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Conflict between incompatible response tendencies is typically followed by control adjustments aimed at diminishing subsequent conflicts, a phenomenon often called conflict adaptation. Dreisbach and Fischer (2015, 2016) recently proposed that it is not the conflict per se but the aversive quality of a conflict that originally motivates this kind of sequential control adjustment. With the present study we tested the causal role of aversive signals in conflict adaptation in a more direct way. To this end, after each trial of a vertical Simon task participants rated whether they experienced the last trial as rather pleasant or unpleasant. Conflict adaptation was measured via lateralized readiness potentials as a measure of early motor-related activation that were computed on the basis of event-related brain potentials. Results showed the typical suppression of automatic response activation following trials rated as unpleasant, whereas suppression was relaxed following trials rated as pleasant. That is, sequential control adaptation was not based on previous conflict but on the subjective affective experience. This is taken as evidence that negative affect even in the absence of actual conflict triggers subsequent control adjustments. [ABSTRACT FROM AUTHOR]

• An explainable framework using CNNs for damage diagnosis is presented. • The LRP algorithm is used to explain a classification CNN and a regression CNN. • The performances using two different databases are discussed. • LRP relevance values are properly aggregated to better interpret the CNNs behaviour. • The CNNs behaviour is compared to the intuition of human experts. Among the maintenance policies adopted to guarantee the safety of structures throughout their service life, condition-based maintenance policies driven by structural health monitoring approaches have progressively gained importance over the last years. Within this field, among the several methods proposed in the literature to diagnose damage affecting thin-walled structures, satisfactory performances have been achieved by adopting tomographic algorithms to process ultrasonic guided waves, even though with some limitations. Recently, such limitations have been overcome by adopting machine learning-based algorithms, even though their implementation in real life applications is still limited because of the mistrust in neural networks determined by their black box-like nature. To date, however, several explainability algorithms have been proposed to interpret the behaviour of neural networks, in particular in the medical and in the military fields, where trust in the tools adopted must be guaranteed. Thus, exploiting the potentialities of explainability frameworks, in this work the layer-wise relevance propagation algorithm is employed to explain the predictions of convolutional neural networks for classification and for regression that characterise damage by processing ultrasonic guided waves excited and sensed by means of a 2-D network of piezoelectric devices. First, the explainability algorithm is applied to give a score to each sample of the acquired ultrasonic guided waves, then such scores are collected by means of a properly developed aggregation strategy to rank the most informative couples of piezoelectric devices. The capabilities of the explainable damage diagnosis framework are demonstrated by means of a numerical, yet realistic, case study involving a metal plate affected by crack-like damage. In particular, the focus is set on the explanation of the behaviour of the neural networks involved, with the aim of building trust in such algorithms and, possibly, revealing damage-related hidden features of ultrasonic guided waves. [ABSTRACT FROM AUTHOR]

In this study, an l -valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE , alaT and ilvA combined with the overexpression of six genes ilvB , ilvN , ilvC , lrp 1 , brnF and brnE . Overexpression of lrp 1 alone increased l -valine production by 16-fold. Deletion of the aceE , alaT and ilvA increased l -valine production by 44-fold. Overexpression of the six genes ilvB , ilvN , ilvC , lrp 1 , brnE and brnF in the triple deletion mutant WCC003 further increased l -valine production. The strain WCC003/pJYW-4- ilvBNC 1 - lrp 1 - brnFE produced 243 mM l -valine in flask cultivation and 437 mM (51 g/L) l -valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l -alanine and l -isoleucine that are usually found in the fermentation of l -valine-producing C. glutamicum . [ABSTRACT FROM AUTHOR]

Abstract: The detection and quantification of specific metabolites in single bacterial cells is a major goal for industrial biotechnology. We have developed a biosensor based on the transcriptional regulator Lrp that detects intracellular l-methionine and branched-chain amino acids in Corynebacterium glutamicum. In assays, fluorescence output showed a linear relationship with cytoplasmic concentrations of the effector amino acids. In increasing order, the affinity of Lrp for the amino acids is l-valine, l-isoleucine, l-leucine and l-methionine. The sensor was applied for online monitoring and analysis of cell-to-cell variability of l-valine production by the pyruvate dehydrogenase-deficient C. glutamicum strain ΔaceE. Finally, the sensor system was successfully used in a high-throughput (HT) FACS screen for the isolation of amino acid-producing mutants after random mutagenesis of a non-producing wild type strain. These applications illustrate how one of nature''s sensor devices – transcriptional regulators – can be used for the analysis, directed evolution and HT screening for microbial strain development. [Copyright &y& Elsevier]

Abstract: Apolipoprotein E (apoE) is recognized as a key actor in brain remodeling. It has been shown to increase after peripheral and central injury, to modulate reparative capacity in neurodegenerative conditions like Alzheimer''s disease (AD) and to be associated with a number of other neurodegenerative diseases. This particular function of apoE has been postulated to underlie the robust association with risk and age at onset of AD. ApoE associations studies with Parkinson''s disease (PD), the second most prevalent neurodegenerative disease, have generated contradictory results but associations with age at onset and dementia in PD stand out. We investigate here whether apoE is involved in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration that models PD-like deafferentation of the striatum in the mouse and participates in compensatory reinnervation mechanisms. We examined the modifications in gene expression and protein levels of apoE and its key receptors, the low density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), as well as the reactive astrocyte marker glial fibrillary acidic protein (GFAP) in different brain structures throughout the degenerative and reactive regenerative period. In the striatum, upregulations of GFAP, apoE and LRP mRNAs at 1day post-treatment were associated with marked decreases in dopamine (DA) levels, loss in tyrosine hydroxylase protein content, as well as to a compensatory increase in dopaminergic metabolism. Subsequent return to near control levels coincided with indications of reinnervation in the striatum: all consistent with a role of apoE during the degenerative process and regenerative period. We also found that this cascade was activated in the hippocampus and more so than in the striatum, with a particular contribution of LDLR expression. The hippocampal activation did not correlate with substantial neurochemical reductions but appears to reflect local subtle alteration of DA metabolism and the regulation of plasticity-related event in this structure. This study provides first evidence of an activation of the apoE/apoE receptors cascade in a mouse model of PD, specifically in the MPTP-induced deafferentation of the striatum. Results are also quite consistent with the postulated role of apoE in brain repair but, raise the issue of possible lesion- and region-specific alterations in gene expression. [Copyright &y& Elsevier]

Abstract: Inhibitory control (IC) is an important contributor to educational performance, and undergoes rapid development in childhood. Age-related changes in IC were assessed using an in-depth analysis of reaction time, the Lateralized Readiness Potential (LRP), and other event-related potential (ERP) measures to control for speed of processing. Five-year-olds, 8-year-olds and adults completed an adapted Stroop task. Both reaction time and ERP results suggest that IC does develop in this age range, over and above changes in speed of processing. The LRP identified two processes that contribute to IC. These processes develop at different rates — an early process, involving how the conflict is initially responded to is mature by age 5, while a later process, involving how the conflict is overcome is still developing after 8years of age. We propose that these early and late processes reflect interference suppression and response inhibition, respectively. Further, a single-trial analysis of the LRP in the incongruent condition provides evidence that the LRP is consistent across trials and functionally similar in each age group. These results corroborate previous findings regarding the development of IC, and present a new and useful tool for assessing IC across development. [Copyright &y& Elsevier]

Abstract: The uropathogenic Escherichia coli colonize the host body by attaching themselves to the epithelial cells through the pyelonephritis-associated pili (pap). The expression of the papBA operon is regulated under a reversible phase-variation mechanism, which partitions the population of cells into those that express the pap and others that do not. The two phases of pap expression are the direct consequences of the two distinct DNA-binding modes exhibited by leucine-responsive regulatory protein (Lrp) in the pap promoter region. In the phase-OFF cells, Lrp occupies the binding sites proximal to the transcription start, blocking transcription initiation. In the phase-ON cells, Lrp occupies the binding sites distal to the transcription start and is thought to promote the CAP (catabolite gene activation protein)-directed transcription initiation. Lrp binds to the proximal binding sites more tightly than to the distal sites, and the switching from phase-OFF to phase-ON requires a local co-regulator, PapI. Here, we used PapI and an isolated DNA-binding domain construct of Lrp to show that there is a DNA co-recognition mechanism by which both proteins acquire enhanced affinity to the distal pap site DNA, to which neither of them binds to an appreciable extent without the other. Also, examination of the binding properties of the Lrp DNA-binding domain presented here led us to propose a new sequence alignment of the six pap Lrp-binding sites. New insights into the design of sequences regulating the pap phase variation as revealed by the pap Lrp-binding site sequences are thus defined and discussed. [Copyright &y& Elsevier]

Abstract: When the bottom halves of two faces differ, people’s behavioral judgment of the identical top halves of those faces is impaired: they report that the top halves are different, and/or take more time than usual to provide a response. This behavioral measure is known as the composite face effect (CFE) and has traditionally been taken as evidence that faces are perceived holistically. Recently, however, it has been claimed that this effect is driven almost entirely by decisional, rather than perceptual, factors (). To disentangle the contribution of perceptual and decisional brain processes, we aimed to obtain an event-related potential (ERP) measure of the CFE at a stage of face encoding () in the absence of a behavioral CFE effect. Sixteen participants performed a go/no-go task in an oddball paradigm, lifting a finger of their right or left hand when the top half of a face changed identity. This change of identity of the top of the face was associated with an increased ERP signal on occipito-temporal electrode sites at the N170 face-sensitive component (∼160ms), the later decisional P3b component, and the lateralized readiness potential (LRP) starting at ∼350ms. The N170 effect was observed equally early when only the unattended bottom part of the face changed, indicating that an identity change was perceived across the whole face in this condition. Importantly, there was no behavioral response bias for the bottom change trials, and no evidence of decisional biases from electrophysiological data (no P3b and LRP deflection in no-go trials). These data show that an early CFE can be measured in ERPs in the absence of any decisional response bias, indicating that the CFE reflects primarily the visual perception of the whole face. [Copyright &y& Elsevier]

Summary: Clusters of complement-type ligand-binding repeats (CRs) in the low-density lipoprotein receptor (LDLR) family are thought to mediate the interactions with their various ligands. Apolipoprotein E (ApoE), a key ligand for cholesterol homeostasis, has been shown to interact with LDLR-related protein 1 (LRP) through these clusters. The segment comprising the receptor-binding portion of ApoE (residues 130–149) has been found to have a weak affinity for isolated CRs. We have fused this region of ApoE to a high-affinity CR from LRP (CR17) for structural elucidation of the complex. The interface reveals a motif that has previously been observed in CR domains with other binding partners, but with several novel features. Comparison to free CR17 reveals that very few structural changes result from this binding event, but significant changes in intrinsic dynamics are observed upon binding. NMR perturbation experiments suggest that this interface may be similar to several other ligand interactions with LDLRs. [Copyright &y& Elsevier]

Abstract: Benjamin Libet has argued that electrophysiological signs of cortical movement preparation are present before people report having made a conscious decision to move, and that these signs constitute evidence that voluntary movements are initiated unconsciously. This controversial conclusion depends critically on the assumption that the electrophysiological signs recorded by are associated only with preparation for movement. We tested that assumption by comparing the electrophysiological signs before a decision to move with signs present before a decision not to move. There was no evidence of stronger electrophysiological signs before a decision to move than before a decision not to move, so these signs clearly are not specific to movement preparation. We conclude that Libet’s results do not provide evidence that voluntary movements are initiated unconsciously. [Copyright &y& Elsevier]

Abstract: Our previous studies have shown that very low-density lipoprotein receptor (VLDLR) is a negative regulator of the Wnt pathway. The present study showed that VLDLR gene knockout (Vldlr−/− ) mice displayed impaired cone ERG responses at early ages. Immunostaining of mid-wavelength cones showed significantly decreased cone densities in the retina and shortened cone outer segments in Vldlr−/− mice. At older ages, Vldlr−/− mice displayed declined rod ERG responses, decreased layers of photoreceptor nuclei, reduced rhodopsin levels and decreased levels of 11-cis retinal, the chromophore of visual pigments. As shown by fluorescein angiography and permeability assay, Vldlr−/− mice had severe retinal vascular leakage. ZO-1, a tight junction protein, was down-regulated in Vldlr−/− mouse retinae, further supporting the impaired blood–retinal barrier. Double staining of pericytes and endothelial cells in retinal sections revealed that neovasculature in Vldlr−/− mice lacks pericyte coverage, suggesting impaired maturation of retinal vasculature in Vldlr−/− mice. Staining of adherent leukocytes in the retinal vasculature revealed significant leukostasis in Vldlr−/− mice. Moreover, Vldlr−/− mice displayed up-regulated expression of multiple pro-inflammatory factors and activated NF-κB and HIF-1α, key regulators of inflammation. These findings suggest that deficiency of VLDLR leads to retinal degeneration and inflammation. [Copyright &y& Elsevier]

Abstract: Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili. [Copyright &y& Elsevier]

Abstract: The present study investigates the event related potential (ERP) components associated with the random version of the Sustained Attention to Response Task (SART). The random SART is a Go/No-Go task in which the No-Go target appears unpredictably and rarely. In the present experiment, the EEG was recorded from 58 electrodes with mastoids as reference. As expected, the N2 and P3 were larger in the No-Go trials compared to the Go trials, with the P3 more frontal for the No-Go trials compared to the Go trials. The functional role of the N2 and P3 in the No-Go trials is discussed comparing our results to the fixed SART results obtained by Dockree et al. [Dockree P. M., Kelly S. P., Robertson I. H., Reilly R. B., & Foxe J. J. (2005). Neurophysiological markers of alert responding during goal-directed behaviour: A high-density electrical mapping study. NeuroImage, 27, 587–601]. In addition, the lateralized readiness potential (LRP) was computed. The absence of an LRP in the No-Go trials suggests that a central inhibitory mechanism intervenes to prevent the preparation and execution of a dominant motor response. [Copyright &y& Elsevier]

Abstract: The endoglycosidase heparanase is the predominant enzyme that degrades heparan sulfate side chains of heparan sulfate proteoglycans, activity that is strongly implicated in tumor metastasis. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Among these is the induction of Akt/PKB phosphorylation noted in endothelial- and tumor-derived cells. Protein domains of heparanase required for signaling were not identified to date, nor were identified heparanase binding proteins/receptors capable of transmitting heparanase signals. Here, we examined the possible function of mannose 6-phosphate receptor (MPR) and low-density lipoprotein-receptor related protein (LRP), recently implicated in cellular uptake of heparanase, as heparanase receptors mediating Akt phosphorylation. We found that heparanase addition to MPR- and LRP-deficient fibroblasts elicited Akt activation indistinguishable from control fibroblasts. In contrast, disruption of lipid rafts abrogated Akt/PKB phosphorylation following heparanase addition. These results suggest that lipid raft-resident receptor mediates heparanase signaling. [Copyright &y& Elsevier]

Abstract: Transcriptional regulatory proteins typically bind specific DNA sequences with ∼103–107-fold higher affinity than non-specific DNA and this discrimination is essential for their in vivo function. Here we show that the bacterial leucine-responsive regulatory protein (Lrp) does not follow this trend and has a ∼20–400-fold binding discrimination between specific and non-specific DNA sequences. We suggest that the dual function of Lrp to regulate genes and to organize DNA utilizes this unique property. A ∼20-fold decrease in binding affinity from specific DNA is dependent upon cryptic binding sites, including the sequence GN2-3TTT and A-tracts. Removal of these sites still results in high binding affinity, only ∼70-fold weaker than that of specific sites. Similar to Lrp''s binding of specific sites in the pap and ilvIH promoters, Lrp binds cooperatively to non-specific DNA; thus, protein/protein interactions are important for both specific and non-specific DNA binding. When considering this cooperativity of Lrp binding, the binding selectivity to specific sites may increase to a maximum of ∼400-fold. Neither leucine nor the pap-specific local regulator PapI alter Lrp''s non-specific binding affinity or cooperative binding of non-specific DNA. We hypothesize that Lrp combines low sequence discrimination and relatively high intracellular protein concentrations to ensure its ability to regulate the transcription of specific genes while also functioning as a nucleoid-associated protein. Modeling of Lrp binding data and comparison to other proteins with regulatory and nucleoid-associated properties suggests similar mechanisms. [Copyright &y& Elsevier]

Abstract: Members of the Wg/Wnt family provide key intercellular signals during embryonic development and in the maintenance of homeostatic processes, but critical aspects of their signal transduction pathways remain controversial. We have found that canonical Wg signaling in Drosophila involves distinct initiation and amplification steps, both of which require Arrow/LRP. Expressing a chimeric Frizzled2-Arrow protein in flies that lack endogenous Wg or Arrow showed that this construct functions as an activated Wg receptor but is deficient in signal amplification. In contrast, a chimeric Arrow protein containing the dimerization domain of Torso acted as a potent amplifier of Wg signaling but could not initiate Wg signaling on its own. The two chimeric proteins synergized, so that their co-expression largely reconstituted the signaling levels achieved by expressing Wg itself. The amplification function of Arrow/LRP appears to be particularly important for long-range signaling, and may reflect a general mechanism for potentiating signals in the shallow part of a morphogen gradient. [Copyright &y& Elsevier]

Abstract: LRP has recently been shown to interact with the regulatory regions of bacterial ribosomal RNA promoters. Here we study details of the LRP–rDNA interaction by gel retardation and high-resolution footprinting techniques. We show that a second regulator for rRNA transcription, H-NS, facilitates the formation of a higher-order LRP–nucleoprotein complex, probably acting transiently as a DNA chaperone. The macromolecular crowding substance ectoine stabilizes the formation of this dynamic complex, while the amino acid leucine, as a metabolic effector, has the opposite effect. DNase I and hydroxyl radical footprint experiments with LRP–DNA complexes reveal a periodic change of the target DNA structure, which implies extensive DNA wrapping reaching into the promoter core region. We show furthermore that LRP binding is able to constrain supercoils, providing a link between DNA topology and regulation. The results support the conclusion that the bacterial DNA-binding protein LRP, assisted by H-NS, forms a repressive nucleoprotein structure involved in regulation of rRNA transcription. The formation of this regulatory structure appears to be directly affected by environmental changes. [Copyright &y& Elsevier]

Abstract: Pyelonephritis-associated pili (pap) allow uropathogenic Escherichia coli to bind to epithelial cells and play an important role in urinary tract infection. Expression of pap is controlled by a phase-variation mechanism, based on the two distinct heritable states that are the result of adenine N6-methylation in either of the two GATC sequences in its regulatory region. The methylation status of these two sequences is sensed by the action of two proteins, Lrp and PapI, and they play a central role in determining pap gene expression in both phase-ON and phase-OFF cells. We used modern NMR techniques to determine the solution structure and backbone dynamics of PapI. We found its overall fold resembles closely that of the winged helix-turn-helix family of DNA-binding proteins. We determined that PapI possesses its own DNA-binding activity, albeit non-sequence-specific, independent of Lrp. PapI appears to bind to DNA with a K d in the 10 μM range. Possible mechanisms by which PapI might participate in the regulation of the pap operon are discussed in light of these new findings. [Copyright &y& Elsevier]

Abstract: It is well-known that leukemic cells of overt leukemia (OL) that have transformed from myelodysplastic syndromes (MDS) are more resistant to chemotherapy as compared with de novo AML cells. Thus, to examine the expression levels of drug-resistant genes and their alterations with the development of OL in MDS, the expression of mRNA for MDR1 and LRP was determined in bone marrow samples from control, de novo AML, MDS, MDS at the time of OL transformation (MDS → OL), and after transformation (OL) by quantitative real-time RT-PCR. The expression of MDR1 in MDS bone marrow at the time of initial diagnosis was as low as that for control subjects. However, the expression level was significantly elevated at the time of the development of OL (MDS → OL) compared with the initial MDS subjects (P < 0.05), while expression was relatively reduced after OL development (OL). The expression of LRP was significantly higher in MDS and MDS → OL samples than control subjects. However, the high expression of LRP in MDS → OL was significantly reduced after OL development (OL). The expression levels of drug-resistant genes in MDS → OL or OL were not significantly higher than those of de novo AML samples, although LRP expression in MDS or MDS → OL was relatively higher than that of de novo AML. Detecting increases in the expression of MDR1 would be useful for predicting OL development in MDS patients. [Copyright &y& Elsevier]

Abstract: The low-density lipoprotein receptor-related protein (LRP) is a large, endocytic receptor involved in intracellular signalling. LRP acts as a co-receptor with the PDGF-receptor (PDGF-r) for platelet-derived growth factor (PDGF). PDGF-r and Src-kinases induce tyrosine-phosphorylation of LRP. We used fluorescence lifetime imaging microscopy (FLIM) to specifically detect LRP phosphorylation, measure its extent and localization in intact cells, and assess its effects upon LRP–APP interaction. Robust phosphorylation of LRP throughout the cell was observed after overexpression of Src-kinase. This depended on LRP’s distal NPXY domain. By contrast, activation of the PDGF-r resulted in phosphorylation of the subpopulation of LRP at or near the cell surface. PDGF activation triggered phosphorylation of endogenous LRP in primary neurons. LRP is also a trafficking receptor for the Alzheimer-related molecule amyloid-precursor-protein (APP). PDGF stimulation did not affect LRP–APP interactions. This approach allows exquisite subcellular resolution of specific LRP post-translational changes and protein-protein interactions of endogenous proteins in intact cells. [Copyright &y& Elsevier]

Abstract: The receptor associated protein (RAP) is a three domain 38kDa ER-resident chaperone that helps folding of LRP and other LDL receptor family members and prevents premature binding of protein ligands. It competes strongly with all known LRP ligands. To further understanding of the specificity of RAP-LRP interactions, the binding of RAP and RAP fragments to two domains (CR7–CR8) from one of the main ligand-binding regions of LRP has been examined by 2D HSQC NMR spectroscopy and isothermal titration calorimetry. We found that RAP contains two binding sites for CR7–CR8, with the higher affinity site (K d ∼1μM) located in the C-terminal two-thirds and the weaker site (K d ∼5μM) in the N-terminal third of RAP. Residues from both CR7 and CR8 are involved in binding at each RAP site. The presence of more than one binding site on RAP for CR domains from LRP, together with the previous demonstration by others that RAP can bind to CR5–CR6 with comparably low affinities suggest an explanation for the dual roles of RAP as a folding chaperone and a tight competitive inhibitor of ligand binding. [Copyright &y& Elsevier]

Abstract: Previous research has shown that subliminally presented stimuli accelerate or delay responses afforded by supraliminally presented stimuli. Our experiments extend these findings by showing that unconscious stimuli even affect free choices between responses. Thus, actions that are phenomenally experienced as freely chosen are influenced without the actor becoming aware of the manipulation. However, the unconscious influence is limited to a response bias, as participants chose the primed response only in up to 60% of the trials. LRP data in free choice trials indicate that the prime was not ineffective in trials in which participants chose the non-primed response as then it delayed performance of the incongruently primed response. [Copyright &y& Elsevier]

Abstract: Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and β-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP +/+ and MVP −/− mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus. [Copyright &y& Elsevier]

Abstract: Objective. : Multidrug resistance (MDR) to chemotherapy is a major obstacle in attempts to improve the clinical outcome of ovarian carcinoma patients. The MDR-related phenotype is associated with over-expression of certain drug transporters, such as P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and lung resistance protein (LRP). The aim of this study was to evaluate the extent and prognostic significance of MDR-related protein expression in ovarian serous carcinomas. In addition, we correlated expression of these proteins with the apoptosis-related proteins p53, bcl-2, and bax. Methods. : Consecutive sections from 60 cases of ovarian serous carcinoma were assessed immunohistochemically for expression of P-gp, MRP1, LRP, p53, bcl-2, and bax. The level of protein expression was scored based on staining intensity and extent. Results. : Strong P-gp expression was observed in 12 (20%), MRP1 in 39 (65%), LRP in 27 (45%), p53 in 45 (75%), bcl-2 in 25 (41.7%), and bax in 30 (50%) of the 60 tumors. MRP1 expression was associated with both p53 and bcl-2 expressions (P = 0.01 and P = 0.03, respectively). Univariate analysis of survival revealed a significant inverse correlation between P-gp expression and patient survival (P = 0.015). Moreover, P-gp expression was significantly increased in tumors of patients unresponsive to chemotherapy (P = 0.009). Multivariate analysis revealed that only FIGO stage and P-gp expression were useful negative independent predictors of survival (P = 0.035 and P = 0.045, respectively). Conclusions. : Our pilot study demonstrates that P-gp expression may be a reliable independent prognostic factor of survival in patients with ovarian serous carcinoma. Moreover, P-gp immunostaining may be useful for dividing ovarian carcinoma patients into chemoresponsive and chemoresistant groups. [Copyright &y& Elsevier]

Abstract: Although ischemia is the leading cause of acute renal failure in human, there is little information on the remodeling the kidney endothelium matrix during ischemic injury. In this study, we investigated the activity and expression of MMP-2 and MMP-9, in an isolated endothelial fraction following an acute in vivo reversible ischemia induced in rats by vascular clamping. Ischemia increased serum creatinine levels 1.4-fold, hallmark of acute renal failure. Isolation of the endothelial cell fraction was performed by affinity chromatography using an anti-PECAM-1 antibody. The isolated fraction was assessed by Western blotting analysis of endothelial cell markers. The positively selected fractions were enriched in the endothelial markers eNOS and PECAM-1 by 128-fold and 44-fold, respectively. Gelatin zymography showed that ischemia strongly stimulated proteolytic activity of proMMP-2 (1.8-fold), proMMP-9 (3-fold) and MMP-9 (4-fold) in the endothelial fractions. Western blot analysis indicated that TIMP-2 protein level increased by 3.2-fold in the endothelial fractions during ischemia. Surprisingly, TIMP-1 was absent from the endothelial preparations but was easily detected in the non-endothelial cells. Levels of the endocytic receptor LRP were increased by 2-fold during ischemia in the endothelial fractions. Occludin, a known in vivo MMP-9 substrate, was partly degraded in the endothelial fractions during ischemia, suggesting that the MMP-9 which was upregulated during ischemia was functional. These data suggest that ischemia in kidney could lead to the degradation of the vascular basement membrane and to increased permeability. This suggests new therapeutic approaches for ischemic pathologies by targeting MMP-9 and its regulators. [Copyright &y& Elsevier]

Abstract: The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the expanding LDL receptor family, and is closely related to LRP. It was discovered as a putative tumor suppressor, and is frequently inactivated in human malignant tissues. However, the expression pattern of LRP1B in normal human tissues was unclear. In the present study, we analyzed LRP1B expression in normal mouse and human tissues. By using RT-PCR, we found that, while mouse LRP1B expression is mostly restricted to the brain, human LRP1B expression is more widespread with highest expression levels detected in the brain, adrenal gland, salivary gland, and testis. Although mouse LRP1B expresses in the forms of both full-length receptor tail and an alternatively spliced form lacking a 33-amino acid insert, human LRP1B is expressed exclusively in the form of full-length receptor tail. Finally, we found that, unlike mouse LRP1B, human LRP1B is cleaved by furin. Taken together, these data demonstrate that there are striking differences between LRP1B expression in mouse and human tissues. The broader expression pattern of LRP1B in human tissues suggests that this putative tumor suppressor may play roles in several types of human cancer. [Copyright &y& Elsevier]

The leucine-responsive regulatory protein (Lrp) of Escherichia coli activates expression of a number of operons and represses expression of others. For some members of the Lrp regulon, exogenous leucine mitigates the effect of Lrp, for some it potentiates the effect of Lrp, and for others it has no effect on Lrp action. For the ilvIH operon that we study, Lrp activates expression in vivo and mediates the repression of the operon by exogenous leucine. We studied Lrp-1, a leucine-insensitive variant, to investigate mechanisms by which leucine alters Lrp action as an activator of ilvIH expression. The Asp114Glu change did not have much effect on the amount of total Lrp-1 in cells but decreased the amount of free Lrp-1 two- to threefold. Lrp monomers associate to form octamers and hexadecamers (hexadecamer form predominates at micromolar concentrations; Kd=5.27×10-8M), and leucine promotes the dissociation of Lrp hexadecamer to a leucine-bound octamer. By contrast, Lrp-1 exists primarily as an octamer in solution (equilibrium dissociation constant 6.5×10-5M) and leucine had little effect on the equilibrium. Thus, the hexadecameric form that Lrp assumes in the absence of DNA is not required for activation of the ilvIH operon. Both leucine and the lrp-1 mutation reduced the apparent affinity of Lrp binding to ilvIH DNA (contains two groups of binding sites separated by 136bp) but they have different effects on intrinsic binding affinity and binding cooperativity. Whereas leucine reduced intrinsic binding affinities and interactions of Lrps bound at upstream and downstream regions of ilvIH DNA, it increased cooperative dimer–dimer interactions of Lrps bound to two adjacent sites. By contrast, the lrp-1 mutation did not have much effect on intrinsic binding affinities but it decreased cooperative adjacent dimer–dimer interactions and enhanced interactions of Lrps bound at upstream and downstream regions of ilvIH DNA. Our analysis is consistent with the idea that leucine enhances dimer–dimer interactions that contribute to octamer formation, concomitantly reducing dimer–dimer interactions that contribute to the longer range interactions of Lrps that are required for activation of the ilvIH promoter. [Copyright &y& Elsevier]

Abstract: Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly. [Copyright &y& Elsevier]

The low-density lipoprotein receptor-related protein (LRP) is a large (>600 kDa) multi-ligand-binding cell surface receptor that is now known to participate in a diverse range of cellular events. To accomplish this diverse role, LRP is composed of repetitive amino acid motifs consisting of complement-type and EGF precursor-type repeats. Within these repeats are six conserved cysteine residues that form the core disulfide bond structure of each repeat. To accommodate the intricate folding that such a complex structure dictates, a specialized chaperone is present in the endoplasmic reticulum (ER) called the receptor-associated protein (RAP) that binds to LRP immediately following its biosynthesis and assists in its exocytic transport. Interestingly, RAP −/− mice show reduced LRP expression in certain cell types, but not a more global affect on LRP expression that was expected. Such a tissue-restricted effect by RAP prompted an investigation if other ER chaperones associate with LRP to assist in its complex folding requirements and compensate for the absence of RAP in RAP −/− cells. Fibroblasts obtained from RAP −/− mice demonstrate similar LRP expression levels and subcellular distribution as RAP +/+ fibroblasts. Moreover, RAP −/− cells show an identical exocytic trafficking rate for LRP as RAP +/+ cells and comparable cell surface internalization kinetics. In RAP −/− cells, three well-known ER chaperones, calnexin, calreticulin, and protein disulfide isomerase (PDI), associate with LRP and likely compensate for the absence of RAP. [Copyright &y& Elsevier]

Studies on multidrug resistance (MDR) require a sensitive and quantitative assay of mRNA expression in clinical tumor samples. Based on the small size, heterogenity, and the possibility of partial degradation of clinical specimens, unambiguous data are often difficult to obtain. The aim of the present study was to develop a multiplex polymerase chain reaction (PCR) in combination with nested PCR for quantitative analyses of mRNA expression of MDR1, MRP (multidrug resistance protein), and DNA topoisomerase IIα in small amounts of tumor tissue. RNA samples extracted from the human cell line RPMI 8226 and its MDR sublines 8226/Dox6 and DOXint40c, that overexpress MDR1 and MRP, respectively, were used as model substrates. In the first step, cDNAs of the three genes as well as of the housekeeping gene β-actin were simultaneously amplified in single tubes using 20 cycles of PCR after random-primed reverse transcription. When necessary, a second amplification step of the preamplified PCR products was employed using nested primer pairs. Primer competition was evaluated by analyses of serially diluted amounts of cDNA and at different numbers of PCR cycles. Based on the results obtained, this multiplex/nested PCR approach may provide a base for quantitative analyses of MDR1, MRP, and topoisomerase IIα mRNA expression in clinical tumor biopsies. [Copyright &y& Elsevier]

Lrp is a global regulator of metabolism in Escherichia coli that helps cells respond to changes in environmental conditions. The action of Lrp as a transcriptional activator or repressor is sometimes affected when the medium contains exogenous leucine. In this study, we examined the thermodynamics of leucine binding to Lrp and to a leucine response mutant, Lrp-1, and leucine-induced dissociation of Lrp hexadecamer to leucine-bound octamer. The results of dynamic light-scattering and fluorescence measurements suggest that Lrp has two leucine-binding sites, one a high-affinity site and the other a low-affinity site that is coupled to the dissociation reaction. The Gibbs free energy change for leucine binding to the high-affinity site is about −7.0 kcal/mol. Binding of two leucine molecules to low-affinity sites on the hexadecamer or one leucine molecule to one octamer induces the dissociation of hexadecamer to leucine-bound octamer. The Gibbs free energy change for leucine binding to the low-affinity site was estimated to be in the range −4.66 to −5.03 kcal/mol for leucine binding to an octamer or −6.01 to −6.75 kcal/mol for leucine binding to a hexadecamer. The thermodynamic parameters derived from this study were used together with other data to estimate the distribution of free Lrp hexadecamer, octamer, leucine-bound hexadecamer, and leucine-bound octamer in cells. Mathematical modeling, employed to simulate modulation of Lrp action in response to growth conditions, gave results that are consistent with known patterns of Lrp action on different operons. [Copyright &y& Elsevier]

Victims of urban violence are at risk of developing Posttraumatic Stress Disorder (PTSD), one of the most debilitating consequences of violence. Considering that PTSD may be associated with inefficient selection of defensive responses, it is important to understand the relation between motor processing and PTSD. The present study aims to investigate the extent to which the severity of posttraumatic stress symptoms (PTSS) is related to motor preparation against visual threat cues in victims of urban violence. Participants performed a choice reaction time task while ignoring a picture that could be threating or neutral. The EEG indices extracted were the motor-related amplitude asymmetry (MRAA) in the alpha frequency range, and the lateralized readiness potential (LRP). We observed a linear relation between longer LRP latency and a slower reaction time, selectively during threat processing (compared to neutral) in low PTSS, but not in high PTSS participants. Alpha MRAA suppression and the PTSS were also linearly related: the smaller the alpha MRAA suppression in the threat condition relative to neutral, the greater the PTSS. These results provide evidence that threatening cues affect motor processing that is modulated by the severity of PTSS in victims of urban violence. [ABSTRACT FROM AUTHOR]