Identification of PrP sequences essential for the interaction between the PrP polymers and Aβ peptide in a yeast-based assay

Alzheimer disease is associated with the accumulation of oligomeric amyloid β peptide (Aβ), accompanied by synaptic dysfunction and neuronal death. Polymeric form of prion protein (PrP), PrP^Sc, is implicated in transmissible spongiform encephalopathies (TSEs). Recently, it was shown that the monomeric cellular form of PrP (PrP^C), located on the neuron surface, binds Aβ oligomers (and possibly other β-rich conformers) via the PrP_23–27 and PrP_90–110 segments, acting as Aβ receptor. On the other hand, PrP^Sc polymers efficiently bind to Aβ monomers and accelerate their oligomerization. To identify specific PrP sequences that are essential for the interaction between PrP polymers and Aβ peptide, we have co-expressed Aβ and PrP (or its shortened derivatives), fused to different fluorophores, in the yeast cell. Our data show that the 90–110 and 28–89 regions of PrP control the binding of proteinase-resistant PrP polymers to the Aβ peptide, whereas the 23–27 segment of PrP is dispensable for this interaction. This indicates that the set of PrP fragments involved in the interaction with Aβ depends on PrP conformational state.     

Vesicular glutamate transporter 2 immunoreactivity in putative vagal mechanosensor terminals of mouse and rat esophagus: indication of a local effector function?

Intraganglionic laminar endings (IGLEs) represent the major vagal afferent structures throughout the gastrointestinal tract. Previous ultrastructural investigations have revealed synaptic contacts of IGLEs on myenteric neurons. Thus, in addtion to functioning probably as mechanosensors, IGLEs may also synaptically influence myenteric neurons. In search of clues for potential transmitters in IGLEs, we investigated, by combined neuronal tracing and immunocytochemistry in the esophagus, the correlation between IGLEs and vesicular glutamate transporter 2 (VGLUT2), which is considered a reliable marker for glutamatergic neurons. In rat esophagus, IGLEs were immunostained with calretinin. In the mouse, anterograde wheat germ agglutinin/horseradish peroxidase (WGA-HRP) tracing from nodose ganglion was used in order to label esophageal IGLEs. Confocal laser scanning microscopy demonstrated that VGLUT2 immunoreactivity was highly colocalized with synaptophysin and that both calretinin and tyramide amplified WGA-HRP in rat and mouse esophagus, respectively. No colocalization was found with calcitonin gene-related peptide, a marker for spinal primary afferents. Thus, VGLUT2 is found in vagal afferent endings in the esophagus, suggesting that glutamate is contained in, and probably released from, synaptic vesicles previously described in IGLEs. Functional evidence pending, this finding is in favor of a local effector function of IGLEs onto myenteric neurons.     

Is the functional interaction between adenosine A2A receptors and metabotropic glutamate 5 receptors a general mechanism in the brain? Differences and similarities between the striatum and the hippocampus

The aim of the present paper was to examine, in a comparative way, the occurrence and the mechanisms of the interactions between adenosine A_2A receptors (A_2ARs) and metabotropic glutamate 5 receptors (mGlu5Rs) in the hippocampus and the striatum. In rat hippocampal and corticostriatal slices, combined ineffective doses of the mGlu5R agonist 2-chloro-5-hydroxyphenylglycine (CHPG) and the A_2AR agonist CGS 21680 synergistically reduced the slope of excitatory postsynaptic field potentials (fEPSPs) recorded in CA1 and the amplitude of field potentials (FPs) recorded in the dorsomedial striatum. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway appeared to be involved in the effects of CGS 21680 in corticostriatal but not in hippocampal slices. In both areas, a postsynaptic locus of interaction appeared more likely. N-methyl-D-aspartate (NMDA) reduced the fEPSP slope and FP amplitude in hippocampal and corticostriatal slices, respectively. Such an effect was significantly potentiated by CHPG in both areas. Interestingly, the A_2AR antagonist ZM 241385 significantly reduced the NMDA-potentiating effect of CHPG. In primary cultures of rat hippocampal and striatal neurons (ED 17, DIV 14), CHPG significantly potentiated NMDA-induced lactate dehydrogenase (LDH) release. Again, such an effect was prevented by ZM 241385. Our results show that A_2A and mGlu5 receptors functionally interact both in the hippocampus and in the striatum, even though different mechanisms seem to be involved in the two areas. The ability of A_2ARs to control mGlu5R-dependent effects may thus be a general feature of A_2ARs in different brain regions (irrespective of their density) and may represent an additional target for the development of therapeutic strategies against neurological disorders.     

Toward a better understanding of the interaction between TGF-β family members and their ALK receptors

Transforming growth factor-beta (TGF-β) proteins are a family of structurally related extracellular proteins that trigger their signaling functions through interaction with the extracellular domains of their cognate serine/threonine kinase receptors. The specificity of TGF-β/receptor binding is complex and gives rise to multiple functional roles. Additionally, it is not completely understood at the atomic level. Here, we use the most reliable computational methods currently available to study systems involving activin-like kinase (ALK) receptors ALK4 and ALK7 and their multiple TGF-β ligands. We built models for all these proteins and their complexes for which experimental structures are not available. By analyzing the surfaces of interaction in six different TGF-β/ALK complexes we could infer which are the structural distinctive features of the ligand-receptor binding mode. Furthermore, this study allowed us to rationalize why binding of the growth factors GDF3 and Nodal to the ALK4 receptor requires the Cripto co-factor, whilst binding to the ALK7 receptor does not.     

Musings on the wanderer: what's new in our understanding of vago-vagal reflexes? I. Morphology and topography of vagal afferents innervating the GI tract

An understanding of the events initiating vago-vagal reflexes requires knowledge of mechanisms of transduction by vagal afferents. Such information presumes an understanding of receptor morphology and location. Anatomic studies have recently characterized two types of vagal afferents, both putative mechanoreceptors distributed in gastrointestinal (GI) smooth muscle. These two receptors are highly specialized in that they 1) are morphologically distinct, 2) have different smooth muscle targets, 3) form complexes with dissimilar accessory cells, and 4) vary in their regional distributions throughout the GI tract. By comparison, information on the architecture and regional distributions of other classes of vagal afferents, notably chemoreceptors, has only begun to accumulate. Progress on the study of the two mechanoreceptors, however, illustrates general principles and delineates experimental issues that may apply to other submodalities of vagal afferents. By extension from morphological and physiological observations on the two species of smooth muscle endings, it is reasonable to hypothesize that additional classes of vagal receptors are also differentiated morphologically and that they vary in structure, accessory cells, regional distributions, and other features. A full appreciation of vago-vagal reflexes will require thorough structural and regional analyses of each of the types of vagal receptors within the GI tract.     

Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility

We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.     

Anatomical evidence for interaction of ACTH1–39 immunostained fibers and hypothalamic paraventricular neurons that project to the dorsal vagal complex

Summary Anatomical evidence is presented for an interaction of ACTH^1–39 immunostained fibers and a specific population of hypothalamic paraventricular (PVN) neurons; these neurons project to the dorsal vagal complex (DVC) of brainstem medulla. Bilateral injection of 10% HRP-WGA into DVC is incorporated into nerve terminals and transported retrogradely to cell bodies in the parvocellular subdivision of PVN, as revealed by standard HRP-WGA histochemistry or antibody to wheatgerm agglutinin followed by immunocytochemical techniques. Labeled cells are localized predominantly in the ventral portion of the caudal medial parvocellular subdivision and ventrolaterally in the posterior subnucleus of PVN. Few labeled cells are seen in the anterior parvocellular PVN, rostrally in the medial parvocellular component and in the dorsal cap. HRP-WGA cells are rarely observed in the magnocellular divisions of PVN. Dual-staining immunocytochemical-retrograde tracing techniques in the same tissue section demonstrate ACTH^1–39 fibers in intimate anatomical proximity to parvocellular PVN neurons that project to DVC. It is suggested that this interaction may partially account for the known cardiovascular effects of opiocorins and supports the role of the paraventricular nucleus in hypothalamie integration and modulation of cardiovascular control.     

Consequences of the interaction between nutrients and plant secondary metabolites on herbivore selectivity: benefits or detriments for plants?

Concentrations of nutrients and plant secondary metabolites (PSM) vary temporally and spatially, creating a multidimensional feeding environment. Interactions between nutrients and PSM are poorly understood because research has relied largely on studying the isolated effects of nutrients or PSM on foraging behavior. Nevertheless, their interactions can influence food selection and the dynamics of plant communities. Our objective was to explore how interactions between nutrients and PSM influence food selection. For 7 d, three groups of lambs received intraruminal infusions of three different doses of a PSM (0=Control; low and high) and 2 h later they were offered two foods that contained either low (high in energy) or high (high in protein) protein/energy ratios. The foods were offered 7 d before (baseline) and 7 d after PSM infusions. We conducted five trials each with a different PSM- terpenoids, cyanogenic glycosides, sodium nitrate, quebracho tannin, and lithium chloride. Lambs consistently preferred the food high in energy to the food high in protein, but toxins modified the degree to which this preference was manifest. Terpenoids, nitrate, and lithium chloride depressed intake of the food high in energy. Cyanogenic glycosides had the opposite effect, and at higher doses they depressed intake of the food high in protein. Tannins enhanced intake of the food high in energy at lower doses and they depressed its ingestion at higher doses. Thus, PSM selectively depressed or enhanced intake depending on the macronutrient composition of the foods. These results imply that the probability of a plant being eaten will depend not only on its chemical defenses but also on the quantity and quality of nutrients in the plant and its neighbors, and that the ability of herbivores to learn associations between nutrients and PSM may have a substantial impact on the way herbivores regulate ecosystem processes.     

The role of the anticodon in the interaction between methionyl-tRNA synthetase and bacterial initiator tRNA?

Complementary and antiparallel oligonucleotides bind to exposed regions of the tRNA molecule. Aminoacylation in the presence of triplets has been used to determine the role of the anticodon in the interaction between methionyl-tRNA synthetase and initiator tRNA. ApUpG has no effect on the charging even when 70% of the tRNA is bound to the triplet, whereas in the presence of GpGpU which binds to the A-C-C sequence adjacent to the 3' terminal adenosine that fraction of the tRNA which is bound to the triplet is completely unavailable for charging. Hence the anticodon is probably not involved in a primary interaction while the A-C-C-A-OH clearly is. This conclusion is supported by the failure of the isolated anticodon loop and stem oligonucleotides to inhibit the aminoacylation reaction.     

Is crossover between the X and Y a regular feature of meiosis in mouse and man?

There has long been an assumption that normal disjunction of the sex chromosomes of all mammals is assured by synapsis of a region of homology between the X and Y and that an obligatory crossover with chiasmata formation follows. Evidence is presented here that much (if not all) observed synapsis between the X and Y in mouse and man is nonhomologous and that crossing over most likely does not occur as a normal event in these organisms. The X and Y have desynapsed to a mere terminal association by the time of pachytene DNA synthesis, generally considered to be associated with crossing over. Recombination nodules or bars observed on the X and Y of human spermatocytes are also present at the wrong substage of pachytene and are insufficient in frequency to accounf for an obligatory crossover between the X and Y and thus assure normal disjunction. Instead it is suggested that orientation and disjunction of the sex chromosomes is mediated in these species by an achiasmatic telomeric association.     

Roles of α- and β-estrogen receptors in mouse social recognition memory: effects of gender and the estrous cycle

Establishing clear effects of gender and natural hormonal changes during female ovarian cycles on cognitive function has often proved difficult. Here we have investigated such effects on the formation and long-term (24 h) maintenance of social recognition memory in mice together with the respective involvement of α- and β-estrogen receptors using α- and β-estrogen receptor knockout mice and wildtype controls. Results in wildtype animals showed that while females successfully formed a memory in the context of a habituation/dishabituation paradigm at all stages of their ovarian cycle, only when learning occurred during proestrus (when estrogen levels are highest) was it retained after 24 h. In α-receptor knockout mice (which showed no ovarian cycles) both formation and maintenance of this social recognition memory were impaired, whereas β-receptor knockouts showed no significant deficits and exhibited the same proestrus-dependent retention of memory at 24 h. To investigate possible sex differences, male α- and β-estrogen receptor knockout mice were also tested and showed similar effects to females excepting that α-receptor knockouts had normal memory formation and only exhibited a 24 h retention deficit. This indicates a greater dependence in females on α-receptor expression for memory formation in this task. Since non-specific motivational and attentional aspects of the task were unaffected, our findings suggest a general α-receptor dependent facilitation of memory formation by estrogen as well as an enhanced long-term retention during proestrus. Results are discussed in terms of the differential roles of the two estrogen receptors, the neural substrates involved and putative interactions with oxytocin.     

Hairpin and duplex forms of a self-complementary dodecamer, d-AGATCTAGATCT, and interaction of the duplex form with the peptide KGWGK: can a pentapeptide destabilize DNA?

Ordered forms of a synthetic dodecamer, d-AGATCTAGATCT, a direct repeat of the BglII recognition sequence, have been investigated using UV, CD, and fluorescence spectroscopy. Complex hairpin-duplex equilibria are manifest in UV thermal transitions, which are monophasic in the presence of very low or high NaCl concentrations but distinctly biphasic at intermediate ionic strengths. In 100 mM NaCl, the 1/Tm vs 1n C curve has a reasonable positive slope, which yields delta H and delta S for duplex formation as -66.2 kcal/mol and -190 cal/mol, respectively. Interaction of the dodecamer in duplex form with a tryptophan-containing peptide, KGWGK, has also been investigated to test the "bookmark" hypothesis (Gabbay et al., 1976) under the uniform structural constraint of the oligonucleotide of defined sequence. CD spectra of the peptide (P), the oligonucleotide (N), and their mixtures at different P/N ratios show a dramatic change in peptide spectrum but little change in nucleic acid dichroism with peptide binding. The Tm of P-N complexes decreases with an increase in peptide binding and levels off at saturation binding of P/N = 2.0. The data are interpreted in terms of a groove-cum-intercalation mode of binding, where intercalation to the tryptophan side chain destabilizes the double helix. A Scatchard plot of the binding data is nonlinear, with best-fit values for an overall association constant K = 4.33 x 10(5) M-1, and the number of binding sites n = 3.23 when fitted to the site-exclusion model of binding.