane anchored molecule to which the soluble adhesion molecule will cross-link provides a means of inducing clustering and leverage around the hinge molecule such that inverse curvature of the membrane is induced and the extracellular driving force of the leverage mediated uptake mechanism is thereby initiated.

In this regard, it should be understood that the soluble adhesion molecule and the hinge molecule may bind to one another prior to their interaction, as a complex, with the membrane anchored molecule and the cell surface, as occurs, for example, for an immune suppressor being taken up by hemocytes in conjunction with lipophorin (Asgari and Schmidt, Insect Biochem Mol. Biol. 32:497-504). Alternatively, the interaction of the soluble adhesion molecule and the hinge molecule may occur after the hinge molecule has become positioned sufficiently proximally to the cell membrane and membrane anchored molecules such that membrane anchored molecule clustering and leveraging over the hinge molecule is facilitated upon binding of the soluble adhesion molecule to the membrane anchored molecule.

(iv) Modulating the interaction of a membrane anchored molecule with a soluble adhesion molecule. Upregulating and/or downregulating this mechanism provides a means for, respectively, upregulating or downregulating the onset of the leverage mediated uptake mechanism and, therefore, the intemalisation of the subject soluble adhesion molecule.

(v) Modulation of the structure of the hinge molecule itself such that its capacity to interact with soluble adhesion molecules, membrane anchored molecules and/or the cell surface membrane are modulated.

(vi) Modulation of LM-mechanisms by biologically active peptides, which can intercalate with other LM-components to mediate pore-formation or modify LM- functions.

Accordingly, in a most prefened embodiment there is provided a method of regulating the uptake of an extracellular molecule by a cell said method comprising modulating one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(iv) the interaction of said membrane anchored molecule with said soluble adhesion molecule;

(v) the lateral clustering of said membrane anchored molecules relative to said hinge molecules;

(vi) the intercalation of biologically active peptides;

(vii) structural modification of LM-proteins by chaperones.

wherein the subject cell's leverage mediated uptake mechanism is modulated.

The extracellular molecule, soluble adhesion molecule, membrane anchored molecule and/or the hinge molecule of the present invention should also be understood to encompass derivatives, homologues, analogues, mimetics and functional equivalents of these molecules.

This aspect of the present invention is essentially directed to a method of upregulating, downregulating or otherwise modulating the uptake of an extracellular molecule by a cell. In this regard, specific reference to modulating the "functioning of said molecule as a soluble adhesion molecule" should be understood as a reference to modulating the physical characteristics of a molecule such that its capacity to interact with one or more hinge molecules and cross link membrane anchored molecules in order to induce inverse curvature of the cell membrane is up-regulated or down-regulated. As detailed hereinbefore, such modulation includes, but is not limited to, coupling the molecule of interest to a canier molecule (such as a multimeric lectin) which can function as a soluble adhesion molecule, thereby leading to uptake of the molecule of interest by virtue of its complexing with the canier molecule. Accordingly, it should be understood that the complexing of an extracellular molecule of interest with a soluble adhesion molecule, such that the molecule of interest can be taken up by a cell via the leverage mediated uptake mechanism, is thereby an example of modulating the functioning of the exfracellular molecule of interest such that it effectively functions as a soluble adhesion molecule in its complexed form with the carrier. Alternatively, the subject molecule may be coupled with agents which inhibit its binding to such a canier molecule, in order to prevent it functioning as a soluble adhesion molecule and thereby prevent its uptake.

In terms of the modulation of the "localisation" of the hinge molecule proximally to both the surface membrane of the cell and one or more membrane anchored molecules, reference to "proximal localisation" should be understood as a reference to said hinge molecule or soluble adhesion molecule-hinge molecule complex interacting with the cell surface membrane at a position which facilitates clustering of membrane anchored molecules around the hinge molecule and their leverage over the hinge molecule subsequently to their interaction with the soluble adhesion molecule. In this regard, it should be understood that the hinge molecule may be located such that it interacts with both one or more membrane anchored molecules and the cell surface membrane prior to interaction of the membrane anchored molecule with the soluble adhesion molecule. Alternatively, the hinge molecule may interact initially with the cell membrane and form an interaction with one or more membrane anchored molecules only subsequently to the interaction of the soluble adhesion molecule with the membrane anchored molecule, wherein membrane anchored molecule clustering around the hinge molecule is induced. As detailed hereinbefore, it should also be understood that the hinge molecule may form interactive bonds with the membrane anchored molecule or it may not. For example, it may be the creation of interactive bonds between the soluble adhesion molecule and the membrane anchored molecules which, in the absence of any significant repulsive forces or steric hindrance, acts to maintain the positioning of the hinge molecule relative to the membrane anchored molecules and cell surface such that membrane anchored molecule leverage can be achieved.

Reference to "lateral clustering" of membrane anchored molecules should be understood as a reference to the positional shifting of one or more membrane anchored molecules such that they bind to the soluble adhesion molecule which is complexed to one or more hinge molecules. In this regard, it is well known that the cell surface membrane is a "fluid" bilayer lipid membrane, the structure of which facilitates the lateral movement of molecules anchored in the membrane. In one example, some membrane anchored molecules are maintained on the cell surface on membrane protmsions ('microspikes' and 'raffling'), which are supported by actin fibers. In this case soluble adhesion molecules sometimes have to overcome actin-anchorage of receptors to cluster membrane anchored molecules on the cell surface. Therefore the regulation of LM-complexes can be performed by regulating the anchorage of the receptors involved in LM-complexes. For example, increasing the stability of receptors on the cell surface may be achieved by proteins that link receptors to cytoplasmic scaffolds or increase actin-cable formation by 'formin' or 'spire' domains. This will increase phagocytosis and angiogenesis. Conversely decreasing receptor-stability on the cell surface by depolymerising actin cytoskeleton will increase LM-complexes and receptor-intemalisation. This will detach cell and prevent adhesive interactions.

Means of modulating any one or more structural or functional elements of the leverage mediated uptake mechanism, such that the uptake of an exfracellular molecule is regulated, would be well known to those of skill in the art and include, but are not limited to:

(i) Modulating the physical characteristics of an exfracellular molecule of interest, hinge molecule or soluble adhesion molecule as hereinbefore described. This can be achieved, for example, by modulating the characteristics of a molecule prior to its introduction to a subject or via the administration of an agent which interacts with the molecule of interest in order to form a complex which effectively modulates its physical characteristics. For example, such an agent may act to either induce or facilitate the functioning of the molecule in issue (such as by introducing a multimeric molecule which complexes with an exfracellular molecule in order to provide the extracellular molecule with the necessary multimeric physical characteristics which are required by a soluble adhesion molecule). For example, dimeric immune suppressors may interact with monomeric lipophorin to produce multimeric lipophorin molecules which can interact with hemocytes to be taken up. Likewise, some chemicals, such as pheromones, or pH-conditions may mediate dimerisation of lipocalins, which are then able to interact with receptors. Alternatively, the multimeric molecule which is introduced may be one which exhibits certain unique characteristics such that it provides for the directed uptake of the molecule to which it is coupled by a particular subtype of cells which express membrane anchored molecules specific for the introduced multimeric molecule) or it may antagonise its functioning (such as introducing a blocking molecule - for example an antibody - which prevents interaction of the extracellular molecule with a hinge molecule and/or membrane anchored molecule).

(ii) Introducing into a subject an agent which agonises or antagonises any one or more of: • the interaction of a soluble adhesion molecule with one or more hinge molecules.

• the localisation of the soluble adhesion molecule/hinge molecule complex proximally to both the surface membrane of the cell and one or more membrane anchored molecules;

• the interaction of a membrane anchored molecule with a soluble adhesion molecule; • the lateral clustering of membrane anchored molecules relative to a hinge molecule.

It should be understood that modulation of the interactions detailed above may be partial or complete. Partial modulation occurs where only some of the subject interactions which would normally occur in a given cell are affected by the method of the present invention (for example, the method of the present invention is applied for only some of the time that the exfracellular molecule of interest is present in a subject) while complete modulation occurs where all interactions are modulated.

The "agent" contemplated herein should be understood as a reference to any proteinaceous or non-proteinaceous molecule which modulates the subject interaction or physical characteristic as detailed above. The agent may be linked, bound or otherwise associated with any other proteinaceous or non-proteinaceous molecule. For example, it may be associated with a molecule which permits targeting to a localised region. The non- proteinaceous agent may be, for example, a nucleotide molecule which is introduced to a cell in order to facilitate the expression of an agent of interest. Alternatively, the molecule may be one which modulates the transcriptional and/or translational regulation of a gene, wherein the subject gene encodes an agent of interest or wherein the subject gene encodes one or more of the components of the leverage mediated uptake mechanism. In yet another example it may be desirable to introduce into a cell a nucleic acid molecule which encodes for a membrane anchored molecule which a given cell may not otherwise express. This provides, for example, a means of genetically inducing expression of a specific membrane anchored molecule such that the delivery of an extracellular molecule of interest (such as a drag) can be target to a specific cell type. Yet another example of non- proteinaceous molecules are hormones, pheromones and odorants that interact with lipoproteins or lipocalins to mediate binding to receptors, which may involve oligomerisation.

The agent, being a proteinaceous or non-proteinaceous molecule, may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening. The non-proteinaceous molecule may also be derived from natural sources, such as for example natural product screening, or may be chemically or otherwise synthesized. For example, the present invention contemplates chemical analogues of any one or more of the components of the leverage mediated uptake mechanism which are capable of acting as agonists or antagonists of the various molecular interactions which occur during the operation of this mechanism. Chemical agonists may not necessarily be derived from a given component of this mechanism but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties of a component of the leverage mediated uptake mechanism. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing the components of the leverage mediated uptake mechanism from interacting. Antagonists include monoclonal antibodies specific for any one or more components of the leverage mediated uptake mechanism or parts thereof. Antagonists also include antisense nucleic acids which prevent transcription or translation of genes or mRNA encoding the subject components, such as dsRNAi mechanisms. Modulation of expression may also be achieved utilising antigens, RNA, ribosomes, DNAzymes, RNAaptamers, antibodies or molecules suitable for use in cosuppression.

Finally, agents could be biologically active peptides, such as amphipathic pore-forming peptides or peptide hormones, such as vaso-active peptide, which has antibacterial activity in prokaryotic membranes but hormone activity in mammalian tissues. These peptides may intercalate within gaps created by LM-components thereby becoming reananged during the LM-process. For example, antibacterial peptides that are active in cholesterol- containing membranes, such as melittin, may have the spatial and configurational requirements to fit between oligomeric adhesion molecules and hinge molecules and become assembled into a pore-forming complex during LM-uptake reactions, which also provide the energy to push the complex into the membrane. Another example may be the spreading factor and growth blocking peptides of lepidopteran plasmatocytes (Strand et al. J. Insect Physiol. 46:817-824, 2000). Interaction with LM-complexes may not lead to pore-formation but to alteration of LM-properties, by weakening or strengthening of intemalisation rates, thereby causing changes in cellular behaviour, such as spreading on a glass surface or cell division.

Screening for the modulatory agents herein defined can be achieved by any one or several suitable methods including, but in no way limited to, contacting a cell culture comprising one or more of the components of the leverage mediated uptake mechanism with an agent and screening for the modulation of the functional activity of a given component or modulation of the activity or expression of a downstream outcome such as the actual uptake (for example, endocytosis) of the molecule. Detecting such modulation can be achieved utilising techniques such as Western blotting, electrophoretic mobility shift assays and/or the readout of reporters of functional activity such as the luciferases, CAT and the like.

It should be understood that the leverage mediated uptake mechanism components which are tested herein may be naturally occuning in the cell which is the subject of testing or the genes encoding them may have been transfected into a host cell for the purpose of testing. Further, the naturally occuning or transfected gene may be constitutively expressed - thereby providing a model useful for, inter alia, screening for agents which downregulate the functioning of a given molecule or the gene may require activation - thereby providing a model useful for, inter alia, screening for agents which modulate functional interactivity under certain stimulatory conditions. Further, to the extent that a nucleic acid molecule encoding a component of the leverage mediated uptake mechanism is transfected into a host cell, that molecule may comprise the entire gene or it may merely comprise a portion of the gene such as the binding site.

In another example, the subject of detection could be a downstream outcome, rather than screening for changes to the functioning of the component of interest itself, such as screening for the presence or absence of an uptake outcome. Yet another example includes utilising binding sites of one or more of the leverage mediated uptake components which are ligated to a minimal reporter. For example, modulation of the interaction of a soluble adhesion molecule with a hinge molecule can be detected by screening for the modulation of a downstream event such as the induction of inverse curvature of the membrane or uptake. This is an example of a system where modulation of the events which are regulated by these molecules are monitored.

These methods provide a mechanism for performing high throughput screening of putative modulatory agents such as the proteinaceous or non-proteinaceous agents comprising synthetic, combinatorial, chemical and natural libraries. The agents which are utilised in accordance with the method of the present invention may take any suitable form. For example, proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules used, linked, bound or otherwise associated with the proteins such as amino acids, lipid, carbohydrates or other peptides, polypeptides or proteins. Similarly, the subject non- proteinaceous molecules may also take any suitable form. Both the proteinaceous and non-proteinaceous agents herein described may be linked, bound otherwise associated with any other proteinaceous or non-proteinaceous molecules. For example, in one embodiment of the present invention said agent is associated with a molecule which permits its targeting to a localised region.

Derivatives include fragments, parts, portions, mutants, variants and mimetics from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of the subject molecule. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins or cyclising the peptide, for example to yield a pharmacologically active form. Derivatives also include fragments having particular epitopes or parts of an entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, a SAM, or derivative thereof may be fused to a molecule to facilitate its localisation to a particular site. Another example would be the fusion of a protein domain with actin nucleation properties, such as formin and spire to a cytoplasmic domain of an adhesive receptor, which is expected to increase adhesive properties due to stabilisation of the receptor on the cell surface and reducing LM-complex formation and receptor- internalisation. Analogues of the molecules contemplated herein include, but are not limited to, modification to side chains, incoφorating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.

Derivatives of nucleic acid sequences which may be utilised in accordance with the method of the present invention may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules. The derivatives of the nucleic acid molecules utilised in the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.

A "variant" or "mutant" of LM-component or a modulatory agent should be understood to mean molecules which exhibit at least some of the functional activity of the form of the molecule of which it is a variant or mutant. A variation or mutation may take any form and may be naturally or non-naturally occuning.

A "homologue" is meant that the molecule is derived from a species other than that which is being treated in accordance with the method of the present invention. This may occur, for example, where it is determined that a species other than that which is being treated produces a form of LM-component or modulatory agent which exhibits similar and suitable functional characteristics to that of the molecule which is naturally produced by the subject undergoing freatment.

Chemical and functional equivalents of the subject LM-components or modulatory agent should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening. For example chemical or functional equivalents can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening. Antagonistic agents can also be screened for utilising such methods.

For example, libraries containing small organic molecules may be screened, wherein organic molecules having a large number of specific parent group substitutions are used. A general synthetic scheme may follow published methods (eg., Bunin BA, et al. (1994) Proc. Natl. Acad. Sci. USA, 91:4708-4712; DeWitt SH, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6909-6913). Briefly, at each successive synthetic step, one of a plurality of different selected substituents is added to each of a selected subset of tubes in an anay, with the selection of tube subsets being such as to generate all possible permutation of the different substituents employed in producing the library. One suitable permutation strategy is outlined in US. Patent No. 5,763,263.

There is cunently widespread interest in using combinational libraries of random organic molecules to search for biologically active compounds (see for example U.S. Patent No. 5,763,263). Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands or interfering with the naturally occuning ligands of a biological target. In the present context, for example, they may be used as a starting point for developing analogues, which exhibit properties such as more potent pharmacological effects. LM- components or a functional part thereof may according to the present invention be used in combination libraries formed by various solid-phase or solution-phase synthetic methods (see for example U.S. Patent No. 5,763,263 and references cited therein). By use of techniques, such as that disclosed in U.S. Patent No. 5,753,187, millions of new chemical and/or biological compounds may be routinely screened in less than a few weeks. Of the large number of compounds identified, only those exhibiting appropriate biological activity are further analysed.

With respect to high throughput library screening methods, oligomeric or small-molecule library compounds capable of interacting specifically with a selected biological agent are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above. In such a method, each member of the library is screened for its ability to interact specifically with the selected agent. In practising the method, a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction. Preferably, the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed for example by any well-known functional or non-functional based method for the detection of substances.

In addition to screening for molecules which mimic the activity of a LM-component, for example, one may identify and utilise molecules which function agonistically or antagonistically to such a molecule in order to up or down-regulate its functional activity. The use of such molecules is described in more detail below. To the extent that the subject molecule is proteinaceous, it may be derived, for example, from natural or recombinant sources including fusion proteins or following, for example, the screening methods described above. The non-proteinaceous molecule may be, for example, a chemical or synthetic molecule which has also been identified or generated in accordance with the methodology identified above. Accordingly, the present invention contemplates the use of chemical analogues of LM-component molecules capable of acting as agonists or antagonists. Chemical agonists may not necessarily be derived from the LM-component molecule but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties of these molecules. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing a LM-component from canying out its normal biological functions. Antagonists include monoclonal antibodies specific for LM-components or parts of LM- components.

Analogues contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.

Derivatives of nucleic acid sequences may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules. The derivatives of the nucleic acid molecules of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-frinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a conesponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuri- benzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri- 4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy- 5 -phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 4.

TABLE 4

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D-valine Dval α-methyl- -aminobutyrate Mgabu

D- -methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate N aabu D-α-methylleucine Dmleu α-napthylalanine Anap

D- -methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycineNbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(l-hydroxyethyl) glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(l -methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(l -methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(/?-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanineMhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine ϊ Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropy 1l)) NNnnbbhhee carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl-Nmbc ethylamino)cyclopropane Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH )_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

It should be understood that the cell which is treated according to the method of the present invention may be treated in vitro or in vivo. A cell which is treated in vitro may be one that has been removed from the body of a mammal. For example, cells or tissue comprising neoplastic cells may be removed from a mammal, treated according to the method of the present invention in order to facilitate uptake of a toxic molecule specifically by the neoplastic cells and then returned to the mammal. This may be of particular use where the subject neoplasm is found in bone marrow or other potential stem cell source wherein ablation of all dividing cells in that population is not desirable. Alternatively, the in vitro cell may be a cell line in respect of which it is sought to either up or downregulate its leverage mediated uptake mechanism in relation to a specific extracellular molecule. In accordance with the prefened aspect of the present invention, the cell is located in vivo and the method of the present invention is applied to a subject in order to modulate the leverage mediated uptake mechanism in relation to the subject cell or population of cells .

Accordingly, the present invention provides a method of regulating the uptake of an exfracellular molecule by a cell in a subject, said method comprising modulating the functioning of any one or more structural or functional elements of said cell's leverage mediated uptake mechanism.

More particularly, there is provided a method of regulating the uptake of an extracellular molecule by a cell in the subject said method comprising modulating one or more of:

wherein the subject cell's leverage mediated uptake mechanism is modulated.

Still more particularly there is provided a method of regulating the uptake of an extracellular molecule by a cell in a subject, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(iv) the interaction of said membrane anchored molecule with said soluble adhesion molecule; and (v) the lateral clustering of said membrane anchored molecules relative to said hinge molecules.

wherein the subject cell's leverage mediated uptake mechanism is modulated.

As detailed hereinbefore, according to the leverage mediated uptake mechanism, ter alia cell signalling, uptake reactions and receptor intemalization are defined by the composition of the complex and the presence or absence of functionally relevant membrane-anchored proteins on the cell surface. As a consequence, it is possible to predict uptake and target cell-type using information about the composition and receptor repertoire of the cell. For example, using a multimeric (but not a monomeric) lectin with specific sugar-specificities it is possible to create uptake reactions in a specific subset of cells which contain a glycoprotein or glycolipid with the appropriate sugar configuration. If the lectin is coupled with other molecules, the mechanism can be used to deliver a cargo, consisting of drags and genes, to a specific set of cells. Accordingly, the elucidation of this externally driven mechanism of, inter alia, uptake reactions, cell-cell interaction, and signalling provides a means for manipulation of these processes in agricultural and human -related biotechnology.

Computer simulation of cell behaviour

This model enables analysis of the role of cytoskeleton-inactivating substances, such as cytochalasins. Where direct or indirect attachments of cytoplasmic receptor-domains to cytoplasmic scaffolding, such as actin-cytoskeleton, prevent receptor-internalisation by LM-reactions, inactivation of F-actin will abolish adhesion and phagocytosis, but not all pinocytosis reactions. Conversely, to maintain their adhesive properties, cells will stabilize receptors on the cell surface by attaching their cytoplasmic domains directly or indirectly to cytoplasmic scaffolds, such as actin-networks or actin-cables on microspikes. In the context of this model, receptors with the ability to make contacts to external binding sites require cytoplasmic stabilisation to remain on the cell surface long enough to carry out attachments to external binding sites (DeMali and Bunidge, J Cell Sci 116, 2389-2397, 2003). Thus transformation of cells with hemomucin receptors with an additional formin peptide attached to the cytoplasmic domain will have adhesive properties, compared to non-transformed cells.

This has implications for computer-modelling of cell functions, since it allows one to describe complex cell behaviour by a simple set of mathematical equations, where receptors can either engage in adhesive interactions with external binding sites or become internalised causing detachment from substrate or other cells. Since similar mechanisms may apply to intracellular processes, such as vesicle transport to the periphery, receptor recycling, and retrograde transport, the complexities of cell behaviour are likely to emerge from sub-routines of existing equations rather than the development of new mathematical concepts. Thus using the existing mathematical description of cell functions, computer algorithms can be developed to predict cell behaviour, which is a precondition for in silico simulation models.

Where cell surface receptors are able to engage in multiple processes, such as adhesion and LM-complex formation, the presence of any given receptor on the cell surface will be a function of infra- and extracellular forces that can, in a first approximation, either stabilise receptors on the cell surface or internalise receptors by an L -reaction (Figure 32).

In this model, the number of receptors on the cell surface (R_s) is determined by the number of newly produced receptors aniving on the surface by exocytosis and those leaving the surface by endocytosis or receptor-intemalisation (Figure 26, 32). With the rate constant for new receptor anivals on the membrane given by ki and that for receptors engaging in the formation of LM-complexes given by k₂, the dynamics of a given receptor on the cell surface follows the simple rate equation

(1) dR_s/dt = k₁R_v - k₂R_s ⁿ,

where R_v represents the number of receptors inside the cell and n is the number of receptors engaged in each LM-complex. Additional classes of LM complexes with varying n can easily be accounted for through additional terms on the right hand side of the above equation. Note also that fø depends on the availability and lateral movement of adhesion molecules to cluster and form complexes. If LM-complexes internalise with a rate of ks, the dynamics of LM-complexes in turn can be described by

(2) dC/dt = k₂/n R_s ⁿ - k₃C,

where the rate of change of C due to complex formation is 1/wth of the rate of change of R_s in equation (1).

Since receptor intemalisation can lead to recycling and retrograde transport, the total rate of change in the internal pool of receptors R_v is determined by the rate of internalised receptors and those that emerge from the cytoplasm, ie.

(3) dR_v/dt = nk₃C - kιR_v

Equations 1, 2 and 3 imply that the total number of receptors per cell is conserved, ie. that the cell is at homeostasis:

(4) R_s + nC + R_v = R = constant

This coupled set of nonlinear differential equations determines the relative allocation of receptors to the cell surface, the cell interior and the LM-complexes at any particular point in time. At equilibrium all fluxes in- and out balance ie. dR_s/dt = dC/dt = dR_v/dt = 0,

yielding coupled algebraic equations for Rs, C and R_v, eg.

(5) R_v = k₂ kιR_s ⁿ

(6) C = k₂/nk₃R_s ⁿ (7) R = k₂(l/k₃ + l/kι)R_s ⁿ + R_s

For a given R_s equations (5) and (6) determine R_v and C uniquely, while Descartes' sign mle may be used to show that equation (7) yields one and only one positive solution for R_s. In other words, the model for the distribution of receptors in an isolated cell predicts a unique stable state for this cell. Manipulations, such as cell fransformation altering the expression of genes, or external application of proteins, will change the balance of forces and depending on the nature of these changes will either stabilise receptors, which will increase adhesive properties, or internalise receptors, which will reduce adhesive properties (Figures 12, 26).

A person skilled in computer-language can translate this basis interdependent relationship into computer algorithms that utilise sub-routines of the mathematical equations to add to the complexity. This will allow one to make predictions by calculating whether any experimental manipulation of a cell or organism will increase adhesive properties, or decrease adhesive properties by increasing receptor-internalisation. For example, systemic over-expression of counter-adhesion molecules will reduce the number of cell surface receptors, leading to tissue destablisation (Mettouchi et al, Molecular & Cellular Biology 17, 3202-3209, 1997) and increased cell detachment. This in turn will enhance cell division and cancer (Huang et al, Cancer Res 61, 8586-8594, 2001; Sargiannidou et al, Experimental Biology and Medicine 226, 726-733, 2001). Conversely, systemic under- expression of counter-adhesion molecules will lead to increased cell adhesion and wound closure (Bradshaw et al, Histochem Cytochem 50, 1-10, 2002). Under the LM-model, the counter-adhesion molecules can act as dynamic driving forces in extracellular space in addition to being signalling molecules (Greenwood and Murphy, 1998 supra).

The development of the method of the present invention has therefore now facilitated its application to a wide range of circumstances including, but not limited to: (i) Delivery ofbiotic and abiotic compounds.

The method of the present invention can be applied towards the delivery and intemalisation of biotic and abiotic compounds, including but not limited to proteins, DNA and dmgs into the intracellular environment of the cell. For example soluble adhesion molecules can be modified to carry cargo, such as fluorescent compounds, into the cell. Specific cell types can be targeted by an LM- mechanism and delivery of substances into the cytoplasm, (e.g. DNA, RNA., peptides, or chemicals) can be attached to the first component, which is a SAM in the LM-mechanism, by covalent or non-covalent binding and detached in the cytoplasm by specific cleavage processes (e.g. attachment by disulfate bridges and cleavage by reducing conditions inside the cell). This delivery mechanism has important implications for the biotechnology of targeted treatment of certain cell types (e.g. inactivation of tumor cells with specific glycodeterminants). Depending on the stmctural nature of soluble adhesion molecules and differential distribution of conesponding membrane anchored molecules in target tissues, the cargo can be applied to specific cells in the body. A major aspect of the present invention is the knowledge of functional requirements for cargo-internalization and target specificity, based on the interaction of three stmctural leverage mediated uptake mechanism components. An implication of the invention is that many stmctural and functional aspects of the invention that are relevant to cell-derived uptake mechanisms are also relevant to abiotic, xenobiotic and modified structural leverage mediated uptake mechanism components. This allows the development of nanotechnologies using existing and modified stmctural elements to manipulate mechanistic and energetic aspects of the biological process in leverage mediated uptake mechanisms and the design of molecular engines that drive cell-shape changes.

The method of the invention is unique and exfremely valuable in that it enables the intracellular delivery of compounds with minimal impact on intracellular signalling.

Specifically, the method of the invention allows one to design means for the intracellular delivery of a molecule via cell surface molecules (MARMs) which are not involved in cytoplasmic signalling events. This can therefore minimise the possibility of unwanted side effects which may be induced by the delivery of agents via non-specific means which may also lead to unwanted signalling events.

(ii) Modulation of cell signalling.

Since attachment of membrane anchored molecules with cytoplasmic domains to intracellular components can be dismpted during leverage mediated uptake, the dislocation of cytoplasmic proteins can constitute a signal. Without limiting the present invention to any one theory or mode of action, signalling is induced when the configurational energy from the extracellular uptake complex is sufficient to tilt membrane anchored molecules and thereby overcome the attachments to intracellular components, such as cytoskeleton (Figure 13B). A major aspect of the present invention is that signalling is a functional part of the LM-uptake or membrane anchored molecule-internalization process and not a precondition as has been predicted in the unrelated receptor mediated endocytosis mechanisms described prior to the advent of the present invention. Many structural and functional aspects of the method of the invention that are relevant to uptake reactions are also relevant to cell-signalling. This allows the development of cell- manipulation techniques (eg. phagocytosis with and without signalling) that target specific cell populations. This technique will additionally achieve this objective without causing an immune response, such as inflammation.

(iii) Pathogen defence strategies.

Since microbes use uptake-processes to gain access to the host cell, the method of the present invention can describe complex pathogen-host interactions that were previously difficult to explain using lectinophagocytosis and receptor mediated endocytosis mechanisms. A major aspect of the present invention is the notion that pathogens gain access to the cell by manipulating extracellular components of the host uptake machinery to induce cell surface changes, which facilitate entry (Figure 21). Accordingly, many structural and functional aspects of the invention that are relevant to uptake mechanisms are also relevant to pathogen invasion into host cells. This allows the development of pathogen defence strategies by interfering with and manipulating pathogen-specific structural elements that mediate pathogen invasion into host cells. Specifically, manipulation of pathogen invasion into the host tissue or uptake by defence cells can be manipulated by targeting host components of the LM-mechanism used by pathogens to enter cells.

(iv) Protection strategies against toxins.

The method of the present invention can be applied to the reduction of host cell damage by pathogens producing toxins such as pore-forming toxins. The toxicity of many pore-forming toxins is possibly based on the membrane-insertion of the channel-forming toxin complex by an uptake mechanism. For example, endotoxin from spore-forming soil bacteria B. thuringiensis aggregate into an oligomeric complex in the absence of lipids (Figure 10, 29). Thus the toxin represents an oligomeric lectin (Burton et al, 1999) with an antibacterial peptide covalently attached (Gerber and Shai, 2000 supra; Szabo et al, 1993 supra). It is thought that the insertion of other pore-forming toxins into the membrane-bilayer is achieved by a similar mechanism (Figure 16). Accordingly, many stmctural and functional aspects of the invention that are relevant to uptake mechanisms are also relevant to the membrane-insertion of pore-forming toxins. This allows the design and implementation of toxin-protection strategies, by using approaches that inactivate toxin molecules by exposure to mimics of membrane anchored molecules and hinge molecules.

(v) Target membrane-receptors which interact with oligomeric adhesion molecules to engage in leverage-mediated uptake reactions.

This can be applied to:

• manipulate phagocytosis (e.g. of apoptotic cells) and uptake reactions (cell- targeting by identification of cell-specific receptors e.g. tumor cells), • use of oligomeric adhesion molecule (weak or strong leverage-mediated components, e.g. strong counter-adhesion molecules in self-incompatibility and immune suppression), • use of oligomeric adhesion molecules for transmission of proteins into the cytoplasm (similar to bipartite toxins), • provide the potential for a wide variety of cargo which can be associated with oligomeric adhesion molecules (dmgs, proteins and DNA) and thereby introduced into a cell), • modify hinge molecules (for example, by engaging oligomeric adhesion molecules with modified lipophorin in coagulation reactions), • modulate attachment/detachment of cells from extracellular matrix or cell-cell connections such as occurs in the context of morphogenesis, tissue sculpturing, wound healing and cell division (Figure 34), • inducing inactivation by forced uptake reactions and destabilisation of the actin cytoskeleton using counter-adhesion molecules,

(vi) Modify leverage-mediate -uptake reactions

This can be applied to: • Inactivate cells (for example by using counter-adhesion molecules, causing immune suppression and self-incompatibility) or by using oligomeric adhesion molecules with amphipathic peptides attached (acting like pore-forming toxins), • break self-incompatibility in pollen by interfering with counter-adhesion property of male factors (e.g. adding biologically active peptide see below).

(vii) Modify the dynamics of exocytosis and leverage-mediated uptake

This can be applied to: • Modulate tip growth (for example in the context of angiogenesis, axon formation pollen tube formation (Figure 37), rootlets in plants) • Manipulate directed tip growth (for example in the context of axon formation, pollen tube etc) by changing exfracellular binding site patterns, or release of soluble counter-adhesion molecules that act like axon growth repellents.

(viii) Modify intracellular protein secretion and quality control

This can be applied to:

• Modulating leverage-mediated uptake vesicle emergence and transport (Figure 38B) by co-synthesising peptides, receptors and oligomeric adhesion molecules that modulate intracellular leverage-mediated uptake processes (eg. ER, Golgi).

(ix) Modify endosome maturation and protein recycling This can be applied to :

• Using proteins as cargo that escape recycling and protease degradation in the sub-endosomal compartment (Figure 30) and are subsequently transported into the cytoplasm (see bi-partite toxins) • Modulate retrograde transport (Figure 38 A) of toxins in bipartite systems by interfering with LM-uptake (using biologically active proteins etc).

(x) Modify cell and tissue compatibility This can be applied to:

• Abolish tissue compatibility by altering receptor composition at the cell surface in one of the two cell types thereby altering the balance of forces between the two cell types (Fig. 14B). • Create tissue compatibility between incompatible cells by expressing similar receptor repertoires (restore balance of forces at the immunological synapse). • Create compatibility between cells and abiotic surface by coating the surface with relevant recombinant receptor repertoires.

The present invention should be understood to extend to all of the applications detailed in points (i)-(x), above.

Accordingly, one embodiment of the present invention is directed to a method for regulating cellular signalling, which cellular signalling is induced and/or otherwise regulated by the assembly of an extracellular complex, said method comprising modulating the functioning of any one or more structural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

More particularly, there is provided a method for regulating cellular signalling, which cellular signalling is induced and/or otherwise regulated by the assembly of an extracellular complex, said method comprising modulating the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(v) the lateral clustering of said membrane anchored molecules relatively to said hinge molecules; wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

(i) the functioning of said molecule as a soluble adhesion molecule;

Still another aspect of the present invention is directed to a method for the intracellular delivery of a molecule to a cell, said method comprising modulating the functioning of any one or more structural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

More particularly, there is provided a method for the intracellular delivery of a molecule to a cell, said method comprising modulating the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

Still more particularly, there is provided a method for the intracellular delivery of a molecule to a cell in a subject, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(ii) the interaction of said soluble adhesion molecule with one or more hinge molecules; (iii) the localisation of the hinge molecule or the complex of (i) proximally to both the surface membrane of said cell and one or more membrane anchored molecules;

Reference to "molecule" in accordance with this aspect of the present invention should be understood to have the same meaning as "exfracellular molecule" hereinbefore defined. For example, the molecule which is the subject of intracellular delivery may be any biotic or abiotic agent, such as a therapeutic drag. The subject molecule may also conespond to a microorganism, such as a virion.

In accordance with this aspect of the present invention, one may seek to either upregulate or downregulate the leverage mediated mechanism depending on the particular circumstances. For example, one would seek to upregulate this mechanism to facilitate uptake of a drag or other therapeutic or prophylactic agent. For instance, the uptake of a drag by neoplastic cells in order to induce apoptosis or to otherwise lyse the subject cell is a particularly prefened embodiment. In another example, it may be particularly desirable to induce the uptake of a drug by a microorganism which has infected a subject (such as the uptake of an antibiotic by a bacterium) in order to facilitate the treatment and/or prophylaxis of the condition which is induced by the colonisation of a subject with the microorganism in issue. In particular, to the extent that microorganisms have developed resistance mechanisms based on preventing the uptake of drags or other such toxic molecules, the method of the present invention now provides a unique and valuable means of overcoming such resistance. In still another example, it may be desirable to downregulate the leverage mediated uptake mechanism in the situation where a subject has been infected by a microorganism which seeks intracellular colonisation, such as a vims. Accordingly, the administration of an agent which interacts with any component of the leverage mediated uptake mechanism, and not necessarily just the vims, may be useful in blocking cellular uptake to the extent that it is required for the vims to become deposited intracellularly. In such a situation, even partial downregulation of the functioning of the leverage mediated uptake mechanism may act to minimise infection.

Accordingly, in another prefened embodiment the present invention is directed to a method for downregulating the microbial infection of a cell, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements downregulates the functioning of the leverage mediated uptake mechanism.

Most particularly, there is provided a method for downregulating the microbial infection of a cell, said method comprising modulating the function of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

Still more particularly there is provided a method for downregulating the microbial infection of a cell in a subject said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

A further aspect of the present invention relates to the use of the invention in relation to the treatment and/or prophylaxis of disease conditions.

Accordingly, another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a subject, which condition is characterised by the abenant, unwanted or otherwise inappropriate cellular uptake of an extracellular molecule, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

More particularly, there is provided a method for the treatment and/or prophylaxis of a condition in a subject, which condition is characterised by the abenant, unwanted or otherwise inappropriate cellular uptake of an exfracellular molecule, said method comprising administering to said subject an effective amount of an agent for a time and under conditions sufficient to modulate one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(iv) the interaction of said membrane anchored molecule with said soluble adhesion molecule; and v

wherein the subject cell's leverage mediated uptake mechanism is modulated.

Reference to "abenant, unwanted or otherwise inappropriate" cellular uptake should be understood as a reference to overactive cellular uptake, to physiological normal cellular uptake which is inappropriate in that it is unwanted or to insufficient cellular uptake. For example, certain conditions are characterised by the deleterious or otherwise vmwanted intracellular uptake of a microorganism or toxin produced therefrom. In such a situation it is desirable to downregulate the leverage mediated uptake mechanism of cells which are susceptible to invasion by the subject microorganism or toxin derived therefrom. Alternatively, the host organism may specifically internalise the receptor used by the abenant uptake. This would deplete the target cells from binding sites for the abenant uptake reaction to proceed. In another example, certain individuals may be susceptible to insufficient or otherwise inadequate uptake of nutrients. The method of the present invention provides a means of either delivering nutrients in a form which would facilitate leverage mediated uptake thereby facilitating cellular uptake of the nutrient or it may be possible to administer an agent which otherwise modulates the functioning of a component of the leverage mediated uptake mechanism which will lead to uptake of nutrient which is present in the individual. Accordingly, the subject modulation may be upregulation or downregulation of the leverage mediated uptake mechanism.

An "effective amount" means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Reference herein to "freatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or onset of a particular condition. "Treatment" may also reduce the severity of an existing condition. Reference to "subject" should be understood as a reference to any organism. In this regard, the organism may be any human or non-human organism. Non-human organisms contemplated by the present invention include primates, livestock animals (eg. sheep, pigs, cows, horses, donkeys), laboratory test animals (eg. mice, hamsters, rabbits, rats, guinea pigs), domestic companion animals (eg. dogs, cats), birds (eg. chicken, geese, ducks and other poultry birds, game birds, emus, ostriches), captive wild or tamed animals (eg. foxes, kangaroos, dingoes), reptiles, fish or prokaryotic organisms. Non-human organisms also include plant sources such as rice, wheat, maize, barley or canola.

Administration of the modulatory agent, in the form of a pharmaceutical composition, may be performed by any convenient means. The modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

The modulatory agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). The modulatory agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as fragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.

Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, infracereberally, intranasally, infusion, orally, rectally, via IN drip patch and implant.

In accordance with these methods, the agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the subject agent may be administered together with an agonistic agent in order to enhance its effects. By "sequential" adminisfration is meant a time difference of from seconds, minutes, hours or days between the adminisfration of the two types of molecules. These molecules may be administered in any order. The present invention further contemplates a combination of therapies.

Another aspect of the present invention contemplates the use of an agent, as hereinbefore defined, in the manufacture of a medicament for the treatment of a condition in a subject, which condition is characterised by abenant, unwanted or otherwise inappropriate cellular uptake of a molecule, wherein said agent modulates the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable earners and/or diluents. Said agents are refened to as the active ingredients.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The canier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the prefened methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible canier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic adminisfration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Prefened compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cheny flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid canier. Narious other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cheny or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector canies a nucleic acid molecule encoding a proteinaceous modulatory agent where it is desired to express the modulatory agent in sites.

Narious methods of transfemng or delivering DΝA to cells for expression of the gene product protein, otherwise refened to as gene therapy, are disclosed in Gene Transfer into Mammalian Somatic Cells in vivo, N. Yang, Grit. Rev. Biotechn. 12(4): 335-356 (1992), which is hereby incorporated by reference.

Many protocols for transfer of regulatory sequences are envisioned in this invention. Transfection of promoter sequences, or other sequences which would modulate the expression and/or activity of the gene encoding a modulatory agent are also envisioned. An example of this technology is found in Transkaryotic Therapies, Inc., of Cambridge, Mass., using homologous recombination to insert a "genetic switch" that turns on an erythropoietin gene in cells. See Genetic Engineering News, Apr. 15, 1994.

Gene transfer methods for gene therapy fall into three broad categories: physical (e.g., electroporation, direct gene transfer and particle bombardment), chemical (lipid-based earners, or other non-viral vectors) and biological (vims-derived vector and receptor uptake). For example, non-viral vectors may be used which include liposomes coated with DNA. Such liposome/DNA complexes may be directly injected intravenously into the patient. Additionally, vectors or the "naked" DNA of the gene may be directly injected into the desired organ, tissue or tumor for targeted delivery of the therapeutic DNA.

Gene therapy methodologies can also be described by delivery site. Fundamental ways to deliver genes include ex vivo gene transfer, in vivo gene fransfer, and in vitro gene transfer.

Chemical methods of gene therapy may involve a lipid based compound, not necessarily a liposome, to feny the DNA across the cell membrane. Lipofectins or cytofectins, lipid- based positive ions that bind to negatively charged DNA, may be used to cross the cell membrane and provide the DNA into the interior of the cell.

Many gene therapy methodologies employ viral vectors such as retroviras vectors to insert genes into cells. A viral vector can be delivered directly to the in vivo site, by a catheter for example, thus allowing only certain areas to be infected by the virus, and providing long-term, site specific gene expression. In vivo gene fransfer using retroviras vectors has also been demonstrated in mammary tissue and hepatic tissue by injection of the altered virus into blood vessels leading to the organs.

Viral vectors may be selected from the group including, but are not limited to, retrovimses, other RNA viruses such as polioviras or Sindbis viras, adenoviras, adeno-associated viras, herpes viruses, SN 40, vaccinia and other DΝA viruses. Replication-defective murine retroviral vectors are the most widely utilized gene transfer vectors and are prefened. Adenoviral vectors may be delivered bound to an antibody that is in turn bound to collagen coated stents.

Mechanical methods of DΝA delivery may be employed and include, but are not limited to, fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion, lipid particles of DΝA incorporating cationic lipid such as lipofectin, polylysine-mediated transfer of DΝA, direct injection of DΝA, such as microinjection of DΝA into germ or somatic cells, pneumatically delivered DΝA- coated particles, such as the gold particles used in a "gene gun," inorganic chemical approaches such as calcium phosphate transfection and plasmid DΝA incoφorated into polymer coated stents. Ligand-mediated gene therapy, may also be employed involving complexing the DΝA with specific ligands to form ligand-DΝA conjugates, to direct the DΝA to a specific cell or tissue.

The DΝA of the plasmid may or may not integrate into the genome of the cells. Νon- integration of the transfected DΝA would allow the transfection and expression of gene product proteins in terminally differentiated, non-proliferative tissues for a prolonged period of time Λvithout fear of mutational insertions, deletions, or alterations in the cellular or mitochondrial genome. Long-term, but not necessarily permanent, transfer of therapeutic genes into specific cells may provide treatments for genetic diseases or for prophylactic use. The DΝA could be reinjected periodically to maintain the gene product level without mutations occuning in the genomes of the recipient cells. Νon-integration of exogenous DΝAs may allow for the presence of several different exogenous DΝA constructs within one cell with all of the constructs expressing various gene products. Gene regulation of a modulatory agent may be accomplished by administering compounds that bind the modulatory agent gene, or control regions associated with the modulatory gene, or conesponding RNA transcript to modify the rate of transcription or translation. Additionally, cells transfected with a DNA sequence encoding a modulatory agent regulator may be administered to a patient to provide an in vivo source of the regulator. For example, cells may be transfected with a vector containing a nucleic acid sequence encoding the regulator.

The term "vector" as used herein means a canier that can contain or associate with specific nucleic acid sequences, which functions to transport the specific nucleic acid sequences into a cell. Examples of vectors include plasmids and infective microorganisms such as vimses, or non-viral vectors such as ligand- DNA conjugates, liposomes, lipid-DNA complexes. It may be desirable that a recombinant DNA molecule comprising a regulator

DNA sequence is operatively linked to an expression control sequence to form an expression vector. The transfected cells may be cells derived from the patient's normal tissue, the patient's diseased tissue, or may be non-patient cells. Patients may be any subject as herein defined, in particular including plants, . Cells may also be transfected by non-vector, or physical or chemical methods known in the art such as electroporation, incoφoration, or via a "gene gun." Additionally, DNA may be directly injected, without the aid of a canier, into a patient.

The gene therapy protocol for transfecting DNA into a patient may either be through integration of the DNA into the genome of the cells, into minichromosomes or as a separate replicating or non-replicating DNA construct in the cytoplasm or nucleoplasm of the cell. Expression may continue for a long-period of time or the DNA may be reinjected periodically to maintain a desired level of expression and/or activity in the cell, the tissue or organ.

The modulated cells are intended to replace existing cells such that the existing development biology or biological function of the cells is modulated or the modulated cells may be used to infiltrate existing problematic regions to halt progression of a disease or unwanted physiological process, for example.

Yet another aspect of the present invention relates to the agent as hereinbefore defined, when used in the method of Hie present invention.

The present invention is further defined by the following non-limiting examples.

EXAMPLE 1 LECTIN-MEDIATED UPTAKE REACTION

Since lectins are involved in cell-spreading, aggregation and uptake reactions, a two-step mechanism was tested, in hemocyte-like Schneider and mbn-2 cells from Drosophila. These cells contain a GalNAc-containing hemomucin receptor that is involved in lectin- mediated uptake reactions and immune-signalling (Theopold et al, Journal of Biological Chemistry 271:12708-15, 1996). While the hexameric Helix pomatia lectin (HPL) is known to induce an immune response (Theopold 1996, supra), it is not always an effective inducer of macropinocytosis (Figure 1, 2). In contrast, tetrameric lectins that are specific for the two glycoforms of hemomucin, are effective at inducing macropinocytosis (Figure 2,3). Moreover, experimental evidence suggests that uptake occurs only with tetrameric, but not with monomeric lectins, even though monomeric lectins compete with oligomeric lectins for the same binding sites (Figure 3).

In addition to lectin-mediated uptake, cells spread extensively on lectin-coated surfaces compared to non-coated glass or poly-Lysine coated surfaces (Figure 33a). Moreover, when spread cells were treated with lectin solutions, the cells slowly detached from the glass surface and showed extensive actin-depolymerisation (Figure 22b, 33b). This is reminiscent of apolipophorin III, which has a hexameric configuration and detach hemocytes when applied in soluble form but increases phagocytosis when immobilised on the object surface (Whitten et al, J Immunol 172, 2177-2185, 2004). Other examples, where one ligand is involved in more than one cell response, are counter-adhesion molecules or matricellular proteins, such as thrombospondin-1 (Adams, 2001, supra). In matricellular protein systems, the two distinct responses to the same ligand have been attributed to the interaction of immobilised and soluble thrombospondin-1 with different sets of cell receptors, causing opposite cellular responses (Chandrasekaran et al, 2000 supra). For example, immobilised thrombospondin-1 interacts with integrin receptors, which instmct the cell to spread, while soluble thrombospondin-1 interacts with CD44, scavenger receptors and proteoglycans to signal detachment. This indicates that lectins used in the experiments with the Drosophila cell line act like counter-adhesion molecules. However, in this cell line, the only lectin-binding glycoprotein on the cell surface is the immune receptor hemomucin. This indicates that lectin-hemomucin interactions can mediate opposite cell behaviour, which is difficult to reconcile with a signalling role of the lectin but is easily explained by an active role of lectin in receptor- internalisation. If the lectin molecules are immobilised on the subsfrate surface the receptors attach but are unable to internalise the complex causing the cell to spread on the substrate by a 'zipper' mechanism (Figure 4). When soluble lectin is added, leverage-mediated lateral cross-linking of receptors lead to local regions of inverse curvature of the membrane and the intemalisation of receptors. Since receptor-intemalisation includes those involved in the turnover of substrate-attached receptors (teeth of the 'zipper'), the 'zipper' is undone, and the cell detaches from the subsfrate.

The present observations indicate a one-step uptake mechanism driven by extracellular driving forces. In this model, soluble adhesion molecules (SAMs), such as tetrameric lectins, cross-link membrane-anchored molecules (MARMs) around lipoproteins or bulky hinge molecules (HMs) tilting MARMs to cause a local inversion of the membrane curvature (Figure 13). This "leverage-mediated uptake" mechanism (LM) involves lateral clustering of MARMs by SAMs, generating the configurational energy, which drives the reaction towards intemalization of the complex (Figure 26).

EXAMPLE 2 UPTAKE-MEDIATED SIGNALLING

There is growing evidence that some extracellular molecules involved in cell adhesion and uptake reactions form an integral part of the signalling process. This model differs from the traditional RME-mechanism (Schwartz, Paediatric Research 38:835-843, 1985), where extracellular signals are a precondition for cytoplasmic-driven endocytosis mechanisms (Hussain, Frontiers in Bioscience 6:D417-D428, 2001; Cardelli, Traffic 2:311-320, 2001). Since tilting of ?MARMs is part of the LM-process, the resulting angle of cytoplasmic MARM-domains against their intracellular attachments produces a signal if the leverage is strong enough to dislodge intracellular proteins (Figure 13B). In this model the signalling capacity of SAMs, such as dimeric and oligomeric adhesion molecules (Table 1) is operationally defined by their ability to form a complex that can dislocate attachment of MARMs from cytoplasmic components

Adhesion molecules are not always SAMs. For example, receptor-specific antibodies may be able to cluster cell surface molecules without initiating intemalisation and signalling. Conversely, GPI-anchored molecules may be MARMs that are involved in uptake without involving a cytoplasmic signal. In the LM-model, signal transduction is not a precondition of cellular uptake, but may be intrinsically linked with the tilting of MARMs with cytoplasmic domains, which create the inverse curvature of the membrane. In LM, an outside signal to the cytoplasm is possible, when the combined leverage of an exfracellular uptake complex supersedes cytoplasmic stabilisation caused by intracellular MARM- associated attachments to the cytoskeleton or other cytoplasmic proteins.

EXAMPLE 3 SAMs THAT ACT AS PORE-FORMING TOXINS.

Pore-forming toxins damage cell membrane integrity by forming oligomeric ion-channels inside the membrane bilayer (Olson et al, 1999, supra). The main issue for understanding bacterial toxicity is how soluble proteins are inserted into the membrane (Lacy et al, 1998, supra). Most models predict membrane insertion of monomeric toxin molecules by a putative receptor-mediated process (Bhakdi et al., Archives of Microbiology 165:73-79, 1996). The assumption is that pore-forming toxins, such as Bt-toxins, assemble into a tetrameric channel-forming complex once monomeric molecules accumulate inside the membrane bilayer (Aronson 2001, supra).

Many pore-forming toxins bind to specific sugar moieties on glycoprotein receptors (Cortajarena et tf/., 2001 supra; Frankel et al, Biochemistry 35:14749-14756, 1996; Hatakeyama et al, Journal of Biological Chemistry 271 : 16915- 16920, 1996; Konska et al , Journal of Biochemistry 116:519-523, 1994; Mercy and Ravindranath, Comparative Biochemistry & Physiology A-Comparative Physiology 109:1075-1083, 1994; Sandros et al, Glycoconjugate Journal 11:501-506, 1994), which could indicate that toxin-insertion into the membrane may be facilitated by an LM-mechanism, where oligomeric toxin complexes interact with glycoprotein receptors like oligomeric lectins (Figure 28, 30). In line with the LM-model we can make several predictions: 1) Pore-forming toxins are functional anti-microbial peptides attached to oligomeric adhesion molecules. This has been demonstrated in the case of endotoxins from Bacillus thuringiensis, which contains functional antibacterial peptides in the pore-forming domains (Gerber and Shai, 2000 supra; Szabo etal, 1993 supra). 2) Pore-forming antimicrobial peptides, such as melittin, interact with LM-complexes to become inserted into the cholesterol-containing membranes (Figure 28). Although this assumption has not been formally tested, results from melittin, its diastereomeric analogs and hybrids with cecropin A peptides (Merrifield et al, 1995 supra), suggest that biologically active peptides utilise specific sterical conformations rather than peptide-peptide interactions to function as pore-forming toxins. This is in agreement with a function protein complex, where peptides can fit into clefts and fractures of the LM-complex to become inserted into the membrane during intemalisation (Figure 28). 3) In the LM- model endotoxins of B. thuringiensis fonn tetrameric channel-forming complexes before interacting with receptors. It is known that a number of protoxins, such as CrylAc, form tetrameric complexes that are stable in the presence of SDS (Figure 29). Insertion of intact toxin channels into the underlying membrane is possible by LM-processes, which provides the configurational energy to insert a water-soluble toxin complex into the underlying membrane.

When pore-forming endotoxins exploit the cellular LM process for the insertion of the pore-forming toxin into the membrane, the formation of oligomeric complexes occurs outside the membrane bilayer in the absence of lipids. CrylAc forms high molecular weight complexes when processed in the presence of gut juice extract, which are stable in SDS at 65°C and revert to low molecular weight proteins at 100°C (Figure 10, 29). To test whether the toxin can form oligomeric complexes in the absence of lipids, the 130 kDa toxin precursor was incubated with trypsin in a lipid-free buffer. Under these conditions a ca 60 kDa band was detected, which is close to the size of the mature toxin and in addition, a ca 230 kDa band, which conesponds in size to an oligomeric toxin-complex (Figure 29). Trypsin-incubation of the protoxin for more than two hours produced smaller diffuse bands, which indicates a proteolytic degradation of the mature toxin, similar to those occasionally observed in gut juice activated protoxin (Figure 29).

Since toxins function as SAMs it is possible to interfere with the process by expressing or applying components which aggregate around toxic SAMs and prevent it from interacting with MARMs (in fact this may be a role of the glycoprotein asialofetuin achieves in vertebrates).

EXAMPLE 4 SAM-HINGE MOLECULE COMPLEXES

A prerequisite of the LM-mediated membrane-insertion of pore-forming toxins is a lipoprotein complex with a lectin-like toxin. When CrylAc was tested in ligand-blotting experiments, the binding pattern was similar to GalNAc-binding lectins indicating that CrylAc interacts with GalNAc-specific glycoproteins like a lectin (Figure 8).

To examine possible toxin-interactions with lipoproteins, gut juice activated protoxin was mixed with cell-free hemolymph and the mixture separated by low-density gradient centrifugation. Oligomeric toxin complexes were detected at low-density regions of the gradient, which remained intact during SDS-extraction at 65°C (Figure 5, fractions 11-15). Tryptic peptide sequences from the oligomeric CrylAc-containing fraction revealed similarities to apolipophorin or hexamerin-like glycoproteins, indicating that Bt-toxins interacts with lipophorin-like molecules to form a soluble toxin-lipoprotein complex. In addition to soluble complexes, CrylAc formed large lipoprotein aggregates (Figure 5, fractions 1-19), which were distributed over the length of the gradient, resembling a coagulum or long threads of vitellin-like molecules. HMs are frequently lipoproteins, which can be Apolipophorin, modified Apolipophorin- like glycoproteins or hexamerin-like glycoproteins. In mammals the possible conesponding genes code for apolipoprotein B100 (or Apo E etc). EXAMPLE 5 MEMBRANE-ANCHORED MOLECULES (MARMs)

MARMs, such as hemomucin (Figure 6), interact with lectin and lipophorin. In the case of Bt-toxin insertion into the membrane aminopeptidase N and cadherin-like molecules. For other pore-forming toxins, other molecules can function as MARMs (Table 2). MARMs are membrane-anchored and fit the structural requirement of providing leverage to the SAMs and HMs (for example, MARMs may be rod-like structures with HM-binding sites at the proximal part of the extracellular domain and adhesion binding-sites, such as glycodeterminants or carbohydrate recognition domains, on a more distal part of the extracellular domain). These molecules can integrate into the membrane by fransmembrane domains or are linked to the membrane by GPI-mediated attachments (Table 1).

In summary, soluble adhesion molecules (SAMs), such as tetrameric lectins, cross-link membrane-anchored molecules (MARMs) around lipoproteins or bulky hinge molecules (HMs), tilting MARMs to cause a local inversion of the membrane curvature (Figure 4, 13 and 16). This "leverage-mediated uptake" mechanism (LM) involves lateral clustering of MARMs by SAMs, generating the configurational energy, which drives the reaction towards intemalization of the complex.

EXAMPLE 6 TRYPANOSOMA CRUZI INVASION

In contrast to lectinophagocytosis of bacterial cells (Ofek et al, Annual Review of Microbiology 49:239-276, 1995; Eichinger, Journal of Eukaryotic Microbiology 48:17-21, 2001), invasion by the protozoan parasite T. cruzi shows some remarkable features that are difficult to understand in the context of "zipper", "trigger" or any other receptor-mediated endocytosis mechanisms. Parasite attachment and invasion is probably the result of a complex reaction involving host and parasite functions. For example, some parasite surface molecules appear to participate in the recognition process, others are involved in enzymatic reactions and there is a requirement for energy on the part of the parasite (Burleigh et al, Annual Review of Microbiology 49:175-200, 1995). The difficulty in understanding T. cruzi invasion by a "trigger" mechanism is that the organism can infect many cell types that have different compilations of surface receptors.

T. cruzi invasion is a significant example of how initial intercellular interactions between parasite and host cells are characterized by a balancing act between cell-cell and leverage- mediated processes (Figure 21). Initial interactions cause the parasite to secrete trans- sialidase. This facilitates the removal of sialic acid residues from mammalian cell surface molecules and their transfer to mucin-like glycoproteins on the parasite surface. The latter process precludes the formation of lectin-linkages between the two organisms - a requirement for a "zipper" -mediated uptake (Ofek 1995, supra). Conversely, sialic acid removal from host glycoproteins promotes lectin-binding among lateral glycoprotein molecules via a "leverage" mechanism, thus producing an inverse membrane curvature on the host cell surface (Figure 21). The observed fusion of cytoplasmic vesicles with the cellular membrane is a response by the cell to restore the balance of cell surface glycoproteins. However, if the fransfer of sialic acid to the parasite continues, the merged vesicles remain in a curved formation until the parasite is completely engulfed by the host cell. While this process is difficult to explain using a zipper or "trigger" model, manipulation of "leverage"-mediated membrane curvatures by the parasite now provides a mechanistic framework which explains the parasite-induced opening of the membrane space. T. cruzi invasion requires relatively few lectin-linkages between the parasite and the cell to drag the parasite into the membrane opening. EXAMPLE 7 Bt-TOXIN BINDS TO A GaLNAc-SPECIFIC PROTEIN

Hemomucin is an immune receptor existing in several glycoforms (Figure 6), one is recognised by Helix pomatia lectin (HPL), which is specific for GalNAc-sugar configurations (the so-called HPL-form), another is recognised by peanut agglutinin (PNA), which is specific for Gal-sugar configurations (the PNA-form). In addition there exists a GlcNAc glycoform in cell lines, which is recognised by ConA (Figure 33). Several observations suggest that the PNA glycoprotein is the immune reactive form, which is not found in circulating glycoproteins and when attempts are made to extract from granules within hemocytes, forming cross-linked protein complexes which are subsequently lost during protein extraction. For example, PNA-forms exist in hemocytes and other tissues but is only recovered from ovaries, where the PNA-form is found in a modified form (Figure 7). Moreover, it is found on the surface of cultured mbn-2 cells (Theopold et al, Insect Biochem Mol R/ø/ 31:189-197, 2001) and when lectins are added to these hemocyte-like cells, PNA induced strong reactions of macropinocytosis with large and multiple vesicles visible inside cells (Figure 27). This indicates that the PNA-form is the immune reactive glycoform of hemomucin in the hemolymph forming covalent linkages with other proteins upon stimulation with lectin.

CrylA binds to hemomucin (Figure 8). CrylA binds to GalNAc sugar configurations (HPL glycoform) on Western blots, indicative of a HPL-like lectin function. However, when whole-mounts of gut tissue were analysed the observed binding patterns of HPL and Bt- toxin differed. Whereas HPL showed strong labelling to vesicles of sub-cellular size inside gut cells (Figure 9), PNA-staining was restricted to extracellular stmctures and intracellular vesicles resembling Golgi vesicles. When CrylA was used as a lectin, staining resembled the PNA-staining pattern. The extracellular stractures may contain phenoloxidase (Figure 11). Since phenoloxidase in lepidopteran is produced exclusively in hemocytes, the presence of phenoloxidase in gut epithelium is an indication that these particles must have migrated from the hemocoel into the gut. Accordingly, CrylA binds in a reversible fashion to GalNAc-containing sugar configurations present on many glycoproteins. However, under in situ conditions, binding in the gut is restricted to a sub-population of glycoproteins which also co-localise with PNA and phenoloxidase. This suggests that CrylA-binding to GalNAc may be a precondition but under non-denaturing conditions the prefened binding is to an immune- active glycoform, such as the PNA-form of a glycoprotein which is localised on microparticles in the gut. This suggests that the CrylA-binding protein in the gut lumen may be a soluble protein, which is also involved in immune reactions. EXAMPLE 8 TOXIN INSERTION INTO THE MEMRANE

Cunent models of Bt-toxicity assume that the toxin molecule is inserted into the membrane as a monomer by receptor-mediated interactions. However, toxin-insertion may occur by a novel uptake mechanism which generates the configurational energy required for the insertion of an oligomeric toxin complex into the membrane. A precondition for LM toxicity is the formation of tetrameric Bt-toxin complexes outside of the membrane in the absence of lipids. CrylAc forms high molecular weight complexes when processed in the presence of gut juice extract, which are stable in SDS at 65°C, but revert to low molecular weight proteins at 100°C (Figure 10, 29). To test whether processed toxin, can form oligomeric complexes in the absence of lipids, the 130 kDa toxin precursor was incubated with trypsin in a lipid-free buffer. Under these conditions a ca 60 kDa band was detected, which is the size of the mature toxin and in addition, a ca 230 kDa band, which conesponds in size to a tetrameric protein-complex (Figure 10, 29). Similar to the gut juice-mediated processed toxin, the complex was stable in SDS at 65°C, but was reduced to a ca 60 kDa protein at 100°C. Trypsin-incubation of the protoxin for more than two hours produced smaller diffuse bands, which suggests a proteolytic degradation of the mature toxin, similar to those occasionally observed in gut juice activated protoxin. This indicates that proteolytic processing of protoxin creates mature toxin, which forms oligomeric complexes before being inserted into the membrane. To further examine complex formation a time course was conducted. When CrylAc crystal was processed for different time periods in the presence of gut juice extracts, high molecular weight complexes were observed at intermediate times. When digestion continued for longer time periods, a 60 kDa protein appeared in addition to the putative mature 69 kDa protein (Figure 29). Under conditions where mixtures of the two proteins co-existed, the high molecular weight complex was separated into several nanow bands above 250 kDa (Figure 29, anows). The relative distribution of these high molecular weight complexes conelated with the relative amounts of the 60 and 69 kDa proteins, which is consistent with the formation of hetero-tetramers of 60 and 69 kDa proteins. Both the complexes and the 60 and 69 kDa proteins stained with anti-toxin antibodies (Figure 29B). The tetrameric complexes were stable in SDS at 65°C but reverted to low molecular weight monomers at 100°C (Figure 10, 29). A similar complex was observed when the 130 kDa toxin precursor was incubated with trypsin in a lipid-free buffer (not shown).

This suggests that Cry 1 Ac-toxin exists as a tetrameric complex with GalNAc-specific lectin properties, which can interact with soluble glycoproteins to form detergent-insoluble aggregates.

EXAMPLE 9 Bt-RESISTANT INSECTS ARE IMMUNE-INDUCED

Where tetrameric toxin interact with soluble gut pro-coagulant proteins (Figure 13C), the expected tolerance to the toxin should be inducible by immune elicitors. Coagulation proteins, such as hexamerin (Scherfer et al, Current Biology 14, 625-629, 2004), apolipophorin (Li et al, 2002) are produced in the fat body and released into the hemolymph as storage proteins and lipid caniers. Hexamerin and lipophorin-like proteins are also involved in immune defence reactions, including detoxification (Kato et al, 1994, Insect Biochemistry & Molecular Biology, 24:547-555; Nilcinskas et al., 1997, supra) and have been identified as a major pro-coagulant in insects (Scherfer et al, 2004 supra; Theopold et al, 2002, supra). It has been determined that lectins and insect toxins form coagulation aggregates and, like immune suppressors (Asgari and Schmidt, Insect Biochem Mol Biol, 32:597-504) are internalized by insect cells after forming a complex with lipophorin. This raises the question as to whether Bt-toxicity and Bt-resistance mechanisms are functionally conelated with a dual function of lipophorin-like molecules (lipid canier and endocytosis versus pro-coagulant and globule formation). In this context, toxin-insertion into the lipid membrane-bilayer may be mediated by an endocytosis reaction of a soluble lipoprotein- toxin complex (Figure 13B, 16), whereas toxin-inactivation may be caused by a coagulation reaction (Figure 13C).

To test this assumption, the immune-status of Bt-resistant and susceptible insects was tested. Genetic resistance to low levels of Bt-endotoxins has been selected in several species in the laboratory, but is common in field populations of diamondback moth (Fene and Nan Rie, 2001, Annual Reviews of Entomology 47:501-533). A Helicoverpa armigera strain was used, which has been selected in the laboratory for resistance to low levels of Bt-toxin. Resistant insect populations were subsequently back-crossed four times with a susceptible population to generate nearly isogenic lines of resistant (ISOC4) and susceptible (AΝGR) insects. Since spontaneous melanisation of hemolymph exposed to air is generally used as a reliable indicator for the immune-status of an insect, melanisation assays were performed with individual resistant and susceptible cateφillars. Cell-free hemolymph from Bt-resistant cateφillars differed from susceptible cateφillars in that the melanisation-reaction started from a higher level and increased at a higher compared to hemolymph from susceptible insects, where the start of the reaction was delayed for several minutes and produced less melanised reaction products (Figure 11). Likewise, hemocytes from resistant cateφillars aggregated more readily in blood smears, whereas hemocytes from susceptible cateφillars remain separated to form layers of individual cells. This indicates that the immune-status of the Bt-resistant strain differs from the susceptible strain.

If the Bt-resistant strain is constitutively immune-induced, Bt-resistance may protect against other pathogens as well. This assumption was tested by feeding cateφillars with baculoviruses. Since baculovims virulence in semi-permissive insects is dependent on the immune-status of the insect (Washburn et al, 1996, Nature 383:767), any differences in virulence between Bt-resistant and susceptible cateφillars can be used as an indication for a conesponding change in the immune-status of the insect. When Autographa cάlifornica M Nucleopolyhedrovims (AcMNPN) was applied to cateφillars from both strains, Bt- resistant cateφillars were less vulnerable to the virus than susceptible cateφillars (Figure 17). Similar results were obtained when virions were injected into the hemocoel. This indicates that resistant insects are not only resistant to the Bt-toxin but to other non-related pathogens as well.

EXAMPLE 10 A MOLECULAR ENGINE THAT DRIVES CELLULAR PROCESSES

The present invention describes the molecular features of a protein complex that has the capacity to generate configurational energy through leverage-mediated (LM) processes, which can produce an inverse curvature of the membrane and dislodge receptors from cytoplasmic attachments (Figure 13,A-C). This is different from cunent models that imply cytoplasmic motor proteins for uptake reactions driven by chemical reactions.

The existence of leverage-mediated protein assemblies that can provide the configurational energy to drive cellular uptake reactions has important implications for the functional role of exfracellular and intracellular molecules involved in these reactions:

(i) The LM-system describes ligands as soluble adhesion molecules (SAM) with multiple binding domains (e.g. oligomeric lectins) that cluster cell surface molecules (receptors), thereby bending the axes of receptors relative to the membrane.

(ii) In the LM-system, oligomerization (including dimerization) of monomeric precursors constitutes an essential step for the regulation of functional properties of SAMs and HMs by intrinsic regulatory cascades. (iii) In this system, receptors are defined by the capacity to interact with SAMs to generate configurational energy via LM-mechanisms.

(iv) Receptors are potentially involved in multiple reactions with SAMs upstream of signalling, such as adhesion to external extracellular binding sites and uptake reactions, creating a dynamic balance of different reactions, such as phagocytosis (zipper-mediated reaction) and induced macropinocytosis (LM-reaction).

(v) In this system, the role of actin-cytoskeleton is to regulate receptor-stability on the cell surface e.g. to prevent receptors from engaging in lateral LM-reactions by keeping receptors stabilised in an upright position to enable interaction with external binding sites.

(vi) The LM-mechanism involves a dynamic interaction of SAMs and receptors in extracellular space, producing cellular shape changes as the result of a balance of complex multiple exfracellular reactions, which do not necessarily require multiple receptor pathways.

The identification of an exfracellular driving force is based on three lines of investigation providing evidence of extracellular complexes that acquire curved or globular shapes through the assembly of conical shaped proteins (Figure 13C, 14A.

(i) A mechanism of cellular immune-suppression in parasitoid-host systems invokes disruption of receptor-actin attachments by suppressor uptake. Immune suppression is based on two observations: Firstly, macropinocytosis is induced by oligomeric lectins in an actin-independent reaction, which indicates uptake reactions driven by an extracellular driving force. Secondly, since these uptake reactions are dependent on lectin-mediated lateral cross-linking reactions of receptors, the putative role of actin-cytoskeleton is to prevent receptors from intemalization and thus allows interactions to external binding sites. Immune suppression is achieved by suppressor-mediated uptake reactions, which dislocate receptor-attachments to actin-cytoskeleton. In the absence of receptors attached to actin, cells are unable to interact with external binding sites, such as subsfrate, other cells and microbes. Since hemomucin is the only lectin-binding receptor in hemocyte-like Drosophila cells, multiple lectin-inducted processes, such as cell adhesion, spreading/detachment (Figure 33), aggregation, induced macropinocytosis (Figure 27), cell proliferation, and immune induction, must all be mediated by hemomucin. The involvement of oligomeric lectins in multiple reactions on the same cell indicates that soluble oligomeric adhesion molecules interact with the same cell surface receptors to produce different outcomes. This indicates that lectins can engage receptors in lateral cross-linking (uptake reactions) or attach to external binding sites (adhesion and spreading) in the same cell (Figure 26). Thus lectins may engage receptors in various exfracellular interactions that produce different outcomes. Some extracellular protein assemblies provide stmctural energy that drives the inverse curvature of the membrane and sculpturing of the membrane.

(ii) The globular stmcture of lectin-mediated coagulation products indicates that aggregation of oligomeric adhesion molecules with coagulation proteins self- assemble into larger stmctures that form a vesicle-like structure. Similar stractures may be formed on the surface if procoagulants and oligomeric lectins interact with receptors to form higher aggregates (Figure 14A). Therefore the assembly is able to create an inverse curvature of the membrane and bent receptors relative to their cytoplasmic anchorage. Changes in the shape of a cell's membrane are integral to numerous cellular processes, including attaching to and detaching from substrates, engulfing particles (phagocytosis) and the uptake of fluid droplets (pinocytosis). In turn, these processes are intimately involved in a diverse range of phenomena, including cellular immune responses, the directional mobility of cells or the directional growth of neurons in animals and pollen tubes in plants. Outcomes from new approaches based on the LM-model

The leverage-mediated uptake (LM) mechanism provides the configurational energy to insert oligomeric adhesion molecules into the membrane. This has implications for the understanding of toxicity and potential resistance mechanisms to pore-forming toxins.

(i) The mechanical stress inherent to LM-processes can produce osmofragility during uptake reactions, which is the basis of membrane-toxicity of pore-forming toxins (Figure 28). The finding is that pore-forming toxins are inserted into the membrane as oligomers (Figure 29).

(ii) Coagulation molecules that form globular coagulation products in the presence of oligomeric adhesion molecules can act as a decoy to inactivate pore-forming toxins in the gut lumen (Figure 9). This is the basis of the immune-inducible resistance mechanism against pore-forming toxins.

EXAMPLE 11 FOCAL ADHESION CLUSTER

Once receptors make contact to external binding sites, the cell membrane will be locally deformed by mechanical stretching forces applied to the membrane area sunounding the receptors. This will dislocate receptors from cytoplasmic anchorage around the adhesion contact and through lateral movement facilitate LM-complexes in the presence of extracellular oligomeric adhesion molecules. The result is that these LM-assemblies create a concave curvature of the membrane upon attachment to external binding sites (Figure 19). Such concentric rings of adhesion molecules during intemalisation have been observed in cells forming so-called glycosynapses (Hakomori, 2002) and immunological synapses (Grakoui et al., 1999).

Mechanical disraptions of receptor-stabilisation will increase lateral receptor-mobility and LM-complex formation at the site of stress thus increasing clustering of LM-complexes with a tendency to create a concave curvature of the membrane (Figure 19).

This provides an integration of mechanical stress and cell behaviour, where several complex cellular processes are conceptually integrated and dependant on the size and shape of the object to which the cell is attached (Figure 26). If the external object is relatively small and can be moved by the action of LM-membrane invaginations around the initial adhesion site (Figure 19), the object will be internalised by a phagocytosis reaction (Figure 4). Depending on the surface properties of the object the uptake could be a 'zipper' or 'velcro-like' engulfment or a loosely fitting membrane vesicle containing the object.

If the external object is another cell, the two cells will attempt to internalise each other by LM-invaginations around the adhesion sites. This will bring the two membranes and their adhesive receptors into close proximity with the result that both cells will increase their adhesive ability and with it their LM-intemalisation capacity. If both cells have similar LM-capabilities the two membranes will form a straight line between the two cells, representing a balance of forces (Figure 14B). If the two cells differ in their ability to interact, the result will be imbalances that eventually lead to phagocytosis or disengagement (Figure 35).

If the external binding site is a subsfrate surface, the cell will attempt to phagocytize this very large object by a 'zipper' or 'velcro' -mechanism. The strength of focal adhesion clusters depends on the recruitment of new adhesive receptors that are able to connect to external binding sites, which may be a function of cytoplasmic receptor-stabilisation and local binding site densities. For example, new receptors aniving at the cell surface will either become attached to external sites or internalised by LM-mechanisms and high densities of binding sites on the subsfrate will increase the chance of receptor-attachments. Likewise, the stabilisation of receptors by cytoplasmic scaffolds will act to keep receptors on the cell surface for longer, increasing the probability they will make contact to external binding sites. A case in point are receptors that facilitate formin-mediated vesicle transport to the cell surface (Higashida et al., 2004), which may also be stabilised against LM-uptake reactions by actin-cables after Golgi-derived vesicles have merged with the cell membrane.

This implies that receptor-stabilisation is a critical part of a dynamic cellular process. For example, the mobilisation of cytoplasmic proteins that enhance receptor-anchorage and attachment to external binding sites decreases LM-complex formation (ki) and receptor- internalisation (fø), thereby increasing the number of adhesive receptors on the cell surface (Figure 32; equation (2)). Likewise, the number of external binding sites determine the likelihood of receptor-stabilisation to subsfrate and thus directional mobility if binding sites are distributed in a gradient (Figure 36). Conversely, the exposure of cells to oligomeric adhesion molecules will increase receptor-uptake and reduce adhesive cell properties, acting as immune suppressors or tip growth repellents.

EXAMPLE 12 CELL-SHAPE CHANGES BASED ON UPTAKE REACTIONS

The model also suggests that the evolutionary processes leading to tissue formation in multicellular organisms were, at least initially, not based on 'cooperative' interactions but rather resembling a tug of war, where one cell tried to phagocytose the other (Figure 14AB). This raises the question how tissue-forming cells dissociate from each other during cell division, differentiation, tissue remodelling, or wound-healing. For a single cell to reduce its cytoplasmic receptor-stabilization unilaterally is not an option, since this would lead to phagocytosis by neighbouring cells (Figure 14B).

One possible mechanism involves the proteolytic cleavage of adhesive connections to neighbouring cells. For example, in the case of neuroblast determination by Notch/Delta interactions (Artavanis-Tsakonas et al., 1999), proteolytic separation of the extracellular Notch domain may release adjacent cells from adhesive connections leading to receptor- uptake reactions in membranes facing the secreting cell (Parks et al., 2000), a prerequisite for LM-signalling (Figure 34). Moreover, as a result of receptor-intemalisation the signalling cell is detached from epithelial connections, a prerequisite for cell delamination and migration (Figure 35).

An alternative pathway for cells to detach from tissues is to secrete oligomeric adhesion molecules into the intercellular space, thus enhancing LM-complex formation (k₂) and receptor-intemalisation (k£), and eventually causing detachment from neighbouring cells. This would involve the secretion of oligomeric adhesion molecules with the ability to internalise receptors, depleting adhesive receptors from the cell surface and eventually detaching cells from their adhesive connections. An example is the action of the matricellular proteins, where dislocation of adhesive receptors from cytoplasmic anchorage is possible through the mechanical stress imposed by contraction of the extracellular matrix, which resembles mechanical stretching, causing receptors to bend and eventually dislocate from their cytoplasmic anchorage (Figure 19). In line with the model, the localised secretion of matricellular proteins into the exfracellular space detaches cells from neighbouring cells or extracellular matrix. Under conditions where matricellular protein production and secretion is restricted to a single cell by lateral inhibition processes, cells will be released from adhesive connectivity without affecting the overall integrity of epithelia or other tissues (Figure 35).

The relaxed adhesive connectivity in turn allows the cell to undergo cell division (Figure 35a) or migrate to new locations. An example for the latter process is the fate of neuroblasts in Notch-expressing cells (Figure 35b), which involves lateral feedback reactions to release a single cell from the adhesive environment, while retaining a balance of forces in the sunounding epithelium. A prediction of this model is that systemic over- expression of counter-adhesion molecules will reduce the number of cell surface receptors, leading to tissue destabilisation (Mettouchi et al., 1997 supra) and increased cell detachment. This in turn will enhance cell division and cancer (Huang et al., 2001 supra; Sargiannidou et al., 2001 supra). Conversely, systemic under-expression of counter- adhesion molecules will lead to increased cell adhesion and wound closure (Bradshaw et al., 2002 supra). Under the LM-model, the counter-adhesion molecules can act as dynamic driving forces in extracellular space in addition to being signalling molecules (Greenwood and Muφhy, 1998 supra).

EXAMPLE 13 CELL MOBILITY

Cells with surface receptors that adhere to the substrate spread by 'zipper' or 'velcro'-like receptor-attachments to substrate binding sites at the leading edge. With the LM-model, the localised secretion of soluble adhesion molecules with matricellular protein properties will cause receptors to internalise faster at the site of secretion, while not affecting the ability of the cell to spread in other areas, thus moving the cell in one direction rather than spreading unilaterally (Figure 36a). Likewise, if cell surface receptors are stabilised by attachments to external sites, any uneven distribution of binding sites will cause the cell to move towards higher densities of binding sites (Figure 36b). Since the two mechanisms are not mutually exclusive, the combination of detachment reactions causing delamination of cells (Figure 35) and the polarised secretion of soluble adhesion molecules can explain directed cell movements in simple terms.

In contrast to actin-driven membrane protmsions (Goldberg, 2001), cellular outgrowth based on tip-growth, observed in filopodia, plant rootlets, pollen tubes (Lord and Russell, 2002) and nerve axons (Goldberg, 2003) are driven by the addition of membrane material at the leading edge. Transport of membrane vesicles to the tip is dependent on formin- mediated actin cables, where membrane vesicles move to and from the tip to exchange material with the cell membrane by exo/endocytosis reactions. Growth is due to the release of Golgi-derived vesicles increasing membrane material at the tip. Under the LM-model, this is a dynamic process, since membrane receptors aniving on the tip surface with new membrane material will also be targeted for intemalisation by LM-mechanisms (Figure 37). Tip growth occurs when more membrane material is added by vesicle exocytosis than internalised by LM-uptake (kjR_v > nk_∑C). In this context, tip growth will be enhanced by the stabilisation of receptors by cytoplasmic protein networks or actin cables, allowing adhesive receptors to be retained on the cell surface longer, thereby increasing the probability of their making contact to external binding sites, thus reducing &₂.

EXAMPLE 14 DIRECTIONAL TIP GROWTH

In the context of the model, adhesive receptors emerging at the tip of a cellular outgrowth are stabilised either by intracellular scaffolds or by attachment to exfracellular binding sites, such as receptors on other cells, adhesive binding sites on substrate or the newly deposited extracellular mafrix or cell wall. This dynamic interaction makes tip growth directly dependent on local densities of binding sites, which provides a conceptual basis for directional growth along gradients of binding determinants. A prediction of this model is therefore that tip growth is enhanced by receptor-attachments to external binding sites causing directional outgrowth at the tip along increasing densities of binding sites. Conversely, the presence of oligomeric adhesion molecules with counter-adhesive properties may slow down or prevent tip growth processes, such as cell spreading and filopodia formation by increasing LM-complex formation (ki) and receptor intemalisation (kj). In the context of this model, the inability of plant pollen to form or extend a tube in the presence of certain stigma-derived factors could result from the protein having extreme counter-adhesion properties that are able to overcome receptor-stabilisation in the pollen cell. During self-incompatibility reactions, pollen outgrowth is zero when (kjR_v = foR_s"), which is achieved when self-incompatible factors (Staiger and Franklin-Tong, 2003) engage pollen cell receptors in LM-assemblies that overcome receptor-stabilisation leading to cellular inactivation by receptor-depletion and actin depolymerisation.

Similar observations in parasitoid-mediated immune suppression of host hemocytes suggest that lectins (Figure 25) and immune suppressors (Asgari et al., 1997) may engage receptors in LM-uptake reactions that force receptor-internalisation and actin-cytoskeleton breakdown (Asgari and Schmidt, 2002). The observed outcomes in parasitised larvae are rounded hemocytes that lack the ability to spread, phagocytize or extend filopodia (Asgari et al., 1997). Immune-suppressed hemocytes may be cytologically similar to pollen cells on incompatible stigmas, which are xmable to extend pollen tubes in the presence of male factors (Staiger and Franklin-Tong, 2003).

Growth of nerve axons and axon guidance may follow similar principles to pollen tube growth. The direction of growth at the tip of never axons is influenced by soluble and substrate molecules with guidance functions (Tessier-Lavigne and Goodman, 1996). Some secreted adhesion molecules, such as SLIT (Brose et al., 1999), interact with cell surface receptors as a repellent, whereas the same molecules may interact with other receptors to promote tip growth (Dickson, 2002). In line with the model, the ability of a receptor to be retained on the cell surface depends on the stmctural features of the conesponding LM- complexes to internalise receptors and remove them from the tip (Figure 37). Gradients of potential counter-adhesion molecules may therefore prevent tip growth in one set, but promote growth in axons with another set of receptors.

Given that receptor densities on the cell surface are a direct consequence of dynamic exo/endocytosis reactions, this raises the question of whether LM-mechanisms play a role in membrane sculpturing in the cytoplasm, such as vesicle formation in the endoplasmatic reticulum. EXAMPLE 15 QUALITY CONTROL, PROTEIN SECRETION AND RECYCLING

LM-assemblies are not confined to the cell surface. In fact, the functional properties of LM-assemblies are likely essential for membrane trafficking in the cytoplasm (Figure 20). Membrane vesicle formation is relevant to membrane traffic from and to the cell periphery (Bonifacino and Glick, 2004), including receptor recycling, retrograde transport (Figure 38a), as well as protein processing in the ER and Golgi (Figure 38b). In fact, the putative function of ER and Golgi-specific LM-assemblies involved in vesicle formation may constitute some of the molecular criteria for quality control of secreted proteins. For example, proteins emerging in the ER that do not readily engage in LM-vesicle formation may be eventually removed by ER-specific proteases. This also applies to retrograde receptor transport and receptor recycling. During the process of endosome formation, functional LM-complexes may be disassembled and after merging with other vesicles, reassembled for recycling and retrograde transport (Hauri et al., 2000). For example, dissociation of LM-complexes may occur at low pH (Rudenko et al., 2002) and calcium concentrations (Clague, 1998), which destabilise some oligomeric adhesion molecules. This may allow receptors to form new LM-complexes after the merger of endosomes with other cytoplasmic vesicles (Mellman, 1996; Rothman, 1994). Again proteins that are able to quickly assemble into new LM-complexes form new vesicles that are transported and recycled, whereas proteins that are left behind are marked, degraded or transfened across the membrane into the cytoplasm for further processing by the proteasome. This indicates that dynamic interactions involving multi-protein assemblies may not be confined to receptor-interactions on the cell surface, but include LM-driving forces and functional check-points inside the cell. Consequently, the dynamics of receptor movements during recycling, retrograde transport and vesicle formation in the ER, Golgi and other cytoplasmic vesicles, have properties that can be described in mathematical terms that are similar to those used to describe receptor alterations on the cell surface. EXAMPLE 16 LECTIN-INDUCED HEMOCYTE INACTIVATION IN INSECTS

HPL can mediate both clustering and detachment reactions

To exclude cell-type-specific lectin-effects, such as plasmatocyte-specific spreading factors (Strand et al, 2000, supra), hemocytes from lepidopteran species were studied, and confirmed that lectin-effects were detected on all cell-types, including granulocytes and plasmatocytes. For example, when spread hemocytes and hemocyte-like cells from the lepidopteran H.armigera were treated with fluorescence-labelled HPL, the first visible changes on the cell surface were small patches of labelling indicating clustering of receptors. These clusters were observed adjacent to existing macropinocytosis vesicles (Figure 15), created previously by constitutive macropinocytosis involving actin-dependant membrane raffling (Johannes and Lamaze, 2000, supra). After prolonged incubation with soluble HPL, detachment from the subsfrate was observed in H. armigera (not shown) and P. rapae hemocytes (Figure 22). This indicates that HPL can interact with glycoprotein receptors and mediate both receptor clustering and detachment in the same cell.

Uptake of HPL can occur independent of F-actin

It is known that cell adhesion and spreading requires an intact actin cytoskeleton (Rogers et al, 2003, supra), with inactivation of F-actin causing detachment of cells or inactivation of hemocytes (Asgari et al, J. Gen. Virol. 78:3061-3070, 1997). Actin-involvement in endocytosis is particularly pronounced in yeast (Shaw et al, Exp Cell Res 271:1-9, 2001) It has been recognised for some time that actin-cytoskeleton also plays a role in phagocytosis

(Helantjaris et al, J. Cell Biol. 69:407-414, 1976), but the exact nature of that role has not been clear. When spread haemocytes from P. rapae were treated with cytochalasin D, cells detached from the glass surface and acquired spindle-shapes (Figure 22, cytD). Similar to hemocyte-like cells, lepidopteran hemocytes that were treated with the oligomeric lectin

HPL also detached and rounded-up, although less quickly and with only few hemocytes acquiring spindle-form cell shapes compared to the cytochalasin D-treated cells (Figure 22, HPL). This indicated that HPL can interfere with cellular attachments to external binding sites in ways that resemble cytochalasin D treatment.

To examine whether lectin-uptake can occur independently of F-actin, Cytochalasin D- treated hemocytes were incubated with TRITC-conjugated HPL and inspected under a confocal microscope after quenching of extracellular fluorescent dye by the addition of toluidine blue. Inspection of median optical cellular sections showed inegular shaped endosomal vesicles mostly in the cortical region of the cell (Figure 22cyt+HPL; Figure 23, anows). The emerging endosomal vesicles remained at the cell periphery and were not transported to the cell interior. This indicates that vesicle formation can occur independently of the actin-cytoskeleton, whereas the processes of vesicle transport and receptor-turnover at the cell surface are impaired when the actin-cytoskeleton has been disrupted.

HPL can overcome F-actin anchorage of receptors

Receptors can be readily internalised by lectins in the absence of intact cytoskeleton. For example, when haemocytes that had been surface stained with HPL were treated with cytochalasin D the surface staining disappeared (see below), This raises the question of whether receptors on the cell surface require cytoplasmic stabilisation to prevent immediate intemalisation.

To examine the presence of cell surface receptors during lectin-mediated uptake and detachment, viable hemocytes were treated with TRITC-conjugated HPL to invoke the uptake and detachment reactions. After hemocytes had detached, cells were fixed and surface-stained with FITC-conjugated HPL. In these cells, the FITC-staining was significantly reduced compared to non-treated spread cells. In those hemocytes where lectin-staining was detected it was either clustered on the cell surface or incoφorated by the hemocytes (Figure 24, FITC) with some of the incoφorated FITC co-localising with TRITC-staining (Figure 24, TRITC). This supports the notion that HPL is able to induce uptake reactions by clustering glycoprotein receptors on the cell surface, and during this process, remove receptors from the cell surface by receptor intemalisation. The observations that internalised receptors were not recycled to the cell surface, or replenished by newly synthesised membrane vesicles, suggest that membrane transport within HPL- treated cells is impaired. Although HPL-treated cells showed a small amount of vesicle movement to the cell interior (Figure 24), it was apparent that TRITC-stained vesicles remained mostly at the cell periphery and that lectin-binding receptors were eventually removed from the cell surface. HPL can induce actin-depolymerization

Since vesicle movement is impaired in HPL-freated cells (Figure 24), whether HPL can disrupt receptor-anchorage during uptake reactions and in the process destabilize the actin- cytoskeleton. We therefore exposed suspended hemocytes to HPL and compared attachment and spreading to hemocytes kept in the absence of lectin. To minimise haemocyte aggregation, hemocytes were diluted into large volumes of buffer and kept continuously in suspension by gentle rotation. Although most cells aggregated in the presence of lectin, only a small fraction of individual cells attached to the surface, whilst most hemocytes remained round and without contact to the surface. Cells that attached and spread to some degree showed partial actin-cytoskeleton breakdown with characteristic FITC-clustering around the nucleus (Figure 25). This suggests that lectin-mediated uptake reactions in suspended hemocytes removed lectin-binding receptors from the surface and caused partial actin-depolymerisation. Similar to small cell aggregates found in spread cells (Figure 24), aggregates formed during lectin incubation retained lectin-binding receptors on cells that were sunounded by other cells and were able to spread after making contact with the surface. In contrast, those cells on the periphery of the aggregate appeared to have a much lower number of remaining cell-surface receptors and as a consequence were precluded from spreading.

EXAMPLE 17 STRUCTURAL SPECIFICITY OF BIOLOGICALLY ACTIVE PEPTIDES

Pore-forming toxins

In the LM-mechanism, the ring-shaped pore complex is formed before or during the assembly of receptors around the oligomeric adhesion molecule (Figure 28), which is different from the cunent assumption that pore-forming toxins, such as crystal endotoxins from Bacillus thuringiensis (Bt-toxins), are inserted into the membrane as a monomer by a receptor-mediated reaction and assembled into pore-forming complexes inside the membrane bilayer (de Maagd et al., Trends in Genetics 17:193-199, 2001). However, some Bt-toxins form tetrameric complexes when processed in vitro (Figure 29). Another intriguing observation is that some plant lectins increase osmofragility (Pande et al., Nature 385:833-838, 1998), which is not damaging to membranes, but may have mechanistic implications. Since Bt-toxins are lectins (Akao et al., Journal of Basic Microbiology 41:3-6, 2001; Burton et al., Journal of Molecular Biology 287:1011-1022, 1999), which recognise mucin-like glycoprotein-receptors (Knight et al, Molecular Microbiology 11:429-436, 1994), it is reasonable to assume that some pore-forming toxins are comprised of oligomeric lectins with covalently linked amphipathic peptides that have antibacterial properties. Indeed, secondary structure predictions, helical wheel/net diagrams and molecular mechanics calculations of membrane- inserting peptides from the Bt-toxin, form a strongly amphiphilic alpha-helix and show haemolytic activity in vitro comparable to that of bee venom peptide melittin (Szabo et al., 1993, supra). Similar results were obtained with the isolated a 4-loop-α 5 haiφin from the toxin, which showed that this peptide is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes (Gerber and Shai, 2000, supra). This indicates that pore-forming peptides are inserted into the membrane together with oligomeric adhesion molecules that interact with membrane receptors to engage in LM-uptake reactions.

With the LM-based insertion of Bt-toxin, insects may become tolerant to the toxin by immune induction. Since LM-assemblies contain lipophorin (Figure 28), which has pro- coagulant activities in insects (Li et al., 2002, supra), an alternative reaction to membrane insertion is a lectin-mediated coagulation in the gut lumen. Increased production of pro- coagulant in the gut lumen may therefore engage the lectin-like mature toxin (Figure 29) in coagulation reactions, instead of interacting with the brush-border membrane. Tolerance of Bt-toxin has been confirmed in laboratory strains of Ephestia kuehniella, where transient immune-induction caused Bt-tolerance, which can be transmitted to the next generation by a maternal effect Rahman et al, Proc Natl Acad Sci (USA) 101:2696-2699. Endosome maturation and receptor recycling

Some pore-forming and bi-partite toxins become active in sub-endosomal compartments (Collier and Young, Annual Review of Cell and Developmental Biology 19:45-70, 2003; Lencer et al., Biochimica et Biophysica Acta - Molecular Cell Research 1450:177-190, 1999; Pless and Wellner, Journal of Cellular Biochemistry 62:27-39, 1996; Sandvig and van Deurs, Annual Review of Cell and Developmental Biology 18:1-24, 2002). LM- mechanisms are not restricted to the cell surface but may play a role in retrograde protein transport and receptor recycling (Hauri et al., EERS Letters 476:32-37, 2000) by re- assembling into new LM-complexes after dissociation under the influence of pH- and ion changes inside the endosome. Many lectins dissociate into monomers in the absence of calcium ions, which release receptors from LM-assemblies allowing new assemblies to emerge after fusion with cytoplasmic vesicles (Figure 38 A). Conversely, some adhesion molecules may not form pores under conditions, where endosome formation occurs, but acquire the ability in post-endosomal compartments under different pH-conditions or with different receptors. Finally, the ability of vesicular proteins to re-organise into new LM- complexes, which form new vesicles can constitute a quality control in the endosomal maturation process. Only useful components, which are able to form new vesicles are retained, whereas proteins that remain in the original space are likely to be digested and degraded by ubiquitin-dependent proteases or transported into the cytoplasm to be digested by the proteasome.

Insertion of antibacterial peptides by an LM-mechanism is conceptually related to multipartite systems, where oligomeric adhesion molecules are vehicles for the insertion of amphipathic peptides into the membrane bilayer. For example, anthrax toxin is comprised of two functional components, the protective antigen (PA), which is a heptameric adhesion molecule that forms after the monomeric PA binds to the cell receptor and is cleaved by a protease. The two toxic components, the edema factor (ΕF) and the lethal factor (LF) bind to the oligomeric PA and are internalized by an endocytosis reaction and fransfened into the cytoplasm by an unknown process (Collier and Young, 2003, supra). In fact, it is the LM-uptake process, which allows the protective antigen to become internalized by an LM- mechanism. Firstly, the cytoplasmic tail of the anthrax toxin receptor is not required for toxin endocytosis (Liu and Leppla, J Biol Chem 278:5227-5234, 2003), an indication that uptake is not dependent on a signal. Secondly, PA induces endocytosis, acidification and ion flux (Zhao et al., J Biol Chem 270:18626-18630, 1995), resembling osmofragility induced by some lectins with amphipathic domains (Pande et al., Nature 385:833-838, 1998). Thirdly, the amphipathic loops of the oligomeric protective antigen are inserted into the membrane like a banel (Petosa et al., Nature 385:833-838, 1997), similar to other endotoxins, such as a-hemolysin and Bt-toxin (Shai, 2002, supra).

In line with the LM-model the EF and LF proteins may be transported into the cytoplasm by re-assembled LM-complexes comprising the protective antigen as a peptide-channel. Or the two proteins may be dissociated from the LM-complex inside the newly formed endosome and subsequently become part of new LM-assemblies in post-endosomal compartments with the configurational requirements to transfer the toxic proteins into the cytoplasm (Collier, Journal of Applied Microbiology 87:283, 1999). Finally, the function of the PA may be restricted to delivering the two toxic proteins into the endosome, where they are able to resist protease digestion and eventually transported into the cytoplasm by cell-derived protein transporters for degradation by the proteasome. If the two toxins survive degradation in the ER and cytoplasm, toxicity in the cytoplasm will occur. Accordingly, the anthrax toxin is internalized by LM-uptake reactions, which provide the configurational energy to transfer associated peptides into the cytoplasm.

Configurational specificity

Since LM-uptake reactions are the driving force for the insertion of pore-forming complexes into and fransfer of peptides across membranes, the specificity of each reaction is less dependent on individual protein-protein interactions, but rather on the configurational properties and functionality of the assembly. Thus the possible interaction of antibacterial peptides with LM-uptake assemblies may depend on stmctural requirements, which allows the peptide to fit into the clefts of oligomeric adhesion molecules, membrane-receptors and proteins that serve as hinges (Figure 28, 30), without damaging the functionality of the complex. This explains the observed peptide-specificity in terms of global protein structure rather than individual protein-protein interactions. This also means that biologically active peptides are able to specifically interact with LM- assemblies without the need to bind to any individual protein or receptors via specific protein-protein interactions. The outcome of this interaction may be the formation of a damaging pore, ion flux or alteration of the LM-uptake potential, which can modify cell behaviour and signalling.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds refened to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Zitzer, A. et al. (2000) Biochimica et Biophysica Acta Biomembranes, 20. TABLE 1

cross-linking oligomeric possible possible References agents status receptors functions lectins dimers, mucins, phagocytosis, cell Castro, 1987;

(C-type, galectins) oligomers glycoprotei adhesion, microbe Pendland, 1996; ns recognition Wilson, 1999; Vasta, 1999 pentraxins pentamers various clearance of Agrawal, 2001; Shrive, 1999; (C-reactive Clq, FcR, immune Swamakar, 2000; proteins, serum LRP complexes, Mold, 1999; amyloid P) regulation of Volanakis, 2001 complement thio-ester proteins pentamers, various clearance of Levashina, 2001 (a-macroglobulins, oligomers LRP immune TEP, complexes complement3) seφin-protease oligomers various clearance of Knauer, 1997; complex LRP immune Janciauskiene, 2001

(seφin-thrombin, complexes plasminogen activating protein) collectins oligomers ClqR, CR3 lectin and C- Ghebrehiwet, 2001; Hansen, 1998; (Mannose-binding όxfrimers pathway Nepomuceno, 1999 lectin, surfactant activation, protein, Clq, clearance & ficollins) recycling of immune components cysteine knot homodimers various growth and Young, 1999; Bergner, 1997; growth factors heterodimers immune Dissing, 2001 (EGF, IL, spaetzle) regulation lipocalins dimers lipocalin chemosensor, Akerstrom, 2000 receptors allergen, retinal- binding protein lactofenin lactofenin- iron-homeostasis McAbee, 2000; Nappi, 2000 receptor cross-linking oligomeric possible possible References agents status receptors functions

asialoglycoproteins multiple O- ASGPR endocytosis, Vandersmissen, glycosylation recycling of 1996; Rotundo, 1998 sites ligands and receptors advanced glycation multiple galectin3 clearance Zhu, 2000 end products glycosylation functions sites chitinase-like dimers, unknown mitogenic Homma, 1996; 999; proteins oligomers activities Kawamura, 1 van der Hoist, 2001 (imaginal disc growth factor)

TABLE 2

toxins oligomeric putative lectin activity references status receptors bact. oligomers glycoproteins Glycoconjugants, Saha, 1997; Zitzer, enterotoxins: (pentamers, (e-g- Gal, Gal/GalNAc, 2000; Cortajarena, hemolysins 2001; Wieckowski, heptamers) glycophorin, sialylated 1998; Sellman, 2001; diphtheria asialofetuin) glycoproteins Sandvig, 1996; toxin, Sellman, 1997; cholera toxin Valeva, 1995; Valeva, 2001; Shogomori, 2001; Barth, 2000; Inoue, 2001

Bacillus tetramer glycoproteins GalNAc Aronson, 2001; thuringiensis (Aminopepti Masson, 1994 toxin dase N, cadherin-like molecules) limulin pentamer CR3 sialic acid residues Armstrong, 1996

CELIII oligomer mucins Gal/GalNAc Kouriki-Nagatomo, 1999 misteletoe dimer/tetramer glycoproteins Gal Tonevitsky, 1996; lectin Srinivas, 2001

ricin dimer IL-2 Frankel, 1996; Fu, 1996; Jackman, 1994

whe ,at .. ge.rm 3xhomodιmer g S^>ly^Jco ^fproteins GlcNAc/NeuNAc/ ^F M^lta^ctⁿsu^eo^s=, ¹1⁹9⁹9⁸6> agglutinm GalNAc concavalin A tetramer glycoproteins GlcNAc Pande, 1998 TABLE 3 Serine-protease cascades controlling SAMs

Function SAM activating reaction references enzymes Gastmlation

Drosophila dorsal- spaetzle defective Toll ligand (1, 2) ventral polarity and snake immvme response easter

Horseshoe crab coagulogen Factor C coagulin (3) hemolymph clotting Factor B Clotting enzyme

Vertebrate C3 Clr C3a (4) complement Cls C3b C2

Vertebrate blood fibrinogen Vlla/Ixa Fibrin (5) clotting Xa thrombin

Claims

CLAIMS:

1. A method of regulating the uptake of an extracellular molecule by a cell, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

2. A method of regulating the uptake of an extracellular molecule by a cell in a subject, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism.

3. The method of claim 1 or 2, wherein said extracellular molecule is a soluble adhesion molecule.

4. A method of regulating the uptake of a soluble adhesion molecule by a cell, said method comprising modulating one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

(iv) the interaction of said membrane-anchored molecule with said soluble adhesion molecule; or

(v) the lateral clustering of said membrane anchored molecules relative to said hinge molecules. wherein the subject cell's leverage-mediated uptake mechanism is modulated.

5. A method of regulating the uptake of a soluble adhesion molecule by a cell in a subject, said method comprising modulating one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

6. The method according to any one of claims 1 to 5, wherein said soluble adhesion molecule is a proteinaceous or non-proteinaceous molecule.

7. The method according to claim 6, wherein said molecule is a multimeric molecule.

8. The method according to claim 6 or 7, wherein said molecule is a drag, hormone, growth factor, antigen, modulator of intracellular signalling, immune regulator or pore forming toxin.

The method according to claim 8, wherein said molecule is selected from:

(i) lectins

(ϋ) C-type lectins

(iii) galectins

(iv) pentraxins

(v) C-reactive proteins

( i) serum amyloid P

(vϋ) thio-ester proteins

(viϋ) a-macroglobulins

(ix) TEP

(x) Complement 3

(xi) seφin-protease complex

(xii) seφin-thrombin

(xiii) plasminogen activating protein

(xiv) collectins

(xv) Mannose-binding lectin

(xvi) surfactant protein

(xvii) Clq

(xviii) ficollins

(xix) cysteine knot growth factors

(xx) EGF

(xxi) IL

(xxii) spaetzle

(xxiii) lipocalins

(xxiv) lactofenin

(xxv) asialoglycoproteins

(xxvi) advanced glycation end products

(xxvii) chitinase-like proteins

(xxviii) imaginal disc growth factor

(xxix) bacterial enterotoxins: hemolysins (xxx) diphtheria toxin (xxxi) cholera toxin (xxxii) Bacillus thuringiensis toxin (xxxiii) limulin (xxxiv) CELIII (xxxv) misteletoe lectin (xxxvi) ricin (xxxvii) wheat germ agglutinin (xxxviii) concavalin A

10. The method according to any one of claims 1 to 5, wherein said MARM is selected

(i) mucins (ϋ) glycoproteins (ϋi) Clq (iv) FcR (v) LRP (vi) ClqR (vii) CR3 (viϋ) lipocalin receptors (ix) lactoferrin-receptor (x) ASGPR (xi) galectin3 (xii) glycophorin (xiii) asialofetuin (xiv) Aminopeptidase N (xv) cadherin-like molecules (xvi) IL-2 receptor

11. The method according to any one of claims 1 to 5, wherein said hinge molecule is an insect lipophorin-like protein, hexamerin-like glycoprotein, lipocalin, pentraxin, apolipoprotein B 100, apolipoprotein E, or macroglobulin.

12. The method according to any one of claims 2 to 11, wherein said modulation is achieved by introducing to said subject an agent which agonises or antagonises any one or more of:

(i) the interaction of a soluble adhesion molecule with one or more hinge molecules;

(ii) the localisation of the soluble adhesion molecule/hinge molecule complex proximally to both the surface membrane of the cell and one or more membrane anchored molecules;

(iii) the interaction of a membrane anchored molecule with a soluble adhesion molecule;

(iv) the lateral clustering of membrane anchored molecules relative to a hinge molecule.

13. A method of modulating cellular functional activity, which activity is induced and/or otherwise regulated by the assembly of an intracellular complex, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

14. A method of modulating cellular functional activity in a subject, which activity is induced and/or otherwise regulated by the assembly of an intracellular complex, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism wherein modulating the functioning of said elements regulates the functioning of the leverage mediated uptake mechanism.

15. The method of claim 13 or 14, wherein said method comprises modulating the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

16. The method according to any one of claims 13 to 15, wherein said soluble adhesion molecule is a proteinaceous or non-proteinaceous molecule.

17. The method according to claim 16, wherein said molecule is a multimeric molecule.

18. The method according to claim 16 or 17, wherein said molecule is a drag, hormone, growth factor, antigen, modulator of intracellular signalling, immune regulator or pore forming toxin.

19. The method according to claim 18, wherein said molecule is selected from: (i) lectins (ϋ) C-type lectins (iϋ) galectins (iv) pentraxins (v) C-reactive proteins (vi) serum amyloid P (vϋ) thio-ester proteins (viϋ) a-macroglobulins (ix) TEP (x) Complement 3 (xi) seφin-protease complex (xii) seφin-thrombin (xiii) plasminogen activating protein (xiv) collectins (xv) Mannose-binding lectin (xvi) surfactant protein (xvii) Clq (xviii) ficollins (xix) cysteine knot growth factors (XX) EGF (xxi) IL (xxii) spaetzle (xxiii) lipocalins (xxiv) lactofenin (xxv) asialoglycoproteins (xxvi) advanced glycation end products (xxvii) chitinase-like proteins (xxviii) imaginal disc growth factor (xxix) bacterial enterotoxins: hemolysins (xxx) diphtheria toxin (xxxi) cholera toxin (xxxii) Bacillus thuringiensis toxin (xxxiii) limulin (xxxiv) CELIII (xxxv) misteletoe lectin (xxxvi) ricin (xxxvii) wheat germ agglutinin (xxxviii) concavalin A

20. The method according to any one of claims 13 to 15, wherein said MARM is selected from (i) mucins (ϋ) glycoproteins (iϋ) Clq (iv) FcR (v) LRP (vi) ClqR (vϋ) CR3 (viii) lipocalin receptors (ix) lactoferrin-receptor (X) ASGPR (xi) galectin3 (xϋ) glycophorin (xiii) asialofetuin (xiv) Aminopeptidase N (xv) cadherin-like molecules (xvi) IL-2 receptor

21. The method according to any one of claims 13 to 15, wherein said hinge molecule is an insect lipophorin-like protein, hexamerin-like glycoprotein, lipocalin, pentraxin, apolipoprotein B 100, apolipoprotein E, or macroglobulin.

22. The method according to any one of claims 14 orl5, wherein said modulation is achieved by introducing to said subject an agent which agonises or antagonises any one or more of:

23. The method according to any one of claims 13 to 22, wherein said cellular functional activity is cellular signalling.

24. The method according to any one of claims 13 to 22, wherein said cellular functional activity is phagocytosis.

25. The method according to any one of claims 13 to 22, wherein said cellular functional activity is the attachment or detachment of said cell from an extracellular matrix.

26. The method according to any one of claims 13 to 22, wherein said cellular functional activity is the formation of a cell-cell interaction.

27. The method according to claim 26, wherein said cell-cell interactions are formed in the context of moφhogenesis, tissue sculpturing, wound healing or cell division.

28. The method according to any one of claims 13 to 22, wherein said cellular functional activity is the inactivation of cellular functioning via destablisation of the actin cytoskeleton.

29. The method according to any one of claims 13 to 22, wherein said cellular functional activity is tip growth.

30. The method of claim 29, wherein said tip growth relates to angiogenesis, axon formation, pollen tube formation or rootlet formation.

31. The method according to any one of claims 13 to 22, wherein said cellular functional activity is intracellular protein secretion.

32. The method according to any one of claims 13 to 22, wherein said cellular functional activity is endosome maturation.

33. The method according to any one of claims 13 to 22 wherein said cellular functional activity is protein recycling.

34. The method according to any one of claims 13 to 22, wherein said cellular functional activity is cell and tissue compatibility.

35. A method for the intracellular delivery of a molecule to a cell, said method comprising upregulating the functioning of any one or more structural or functional elements of said cell's leverage mediated uptake mechanism wherein upregulating the functioning of said elements upregulates the functioning of the leverage mediated uptake mechanism.

36. A method for the infracellular delivery of a molecule to a cell in a subject, said method comprising upregulating the functioning of any one or more stractural or functional elements of said cell's leverage mediated uptake mechanism wherein upregulating the functioning of said elements upregulates the functioning of the leverage mediated uptake mechanism.

37. The method according to claim 35 or 36, said method comprising upregulating the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

38. The method according to any one of claims 35 to 38, wherein said molecule is a biotic or abiotic compound.

39. The method according to claim 38, wherein said compound is a therapeutic or prophylactic drag or microorganism.

40. The method according to claim 39, wherein said microorganism is a virion.

41. The method according to claim 38, wherein said cell is a neoplastic cell and said drag is a pro-apoptotic drag.

42. The method according to claim 38, wherein said cell is an infecting microorganism and said dmg is a toxic compound.

43. A method for downregulating the microbial infection of a cell, said method comprising downregulating the functioning of any one or more stractural or functional elements of said cell's leverage mediated uptake mechanism wherein downregulating the functioning of said elements downregulates the functioning of the leverage mediated uptake mechanism.

44. A method for dowmegulating the microbial infection of a cell in a subject, said method comprising downregulating the functioning of any one or more structural or functional elements of said cell's leverage mediated uptake mechanism wherein dowmegulating the functioning of said elements dowmegulates the functioning of the leverage mediated uptake mechanism.

45. The method according to claim 43 or 44, said method comprising downregulation the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

46. The method according to any one of claims 43-45, wherein said cell is an infecting microorganism and said drag is a toxic compound.

47. A method for reducing pore-forming toxin induced cellular damage, said method comprising dowmegulating the functioning of any one or more stractural or functional elements of said cell's leverage mediated uptake mechanism wherein downregulating the functioning of said elements downregulates the functioning of the leverage mediated uptake mechanism.

48. A method for modulating pore-forming toxin induced cellular damage in a subject, said method comprising modulating the functioning of any one or more stmctural or functional elements of said cell's leverage mediated uptake mechanism wherein dowmegulating the functioning of said elements downregulates the functioning of the leverage mediated uptake mechanism and upregulating the functioning of said elements upregulates the functioning of the leverage mediated uptake mechanism.

49. The method according to claim 47 or 48 wherein said toxin is the endotoxin produced by B. thuringiensis and said modulation is upregulation of cellular damage in insects.

50. The method according to claim 47, 48 or 49, said method comprising modulating the functioning of any one or more of:

(i) the functioning of said molecule as a soluble adhesion molecule;

51. The method according to claim 50 wherein said modulation is downregulation of pore-forming toxin induced cellular damage and said downregulation is achieved by exposure of the extracellular toxin to a mimic of said MARM or hinge molecule.

52. A method for the treatment and/or prophylaxis of a condition in a subject, which condition is characterised by the abenant, unwanted or otherwise inappropriate cellular uptake of an extracellular molecule, said method comprising modulating the functioning of any one or more stractural or functional elements of said cell's leverage mediated uptake mechanism.

53. A method for the treatment and/or prophylaxis of a condition in a subject, which condition is characterised by the abenant, unwanted or otherwise inappropriate assembly of an infracellular complex, said method comprising modulating the functioning of any one or more structural or functional elements of said cell's leverage mediated uptake mechanism.

54. The method according to claim 52 or 53 wherein said condition is a viral infection and said modulation is down-regulation of the leverage mediated uptake mechanism for down-regulating the cellular uptake of the virus.

55. The method according to claim 52 wherein said condition is a neoplasia or metastises and said modulation is upregulation of the leverage mediated uptake mechanism for facilitating the delivery of a toxin.

56. The method according to claim 52 wherein said condition is a bacterial infection and said modulation is upregulation of the leverage mediated uptake mechanism for facilitating the delivery of an antibiotic.

57. The method according to claim 52 or 53 wherein said condition is insect cellular resistance to B. thurigiensis and said modulation is upregulation of the leverage mediated uptake mechanism for facilitating the enfry of said B. thurigiensis to said insect cells.

58. The method according to claim 52-57, said method comprising modulating the functioning of any one or more of: