PTPσ promotes retinal neurite outgrowth non‐cell‐autonomously

The receptor‐like protein tyrosine phosphatase (RPTP) PTPσ controls the growth and targeting of retinal axons, both in culture and in ovo. Although the principal actions of PTPσ have been thought to be cell‐autonomous, the possibility that RPTPs related to PTPσ also have non‐cell‐autonomous signaling functions during axon development has also been supported genetically. Here we report that a cell culture substrate made from purified PTPσ ectodomains supports retinal neurite outgrowth in cell culture. We show that a receptor for PTPσ must exist on retinal axons and that binding of PTPσ to this receptor does not require the known, heparin binding properties of PTPσ. The neurite‐promoting potential of PTPσ ectodomains requires a basic amino acid domain, previously demonstrated in vitro as being necessary for ligand binding by PTPσ. Furthermore, we demonstrate that heparin and oligosaccharide derivatives as short as 8mers, can specifically block neurite outgrowth on the PTPσ substrate, by competing for binding to this same domain. This is the first direct evidence of a non‐cell‐autonomous, neurite‐promoting function of PTPσ and of a potential role for heparin‐related oligosaccharides in modulating neurite promotion by an RPTP. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

α,α′-trehalose 6,6′-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120-2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir-Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.     

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a β-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.     

Fab MOR03268 triggers absorption shift of a diagnostic dye via packaging in a solvent-shielded Fab dimer interface

Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody.     

Cytokinin deficiency causes distinct changes of sink and source parameters in tobacco shoots and roots

Cytokinin deficiency causes pleiotropic developmental changes such as reduced shoot and increased root growth. It was investigated whether cytokinin-deficient tobacco plants, which overproduce different cytokinin oxidase/dehydrogenase enzymes, show changes in different sink and source parameters, which could be causally related to the establishment of the cytokinin deficiency syndrome. Ultrastructural analysis revealed distinct changes in differentiating shoot tissues, including an increased vacuolation and an earlier differentiation of plastids, which showed partially disorganized thylakoid structures later in development. A comparison of the ploidy levels revealed an increased population of cells with a 4C DNA content during early stages of leaf development, indicating an inhibited progression from G2 to mitosis. To compare physiological characteristics of sink leaves, source leaves and roots of wild-type and cytokinin-deficient plants, several photosynthetic parameters, content of soluble sugars, starch and adenylates, as well as activities of enzymes of carbon assimilation and dissimilation were determined. Leaves of cytokinin-deficient plants contained less chlorophyll and non-photochemical quenching of young leaves was increased. However, absorption rate, photosynthetic capacity (F(v)/F(m) and J(CO2 max)) and efficiency (Phi CO(2 app)), as well as the content of soluble sugars, were not strongly altered in source leaves, indicating that chlorophyll is not limiting for photoassimilation and suggesting that source strength did not restrict shoot growth. By contrast, shoot sink tissues showed drastically reduced contents of soluble sugars, decreased activities of vacuolar invertases, and a reduced ATP content. These results strongly support a function of cytokinin in regulating shoot sink strength and its reduction may be a cause of the altered shoot phenotype. Roots of cytokinin-deficient plants contained less sugar compared with wild-type. However, this did not negatively affect glycolysis, ATP content, or root development. It is suggested that cytokinin-mediated regulation of the sink strength differs between roots and shoots.     

Leukocyte extravasation: an immunoregulatory role for alpha-L-fucosidase?

Fucosylated oligosaccharides and glycoconjugates have been implicated in several biological events, including the cell-cell adhesion processes that mediate inflammation. Alpha-L-fucosidase (ALF) is an exoglycosidase that is involved in the hydrolytic degradation of alpha-L-fucose from glycoconjugates. In this study, we investigated the potential role of ALF in regulation of leukocyte migration. Measurement of transendothelial migration in response to CCL5 demonstrated that pretreatment of monocytic cells with ALF reduced migration (p = 0.0004) to a greater extent than treatment of the endothelial monolayer (p = 0.0374). Treatment with ALF significantly reduced the adhesion of monocytic cells to immobilized P-selectin.Fc. A murine model of experimental autoimmune uveitis was then used to show that treatment of splenic cells with ALF produced an 8.6-fold decrease in rolling and a 3.2-fold decrease in cell migration across the retinal vasculature. Further in vitro studies demonstrated that treatment of monocytes with the chemokines CCL3 or CCL5 increased the level of mRNA encoding ALF; this was accompanied by the detection of significant increases in both the 51- and 56-kDa components of ALF by Western blotting. Treatment of monocytic cells with ALF for 2 h significantly reduced the cell surface expression of CD31, with a further decrease in expression observed after 5 h (p = 0.002). Thus, CD31 and fucosylated ligands of P-selectin seem to be the candidates through which ALF mediates its effect in vitro. These data identify a previously unrecognized immunoregulatory role for ALF in late stages of inflammation.     

Cytochromes P450 from family 4 are the main omega hydroxylating enzymes in humans: CYP4F3B is the prominent player in PUFA metabolism

Human CYP450 omega-hydroxylases of the CYP4 family are known to convert arachidonic acid (AA) to its metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). This study deals with hydroxylations of four PUFAs, eicosatrienoic acid (ETA), AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) by either human recombinant CYP4s enzymes or human liver microsomal preparations. CYP4F3A and CYP4F3B were the most efficient omega-hydroxylases of these PUFAs. Moreover, the differences in the number of unsaturations of ETA, AA, and EPA allowed us to demonstrate a rise in the metabolic rate of hydroxylation when the double bond in 14-15 or 17-18 was missing. With the CYP4F enzymes, the main pathway was always the omega-hydroxylation of PUFAs, whereas it was the (omega-1)-hydroxylation with CYP1A1, CYP2C19, and CYP2E1. Finally, we demonstrated that the omega9 and omega3 PUFAs (ETA, EPA, and DHA) could all be used as alternative substrates in AA metabolism by human CYP4F2 and -4F3B. Thus, they decreased the ability of these enzymes to convert AA to 20-HETE. However, although ETA was the most hydroxylated substrate, EPA and DHA were the most potent inhibitors of the conversion of AA to 20-HETE. These findings suggest that some physiological effects of omega3 FAs could partly result from a shift in the generation of active hydroxylated metabolites of AA through a CYP-mediated catalysis.     

Arf family GTP loading is activated by, and generates, positive membrane curvature

Small G-proteins belonging to the Arf (ADP-ribosylation factor) family serve as regulatory proteins for numerous cellular processes through GTP-dependent recruitment of effector molecules. In the present study we demonstrate that proteins in this family regulate, and are regulated by, membrane curvature. Arf1 and Arf6 were shown to load GTP in a membrane-curvature-dependent manner and stabilize, or further facilitate, changes in membrane curvature through the insertion of an amphipathic helix.     

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1-AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN(+/-) mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN(+/-) mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1-AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.