The low density lipoprotein receptor-related protein (LRP) is a novel beta-secretase (BACE1) substrate

BACE is a transmembrane protease with beta-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for gamma-secretase, leading to release of amyloid-beta peptide (Abeta), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.     

Mechanisms and function of flower and inflorescence reversion

Flower and inflorescence reversion involve a switch from floral development back to vegetative development, thus rendering flowering a phase in an ongoing growth pattern rather than a terminal act of the meristem. Although it can be considered an unusual event, reversion raises questions about the nature and function of flowering. It is linked to environmental conditions and is most often a response to conditions opposite to those that induce flowering. Research on molecular genetic mechanisms underlying plant development over the last 15 years has pinpointed some of the key genes involved in the transition to flowering and flower development. Such investigations have also uncovered mutations which reduce floral maintenance or alter the balance between vegetative and floral features of the plant. How this information contributes to an understanding of floral reversion is assessed here. One issue that arises is whether floral commitment (defined as the ability to continue flowering when inductive conditions no longer exist) is a developmental switch affecting the whole plant or is a mechanism which assigns autonomy to individual meristems. A related question is whether floral or vegetative development is the underlying default pathway of the plant. This review begins by considering how studies of flowering in Arabidopsis thaliana have aided understanding of mechanisms of floral maintenance. Arabidopsis has not been found to revert to leaf production in any of the conditions or genetic backgrounds analysed to date. A clear-cut reversion to leaf production has, however, been described in Impatiens balsamina. It is proposed that a single gene controls whether Impatiens reverts or can maintain flowering when inductive conditions are removed, and it is inferred that this gene functions to control the synthesis or transport of a leaf-generated signal. But it is also argued that the susceptibility of Impatiens to reversion is a consequence of the meristem-based mechanisms controlling development of the flower in this species. Thus, in Impatiens, a leaf-derived signal is critical for completion of flowering and can be considered to be the basis of a plant-wide floral commitment that is achieved without accompanying meristem autonomy. The evidence, derived from in vitro and other studies, that similar mechanisms operate in other species is assessed. It is concluded that most species (including Arabidopsis) are less prone to reversion because signals from the leaf are less ephemeral, and the pathways driving flower development have a high level of redundancy that generates meristem autonomy even when leaf-derived signals are weak. This gives stability to the flowering process, even where its initiation is dependent on environmental cues. On this interpretation, Impatiens reversion appears as an anomaly resulting from an unusual combination of leaf signalling and meristem regulation. Nevertheless, it is shown that the ability to revert can serve a function in the life history strategy (perenniality) or reproductive habit (pseudovivipary) of many plants. In these instances reversion has been assimilated into regular plant development and plays a crucial role there.     

Identification of a signaling network in lateral nucleus of amygdala important for inhibiting memory specifically related to learned fear

We identified the Grp gene, encoding gastrin-releasing peptide, as being highly expressed both in the lateral nucleus of the amygdala, the nucleus where associations for Pavlovian learned fear are formed, and in the regions that convey fearful auditory information to the lateral nucleus. Moreover, we found that GRP receptor (GRPR) is expressed in GABAergic interneurons of the lateral nucleus. GRP excites these interneurons and increases their inhibition of principal neurons. GRPR-deficient mice showed decreased inhibition of principal neurons by the interneurons, enhanced long-term potentiation (LTP), and greater and more persistent long-term fear memory. By contrast, these mice performed normally in hippocampus-dependent Morris maze. These experiments provide genetic evidence that GRP and its neural circuitry operate as a negative feedback regulating fear and establish a causal relationship between Grpr gene expression, LTP, and amygdala-dependent memory for fear.     

Identification of a monoclonal antibody specific for a neoantigenic determinant on alpha 2-macroglobulin: use for the purification and characterization of binary proteinase-inhibitor complexes

A monoclonal antibody was obtained from the fusion of spleen cells of mice, immunized with methylamine-treated alpha 2-macroglobulin (alpha 2M), with the myeloma cell line P3-X63-Ag8.653. A competitive binding assay demonstrated that the antibody was specific for a neoantigen expressed on alpha 2M when the inhibitor reacts with proteinases or with methylamine. When immobilized, the monoclonal antibody retained its ability to specifically bind alpha 2M-proteinase complexes or methylamine-treated alpha 2M, both of which could be quantitatively recovered from the immunoaffinity column by lowering the pH to 5.0. Binary alpha 2M-proteinase complexes of trypsin, plasmin, and thrombin, prepared by incubating large amounts of alpha 2M with a small amount of enzyme, were isolated by immunoaffinity chromatography. Each purified complex was characterized with regard to proteinase content, extent of alpha 2M subunit cleavage, extent of thiol ester hydrolysis, and extent of conformational change. Each complex contained 0.8-0.9 mol of proteinase/mol of inhibitor. In the alpha 2M-thrombin, alpha 2M-plasmin, and alpha 2M-trypsin complexes, approximately 50%, 60%, and 75% of the subunits are cleaved, respectively. Titration of sulfhydryl groups revealed that all purified binary complexes contained 2 +/- 0.5 mol of thiol/mol of complex, suggesting that each complex retains two intact thiol ester bonds. When the purified complexes were incubated with excess trypsin or with methylamine, an additional 1-2 mol of sulfhydryl/mol of complex could be titrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-reversion of Impatiens in the absence of meristem commitment

Purple-flowered plants of Impatiens balsamina maintained floral development on transfer from inductive short days (SD) to long days (LD), a treatment in which red-flowered plants of Impatiens are known to revert to leaf production. An investigation into the non-reverting nature of purple-flowered plants was carried out to establish whether these plants achieved meristem commitment or whether their non-reverting state was controlled by the leaves. When the leaves that had unfolded during the inductive SD treatment were removed at the time of transfer to LD, the purple-flowered plant did revert. This result suggests that, as in red-flowered Impatiens, meristem commitment is absent, but that purple-flowered plants maintain flowering in LD conditions because of a more permanent supply of signal from their leaves than occurs in red-flowered plants. A working hypothesis is proposed to explain how a signal from the leaves can retain a controlling role during flower development.Key words: Floral commitment, Impatiens, floral reversion, floricaula.      

Calcium-dependent phospholipid-binding proteins in plants

There is evidence that Ca²⁺ can regulate vesicle-mediated secretion in plant cells, but the mechanism for this is not known. One possibility is that Ca²⁺ -dependent phospholipid-binding proteins (annexins) couple the Ca²⁺ stimulus to the exocytotic response. Using a protocol developed for the isolation of animal annexins we have identified proteins in maize (Zea mays L.) coleoptiles that have similar characteristics to annexins. The predominant polypeptide species run as a doublet of relative molecular mass (M_r) 33000–35000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); another less-abundant protein of M_r 23000 is also present. In the presence of Ca²⁺ these proteins bind to liposomes composed of acidic phospholipids. Calcium-sensitivity of binding differs for each protein and is also influenced by the pH of the buffer used for the liposome-binding assay. Antiserum raised to the 33 to 35-kDa doublet purified on SDS-PAGE recognises the doublet in crude extracts from maize and proteins of similar M_r in Tradescantia virginiana and tobacco Nicotiana tabacum L. The antiserum also recognises p68 (Annexin VI) from chicken gizzard extracts, indicating homology between animal annexins and the maize proteins. For the maize proteins to be involved in the regulation of exocytosis, binding to phospholipids would be expected to occur at physiological levels of Ca²⁺. The characteristics of the maize annexin-like proteins are described and attention drawn to the marked effect of pH in lowering the requirement for Ca²⁺ for phospholipid binding.Abbreviations DEAE diethylaminoethyl - EGTA ethylene glycol-bis (-aminoethyiether)-N,N,N,N-tetraacetic acid - kDa kilodalton(s) - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This work was funded by the Agricultural and Food Research Council. Our thanks also to Professor P. Lowry and Dr R. Woods, Department of Biochemistry, University of Reading for facilities and advice for antiserum production, and C. Boustead, Department of Biochemistry, University of Leeds for advice on immunoblotting and phospholipid-binding assays.     

Functional characterization of human bitter taste receptors

The T2Rs belong to a multi-gene family of G-protein-coupled receptors responsible for the detection of ingested bitter-tasting compounds. The T2Rs are conserved among mammals with the human and mouse gene families consisting of about 25 members. In the present study we address the signalling properties of human and mouse T2Rs using an in vitro reconstitution system in which both the ligands and G-proteins being assayed can be manipulated independently and quantitatively assessed. We confirm that the mT2R5, hT2R43 and hT2R47 receptors respond selectively to micromolar concentrations of cycloheximide, aristolochic acid and denatonium respectively. We also demonstrate that hT2R14 is a receptor for aristolochic acid and report the first characterization of the ligand specificities of hT2R7, which is a broadly tuned receptor responding to strychnine, quinacrine, chloroquine and papaverine. Using these defined ligand-receptor interactions, we assayed the ability of the ligand-activated T2Rs to catalyse GTP binding on divergent members of the G(alpha) family including three members of the G(alphai) subfamily (transducin, G(alphai1) and G(alphao)) as well as G(alphas) and G(alphaq). The T2Rs coupled with each of the three G(alphai) members tested. However, none of the T2Rs coupled to either G(alphas) or G(alphaq), suggesting the T2Rs signal primarily through G(alphai)-mediated signal transduction pathways. Furthermore, we observed different G-protein selectivities among the T2Rs with respect to both G(alphai) subunits and G(betagamma) dimers, suggesting that bitter taste is transduced by multiple G-proteins that may differ among the T2Rs.