Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development.

SummaryThe evolution of the seed system provides enormous adaptability to the gymnosperms and angiosperms, because of the properties of dormancy, nutrient storage and seedling vigour. Many of the unique properties of seeds can be exploited in molecular farming applications, particularly where it is desirable to produce large quantities of a recombinant protein. Seeds of transgenic plants have been widely used to generate a raw material for the extraction and isolation of proteins and polypeptides, which can be processed into valuable biopharmaceuticals. The factors that control high-level accumulation of recombinant proteins in seed are reviewed in the following paragraphs. These include promoters and enhancers, which regulate transcript abundance. However, it is shown that subcellular trafficking and targeting of the desired polypeptides or proteins play a crucial role in their accumulation at economically useful levels. Seeds have proven to be versatile hosts for recombinant proteins of all types, including peptides or short and long polypeptides as well as complex, noncontiguous proteins like antibodies and other immunoglobulins. The extraction and recovery of recombinant proteins from seeds is greatly assisted by their dormancy properties, because this allows for long-term stability of stored products including recombinant proteins and a decoupling of processing from the growth and harvest cycles. Furthermore, the low water content and relatively low bioload of seeds help greatly in designing cost-effective manufacturing processes for the desired active pharmaceutical ingredient. The development of cGMP processes based on seed-derived materials has only been attempted by a few groups to date, but we provide a review of the key issues and criteria based on interactions with Food and Drug Administration and European Medicines Agency. This article uses 'case studies' to highlight the utility of seeds as vehicles for pharmaceutical production including: insulin, human growth hormone, lysozyme and lactoferrin. These examples serve to illustrate the preclinical and, in one case, clinical information required to move these plant-derived molecules through the research phase and into the regulatory pathway en route to eventual approval.

SummaryThe increased incidence of diabetes, coupled with the introduction of alternative delivery methods that rely on higher doses, is expected to result in a substantial escalation in the demand for affordable insulin in the future. Limitations in the capacity and economics of production will make it difficult for current manufacturing technologies to meet this demand. We have developed a novel expression and recovery technology for the economical manufacture of biopharmaceuticals from oilseeds. Using this technology, recombinant human precursor insulin was expressed in transgenic plants. Plant-derived insulin accumulates to significant levels in transgenic seed (0.13% total seed protein) and can be enzymatically treated in vitro to generate a product with a mass identical to that of the predicted product, DesB 30 -insulin. The biological activity of this product in vivo and in vitro was demonstrated using an insulin tolerance test in mice and phosphorylation assay performed in a mammalian cell culture system, respectively.

CD59 is a glycosylphosphatidylinositol-anchored cell surface glycoprotein involved in protecting cells from host-mediated complement attack. Studies have shown that antibody cross-linking of CD59 induces a series of intracellular signaling events including the activation of protein-tyrosine kinases (PTK). To further characterize these events, antibodies and complement 8, one of the natural ligands of CD59, were used to activate CD59. Antibody-induced cross-linking of CD59 on the surface of THP-1 and U937 hematopoietic cell lines as well as exposure to complement 8 induces a rapid increase in the tyrosine phosphorylation of several proteins within the cell. Consistent with an early role for the Src family PTKs in these signaling events, we found that transient activation of Hck-and CD59-mediated signaling was abrogated in the presence of the Src family PTK-selective inhibitor PP1. Although the molecular mechanism by which CD59 communicates to Hck is unknown, cellular fractionation studies indicated that both CD59 and Hck are compartmentalized in plasma membrane microdomains. We also detected tyrosine phosphorylation of the adaptor proteins p120 cbl and Shc, and the cytoplasmic nonreceptor tyrosine kinase Syk. The identification of CD59-mediated signaling events may help explain why paroxysmal nocturnal hemoglobinuria patients, who are deficient in glycosylphosphatidylinositol-linked proteins including CD59, are susceptible to proliferative disorders.CD59 is one of a large family of proteins that are attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) 1 moiety attached to their C-terminal ends (1). It is abundantly expressed on a wide variety of cells, including many cells of hematopoietic lineage (2-5), and functions to inhibit complement-mediated lysis by interfering with the assembly of the membrane attack complex and the insertion of complement 9 into the cell membrane (reviewed in Ref.6). The natural ligands for CD59 include the ␣-chain of complement 8 (C8) and the "b" domain of complement 9 (7).Antibody-mediated cross-linking of CD59 on various hematopoietic cells induces a cascade of intracellular signaling activities including events such as calcium influx and release of calcium from intracellular stores, generation of reactive oxygen intermediates (8 -12), and induction of tyrosine phosphorylation (13,14). These events are not unique to CD59, since many other GPI-anchored proteins induce a series of similar intracellular signaling events when cross-linked or upon binding of appropriate ligand. Other GPI-linked molecules expressed on hematopoietic cells include the lipopolysaccharide receptor (CD14) (15, 16) and THY-1, a regulatory molecule in T-cell activation (17, 18).Since GPI-anchored proteins adhere to cells through their acyl chains and do not span the membrane, the mechanism by which this family of proteins transduce their signal into the cell remains unknown. However, GPI-linked proteins have been shown to cluster in detergent-insoluble glycolipid-enriche...

SummaryApolipoprotein AI Milano (ApoAI Milano ) was expressed as a fusion protein in transgenic safflower seeds. High levels of expression corresponding to 7 g of ApoAI Milano per kilogram of seed have been identified in a line selected for commercialization.The ApoAI Milano fusion protein was extracted from seed using an oilbody-based process and matured in vitro prior to final purification. This yielded a Des-1,2-Apo-AI Milano product which was confirmed by biochemical characterization including immunoreactivity against ApoAI antibodies, isoelectric point, N-terminal sequencing and electrospray mass spectrometry. Purified Des-1,2-ApoAI Milano readily associated with dimyristoylphosphatidylcholine in clearance assays comparable to Human Apo-AI. Its biological activity was assessed by cholesterol efflux assays using Des-1,2-Apo-AI Milano :1-palmitoyl-2-oleoyl phosphatidylcholine complexes in vitro and in vivo. This study has established that high levels of biologically functional ApoAI Milano can be produced using a plant-based expression system.

The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates its activity through binding to cell surface receptors. The receptor for GM-CSF belongs to a superfamily of cytokine receptors characterized by a conserved extracellular motif. The high affinity GM-CSF receptor (GMR) consists of two transmembrane anchored subunits; a ligand binding alpha subunit (transmembrane GMRalpha) and a signal transducing beta subunit (GMRbeta), both of which belong to the cytokine receptor superfamily. The human GM-CSF receptor alpha subunit also exists in a soluble form (solGMRalpha), which antagonizes GM-CSF activity in vitro. We directly tested the potential for solGMRalpha to interact with GMRbeta in vitro. Our experiments demonstrated that exogenous solGMRalpha, even in the presence of GM-CSF, does not interact with GMRbeta on the cell surface. However, when solGMRalpha and GMRbeta are co-expressed in baby hamster kidney cells, solGMRalpha is retained on the cell surface and forms a functional intermediate affinity GM-CSF binding complex (Kd = 331 pM). In addition, the cell surface expression of solGMRalpha is independent of the presence of GM-CSF as demonstrated using flow cytometry. Cells expressing only solGMRalpha do not show cell surface retention or form functional GM-CSF cell surface binding complexes. Sequencing of our GMRbeta clone revealed a nucleotide substitution (A --> C) resulting in the substitution of Ala for Glu at position 9 from the amino terminus of the mature GMRbeta peptide. Because the GMRbeta (A --> C) clone is capable of forming functional high affinity receptors with transmembrane GMRalpha (Kd = 64 pM), we feel that the cell surface retention of solGMRalpha is independent of the GMRbeta mutation. We suggest that the co-expression and interaction of solGMRalpha and GMRbeta represents a previously unrecognized GM-CSF receptor complex and a novel, ligand-independent mechanism of cytokine receptor assembly.

A new diagnostic technique based on DNA heteroduplex analysis has been used to identify specific point mutations in the von Willebrand's factor (vWF) gene of patients with von Willebrand's disease type 2B. Molecular analysis in these patients has shown previously that their mutations are clustered in a short region of sequence in exon 28 of the vWF gene. The principle of the method involves heteroduplex formation between amplified genomic sequence containing the defect and an exon 28 vWF gene universal heteroduplex generator (UHG). The UHG is a synthetic vWF gene exon 28 homologue which contains a number of sequence mismatches designed to generate allele specific heteroduplexes for each type 2B mutation. Individual mutant genotypes are identified by characteristic banding patterns following polyacrylamide minigel electrophoresis. The technique is rapid, simple, inexpensive, and is ideally suited for adoption by non-specialist haematology laboratories for screening purposes.

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