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Find video protocols related to scientific articles indexed in Pubmed.
Particularities and Universalities of the Emergence of Inductive Generalization.
Integr Psychol Behav Sci
PUBLISHED: 09-14-2014
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Inductive generalization is the primary way by which human beings arrive at the construction of knowledge. Usually, it is assumed that it operates in a linear manner-each new feature becomes "piled up" in the inductive accumulation of evidence. We question this view, and otherwise claim that inductive generalization is essentially a non-linear dynamic process that fits the theoretical premises of the Dynamic Systems Theory. In our study, we explore the ability that young infants have when making inductive generalizations -previous studies show the existence of this capacity not earlier than at the age of 14 months. These studies have been cross-sectional in nature, but they do not offer an answer to the question of emergence of cognitive capabilities, therefore, a short-term longitudinal study is needed. Based on 3 case studies carried out longitudinally in infants ranging from 9 to 14 months, we demonstrate how the process of inductive generalization occurs from a conceptualization of nonlinear dynamic systems. We use Min - Max and State Space techniques, which allow us to show how the infant uses diverse pathways of actions with everyday objects to facilitate inductive generalization. The identified paths are not the same, they present differential and common moments that confirm the dynamic nature of development, and provide empirical evidence on the emergence of non-linear, non-sequential or inductive generalization.
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Structural and functional characterization of cargo-binding sites on the ?4-subunit of adaptor protein complex 4.
PLoS ONE
PUBLISHED: 01-01-2014
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Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (?1-?4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in ? subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the ?4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of ?4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type ?4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered ?4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of ?4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non-canonical site of ?4.
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Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration.
Biochem. Biophys. Res. Commun.
PUBLISHED: 10-29-2013
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Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.
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Structural basis for the recognition of tyrosine-based sorting signals by the ?3A subunit of the AP-3 adaptor complex.
J. Biol. Chem.
PUBLISHED: 02-12-2013
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Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous ?1, ?2, ?3, and ?4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on ?2 and ?4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the ? family, we solved the crystal structure at 1.85 ? resolution of the C-terminal domain of the ?3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The ?3A C-terminal domain consists of an immunoglobulin-like ?-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on ?3A subdomain A, at a location similar to the YXXØ-binding site on ?2 but not ?4. The binding sites on ?3A and ?2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the ?3A site and show that this protein binds YXXØ signals with 14-19 ?m affinity. The surface electrostatic potential of ?3A is less basic than that of ?2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.
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Turnover of C99 is Controlled by a Crosstalk between ERAD and Ubiquitin-Independent Lysosomal Degradation in Human Neuroglioma Cells.
PLoS ONE
PUBLISHED: 01-01-2013
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Alzheimers disease (AD) is characterized by the buildup of amyloid-? peptides (A?) aggregates derived from proteolytic processing of the ?-amyloid precursor protein (APP). Amyloidogenic cleavage of APP by ?-secretase/BACE1 generates the C-terminal fragment C99/CTF? that can be subsequently cleaved by ?-secretase to produce A?. Growing evidence indicates that high levels of C99/CTF? are determinant for AD. Although it has been postulated that ?-secretase-independent pathways must control C99/CTF? levels, the contribution of organelles with degradative functions, such as the endoplasmic reticulum (ER) or lysosomes, is unclear. In this report, we investigated the turnover and amyloidogenic processing of C99/CTF? in human H4 neuroglioma cells, and found that C99/CTF? is localized at the Golgi apparatus in contrast to APP, which is mostly found in endosomes. Conditions that localized C99/CTF? to the ER resulted in its degradation in a proteasome-dependent manner that first required polyubiquitination, consistent with an active role of the ER associated degradation (ERAD) in this process. Furthermore, when proteasomal activity was inhibited C99/CTF? was degraded in a chloroquine (CQ)-sensitive compartment, implicating lysosomes as alternative sites for its degradation. Our results highlight a crosstalk between degradation pathways within the ER and lysosomes to avoid protein accumulation and toxicity.
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Crystallographic and functional analysis of the ESCRT-I /HIV-1 Gag PTAP interaction.
Structure
PUBLISHED: 07-14-2010
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Budding of HIV-1 requires the binding of the PTAP late domain of the Gag p6 protein to the UEV domain of the TSG101 subunit of ESCRT-I. The normal function of this motif in cells is in receptor downregulation. Here, we report the 1.4-1.6 Å structures of the human TSG101 UEV domain alone and with wild-type and mutant HIV-1 PTAP and Hrs PSAP nonapeptides. The hydroxyl of the Thr or Ser residue in the P(S/T)AP motif hydrogen bonds with the main chain of Asn69. Mutation of the Asn to Pro, blocking the main-chain amide, abrogates PTAP motif binding in vitro and blocks budding of HIV-1 from cells. N69P and other PTAP binding-deficient alleles of TSG101 did not rescue HIV-1 budding. However, the mutant alleles did rescue downregulation of endogenous EGF receptor. This demonstrates that the PSAP motif is not rate determining in EGF receptor downregulation under normal conditions.
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AGAP2 regulates retrograde transport between early endosomes and the TGN.
J. Cell. Sci.
PUBLISHED: 06-15-2010
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The retrograde transport route links early endosomes and the TGN. Several endogenous and exogenous cargo proteins use this pathway, one of which is the well-explored bacterial Shiga toxin. ADP-ribosylation factors (Arfs) are approximately 20 kDa GTP-binding proteins that are required for protein traffic at the level of the Golgi complex and early endosomes. In this study, we expressed mutants and protein fragments that bind to Arf-GTP to show that Arf1, but not Arf6 is required for transport of Shiga toxin from early endosomes to the TGN. We depleted six Arf1-specific ARF-GTPase-activating proteins and identified AGAP2 as a crucial regulator of retrograde transport for Shiga toxin, cholera toxin and the endogenous proteins TGN46 and mannose 6-phosphate receptor. In AGAP2-depleted cells, Shiga toxin accumulates in transferrin-receptor-positive early endosomes, suggesting that AGAP2 functions in the very early steps of retrograde sorting. A number of other intracellular trafficking pathways are not affected under these conditions. These results establish that Arf1 and AGAP2 have key trafficking functions at the interface between early endosomes and the TGN.
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Sorting of the Alzheimers disease amyloid precursor protein mediated by the AP-4 complex.
Dev. Cell
PUBLISHED: 01-08-2010
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Adaptor protein 4 (AP-4) is the most recently discovered and least well-characterized member of the family of heterotetrameric adaptor protein (AP) complexes that mediate sorting of transmembrane cargo in post-Golgi compartments. Herein, we report the interaction of an YKFFE sequence from the cytosolic tail of the Alzheimers disease amyloid precursor protein (APP) with the mu4 subunit of AP-4. Biochemical and X-ray crystallographic analyses reveal that the properties of the APP sequence and the location of the binding site on mu4 are distinct from those of other signal-adaptor interactions. Disruption of the APP-AP-4 interaction decreases localization of APP to endosomes and enhances gamma-secretase-catalyzed cleavage of APP to the pathogenic amyloid-beta peptide. These findings demonstrate that APP and AP-4 engage in a distinct type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network (TGN) to endosomes, thereby reducing amyloidogenic processing of the protein.
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Analysis of articulation between clathrin and retromer in retrograde sorting on early endosomes.
Traffic
PUBLISHED: 10-05-2009
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Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans-Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes-TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069-7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.
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Human immunodeficiency virus type 1 Nef protein targets CD4 to the multivesicular body pathway.
J. Virol.
PUBLISHED: 04-29-2009
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The Nef protein of human immunodeficiency virus type 1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. Herein, we report that Nef has the additional function of targeting CD4 to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. This targeting involves the endosomal sorting complex required for transport (ESCRT) machinery. Perturbation of this machinery does not prevent removal of CD4 from the cell surface but precludes its lysosomal degradation, indicating that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. We also show that both CD4 and Nef are ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway.
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Adaptor protein 2-mediated endocytosis of the ?-secretase BACE1 is dispensable for amyloid precursor protein processing.
Mol. Biol. Cell
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The ?-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER-Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.
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