Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNA^Val (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA^Val gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA^Val is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA^Val species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.     

Measurements of conformational changes during adhesion of lipid and protein (polylysine and S-layer) surfaces

The adhesion forces between various surfaces were measured using the "surface forces apparatus" technique. This technique allows for the thickness of surface layers and the adhesion force between them to be directly measured in controlled vapor or liquid environments. Three types of biological surfaces were prepared by depositing various lipid-protein monolayers (with thicknesses ranging from 1 to 4 nm) on the inert, molecularly smooth mica surface: (i) hydrophobic lipid monolayers; (ii) amphiphilic polyelectrolyte surfaces of adsorbed polylysine; and (iii) deposited bacterial S-layer proteins. The adhesion, swelling, and wetting properties of these surfaces was measured as a function of relative humidity and time. Initial adhesion is due mainly to the van der Waals forces arising from nonpolar (hydrophobic) contacts. Following adhesive contact, significant molecular rearrangements can occur which alter their hydrophobic-hydrophilic balance and increase their adhesion with time. Increased adhesion is generally enhanced by (i) increased relative humidity (or degree of hydration); (ii) increased contact time; and (iii) increased rates of separation. The results are likely to be applicable to the adhesion of many other biosurfaces, and show that the hydrophobicity of a lipid or protein surface is not an intrinsic property of that surface but depends on its environment (e.g., on whether it is in aqueous solution or exposed to the atmosphere), and on the relative humidity of the atmosphere. It also depends on whether the surface is in adhesive contact with another surface and-when considering dynamic (nonequilibrium) conditions-on the time and previous history of its interaction with that surface. (c) 1993 John Wiley & Sons, Inc.     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The amino acid sequence of wheatwin1, a monomeric protein of 125 residues isolated from wheat kernel (variety S. Pastore), is reported. Wheatwin1 is highly homologous (95%) to barwin, a protein from barley seed, which was shown to be related to the C-terminal domain of two proteins encoded by the wound-induced geneswin1 andwin2 in potato and to a protein encoded by the same domain of the hevein gene (hev1) in rubber tree. Similarly to barwin, wheatwin1 contains six cysteine residues all linked in disulfide bridges and the N-terminal residue is pyroglutamate. Moreover, structural studies performed on wheatwin1 andwin1 protein by predictive methods demonstrated that these proteins and barwin are closely related in the secondary structure also. The high level of homology found with the product ofwin1,win2, andhev1 genes strongly suggests that barwin and wheatwin1 play a common role in the mechanism of plant defence.     

Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

The effect of salinity on the reproduction of coastal submerged macrophytes in experimental communities

The effects of salinity on the reproduction of coastal submerged macrophyte species were studied on samples of communities from six seasonal marshes in two outdoor experiments performed in autumn and in spring. The submerged macrophyte communities were submitted to five different salinity levels (0, 1, 2, 4 and 6 g/1 Cl^?1). In a companion paper (Grillas, van Wijck & Bonis 1993) three groups of species were distinguished on the basis of their biomass production over the salinity range 0 to 6 g/1 Cl^?1: (1) glycophytes (non-salt-tolerant species), (2) salt-tolerant species and (3) halo-phytes. This part of the study describes the impact of salinity on the reproduction of the individual species during the two experiments. The species differ in their capacity to reproduce in the autumn; only Zannichelliapedunculata and Tolypella hispánica were able to produce fruits in that season. For all species reproduction was greater in spring and strongly correlated with biomass, except for Chara canescens. Differences in reproductive effort over the salinity range amplified the halophytic nature of Ruppia marítima and Chara canescens and the intolerance of Callitriche truncata and Chara contraria. For the other species, reproductive effort did not differ significantly over the salinity range. Regarding the effect of salinity on biomass and reproductive effort of individual species, there were large differences in the total weight of propagules produced at the community level and in the relative contribution of individual species. The resulting quantitative changes in the species composition of the seed bank could affect the structure of the communities by their effects on the establishment and survival of species populations.     

Ecological and behavioral consequences of digestion in frugivorous animals

The postingestional effect of seed size and mass and nutrient composition on fruit profitability are reviewed. It is emphasized that profitability results from the interaction between fruit characteristics and the physiological and morphological traits of frugivores. The processes by which frugivores regurgitate or defecate seeds and the differential processing of the nutrient rich pulp and ballast in fruit are strongly dependent on the interaction between frugivore gut morphology and seed size. Euphonias that lack a functional gizzard defecate seeds, whereas tanagers that have a delete a functional gizzards regurgitate seeds. Some frugivores separate seeds from pulp and exocarp in the gizzard. It is hypothesized that the gizzard plays an important role in determining the postigestional fate of different pulp components. Although fruit nutrient content is often invoked as a determinant of frugivore feeding choices, studies that rely on proximal nutrient analysis, have often failed to find clear nutrient composition-preference correlations. It is argued that a partial reason for this failure is that proximal nutrient analysis ignores the complexities of fruit digestion. The ecological and physiological correlates of lipid and sugar assimilation are used to identify the limitations of traditional proximal nutrient analyses in fruit-frugivore studies. We suggest that recognizing the intricacies of the digestive characteristics of frugivores may reveal a much richer patterning in the interaction of frugivores with plants than has been previously hypothesized.     

Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type1

Cytotoxic necrotizing factor type 1 (CNF1) from strains of pathogenic Escherichia coli induces in human epithelial HEp-2 cells, a profound reorganization of the actin cytoskeleton into prominent stress fibres and membrane ruffles. We report here that this process is associated with induction of phagocytic-like activity. CNF1-treated cells acquired the ability to ingest latex beads as well as non-invasive bacteria such as Listeria innocua, which were taken as a model system. Uptake of bacteria was similar to pathogen-induced phagocytosis, since L. innocua transformed with DNA coding for the pore-forming toxin listeriolysln O behaved, with respect to intracellular growth, like the invasive, pathogenic species L. monocytogenes. Our results raise the possibility that, in vivo, pathogenic CNF1 -producing E. coli may invade epithelia by this novel induced phagocytic-like mechanism.     

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.