Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.     

A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth

Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control.     

Cholinesterases and cell proliferation in “nonstratified” and “stratified” cell aggregates from chicken retina and tectum

Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - iso-OMPA specific inhibitor of BChE - BW 284C51 specific inhibitor of AChE - IPL inner plexiform layer - OPL outer plexiform layer     

Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.     

Acetylcholinesterase in cell adhesion, neurite growth and network formation

The expression of acetylcholinesterase is not restricted to cholinergically innervated tissues and relates to both neurotransmission and multiple biological aspects, including neural development, stress response and neurodegenerative diseases. Therefore, the classical function of acetylcholinesterase has to be distinguished from its non-classical, e.g. enzymatic from non-enzymatic, functions. Here, the roles of acetylcholinesterase in cell adhesion, promoting neurite outgrowth and neural network formation are reviewed briefly, together with potential mechanisms to support these functions. Part of these functions may depend on the structural properties of acetylcholinesterase, for example, protein-protein interactions. Recent findings have revealed that laminin-1 is an interaction partner for acetylcholinesterase. The binding of acetylcholinesterase to this extracellular matrix component may allow cell-to-cell recognition, and also cell signalling via membrane receptors. Studies using monolayer and 3D spheroid retinal cultures, as well as the acetylcholinesterase-knockout mouse, have been instrumental in elaborating the non-classical functions of acetylcholinesterase.     

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.     

Effect of serotonergic agonists in the nucleus accumbens on d-amphetamine-stimulated locomotion.

Serotonergic projections from the raphe nuclei are thought to modulate locomotor activity in the rat, and serotonin injection into the nucleus accumbens attenuates the hypermotility elicited by amphetamine. The purpose of the present study was to characterize the effects of various classes of serotonergic agonists administered into the nucleus accumbens on d-amphetamine-stimulated locomotor activity in order to determine which serotonin receptor subtypes are involved. Administration of the nonselective 5-HT agonist quipazine, the 5-HT-1 agonist mCPP, the 5-HT-1a agonist 8-OH-DPAT, the 5-HT-1b agonist CGS-12066B, and the 5HT-1c/2 agonist DOI did not inhibit d-amphetamine-stimulated locomotor activity. Pronounced lateral head weaving was noted after 8-OH-DPAT administration. The combination of the 5-HT-1a agonist 8-OH-DPAT and the 5-HT-1b agonist CGS-12066B, however, did inhibit d-amphetamine-stimulated locomotor activity. In contrast, the 5-HT-3 agonist 1-phenylbiguanide enhanced the locomotor effect of d-amphetamine. This effect was partially reversed by the 5-HT-3 antagonist MDL-7222. These studies suggest that serotonin has complex and multiple effects on the regulation of locomotor activity within the nucleus accumbens.