Quantification of apolipoprotein E receptors in human brain-derived cell lines by real-time polymerase chain reaction

Apolipoprotein (apo) E4 is a risk factor for Alzheimer's disease (AD) and other neurodegenerative diseases, compared to wild-type apoE3. The mechanism(s) is unknown. One possibility, demonstrated in peripheral tissue cell lines, is that apoE stimulates nitric oxide synthase (NOS) via a receptor-dependent signalling pathway and that apoE4 generates inappropriate amounts of nitric oxide (NO) compared to apoE3. Prior to biochemical investigations, we have quantified the expression of several candidate receptor genes, including low-density lipoprotein-receptor (LDL-r) family members and scavenger receptor class B, types I and II (SR-BI/II), as well as the three NOS isoenzymes and protein kinase B (Akt), in 38 human cell lines, of which 12 derive from brain. Expression of apoE receptor 2 (apoER2), a known signalling receptor in brain, was readily detected in SH-SY-5Y and CCF-STTG1 cells, common models of neurons and astrocytes, respectively, and was highest in H4 neuroglioma, NT-2 precursor cells and IMR-32 neuroblastoma cells. Transcripts of the other lipoprotein receptors were widely, but variably, distributed across the different cell types. Of particular note was the predominant expression of SR-BII over SR-BI in many of the brain-derived cells. As the C-terminus of SR-BII, like apoER2, contains potential SH3 signalling motifs, we suggest that in brain SR-BII functions as a signal transducer receptor. (c) 2004 Elsevier Inc. All rights reserved

Children, family and the state : revisiting public and private realms

The state is often viewed as part of the impersonal public sphere in opposition to the private family as a locus of warmth and intimacy. In recent years this modernist dichotomy has been challenged by theoretical and institutional trends which have altered the relationship between state and family. This paper explores changes to both elements of the dichotomy that challenge this relationship: a more fragmented family structure and more individualised and networked support for children. It will also examine two new elements that further disrupt any clear mapping between state/family and public/private dichotomies: the third party role of the child in family/state affairs and children's application of virtual technology that locates the private within new cultural and social spaces. The paper concludes by examining the rise of the 'individual child' hitherto hidden within the family/state dichotomy and the implications this has for intergenerational relations at personal and institutional levels

Chemoselective polymerizations from mixtures of epoxide, lactone, anhydride, and carbon dioxide

Controlling polymer composition starting from mixtures of monomers is an important, but rarely achieved, target. Here a single switchable catalyst for both ring-opening polymeri-zation (ROP) of lactones and ring-opening copolymerization (ROCOP) of epoxides, anhydrides and CO2 is investigated, using both experimental and theoretical methods. Different combinations of four model monomers: -caprolactone, cyclohexene oxide, phthalic anhydride and carbon dioxide are investigated using a single dizinc catalyst. The catalyst switches between the distinct polymerization cycles and shows high monomer selectivity resulting in block sequence control and predictable compositions (esters and car-bonates) in the polymer chain. The understanding gained of the orthogonal reactivity of monomers, specifically con-trolled by the nature of the metal-chain end group, opens the way to engineer polymer block sequences

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope

AbstractBackground: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60 kD importin protein (importin 60) as a single polypeptide.Results We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)–nitrolotriacetic acid–Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin–agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors.Conclusion Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope

Fast continuous alcohol amination employing a hydrogen borrowing protocol

A continuous flow method for the direct conversion of alcohols to amines via a hydrogen borrowing approach is reported.The authors are grateful to Eli Lilly & Co. (RL) and the EPSRC (SVL, grants EP/K009494/1, EP/M004120/1 and EP/K039520/1) for financial support. This work was supported by Eli Lilly & Co. through the Lilly Research Award Program (LRAP)

MetaCAM: Ensemble-Based Class Activation Map

The need for clear, trustworthy explanations of deep learning model predictions is essential for high-criticality fields, such as medicine and biometric identification. Class Activation Maps (CAMs) are an increasingly popular category of visual explanation methods for Convolutional Neural Networks (CNNs). However, the performance of individual CAMs depends largely on experimental parameters such as the selected image, target class, and model. Here, we propose MetaCAM, an ensemble-based method for combining multiple existing CAM methods based on the consensus of the top-k% most highly activated pixels across component CAMs. We perform experiments to quantifiably determine the optimal combination of 11 CAMs for a given MetaCAM experiment. A new method denoted Cumulative Residual Effect (CRE) is proposed to summarize large-scale ensemble-based experiments. We also present adaptive thresholding and demonstrate how it can be applied to individual CAMs to improve their performance, measured using pixel perturbation method Remove and Debias (ROAD). Lastly, we show that MetaCAM outperforms existing CAMs and refines the most salient regions of images used for model predictions. In a specific example, MetaCAM improved ROAD performance to 0.393 compared to 11 individual CAMs with ranges from -0.101-0.172, demonstrating the importance of combining CAMs through an ensembling method and adaptive thresholding.Comment: 9 page

The Drosophila snr1 and brm Proteins are Related to Yeast SWI/SNF Proteins and are Components of a Large Protein Complex

During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin

The Drosophila snr1 and brm Proteins are Related to Yeast SWI/SNF Proteins and are Components of a Large Protein Complex

During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin

Impact of PNKP mutations associated with microcephaly, seizures and developmental delay on enzyme activity and DNA strand break repair

Microcephaly with early-onset, intractable seizures and developmental delay (MCSZ) is a hereditary disease caused by mutations in polynucleotide kinase/phosphatase (PNKP), a DNA strand break repair protein with DNA 5'-kinase and DNA 3'-phosphatase activity. To investigate the molecular basis of this disease, we examined the impact of MCSZ mutations on PNKP activity in vitro and in cells. Three of the four mutations currently associated with MCSZ greatly reduce or ablate DNA kinase activity of recombinant PNKP at 30°C (L176F, T424Gfs48X and exon15Δfs4X), but only one of these mutations reduces DNA phosphatase activity under the same conditions (L176F). The fourth mutation (E326K) has little impact on either DNA kinase or DNA phosphatase activity at 30°C, but is less stable than the wild-type enzyme at physiological temperature. Critically, all of the MCSZ mutations identified to date result in ∼10-fold reduced cellular levels of PNKP protein, and reduced rates of chromosomal DNA strand break repair. Together, these data suggest that all four known MCSZ mutations reduce the cellular stability and level of PNKP protein, with three mutations likely ablating cellular DNA 5'-kinase activity and all of the mutations greatly reducing cellular DNA 3'-phosphatase activity